Citation
Characterization and quantification of cuticular hydrocarbons in the harvester ant pogonomyrmex occidentalis and the pavement ant

Material Information

Title:
Characterization and quantification of cuticular hydrocarbons in the harvester ant pogonomyrmex occidentalis and the pavement ant
Creator:
Chen, Chao Yu
Publication Date:
Language:
English
Physical Description:
x, 155 leaves : ; 28 cm

Subjects

Subjects / Keywords:
Harvester ants ( lcsh )
Pavement ant ( lcsh )
Cuticle ( lcsh )
Hydrocarbons -- Analysis ( lcsh )
Cuticle ( fast )
Harvester ants ( fast )
Hydrocarbons -- Analysis ( fast )
Pavement ant ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 152-155).
Statement of Responsibility:
by Chao Yu Chen.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
318448349 ( OCLC )
ocn318448349
Classification:
LD1193.L46 2008m C43 ( lcc )

Downloads

This item has the following downloads:


Full Text
CHARACTERIZATION AND QUANTIFICATION OF CUTICUFAR HYDROCARBONS IN THE HARVESTER ANT Pogonomyrmex occidentalis AND THE PAVEMENT ANT Tetramorium caespitum
by
Chao Yu Chen
B.S., Normal Kaohsiung National University, Taiwan. 2005
A thesis submitted to the University of Colorado Denver in partial fulfillment of the requirements for the degree of Master of Science
Chemistry
2008


This thesis for the Master of Science
degree by Chao Yu Chen has been approved by
try avV .
Dr. Marc A. Donsky, Ph.D.
tr


Chen, Chao Yu (Master of Science)
Characterization and Quantification of Cuticular Hydrocarbons in the Harvester Ant Pogonomyrmex occidentalis and the Pavement Ant Tetramorium cespitum.
Thesis directed by Douglas F. Dyckes and Marc A. Donsky.
These recognition cues are often present in the suite of hydrocarbon molecules present on the cuticle, known as a hydrocarbon profile. Social insects can use variation in hydrocarbon profiles as recognition cues in order to recognize group membership of other individuals they interact with. The cuticular hydrocarbons of the western harvester ant Pogonomyrmex occidentalis and the pavement ant Tetramorium cespitum were characterized and quantified. Five ant colonies and bulk ant samples were analyzed by GC/MS with both electron ionization and methane chemical ionization. Oleic acid is present in all harvester ant colonies but only tin two pavement ant colonies, which could be an indication that diet does change their cuticular lipid composition. Genetic relative or close colony locations may be similar in their cuticular hydrocarbon patterns. The similarity of hydrocarbon profiles might be related to the aggression levels against non-nestmate conspecific species or heterospecific species. The range in total carbon length of harvester ant P occidentalis is shorter and P. bartatus. Recognition behavior may correlate to one or some internally methyl alkanes or the percent composition in the shared common internally methyl alkanes. Possible recognition cues were listed on the tables for each species. The cuticular hydrocarbon profiles in pavement ant T. cespitum are more consistent than in harvester ant P. occidentalis, which may be relative to the unicolonial polygyny in pavement ant T. cespitum. The results give some interesting ideas for future directions. More bioassays are necessary to investigate the roles of individual hydrocarbon compounds in chemical communication.
This abstract accurately represents the content of the candidate's thesis. I recommend its publication.
ABSTRACT
Signed


ACKNOWLEDGEMENT
I would like to express my gratitude to Dr. Marc Donsky, Dr. Mike Greene and Dr. Douglas Dyckes who guided me the direction of my thesis; Dr. Karen Jonscher and Dr. Touraj Shokati who provided assistance on instruments for my research; Jeff Boon and Dr. Larry Anderson who taught me and made analytical chemistry interesting.


CONTENTS
Figures.........................................................................viii
Tables..........................................................................ix
Chapter
1. Introduction...................................................................1
1.1 Research Aims..............................................................1
1.2 Composition of Social Insect Lipids........................................2
1.3 Nestmate and Task Recognition..............................................2
1.4 Weatern Harvester Ant and Pavement Ant.....................................3
2. Chemistry Background...........................................................6
2.1 Gas Chromatography Mass Spectrometry.......................................6
2.1.1 Electron Ionization...................................................6
2.1.2 Chemical Ionization...................................................6
3. Compound Identification........................................................7
3.1 Retention Index Systems....................................................7
3.1.1 Kovats Index..........................................................8
3.1.2 Equivalent Chain Length...............................................9
3.2 Mass Spectra of Aliphatic Hydrocarbons
10


3.2.1 Saturated Compouds..................................................10
3.2.2 Unsaturated Compounds...............................................11
3.2.3 Positive Chemical Ionization Mass Spectra...........................13
4. Ant Cuticular Hydrocarbon Profile Analyses...................................15
5. Experimental.................................................................16
5.1 Reagents.................................................................16
5.2 Field Sites and Ant Collection...........................................16
5.3 Methods..................................................................16
5.3.1 Ant Sample Extraction and Sample Separation.........................16
5.3.2 Chemical Analysis...................................................17
5.3.3 Data Analysis.......................................................17
6. Results and Discussions......................................................19
6.1 Results for Research Aim 1...............................................19
6.1.1 Mass Spectra and Characterization of Harvester Ant Cuticular
Hydrocarbons........................................................19
6.1.2 Quantification of Harvester Ant Cuticular Hydrocarbons..............22
6.2 Results for Research Aim 2...............................................29
6.3 Results for Research Aim 3...............................................34
6.4 Results for Research Aim 4...............................................38
vi


6.4.1 Mass Spectra and Characterization of Pavement Ant Cuticular
Hydrocarbons.........................................................38
6.4.2 Quantification of Pavement Ant Cuticular Hydrocarbons...............39
6.5 Results for Research Aim 5...............................................45
7. Conclusion...................................................................51
8. Future Direction.............................................................54
Appendix
A...............................................................................56
B..............................................................................102
References.....................................................................152
vii


LIST OF FIGURES
Fig. 1.4.1 The Western Harvester Ant P. occidentalis...............................5
Fig. 1.4.2 The Pavement Ant T. caespitum...........................................5
Fig. 3.1 The linear relationship of the logarithm of the retention time and
carbon chain length.......................................................8
Fig. 3.2.1 Mass spectrum of n-hexadecane..........................................11
Fig. 3.2.2.1 Allylic cleavage in a mono-olefin....................................12
Fig. 3.2.2.2 Mass spectra of 1-butene and 2-butene................................12
Fig. 3.2.3 Methane Cl mass spectra of n-decene isomers............................14
Fig. 5.3.3 Integration parameters for ant cuticular hydrocarbon quantification....18
Fig. 6.1.1.1 Tinear relationship between ECL and identified n-alkanes.............21
Fig. 6.1.1.2 Linear relationship between 1 and identified n-alkanes...............21
Fig. 6.2.1 Shared cuticular hydrocarbons of harvester ant colonies...............31
Fig. 6.2.2 Shared cuticular hydrocarbons of individual harvester ant colonies....32
Fig. 6.5.1 Shared cuticular hydrocarbons of pavement ant colonies................47
Fig. 6.5.2 Shared cuticular hydrocarbons of individual pavement ant colonies.....48
viii


LIST OF TABLES
Table 6.1.1.1 Averages and standard deviations of shared rc-alkanes of harvester ant
cuticular hydrocarbon profiles....................................20
Table 6.1.2.1 Percent composition of harvester ant cuticular hydrocarbons
from colony #1....................................................22
Table 6.1.2.2 Percent composition of harvester ant cuticular hydrocarbons
from colony #2.....................................................23
Table 6.1.2.3 Percent composition of harvester ant cuticular hydrocarbons
from colony #4.....................................................24
Table 6.1.2.4 Percent composition of harvester ant cuticular hydrocarbons
from colony #B.....................................................25
Table 6.1.2.5 Percent composition of harvester ant cuticular hydrocarbons
from colony #C.....................................................26
Table 6.1.2.6 Percent composition of harvester ant cuticular hydrocarbons
of Bulk Ant II.....................................................27
Table 6.1.2.7 Percent composition of harvester ant cuticular hydrocarbons
of Bulk Ant I......................................................28
Table 6.2.1.1 Percent composition of seven harvester ant P. occidentalis
samples............................................................29
Table 6.2.1.2 The percent composition differences in cuticular hydrocarbons
among harvester ant colonies.......................................33
Table 6.3.1 Hydrocarbon percent compositions in the harvester ant P. occidentalis
and P. bartatus......................................................35
Table 6.3.2 The percent composition differences in cuticular hydrocarbons
of harvester ant P. occidentalis and P. bartatus.....................36
IX


Table 6.3.3 Percent composition of hydrocarbons from laboratory maintained
harvester ant P. bar bat us........................................37
Table 6.4.2.1 Percent composition of pavement ant cuticular hydrocarbons
from colony SP3..................................................39
Table 6.4.2.2 Percent composition of pavement ant cuticular hydrocarbons
from colony Speer 3..............................................40
Table 6.4.2.3 Percent composition of pavement ant cuticular hydrocarbons
from colony SP4..................................................41
Table 6.4.2.4 Percent composition of pavement ant cuticular hydrocarbons
from colony NC-3.................................................42
Table 6.4.2.5 Percent composition of pavement ant cuticular hydrocarbons
from colony K.C-1................................................43
Table 6.4.2.6 Percent composition of pavement ant cuticular hydrocarbons
from Bulk Ant 1..................................................44
Table 6.5.1 Percent composition of pavement ant Tetramorium caespitum
cuticular hydrocarbons...........................................45
Table 6.5.2 The percent composition differences of cuticular hydrocarbons among
pavement ant colonies..............................................49


1. Introduction
1.1 Research aims
All insects envelop various polymeric components and their surfaces are usually covered with complex mixtures of long chain compounds. The outer layer of the insects cuticle is coated with lipids that consist of mainly aliphatic compounds. The large ratio of insects surface lipids to cuticle volume prevents excessive transpiration of water. Early studies on insect surface lipids primarily focused on their function and physiology, which have evolved to prevent desiccation, to form a barrier to microorganisms, and to prevent the absorption of toxic chemicals from the environment (Jackson and Blomquist, 1976).
More recently cuticular hydrocarbons have been recognized as having a key role in chemical communication, including species recognition, task allocation, and nestmate recognition in social insects (Nelson et al., 2001). The goal of this study was to structurally identify, chemically characterize, and quantify the cuticular hydrocarbons of both the Western Harvester Ant Pogonomyrmex occidentalis and the Pavement Ant Tetramorium cespitum.
The following research aims were pursued:
Research Aim 1: To characterize and quantify the western harvester ant cuticular hydrocarbons.
Research Aim 2: To investigate the composition of harvester ant cuticular hydrocarbons among colonies.
Research Aim 3: To compare my results of harvester ant cuticular hydrocarbons with previously published results.
1


Research Aim 4: To characterize and quantify pavement ant cuticular hydrocarbons.
Research Aim 5: To investigate the composition of pavement ant cuticular hydrocarbons among colonies.
1.2 Composition of social insect surface lipids
The major components of social insect cuticle lipids are hydrocarbons with a small amount of fatty acids, alkyl esters, alcohols, glycerides, sterols, aldehydes, and ketones (Lockey, 1988). There are three main classes of insect cuticular hydrocarbons: saturated straight-chain alkanes, methyl-branched alkanes and alkenes. n-Alkanes and branched alkanes are usually the most abundant of the cuticular hydrocarbons, ranging from about 11 to 43 carbon chain length (Howard and Blomquist, 2005).
-Tricosane, n-pentacosane, -heptacosane, -nonacosane and ^-hentriacontane are often predominant in n-alkanes of insect cuticular hydrocarbons. Mono-methyl alkanes, dimethylalkanes and trimethylalkanes, referred to as methyl-branched alkanes, and their isomers exist in all insect cuticular lipids (Nelson. 1978; Lockey, 1988). The major hydrocarbon components found on the surface lipids are mostly methyl-branched alkanes. However, ^-alkanes are the most abundant of the cuticular hydrocarbons in some species or at different stages (Nelson, 1993).
1.3 Nestmate and task recognition
Cuticular hydrocarbons contain nestmate recognition cues. Wagner showed a connection between cuticular hydrocarbons and nestmate recognition. Small glass blocks were coated with whole cuticular lipids and the purified hydrocarbons to test on harvester ant
2


colonies. The result revealed that the ants had aggression to the glass blocks with non-nestmate cuticular hydrocarbons (Wagner et al., 2000). Within the similar compositions of n-alkanes in insect cuticular hydrocarbons, Lucas suggested that methyl-branched alkanes have strong involvement in insect recognition behavior (Lucas, 2005) due to their diverse variations in the position or number of methyl groups. In a harvester ant colony, patrollers go out in the early morning to locate seed sources. The patrollers return to the nest and inform the foragers who go out. bring the food back, and store it in the nest. Nest maintenance workers repair and maintain the chamber, stuff the walls with moist soil to dry and firm the nest, and carry the excavated dry soil out of the nest. Thus, patrollers and foragers, spending most of their time outside of the nest, were found to have a higher proportion of straight-chain alkanes in their cuticular hydrocarbons than the nest maintenance workers (Wagner et al., 1998). A subsequent study revealed that hot and dry conditions increase the proportion of 77-alkanes in ant cuticular hydrocarbons (Wagner et al, 2001). Further experiments investigated responses in different task groups in the whole colony. Greene and Gordon mimicked the flow of returning patrollers by dropping the glass beads coated with cuticular hydrocarbons of patrollers, cuticular lipids of patrollers, cuticular hydrocarbons of nest maintenance workers, and a solvent blank test. In the results, the patrollers' cuticular hydrocarbons were sufficient to draft foragers out of the nest, while blank and nest maintenance workers' hydrocarbons did not elicit the foraging response (Greene and Gordon, 2003).
1.4 Western harvester ant Pogonomyrmex occidentalis and pavement ant
Tetramorium caespitum
The western harvester ant (Fig. 1.4.1.) is very common in the southwest United States and throughout Arizona desert. The size of a harvester ant is about 5-7 mm, and seeds are their main source of food. The size of a harvester ant colony consisting of one single queen is about 10,000 to 12,000 ants. This population makes the harvester ant a good


model for cuticular hydrocarbon communication studies (Howard and Blomquist, 2005).
The pavement ant Tetramorium caespitum (Fig. 1.4.2.) is native to Europe and was introduced to the United States during 17th century. It is now a common household pest in the United States. The pavement ant is dark brown to black in color and around 3.2 mm in length. Their sources of food are almost anything, such as honey, insects, seeds, fruits, cheese, and grease. Their name comes from their tendency to nest under stones, sidewalks or wood boards. They are very aggressive and invade other colonies, resulting in huge battles.
4


Figure 1.4.1. The western harvester ant Pogonomyrmex occidentalis Picture taken from http://www.alexanderwild.com/
a. b.
Figure 1.4.2. The pavement ant Tetramorium caespitum Identifying characteristics of pavement ant Tetramorium caespitum. Picture taken from http://www.ipm.ucdavis.edu/TOOLS/ANTKEY b Picture taken from http://www.pbase.com/tmurrav74/image/57390305
5


2. Chemistry Background
2.1 Gas chromatography mass spectrometry (GC-MS)
Gas chromatography combined with a mass spectrometer is the most useful tool for environmental analyses. It has been used for separating, characterizing, and quantifying insect cuticular hydrocarbons in the past years. Both electron impact ionization (El) and chemical ionization (Cl) are used for the analysis of cuticular hydrocarbon.
2.1.1 Electron impact ionization (El)
Vaporized sample molecules entering the ion source of the mass spectrometer collide with a beam of high-energy (70eV) electrons from the electron-generating filament and form positive ions. The energy is transferred from electrons to sample molecules and produce fairly predictable fragment ions from the molecular ions [M]+ (Reaction 2.1.1). These positive ions are pushed out of the source by a repeller potential and accelerated into the mass analyzer.
M + e~ + + 2e (Reaction 2.1.1)
2.1.2 Chemical ionization (Cl)
Chemical ionization is another technique to create ions used in mass spectrometry analyses. In chemical ionization, large amounts of reagent gas are introduced into the ionization chamber and react with emitted electrons to form reagent gas ions. Then they react with sample molecules to form sample ions. Methane is the most common chemical ionization reagent gas due to its characteristic fragment patterns and its ability to react and yield ions with almost every sample molecule. Other reagent gases produce different fragment patterns and may result in better sensitivity for some samples.
6


3. Compound Identification
Insect cuticle extracts contain a mixture of hydrocarbons. The use of a capillary column and temperature programming gas chromatography is a powerful technique for separating the components. Retention time is the major information for identification in gas chromatography. Many compounds may be specified by their retention time in a gas chromatogram. However, if two compounds have close retention times, the identification and differentiation becomes difficult. A mass spectrometer provides additional information for compound identification. Molecular weight and fragmentation patterns can be obtained from mass spectra, so compounds can be identified unambiguously by using retention indices, molecular weight and fragmentation patterns.
3.1 Retention index systems
Kovats Index and Equivalent Chain Length are the two most useful tools for the comparison of retention times between different studies and conditions in GC/MS analyses. The simplest way to identify compounds is to compare retention times of unknown peaks with standard peaks in the chromatograms under the same experimental conditions. However, not all the unknown compound peaks have the same retention times with standards. The relative retention of a component is the ratio of the retention time of the component to that of the chosen standard. A linear relationship is observed when the logarithm of the retention time is plotted against the number of carbon atoms for hydrocarbon analysis (Figure 3.1). Retention index gives us an idea of the total carbon number in hydrocarbon analysis.
7


U)
o
Log t vs n-alkanes
TS14-5ug/ml TS13-1ug/ml
Carbon Chain Length
Fig 3.1 The linear relationship of the logarithm of the retention time and carbon chain length of 77-alkanes in GC/MS analysis.
3.1.1 Kovats Index (I.)
The Kovats Index, proposed by E. Kovats, expresses that in the homologous series of 17-paraffins in gas chromatography, the retention index (/) of an analyte is obtained by interpolation (Equation 3.1). Under the same stationary phase and isothermal temperature T, the logarithm of the ratio of retention value of the compound of interest 5 to the 77-alkane with z carbon atoms is divided by the logarithm of the ratio of retention values of the two 77-alkane reference compounds. The number is added to the number of carbon atoms of 77-alkane z. To avoid decimal numbers, the quantity is further multiplied by 100. This equation was based on the linear relationship between the logarithm of retention values and the number of carbon atoms of straight-chain saturated fatty acids. The retention indices of straight chain alkane standards are by definition as 100 times of the number of carbon atoms (Kovats, 1958). The Kovats Index is also used for the characterization of branched or unsaturated fatty acids.
8


(3.1)
Equation 3.1 X is the retention value, which refers the adjusted retention time or volume (Ettre, 2003); n is the difference in carbon number of two w-alkanes taken as standards, s is the compound of interest, and z is the number of carbon atoms of the -alkane eluting before the peak of interest.
In Temperature Programming Gas Chromatography (TPGC). the Linear Retention Index equation is applied. Total retention times measured under the same temperature programming conditions are used instead of their logarithm values in this equation.
w-alkanes taken as standards, s is the compound of interest, and z is the number of carbon atoms of the n-alkane eluting before the peak of interest.
3.1.2 Equivalent Chain Length (ECL)
This system is similar to Kovats Index, but with the advantage in representing the number of carbon atoms directly (Miwa. 1963). ECL is expressed as the following equation:
TPGC
= lOOn tBW~tRW + z
(3.2)
_tR(z+n) tR(z)
Equation 3.2 tR is the gross retention time of a substance, n is the difference in carbon number of two
9


Figure 3.2.1 Mass spectrum of rz-hexadecane. (Adapted from NIST Mass Spec Data Center http://webbook.nist.uov.)
Chain branching causes a lower abundance of molecular ion and increases the abundances of CnH2n+i+ and CnH2n* ions through cleavage to form more stable carbon cations and the charge retention at the branched carbon with the loss of the largest alkyl radical is favored.
3.2.2 Unsaturated compounds
The mass spectra of mono-olefins show clusters of CnH2n-i+ and Cnfhn* ions also gradually decreasing in abundance with increasing mass. The molecular ion is more prominent than in saturated alkanes for compounds of lower molecular weight, owing to better stabilization of the positive charge by removal of one of the 71-electrons which leaves the carbon skeleton undisturbed. Cis and trans isomers usually have very similar mass spectra.
Alkene ions exhibit allylic cleavage (most favored) (Figure 3.2.2) and vinylic cleavage (less favored). However, a strong tendency of hydrogen rearrangement in the molecular
II


ions results in migration of the double bond along the chain. Hence mass spectra of mono-olefins with double bonds in different positions are generally very similar (Figure 3.2.3). The location of double bond position needs to be further studied.
R
M-R
h2
-c -
M-R,
C=
Ri
-c-
h2
-c -
-R,
Fig. 3.2.2.1 Allylic cleavage in a mono-olefin.
Fig. 3.2.2.2 Mass spectra of 1-butene (left) and 2-butene (right). The mass spectra were adopted from NIST Mass Spec Data Center http://webbook.nist.gov.
3.2.3 Positive chemical ionization mass spectra
12


Chemical ionization involves much lower energy transfer than electron ionization, which makes chemical ionization a softer ionization technique. Therefore, chemical ionization is often used to determine the molecular weight of sample compounds. There are four common positive chemical ionization processes in an ion source with reagent pressure at 0.8-2.0 Torr: proton transfer, hydride abstraction, addition, and charge exchange. The thermodynamics are favorable for hydride abstraction in positive chemical ionization if the hydride-ion affinity of methane reagent gas ions is higher than the hydride-ion affinity of the ion formed by the analytes loss of H'. Hydride abstraction is the main reaction in hydrocarbons because of the low proton affinity of methane. When methane is used as the reagent gas, both CH3+ and C3H5+ reagent ions are capable of hydride abstraction (Reaction 3.2.3.2-3). CHs+ and C2H5+have large hydride ion affinities, which results the loss of H for long-chain hydrocarbons. This reaction is exothermic so the fragmentation of [M-H]+ is abundant as the base peaks in the Cl mass spectrum. Here we denote [M-H]+ ions as quasimolecular ions since molecular ions represent [M*]+ in El spectrum (Gross, 2004)
CH4 + e -> [CH4]+ + 2 e' or (Reaction 3.2.3.1)
CH4 + e -> CH3+ + H" + e"
[CH4*]+ + CH4 CH5+ +CH3* (Reaction 3.2.3.2)
CH3+ + CH4 ->C2H7~ ->C2H5+ + H2 (Reaction 3.2.3.3)
R+ + M -> [M-H]+ + RH (Reaction 3.2.3.4)
(R= CH5+, C2H5+; M=hydrocarbon molecules)
The Cl spectrum shows not only a method of determination for the molecular weight of hydrocarbons; it also provides structural information to characterize of the hydrocarbons. Howard and his colleges investigated the ratios of Cl generated [M-15]+ to the base peak
13


of normal alkane, monomethyl alkane and dimethyl alkane standards. For straight chain alkanes, [M-15]+/base peak= 3-5%, monomethyl alkanes the ratio is about 6-10%. and dimethyl alkanes is 11-20% (Howard et al, 1980).
The methane chemical ionization spectra of mono-olefins consist two ion series:
CnH2n+r alkyl ions from the [M+H]+ ion and CnH2n-i+ alkenyl ions from the [M-H]+ ion. The CnH2n-i+ alkenyl ions are usually more prominent than CnH2n+i+ alkyl ions in the spectra since allylic hydrogens are the most abstracted for rearrangement. An example of methane Cl mass spectra of -decenes is shown in Figure 3.2.3. The clusters of peaks reach the maximum intensity around C/t-Cg (m/z at 55-111) and gradually decrease to the higher mass region. Quasimolecular ions [M+H]+and [M-H]+ can be seen easily in the spectra. Like alkenes in El spectra, there is no reliable indication of the double bond location in methane Cl spectra of olefins (Harrison, 1992).
Fig. 3.2.3 Methane Cl mass spectra of /7-decene isomers (adapted from Harrison, 1992).
14


4. Ant cuticular lipids analyses
In previous studies of insect cuticular hydrocarbon gas chromatography analysis, the presence of branchings or double bonds in alkanes elutes earlier than the n-alkanes with the same total number of carbon atoms. Branching positions in the alkanes also have some effects upon retention times. Internally branched alkane isomers (such as branching position at 15-, 13-, 11-, 9-, or 7-methyl alkanes) elute earlier than the terminally branched alkane isomers (such as 3-, or 2-methyl alkanes) (Lockey, 1988). However, the separation of isomers is not always seen in gas chromatography. Sometimes the isomer mixtures co-elute as one peak in the total ion chromatogram. As an example in the red harvester ant cuticular hydrocarbon profile (Table 6.3.2, page 30), peak 27A contains three methylnonacosane isomers: 15-, 13-, and 9-methylnonacosane.
Fatty acid mixtures are very common components of insect cuticular lipids. The fatty acids contain primary even carbon atoms ranging from 10-36, but shorter in homologous ranging from 14-18 carbons. Laurie acid (12:0), myrystic acid (14:0), palmitic acid (16:0) and stearic acid (18:0) are the most common saturated carboxylic acids. Palmitoleic acid (16:1), linoleic acid (18:2) and oleic acid (18:1) are the most commonly reported unsaturated acids in insect lipids (Lockey, 1988).
15


5. Experimental
5.1 Reagents
-alkane standard mixture 50mg/l in /7-heptane (Sigma-Aldrich, contains Cio, C20, C22, C24, C26, C28, C30, C32, C34, C36, C38, and C40)
Chloroform HPLC grade (Fisher Scientific)
Pentane HPLC grade (Sigma-Aldrich)
5.2 Field sites and ant collection
Ants were collected by Greene and Sano. Harvester ants were collected in the summer of 2007 from colony nest mounds at the permanent field site next to the Denver Children's Museum and the South Platte River using gloved-hand then placed into collection tubes. Pavement ants were collected in 2006 from nests found on the University of Colorado Denver Campus and Downtown Denver area using an aspirator from foraging trails or by using honey bait to lure foragers out of the nest.
5.3 Methods
5.3.1 Ant sample extraction and sample preparation
Cuticular hydrocarbons were extracted by Greene. Ants from all colonies were collected in the field and killed by freezing at -20C. Cuticular lipids were extracted by soaking ants in 1.0ml 100% /7-pentane. Gently shaken for the first 1 minute of soaking step, after 10 minutes of the soaking step we transferred the lipid extracts onto a solid phase of 2 cm
16


silica gel (70-230 mesh, average pore diameter: 60 A. SIGMA8) in a pasteur pipette. Cuticular hydrocarbons were separated from polar surface lipids by elution from the silica gel using 2-3ml 100% ^-pentane. Extracts were collected in clean glass vials and dried under a stream of nitrogen. The residue was redissolved in 1 ml of chloroform.
5.3.2 Chemical analysis
The cuticular extract samples were analyzed by using gas chromatography mass spectrometry (Agilent 6890N/5973 MSD) in the Anesthesiology Department of the University of Colorado Denver. 2pl of samples were injected by spitless injection onto a capillary column (OV-5 fused silica capillary column, 30m, 0.32mm ID, 0.5pm; Ohio Valley). Samples were purged after 1 min. The carrier gas was helium with the flow rate set to 1.1 ml/min. The injection temperature was 280C. For electron ionization, the initial oven temperature was 170C for 1 min, and then the temperature was increased from 170C to 300C at 100C /min, then more slowly from 300C to 330C at 15C /min. To ensure of the consistency of retention time between runs, a mixture of rc-alkane standard (containsCio, C2o, C22, C24, C26, C28, C30, C32, C34, C36, C38, and C40, Sigma-Aldrich) were injected before sample runs. For chemical ionization GC, the initial temperature was 75C. after 2 minutes the temperature was increased from 75 C to 330C at 15C /min and held at 330C for 10 minutes.
5.3.3 Data analysis
The ant cuticular hydrocarbons were quantitatively analyzed by measuring the peak areas of the resulting chromatograms using Agilent MSD ChemStation Software. RTE Integrator was selected for integration. Only the peak area >1% of the largest peak area were selected for integration for quantitation. The idea of using relative abundance in insect cuticular hydrocarbons is to take out variability of the number of ants in the sample and compare the percent composition for further studies. The integration
17


parameter settings are shown in Fig. 5.3.3.
RTE Integrator Parameters
Detector
D.ata point sampling [T R? Smoothing
Detection filtering 5 point _J
Start threshold 0.200
Stop threshold 0.000
U utpi.it ---------------------
Minimum peak area [ 1.0
% of largest Peak r Area counts
Peak location [Top Maximum number of peaks [250
Baseline Allocation
fiaseline reset (tt points) > [O If leading or trailing edge < |l00.0
B aseline Preference Baseline drop else tangent
Fig. 5.3.3. Integration parameters for ant cuticular hydrocarbon quantitation.
18


