Intra- and interspecific variation of cuticular hydrocarbon composition in two Ectatomma species (Hymenoptera: Formicidae) based on Fourier transform infrared photoacoustic spectroscopy

We have been able to discriminate different castes and sexes of ants in the same colony by measuring cuticular hydrocarbon levels with Fourier transform infrared photoacoustic spectroscopy, compared by canonical discriminant function analysis. We have now applied this methodology to various colonies of two species of ants of the genus Ectatomma in the Brazilian Cerrado. There were clear interspecific differences in cuticular hydrocarbons of these ants, with a small intraspecific variation. The differences between colonies were greater in E. brunneum than in E. vizottoi. Genetic differences among the colonies and species were well estimated by Fourier transform infrared photoacoustic spectroscopy and statistical analyses.     

Cuticular hydrocarbons in workers of the slave-making ant Polyergus samurai and its slave, Formica japonica (Hymenoptera: Formicidae)

Comparisons of cuticular hydrocarbons between workers of the dulotic ant Polyergus samurai and its slave, Formica japonica, were carried out. Gas chromatography–mass spectrometry showed that the slave‐maker and its slave shared the major cuticular hydrocarbon compounds, but possessed several minor products unique to each species. No difference in hydrocarbon composition was detected between enslaved and free‐living F. japonica workers, suggesting that association with P. samurai has no qualitative effect on hydrocarbon composition in these ants. Principal component analyses of the cuticular hydrocarbon profiles (CHP) revealed that (i) CHP was species specific in a given mixed colony; and (ii) among mixed colonies, P. samurai workers had species‐colony specific CHP, while the same feature was not always found in enslaved and free‐living F. japonica workers. Therefore, a ‘uniform colony odor’ in terms of CHP is not achieved in naturally mixed colonies of P. samurai nor those of its slaves, F. japonica.     

Correlation between mandibular gland secretion and cuticular hydrocarbons in the stingless bee Melipona quadrifasciata

We investigated whether Melipona quadrifasciata worker mandibular gland secretions contribute directly to their cuticular hydrocarbon profile. The mandibular gland secretion composition and cuticular surface compounds of newly emerged worker bees, nurse bees, and foragers were determined by gas chromatography and mass spectrometry and compared. Both the mandibular gland secretions and the cuticular surface compounds of all worker stages were found to be composed almost exclusively of hydrocarbons. Although the relative proportion of hydrocarbons from the cuticular surface and gland secretion was statistically different, there was a high similarity in the qualitative composition between these structures in all groups of bees.     

Two genetically distinct lineages of the sika deer, Cervus nippon, in Japanese islands: comparison of mitochondrial D-loop region sequences

To investigate genetic diversity among populations of the sika deer, Cervus nippon, nucleotide sequences (705-824 bases) of the mitochondrial D-loop regions were determined in animals from 13 localities in the Japanese islands. Phylogenetic trees constructed by the sequences indicated that the Japanese sika deer is separated into two distinct lineages: the northern Japan group (the Hokkaido island and most of the Honshu mainland) and the southern Japan group (a part of the southern Honshu mainland, the Kyushu island, and small islands around the Kyushu island). All sika deer examined in this study shared four to seven units of repetitive sequences (37 to 40 bases each) within the D-loop sequences. The number of tandem repeats was different among the populations, and it was specific to each population. Six or seven repeats occurred in populations of the northern Japan group, while four or five repeats occurred in populations of the southern Japan group. Each repeat unit included several nucleotide substitutions, compared with others, and 26 types were identified from 31 animals. Sequences of the first, second, and third units in arrays were clearly different between the northern and the southern groups. Based on these D-loop data, colonization and separation of the sika deer populations in the Japanese islands were estimated to have occurred less than 0.5 million years before present. Our results provide an invaluable insight into better understanding the evolutionary history, phylogeny, taxonomy, and population genetics of the sika deer.     

Chemical basis for inter-colonial aggression in the stingless bee Scaptotrigona bipunctata (Hymenoptera: Apidae)

Inter-colonial aggression was tested using three colonies of Scaptotrigona bipunctata in a natural setting when their nests were moved and by artificial contact between individuals. Examination of the cuticular lipids of individuals from two colonies kept under identical conditions showed clear differences in their cuticular hydrocarbon profiles. The cuticular lipids were a mixture of hydrocarbons (saturated and unsaturated alkanes and alkenes) within the range of C23-C29. The use of multivariate analysis (PCA and discriminant analysis) showed that seven of the identified surface compounds are enough to separate workers from colonies A and B from each other.     