6. Results and Disscussion
6.1 Results of research aim 1: To characterize and quantify harvester ant cuticular hydrocarbons
6.1.1 Mass spectra and characterization of harvester ant cuticular hydrocarbons
All structural characterizations were mainly based upon both of El and Cl mass spectra and methods described in Chapter 3. Examples of TIC, mass spectra and structure characterizations of Harvester Bulk Ant I sample are shown in APPENDIX A.
Five harvester ant colonies and two bulk ant samples were analyzed. The cuticular hydrocarbons of harvester ants range in length from 23 to 36 carbon atoms. Six tt-alkanes, eleven mono-methyl alkanes and one dimethyl alkanes were found in five harvester ant colonies. Compounds that are identified are -pentacosane (U-C25), /7-hexacosane (H-C26), 17-heptacosane (n-C27). -octacosane(-C2s), ft-nonacosane (-C29)and /r-hentriacontane (17-C31) in straight chain alkanes; 7-, 3-methylpentacosane (7-, 3-meC25). 8-methylhexacosane (8-meC26), 13-, 7-methylheptacosane (13-, 7-meC27), 13-, 11 9-methylnonacosane (13-, 11 9-meC29), 15-, 13-, and 7-methylhentriacontane (15-, 13-, 7-
meCji) in mono-methyl branched alkanes, and 7, 13-dimethylheptacosane (7, 13-dimeC27) in dimethyl branched alkanes. Trimethyl alkanes or alkenes in the western harvester ant cuticular hydrocarbons were either missing or less than 1% of the largest peak in chromatograms. Fatty acids were found in harvester ant samples except for the Bulk Ants I sample, which passed through the silica gel column to isolate fatty acids from hydrocarbons. Other samples did not pass through the silica gel column so the fatty acids were shown and integrated in total ion chromatograms (Table 6.2.1.1, page 27).
19


Both Kovats Index and ECL retention indices of each identified compound were listed in Tables 6.1.2.1 to 6.1.2.7. The average and standard deviation of shared n-alkanes between harvester ant samples were listed in Table 6.1.1.1. Kovats Index has much bigger standard deviation than ECL due to 100 was multiplied to avoid decimal point. Regardless the factor, the standard deviations of both retention indices is close to each other. Figure 6.1.1.1 and 6.1.1.2 show the linear relationship of straight chain alkanes and their ECL and Kovats Index, respectively. Generally, straight alkanes have number of carbon atoms as ECL and 100 times of the number of carbon atoms as Kovats Index 1.
For instance, the ECL of /7-pentacossane {n-C25) is 25 and / is 2500. Linear relationship does exist before 77-C28 between ECL and 17-alkanes, however for Kovats Index it is off from 77-C26 to 77-C31. Hydrocarbon structurally characterization are primarily based on both El and Cl mass spectra while using retention indices as references.
Avg. STDEV
/7-alkanes / ECL / ECL
H-C25 2503.12 24.96 3.65 0.03
/7-C26 2618.12 25.98 3.65 0.03
/7-C27 2692.22 26.95 3.94 0.04
/7-C28 2796.47 27.97 2.57 0.02
/7-C29 2890.43 28.93 2.57 0.03
77-C31 3091.24 30.95 1.62 0.02
Table 6.1.1.1 Averages and standard deviations of shared /7-alkanes of harvester ant cuticular hydrocarbon profiles.
20


Figure 6.1.1.1 Linear relationship between ECL and identified ^-alkanes.
n-C25 n-C26 n-C27 n-C28 n-C29 n-C31
#2
-*-#4
*#c
BA II BA I
Figure 6.1.1.2 Linear relationship between I and identified ^-alkanes.
21


6.1.2 Quantification of harvester ant cuticular hydrocarbons
Percent composition was calculated from the integrated TIC area as described in Data analysis. Tables 6.1.2.1 to 6.1.2.7 show all the characterized compounds with their relative abundance of harvester ant samples.
Harvester ant Colony #1
Peak R.T.* Pet Total 1.* ECL* Compounds
1 4.505 3.83 2223 22.24 Oleic acid, trimethylsilyl ester
2 4.681 0.55 2298 23.01 fl-Tricosane
3 5.167 4.26 2507 24.99 /7-Pentacosane
4 5.276 5.05 2554 25.41 7-meC25
5 5.368 1.14 2593 25.76 3-meC25
6 5.435 1.89 2622 26.01 -Hexacosane
7 5.544 1.88 2635 26.37 8-meC26
8 5.728 15.94 2694 26.97 -Heptacosane
9 5.837 27.18 2729 27.31 13-, 11-, 7-meC27
10 5.963 3.61 2769 27.71 7,13-dimeC27
11 6.055 1.04 2799 27.99 -Octacosane
12 6.173 1.76 2829 28.31 14-meC28
13 6.424 2.75 2892 28.95 -Nonacosane
14 6.558 14.08 2926 29.29 13-, II-. 9-meC29
15 6.718 3.09 2966 29.68 7,13-dimeC29
16 6.994 1.01 3027 30.29 14-meC3o
17 7.338 1.26 3093 30.96 -Hentriacontane
18 7.506 4.13 3125 31.28 15-, 13-meC3i
19 7.556 2.92 3134 31.38 7-meC3i
20 7.723 1.48 3166 31.68 7,13-dimeC3i
21 8.796 0.37 3325 33.29 1 l-meC33
22 8.855 0.79 3334 33.37 7-meC33
Table 6.1.2.1 Percent composition of harvester ant cuticular hydrocarbons from colony #1.
* R.T. is the retention time shown in GC total ion chromatogram; I. is Kovats Index, and ECL is Equivalent Chain Length.
22


Harvester ant Colony #2
Peak R.T.* Pet Total I.* ECL* Compounds
1 4.102 2.94 2044 20.45 Hexadecanoic acid, trimethylsilyl ester
2 4.488 21.15 2216 22.16 Oleic acid, trimethylsilyl ester
3, 5.150 3.47 2500 24.93 w-Pentacosane
4 5.259 3.04 2546 25.35 7-meC25
5 5.351 0.91 2586 25.69 3-meC25
6 5.418 2.10 2614 25.94 n-Hexacosane
7 5.527 1.14 2629 26.31 8-meC26
8 5.712 21.93 2689 26.92 -Heptacosane
9 5.821 17.15 2724 27.26 13-, 11-, 7-meC27
10 5.946 1.87 2764 27.66 7,13-dimeC27
11 6.039 1.36 2794 27.94 n-Octacosane
12 6.156 1.04 2825 28.26 14-. 8-meC28
13 6.407 4.78 2888 28.91 n-Nonacosane
14 6.541 8.25 2922 29.25 13-, 11-, 9-meC29
15 6.701 0.89 2962 29.64 9,13-dimeC29
16 7.321 2.49 3090 30.93 n-Hentriacontane
17 7.480 2.05 3120 31.23 15-, 13-meC3i
18 7.539 2.04 3131 31.34 7-meC3i
19 8.268 1.41 3252 32.55 unknown
Table 6.1.2.2 Percent composition of harvester ant cuticular hydrocarbons from colony #2.
* R.T. is the retention time shown in GC total ion chromatogram; I. is Kovats Index, and E.C.L. is Equivalent Chain Length.
23


Harvester ant Colony #4
Peak R.T.* Pet Total 1.* ECL* Compounds
1 4.488 0.41 2216 22.16 11-Cis-Octadecenoic acid
2 4.664 0.70 2291 22.94 n-Triacosane
3 5.150 7.20 2500 24.93 77-Pentacosane
4 5.259 3.69 2546 25.35 7-meC25
5 5.351 0.84 2586 25.69 3-meC25
6 5.418 3.53 2614 25.94 rc-Hexacosane
7 5.527 1.31 2629 26.31 8-meC26
8 5.712 30.59 2689 26.92 77-Heptacosane
9 5.821 20.43 2724 27.26 13-, 9-, 7-meC27
10 5.946 2.28 2764 27.66 7,13-dimeC27
11 6.039 1.69 2794 27.94 w-Octacosane
12 6.164 1.24 2827 28.28 8-meC28
13 6.407 5.40 2888 28.91 77-Nonacosane
14 6.541 9.23 2922 29.25 15-, 13-. 11-, 9-, 7-meC29
15 6.701 1.31 2962 29.64 9,13-; 7,13-dimeC29
16 7.321 2.42 3090 30.93 77-Hentriacontane
17 7.489 2.10 3122 31.25 15-, 13-meC3i
18 7.539 3.63 3131 31.34 7-meC3i
19 7.698 0.54 3162 31.64 14-, 7-meC32; 7,13-dimeC3i
20 8.847 1.46 3333 33.36 7-meC33
Table 6.1.2.3 Percent composition of harvester ant cuticular hydrocarbons from colony #4.
* R.T. is the retention time shown in GC total ion chromatogram; 1. is Kovats Index, and E.C.L. is Equivalent Chain Length.
24


Harvester ant Colony #B
Peak R.T.* Pet Total 1/ ECL* Compounds
1 4.496 0.35 2219 22.20 Oleic acid TMS
2 4.664 0.55 2291 22.94 -Tricosane
3 5.150 5.48 2500 24.93 /7-Pentacosane
4 5.259 3.43 2546 25.35 13-, 11 9-, 7-meC25
5 5.351 0.79 2586 25.69 3-meC25
6 5.418 2.57 2614 25.94 n-Hexacosane
7 5.527 1.27 2629 26.31 8-meC26
8 5.712 27.17 2689 26.92 n-Heptacosane
9 5.821 19.29 2724 27.26 13-, 11-, 9-, 7-meC27
10 5.946 2.32 2764 27.66 7,13-dimeC27
11 6.039 1.65 2794 27.94 -Octacosane
12 6.164 1.30 2827 28.28 14-. 8-meC28
13 6.407 5.93 2888 28.91 w-Nonacosane
14 6.541 11.18 2922 29.25 15-, 13-. 11-, 9-. 7-meC29
15 6.701 1.80 2962 29.64 9,13-; 7,13-dimeC29
16 6.826 0.42 2994 29.94 w-Triacontane
17 6.977 0.80 3028 30.30 14-meC3o
18 7.321 3.30 3090 30.93 n-Hentriacontane
19 7.480 3.79 3120 31.23 15-, 13-, 1 l-meC3i
20 7.539 3.70 3131 31.34 7-meC3i
21 7.698 0.86 3162 31.64 14-, 7-meC32; 7,13-dimeC3]
22 8.536 0.76 3289 32.93 -Tritriacontane
23 8.838 1.34 3331 33.35 7-meC33
Table 6.1.2.4 Percent composition of harvester ant cuticular hydrocarbons from colony #B.
* R T. is the retention time shown in GC total ion chromatogram; I is Kovats Index, and E.C.L. is Equivalent Chain Length.
25


Harvester ant Colony #C
Peak R.T.* Pet Total i: ECL* Compounds
1 4.496 0.30 2219 22.20 Oleic acid, trimethylsilyl ester
2 4.672 0.47 2294 22.97 -Tricosane
3 5.158 4.88 2503 24.96 tt-Pentacosane
4 5.267 3.53 2550 25.38 13-, 11-, 9-, 7-meC25
5 5.360 0.99 2590 25.73 3-meC25
6 5.427 2.56 2618 25.98 n-Hexacosane
7 5.536 1.48 2632 26.34 8-meC26
8 5.720 24.70 2691 26.94 -Heptacosane
9 5.829 19.74 2726 27.29 13-, I1-. 9-, 7-meC27
10 5.955 2.29 2767 27.68 7,13-dimeC27
11 6.047 1.68 2796 27.97 -Octacosane
12 6.173 1.55 2829 28.31 14-, 13-. 12-, 8-meC28
13 6.416 5.73 2890 28.93 tf-Nonacosane
14 6.550 12.05 2924 29.27 13-, 11-. 9-, 7-meC29
15 6.709 1.27 2964 29.66 9,13-; 7,13-dimeC29
16 6.835 0.50 2996 29.96 n-Triacontane
17 6.986 1.09 3026 30.27 14-meC3o
18 7.329 3.50 3091 30.95 tt-Hentriacontane
19 7.497 4.12 3123 31.27 15-, 13-, 1 l-meC3i
20 7.547 4.01 3133 31.36 7-meC3i
21 7.707 0.92 3163 31.65 7.13-dimeC3i
22 8.545 0.84 3290 32.94 -Tritriacontane
23 8.847 1.81 3333 33.36 7-meC33
Table 6.1.2.5 Percent composition of harvester ant cuticular hydrocarbons from colony #C.
* R.T. is the retention time shown in GC total ion chromatogram; 1. is Kovats Index, and E.C.L. is Equivalent Chain Length.
26


Harvester Bulk Ant II
Peak R.T.* Pet Total i: ECL* Compounds
1 4.505 0.42 2223 22.24 11-Trans-Octadecenoic acid 1TMS
2 4.681 0.67 2298 23.01 w-Tricosane
3 5.167 7.36 2507 24.99 -Pentacosane
4 5.276 3.14 2554 25.41 13-, I1-. 9-. 7-meC25
5 5.368 0.79 2593 25.76 5-, 3-meC25
6 5.435 2.93 2622 26.01 w-Hexacosane
7 5.544 0.96 2635 26.37 8-meC26
8 5.728 33.57 2694 26.97 tt-Heptacosane
9 5.837 16.46 2729 27.31 13-, 11-, 9-. 7-meC27
10 5.963 2.18 2769 27.71 7,13-dimeC27
11 6.055 1.72 2799 27.99 M-Octacosane
12 6.173 0.85 2829 28.31 14-. 8-meC28
13 6.424 6.71 2892 28.95 n-Nonacosane
14 6.558 8.20 2926 29.29 13-, 11-. 9-, 7-meC29
15 6.718 1.39 2966 29.68 9.13-; 7,13-dimeC29
16 6.843 0.47 2998 29.98 w-Triacontane
17 6.994 0.62 3027 30.29 14-meC3o
18 7.338 3.55 3093 30.96 -Hentriacontane
19 7.505 2.12 3125 31.28 15-, 13-, 1 l-meC3i
20 7.556 3.32 3134 31.38 7-meC3i
21 7.723 0.47 3166 31.68 7,13-dimeC3i
22 8.553 0.73 3291 32.96 rc-Tritriacontane
23 8.855 1.37 3334 33.37 7-meC33
Table 6.1.2.6 Percent composition of harvester ant cuticular hydrocarbons of Bulk Ant II.
* R.T. is the retention time shown in GC total ion chromatogram; I. is Kovats Index, and E.C.L. is Equivalent Chain Length.
27


Harvester Bulk Ant I
Peak R.T.* Pet Total 1.* ECL* Compounds
1 4.681 0.73 2298 23.01 77-Tricosane
2 4.915 0.23 2399 23.99 w-Tetracosane
3 5.167 6.16 2507 24.99 /7-Pentacosane
4 5.276 4.19 2554 25.41 13-, 11-, 9-. 7-meC25
5 5.368 1.03 2593 25.76 3-meC25
6 5.435 2.82 2622 26.01 -Hexacosane
7 5.544 1.56 2635 26.37 8-meC26
8 5.745 16.78 2699 27.02 -Heptacosane
9 5.846 20.09 2732 27.34 13-, 11-. 9-, 7-meC27
10 5.963 2.68 2769 27.71 7,13-dimeC27
11 6.055 1.77 2799 27.99 n-Octacosane
12 6.173 1.55 2829 28.31 14-, 13-, 12-. 8-meC28
13 6.432 6.11 2894 28.97 n-Nonacosane
14 6.558 12.35 2926 29.29 13-, 11-, 9-, 7-meC29
15 6.717 2.14 2966 29.68 9,13-; 7,13-dimeC29
16 6.843 0.51 2998 29.98 -Triacontane
17 6.994 1.15 3027 30.29 14-meC3o
18 7.338 3.86 3093 30.96 w-Hentriacontane
19 7.505 4.38 3125 31.28 15-, 13-, 1 l-meC3i
20 7.556 3.81 3134 31.38 7-meC3i
21 7.715 1.27 3165 31.67 7,13-dimeC3i
22 7.891 0.34 3198 31.99 77-Dotriacontane
23 8.553 1.01 3291 32.96 77-Tritriacontane
24 8.788 0.61 3324 33.28 15-, 13-, 1 l-meC33
25 8.863 2.15 3335 33.38 7-meC33
26 9.064 0.33 3363 33.65 7,13-dimeC33
27 10.523 0.12 3522 35.26 13-meC35
28 10.640 0.30 3534 35.38 7-meC35
Table 6.1.2.7 Percent composition of harvester ant cuticular hydrocarbons of Bulk Ant I.
* R.T. is the retention time shown in GC total ion chromatogram; I. is Kovats Index, and E.C.L. is Equivalent Chain Length.
28


6.2 Results of research aim 2: To investigate harvester ant cuticular hydrocarbons among colonies.
There are about 21 peaks identified in each colony. Seven /7-alkanes, eleven monomethyl alkanes and one dimethyl alkanes were found in five harvester ant colonies. Compounds that were identified included /7-tricosane (/7-C23), -pentacosane (17-C25), -hexacosane (17-C26). /7-heptacosane (/7-C27), /?-octacosane (/7-C28), /7-nonacosane (77-C29)and /7-hentriacontane (77-C31) in straight chain alkanes; 7-, 3-methylpentacosane (7-, 3-meC25), 8-methylhexacosane (8-meC26), 13-, 7-methylheptacosane (13-, 7-meC27), 13-, 11-. 9-methylnonacosane (13-, 11-, 9-meC29), 15-, 13-. and 7-methylhentriacontane (15-, 13-, 7-meC3i) in mono-methyl branched alkanes, and 7, 13-dimethylheptacosane (7. 13-dimeC27) in dimethyl branched alkanes. /7-Heptacosane is the most abundant peak in harvester ant colonies #2, #4, #B. #C. Mono-methyl branched heptacosane and nonacosane are the second and the third largest peaks, respectively. Table 6.2.1.1 shows the proportions of straight chain alkanes, methyl branched alkanes, and fatty acids in harvester ant samples.
Colony #1 ' Colony #2 Colony #4 Colony #B * Colony #C Bulk Ants II Bulk Ants I
Fatty acids3 3.83 24.09 0.41 0.35 0.30 0.42 fb
/7-Alkanes 27.69 36.13 51.53 47.81 44.87 57.71 40.30
Mono-methyl alkanes 60.31 35.61 44.47 47.73 50.35 37.83 53.29
Dimethyl alkanes 8.17 2.76 3.59 4.12 4.48 3.57 6.41
Table 6.2.1.1 Percent composition of seven harvester ant P. occidentalis samples.
a. The fatty acids in Bulk Ants I sample were either missing or less than 1% of the largest peak area while they were shown and integrated in other harvester ant samples, which did not pass through the silica gel column to isolate fatty acids from cuticular hydrocarbons.
29


b. A t indicates the fatty acids were either missing or its less than 1% of the largest peak area in the chromatogram.
The harvester ant cuticular lipids contain mainly n-alkanes and mono-methyl branched alkanes (>70%), with small amount of dimethyl alkanes and fatty acids. Fatty acids were present in small quantity (ca. 0.37%) except in colonies #1 and #2, where fatty acid concentrations were observed in 10-100 folds bigger than in other samples. Oleic acid (18:1) is the most common fatty acid among harvester ant samples. Oleic acid concentrations were 3.83% in colony #1, 21.15% in colony #2, 0.35% in colony #B. and 0.30% in colony #C while 11-octadecenoic acid concentrations were 0.41% in colony #4 and 0.42% in Bulk Ant II. Also, hexadecenoic acid was found in colony #2 (2.94%).
Colony #2 has extremely high concentrations of oleic acid (88% of its total fatty acids), which is present in many of the seeds that the harvester ants eat. But what causes the ants produce such a high abundance of oleic acid on their cuticles? It has been demonstrated that diet alters insects' cuticular hydrocarbon profiles. The source of food of colony #2 might contain excessive oleic acid, reflected in their cuticular lipid composition. It could be a chemical signal of necrophoric behavior. Oleic acid is released from the decaying bodies of dead harvester ants, triggering midden workers' instincts to remove dead bodies from the nest (Gordon, 2000). Another interesting point of view is fatty acids can also be converted to n-alkanes in some insects (Blomquist et al., 1987). Although the biosynthesis pathways of cuticular lipids are not clear, events such as expanding the colony, switching tasks and the responses to diet or to environmental conditions may induce biosynthetic factors that alter the cuticular hydrocarbon profile.
Although most harvester ant cuticular hydrocarbons are detected in all colonies, their relative quantities differ (Figure 6.2.2). Methyl branched alkanes (-68%) in colony #1 are much higher than r?-alkanes (28%) compare to other colonies. In addition, monomethyl heptacosane (-27%) and mono-methyl nonacosane (-14%) are the major contributors in the high abundant methyl alkanes.
30


#2
#4
#B
#C
Figure 6.2.1 Shared cuticular hydrocarbons of harvester ant colonies.
31


#1
30
25
20
15
10
5
0
27.176
T 1
15.941 I 14.084
I 1
I
1 1 B
rA A? A rO? A A A A A A A A A A
A & & A & S> A AAA A A

v v
\''
N A' A &

V'
#2
25
20
15
10
5
0
20X
I 77:151
1 8.25
1 I 1
1 Li 1 H a a
rh rh rfl rfo Jo p,A pA rA rfb rP) rPl r^S ->N
,o y A A A A A A' A A A A A A V"V V* A A V* VV
o'A a N
a
35
30
25
20
15
10
5
0
#4
30.589
20.434

1
9.232

i i.. u u 1 B |
rh A) rh Ao AO rA rA <"A rA> rP rA AS AS .AS
A A A A A A A A A A A A A A
A y y A y A .A? A ^
V V V A' >* A' V

y
r

y
30
25
20
15
10
5
0
#B
27.173 -
19.287

TT.T75
-I
1 1 . H B 1 1 H 1 1
Ap A~) rS) Ao Ao rA rA rA Ak rP rp AS AS AS
A A A A A A A A A A A A A A
A' V V A'\ >N A' x\ A'
?' ?'\y Vv
NV Nv Nv v>
NV Nv N

y~
30
25
20
15
10
5
0
#C
24.704
gj 19.741
1 i 12.045
1 1
1 1 _ 8
1 - 1 1 IB a fii M M
Al A~) rh 0*0 A 'A' A.A.Aa\Aa\A^^A\Aj*J>A
a' A'
A' v V A'\ ,S> 01 S
Nv- V
* V" y /
^ NV-
\
.V
A'
Figure 6.2.2 Shared cuticular hydrocarbons of individual harvester ant colonies.
32


Lucas mentioned that the presence of internally methyl alkanes have strongly correlated with recognition behavior (Lucas, 2005). Eighteen, fifteen, twenty one, twenty eight, and twenty seven internally methyl alkanes were identified in harvester ant colony #1, #2, #4, #B, and #C, respectively. After subtracting those common internally methyl alkanes, the differences in internally methyl alkanes among five harvester ant colonies are shown in Table 6.2.1.2.
#1 #2 #4 #B #C
//-Alkane
/7-C33 0.76% 0.84%
Methyl branched alkanes
1 l-meC27 Pa P P P
9-meC27 P P P
14-meC28 P b. 1.04% 1.30% 1.55%
8-meC28 1.24%
13-meC28
12-meC28
15-meC29 P P
9,13-dimeC29 0.89% 1.31% 1.80% 1.27%
7,13-dimeC29 3.09%
14-meC3o P 0.80% 1.09%
1 l-meC3i P P
7,13-dimeC3i 1.48% 0.54% 0.86% 7.07%
14-meC32
7-meC32
1 l-meC33 0.37%
7-meC33 0.79% 1.46% 1.34% 1.81%
Table 6.2.1.2 The percent composition differences in cuticular hydrocarbons among
33


harvester ant colonies.
A P represents the compound was identified by El mass spectrum, but co-eluted with its isomers or other compounds so the percent composition itself can not be determined.
The percentage with bold borders represents the isomers or compounds co-eluted and integrated as one peak in TIC, which is the total percent composition of these compounds.
In colony #1, 11-methyltritriacontane (ll-meC33) was only compound detected in colony #1, and 7,13-dimethyltritriacontane (7,13-dimethylC33) in colony #C is relatively high in percent composition while in colonies #4 and #B are below 1%. The compounds may be their chemical cues involved in nestmate recognition. Colonies #4, #B and #C have similar cuticular hydrocarbon expression profiles, which might be an indication of they are genetic-related or experience the similar environmental conditions. Task-recognition in harvester ant P. barbatus has been investigated. Task groups have significantly different relative proportions in the classes of hydrocarbons and in the individual compounds (Wagner et al., 1998). Larger ant colony size might require more compounds as task-recognition cues to distinguish different tasks performed. Colonies #B and #C contain the most differences in cuticular hydrocarbons and colony #2 has the fewest individual compounds, which could relate to bigger colony size and older colony age of colonies #B and #C, and smaller colony size and younger colony age of colony #2.
6.3 Results of research aim 3: The comparisons of my results of harvester ant Pogonomyrmex occidentalis cuticular hydrocarbons with previous studies of the red harvester ant Pogonomyrmex barbatus.
Around 42 peaks were detected that are >0.1% of total area in the red harvester ant sample. The carbon chain length of red harvester ant Pogonomyrmex barbatus ranges from 23 to 49 carbon atoms. Table 6.3.3 shows the red harvester ants cuticular hydrocarbons (Nelson et al., 2000). In the western harvester ant (Table 6.1.2.7), only 28 peaks were integrated and identified, which range from C23 to C36. As summarized in
34


Table 6.3.1, the cuticular hydrocarbon profiles of P. occidentalis contain ca. 40 % 17-alkanes. ca. 53% mono-methyl branched alkanes, ca. 6% dimethyl alkanes. Trimethyl alkanes or alkenes were either missing or less than 1% of the largest peak area. P. bartatus has equally represented 77-alkanes and mono-methyl alkanes were present at lower abundance (13%). Dimethyl alkanes, trimethyl alkanes and alkenes only constituted 1-2% of the cuticular hydrocarbon composition. Both of the harvester ant species contain mainly saturated alkanes (>95%) in their cuticular hydrocarbon profiles. The major 77-alkane is 77-heptacosane in P. occidentalis and 77-pentacosane in P bartatus. The major mono-methyl alkane is mono-methyl heptacosane in P. occidentalis and mono-methyl hentriacontane in P. bartatus. Internally methyl branched alkanes are related to recognition behavior. Table 6.3.2 lists the differences in cuticular hydrocarbons of harvester ant P. occidentalis and P. bartatus. Species-specific recognition compound(s) may be one or more of internally methyl alkanes listed on the table. Within a species, ants share the same hydrocarbon components, but the abundance of each compound relative to the others varies between different colonies. However, different species have different cuticular hydrocarbon profiles.
P. occidentalis P. bartatus
77-Alkanes 40.3 37.1
Methyl alkanes 53.3 36.2
Dimethyl alkanes 6.4 13.2
Trimethyl alkanes * t 1.6
Alkenes t 2.3
Table 6.3.1. Hydrocarbon percent composition of the harvester ant P. occidentalis and P. bartatus.
* A t indicates the compounds were either missing or its less than 1% of the largest peak area in the chromatogram.
35


P. occidentalis P. bartatus
n-Alkanes
n-C24 0.2%
-C33 0.7%
Methyl branched alkanes
1 l-meC25 Pa
7,13-dimeC25 P
1 l-meC27 P
5-meC27b 0.5%
9,13-dimeC27 0.8%
3-meC27b P
14-. 13-, 12-, 8-meC28 1.6%
2-meC28 b 0.5%
15-meC29 P
5-meC29b 0.8%
11,15-; 13.17-dimeC29 P
3-meC29b P
15-, 13-, 11-, 8-, 7-meC30 1.1%
2-meC3ob 1.1%
9-meC3i P
13,17-; 11,15-; 9,13-dimeC31 3.8%
11,15,19-C3, 0.5%
15-, 14-, 13-meC32 0.6%
14,18-; 13,x-dimeC32 ' 0.5%
17-. 9-meC33 P
15,19-; 13,19-; 1 l,21-dimeC33 P
13,17,21-; 11,15,21-trimeC33 0.5%
13-meC35 0.1%
7-meC35 0.3%
7.13-dimeC35 0.5%
15,21-dimeC47 0.5%
Table 6.3.2 The percent composition differences in cuticular hydrocarbons of harvester ant P. occidentalis and P. bartatus.
a. A P represents the compound was identified by El mass spectrum, but co-eluted with its isomers or other compounds so the percent composition itself can not be determined.
b. These compounds are considered to be externally methyl alkanes.
36


Peak Pet Total3 Compounds
23 6 r?-Tricosane
25 20.1 n-Pentacosane
25A 2.6 13-. t9-meC25b
25A 1.5 7-meC25
25A\ B 0.7 3-meC25: 7.13-dimeC25
26 1.4 n-Hexacosane
27:1 0.7 rc-Heptacosene
27 4.7 n-lleptacosane
27A 4.3 13-. t9-meC27
27A 3.5 7-meC27
21A 0.5 5-meC27
27B 0.8 9.13-dimeC27
27B 3.0 7,13-dimeC27: t3-meC27
28 0.5 rc-Octacosane
28A' 0.5 2-meC2g
29:1 0.6 -Nonacosene
29 2.2 n-Nonacosane
29A 5.3 15-.U3-. ll-meC29
29A 0.9 9-meC29
29A 1.7 7-meC29
29A 0.8 5-meC29
29B 0.3 11.15-: 13.17-dimeC29
29B 1.0 9.13-dimeC29
29B 1.9 7,13-dimeC29; t3-meC29
30 0.6 n-Triacontane
30A 1.1 15-. 14-. 13-. 11-. 8-. 7-meCjo
30 A' 1.1 2-meC30
31:1 1.0 n-\ lentriacontene
31 1.6 rc-Hentriacontane
31A 5.9 15-. 13-. 11-. 9-meC,,
31A 1.1 7-meC31
31B 3.8 13.17-; 11,15-; 9,13-dimeC3l
31B 0.7 7.13-dimeC3,
31C 0.5 11.15. 19-trimeC
32A 0.6 15-, 14-. 13-meC32
32B 0.5 14,18-; 13,?-dimeC32 and isomers
33A 2.0 17-. 15-. 13-. 11-. 9-. 7-meC33
33B 2.8 15.19-; 13.19-; 11,21-; 7.13-dimeC33
33C 0.5 13, 17,21-; 11. 15, 21-trimeC33
35B 0.5 7.13-dimeC35
45C 0.6 ?
47B 0.5 15.21-dimeC47
Table 6.3.3 Percent composition of hydrocarbons from laboratory maintained harvester ant P. bar bat us.
37


a' Percent composition was calculated from the integrated TIC areas, only the percent composition >0.1% of total area is shown on the table.
b' A t indicates that a trace amount of that isomer was present based on the presence of diagnostic ions in the mass spectra. c This table was adapted from Nelson el al., 2000.
6.4 Results of research aim 4: To characterize and quantify pavement ant cuticular hydrocarbons.
6.4.1 Mass spectra and characterization of pavement ant cuticular hydrocarbons
All structural characterizations are based on the methods described in Chapter 3. Examples of mass spectra and characterizations of the Pavement Bulk Ant I sample are shown in APPENDIX B.
Five ant colonies and one bulk ant sample were analyzed. There are about 22 peaks detected in each pavement ant colony. The chain length in pavement ant cuticular hydrocarbon ranges from 15 to 31 carbon atoms. Straight chain alkanes, mono-methyl, dimethyl and trimethyl alkanes, and alkenes were characterized in pavement ant cuticular hydrocarbon profiles. Six alkanes, ten mono-methyl alkanes and two alkenes were found in all five pavement ant colony samples. The compounds present in all pavement colonies are n-pentadecane (/7-C15), tt-tricosane (n-C23), ft-tetracosane (H-C24), n-pentacosane (n-C25), tf-hexacosane (n-Cx), and n-heptacosane (n-C21) in normal alkanes; 11 -, 3-methyltricosane (11-, 3-meC23), 13-, 11-methyltetracosane (13-, ll-meC24), 11 -, 3-methylpentacosane (11-, 3-meC2s), 13-, 4-methylhexacosane (13-, 4-meC26), 13-, and 3-methylheptacosane (13-, 3-meC27) in mono-methyl branched alkanes; heptacosene and nonacosene in alkenes. Small amount (~1%) of fatty acids was found in pavement ant colonies Speer 3 and NC-3.
38


6.4.2 Quantification of pavement ant cuticular hydrocarbons
Percent composition was calculated from the integrated TIC area as described in Data analysis. Tables 6.4.2.1 to 6.4.2.6 list all the characterized compounds and relative abundance for each pavement ant sample.
Pavement ant Colony SP3
Peak R.T.* Pet Total I.* ECL* Compounds
1 2.770 4.80 -Pentadecane
2 4.672 2.50 2302 23.04 w-Tricosane
3 4.756 0.67 2338 23.40 1 l-meC23
4 4.848 1.32 2374 23.75 3-meC23
5 4.907 1.09 2400 24.00 -Tetracosane
6 4.999 0.91 2436 24.37 13-, 1 l-meC24
7 5.100 0.67 2476 24.77 3-meC24
8 5.159 10.00 2502 25.03 /7-Pentacosane
9 5.251 18.60 2536 25.37 1 l-meC25
10 5.360 12.09 2575 25.75 3-meC25
11 5.427 1.12 2600 26.00 77-Hexacosane
12 5.460 1.18 2608 26.08 8,1 l-dimeC25
13 5.527 2.65 2634 26.34 13-meC26
14 5.603 0.57 2659 26.61 4-meC26
15 5.670 13.37 2680 26.82 Heptacosene
16 5.720 6.27 2702 27.03 77-Heptaconsane
17 5.829 11.43 2734 27.36 13-meC27
18 5.972 7.33 2775 27.76 3-meC27
19 6.089 1.36 2807 28.07 10,13-dimeC27
20 6.290 0.61 2865 28.66 8,12-dimeC29
21 6.366 1.45 2882 28.83 Nonacosene
Table 6.4.2.1 Percent composition of pavement ant cuticular hydrocarbons from Colony SP3.
* R.T. is the retention time shown in GC total ion chromatogram; I. is Kovats Index, and E.C.L. is Equivalent Chain Length.
39


Pavement ant Colony Speer 3
Peak R.T.* Pet Total 1/ ECL* Compounds
1 2.769 3.81 n-Pentadecane
2 4.505 0.57 2223 22.24 Oleic acid. TMS
3 4.681 3.40 2298 23.01 tt-Tricosane
4 4.764 0.63 2334 23.36 1 l-meC23
5 4.857 1.02 2374 23.75 3-meC23
6 4.915 1.26 2399 23.99 n-Tetracosane
7 4.999 0.69 2431 24.33 13-, 1 l-meC24
8 5.167 15.61 2496 24.99 n-Pentacosane
9 5.251 17.16 2529 25.31 1 l-meC25
10 5.368 11.02 2574 25.75 3-meC25
11 5.435 1.63 2600 26.00 n-Hexacosane
12 5.460 0.97 2608 26.08 8.1 l-dimeC25
13 5.527 1.98 2629 26.31 13-meC26
14 5.603 0.45 2654 26.56 4-meC26
15 5.678 15.64 2678 26.81 Heptacosene
16 5.728 6.73 2694 26.97 n-Heptacosane
17 5.829 9.30 2726 27.29 13-meC27
18 5.971 6.68 2772 27.73 3-meC27
19 6.298 0.73 2861 28.63 8,12-dimeC29
20 6.365 0.74 2877 28.80 Nonacosene
Table 6.4.2.2 Percent composition of pavement ant cuticular hydrocarbons from Colony Speer 3.
* R.T. is the retention time shown in GC total ion chromatogram; I. is Kovats Index, and E.C.L. is Equivalent Chain Length.
40


Pavement ant Colony SP4
Peak R.T.* Pet Total 1/ ECL* Compounds
1 2.770 4.93 n-Pentadecane
2 4.681 3.54 2298 23.01 -Tricosane
3 4.764 0.41 2334 23.36 1 l-meC23
4 4.857 1.01 2374 23.75 3-meC23
5 4.924 1.04 2402 24.02 n-Tetracosane
6 5.008 0.55 2435 24.36 13-, 1 l-meC24
7 5.108 0.28 2473 24.76 3-meC24
8 5.167 18.94 2496 24.99 n-Pentacosane
9 5.259 14.61 2532 25.34 11 -meC25
10 5.368 11.74 2574 25.75 3-meC2s
11 5.435 1.77 2600 26.00 n-Hexacosane
12 5.469 0.82 2611 26.11 8,11 -dimeC25
13 5.536 1.73 2632 26.34 13-meC26
14 5.611 0.42 2656 26.59 4-meC26
15 5.687 9.38 2681 26.84 Heptacosene
16 5.737 10.14 2697 27.00 n-Heptacosane
17 5.837 9.19 2729 27.32 13-, 1 l-meC27
18 5.980 7.78 2775 27.76 3-meC27
19 6.097 0.80 2810 28.10 8,11 -dimeC27
20 6.298 0.42 2861 28.63 6, 12, 21-trimeC28
21 6.374 0.50 2880 28.83 Nonacosene
Table 6.4.2.3 Percent composition of pavement ant cuticular hydrocarbons from Colony SP4.
* R.T. is the retention time shown in GC total ion chromatogram; 1. is Kovats Index, and E.C.L. is Equivalent Chain Length.
41


Pavement ant Colony NC-3
Peak R.T.* Pet Total I.* ECL* Compounds
1 2.736 2.73 n-Pentadecane
2 4.094 0.70 2040 20.42 9-Tetradecenoic acid, trimethylsilyl ester
3 4.505 0.71 2223 22.24 Oleic acid, trimethylsilyl ester
4 4.681 3.44 2298 23.01 n-Tricosane
5 4.773 0.75 2338 23.40 1 l-meC23
6 4.865 1.42 2377 23.78 3-meC23
7 4.924 1.10 2402 24.02 /7-Tetracosane
8 5.008 0.77 2435 24.36 13-, 11 -meC24
9 5.066 0.22 2457 24.59 6-, 4-meC24
10 5.167 15.08 2496 24.99 n-Pentacosane
11 5.259 17.44 2532 25.34 1 l-meC25
12 5.376 12.69 2577 25.78 3-meC25
13 5.443 1.15 2602 26.03 n-Hexacosane
14 5.469 1.29 2611 26.11 12-, 10-. 8-, 3-meC26
15 5.536 1.62 2632 26.34 13-meC26
16 5.619 0.48 2659 26.61 4-meC26
17 5.687 21.88 2681 26.83 Heptacosene
18 5.737 4.55 2697 27.00 n-Heptacosane
19 5.837 5.67 2729 27.31 13-, 1 l-meCr?
20 5.938 0.46 2761 27.63 9, x-dimeC27
21 5.988 4.36 2778 27.79 3-meC27; 5,10-dimeC27
22 6.097 0.57 2810 28.10 10,16-dimeC27
23 6.374 0.95 2880 28.83 Nonacosene
Table 6.4.2.4 Percent composition of pavement ant cuticular hydrocarbons
from colony NC-3.
* R.T. is the retention time shown in GC total ion chromatogram; I. is Kovats Index, and E.C.L. is Equivalent Chain Length.
42


Pavement ant colony KC-1
Peak R.T.* Pet Total I.* ECL* Compounds
1 2.769 1.27 /7-Pentadecane
2 4.681 1.55 2298 23.01 77-Tricosane
3 4.764 0.44 2334 23.36 1 l-meC23
4 4.865 0.78 2377 23.78 3-meC23
5 4.915 0.68 2399 23.99 n-Tetracosane
6 5.007 0.63 2434 24.36 13-, 1 l-meC24
7 5.108 0.27 2473 24.76 3-meC24
8 5.167 9.92 2496 24.99 -Pentacosane
9 5.259 15.82 2532 25.34 1 l-meC25
10 5.376 9.44 2577 25.78 3-meC25
11 5.435 1.35 2600 26.00 rt-Hexacosane
12 5.468 1.04 2610 26.11 12-, 10-, 3-meC26
13 5.536 2.56 2632 26.34 13-meC26
14 5.611 0.60 2656 26.59 4-meC26
15 5.686 15.70 2681 26.83 Heptacosene
16 5.737 8.43 2697 27.00 n-Heptacosane
17 5.779 0.54 2710 27.13 unknown
18 5.837 13.96 2729 27.31 13-meC27
19 5.98 9.23 2775 27.76 3-meC27; 5,10-dimeC27
20 6.097 0.98 2810 28.10 3.1 l-dimeC27
21 6.181 0.60 2831 28.33 16-, 14-. 12-, 10-meC28
22 6.298 0.51 2861 28.63 6,12,21-trimeC28
23 6.374 2.02 2880 28.83 Nonacosene
24 6.432 0.39 2894 28.97 77-Nonacosane
25 6.558 0.72 2926 29.29 13-meC29
26 6.751 0.33 2975 29.76 3-meC29
27 6.877 0.23 3005 30.05 /7-Triacontane
Table 6.4.2.5 Percent composition of pavement ant cuticular hydrocarbons from colony KC-1.
* R.T. is the retention time shown in GC total ion chromatogram; 1. is Kovats Index, and E.C.L. is Equivalent Chain Length.
43


Pavement Bulk Ants I
Peak R.T.* Pet Total i: ECL* Compounds
1 2.770 1.10 n-Pentadecane
2 4.412 0.19 2182 21.83 3-meC2i
3 4.656 0.28 2288 22.9 9-Tricosene
4 4.697 2.81 2305 23.08 w-Tricosane
5 4.781 1.30 2341 23.43 9-, 1 l-meC23
6 4.873 2.40 2381 23.82 3-meC23
7 4.932 0.72 2405 24.06 -Tetracosane
8 4.957 0.36 2415 24.16 12-meC24
9 5.024 1.21 2441 24.43 13-, 1 l-meC24
10 5.083 0.36 2464 24.66 4-meC24
11 5.142 2.13 2487 24.89 Tetracosene
12 5.192 7.77 2506 25.08 n-Pentacosane
13 5.276 15.38 2538 25.41 1 l-meC25
14 5.393 12.41 2584 25.84 3-meC25
15 5.460 0.79 2608 26.08 /7-Hexacosane
16 5.485 2.15 2616 26.17 12-, 10-. 8-, 3-meC26
17 5.552 3.13 2637 26.39 13-meC26
18 5.636 0.81 2664 26.67 4-meC26
19 5.661 0.72 2672 26.75 8, 10, 14-trimeC26
20 5.712 18.40 2689 26.92 Heptacosene, -Heptacosane
21 5.804 0.76 2718 27.21 Octacosene
22 5.863 9.55 2737 27.4 13-meC27
23 6.005 6.65 2783 27.84 3-meC27; 5,10-dimeC27
24 6.114 1.53 2814 28.15 3,1 l-dimeC27
25 6.215 0.67 2840 28.42 14-. 5-meC2g
26 6.315 0.77 2865 28.68 6, 12, 21-trimeC28
27 6.391 1.16 2884 28.87 Nonacosene
28 6.449 2.90 2899 29.02 n-Nonacosane
29 6.583 0.29 2932 29.35 13-, 1 l-meC29
30 6.793 0.28 2985 29.86 5,12-; 5, 7-dimeC29
31 6.835 0.22 2996 29.96 5, 10, 22-trimeC29
32 6.885 0.37 3006 30.07 3-meC29
33 7.355 0.43 3092 30.96 -Henitricontane
Table 6.4.2.6 Percent composition of pavement ant cuticular hydrocarbons
44


from Bulk Ant I.
* R.T. is the retention time shown in GC total ion chromatogram; I. is Kovats Index, and E.C.L. is Equivalent Chain Length.
6.5 Results of research aim 5: To investigate pavement ant cuticular hydrocarbons among colonies.
There are about 22 peaks in each pavement ant colony. The chain length in pavement ant cuticular hydrocarbon ranges from 15 to 31 carbon atoms. Six alkanes, ten mono-methyl alkanes and two alkenes were found in all five pavement ant colony samples. The compounds present in all pavement colonies are /7-pentadecane (77-C15), rc-tricosane (n-C23), rc-tetracosane {n-C24), -pentacosane (n-C25), rc-hexacosane (n-C26), and n-heptacosane (n-C27) in normal alkanes; 11-, 3-methyltricosane (11-, 3-meC23), 13-, 11-methyltetracosane (13-, ll-meC24), 11 3-methylpentacosane (11 -, 3-meC2s), 13-, 4-methylhexacosane (13-, 4-meC26), 13-. and 3-methylheptacosane (13-, 3-meC27) in mono-methyl branched alkanes; heptacosene and nonacosene in alkenes. Table 6.5.1 shows the proportions of straight chain alkanes, branched alkanes, alkenes and fatty acids in pavement ant samples.
SP3 Speer 3 SP4 NC-3 K.C-1 Bulk Ants I
Fatty acids* * t 0.57 t 1.41 t t
-Alkanes 25.78 32.44 40.37 28.04 23.83 22.04
Mono-methyl alkanes 56.25 48.92 47.72 44.52 51.80 53.91
Dimethyl alkanes 3.16 1.70 1.62 3.21 5.58 5.13
Trimethyl alkanes t t 0.42 t 0.51 1.71
Alkenes 14.82 16.37 9.88 22.82 18.26 17.21
Table 6.5.1 Percent composition of pavement ant Tetramorium caespitum cuticular hydrocarbons.
45


* Only pavement Bulk Ants I sample pass through silica gel column prior GC-MS analysis to separate fatty acids from cuticular hydrocarbons.
* A indicates the compounds were either missing or the peak area is less than 1% of the largest peak area in the total ion chromatogram.
The pavement ant cuticular lipids contain n-alkanes (16-40%), mono-methyl alkanes (45-56%) and alkenes (10-20%), with small amounts of dimethyl, trimethyl alkanes and fatty acids. Cuticular hydrocarbons in pavement ant T. caespitum are more consistent than in harvester ant P. occidentalis. The most abundant cuticular hydrocarbon class was monomethyl alkanes; the second was normal alkanes, and the third was alkenes in all five pavement ant colonies. The most abundant compound was 11 -methylpentacosane (11-meC25) in mono-methyl alkanes; -pentacosane in n-alkanes, and heptacosene in alkenes.
Fatty acids were found in pavement ant colonies Speer 3 and NC-3. The fatty acid that is present in both colonies was oleic acid (ca. 0.65%). Pavement ants eat pretty much everything but prefer greasy food. Without prominent oleic acid in their major food source, small amount of fatty acids can still present on insect cuticles. Most compounds found in pavement ant cuticular hydrocarbons are the same but with only slightly different percent composition. Shared cuticular hydrocarbons of pavement ant colonies with relative abundances were shown in Figure 6.5.1. The differences in hydrocarbons among pavement ant colonies were listed in Table 6.5.2.
46


SP3 Speer 3
SP4
NC3
KC1

n^O njo r4o n\ r\\
^ £ & J> & & & J> > < <$? <> 0
0* J? o ^ ^
v

Fig. 6.5.1 Shared cuticular hydrocarbons of pavement ant colonies.
47


-0.
oo

n-C15 n-C23 11 -meC23 3-meC23 n-C24 13-, 11 -meC24 n-C25 1 l-meC25
3- meC25 n-C26
13-meC26
4- meC26 Heptacosene
n-C27
13-meC27
3-meC27;.
Nonacosene
*
n
n-C!5 n-C23 1 l-meC23 3-meC23 n-C24 13-, 11 -meC24 n-C25 1 l-meC25
3- meC25
n-C26
13-meC26
4- meC26 Heptacosene
n-C27 13-, 1 l-meC27 3-meC27 Nonacosene
t/3
-o
u
n-C15 n-C23 11 -meC23 3-meC23 n-C24 13-, 1 l-meC24 n-C25 1 l-meC25
3- meC25
n-C26
13-meC26
4- meC26 Heptacosene
n-C27
13-meC27
3-meC27
Nonacosene
C/3
13
05
3
f
n
o_
o
3_
?e
tzi
=r -n
v -
D. tp
O
o
05
1
O'
o
3
V5
3
CO-
OS
Lai
k>
C/2
3
05
i-t
rt>
D-
o
3
D-
3
£5.
73
05
<
3

3
o
3
%
25 20 15 10 5 0 OM-^ONOOOM-fcsO'OOO
n-C15 n-Cl 5
n-C23 m n-C23
1 l-meC23 a 11 -meC23 a
3-meC23 B 3-meC23
n-C24 B n-C24
13-, 1 l-meC24 1 13-, 11 -meC24 a m n X/l
n-C25 2 n-LZj z z Ml. J
1 l-meC25 1 n 11 -meC25 z £ * ft rt
3-meC25 3-meC25 D
n-C26 13-meC26 4-meC26 Heptacosene n-C27 D B B n-C26 13-meC26 4-meC26 Heptacosene J -
n-C27 13-meC27 z ft
13-, 1 l-meC27 1 3-meC27 gg E
3-meC27; 5, 10-.. Nonacosene i
Nonacosene


SP3 Speer 3 SP4 NC-3 KC-1
//-Alkanes
n-C29 0.39%
H-C30 0.23%
Methyl branched alkanes
3-meC24 0.67% 0.28% 0.27%
6-, 4-meC24 0.22%
8.1 l-dimeC25 1.18% 0.97% 0.82%
12-, 10-, 3-meC26 1.29 b% 1.04%
8-meC26
1 l-meC27 Pa P
9, x-dimeC27 0.46%
5,10-dimeC27 P P
10,13-dimeC27 1.36%
8.1 l-dimeC27 0.80%
10,16-dimeC27 0.57%
6,12,21-trimeC28 0.42% 0.51%
13-meC29 0.72%
3-meC29 0.33%
8,12-dimeC29 0.61% 0.73%
Table 6.5.2 The percent composition differences of cuticular hydrocarbons among pavement ant colonies.
a A P represents the compound was identified by El mass spectrum, but co-eluted with its isomers or other compounds so the percent composition itself can not be determined.
b' The percentage with bold borders represents the isomers or compounds co-eluted and integrated as one peak in TIC, which is the total percent composition of these compounds.
49


Sano has demonstrated the unicoloniality of pavement ant T. caespitum (Sano, 2007), where individuals can move freely between physically separated colonies and there is the presence of more than one egg-laying queen in a colony. The consistency of cuticular hydrocarbons in five pavement ant colonies may be an indication of the unicoloniality. Colonies SP3 and Speer 3 have similar cuticular hydrocarbon profiles that might be because the unicolonial polygyny. In other words, ants in colonies SP3 and Speer 3 are likely conspecific species.
Species-specific and nestmate-specific recognition behaviors exist in Pavement ant T. caespitum (Sano, 2007). In the current study, colonies SP3 and Speer 3 have somewhat similar patterns in cuticular hydrocarbons, 8,11-dimethylpentacosane (8.11-dimeC25) and 8,12-dimethylnonacosane (8,12-dimeC29) are present in both ant colonies. However, 3-methyltetracosane (3-meC24) and 10.13-dimethylheptacosane (10,13-dimeC27) are present in colony SP3 but not in colony Speer 3. 8,11-Dimethylheptacosane (8.11 -dimeC27) was found only in pavement ant colony SP4; 6- and 4-methyltetracosane (6-, 4-meC24), 8-methylhexacosane (8-meC26), 9,x-dimethylheptacosane (9,x-dimeC27), and 10.16-dimethylheptacosane (10,16-dimeC27) were exclusive to colony NC-3; n-nonacosane (77-C29), n-tritriacontane (77-C31), 6,12,21-trimethyloctacosane (6,12,21-trimeC28), 13-and 11-methylnonacosane (13-, ll-meC29) were present only in colony KC-1. The differentiality expressed compounds could be the chemical cues for nestmate recognition. Further work must be done to determine whether compounds provide nestmate recognition cues or if only internally methyl and dimethyl branched alkanes possess that functionality.
50


7 Conclusion
The chain length of cuticular hydrocarbons was found to range from 23-36 carbon atoms in the harvester ant and from 15-31 carbon atoms in the pavement ant. Compounds that were detected in both ant cuticular hydrocarbons include 77-tricosane (77-C23), -pentacosane (77-C25), 77-hexacosane (n-Cid), /7-heptacosane (77-C27), 77-nonacosane (77-C29), 77-hentriacosane (77-C31), 3-methylpentacosane (3-meC25) and 13-methylheptacosane (13-meC27). The odd-number positions of methyl alkanes were characterized more than of even-number methyl alkanes in all ant samples. 77-Heptacosane were found to be at least three times bigger than 77-pentacosane in harvester ant cuticular hydrocarbon profiles while 77-heptacosane is as much as 77-pentacosane in pavement cuticular hydrocarbon profiles.
The approaches of structurally identification for cuticular hydrocarbons were described in Chapter 3. 77-Alkanes were primarily determined by [M-15]+/base peak= 3-5% in Cl spectra and El mass spectra library search with ECL and Kovats Index / retention indecis as reference. Mono-methyl alkanes were first identified by [M-15]+/base peak= 6-10% in Cl spectra and El mass spectra. Dimethyl alkanes were characterized by [M-15]+/base peak= 11-20% in Cl mass spectra and El mass spectra. Quantitative GC/MS analysis of ant cuticular hydrocarbons revealed that the percent composition of mono-methyl alkanes is more abundant than 77-alkanes in the pavement ant T. cespitum. In contrast, the relative abundance of 77-alkanes and mono-methyl alkanes varies among colonies in the harvester ant P. Occident alls.
Oleic acid is extremely abundant in one of the harvester ant colonies, it could relate to the source of food, the death of ants, or the conversion of fatty acids to hydrocarbons. However, it could simply be an error due to the one-time experiment analysis of each sample. The differential expression of hydrocarbons in harvester ant (Table6.2.1.2, page 29) shows possible compounds to investigate further for nestmate recognition cues. The
51


similarity of cuticular hydrocarbon patterns may imply the ant colonies are genetic related, and the number of hydrocarbons might relate to the size and the age of the colonies.
Species have unique hydrocarbon profiles, which differ from other species in the presence or absence of hydrocarbons and quantitatively in shared molecules. In the comparison of harvester ant P. occidentalis and P. bartatus, the former has shorter carbon chain length (23-36) in cuticular hydrocarbons than the latter (23-49). Trimethyl alkanes and/or alkenes were either missing or less than 1 % of the largest peak area in P. occidentalis, but about 1.6% trimethyl alkanes and 2.3% alkenes were present in P bartatus. P. occidentalis has more abundant mono-methyl alkanes than straight chain alkanes while P. bartatus has fairly equal abundance in /7-alkanes and mono-methyl alkanes. Patrollers and foragers in a harvester ant colony contain higher proportion of n-alkanes relative to methyl alkanes and alkenes (Wagner et al., 1998). Therefore, the number of patrollers and foragers collected may change the percent composition of hydrocarbon classes in the tested sample. Nestmate recognition compounds could be either unique to a certain colony or shared compounds between colonies but detected with different percent composition. Possible harvester ant P. occidentalis nestmate recognition cues are listed from page 31 to 33.
The cuticular hydrocarbon profdes in pavement ant T. cespitum are more consistent between colonies than in harvester ant P. occidentalis, which may be due to the unicolonial polygyny in pavement ant T. cespitum. Similiarity in cuticular hydrocarbon profiles might also be an indication of conspecific ants, also could be the information correlates to the aggression against non-nestmate conspecific or heterospecific species. Species and nestmate recognition behavior have been demonstrated in the pavement ant T. cespitum (Sano, 2007).The differential hydrocarbon profiles in pavement ant (page 47-49) provides possible nestmate recognition cues in pavement ant T. cespitum.
52


This is a preliminary research for the western harvester ant P. occidentalis and the pavement ant T. cespitum. Duplicates of samples and standards were not performed to ensure the accuracy and reproduciblity of GC quantitation results. This needs to be done for the future work. Although the ecology and biochemistry of the insect cuticular hydrocarbons has been intensively investigated in the last twenty years, the questions in some areas are still need to be answered. The stereochemistry of the methylbranched cuticular hydrocarbons and the effect of diet or the environmental conditions on insect cuticular hydrocarbons will be studied. Also, the biological activities of individual component need to be investigated.
53