Two Genetically Distinct Lineages of the Sika Deer, Cervus nippon, in Japanese Islands: Comparison of Mitochondrial D-Loop Region Sequences

To investigate genetic diversity among populations of the sika deer, Cervus nippon, nucleotide sequences (705–824 bases) of the mitochondrial D-loop regions were determined in animals from 13 localities in the Japanese islands. Phylogenetic trees constructed by the sequences indicated that the Japanese sika deer is separated into two distinct lineages: the northern Japan group (the Hokkaido island and most of the Honshu mainland) and the southern Japan group (a part of the southern Honshu mainland, the Kyushu island, and small islands around the Kyushu island). All sika deer examined in this study shared four to seven units of repetitive sequences (37 to 40 bases each) within the D-loop sequences. The number of tandem repeats was different among the populations, and it was specific to each population. Six or seven repeats occurred in populations of the northern Japan group, while four or five repeats occurred in populations of the southern Japan group. Each repeat unit included several nucleotide substitutions, compared with others, and 26 types were identified from 31 animals. Sequences of the first, second, and third units in arrays were clearly different between the northern and the southern groups. Based on these D-loop data, colonization and separation of the sika deer populations in the Japanese islands were estimated to have occurred less than 0.5 million years before present. Our results provide an invaluable insight into better understanding the evolutionary history, phylogeny, taxonomy, and population genetics of the sika deer.     

VARIABILITY OF CELL WALL STRUCTURE AND HYDROCARBON TYPE IN DIFFERENT STRAINS OF BOTRYOCOCCUS BRAUNII1

Different samples of Botryococcus braunii Kütz., freshly collected from nature or laboratory-grown from culture collection strains, were studied by electron microscopy and their hydrocarbon content analyzed. Although the general internal structure of the cells was rather constant, the organization of the outer walls forming the hydrocarbon-rich matrix of the colonies differed greatly from one sample to another. In the majority of cultivated strains, the colonies were rather small, the different successive external walls remained distinct and all strains contained dienic or trienic hydrocarbons. In contrast, most of the collected samples possessed large colonies with a rather compact matrix formed by the hydrocarbon-rich part of the successive closely appressed external wall layers. These samples contained polyunsaturated hydrocarbons, i.e. botryococcenes. Well defined cell caps which sheared off the cells were observed only in those strains with a compact matrix. The Austin strain and some collected samples, however, were intermediate with rather small colonies, dense matrix, definite cell caps and dienic hydrocarbons. Thus, the hydrocarbon composition did not correlate directly with the variations in wall structure; however, the occurence of dienic and botryococcene-like hydrocarbons together in one strain was never observed, although analyzed at various stages of growth. Thus, the existence of distinct strains of Botryococcus braunii, some synthesizing dienes, others botryococcenes, appears highly probable.     

Effect of whitefly parasitoids on the cuticular lipid composition of Bemisia argentifolii (Homoptera: aleyrodidae) nymphs

Experiments were conducted to determine the effects of whitefly parasitoids on the cuticular lipid composition of the silverleaf whitefly, Bemisia argentifolii Bellows and Perring [=sweetpotato whitefly, Bemisia tabaci (Gennadius), Biotype B] nymphs. The cuticular lipids of B. argentifolii nymphs that had been attacked by parasitic wasps, either Eretmocerus mundus Mercet or Encarsia pergandiella Howard, were characterized by capillary gas chromatography and CGC-mass spectrometry and the results compared with the cuticular lipids of unparasitized nymphs. Previous studies with B. argentifolii nymphs had shown that wax esters were the major components of the cuticular lipids with lesser amounts of hydrocarbons, long-chain aldehydes, and long-chain alcohols. No appreciable changes in lipid composition were observed for the cuticular lipids of E. pergandiella-parasitized nymphs as compared to unparasitized controls. However, the cuticular lipids from nymphs parasitized by E. mundus contained measurable quantities of two additional components in their hydrocarbon fraction. Analyses and comparisons with an authentic standard indicated that the two hydrocarbons were the even-numbered chain length methyl-branched alkanes, 2-methyltriacontane and 2-methyldotriacontane. The occurrences and possible functions of 2-methylalkanes as cuticular lipid components of insects are discussed and specifically, in regard to host recognition, acceptance, and discrimination by parasitoids. Published 2000 Wiley-Liss, Inc.     