8. Future Direction
8.1 Identification of double bond positions in alkenes
It was difficult to locate double bond positions in alkenes and determine their stereochemistry by simply interpreting mass spectra of ant samples. Three methods have been developed to solve this problem. Methylthiolation with dimethyl disulfide to derivatize alkenes and form methylthioalkanes (Francis and Veland. 1981) gives prominent fragment ions in electron ionization spectra. Additionally, molecular ions are always detected in El spectra. This method can be applied for the determination of double bond positions in alcohols, aldehydes, and carboxylic acid methyl esters, as well as alkenes (Leonhardt and DeVilbiss, 1985).
Another approach to locate double bond positions in alkenes makes use of GC chemical ionization mass spectrometry. The use of ammonia in positive chemical ionization converts monoenes to methoxyesters. Pechine characterized alkenes from Drosophila melanogaster and Drosophila simulans (Pechine et al., 1985) and Lange analyzed alkenes from termites, ants and flies (Lange et al., 1989). Later Pechine explored another approach by epoxidation of alkenes with metachloroperbenzoic acid, then analyzing samples in negative chemical ionization and using 90% methane-10% nitrous oxide as reagent gas (Pechine et al., 1988).
8.2 The roles of individual hydrocarbon components as chemical communication
After characterizing the cuticular hydrocarbons, we know there are at least 20 compounds in each colony. Is every one of them involved in the communication message
54


or are only certain hydrocarbon compounds? The biological activities of individual hydrocarbon components are still unclear. In order to answer these questions, the acquisition of individual compound by either chromatography separation or organic synthesis, along with numbers of bioassays must be conducted (Howard, 1993).
8.3 Hydrocarbon stereochemistry
Although the insect cuticular hydrocarbons were characterized and identified, all methyl branched alkanes could have chiral centers. There was no study of identification the stereochemistry in insect hydrocarbons. The first question is: are those methyl alkane isomers all chrial? To investigate the chirality of insect hydrocarbons, we have to either synthesize pure chiral compounds then develop the bioassays to yield distinguishable results, or derivatize hydrocarbons into polar compounds that give us optical rotation values (Howard. 1993).
55


APPENDIX A
Mass Spectra of Harvester Bulk Ant I
56


> Peak 1: EI=4.681; CI=15.455
n-Tricosane
Abundance
m/z>
Fig. A.1.1. El mass spectrum of rz-tricosane.
Name Tricosane (CAS) $$ n-Tricosane CAS Number 000638-67-5 Entry Number 275689 Molecular Formula C23H48
Misc Information QI=836, Source=WS-1986-23-0, WLN=23H
Match Quality 99
Company ID 0
Retention Index 0
Melting Point
Boiling Point
Molecular Weight 324.38
57


Abundanc
Fig. A.1.2. Cl mass spectrum of fl-tricosane. [M-15]+/base peak:
= (m/z at 309.50) / (m/z at 323.45)
= 8088.0 /384580.0 = 2.10%
> Peak 2: EI=4.92; CI=16.010 n-Tetracosane
Fig. A.2.1. El mass spectrum of /7-tetracosane.
58


Name Tetracosane
CAS Number 000646-31-1
Entry Number 288781
Molecular Formula C24H50
Misc Information QI=900, Source=NS-12-7042-0
Match Quality 99
Company ID 0
Retention Index 0
Melting Point
Boiling Point
Molecular Weight 338.39
Fig. A.2.2. Cl mass spectrum of/7-tetracosane
[M-15]+/ base peak:
= (m/z at 323.40) / (m/z at 337.50) = 2835.0/90731.0 = 3.12%
59


> Peak 3: EI=5.167; CI=16.559 n-Pentacosane
Fig. A.3.1. El mass spectrum of -pentacosane.
Name Pentacosane (CAS) $$ n-Pentacosane CAS Number 000629-99-2 Entry Number 300503 Molecular Formula C25H52
Misc Information QI=621, Source=PG-1982-1717-0, WLN=25H
Match Quality 99
Company ID 0
Retention Index 0
Melting Point
Boiling Point
Molecular Weight 352.41
60


Abundance
Fig. A.2.2. Cl mass spectrum of -pentacosane.
[M-15]+/ base peak:
= (m/z at 337.50) / (m/z at 351.50)
= 79938.0/2760456.0 = 2.90 %
> Peak 4: EI=5.276; CI=16.770
281 -
Cl8
253~
-113
li
c|-c6
85
7-meC25
13-, 11-, 9-, 7-methylpentacosane
61


Abundance
Fig. A .4.1. El mass spectrum of 7-methylpentacosane.
197-
-197
225-
C-12 ; CJ C12 169 ' 169
l3-meC25
169
Tl
C|c.
253-
-141
^14 I'-i ^10
197 141
1 l-meC25
C!-Cr
-16
225
9-meC25
113
Fig. A.4.2. El mass spectrum of 13-, 11-, 9-methylpentacosane.
62


Abundance
Fig. A.4.3. Cl mass spectrum of 13-, 11-, 9-, 7-methylpentacosane. [M-15]+/base peak:
= (m/z at 351.50) / (m/z at 365.60)
= 87901.0/ 1194176.0 = 7.36 %
> Peak 5: EI=5.368; CI=16.936 3-methylpentacosane
337
C22~
309
,57
ici : I!
ic;-c2
29
3-meC25
63


Abundance
Fig. A.5.1. El mass spectrum of 3-methylpentacosane.
Abundance
Fig. A.5.2. Cl mass spectrum of 3-methylpentacosane. [M-15]+/ base peak:
= (m/z at 351.50) / (m/z at 365.60)
= 38359.0/454638.0 = 8.44 %
64
*


r Peak 6: EI=5.435; CI=17.067 n-Hexacosane
Fig. A.6.1. El mass spectrum of n-hexacosane.
Name Hexacosane $$ n-Hexacosane
CAS Number 000630-01-3
Entry Number 311169
Molecular Formula C26H54
Misc Information QI=540, Source=NS-8-7573-0
Match Quality 99
Company ID 0
Retention Index 0
Melting Point
Boiling Point
Molecular Weight 366.42
65


Abundance
Scan 2185 (17 067 min): TS17.D (-2 173) (-)
Fig. A.6.2. Cl mass spectrum of -hexacosane.
[
M-l5]+/ base peak:
= (m/z at 351.50) / (m/z at 365.60)
= 37720.0/ 1373940.0 = 2.75 %
> Peak 7: EI=5.544; CI=17.262
281
8-meC26
8-methylhexacosane
66


Abundam
Fig. A.7.1. El mass spectrum of 8-methylhexacosane.
Abundance
Scan 2219 (17.262 min): TS17.D (-2205) (-)
m/z>
Fig. A.7.2. Cl mass spectrum of 8-methylhexacosane.
[M-15]"/ base peak:
= (m/z at 365.60) / (m/z at 379.60)
67


= 33063.0/ 526880.0 = 6.28 %
r Peak 8: EI=5.745; CI=17.593 n-Heptacosane
Fig. A.8.1. El mass spectrum of n-heptacosane.
Name Heptacosane
CAS Number 000593-49-7
Entry Number 320678
Molecular Formula C27H56
Misc Information QI=898, Source=NS-12-7866-0
Match Quality 99
Company ID 0
Retention Index 0
Melting Point
Boiling Point
Molecular Weight 380.44
68


Abundance
Fig. A.8.1. Cl mass spectrum of -heptacosane.
[M-15]+/ base peak:
= (m/z at 365.55) / (m/z at 379.60)
= 168988.0/5812960.0 = 2.91 %
> Peak 9: EI=5.846; CI=17.742 13-, 11-, 9-, 7-methylheptacosane
r197 225 1 r169 ?53 281 r141 i
: CM T~ 0 1 -o' T- o 1 i Cl6 C| C10 C-18" 00 0 1 -o
19r^ 169 225 '141 253 113
13-meC27 ll-meC27 9-meC27
69


to
oc O o

1 o
"O 1 1
3 o o 1
a I
n N> f 00 o o> OJ

-O
O


Fig. A.9.1. El mass spectrum of 13-. 11-. 9-heptacosane.
3
N
I
v -*NJU^UlClSCDt0O-kWU^UlCl'JCI(0O-k 000000000000000000000 000000000000000000000 000000000000000000000 000000000000000000000
>
V
c
3
a
D
1


Abundanc
Fig. A.9.2. El mass spectrum of 7-heptacosane.
Fig. A.9.3. Cl mass spectrum of 13-, 11-. 9-, 7-heptacosane.
[M-15]+/ base peak:
= (m/z at 379.60) / (m/z at 393.60)
71


= 228241.0/ 3426264.0 = 6.66 %
Peak 10 EI=5.963; C
197 225 295^ 323i
"1 c ' C | 1
14 j C - 1 1 C 5C ;
211 183 113
7, I 3-dimeC27
-c,
85
7,13-dimeC27
72



Fig. A.10.2. Cl mass spectrum of 7.13-dimethylheptacosane. [M-15]+/base peak:
= (m/z at 393.60) / (m/z at 407.60)
= 104587.0/400931.0 = 26.09 %
> Peak 11: EI=6.055; CI=18.044 n-Octacosane
Scan 415 (6.055 min): TS22 D (-425) (-)
Fig. A.ll.l. El mass spectrum of n-octacosane.
73


Name Octacosane (CAS) $$ n-Octacosane CAS Number 000630-02-4 Entry Number 329269 Molecular Formula C28H58
Misc Information QI=555, Source=PG-1982-1720-0, WLN=28H
Match Quality 99
Company ID 0
Retention Index 0
Melting Point
Boiling Point
Molecular Weight 394.45
Abundance
m/z>
Fig. A. 11.2. Cl mass spectrum of n-octacosane.
[M-15]+/ base peak:
= (m/z at 379.55) / (m/z at 393.60) = 31453.0/ 1012280.0 = 3.11 %
74


> Peak 12: EI=6.173; CI=18.193 14-, 13-, 12-, 8-meC28
,211 ,197 ,183
1 239- 1 253 1
i¥i : c; I I !C| 1 l
1 i I C[c13 ^15 1 1 1 -:c;-c12 ^16 ' 1! -ICj-Cn
197 ' 183 211 ' 169 225 ' 155
14-meC28 13-meC28 12-meC28
Fig. A.12.1. El mass spectrum of 14-, 13-, 12-methyloctacosane
309-
-127
9!
^20__[Cj C7
281 ' 1 99
8-meC28
75


) <->

Fig. A.12.2. El mass spectrum of 8-methyloctacosane.
Fig. A.12.3. Cl mass spectrum of 14-, 13-, 12-, 8-methyloctacosane.
[M-15]+/ base peak:
= (m/z at 393.60) / (m/z at 407.60)
76


= 32993.0/474299.0 = 6.96 %
> Peak 13: El=6.432; CI=18.513 n-Nonacosane
Fig. A.13.1. El mass spectrum of n-nonacosane.
Name Nonacosane (CAS) $$ n-Nonacosane $$ Celidoniol, deoxy- (CAS) CAS Number 000630-03-5 Entry Number 337002 Molecular Formula C29H60
Misc Information QI=623, Source=PG-1982-1721-0, WLN=29H
Match Quality 99
Company ID 0
Retention Index 0
Melting Point
Boiling Point
Molecular Weight 408.47
77


Fig. A.13.2. Cl mass spectrum of -nonacosane.
[M-15]+/ base peak:
= (m/z at 393.55) / (m/z at 407.60) = 93528.0/3203453.0 = 2.92 %
> Peak 14: El=6.558; Cf= 18.662 IS-, 11-, 9-, 7-methylnonacosane
253'
-197
C-16
225
-169
i ?! 281 - 309-^l 1 1 1
11 C|c12 C-I8 ._l_ o- 1 p o i 1! C20 c!
'169 253
'141 281"
-141
C8
113
13-meC29
1 l-meC29
9-meC29
78


Fig. A.14.1. El mass spectrum of 13-, 11-, 9-methylnonacosane.
309
13
C6
85
7-meC29
79


Fig. A. 14.2. El mass spectrum of 7-methylnonacosane.
Fig. A. 14.3. Cl mass spectrum of 13-, 11-, 9-, 7-methylnonacosane.
[M-15]+/ base peak:
= (m/z at 407.60) / (m/z at 421.60)
80


= 215983.0/3171496.0 = 6.81 %
> Peak 15: El6.717; CI= 18.822 9,13-; 7,13-dimethylnonacosane
225 253 ~i c i 295 32^ ~i C | 225 253 "7 c ; 323 351
ii i i i ,6 i Ci C3;-Cj -c8 c16-;-c-|-c5-i-c-i-c
211 183 141 113 211 183 113 85
9,13-dimeC29
7,13-dimeC29
Fig. A.15.1. El mass spectrum of 9.13- and 7,13-dimethylnonacosane.
81


Abundan
2492 (1 8 822 min): TS'IT.D (-2452) (-)
500000
450000
-400000
350000
300000
250000
200000
150000
1OOOOO
1 ' 3 1 55 1 33 2 1 2 3 2 C 1 3: 3 3 5 1
I 37
, i 1 9 a U, >5 2 aa
1 OO 120 1-40 160 1 SO 200 220 240 200 280 300 320 340 35 0 3QO
Fig. A.15.2. Cl mass spectrum of 9,13- and 7,13-dimethylnonacosane.
[M-15]+/ base peak:
= (m/z at 421.65) / (m/z at 435.65) = 70906.0/512533.0 = 13.83 %
> Peak 16: EI=6.843; CI=18.948 n-Triacontane
82


Fig. A.16.1. El mass spectrum of n-triacontane.
Name Triacontane $$ n-Triacontane
CAS Number 000638-68-6
Entry Number 343923
Molecular Formula C30H62
Misc Information QI=895, Source=NS-9-5920-0
Match Quality 97
Company ID 0
Retention Index 0
Melting Point
Boiling Point
Molecular Weight 422.49
83


Fig. A.16.2. Cl mass spectrum of tt-triacontane.
[M-15]+/base peak:
= (m/z at 407.60) / (m/z at 421.60) = 12190.0/344355.0 = 3.54 %
> Peak 17: EI=6.994; CI=19.085 14-methyltriacontane
253
Ci6
225
r211
1
C-|3
' 183
14-meC30
84


Abundanci
Fig. A.17.1. El mass spectrum of 14-methyltriacontane.
Fig. A.17.2. Cl mass spectrum of 14-methyltriacontane.
[M-15]+/ base peak:
= (m/z at 421.60) / (m/z at 435.70)
85


= 29268.0/385967.0 = 7.58 %
y Peak 18: EI=7.338; CI=19.410 n-Hentriacontane
Fig. A.18.1. El mass spectrum of /7-hentriacontane.
86


Fig. A. 18.2. Cl mass spectrum of w-hentriacontane.
[M-15]+/ base peak:
= (m/z at 421.55) / (m/z at 435.70) = 72746.0/ 2292773.0 = 3.17%
> Peak 19: EI=7.505; Cl=19.559 15-, 13-, 11 -methylhentriacontane
t225 ,197 ,169
7-1 281i 309r
1 281- 1 309- 1
iVi 1 i
o- .... o 4^ ^18 1 i -:c!-c12 C20 0 T 0 1 -O _
225 197 253 ' ' 169 281 ' ' 141
15-meC3i 13-meC3| ll-meC3I
Fig. A. 19.1. El mass spectrum of 15-, 13-, 11-methylhentriacontane.
87


Abundance
Fig. A. 19.2. Cl mass spectrum of 15-, 13-, 11-methylhentriacontane.
[M-15]+/ base peak:
= (m/z at 435.70) / (m/z at 449.65) = 122240.0/ 1784479.0 = 6.85 %
> Peak 20: EI= 7.556; CI= 19.610
365
C6
85
7-meC3]
7-methylhentriacontane
88


Fig. A.20.1. El mass spectrum of 7-methylhentriacontane.
Fig. A.20.2. Cl mass spectrum of 7-methylhentriacontane.
[M-15]+/ base peak:
= (m/z at 435.65) / (m/z at 449.70)
89


= 126011.0/ 1876117.0 = 6.72 %
r- Peak 21: EI=7.715; Cl=19.742 7,13-dimethylhentriacontane
253
C,8-
281 351 379
211 183 113
-c6
85
7,13-dimeC31
Fig. A.21.1. El mass spectrum of 7.13-dimethylhentriacontane.
90


Full Text

PAGE 1

CHARACTERIZATION AND QUANTIFICATION OF CUTICULAR HYDROCARBONS IN HARVESTER ANT AND PAVEMENT ANT by Chao Yu Chen B.S. ormal Kaohsiung National University Taiwan 2005 A thesis submitted to the University of Colorado Denver in partial fulfillment of the requirements for the degree of Master of Science Chemistry 2008

PAGE 2

This thesis for the Master of Science degree b y Chao Yu Chen has been appro v ed by Dr. Marc Donsk y Ph.D. Dat e

PAGE 3

Chen Chao Yu (Master of Science) Characterization and Quantification of Cuticular Hydrocarbons in the Harvester Ant and the Pavement Ant Thesis directed by Douglas F. Dyckes and Marc A. Donsky. These recognition cues are often present in the suite of hydrocarbon molecules present on the cuticle known as a hydrocarbon profile. Social insects can use variation in hydrocarbon profiles as recognition cues in order to recognize group membership of other individuals they interact with. The cuticular hydrocarbons of the western harvester ant and the pavement ant were characterized and quantified. Five ant colonies and bulk ant samples were analyzed by with both electron ionization and methane chemical ionization. Oleic acid is present in all harvester ant colonies but only tin two pavement ant colonies which could be an indication that diet does change their cuticular lipid composition. Genetic relative or close colony locations may be similar in their cuticular hydrocarbon patterns. The similarity of hydrocarbon profiles might be related to the aggression levels against non nestmate conspecific species or heterospecific species. The range in total carbon length of harvester ant is shorter and Recognition behavior ma y correlate to one or some internally methyl alkanes or the percent composition in the shared common internally methyl alkanes. Possible recognition cues were listed on the tables for each species The cuticular hydrocarbon profiles in pavement ant are more consistent than in harvester ant which may be relative to the unicolonial polygyny in pavement ant The results give some interesting ideas for future directions. More bioassays are necessary to investigate the roles of individual hydrocarbon compounds in chemical communication. This abstract accurately represents the content of the candidate s thesis. I recommend its publication. Signed

PAGE 4

I would like to express my gratitude to Dr. Marc Donsky Dr. Mike Greene and Dr. Douglas Dyckes who guided me the direction of my thesis; Dr. Karen Jonscher and Dr. Touraj Shokati who provided assistance on instruments for my research; Jeff Boon and Dr Larry Anderson who taught me and made analytical chemistry interesting.

PAGE 5

Figures ................. ...... .................................................................................. viii Tables .................................................................................................... ... ....... ix Chapter 1. Introduction ............................... ..... .... ................. .......................... ..... .... .... ........ ........... 1 1.1 Research Aims ..... ....... ..... . ....... ... ........... .... ......... ........ .......... ..... ......... ....... 1 1.2 Composition of Social Insect Lipids ........ ........ .. ... .... .......... ................. .. .............. 2 1.3 Nestmate and Task Recognition ..... ... . .... ........ ........ .... ..... ......... ..................... .... 2 1.4 Weatern Harvester Ant and Pavement Ant.. .................... .... ....... .... ................ .......... 3 2. Chemistr y Background .. ......... ..... ...... ...... ........ ... ................ .... ............. ............. .... 6 2.1 Gas Chromatograph y Mass Spectrometr y .... ................. ..... ......................... ...... 6 2 .1.1 E lectron Ionization .. .. ...................... ..... .... ..... ........ .... ........ .... ............... ...... 6 2.1.2 Chemical Ionization ........... ..... .................. .... ................ .... ...... ........... ..... .... 6 3. Compound Identification ....... .... .... ...... .... ..... ..... ............ ....... ........ ... ........... ..... ..... 7 3 1 Retention Index S y stems ..................... ..... ...... ... ....... ... ........ ........ ...... .... ......... ........ 7 3.1.1 Kovats Inde x ................................ .. ...... ..... ....... ...... ............. ..... ....... .... ..... 8 3 1.2 Equi v alent Chain Length .... ........ ..... ........ ............ .......... .............. .... ..... 9 3 2 Mass Spectra of Aliphatic H y drocarbons .... ........ .......... .... ........... ................. 10 v

PAGE 6

3.2.1 Saturated Compouds ........ ...... ........ ...................... .............................. ...... 10 3.2.2 U n sa turated Compounds .................. ..... ......... .... ........... ......... ..... ..... .... .... .... 3.2.3 Positi ve Chemica l Ionization Mass Spectra ............................................ .... 13 4. Ant Cuticu l ar Hydrocarbon Profile Analyses ..... ..... ............... ..... ......... ............... 15 5. Exper imental ........ ... ............... ............... ...... ..... ..................... ........... ............... .... 16 5.1 Reagent s ............. ......................... ....... .......... ....... ............ .... ........................ ........ 16 5.2 Field Sites and Ant Collection . ...... . ..... ..... .................................................... 16 5.3 Methods ............... ..... ...... ........ .............. ..... ......... .................. ... ............. .......... .... 16 5.3.1 Ant Sample Ex traction and Sample Separation .......... .... ................. .... ... ..... 16 5 3.2 Chemical Analysis ....................... .. .......... ............. ...... ...... ........ ... ......... ........ 17 5.3.3 Data Analysis ....... ... ................................. .... ..... ........... ........... ..... ... ... ............ 17 6. Re s ults and Discussions ... ................ ..... .......... ..... .... .......... .. .. .. ...... ............. ................ 19 6.1 Results for Research Aim 1 .......... ... ........ ............................. . ....... ....... ........ .... 19 6.1.1 Mass Spectra and Characterizatio n of Har ves ter Ant Cuticular H y drocarbons .... ..... ............... ..... ......... ........ ....................... ...... ........ ...... 19 6.1.2 Quantification of Har veste r Ant Cuticular Hydrocarbons ........... ..... ........... 22 6.2 Results for Research Aim 2 .... ...................... .... .... .... .... ... ...... .................. .... ........ 29 6.3 Results for Research Aim 3 ......... ..... ............. ..... .................... ....... .... ............. ...... 34 6.4 Results for Re searc h Aim 4 ...... .... ........ ......... ..... . ................................... .......... 38 V I

PAGE 7

......... ... ...... . ... ... ... ....... ......... ... .... .... .... ........ .... 5 ... ....... . ............. ........ ..... ....... ... .... ... ... ........ ..... ....... . .... .............. ...... ... ... .... .... .... ... .. ... ...... ... ... ... ...... ... ............. ..... ......... ... ... ... ...... .... .... A .... ....... ....... .......... ...... ....... ........ ... .. .......... ............... . ....... .... ..... .... ........ ....... ...... 56 B .... ..... . . ..... ........... ............. ...... .............. .... .... ..... .... ......... .... ..... ........ ..... ......... 102 .... ...... . ......... ... ... ... ..... . ... ........ . ... ... .... .... .... ......... ... ........ ...

PAGE 8

Fi g 1.4 1 The Western Har v ester Ant .............. 5 Fig. 1.4 .2 The Pa v em e nt Ant ...... ..... . .... ..... ...... .... ..... ............ ....... .......... 5 Fig 3 1 T he l inear relationship of the logarithm of the retention time and carbon chain l en g th ... .... .... .... ..... ..... ....... .... ........ . ...... . .... .... .......... .......... . 8 Fig. 3 .2.1 Mass spectrum of n-he x adecane .. ...... ........... . .............. ........ .... .......... .... . 11 Fig 3.2 .2.1 All y lic clea v a g e in a mono olefin .......... . ............. . ..... ....... ....... ............ 1 2 Fig 3.2 2.2 Mass spectra o f I butene and 2-butene .......... .... .... . .... .... ....... ... ..... ... 12 Fi g 3.2 3 Methane CI mas s spectra of n-decene i s omer s ...... .. ...... .... ........................... 14 F ig. 5.3. 3 Integration parameter s for ant cuticu l ar h y drocarbon quantification .... ..... 18 Fig. 6 1 1 1 Linear relationship between E CL a nd ide ntified n alkane s ....... ........ .... ....... 2 1 Fig. 6.1.1 2 Linear relation s h ip between I a n d identified n a l kanes ....... ..... ......... ..... ... 21 Fig. 6 .2.1 Shared cuticular hydrocarbons of har ves ter ant colonies . ........ . ......... ...... 31 Fig. 6.2.2 Shared cuticular h y drocarbons o f i ndividual harvester ant colonie s ...... .... .... 32 F ig. 6 5 1 Shared cuticular hydrocarbons of pav ement ant co l onie s .... .... ..... ... ... ......... 47 Fig. 6 5.2 Shared cuticu l ar hydrocarbo n s of individual pavement ant colonies . ... ... ... .48

PAGE 9

Table 6.1.1.1 Averages and standard deviations of shared n-alkanes of harvester ant cuticular h y drocarbon profiles .................................................................... 20 Table 6.1.2.1 Percent composition of harvester ant cuticular hydrocarbons froIll colony # 1 ...... ...... ..... ...... .... ....... ....... .... .......... ........... ...... ......... ..... 22 Table 6.1.2.2 Percent composition of har ves ter ant cuticular h y drocarbons from colony #2 ..... ... .... .... ...... ......... .... ................ ...... ................ ........... .... 23 Table 6.1.2.3 Percent composition of harvester ant cuticular h y drocarbon s from colony # 4 ... ..................... ....... ..... ........... .... ...... ... ........... ...... ............ 24 Table 6.1.2.4 Percent composition of harvester ant cuticular hydrocarbons from colony # B ........ ... ........... ...................... ..... ...................... ................... 25 Table 6.1.2.5 Percent composition of harvester ant cuticular hydrocarbons from colon y #C ....... ........ . ......... ................ ......................... ................ 26 Table 6.1.2.6 Percent composition of harvester ant cuticular h y drocarbons of Bulk Ant 27 Table 6.1.2.7 Percent composition of harvester ant cuticular h y drocarbons of Bulk Ant 28 Table 6.2.1. 1 Percent composition of seven har ves ter ant samples .............. ..... ........................................ ..... ............ ...... .... ..... ......... 29 Table 6.2.1.2 The percent composition differences in cuticular h y drocarbons among har ves ter ant colonies .... ..... ..... ..... ....... ........... .... ... .... ..... .......... 33 Table 6.3.1 Hydrocarbon percent compositions in the har ves ter ant and .... ..... ...... ............................. ........................ .. ................... .... 35 Table 6.3.2 The percent composition differences in cuticular h y drocarbons of har ves ter ant and .... ... .................... ................ 36 i x

PAGE 10

Table 6.3.3 Percent composition of h y drocarbons from laboratory maintained harvester ant .......... ............................................................ ....... 37 Table 6.4.2.1 Percent composition of pavement ant cuticular hydrocarbons from colony SP3 .................................... ................................... ......... ....... 39 Table 6.4.2.2 Percent composition of pavement ant cuticular hydrocarbons from colony Speer 3 ...... ........ ............ ......... .... ......... ........ ......... ............ 40 Table 6.4.2.3 Percent composition of pavement ant cuticular hydrocarbons from colony SP4 .......................................... .............. ... ............ ..... ............ 41 Table 6.4.2.4 Percent composition of pavement ant cuticular hydrocarbons from colony NC-3 ....................... ......... ........... .... ........... .... ................. 42 Table 6.4.2.5 Percent composition of pavement ant cuticular hydrocarbons from colony KC-1 ....................................................................................... 43 Table 6.4.2.6 Percent composition of pave ment ant cuticular hydrocarbons from Bulk Ant I .... .......... .... ........................ .................. ..... ... ... ........ ....... 44 Table 6.5.1 Percent composition of pavement ant cuticular h y drocarbons .................... ........ ........ .... .... ........ ..... .............. .... 45 Table 6.5.2 The percent composition differences of cuticular hydrocarbons among pavement ant colonies ......................................................... .......................... 49

PAGE 11

All insects envelop various polymeric components and their surfaces are usually covered with complex mixtures of long chain compounds. The outer layer of the insects cuticle is coated with lipids that consist of mainly aliphatic compounds. The large ratio of insects surface lipids to cuticle volume prevents excessive transpiration of water Early studies on insect surface lipids primarily focused on their function and physiology which have evolved to prevent desiccation to form a barrier to microorganisms and to prevent the absorption of toxic chemicals from the environment (Jackson and Blomquist 197 6). More recently cuticular hydrocarbons have been recognized as having a key role in chemical communication including species recognition task allocation and nestmate recognition in social insects (Nelson 2001) The goal of this study was to structurally ident i fy chemically characterize and quantify the cuticular hydrocarbo n s of both the Western Harvester Ant and the Pavement Ant The following research aims were pursued : Research Aim 1: To characterize and quantify the western harvester ant cuticular hydrocarbons. Research Aim 2: To investigate the composition of harvester ant cuticular hydrocarbons among colonies. Research Aim 3: To compare my results of harvester ant cuticular hydrocarbons with previously published results.

PAGE 12

Research Aim 4: To character i ze and quantify pavement ant cuticular hydrocarbons Research Aim 5: To investigate the composition of pavement ant cuticular hydrocarbons among co l on i es. The major components of social insect cuticle lipids are hydrocarbons with a small amount of fatty acids alkyl esters alcohols glycerides stero l s aldehydes and ketones (Lockey 1988). There are three main classes of insect cuticular hydrocarbons: saturated straight chain alkanes methyl branched alkanes and alkenes. n-Alkanes and branched alkanes are usually the most abundant of the cuticular hydrocarbons ranging from about to 43 carbon chain length (Howard and Blomquist, 2005). Tricosane n-pentacosane n-h eptacosane n-nonacosane and n-hentriaconta n e are often predominant in n-alkanes of insect cuticular hydrocarbons. Mono methyl alkanes dimethylalkanes and trimethy l alkanes referred to as methyl-branched alkanes and their isomers exist in all insect cuticular lipids (Ne l son 1978 ; Lockey 1988). The major hydrocarbon components found on the surface l ipids are mostly methyl-branched alkanes. However n-a lkanes are the most abundant of the cuticular h ydroca r bons in some species or at different stages (Nelson, 1993). Cuticular hydrocarbons contain nestmate recognition cues. Wagner showed a connection between cuticular hydrocarbons and nestmate recognition Small glass blocks were coated with whole cuticular lipids and the pur i fied hydrocarbons to test on harvester ant 2

PAGE 13

colonies. The result revealed that the ants had aggression to the glass blocks with non nestmate cuticular hydrocarbons (Wagner 2000). Within the similar compositions of n-alkanes in insect cuticular hydrocarbons Lucas suggested that methyl branched alkanes have strong involvement in insect recognition behavior (Lucas 2005) due to their diverse variations in the position or number of methyl groups. In a harvester ant colony patrollers go out in the early morning to locate seed sources The patrollers return to the nest and inform the foragers who go out bring the food back and store it in the nest. est maintenance workers repair and maintain the chamber stuff the walls with moist soil to dry and firm the nest and carry the excavated dry soil out of the nest. Thus patrollers and foragers spending most of their time outside of the nest were found to have a higher proportion of straight-chain alkanes in their cuticular hydrocarbons than the nest maintenance workers (Wagner 1998). A subsequent study revealed that hot and dry conditions increase the proportion of n alkanes in ant cuticular hydrocarbons (Wagner 2001). Further experiments investigated responses in different task groups in the whole colony. Greene and Gordon mimicked the flow of returning patrollers by dropping the glass beads coated with cuticular hydrocarbons of patrollers cuticular lipids of patrollers cuticular hydrocarbons of nest maintenance workers and a solvent blank test. the results the patrollers cuticular hydrocarbons were sufficient to draft foragers out of the nest while blank and nest maintenance workers hydrocarbons did not elicit the foraging response (Greene and Gordon 2003) The western harvester ant (Fig. 1.4.1.) is very common in the southwest United States and throughout Arizona desert. The size of a harvester ant is about 5-7 mm and seeds are their main source of food. The size of a harvester ant colony consisting of one single queen is about 10, 000 to 12, 000 ants. This population makes the harvester ant a good 3

PAGE 14

model for cuticular hydrocarbon communication studies (Howard and Blomquist, 2005). The pavement ant (Fig. 1.4.2 ) is native to Europe and was introduced to the United States during 1 century. It is now a common household pest in the United States. The pavement ant is dark brown to black in color and around 3.2 mm in length. Their sources of food are almost anything such as honey insects seeds fruits cheese and grease Their name comes from their tendency to nest under stones sidewalks or wood boards. They are very aggressive and invade other colonies resulting huge battles. 4

PAGE 15

T h e western h a r veste r ant Picture taken from http: // www.alexanderwild .com/ a. b. The pavement ant a. Identifying characteristics of pave m ent ant Picture taken from http ://www. ipm ucdavis.edu /TOOLS/ANTKEY b Picture taken from 5

PAGE 16

Gas chromatography combined with a mass spectrometer is the most useful tool for environmental analyses. has been used for separating, characterizing and quantifying insect cuticular hydrocarbons in the past years. Both electron impact ionization (El) and chemical ionization (CI) are used for the analysis of cuticular hydrocarbon Vaporized sample molecules entering the ion source of the mass spectrometer collide with a beam of high-energy (70e V) electrons from the electron generating filament and form positive ions. The energy is transferred from electrons to sample molecules and produce fairly predictable fragment ions from the molecular ions (Reaction 2.1.1). These positive ions are pushed out of the source by a repeller potential and accelerated into the mass analyzer. (Reaction 2.1.1) Chemical ionization is another technique to create ions used in mass spectrometry analyses. chemical ionization large amounts of reagent gas are introduced into the ionization chamber and react with emitted electrons to form reagent gas ions. Then they react with sample molecules to form sample ions. Methane is the most common chemical ionization reagent gas due to its characteristic fragment patterns and its ability to react and yield ions with almost every sample molecule. Other reagent gases produce different fragment patterns and may result in better sensitivity for some samples 6

PAGE 17

Insect cuticle extracts contain a mixture of hydrocarbons. The use of a capillary column and temperature programming gas chromatography is a powerful technique for separating the components Retention time is the major information for identification in gas chromatography. Many compounds may be specified by their retention time in a gas chromatogram. However if two compounds have close retention times the identification and differentiation becomes difficult. A mass spectrometer provides additional information for compound identification Molecular weight and fragmentation patterns can be obtained from mass spectra so compounds can be identified unambiguously by using retention indices molecular weight and fragmentation patterns Kovats Index and Equivalent Chain Length are the two most useful tools for the comparison of retention times between different studies and conditions in analyses The simplest way to identify compounds is to compare retention times of unknown peaks with standard peaks in the chromatograms under the same experimental conditions. However not all the unknown compound peaks have the same retention times with standards. The relative retention of a component is the ratio of the retention time of the component to that of the chosen standard. A linear relationship is observed when the logarithm of the retention time is plotted against the number of carbon atoms for hydrocarbon analysis (Figure 3.1). Retention index gives us an idea of the total carbon number in hydrocarbon analysis 7

PAGE 18

1.4 1. 2 ... 0 8 0 0 6 ...J 0.4 0 2 0 20 22 24 26 28 = R"= 0 .973 30 32 34 36 38 40 F i g 3 1 The linear relationship of the logarithm of the retention time and carbon chain length of n alkanes in analysis The Kovats Index proposed by E. Kovats expresses that in the homologous series of paraffins in gas chromatography the retention index (1) of an analyte is obtained b y interpolation (Equation 3.1) Under the same stationary phase and isothermal temperature the logarithm of the ratio of retention v alue of the compound of interest s to the alkane with carbon atoms is divided by the l ogarithm of the ratio of retention values of the two n-alkane reference compounds The number is added to the number of carbon atoms of n-alkane To avoid decimal numbers the quantity is further multiplied b y This equation was based on the linear relationship between the logarithm of retention values and the number of carbon atoms of straight chain saturated fatty acids. The retention indices of straight chain alkane standards are b y definition as times of the number of carbon atoms (Kovats 1958). The Kovats Index is also used for the characterization of branched or unsaturated fatty acids. 8

PAGE 19

(3.1) X is the retention value, which refers the a dju s ted retention time or volume (Ettre 2003) ; is the difference in carbon number of two n-alkanes taken as sta ndard s, s is the compound of interest and is the number of carbon atoms of the n-alkane eluting before the peak of intere st. Temperature Programming Gas Chromatography (T PGC) the Linear Retention Index equation is applied. Total retention times measured under the same temperature programming conditions are used instead of their logarithm values in this equation. (3.2) is the g ro ss retention time of a substance. is the difference in carbon number of two n-alkanes taken as standards s is the compound of interest and is the number of carbon atoms of the alkane e lutin g before the p eak of inter est. This system is similar to Kovats Index but with the advantage in representing the number of carbon atoms directly (Miwa, 1963). ECL is expressed as the following equation: 9

PAGE 20

100 5 7 80 43 n-hexadecane 71 c 60 -0 c ::::l ..CI 4 0 85 2 0 29 [Ml' 197 226 0 0 0.0 50 100 150 200 m / z Mass spectrum of n-hexadecane. (Adapted from NIST Mass Spec Data Center http: // webbook.nist.gov.) 250 Chain branching causes a lower abundance of molecular ion and increases the abundances of C n H2n 1 and C n H 2,/ ions through cleavage to form more stable carbon cations and the charge retention at the branched carbon with the loss of the largest alkyl radical is favored. The mass spectra of mono-olefins show clusters of C n H 2 n -1 and C n H 2 ne ions also gradually decreasing in abundance with increasing mass. The molecular ion is more prominent than in saturated a l kanes for compounds of lower molecular weight owing to better stabilization of the positive charge by removal of one of the n-electrons which leaves the carbon skeleton undisturbed. and isomers usually have very similar mass spectra. Alkene ions exhibit allylic cleavage (most favored) (Figure 3.2.2) and vinylic cleavage (less favored). However a strong tendency of hydrogen rearrangement in the molecu l ar 11

PAGE 21

ions results in migration of the double bond along the chain. Hence mass spectra of mono-olefins with double bonds in different positions are generally very similar (Figure 3.2.3 ) The location of double bond position needs to be further studied. .------, : H2 H2 : R-:-C -c====c-c -:-R 3 F ig. 3 2.2 1 Allylic cleavage in a mono-olefin 41 00 41 1 00 c=c/ eo., .., c: c: .. = 1J c: c: .Q 60 .Q .. .?: ;; ;; 0 a: 10 20 20 1 20 25 30 4() .$ 50 55 m l e m l e Fig. 3 2.2 2 Mass spectra of I-butene (left) and 2-butene (right). The mass spectra were adopted from NIST Mass Spec Data Center http: // webbook.nist.gov

PAGE 22

Chemical ionization involves much lower energy transfer than electron ionization, which makes chemical ionization a softer ionization technique. Therefore chemical ionization is often used to determine the molecular weight of sample compounds. There are four common positive chemical ionization processes in an ion source with reagent pressure at 0.8-2.0 Torr: proton transfer hydride abstraction, addition, and charge exchange. The thermodynamics are favorable for hydride abstraction in positive chemical ionization if the hydride ion affinity of methane reagent gas ions is higher than the hydride-ion affinity of the ion formed by the analyte s loss of Hydride abstraction is the main reaction in hydrocarbons because of the low proton affinity of methane. When methane is used as the reagent gas, both + and C 2HS + reagent ions are capable of hydride abstraction (Reaction 3.2.3.2 3). CH5+ and C 2 H5+ have large hydride ion affinities, which results the loss of for long-chain hydrocarbons. This reaction is exothermic so the fragmentation of [M-Ht is abundant as the base peaks in the CI mass spectrum. Here we denote [M -Ht ions as quasimolecular ions since molecular ions represent [M e r in EI spectrum (Gross 2004) CH4 + e[CHd + + 2 e-or CH4 eCH3 + H-e-R + M [M-H] + RH (R = CH s + C2H/; M = hydrocarbon molecules) (Reaction 3 2.3.1) (Reaction 3.2.3.2) (Reaction 3.2.3.3) (Reaction 3.2.3.4) The CI spectrum shows not only a method of determination for the molecular weight of hydrocarbons ; it also provides structural information to characterize of the hydrocarbons. Howard and his colleges investigated the ratios of CI generated [M-1Sr to the base peak

PAGE 23

of norma l a l kane monomethyl a l kane and dimethyl alkane standards. For straight chain alkanes [M -15t/ base peak = 3-5 %, monomethyl alkanes the ratio is about 6 -10%, and dimeth y l alkanes is 11-20% (Howard et aI, 1980). The methane chemical ionization spectra of mono olefins consist two ion series: C n H 2 n + lalkyl ions from the [M +Ht ion and C n H 2n-l+ alkenyl ions from the ion. The C n H 2 n -1 + a l kenyl ions are usually more prominent than C n H2n1 + alkyl ions in the spectra since allylic hydrogens are the most abstracted for rearrangement. An example of methane CI mass spectra of n decenes is shown in Figure 3.2.3. The clusters of peaks reach the maximum intensity around C4C g at 55-111 ) and gradually decrease to the higher mass region Quasimolec u lar ions and can be seen easily in the spectra Like alkenes in EI spectra there is no reliable indication of the doub l e bond location in methane CI spectra of olefins (Harrison 1992) 5 Methane CI mass spectra of n-decene isomers (adapted from Harrison 1992).

PAGE 24

In previous studies of insect cuticular hydrocarbon gas chromatography analysis the presence of branchings or double bonds in alkanes elutes earlier than the n-alkanes with the same total number of carbon atoms. Branching positions in the alkanes also have some effects upon retention times. Internally branched alkane isomers (such as branching position at 15, 13, 11, 9, or 7-methyl alkanes) elute earlier than the terminally branched alkane isomers (such as 3, or 2-methyl alkanes) (Lockey 1988) However the separation of isomers is not always seen in gas chromatography. Sometimes the isomer mixtures co elute as one peak in the total ion chromatogram. As an example in the red harvester ant cuticular hydrocarbon profile (Table 6.3.2 page 30), peak 27 A contains three methylnonacosane isomers: 15, 13, and 9-methylnonacosane Fatty acid mixtures are very common components of insect cuticular lipids. The fatty acids contain primary even carbon atoms ranging from 10-36 but shorter in homologous ranging from 14-18 carbons. Lauric acid (12:0) myrystic acid (14 : 0) palmitic acid (16:0) and stearic acid (18 :0) are the most common saturated carboxylic acids. Palmitoleic acid (16 :1), linoleic acid (18:2) and oleic acid (18:1) are the most commonly reported unsaturated acids in insect lipids (Lockey, 1988).

PAGE 25

n-alkane standard mixture 50mg/1 in n-heptane (Sigma-Aldrich contains C C20, CC24, C 26, C28, C30, C32, C34, C36, C38, and C40) Chloroform HPLC grade (Fisher Scientific) Pentane HPLC grade (Sigma-Aldrich) Ants were collected b y Greene and Sano. Harvester ants were collected in the summer of 2007 from colony nest mounds at the permanent field site next to the Denver Children s Museum and the South Platte River using gloved-hand then placed into collection tubes. Pavement ants were collected in 2006 from nests found on the University of Colorado Denver Campus and Downtown Denver area using an aspirator from foraging trails or by using honey bait to lure foragers out of the nest. Cuticular hydrocarbons were extracted by Greene. Ants from all colonies were collected in the field and killed by freezing at -20 C. Cuticular lipids were extracted by soaking ants in 1.0ml 100 % n-pentane. Gently shaken for the first 1 minute of soaking step after 10 minutes of the soaking step we transferred the lipid extracts onto a solid phase of2 cm

PAGE 26

silica gel (70 230 mesh average pore diameter: 60 A. SIGMA ) in a pasteur pipette. Cuticular hydrocarbons were separated from polar surface lipids by elution from the silica gel using 2-3ml 100% n-pentane. Extracts were collected in clean glass vials and dried under a stream of nitrogen. The residue was redissolved in 1 ml of chloroform The cuticular extract samples were analyzed by using gas chromatography mass spectrometry (Agilent MSD) in the Anesthesiology Department of the University of Colorado Denver. 2, d of samp l es were injected by spitless injection onto a capillar y column (OV-S fused silica capillary column 30m 0.32rnm ID Ohio Valley). Samples were purged after 1 min. The carrier gas was helium with the flow rate set to 1.1 mllmin. The injection temperature was 280 C. For electron ionization the initial oven temperature was 170 C for 1 min and then the temperature was increased from l70 C to 300 C at lOO C then more slowly from 300 C to 330 C at lS O C To ensure of the consistency of retention time between runs a mixture of n-alkane standard (containsCIO, C20 C22 C2 4 C26 C2 8 C30 C32 C34, C36 C3 8 and C4 0 Sigma Aldrich) were injected before sample runs. For chemical ionization GC the initial temperature was 7So C after 2 minutes the temperature was increased from 7So C to 330 C at ISO C I min and held at 330 C for 10 minutes. The ant cuticular hydrocarbons were quantitatively analyzed by measuring the peak areas of the resulting chromatograms using Agilent MSD ChemStation Software. RTE Integrator was selected for integration. Only the peak area > 1 % of the largest peak area were selected for integration for quantitation The idea of using relative abundance in insect cuticular hydrocarbons is to take out variability of the number of ants in the sample and compare the percent composition for further studies. The integration

PAGE 27

parameter settings are shown in Fig 5 3.3. 1 p 2] ITop .s. 1 0 .200 2 50 % J Fig. 5 3 3. Integration parameters for ant cuticular hydrocarbon quantitation.

PAGE 28

All structural characterizations were mainly based upon both ofEI and CI mass spectra and methods described in Chapter 3. Examples of TIC mass spectra and structure characterizations of Harvester Bulk Ant I sample are shown in APPENDIX Five harvester ant colonies and two bulk ant samples w ere analyzed. The cuticular hydrocarbons of har v ester ants range in length from 23 to 36 carbon atoms. Six n alkanes eleven mono-methyl alkanes and one dimethyl alkanes were found in five harvester ant colonies Compounds that are identified are n-pentacosane n-hexacosane n-heptacosane n-octacosane(n-C2 8 ) n-nonacosane (n-C29)and n-hentriacontane in straight chain alkanes ; 7, 3-methylpentacosane (7, 3-meC2S) 8 methylhexacosane ( 8-meC26 ) 13, 7-methylheptacosane (13, 7-meC27 ) 13, 11-, 9methylnonacosane (13 11, 9-meC2 9 ) 15, 13, and 7-methylhentriacontane (15 13, 7meC in mono-methyl branched alkanes and 7 13-dimethylheptacosane (7, 13dimeC27 ) in dimethyl branched alkanes. Trimethyl alkanes or alkenes in the western harvester ant cuticular hydrocarbons were either missing or less than 1 of the largest peak in chromatograms Fatty acids were found in har v ester ant samples except for the Bulk Ants I sample which passed through the silica gel column to isolate fatty acids from hydrocarbons. Other samples did not pass through the silica gel column so the fatty acids were shown and integrated in total ion chromatograms (Table 6.2.1.1 page 27).

PAGE 29

Both Kovats Index and retention indices of each identified compound were listed in Tables 6 1.2.1 to 6 1.2.7 The average and standard deviation of shared n-alkanes between harvester ant samples were listed in Table 6.1.1.1. Kovats Index has much bigger standard deviation than due to 100 was multiplied to avoid decimal point. Regardless the factor the standard deviations of both retention indices is close to each other Figure 6.1.1.1 and 6.1. 1.2 show the linear relationship of straight chain alkanes and their and Kovats Index respectively Generally, straight alkanes have number of carbon atoms as and 100 times of the number of carbon atoms as Kovats Index 1. For instance the of n-pentacossane is 25 and is 2500. Linear relationship does exist before between and n-alkanes however for Kovats Index it is off from to H y drocarbon structurally characterization are primarily based on both EI and CI mass spectra while using retention indices as references. Avg. STDEV n-alkanes 1 2503.12 24.96 3 .65 0 .03 2618.12 25.98 3.65 0.03 2692.22 26 .95 3.94 0.04 2796.47 27.97 2.57 0.02 2890.43 28.93 2 57 0.03 3091.24 30 .95 1.62 0.02 Table 6 .1.1.1 Averages and standard deviations of shared n-alkanes of harvester ant cuticular hydrocarbon profiles 20

PAGE 30

32 31 30 = 1.135x 23.68 R 2 = 0.98 29 Cj 28 -'-# 4 27 26 25 BA II 24 n-C25 n-C26 n-C27 n-C28 n-C29 n-C31 Figure 6.1.1.1 Linear relationship between and identified n-alkanes. 3200 3100 Y = 109.9 x 2384 3000 R 2 = 0.975 ---# 2 2900 -.-# 4 2800 2700 #C 2600 BA II 2500 BA 1 n-C25 n-C26 n-C27 n-C28 n-C29 n-C31 Figure 6.1.1.2 Linear relationship between and identifi ed n-alkanes. 21

PAGE 31

Percent compos ition was c alculat e d from the integrat e d T I C area as desc r ib e d in Data an a l y si s T abl es 6.1. 2.1 t o 6 .1.2. 7 s ho w all th e charact e rized compounds w ith th e ir rel a ti v e abundance of h a rvest e r an t samples. H arves ter ant Colony 1 Peak R.T. Pet T o t a l ECL C o mp o und s 1 4.505 3.8 3 2223 22 2 4 Oleic acid trim e th y l sily l e s t e r 2 4 .681 0 5 5 2298 2 3 .01 T ric os an e 3 5 .167 4 .26 2 507 2 4 99 P e n t ac os an e 4 5.2 76 5.05 2 554 2 5.41 7-m eC25 5 5 3 68 1.14 2 593 25. 76 3 -meC25 6 5.4 3 5 1.89 2 6 22 2 6.01 n-H e xac os an e 7 5.544 1.88 2 6 3 5 26.3 7 8 m eC26 8 5.728 15. 9 4 2 694 2 6.97 H e ptac o san e 9 5.83 7 27 .18 2 729 2 7.31 1 3 11-, 7-m eC27 10 5.9 6 3 3 6 1 2 769 2 7.71 7 1 3 -dim e C27 6.055 1.0 4 2799 2 7.99 n-O c t a c osa n e 1 2 6.17 3 1.76 2 8 2 9 28.3 1 1 4 -m e C28 1 3 6.424 2 .75 2892 2 8.95 n-N o nac os an e 14 6.558 14. 08 2 926 2 9.29 1 3 11, 9-m eC29 1 5 6.718 3 09 2 966 2 9.68 7 1 3 -dime C29 16 6.9 94 1.0 1 3 0 2 7 3 0.29 1 4 -m eC3o 17 7.33 8 1.2 6 3093 30.96 n-H e ntriacontan e 18 7.506 4 1 3 3125 3 1.28 15, 1 3 -m e C31 19 7.556 2 92 3 1 3 4 31.3 8 7-m eC3 1 2 0 7.72 3 1.48 3166 3 1 68 7 1 3 -dimeC3 1 2 1 8 796 0.37 3 325 33.2 9 11-m e C33 22 8 855 0 7 9 3 3 3 4 33. 3 7 7-m eC33 P e rc e nt compos ition of harves t e r ant cuticul a r h y drocarbons from colon y #1. R.T. i s t h e r e t entio n tim e s hown in GC t o t a l i o n c hromatogram; i s K ova t s Index, and ECL i s Equival ent C h ain L e n gth. 22

PAGE 32

Harvester ant Col ony #2 Peak R.T. P et Tota l ECL' Co mp ounds 4.102 2 94 2044 20.45 Hexa d eca n oic acid trimet h ylsilyl ester 2 4.488 21.15 22 1 6 22 1 6 O l eic ac i d trimethy l sily l ester 3 5.150 3.47 2500 24.93 Pe n tacosane 4 5.259 3.04 2546 25 35 7 meC25 5 5.351 0.91 2586 25.69 3 meC25 6 5.418 2.1 0 26 1 4 25.94 7 5.527 1.14 2629 26.31 8-meC26 8 5.712 21.93 2689 26 92 n Heptacosane 9 5.82 1 17.15 2724 27.26 13, 11-, 7 -meC27 10 5.946 1.87 2764 27 66 7 ,13dimeC27 6.039 1.36 2 7 94 2 7 .94 12 6 .156 1.04 2825 28.26 14, 8 meC28 1 3 6.40 7 4.78 28 88 28.9 1 14 6 .541 8 25 2922 29.25 13, 11-, 9-meC29 15 6 70 1 0.89 2962 29 64 9 1 3 d i meC29 16 7 .321 2.49 3090 30.93 n-Hentriacontane 1 7 7.4 8 0 2 .05 3 1 20 3 1 .23 15, 1 3 meC18 7.539 2.04 3 131 31.34 7-meC 1 9 8 268 1.41 3252 32.55 un known Percent composition of harvester ant cuticular hydrocarbons from colony # 2 R.T. i s the retention time s hown in GC total i o n chro mato gram; i s Kovat s Index, and E .C.L. i s Equivalent Chain Len gth. 23

PAGE 33

Harves ter a n t Colony # 4 P eak R.T." P et Tota l I." ECL Co mp o und s 4.488 0.41 22 1 6 22.16 ll-C i s -O ctadece n o i c aci d 2 4. 6 64 0 70 229 1 22.94 Triacosane 3 5 1 50 7 .20 2 500 24. 9 3 P e n tacosa n e 4 5.2 5 9 3.69 2 54 6 25.35 7-m eC25 5 5.3 5 1 0 8 4 25 8 6 25. 6 9 3 -m eC25 6 5.418 3.53 2614 25.94 nH exacosa n e 7 5.5 27 1.3 1 2629 26.3 1 8 m eC26 8 5.712 30.59 2 68 9 26 92 n-H e pt acosa n e 9 5.8 2 1 20.43 2 72 4 27.26 1 3 9 7-m eC 1 0 5.946 2 2 8 2 7 64 27.66 7 1 3 -dim eC1 1 6 0 39 1.69 2 7 94 2 7 .94 nOct a c osa n e 12 6.164 1.24 2 8 2 7 28.2 8 8-m eC28 13 6.40 7 5.40 2 888 28.9 1 n -Non acosa n e 1 4 6.54 1 9.23 2922 29.25 1 5 1 3 11-, 9 7-m eC29 1 5 6.701 1.3 1 2 9 62 29.64 9 1 3 ; 7 1 3-d i meC29 1 6 7 .32 1 2.42 3 090 30.93 n-He ntri aco n ta n e 17 7 .489 2 1 0 3 1 22 3 1 .25 1 5, 1 3 -m eC31 1 8 7 .539 3.63 3 131 3 1 .34 7-m eC31 1 9 7 .6 98 0.54 3 1 62 3 1 .64 1 4 7-m eC32 ; 7 1 3 -dim eC3 1 20 8 8 4 7 1 .46 3333 33.36 7-m eC33 6.1.2.3 P erce n t composition of h arves ter ant c u tic ul ar h y d rocar bons fro m colony # 4. R .T i s t h e r e t entio n time s h o w n i n GC t o t a l i o n c hrom a togr a m ; i s K o v a t s Index, and E .C.L. i s Equival ent C h a i n L e n gth. 24

PAGE 34

Harvester ant Colony # B Peak R.T: Pct Total : ECL Compounds 4.496 0 .35 2219 22.20 Oleic acid TMS 2 4.664 0.55 229 1 22.94 T rico sane 5.150 5.48 2500 24.93 Pentacosane 4 5.259 3.43 2546 25.35 13-, 11-, 9, 7-meC25 5 5.351 0.79 25 86 25.69 3-meC25 6 5.418 2.5 7 2614 25.94 n-Hexacosane 7 5.527 1.27 2629 26.3 1 8-meC26 8 5.712 2 7 .17 26 89 26 .92 n-Heptacosane 9 5.821 19.29 2724 2 7.26 1 3 11-, 9, 7 -meC 10 5.946 2.32 2 764 2 7.66 7 1 3 -dimeC27 6.039 1.65 2 794 27.94 n-Octacosane 12 6 164 1.30 2827 28.28 14-, 8-meC28 1 3 6.407 5.93 2 888 28 .91 n-Nonacosane 14 6 .541 11.18 2922 29.25 15, 13-, 11-, 9, 7 -me C29 15 6.701 1.80 2962 29. 64 9 13; 7 13-dime C29 16 6.826 0.42 2994 29.94 Triacontane 17 6.977 0.80 302 8 30.30 14-meC3o 18 7.321 3.30 309 0 30.93 n-Hentriacontan e 19 7.480 3. 79 3 120 31.23 15, 13-, 11-m eC31 20 7.539 3 .70 3131 3 1 .34 7-meC31 21 7 .69 8 0.86 3162 3 1 .64 14, 7-meC32 ; 7 1 3 -dimeC31 22 8.536 0 76 3289 32.93 Tritriacontane 23 8.838 1.34 333 1 33.35 7-meC33 Perc ent composition of harvester ant cuticular hydrocarbons from colony #B. R.T. is the r e t e nti on tim e s hown in GC t ota l ion c h roma togram ; 1. is Kova t s Ind ex and E.C.L. i s Equival e nt C hain Length 25

PAGE 35

H arves t e r ant C olon y #C Pea k R.T." Pet T o tal I." E CL Compound s 1 4.496 0.30 2219 22. 20 Oleic acid trimeth y lsil y l e s t e r 2 4 .672 0.47 2294 22. 97 Tric os an e 3 5 158 4 88 2 50 3 24.96 Pentac os an e 4 5.267 3.53 2550 2 5.38 13, 11-,9-, 7-meC25 5 5.360 0 99 2590 2 5 7 3 3 -meC25 6 5.4 2 7 2 .56 2618 2 5.98 n-He x ac os ane 7 5 .536 1.48 26 3 2 2 6.34 8-meC26 8 5 720 2 4.70 2691 2 6.94 n-Heptac os an e 9 5.829 19.74 2726 2 7.29 13, 11-, 9, 7-meC27 10 5.955 2 2 9 2767 27.68 7 1 3 -dim e C11 6.047 1.68 2 796 27.97 n-Octac osane 1 2 6 .173 1.55 2829 2 8 .31 14-, 13-, 1 2 8-meC28 1 3 6.416 5 7 3 2890 28.9 3 n-Nonac os an e 14 6 .550 1 2.05 2 924 2 9 27 13, 11-, 9, 7 -m eC2 9 15 6.709 1.2 7 2964 2 9.66 9 13; 7 1 3 -dime C29 16 6 835 0 50 2996 2 9.96 T riac o ntan e 17 6.986 1.09 3026 3 0.27 14-meC3o 18 7.329 3 50 3091 3 0 .95 n-Hentriac o ntan e 19 7.497 4.12 3123 31.2 7 15-, 1 3 Il-me C 2 0 7 .547 4.01 3133 31.3 6 7-meC2 1 7.707 0 9 2 3163 3 1.65 7 13-dimeC22 8 .545 0.84 3290 32 .94 Tritriac o nt a n e 23 8.847 1.81 3333 33. 3 6 7-meC33 6.1.2.5 Perc e nt compos ition o f har v e s ter ant cuticular hydrocarbons f rom colon y #c. R.T. i s th e rete nt i o n time s hown i n GC tota l i on c hromat ogram ; is Kova t s I n dex a n d E C.L. is Equivalen t C h a i n Length 2 6

PAGE 36

Harvester Bulk Ant II Peak Pet Total ECL Compounds I 4.505 0.42 2223 22.24 11-Trans-Octadecenoic acid 1 TMS 2 4.681 0.67 2298 23.01 Tricosane 3 5.167 7.36 2507 24.99 n-Pentacosane 4 5.276 3.14 2554 25.41 13-, 11-, 9, 7-meC25 5 5.368 0.79 2593 25.76 5-,3-meC25 6 5.435 2.93 2622 26.01 n-Hexacosane 7 5.544 0.96 2635 26 37 8-meC26 8 5.728 33.57 2694 26.97 n-Heptacosane 9 5.837 16.46 2729 27 .31 13-, 11-, 9, 7-meC27 10 5.963 2.18 2769 27.71 7 13-dimeCII 6.055 1.72 2799 27.99 n-Octacosane 12 6.173 0.85 2829 28.31 14, 8-meC28 13 6.424 6.71 2892 28.95 n-Nonacosane 14 6.558 8.20 2926 29.29 13-, 11-, 9, 7-meC29 15 6.718 1.39 2966 29.68 9 ,13-; 7 13-dimeC29 16 6.843 0.47 2998 29.98 Triacontane 17 6 994 0 62 3027 30.29 14-meC18 7.338 3.55 3093 30.96 n-Hentriacontane 19 7.505 2.12 3125 31.28 15-, 13-, II-meC 20 7.556 3 32 3134 31. 38 7-meC 2 1 7.723 0.47 3166 31. 68 7 13-dimeC22 8.553 0 .73 3291 32.96 Tritriacontane 23 8.855 1.37 3334 33. 37 7-meC6.1.2.6 Percent composition of harvester ant cuticular hydrocarbons of Bulk Ant II R.T. is the r e t entio n time s hown in GC t o t a l ion c hrom atog r a m ; i s Kovats Index, and E .C.L. i s Equival ent C h ain Length

PAGE 37

Harvester Bulk Ant Peak R .T.' Pet Total 1.' ECL Compounds 4 .681 0.73 2298 23.01 Tricosane 2 4 915 0 .23 2399 23.99 Tetracosane 3 5.167 6 .16 2507 24.99 Pentacosane 4 5.276 4 .19 2554 25.41 13, 11-, 9, 7 -meC25 5 5.368 1.03 2593 25.76 3-meC25 6 5.435 2 82 2622 26.01 n-Hexacosane 7 5 544 1.56 2635 26.37 8-meC26 8 5.745 16. 78 2699 27.02 n-Heptacosane 9 5.846 20.09 2732 27.34 13, 11-, 9, 7-meC27 10 5 963 2 68 2769 27 .71 7 13-dimeC27 11 6.055 1.77 2799 27.99 n-Octacosane 12 6.173 1.55 2829 28.31 14, 13, 12, 8-meC28 13 6.432 6.11 2894 28 97 n-Nonacosane 14 6.558 12. 35 2926 29 29 13, 11-, 9, 7-meC29 15 6 717 2.14 2966 29 68 9 13; 7 13-dimeC29 16 6.843 0 .51 2998 29.98 Triacontane 17 6.994 1.15 3027 30.29 14-meC3o 18 7.338 3 86 3093 30 96 n-Hentriacontane 19 7.505 4 38 3125 31.28 15, 13, II-meC31 20 7.556 3.81 3134 31.38 7-meC3 1 21 7 715 1.27 3165 31.67 7 13-dimeC3 1 22 7.891 0 34 3198 31.99 n-Dotriacontane 2 3 8 553 1.01 3291 32.96 Tritriacontane 24 8.788 0 .61 3324 33.28 15, 13, 11-meC33 25 8 863 2 .15 3335 33.38 7-meC33 26 9.064 0.33 3363 33.65 7 I 3-dimeC33 27 10.523 0 .12 3522 35.26 13-meC35 28 10.640 0.30 3534 35.38 7-meC35 Percent composition of harvester ant cuticular hydrocarbons of Bulk Ant R.T i s the r e t entio n time s h own in GC t o t a l i o n c hr o m a t og r a m ; i s Kovat s Index, and E.C.L. i s Equival ent Chain Length 2 8

PAGE 38

There are about 21 peaks identified in each colony. Seven n-alkanes eleven mono meth y l alkanes and one dimethy l alkanes were found in five harvester ant colonies Compounds that were identified included n-tricosane n-pentacosane hexacosane n-heptacosane n-octacosane n-nonacosane C29 )and n-hentriacontane in straight chain alkanes; 7, 3-methylpentacosane (7 3-meC2S) 8-methylhexacosane (8-meC26 ) 13, 7-methylheptacosane (13, 7-meC27) 13, 11, 9-methylnonacosane (13, 11, 9-meC29) 15, 13, and 7-methylhentriacontane (15, 13, 7-meC 31) in mono-methyl branched alkanes and 7 13-dimethylheptacosane (7 13dimeC27) in dimethyl branched alkanes n-Heptacosane is the most abundant peak in harvester ant colonies # 2 # 4 # B #c. Mono-methyl branched heptacosane and nonacosane are the second and the third largest peaks respectively Table 6.2 1 1 shows the proportions of straight chain alkanes methyl branched alkanes and fatty acids in harvester ant samples Colony Colony Colony Colony Colony Bulk Ants [] Bulk Ants I #2 # 4 # B # C Fatt y acidsa 3 .83 24.09 0.41 0.35 0.30 0.42 b n-Alkanes 27.69 36.13 51.53 47.81 44.87 57.71 40.30 Mono-methyl 60.31 35.61 44.47 47.73 50.35 37.83 53.29 alkanes Dimethyl 8.17 2.76 3.59 4.12 4.48 3.57 6.41 alkanes Percent composition of seven harvester ant samples a T h e fatty a cids in Bulk Ant s I sample wer e eithe r missing o r l ess than I of the l a rgest p ea k area while they w e r e s h own and int eg rat e d in othe r h arves t e r ant s amples, which did n o t pass through the silica gel c o lumn t o i o l a t e f att y acid s fro m cuticula r h y d rocarbo n s 29

PAGE 39

b A t indic a tes the fatty acids were eithe r missing o r it' s less tha n 1 o f the largest p ea k area in the c hromatogram. The harvester ant cuticular lipids contain main l y n-alkanes and mono methyl branched alkanes (>70%), with small amount of dimethy l alkanes and fatty acids. Fatty acids were present in small quantity (ca. 0.37%) except in colonies # 1 and #2, where fatty acid concentrations were observed in 10-100 folds bigger than in other samples. Oleic acid (18: 1) is the most common fatty acid among harvester ant samples. Oleic acid concentrations were 3.83% in colony # 1 21.15% in colony # 2 0.35% in colony # B and 0 30 % in colony # C while II-octadecenoic acid concentrations were 0.41 % in colony # 4 and 0.42% in Bulk Ant II. Also hexadecenoic acid was found in colony #2 (2.94%). Colony # 2 has extremely high concentrations of oleic acid (88% of its total fatty acids) which is present in many of the seeds that the harvester ants eat. But what causes the ants produce such a high abundance of oleic acid on their cuticles ? It has been demonstrated that diet alters insects cuticular hydrocarbon profiles. The source of food of colony #2 might contain excessive oleic acid reflected in their cuticular lipid composition. It could be a chemical signal of necrophoric behavior Oleic acid is released from the decaying bodies of dead harvester ants triggering midden workers' instincts to remove dead bodies from the nest (Gordon 2000). Another interesting point of view is fatty acids can also be converted to n-alkanes in some insects (Blomquist Although the biosynthesis pathways of cuticular lipids are not clear events such as expanding the colony switching tasks and the responses to diet or to environmental conditions may induce biosynthetic factors that alter the cuticular hydrocarbon profile Although most harvester ant cuticular hydrocarbons are detected in all colonies, their relative quantities differ (Figure 6.2 .2) Methyl branched alkanes in colony # 1 are much higher than n-alkanes (28 %) compare to other colonies. In addition mono methyl heptacosane and mono-methyl nonacosane are the major contributors in the high abundant methyl alkanes. 30

PAGE 40

35 30 25 2 0 15 # 2 1 0 -#4 # 8 5 # C 0 F i g ure 6.2. 1 Shar e d c uticular h y droc a rbon s o f h a r ves t e r a nt colonies. 3 1

PAGE 41

30 25 20 1 5 1 0 5 o 35 30 25 20 1 5 1 0 5 0 30 25 20 1 5 10 5 o 32 25 20 1 5 1 0 5 o 30 25 20 1 5 1 0 6.2.2

PAGE 42

Lucas mentioned that the presence of internally methyl alkanes have strongly correlated with recognition beha v ior ( Lucas 2005) Eighteen fifteen twenty one twenty eight and twent y seven internally methyl alkanes were identified in harvester ant colony # 1 # 2 # 4 # B and # C respectivel y After subtracting those common internally meth y l alkanes the differences in internally methyl alkanes among five harvester ant colonies are shown in Table 6.2 1 .2. #1 #2 #4 # B #C nA lkane 0.76% 0.84% Methyl branch ed a lkan es II-meC 9-meC14-meC28 [;J 1.04% 1.30% 8-meC28 1.24% 1.55% 13meC28 12-meC 2 8 15-meC 2 9 9 ,13 -dimeC29 0.89% 1.31% 1.80% 1.27% 7 ,13 -dimeC29 3.09% 14-meC3o 0.80% 1.09% I1-meC 3 1 7 13-dimeC 3 1 1.48% 7.07% 14-meC32 0 .54% 0.86% 7meC32 II-meC33 0.37% 7-meC33 0.79% 1.46% 1.34% 1.81% Table 6.2.1.2 The percent composition differences in cuticular hydrocarbons among 33

PAGE 43

harvester ant colonies. a A P r e pr ese nt s the co mp o und was ide ntifi e d b y E I m ass s p ec trum but co e lut e d w ith it s i so m e r s o r othe r co mp o und s so the pe r ce nt co mp ositio n itse l f c an n o t b e d e t e rmin e d b T h e p e r centag e with b o ld b o rd e r s r e pr esents the i so m e r s o r co mp o und s co e lut e d and int eg r a ted as o n e peak in TI C whi c h i s the t o t a l p erce nt c o mpo sitio n o f these co mp o llnd s In colony # 1 11-methyltritriacontane (ll-meC33 ) was only compound detected in colony # 1 and 7 ,13dimethyltritriacontane (7 13dimethy I C33) in colony # C is relatively high in percent composition while in colonies # 4 and # B are below 1 % The compounds may be their chemical cues involved in nestmate recognition Colonies # 4 # B and # C have similar cuticular hydrocarbon expression profiles which might be an indication of they are genetic-related or experience the similar environmental conditions. Task recognition in harvester ant has been investigated. Task groups have significantly different relative proportions in the classes of hydrocarbons and in the individual compounds (Wagner 1998). Larger ant colony size might require more compounds as task-recognition cues to distinguish different tasks performed. Colonies # B and #C contain the most differences in cuticu l ar hydrocarbons and co l ony # 2 has the fewest individual compounds which could relate to bigger colony size and older colony age of colonies # B and #C and smaller colony size and younger colony age of colony #2. Around 42 peaks were detected that are >0.1 of total area in the red harvester ant sample The carbon chain length of red harvester ant ranges from 23 to 49 carbon atoms. Table 6.3 3 shows the red harvester ants cuticular hydrocarbons (Nelson 2000). In the western harvester ant (Table 6.1.2 7) only 28 peaks were integrated and identified which range from C23 to C3 6 As summarized in 34

PAGE 44

Table 6.3.1 the cuticular hydrocarbon profiles of contain ca. 40 alkanes ca. 53% mono-methyl branched alkanes ca. 6% dimethyl alkanes. Trimethyl alkanes or alkenes were either missing or less than 1 of the largest peak area. has equally represented n-alkanes and mono-methyl alkanes were present at lower abundance (13%). Dimeth y l alkanes trimethyl alkanes and alkenes only constituted 1-2 % of the cuticular hydrocarbon composition Both of the harvester ant species contain mainly saturated alkanes ( > 95%) in their cuticular h y drocarbon profiles. The major n-alkane is n-heptacosane in and n-pentacosane in The major mono-methyl alkane is mono-methyl heptacosane in and mono-methyl hentriacontane in Internall y methyl branched alkanes are related to recognition behavior. Table 6.3.2 lists the differences in cuticular hydrocarbons of harvester ant and Species-specific recognition compound(s) ma y be one or more of internally methyl alkanes listed on the table. Within a species ants s hare the same hydrocarbon components but the abundance of each compound relative to the others varies between different colonies. However different species have different cuticular hydrocarbon profiles. n-Alkanes 40 3 37.1 Methyl alkanes 53.3 36.2 Dimethyl alkanes 6.4 13.2 1.6 Trimethyl alkanes Alkenes 2.3 Hydrocarbon percent composition of the harvester ant and A indicate s the c o mp o und s were e ither missing o r i t's l ess than 1 % of th e l a r ges t p ea k ar ea in th e chromatogram. 35

PAGE 45

n-Alkanes 0.2% 0.7% Methy l branched alkanes I I -meC25 7 1 3 -di meC25 II-meC27 S -m eCb O.S% 9 1 3 -dim eC27 0.8% 3 m eC27 b 1 4 13, 1 2 8-m eC28 1.6% 2 -m eC28 b O.S% lS -m eC29 S-m eC29 b 0.8% II, I S ; 1 3 17-dim eC29 3 -m eC29 b I S 1 3 11, 8, 7 meC3 0 1.1% 2 -m eC30 b 1.1% 9 -m eC31 1 3 17; 11, I S ; 9 1 3 -dim eC3 1 3.8% 11, I S 1 9 C3 1 O.S% I S 1 4 1 3 -m eC32 0.6% 1 4 1 8; 1 3 x -dim eC32 O.S% 17, 9-meC33 I S 1 9 ; 1 3 1 9 ; II 2 1-dim eC33 1 3 ,17, 21-; ll, I S 21-tri m eC33 O.S% 1 3 meC35 0.1% 7-m eC35 0.3% 7 1 3 -dim eC35 O.S% I S ,2 1 -dim eC47 O.S% Table 6.3.2 T h e p e r ce nt composi t i o n di ffe r ences in c u t i c ul ar h ydrocarb o n s of h arves t e r an t a nd a A P r e pr ese nt s the co mp o u nd was ide ntifi ed b y E I m ass s pec t rum but co e lu t e d with its i so m e r s o r othe r co mp o und s so the pe r ce nt co mp ositio n it se l f ca n no t b e d e t e rmin ed b. T h ese co mp oun d s are co nside r e d t o be ex t ernally m e t h y l a lk anes 3 6

PAGE 46

Peak Pet Total Compounds 6 r Tr i cosa n e 20 1 r-P e nt acosa n e 2 6 1 3 t9 -m eC25 b 7 -m eC2S B 0.7 3 -m eC2S; 7 1 3 -dim eC25 1.4 r-H exacosa n e 27:1 0.7 r-He pt acose n e 4 7 r H e pt acosa n e 27A 4 3 1 3 t 9 -m eC27 27A 3.5 7 meC27 27A O S S-m eC27 27 B 0.8 9 1 3 -dim eC27 27 B 3.0 7 1 3 -dim eC27 : t3-m eC27 28 0 5 2 8A' O.S :2-m eC28 2 9:1 0 .6 1-No n a c os en e 2 9 2.2 n-No n acosa n e 29A S 3 IS. t 1 3 II-meC29 29A 0.9 9 -m eC29 29A 1.7 7 -m eC29 0 8 S-m eC29 29 B 0 3 11, 1 5 ; 1 3 1 7 -dim eC29 29 B 1.0 9.1 3d i m eC29 29 B 1.9 7 1 3 -dim eC29 ; t 3 meC29 30 0 6 T r iaco nt ane 30A 1.1 IS. 1 4 1 3 11-, 8 7 -m eC30 30A' 1.1 2 -m eC30 3 1 : I 1.0 nH e ntri a conte n e 3 1 1.6 nH e ntri aco nt ane 3 1 A S.9 15. 1 3 11-. 9 -m eC31 3 1 A 1.1 7 -m eC3 1 31B 3.8 1 3. 1 7 ; II IS; 9 1 3 -dim eC3 1 3 1 B 0.7 7 1 3 -dim eC3 1 3 1 C O.S II. 1 5 1 9 -trim eC3 1 32A 0.6 IS, 1 4 1 3 -m eC32 3 2B 0.5 1 4. 1 8 ; 13, ? -dim eC32 an d i so m e r s 33A 2.0 1 7 IS. 13, 11. 9 7 -m eC33 33B 2 8 15, 1 9-: 1 3 1 9 ; 11.21; 7 1 3 -dim eC33 O.S 1 3, 17,21-; II. IS, 2 1 trim eC33 3SB 0.5 7 1 3dim eC35 0.6 ? 147B 0.5 15.21-dimeC4 7 6.3.3 Percent compo s ition o f h y drocarbons from laborator y maintained harvester ant 37

PAGE 47

a. P e rcent c ompositio n was cal cula t e d fro m the integ r a t e d T I C a reas, only the p e rcent compositio n > 0 1 o f t o t a l a rea i s s h own o n the t able b A t indicates that a trace a m o unt o f tha t isom e r was present based o n the presence of diagnostic i o n s in the mass s p e ctr a c This tabl e was ad apte d from Nel so n All structural characteri z ations are based on the methods described in Chapter 3 Examples of mass spectra and characterizations of the Pavement Bulk Ant I sample are shown in APPENDIX B Fiv e ant colonies and one bulk ant sample were anal y zed There are about 22 peaks detected in each pavement ant colony. The chain length in pavement ant cuticular hydrocarbon ranges from 15 to 31 carbon atoms. Straight chain alkanes mono methyl dimeth y l and trimeth y l alkanes and alkenes were characterized in pavement ant cuticular hydrocarbon profiles Six alkanes ten mono-methyl alkanes and two alkenes were found in all fiv e pavement ant colon y samples. The compound s present in all pavement colonies are n-pentadecane 1 5 ) n-tricosane n-tetracosane pentacosane and n-heptacosane in normal alkanes ; 11-, 3-meth y ltricosane (11, 3-meC23), 13, 11methyltetracosane (13, 11-meC 11, 3meth y lpentacosane (11, 3-meC25), 13, 4-methy lhe x aco s ane (13, 4-meC26), 13, and 3meth y lheptacosane (13, 3-meC27 ) in mono-meth y l branched alkanes ; heptacosene and nonacosen e in alkenes. Small amount % ) of fatt y acids was found in pavement ant colonies Speer 3 and N C-3 3 8

PAGE 48

Percent composition was calculated from the integrated TIC area as described in Data analysis. Tables 6.4.2.1 to 6.4.2.6 list all the characterized compounds and relative abundance for each pavement ant sample. Pavement ant Colony SP3 Peak Pet Total ECL Compounds I 2.770 4.80 n-Pentadecane 2 4.672 2.50 2302 23.04 Tricosane 3 4 756 0.67 2338 23.40 II-meC23 4 4.848 1.32 2374 23.75 3-meC2 3 5 4.907 1.09 2400 24.00 Tetracosane 6 4.999 0 .91 2436 24.37 13-, 11-meC24 7 5.100 0.67 2476 24.77 3-meC2 4 8 5.159 10. 00 2502 25.03 n-Pentacosane 9 5.251 18.60 2536 25.37 11-meC25 10 5.360 12.09 2575 25.75 3-meC2 5 5.427 1.12 2600 26.00 n-Hexaco s ane 12 5.460 1.18 2608 26.08 8 II-dimeC25 13 5.527 2.65 2634 26.34 13-meC26 14 5.603 0.57 2659 26 .61 4-meC26 15 5.670 13.37 2680 26.82 HeRtacosene 16 5.720 6 27 2702 27.03 Heptaconsane 17 5.829 11.43 2734 27.36 13-meC18 5 972 7.33 2775 27.76 3-meC27 19 6.089 1.36 2807 28.07 10, 13-dimeC 20 6.290 0 .6 1 2865 28.66 8 12-dimeC29 21 6 366 1.45 2882 28.83 Nonacosene Percent composition of pavement ant cuticular hydrocarbons from Colony SP3. RT is the r etention time s hown in GC t ota l i o n chromatogram : i s Kovats lndex, and E .C.L. is Equivalent C hain Len g th 39

PAGE 49

Pavement ant Colon y Speer 3 Peak R .T." Pct Total I." ECl' Compounds 1 2.769 3 .81 n-Pentadecane 2 4 .505 0 57 2223 22.24 Oleic acid TMS 3 4 .681 3.40 2298 23.01 Tricosane 4 4 .764 0 6 3 2334 23.36 II-meC23 5 4 857 1.0 2 2374 23.75 3-meC23 6 4 915 1.26 2 399 23. 99 Tetracosane 7 4.999 0.69 2431 24.33 13-, II-meC24 8 5.167 15.61 2496 24 99 Pentaco s ane 9 5 .251 17.16 2529 25.31 II-meC25 10 5 368 11. 02 2574 25.75 3-meC25 5.435 1.63 2600 26.00 n-Hexacosane 12 5.460 0 .97 2608 26.08 8 II-dimeC25 13 5 527 1.98 2629 26.31 13-meC26 14 5.603 0.45 2654 26 56 4-meC26 15 5.678 15.64 2678 26 .81 Heptacosene 16 5.728 6.73 2694 26.97 n-Heptaco s ane 17 5 829 9.30 2726 27.29 13-meC27 18 5.971 6 .68 2772 27.73 3meC27 19 6.298 0 7 3 2861 28.63 8 12-dimeC29 20 6.365 0 .74 2877 28.80 Percent composition of pav ement ant cuticular hydrocarbons from Colony Speer 3 R.T. i s the r e t entio n t i m e s hown in GC t o t a l ion c hrom a togr a m ; i s Kovat s Index, and E .C.L. i s Equival e nt C h ain Len gth. 40

PAGE 50

Pavement ant Colony SP4 Peak R.T. Pet Total E CL Compound s 1 2.770 4 .93 n-Pentadecane 2 4.681 3.54 2298 23 .01 Tricosane 3 4 764 0.41 2334 23.36 II-meC23 4 4.857 1.01 2374 23.75 3meC23 5 4.924 1.04 2402 24.02 T etrac os an e 6 5 008 0 .55 2435 24 36 13, II-meC24 7 5 108 0 28 2473 24 76 3-meC24 8 5 167 18. 94 2496 2 4.99 Pentaco s an e 9 5.259 14.61 2532 25.34 II-meC25 10 5 368 11.74 2574 2 5.75 3-meC25 11 5.435 1.77 2600 26 00 n-Hexaco s an e 1 2 5.469 0 82 2611 26 .11 8 ,II-dimeC25 1 3 5.536 1.73 263 2 26 34 13-meC26 14 5.611 0.42 2656 2 6 59 4-meC26 15 5 687 9 3 8 2681 26.84 Heptaco s en e 16 5 737 10.14 2697 27 00 Heptac os ane 17 5.837 9.19 2729 27.32 13, 11-meC27 18 5 980 7 78 2775 27 76 3meC27 J9 6 097 0 80 2810 28.10 8 ,II-dimeC27 20 6 298 0.42 2861 2 8 6 3 6 12, 21-trimeC28 21 6 374 0.50 2880 28.83 Nonacosene 6.4.2 3 P e rcent composition o f pavement ant cuticular hydrocarbons from Colony SP4. RT is the r e t ent ion time shown i n GC t o t a l ion chroma t og r a m ; is Kovat s Index, and E .C.L. is Equivalent C h a i n Len gth.

PAGE 51

Pavement ant Colony NC-3 Peak R .T." Pet Total I." ECL Compounds 2.736 2.73 n-Pentadecane 2 4 094 0 70 2040 20.42 9-Tetradecenoic acid trimethylsilyl ester 3 4.505 0.71 2223 22.24 Oleic acid, trimethylsilyl ester 4 4.681 3.44 2298 23.01 Tricosane 5 4.773 0.75 2338 23.40 II-meC23 6 4.865 1.42 2377 23.78 3-meC23 7 4.924 1.10 2402 24.02 Tetracosane 8 5.008 0.77 2435 24 .36 13-, II-meC24 9 5.066 0.22 2457 24.59 6, 4-meC24 10 5.167 15.08 2496 24.99 Pentacosane 11 5.259 17.44 2532 25.34 II-meC25 12 5.376 12. 69 2577 25. 78 3-meC25 13 5.443 1.15 2602 26.03 n-Hexacosane 14 5.469 1.29 2611 26.11 12, 10, 8, 3 -meC26 15 5.536 1.62 2632 26 34 13-meC26 16 5.619 0.48 2659 26.61 4-meC26 17 5.687 21.88 2681 26.83 Heptacosene 18 5.737 4.55 2697 27.00 H eptacosane 19 5.837 5.67 2729 27.31 13, II-meC27 20 5 938 0.46 276 1 27 .63 9 xdimeC27 21 5.988 4 36 2778 27.79 3-meC27 ; 5 10-dimeC27 22 6.097 0.57 2810 28.10 10, 16-dimeC23 6.374 0.95 2880 28.83 lNonacosene Percent composition of pavement ant cuticular hydrocarbons from colony NC-3. R.T. i s the retention time s hown in GC t otal i o n chromatogram ; is Kovats Index, and E .C.L. is Equival e nt Chain Length. 42

PAGE 52

Pavement ant co lony KC l P eak R .T." P et T o ta l I." ECL Co mp ounds 2.7 69 1.27 nP e n ta d ec a n e 2 4.681 1.55 2298 23 0 1 Tricosa n e 3 4.764 0.44 2334 2 3 .36 I I-m eC23 4 4.865 0 78 2377 23 78 3 -m eC23 5 4.9 1 5 0 .68 2399 2 3 .99 Tet raco s ane 6 5 00 7 0 .63 2434 24.36 1 3 11-meC24 7 5.10 8 0 2 7 24 7 3 24.76 3 -m eC24 8 5.1 6 7 9 92 2496 24.99 nP e n taco s a n e 9 5 259 15. 82 253 2 25. 34 II-meC25 10 5.3 7 6 9.44 2577 25.7 8 3m eC25 II 5.435 1.35 2600 26 00 n-Hexacosa n e 12 5.468 1.04 26 1 0 26 .11 1 2 1 0 3 -m eC26 1 3 5.536 2.56 2632 26.34 1 3 -m eC26 14 5 .611 0 .60 2656 2 6 .59 4 -m eC26 1 5 5.6 8 6 1 5 7 0 2681 26 8 3 H eQtacose n e 1 6 5 7 3 7 8.4 3 269 7 27.00 n-Heptacos a ne 1 7 5 .77 9 0 54 2710 2 7 .13 unkn ow n 18 5 .837 1 3. 96 2729 2 7 3 1 1 3 -m eC27 1 9 5 9 8 9 23 2 77 5 2 7 7 6 3 -m eC27; 5 1 O -dimeC27 20 6 09 7 0.9 8 28 1 0 28.10 3 ,II-dimeC27 2 1 6 .181 0.60 2 8 3 1 2 8 .33 1 6 1 4 1 2 10-m eC28 22 6 298 0 .5 1 286 1 28 .63 6 1 2 2 1-t rimeC28 23 6 3 7 4 2 02 2 8 80 2 8 8 3 No n acose n e 24 6.432 0 39 2894 28 9 7 n No n acosane 25 6 .558 0 72 2926 29 29 1 3 -m eC29 26 6.75 1 0 .33 29 7 5 29 7 6 3 -m eC29 6.877 0.23 3005 30.05 Triaco n tane 6.4.2.5 P e r cent co m pos i t i o n of pavemen t ant c u ticu l ar h ydroca r bons from co lony KC-I R.T. is the r e t e n t i o n time s hown in GC t o t a l i o n c hrom a togr a m ; is Kovat s Index, and E .C.L. i s Equival ent C h ain Len gt h 4 3

PAGE 53

Pavement Bulk Ants I Peak R.T." Pct T o tal ECL Compounds I 2 770 1.10 Pentadecane 2 4.412 0.19 2182 21.83 3-meC 3 4.656 0.2 8 2288 22.9 9Tricosene 4 4.697 2.81 2305 23 08 Tricosane 5 4.781 1.3 0 2341 23.43 9, II-meC23 6 4 873 2.40 2381 23.82 3-meC 7 4.932 0 72 2405 24.06 Tetracosane 8 4.957 0 36 2415 24 .16 12-meC 9 5.024 1.21 2441 24.43 13, II-meC10 5 083 0 36 2464 24.66 4-meC II 5.142 2 .13 2487 24.89 Tetracosene 12 5 .192 7.77 2506 25.08 Pentacosane 13 5 276 15.38 2538 25.41 II-meC 14 5 393 12.41 2584 25.84 3-meC 15 5.460 0 79 2608 26.08 n-Hexacosane 16 5.485 2 .15 2616 26.17 12-, 10-, 8, 3-meC 17 5 .552 3.13 2637 26.39 13-meC 18 5.636 0.81 2664 26.67 4-meC 19 5 .661 0.72 2672 26 .75 8 10, 14-trimeC20 5 712 18.40 2689 26.92 Heptac o sene n-Heptaco s ane 21 5.804 0.76 2718 27.21 Octacosene 22 5 863 9.55 2737 27.4 13-meC27 23 6 005 6.65 2783 27.84 3-meC 5 I O-dimeC27 24 6.114 1.53 2814 28.15 3 II-dimeC27 25 6.215 0.67 2840 28.42 14-, 5-meC 26 6.315 0.77 2865 28.68 6 12, 21-trimeC 27 6 .391 1.16 2884 28.87 lNonacosene 28 6.449 2.90 2899 29 02 n-Nonaco s ane 29 6 583 0.29 2932 29 .35 13, II-meC30 6 793 0 28 2985 29.86 5 12; 5 7-dimeC 31 6 835 0.22 2996 29 96 5 10, 22-trimeC 32 6.885 0.37 3006 3 0 07 3-meC 33 7 355 0.43 3092 30.96 n-Henitricontane 6.4.2.6 Percent composition of pavement ant cuticular h y drocarbons 4 4

PAGE 54

from Bulk Ant I. R .T. i s the r e t entio n time s hown in GC t otal i o n c hrom a t og r a m: is K ova t s Index, and E .C.L. i s Equiva l ent C h ain Len gth. There are about 22 peaks in each pavement ant colony The chain length in pavement ant cuticular hydrocarbon ranges from 15 to 31 carbon atoms Six alkanes ten mono-methyl alkanes and two alkenes were found in all five pavement ant colony samples. The compounds present in all pavement colonies are n-pentadecane n-tricosane C23) n-tetracosane n-pentacosane n-hexacosane and heptacosane in normal alkanes ; 11-, 3-methyltricosane (11, 3-meC23 ) 13, 11-methyltetracosane (13, II-meC2 4 ) 11, 3-methylpentacosane (11, 3-meC25), 13, 4methylhexacosane (13, 4-meC26 ) 13, and 3-methylheptacosane (13, 3-meC27 ) in mono-methyl branched alkanes ; heptacosene and nonacosene in alkenes Table 6.5.1 shows the proportions of straight chain alkanes branched alkanes, alkenes and fatty acids in pavement ant samples SP3 Speer 3 SP4 NC-3 KC-l Bulk Ants Fatty acids 0.57 1.41 n-Alkanes 25.78 32.44 40.37 28 04 23.83 22 04 Mono-methyl 56.25 48.92 47.72 44.52 51.80 53.91 alkanes Dimethyl 3.16 1.70 1.62 3.21 5.58 5.13 alkanes Trimethyl 0.42 0.51 1.71 alkanes Alkenes 14.82 16.37 9.88 22.82 18.26 17.21 Percent composition of pavement ant cuticular hydrocarbons. 45

PAGE 55

Only pavem ent Bulk Ant s I sample pass thro u g h silica gel column prio r GCM S analysi s t o sep a r a t e fatty acids from cuticula r h ydrocarbons A / indic a tes the compounds w e r e eithe r missing o r the peak a rea i s less than 1 % of the l arges t peak are a i n the t o t a l i o n c hrom a togr a m The pavement ant cuticular lipids contain n-alkanes (16-40 % ) mono-methyl alkanes (45-56%) and alkene s ( 10-20 % ) with small amounts of dimeth y l trimeth y l alkanes and fatt y acid s Cuticular hydrocarbons in pa v ement ant are more consistent than in har v ester ant The most abundant cuticular h y drocarbon class was mono meth y l alkanes ; the second was normal alkanes and the third was alkenes in all five pa ve ment ant colonies. The most abundant compound wa s II-methy lpentacosane (11meC25 ) in mono-meth y l alkanes ; n pentacosane in n alkanes and heptacosene in alkenes Fatt y acids were found in pavement ant colonies Speer 3 and NC-3. The fatt y acid that is present in both colonies was oleic acid (ca 0.65 %) Pa v ement ants eat pretty much everything but prefer g reas y food. Without prominent oleic acid in their major food source small amount of fatty acids can still present on insect cuticles Most compounds found in pa v ement ant cuticu l ar h y drocarbons are the s ame but with onl y slightl y different percent composition. Shared cuticular h y drocarbons of pav ement ant colonies with relative abundance s were shown in Figure 6.5 .1. The differenc es in h y drocarbons among pa v ement ant colonie s were lis ted in Table 6 .5.2 46

PAGE 56

25 2 0 Speer 3 C 3 5 .a.I ....... o Fig. 6.5.1 Shared cuticular h y drocarbons of pa v ement a nt colonies. 4 7

PAGE 57

ON.,f::I. 0\000 N.+::o.o\OO n-C 1 5 n C23 )I II m e C23 3-meC23 D n-C24 1 3, 11-l11e C24 n-C25 II-m e C25 3-l11e C25 n-C26 1 3-me C 26 .. 4-meC26 H eptaco s e n e n-C27 1 3-l11eC27 3 -l11e C27; .. N o n acose n e .. .., ::r' ...... 0.. a'Q. 0\ o ::::s ::r' o ......, 20.. C ....... 0.. C C -.., 3 ::::s ...... ('j n-C 1 5 n-C23 II m eC23 3-l11eC23 n-C2 4 1 3 1 l m eC24 n-C25 11-l11e C25 3-l11eC25 n-C 26 1 3-meC26 4 -l11eC26 Hept acose n e n-C27 1 3 II -l11eC27 3-l11e C 27 Non acose n e o V> n C 1 5 n C 2 3 11-l11cC23 3 -l11e C 2 3 n-C24 1 3 II-m eC24 n-C25 11-l11e C25 3-me C25 n-C 26 1 3-l11e C26 4-l11cC 26 1 3, 11-l11e C 27 3-l11e C27; 5 10. N o n acose n e z ('j n-C 1 5 n C23 11-l11e C23 3-l11eC23 n C24 1 3 II-m cC24 n C25 11-l11cC 2 5 3m cC25 n C 26 1 3-l11e C 26 4-m cC 26 H eptaco s e n e n C 27 1 3 -l11e C 27 3-l11cC 27 Non acosc n e -----N n C 1 5 n C23 11-l11e C 2 3 3-meC23 n-C24 1 3 11-l11e C 24 n-C25 11-l11e C25 3 m cC25 n-C 26 1 3 -l11e C26 4-mcC 26 1 3-l11e C27 3m e C27 Non acosc n e [/) W

PAGE 58

SP3 S p eer 3 SP4 NC3 KCl 0.39 % 0 .23% 3 -meC24 0 6 7 % 0.28 % 0.27 % 6 4-meC24 0 .22% 8 ,II-d imeC25 1.18 % 0.97 % 0 8 2% 12, 10 3 meC26 1.04 % 1 2 9 8 -meC26 I1 meC27 9 x dimeC27 0.46 % 5 1 O -dimeC27 10 ,13dimeC27 1.36 % 8 ,IIdimeC27 0 80 % 10 16 di m eC27 0.57 % 6 1 2 ,21tr im eC2 8 0.4 2% 0 .51% 13-meC2 9 0 72 % 3 -meC29 0.33 % 8 1 2 d i meC2 9 0.61% 0.7 3% The percent composition differe n ces of cuticu l ar hydrocarbons among pavement ant co l on i es. a A P r e pr ese nt s the com p ound was ide ntifi ed by E I m ass spec trum but co -elut ed w ith its i so m ers o r oth er co mp o und s so the percent co mpositio n itself can n ot be determined T h e percen t age with bold borders r e pr esen t s the iso m e r s o r co mp o und s co e lut ed and int eg r a t e d as one peak in T I C w hich is the t o tal percent co mp ositio n of these compounds 49

PAGE 59

Sano has demonstrated the unicoloniality of pavement ant (Sano 2007) where individuals can move freely between physically separated colonies and there is the presence of more than one egg-laying queen in a colony. The consistency of cuticular hydrocarbons in five pavement ant colonies may be an indication of the unicoloniality Colonies SP3 and Speer 3 have simi lar cuticular hydrocarbon profiles that might be because the unicolonial polygyny. other words ants in colonies SP3 and Speer 3 are likely conspecific species. Species-specific and nestmate-specific recognit ion behaviors exist in Pavement ant (Sano 2007). the current study colonies SP3 and Speer 3 have somewhat similar patterns in cuticular hydrocarbons 8 II-dimethylpentacosane (8, II-dimeCand 8 12-dimethylnonacosane (8, 12-dimeC2 9 ) are present in both ant colonies. However 3methyltetracosane (3-meC2 4 ) and 10, 13-dimethylheptacosane (10,13-dimeC27) are present in colony SP3 but not in colony Speer 3 8 11-Dimethylheptacosane (8,11-dimeC27) was found only in pavement ant colony SP4; 6and 4-methyltetracosane (6-, 4meC24 ) 8-methylhexacosane (8-meC26 ) 9 x-dimethylheptacosane (9 x -dim eC27) and 10, 16-dimethylheptacosane (1 0 16-dimeC27) were exclusive to colony NC-3 ; nonacosane n-tritriacontane 6 12, 21-trimethy loctaco sane (6 ,12, 21trimeC2 8 ) 13-and 11-methylnonacosane (13, 11-meC29 ) were present only in colony KC-l. The differentiality expressed compounds could be the chemical cues for nestmate recognition Further work must be done to determine whether compounds provide nestmate recognition cues or if only internally methyl and dimethyl branched alkanes possess that functionality. 50

PAGE 60

7 The chain length of cuticular hydrocarbons was found to range from 23-36 carbon atoms in the harvester ant and from 15-31 carbon atoms the pavement ant. Compounds that were detected in both ant cuticular hydrocarbons include n-tricosane pentacosane n-hexacosane n-heptacosane n-nonacosane n-hentriacosane 3-methylpentacosane (3-meC25) and 13-methylheptacosane (13meC27). The odd-number positions of methyl alkanes were characterized more than of even-number methyl alkanes in all ant samp les. n-Heptacosane were found to be at least three times bigger than n-pentacosane in har vester ant cuticular hydrocarbon profiles while n-heptacosane is as much as n-pentacosane in pavement cuticular hydrocarbon profiles. The approaches of structurally identification for cuticular hydrocarbons were described in Chapter 3. n-Alkanes were primarily determined by [M-15t/base peak= 3-5% in CI spectra and EI mass spectra library search with and Kovats Index retention indecis as reference Mono-methyl alkanes were first identified by /base peak= 6-10 % in CI spectra and EI mass spectra. Dimethyl alkanes were characterized by /base peak= 11-20% in CI mass spectra and EI mass spectra. Quantitative analysis of ant cuticular hydrocarbons revealed that the percent composition of mono-methyl alkanes is more abundant than n-alkanes in the pavement ant In contrast the relative abundance of n-alkanes and mono-methyl alkanes varies among colonies in the harvester ant Oleic acid is extremely abundant in one of the harvester ant colonies it could relate to the source of food the death of ants or the conversion of fatty acids to hydrocarbons However it could simpl y be an error due to the one-time experiment analysis of each sample. The differential expression of hydrocarbons in harvester ant (Table6 2.1.2 page 29) shows possible compounds to investigate further for nestmate recognition cues. The

PAGE 61

similarity of cuticular hydrocarbon patterns may imply the ant colonies are genetic related and the number of hydrocarbons might relate to the size and the age of the colonies Species have unique hydrocarbon profiles which differ from other species in the presence or absence of hydrocarbons and quantitatively in shared molecules. In the comparison of harvester ant and the former has shorter carbon chain length (23 36) in cuticular hydrocarbons than the latter (23-49). Trimethyl alkanes and / or alkenes were either missing or less than 1 % of the largest peak area in but about 1.6% trimethyl alkanes and 2.3% alkenes were present in has more abundant mono-methyl alkanes than straight chain alkanes while has fairly equal abundance in n-alkanes and mono-methyl alkanes. Patrollers and foragers in a harvester ant colony contain higher proportion of alkanes relative to methyl alkanes and alkenes (Wagner 1998). Therefore, the number of patrollers and foragers collected may change the percent composition of hydrocarbon classes in the tested sample. Nestmate recognition compounds could be either unique to a certain colony or shared compounds between colonies but detected with different percent composition. Possible harvester ant nestmate recognition cues are listed from page 31 to 33. The cuticular hydrocarbon profiles in pavement ant are more consistent between colonies than in harvester ant which may be due to the unicolonial polygyny in pavement ant Similiarity in cuticular hydrocarbon profiles might also be an indication of conspecific ants also could be the information correlates to the aggression against non-nestmate conspecific or heterospecific species. Species and nestmate recognition behavior have been demonstrated in the pavement ant (Sano, 2007).The differential hydrocarbon profiles in pavement ant (page 47-49) provides possible nestmate recognition cues in pavement ant 52

PAGE 62

This is a preliminary research for the western harvester ant and the pavement ant Duplicates of samples and standards were not performed to ensure the accuracy and reproduciblity of GC quantitation results This needs to be done for the future work. Although the ecology and biochemistry of the insect cuticular hydrocarbons has been intensively investigated in the last twenty years the questions in some areas are still need to be answered The stereochemistry of the methyl branched cuticular hydrocarbons and the effect of diet or the environmental conditions on insect cuticular hydrocarbons will be studied. Also the biological activities of individual component need to be investigated. 5 3

PAGE 63

was difficult to l ocate double bond positions in alkenes and determine their stereochemistry by simply interpreting mass spectra of ant samples. Three methods have been developed to solve this problem. Methylthiolation with dimethyl disulfide to derivatize alkenes and form methylthioalkanes (Francis and Veland 1981) gives prominent fragment ions in electron ionization spectra Additionally molecular ions are always detected in EI spectra This method can be applied for the determination of doub l e bond positions in alcoho l s aldehydes and carboxylic acid methyl esters as well as alkenes (Leonhardt and DeVilbiss 1985). Another approach to locate double bond positions in alkenes makes use of GC chemical ionization mass spectrometry. The use of ammonia in positive chemical ionization converts monoenes to methoxyesters. Pechine characterized alkenes from and (Pechine 1985) and Lange analyzed alkenes from termites ants and flies (Lange 1989) Later Pechine explored another approach by epoxidation of a l kenes with metachloroperbenzoic acid then ana l yzing samples in negative chemical ionization and using 90 % methane l 0% nitrous oxide as reagent gas (Pechine After characterizing the cuticular hydrocarbons we know there are at least 20 compounds in each colony Is everyone of them involved in the communication message 54

PAGE 64

or are only certain hydrocarbon compounds ? The biological activities of individual hydrocarbon components are still unclear In order to answer these questions the acquisition of individual compound by either chromatography separation or organic synthesis along with numbers of bioassays must be conducted (Howard 1993) Although the insect cuticular hydrocarbons were characterized and identified all methyl branched alkanes could have chiral centers. There was no study of identification the stereochemistry in insect hydrocarbons. The first question is : are those methyl alkane isomers all chrial ? To investigate the chirality of insect hydrocarbons we have to either synthesize pure chiral compounds then develop the bioassays to yield distinguishable results or derivatize hydrocarbons into polar compounds that give us optical rotation values (Howard 1993) 55

PAGE 65

APPENDIX A 16e+07 1 5e+07 1 1 .. 07 7 000000 4000000 200000O 1000000 500 6 00 7 00 800 1000 1100 56

PAGE 66

1.oljJ EI mass spectrum of n-tricosane. Name Tricosan e (CAS) $$ nTricosane CAS Number 000638-67-5 Entry Number 275689 Molecular Formula C23H48 Misc Information QI = 836 Source = WS-1986-23-0 WLN = 23 H Match Quality 99 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 324.38 57

PAGE 67

3 3 113 100 a 100 1 S 0 CI mass spectrum of n tricosane. [M-15r / base peak: at 309 50) / at 323.45) 8088 0 / 384580.0 =2.10% 2: 51' 8 5 EI mass spectrum of n-tetracosane. 58

PAGE 68

Name Tetracosane CAS Number 000646-31-1 Entry Number 288781 Molecular Formula C24H50 Misc Information QI =900, Source =NS-127042-0 Match Quality 99 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 338.39 aOODO 100 0 3 7 100 1 2 0 140 1 6 0 1 B O 200 220 240 260 2BO 300 320 340 360 mass spectrum of n-tetracosane [M I base peak: at 323.40) (rn/z at 337 50) = 2835.0 9073 1.0 = 3.12 59

PAGE 69

3: C 1 = 16.559 57 200000 I 141 J :3 __ 40 EI mass spectrum of n-pentacosane. Name Pentacosane (CAS) $$ n-Pentacosane CAS Number 000629-99-2 Entry Number 300503 Molecular Formu l a C25H52 Misc Inform atio n QI = 621 Source = PG-198 2-1717 -0 WLN = 25H Match Quality 99 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 352.4 1 60

PAGE 70

3 1 295 A.2.2. CI mas s s pectrum of n-pentacosane. [M-15 r / base peak: a t 337 50) / at 35 1 50) = 79938 0 / 2760456.0 = 2.90 % 4 : 1 = 5 2 7 6 ; r-113 C1S: C:-Cs -oJ 253 85 7-meC25 9-, 61

PAGE 71

7 900000 800000 700000 600000 500000 rn/z--> EI ma ss s p ec trum of 7-meth y lpentaco s an e r-197 C12-: C:-C12 169--' '--16 9 13-meC2 5 r-169 r-14 1 C14: C:-C10 C1S-: C:-Cs 197--' '--14 1 225--' '--I 1 3 II-meC25 9-meC25 ma ss s p e ctrum o f 13, 11-, 9-meth y lpentaco sa ne. 6 2

PAGE 72

Scan 2133 (16.770 m i n ) : TS1 7 D ( 2115) ( ) 3 5 1100000 1 000000 900000 800000 700000 600000 500000 400000 300000 200000 100000 100 120 140 1 6 0 1 8 0 200 220 240 260 280 300 320 340 360 380 ma ss s p e ctrum of 13-, 11, 9, 7-m e th y lp e nt a co s ane. [ M -15r / b ase peak : (m/z a t 35 1.50 ) / ( rn/z a t 365.60) = 8 790 1 0 /119 417 6.0 = 7 36 % 5 : 29 3 meC25 63

PAGE 73

71 3 E I ma ss s p e ctrum o f 3 -m e th y lpent a co sane. Abundance 450000 Scan 2162 (16.936 TS17. D (-2145) (-) 3 5 400000 350000 300000 250000 200000 150000 100000 50000 100 393 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 C I m ass s p ec trum of 3 -m e th y lp e nt a c os ane. [M-15r / base p eak: ( m / z a t 351 .50) / (m/z at 365.60) 38359 0 / 45463 8.0 8.44 % 64

PAGE 74

6: mass spectrum of n-hexacosane. Name Hexacosane $$ n-Hexacosane CAS Number 000630-01-3 Entry Number 311169 Molecular Formula C26H54 Misc Information QI = 540 Source = NS-8-7573-0 Match Quality 99 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 366.42 65

PAGE 75

3 S 800000 700000 600000 SOOOOO 300000 1 3 2 7,4" SS1 69, 8 3 1 9 7 2 1 12252392S326728 1 29S 3 0 9 CI mass spectrum of n-hexacosane. base peak: at 351.50) at 365 60) 7 : ,-127 253---l 1--99 8-meC26 66

PAGE 76

mass spectrum of 8-methylhexacosane. Abundance Scan 2219 (17.262 min) : TS17. D ( -2205) ( ) 500000 3 9 450000 400000 350000 300000 250000 200000 150000 100000 1131271411551691 50000 -.:>09323 365 100 337351 393 rn/z--> 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 mass spectrum of 8-methylhexacosane. [M-15 r base peak : at 365 60) at 379 60) 67

PAGE 77

= 33063 0 / 526880.0 = 6.28 % 8: /=5.745; .., EI mass spectrum of n-heptacosane. Name Heptacosane CAS Number 000593-49-7 Entry Number 320678 Molecular Formula C27H56 Misc Information QI = 898 Source = NS-12-7866 0 Match Quality 99 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 380.44 68

PAGE 78

Abunda nce Scan 2277 (17 5 9 3 .."in): IS17. D ( -224 5 ) ( ) 3 9 5500000 5000000 4 500000 4000000 3500000 3000000 2500000 2000000 1 500000 1000000 500000 113127141155169 183197211225239253267 281295 3 0 9323 1 DO 3373513 6 5 100 1 2 0 140 1 6 0 180 200 220 240 260 280 300 320 340 360 380 Fig. A.S.l. CI mass spectrum of n-heptacosane. base peak: at 365.55) at 379 60) = 2.9 1 9 : C1= 1 7.7 42 r--169 r--141 253-----, 281-----, 14 : 12 1S-: 10 18-1 18 19r L-169 L-141 253---.J L-113 13-meC11-meC9-meC69 399

PAGE 79

Abu...,dco...,c_ ...,.,1...,): 8 5 ...,.,/:01:--:_ Fig. A.9.1. E I ma ss s p ec trum of 1 3 11-. 9-hepta c osane r-113 C20-: C:-Cs 281 85 7-m eC27 70

PAGE 80

7 8 S 140 A.9.2. EI mass spectrum of7-heptacosane. A.9.3. CI mass spectrum of 13-, 11-.9, 7-heptaco sane. [M-ISf / base p eak: = (m/z at 379.60) / (mlz at 393.60)

PAGE 81

1.\1 2 as s:: ro r/) 0 U C""J ro ...... 0... ...s:: ...s:: 01 6 c :.a ........ r---0 ........ 6 t3 l-< 0... '0 r/) r/) 0 o.r) V) r/) 0\ on ro ..,f 01 6 01 >-< '" U.J 01 6 -0 '
PAGE 82

___ CI mass spectrum of 7 13-dimethylheptacosane. [M-15r base peak: (m/z at 393.60) (m/z at 407.60) 26 09 % 11: 200000 s 00 0: .... 3T./I\-... EI mass spectrum of n-octacosane. 73

PAGE 83

Name Octacosane (CAS) $$ n-Octacosane CAS Number 000630-02-4 Entry Number 329269 Molecular Formula C28H58 Misc Information QI = 555 Source = PG-I 982-1 720-0 WLN = 28H Match Quality 99 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 394.45 3 3 CI mass spectrum of n-octacosane [M-15r / base peak: = (rn/z at 379.55) / (rn/z at 393 60) = 31453.0 / 1012280 0 =3.11 % 74

PAGE 84

12 : C 1 = 18.193 14, 13, 1 2, r--211 r--197 r--183 C14: C:-C13 C1S: C:-C12 C1S-: C:-C11 ---1 L-----1 L-----1 L-197 183 211 169 225 ISS 14-meC28 13-meC28 1 2 -meC28 Fig. A.12.t. E I ma ss s p ect rum of 14, 13-, 1 2 -meth y loctacosane 75

PAGE 85

c o 0' o o o o o o o 0 VJ o ro o ...c E o 0... VJ VJ VJ ro o c 1) c J o c E C m o N N o 0 ,1+ 0 V V ufi W 0 W 0 V o 0 N W 0 W 0 V N 0 0 0 m F. o W o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 0 o o o A VJ o o ;>-. ...c o 2 0... VJ VJ VJ ro E ...... -,-.., o r-: ';;j .;.:'--' II) ,-.., 0
PAGE 86

= 32993.0 / 474299.0 = 6 96 % 13: mass spectrum of n-nonacosane Name Nonacosane (CAS) $$ n-Nonacosane $$ Celidoniol deoxy(CAS) CAS Number 000630-03-5 Entry Number 337002 Molecular Formula C29H60 Misc Information QI = 623 Source = PG-1982-1721-0 WLN = 29H Match Quality 99 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 408.47 77

PAGE 87

'30"'00::.0::.0::.0::. 0::-0000::.0 :;011 ... F ig. A 13.2. CI ma ss s p e ctrum o f n-nonaco s an e [M1 5 r / b ase p ea k : = (m/z a t 393 55 ) / (m/z a t 40 7 6 0 ) = 9 352 8 0 / 32 0 3 45 3 0 2.9 2 % 1 4 : r--197 r--169 r--141 253 ----, 281 ----, 309 ----;---oC C1 S : C :-C12 C1S-: C :-C10 C20 : C :-Cs L....-L....225 169253 141281 113 13meC2 9 II-meC29 9-meC29

PAGE 88

mass spectrum of 13, 11-, 9-methylnonacosane. 309---1 L--8 5 7 meC29 79

PAGE 89

Q Q V N o 0 v 0 Q 0 o o v o o o o o o o o o o o o ...... 6 r--o 0.. 6 w.l ...,. o! C o f! $ o 6 C f N 5 c o 00000000000000000000000000000000 0000000000000000000000000000000 0000000000000000000000000000000 0000000000000000000000000000000 0000000000000000000000000000000 o o 6 r---0\ M o 0.. 6 \0 ..:.:'-' '" 0 "'0 '"7E o 00

PAGE 90

= 215983 0 / 3 1 7 1 496 0 = 6 8 1 1 5 : 9 1 3 ; 225 253 295 323 --, --,--, C C CI6-:-C-:-C3-:-C-:-C8 211 183 1 4 1 113 225 253 323 351 C C C -:-C-:-C -:-C-:-C 5 6 211 18 3 113 8 5 9 ,13-di meC2 9 7 1 3 -dimeC2 9 65 EI mass spec t r um of 9 1 3 a nd 7 ,13-dim e t hy l no n acosa n e 8 1

PAGE 91

4 e 113 155 163 211 323 351 e o 4 5 1 CI mass spectrum of9, 13and 7 13-dimeth y lnonacosane. [M-15f base p eak: = (m/z at 421.65) at 435.65) 512533.0 13.83 % 16: 1 = 6.843; 82

PAGE 92

. ,. EI mass spectrum of n-triacontane. Name Triacontane $$ n Triacontane CAS Number 000638-68-6 Entry Number 343923 Molecular Formula C30H62 Misc Information QI = 895 Source = NS-9-5920-0 Match Quality 97 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 422.49 83

PAGE 93

..... ) ; CI mass spectrum of n-triacontane. [M-15r ba se peak : at 407.60) (m/z at 421.60) = = 3.54 1 7 : ,---211 253 ----;c1 225 183 14-meC30 84

PAGE 94

0 a) a) t:: IJJ ro t:: t:: 0 0 C ro I-< "t:: ....... V) >-. >-. 00 ...c:: ...c:: ....... S S -.::t -.::t c ....... m 0 0 0 IJJ S S ;::l /""-;::l I-< I-< 0 v) "
PAGE 95

% 1 8: (7' ..,....,i,...,,) t::::'O Fig. A.18.I. E 1 mas s s pectrum of n -h entriacontane. 2200000 900000 1 aooooo 500000 ., ..... 00000 ""00000 1000000 500000 400000 :2.00000 86

PAGE 96

Fig. A .18.2. CI mass spectrum of n-hentriacontane. [M-15 f / ba s e peak : a t 42 1 55 ) / a t 4 35 70 ) = 72 74 6.0 / 2 29277 3. 0 = 3 .17% 19: C l = 19 559 1 5, 13 r--225 r--197 r--169 C1S-: C :-C14 C1S-: C :-C12 C20-: C: C10 15-meC 13meC I 1 meC A.bl...lr'l d _rlC-E!I ,52::2.. ( -S81 ) ( ) 600000 5 550000 500000 350000 85 1 50000 1 00000 0 40 6 0 rrl/:z::-->_ Fig. A. 19.1. E I mas s spectrum of 15, 13, I1-methylhentriaco ntane. 87

PAGE 97

Abund a nce 1 700000 1 600000 1 500000 1400000 1 300000 1 200000 1100000 1000000 900000 800000 700000 600000 500000 400000 300000 200000 100000 Scan 2621 ( 1 9 .559 min ) : T S 1 7 D ( 2609 ) ( ) 4 0 10012014016018 0 200220 240260280 300320 34036038040042044046 0 Fig. A.19.2. C I m ass s p e ctrum o f 1 5 1 3 II-me th y lh e ntri a contane. [M-15 r b ase p eak : at 435 70) at 449 65) = 6 .85 1=7.556; r-113 C24 : C:-Cs 337----.J 1--85 7-m eC 88

PAGE 98

S;:Co,eo,.., 7c>c>OOO F i g A .20.t. EI mass spectrum of 7 methylhentriacontane A.bundance Scan 2630 ( 1 9 610 min): 8 1 7 0 ( -2610) ( ) .... 0 1 800000 1700000 1 600000 1 500000 1400000 1 300000 1 200000 1100000 1000000 900000 800000 700000 600000 500000 400000 300000 200000 113 100000 F i g A .20.2. CI mass spectrum of7methylhentriacontane. [M-15r / base peak: (m/ z at 435 65) / (m/z at 449 70) 89

PAGE 99

= 6 .72 % 2 1: 253 281 351 379 CI8-:-C-:-Cs-:-C-:-C6 211 183 113 85 7 13-dim e C 85 E I ma ss s p e ctrum of 7 13-dimeth y lh e ntriac o ntan e 90

PAGE 100

-,..,... CI mass spectrum of 7 13-dimethylhentriacontane. [M-15f base peak : (m/z at 449 70) (m/z at 463 70) = 11.34 % 22: Scan 634 (7.B91 min): TS22. D ( -651) ( ) 5 50000 45000 40000 35000 30000 25000 20000 15000 5000 0 295 323 351 393 450 40 60 B01001201401601B02002202402602B03003203403603B0400420440 mass spectrum of n-dotriacontane.

PAGE 101

Name Dotriacontane CAS Number 000544-85-4 Entry Number 355041 Molecular Formula C32H66 Misc Information QI = 895 Source = NS-12-86 14-0 Match Quality 95 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 450 52 mass spectrum of n-dotriacontane. [M-15r / base peak: at 435.70) / (rn/z at 449.65) 7287 0 / 211385 0 3.45 % 92

PAGE 102

23: 57 85 0 113 J 197 225,,,,....g>67 295 .... 6 .... .... 0 60 mass spectrum of n-tritriacontane. Name Tritriacontane $$ n-Tritriacontane CAS Number 000630-05-7 Entry Number 359681 Molecular Formula C33 H68 Misc Information QI = 885 Source = NS-1 0-56-0 Match Qualit y 95 Company ID 0 Retention Index 0 Meltin g Point Boilin g Point Molecular Weight 464 .53 93

PAGE 103

500000 4 50000 400000 350000 3 0 0000 250000 2 0 0000 1 50000 1"'5 o 100 1 201401e 0 180200220 2 4 02e0 28030032 0 3403e0 380400420440 0 F i g A .23.2. CI mass spectrum of n-tritriacontane. [M-15 r ba se peak : ( m/z a t 449 65 ) (m/z a t 463.65 ) = = 3 .16% 15 13, 281 1 97 169 : C: : C : :C: C18-: C :-C14 C20 : C :-C1 2 C22 : C :-C10 1.-1 97 1.-169 1.--141 I5 meC33 I3-meC33 IImeC33 9 4 480

PAGE 104

25000 16 113 5000 0 365 463 40 6 0 EI mass spectrum of 15-, 13, ll-methyltritriaco ntane. 1 20000 4-0000 CI mass spect rum of 15, 13, II-methyltritriacontane. [M-15r / base peak: (m/z at 463 65) / (m/z at 477.70) 95

PAGE 105

= 18517.0/256699 0 = 7 .21 25 : mass spectrum of 7-methyltritriacontane 96

PAGE 106

Abunda nce 900000 Scan 2 8 1 8 ( 2 0 6 8 5 min): TS1 7 D ( -2794 ) ( ) 4 8 800000 700000 600000 500000 400000 300000 200000 100000 393 4 9 8 o 1001 2 0 1401 6018 0 200220 240260 280300320 340360 380400420440460480 Fig. A.2S.2. C I m ass s pectrum o f 7 -m e th y ltritri a c o nt a ne. [M-15 r b ase p eak: at 463.65) at 477.70) 6.83 % 26: 1 = 9 281 3 0 9 3 7 9 40 7 .--, .--,.--, L...-L...-L...-L...-211 183 113 8 5 7 1 3 -dim eC33 97

PAGE 107

7 85 463 0 40 60 EI mass spectrum of 7,13-dimeth y ltritriacontane. 4 2 55000 50000 35000 113 5 1 6 o CI mass spectrum of 7 13-dimethyltritriacontane. [M-IS r / base peak : at 477.70) / at 491. 80) 98

PAGE 108

= 13639 0 / 64751.0 = 21.06 % 27: : C : C22 : C:-C12 13-meC35 .A.bundance 1 3000 7 1 2000 11000 10000 9000 7000 6000 5000 4000 3000 ,52.2. 0 71 85 9 Fig. A .27.1. EI mass spectrum of 13methy l pentatriacontane. 99

PAGE 109

CI mass spectrum of 13-methylpentatriacontane. [M-IS f base peak : at 491 70) at SOS. 80) 4S392 0 = 7 .SI % 28: ----l 393 85 7-meC3 5 100

PAGE 110

26000 24000 22:000 2:0000 1 2 8 5 EI mass spectrum of7-methylpentatriacontane. 1 20000 5 6 110000 100000 90000 BOOOO 70000 60000 50000 40000 30000 20000 10000 113 422 100 1 5 0 200 250 300 350 400 CI mass spectru m of 7-methylpentatriacontane. [M-IS r base peak: at 491. 6S) (m/z at 101 4 5 0 500

PAGE 111

of Pavement : : ; .. "-.....

PAGE 112

1 : r-57 C1S: C:-C2 3-meC 71 8 S 99 EI mass spectrum of 3-methylhenicosane.

PAGE 113

113 155 197 225 1 9 1 0 22r 337 429 o 10012014016018 0 200220 240260 280300320 34036038040042 0 CI mass spectrum of 3 -m ethy lh enicosane. [M-15f / base peak: at 295 50) / (m/z at 309 50) = 9838.0 / 117477.0 8 37 % 2 : 55 8 3 97 69 111 125 EI mass spectrum of9-tricosene. 104

PAGE 114

Name 9Tricosene (Z)-$$ (Z)-9Tricosene $$ cis-9 Tricosene $$ Muscalure $$ (9Z)Tricosene CAS Number 027519-02-4 Entry Number 273778 Molecular Formula C23H46 Misc Information QI = 897 Source =NS7-7807-0 Match Quality 99 Company ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight 322.36 mass spectrum of 9 tricosene. 105

PAGE 115

3: Scan 253 (4.697 min): TS30. D (-240) ( ) 1300000 57 1200000 1100000 1000000 71 900000 700000 600000 500000 400000 300000 99 200000 100000 60 BO 100 120 140 160 1 BO 200 220 240 260 300 320 340 EI mass spectrum of Name Tricosane (CAS) $$ nTricosane CAS Number 0006 3 8-67-5 Entry Number 275689 Molecular Formula C23 H48 Misc Information QI = 836 Source = WS-1986-23-0 WLN = 23H Match Quality 94 Company ID 0 Retention Ind ex 0 Melting Point Boilin g Point Molecul a r Weight 324.38 106

PAGE 116

Abundance Scan 1904 (15.461 min): TS25. D (-1890) ( ) 3 3 1600000 1400000 1200000 1000000 800000 600000 400000 200000 113127 141155169183197211225239 253 267 100 281 295309 337 100 120 140 160 180 200 220 240 260 280 300 320 rn/z--> Fig. B.3 2. CI mass spectrum of n-tricosane [M-15r base peak : (m/z at 309.50) (m/z at 323 50) % 4: r-169 C12-: C:-C1 0 169 141 11-meC23 r-141 C14-: C:-Cs 197---1 113 9-meC23

PAGE 117

Abundance Scan 263 (4.781 min): TS30. D ( -259) ( ) 550000 500000 450000 400000 71 350000 300000 85 250000 200000 150000 100000 50000 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 rn/z--> EI mass spectrum of 11-, 9-methyltricosane. :3 "7 CI mass spectrum of 1] 9-methyltricosane. [M-15f / base peak: at 323.45) / at 337 50)

PAGE 118

= 56895 0 / 777832 0 = 7 .31 % 5 : 57 C20 : C :-C2 --.J 281 29 3-meC23 Abundance Scan 274 min): TS30. D ( -269) ( ) 1000000 900000 700000 71 600000 500000 B5 400000 300000 200000 99 100000 113 127141 40 60 BO 100 120 140 160 1 BO 200 220 240 260 300 320 340 rn/z--> F i g B. S 1. EI mass spectrum of 3-methyltricosane. 109

PAGE 119

Scan 1976 (15.872 rTlin): TS25. D ( -1957) ( ) 1200000 3 7 1100000 1000000 900000 800000 700000 600000 500000 400000 300000 200000 100000 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 CI mass spectrum of 3 -meth yltricosane. [M-15r / base peak : at 323 50) / at 337 50) = 112467 0 / 1255040 0 = % 6 : Scan 281 (4.932 rTlin): TS30. D ( -278) ( ) 57 300000 250000 71 200000 85 150000 100000 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 EI mass spectrum of n-tetracosane. 110

PAGE 120

Name Tetracosane CAS Number 000646 3 I-I Entry Number 288784 Molecular Formula C24H50 Misc Information Q I = 844 Source = NS-12-7043-0 Match Qual i ty 98 Compan y ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weight Abundance 338.39 500000 Scan 2001 (16.015 min): TS25. D ( -1988) ( ) 3 7 450000 400000 350000 300000 250000 200000 150000 100000 50000 113127141 15516918319721122B23S253 267 281 100 29530g323 o 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 rn/z--> mass spectrum of n tetracosane. [M-15 r base peak: at 323 50) at 337 50) 2.73 % III

PAGE 121

7: r-183 12-meC24 65000 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 Scan 284 (4.957 min): TS30. D ( -283) ( ) 71 85 5000 323 211225239254 282295;309 337 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 mass spectrum of 12-methyltetracosane.

PAGE 122

Scan 2009 (16.061 rTlin): TS25. D (-2005) ( ) 150000 140000 130000 120000 110000 100000 90000 70000 60000 50000 40000 30000 20000 10000 o 100.1 100 120 140 160 200 220 240 260 300 320 340 360 Fig. B.7.2 CI mass spectrum of 12-methyltetracosane [M-15 f / ba se peak : at 337.50) / at 351.50) = 838 4 0 / 156776.0 5.35 methylalkane Due to co-eluting peak with n-tetraco sa ne so the abundance ofmlz 337.50 i s actually bigger than 8384 0 and the percent age of[m-15flbase peak i s > 5.35% 8: C13-: C:-C10 183----1 '--141 ll-meC24 113

PAGE 123

Abundance Scan 292 (5.024 min): TS30.D (-288) (-) 400000 350000 71 300000 250000 85 200000 150000 100000 99 50000 352 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 EI mass spectrum of ll-methyltetracosane. 3 1 CI mass spectrum of ll-methyltetracosane. [M-15r / base peak: at 337 50) / at 351. 50) 58593.0 / 811336.0

PAGE 124

= 7 22 % 9: ,-71 C20 : C:-C3 281----l 1--43 4-meC24 Abunda nce 110000 100000 90000 aoooo 70000 60000 50000 5 4-0000 30000 3 0 20000 9 9 10000 a 40 6 0 eo 100120140160180200220 240260280 300320 340360 380400 Fig. B.9.1. EI mass spectrum of 4-methyltetracosane.

PAGE 125

Abundance 220000 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 Scan 2057 (16 .335 min) : TS25. D (-2048) (-) 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 CI m ass spectrum of 4-methyltetracosane [M-15 r / base p eak : at 337.50) / at 351.50) = 1 5812 0 / 220272 0 = 7 .18% 111 125 ao EI mass spect rum oftetracosene. 116

PAGE 126

BOOOO 125 139 153 1 6 7 351 J J 3BO CI mass spectrum of tetracosene. 11: 281 352 40 EI mass spectrum of n-pentacosane

PAGE 127

Na m e P e nt acosa n e (CAS) $$ n -P e nt acosa n e CAS umb e r 000629 99-2 E n try N umb e r 300503 Mo l ecula r F o rmul a C25 H 52 M isc Inform a ti o n Q I = 621 So ur ce = P G 1982-17 1 7-0 WL N = 25 H Ma t c h Q u a l ity 99 C omp a n y ID 0 R ete n tio n Ind ex 0 M elting P o int B oili n g P oi nt M o l ecula r Wei g ht Abundance 3500000 3000000 2500000 2000000 1500000 1000000 352.4 1 Scan 2097 (16.564 min) : TS25. D (-2086) (-) 500000 83197211225239253:267 281295 3 1 1 00 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m ass s p ec trum o f n-p e nt ac o sa n e [M-15r b ase p eak : at 337 50) a t 35 1 50) = % 118

PAGE 128

= 5 2 7 6 ; r-169 C14-: c:-c10 ---.J 197 141 11-meC25 ,sc:=EOO ...... 322 < 5 .27 ........... ,..-.): ( 3'8) ( ) Fig. EI mass spectrum of 11-meth ylpentacosane.

PAGE 129

2 1 3 1 (10 .7"158 ( _:211C 150<>00<:>0 04-00000 15200000 150000C>C> 4-6C>C>C>C>C> """ eoococ>o """4-0C>OC>C> """ 20C>C>COC> """ OOc>c>c>O 3600CO<:>C> ::3 Oc>c>c>c> 3 00000 320000<:) 3000000 2_C>0C>00 2 00000 24-00000 2200000 2000000 1 13C>0C>C>C> 1 0C>C>00C> 1 """000c>0 1 2C>C>C>0C> 100c>c>c>0 6C>C>C>C>C> 600000 4-0C>C>C>C> 200COOO Fig. B.12.2. C I m ass s pec trum of Il-me th ylpe nt acos ane. [M-15r base p eak: at 351 .50) at 365.50) 7.28 % 13 : r--57 C22-: C:-C2 309 29 3 -m eC25 1 20 ( ) .3

PAGE 130

E I m ass s p e ctrum of 3-m e th y lp e nt acos ane. o 3 5 379 C I m ass s p e ctrum of 3 -m e th y lp e nta cosa ne. [M-15t b ase p eak : a t 351.50 ) at 365.5 0 ) = = 8 55 % 1 2 1

PAGE 131

14: 8 5 mass spectrum of n-hexacosane. Name Hexacosane CAS Number 000630-01-3 Entry Number 311165 Molecular Formula C26H54 Misc informationQI= 900 Source = NS-12-7637-0 Match Quality 95 Company 10 0 Retention Ind ex 0 Melting Point Boiling Point Molecular Weight 366.42 122

PAGE 132

CI mass spectrum of n-hexacosane [M-15r / base peak: = (m/z at 351.45) / (m/z at 365 60) = 21574 0 / 629216 0 = 3.43 % 15: = 5.485 ; 12meC26 10-meC26 3-meC26 123

PAGE 133

8S EI mass spectrum of 12, 10-, 3-methylhexacosane 3 127 183 225 155 281 1 1 1 1 f08 3 r 31. 3r 3",'" ... ,7 CI mass spectrum of 12-, 10, 3-methylhexacosane [M-15 r base peak: at 365.60) at 379.60)

PAGE 134

287 1 2.0 / 570240.0 5 04 %-+mo n o -m e th y l a lka n e Du e to c o e lutin g p eak w i t h n-h exacosa n e so th e a bun da n ce of 365.60 i s ac tu ally bigge r t h an 28712.0 a n d t h e p e r ce n tage of[m-15 ] + lbase pea k is > 5.04% 1 6 : 1 = 5 552,C1= 17.238 r-1 9 7 183---.J '--169 13meC26 40 EI mass spec tru m of 1 3 me th y lh exacosane 125

PAGE 135

Fig. A.16.2. CI mass spectrum of 13-methylhexacosane [M-15r ba se p eak : (m/z at 365.55) (m/z at 379 60) = 7.09 % 1 7 : ,--71 337---;ci C C:-C309--l L-43 4-meC26

PAGE 136

...... 3 mass spectrum of 4-methylhexacosane. ma ss spectrum of 4-methylhexacosane. [M-15 f / base peak: = (m/z at 365.50) / (m/z at 379.60)

PAGE 137

% 1 8: = 5 .661; 169 !21, L--L--239 211 169 141 127 99 8, Fig. A.18.I. EI mass spectrum of 8 ,10, 14-trimethylhexacosane.

PAGE 138

127 50000 155 4 5000 183 239 40000 211 35000 267 30000 25000 295 20000 407 323 10000 5000 ri O 31' 3r 432 469 o 100120140160180200220 240260280 300320 340360380400420440460480 rn/z--> mass spec trum of 8 ,10, 1 4 -t rimethylhexacosane 1 9 : 1 = 5.71 2 ; mass spectrum of heptacosene 4 99

PAGE 139

15 15 c> c> c> c> :2 15 c=-c>c>c> CI mass spectrum of heptacosene. EI mass spectrum of n-heptacosane. Name Heptacosane $$ n-Heptacosane CAS Number 000593-49-7 Entry Number 320679 1 3 0

PAGE 140

Molecular For mul a C27 H 56 Misc In formatio n Q I = 89 1 So u rce =NS8-9992 0 Match Qual ity 99 Co mp a n y ID 0 R etention Index 0 Me l t i ng Point B oiling Po i nt Molecular Wei ght 380.44 97 125 210 7 2 r 327 o 356 3 r 4 2 9 El m ass s p ec trum of u nknow n 1 3 1

PAGE 141

'52S. D 1 1 139 393 1 6 7 195 223 lL 31' 3 4 9 4 a 5 9 Fig. A.20.2. CI m ass s p ect ru m of u nknown 21: .--197 225----, :C: C14: C :-C12 169 13meC

PAGE 142

7 as 113 379 40 EI mass spectrum of 13-methylheptacosane 600000 113 3 3 CI mass spectrum of 13-methylheptacosane. [M-IS f / ba se peak : a t 379 60) / at 393 60 ) 1 3 3

PAGE 143

= 7 1 0 % 22 : 5 ,---57 337--.J 1-29 3 -m eC 7 a 6 40 6 0 E 1 m ass s p e ctrum o f 3m e th y lh e pt a c osan e 134

PAGE 144

3 3 127 169 197 267 3 0 9 239 100 '>12 1 3 r 1 3 m ass s p ect rum of 3 -m et h y lh e pt acosane. [ M-15f b ase p eak: ( m / z at 379.60) (m/z at 393 60) 8.03 % 239 267 323 351 -, -,---, C C 169 85 5 7 5 10-dim eC1 35

PAGE 145

( t o t ( (I) V t:: 0 8 0 CJi ...... 0-(I) (1) ...s:: ...s:: (I) ...... (1) 0 lr) 0 0 CJi ;::::l (1) 0-n 0 0 % Q t ...... 0-(1) ...s:: '-0 ...s:: M ...... (1) lr) S ,.-.. 0 ...... r-(1) 0 0-'
PAGE 146

132755 0 / 740176.0 1 7.94 23: 225 253 351 379 --, --,--, L.....-L.....L.....L.....183 155 57 29 3 11-dimeC27 8S EI mas s spectrum of 3 I1-dimethylheptacosane 1 37

PAGE 147

23es3 ..... 2"7 e<> '9"7 225 25'" 2<>5 323 2 3r "'1<> 2 m ass s pectrum o f 3 Il-dime th y lh e pt acos an e [ M -15f b ase p eak : at 393 6 0 ) (m/z at 407 60) 1 1.85 24 : 197---l L-183 323---l L-57 1 4 -m eC28 5 -m eC28 1 38 4 "7

PAGE 148

EI mass spectrum of 14, 5-methyloctacosane. "7 113 183 141 253 211 3 0 9 o CI mass spectrum of 14-, 5-methyloctacosane. [M-15r / base peak : at 393 55) / at 407 60) 1 39

PAGE 149

8.38 % 25: /= 6 315 ; C/= 18 341 6 12 ....... EI mass spectrum of 6 12,21trimethyloctacosane.

PAGE 150

CI m ass s p e ctrum o f 6 12,21-trim e th y loct a cos a ne. 2 6: 97 125 a E I ma ss s p e ctrum o f non ac o se ne.

PAGE 151

405 1 6 7 :2 1 o m/:z:--::.Fig A 26 2. CI mass spectrum of nonacosene. 2 7 : = 6.449 Fig. A .27 1. EI mass spectrum of n-nonacosa n e

PAGE 152

Name Nonacosane $$ n-Nonacosane CAS Number 000630-03-5 Entry Number 336999 Molecular Formula C29H60 Misc Information QI = 900 Source =NS-9-4l91-0 Match Quality 99 Compan y ID 0 Retention Index 0 Melting Point Boiling Point Molecular Weig ht 408.47 mass spectrum of n-nonacosane. base peak: at 393.55) (rn/z at 407.60) = = 3 .01 143

PAGE 153

28: = 6.583 r--197 r--169 C1S-: C:-C12 C1S-: C:-C10 L--169 L--14 1 13-meC29 11-meC29 Fig. A.2S.!, EI mass spectrum of 13, 11-methylnonacosane.

PAGE 154

__ mass spectrum of 13-, II-methylnonacosane. [M-15 f base peak : at 407 60) at 421.55 ) = 10563.0 / 170812 0 = 6 .18% 29: = 6.793 ; 5 12-; 309 337 351 379 L-L--L-L-127 99 85 57 239 267 351 379 L-.L-.197 169 85 57 5 12-dimeC29 5 7-dimeC29 145

PAGE 155

oj oj c: c: ro ro G 0 0 ro ro c: c: 0 0 c: c: ....... ....... 0.-< :.a \0 G r--r-V) V) V) V) 0 0 0 v) C
PAGE 156

14.15% 99 127 281 309 365 393 ---, ---, --, ---, 7 II 4 L....-351 323 169 85 57 mass spectrum of 5 1 0 22-trimeth y lnonacosane. 147

PAGE 157

ll. o m ass s p ect rum of 5 1 O 22 t rim e th y ln onacosa ne. [ M-15 f / b ase peak : at 435 70) / at 449 .70) = 15954 0 / 38408 0 =41.54% 3 1 : r--57 365 ---1 1..-29 3 meC29 1 48

PAGE 158

....... 127 E I ma ss s pectrum o f 3 -meth y lnonaco s ane -rS,2S. C I m ass s p e ctrum o f 3 -m e th y lnon a co san e [ M-15r b ase p eak : a t 4 0 7 65) (m/z at 42 1 7 0 ) 1 49

PAGE 159

= 5283.0 / 59451.0 = 8.87% 32: 7. 355 ; C 1 =19.399 EI mass spectrum of n-hentricontane. 150 ......

PAGE 160

C I ma ss spectrum o f n-h e nt r icont a ne. [M-15r b ase p eak: = (m/z at 42 1 60) (m/z a t 435 70) = 154 1 9.0 / 4977 1 7.0 = 3. 1 0 % 151

PAGE 161

Blomquist G 1., Nelson D R., Renobales M d (1987) Chemistry biochemistry and physiology of insect cuticular lipids. 6 227 265. Dani R. (2006) Cuticular lipids as semiochemicals in paper wasps and other social insects. 43 500-514 Ettre S (2003 ) Retention Index Ex pressions 5 8, 491494. Francis G. W Veland K. (1981) Alkylthiolation for the determination of double bond positions in linear alkenes. Gordon D M. (2000) Ants at work: How an insect societ y is organized W W Norton Company Greene M. J., Gordon D M (2003) Cuticular hydrocarbons inform task decisions. 4 23 32. Gross 1. (2004) Chemical ionization. In mass spectrometrya textbook Springer Verlag Heidelberg pp 331-354 Harrison G (1992) Chemical ionization mass spectrometry. 2ed CRC Press Howard R. W. (1993 ) Cuticular hydrocarbons and chemical communication In insect lipids: Chemistry, biochemistry and biology. Stanley-Samuelson D. W., Nelson D. R., ed U niversity of Nebraska Press Lincoln pp. 180-226. Howard R. W., Blomquist G J (2005) Ecological behavioral and biochemical aspects 152

PAGE 162

of insect hydrocarbons. Howard R. W McDaniel, A. Nelson, D. R. Blomquist, G. 1. (1980) Chemical ioni z ation mass spectrometry. Application to insect-deri v ed cuticular alkanes 6 609-623 Jackson, L.L. Blomquist G. 1. (1976 ) Insect waxe s In Chemistr y and Biochemistr y of Natural Waxes.Kolattukudy P.E. ed. Elsevier Amsterdam, pp. 201-233. Kovats E (1958) Gas-chromatographische charakterisierung organischer verbindungen Teil 1 : Retentionsindice s aliphatischer halogenide alkohole aldeh y de und ketone 41, 1915-1932. Lan g e Basselier 1. 1., Bag neres A. G. E scoubas P., Lemaire M. Lenoir A. Clement J.-L. Bonav ita-Cougourdan A. Trabalon M. Campan, M., Courgourdan A. B. (1989 ) Strategy for the anal y sis of cuticular hydrocarbon waxes from insects using gas chromatograph y/ mass spectrometr y with electron impact and chemical ionization. 1 8, 787-800. Leonhardt B. A. DeVilbiss E .D (1985) Separation and double-bond determination on nanogram quantities of aliphatic monounsaturated alcohols aldehydes and carbox y lic acid methyl esters 322, 484. Locke y, K. (1988) Lipids of the insect cuticle: Origin composition and function. Lucas Pho, D. B. JaBon J. M. F resneau D (2005) Role of cuticular h y drocarbons in the chemical recognition between ant species in the species complex. 51 1148-1157.

PAGE 163

McLafferty, F. W. (1980) Interpretation of mass spectra. TUff 0 N. 1., ed. University Science Books Mill Valley, California. Miwa, K. (1963) Identification of peaks in gas-liquid chromatography. Nelson D. R. (1978) Long-chain methyl-branched hydrocarbons: Occurrence biosynthesis and function. 13 1-33. Nelson D.R. (1993) Methyl-branched lipids in insects. Insect I ipids. Ed. by Stanley-Samuelson D.W. and Nelson D.R. University of Nebraska Press Lincoln, pp. 271-315. Nelson D. R., Tissot, M., Nelson, L. 1., FatIand L., Gordon, D. M (2001) Novel wax esters and hydrocarbons in the cuticular surface lipids of the red harvester ant, Pechine 1. M. Perez F., Antony C, ]allon 1.-M. (1985) A further characterization of cuticular monoenes using a mass spectrometry method to localize double bonds in complex mixtures. 145 177-182. Pechine 1. M., Antony C., lallon 1.-M. (1988) Precise characterization of cuticular compounds in young by mass spectrometry. 1071-1085. Sano (2007) The role of cuticular hydrocarbons as social recognition cues in the pavement ant

PAGE 164

Wagner D., Brown M 1. F., Broun P., Cuevas W., Moses, L. E., Chao D. L. and Gordon D. M. (1998) Task-related differences in the cuticular hydrocarbon composition of harvester ants 24,2021 2037. Wagner D., Tissot M., Cuevas W., Gordon D M. (2000) Harvester ants utilize cuticular hydrocarbons in nestmate recognition. 26 2245-2257.