Dynamics of cuticular chemical profiles of Polistes dominulus workers in orphaned nests (Hymenoptera, Vespidae)

We analysed changes in cuticular hydrocarbon signatures of workers in orphaned colonies of the paper wasp Polistes dominulus. In natural conditions, workers and foundresses possess characteristic cuticular signatures, and foundresses are further distinguishable, both behaviourally and chemically, on the basis of their rank in a reproductive dominance hierarchy. In our study, several workers were found to develop their ovaries and produce cuticular signatures resembling those of dominant foundresses, while remaining workers possessed undeveloped ovaries and had cuticular blends characteristic of subordinate foundresses. Workers that did not develop their ovaries had changed epicuticular signatures, demonstrating that the mixture of hydrocarbons of worker individuals is strongly dependent on social role and environment. Our results suggest that the composition of epicuticular lipids is not determined at the pre-imaginal stage, and that physiological pathways leading to cuticular chemical changes are similar in foundresses and workers of P. dominulus.     

Intraspecific Genetic Diversity of Undaria pinnatifida in Japan, based on the Mitochondrial cox3 Gene and the ITS1 of nrDNA

The intraspecific genetic diversity of the kelp Undaria pinnatifida (Harvey) Suringar (Laminariales, Phaeophyceae) was investigated using DNA sequences of the mitochondrial cytochrome oxidase subunit 3 (cox3) gene and internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA in plants collected from 21 localities along the Japanese coast between 2001 and 2003. Morphological variation was also examined and compared with the genetic diversity. Cox3 analyses of 106 plants revealed 9 haplotypes (I–IX) that differed from each other by 1–7 bp (all synonymous substitutions). Haplotype I was distributed in Hokkaido and the northern Pacific coast of Honshu, while haplotype III was found along the Sea of Japan coast of Honshu. Other types were found along the central and southern coast of Honshu. ITS1 analyses of 42 plants revealed 0–1.7% nucleotide differences, but plants from the Sea of Japan coast and northern Japan had similar sequences. The lower genetic differentiation along the Sea of Japan and northern coasts might be due to the recent establishment (after the middle of the last glacial period) of the Sea of Japan flora. The cox3 haplotype of cultivated plants was found in natural populations occurring close to cultivation sites (Naruto, Tokushima Pref., and Hokutan, Hyogo Pref.). This suggests that cultivated plants possibly escaped and spread or crossed with plants of natural populations. Morphological analyses of variation in 10 characters were conducted using 66 plants. The results showed no significant local variation owing to the wide variation in each population and did not support any forma previously described. No correlations between the morphological characters and cox3 haplotypes were detected.     

Disentangling environmental and heritable nestmate recognition cues in a carpenter ant

Discriminating between group members and strangers is a key feature of social life. Nestmate recognition is very effective in social insects and is manifested by aggression and rejection of alien individuals, which are prohibited to enter the nest. Nestmate recognition is based on the quantitative variation in cuticular hydrocarbons, which can include heritable cues from the workers, as well as acquired cues from the environment or queen-derived cues. We tracked the profile of six colonies of the ant Camponotus aethiops for a year under homogeneous laboratory conditions. We performed chemical and behavioral analyses. We show that nestmate recognition was not impaired by constant environment, even though cuticular hydrocarbon profiles changed over time and were slightly converging among colonies. Linear hydrocarbons increased over time, especially in queenless colonies, but appeared to have weak diagnostic power between colonies. The presence of a queen had little influence on nestmate discrimination abilities. Our results suggest that heritable cues of workers are the dominant factor influencing nestmate discrimination in these carpenter ants and highlight the importance of colony kin structure for the evolution of eusociality.     

Colonial and geographic variations in agonistic behaviour,cuticular hydrocarbons and mtDNA of Italian populations of <Emphasis Type="Italic">Reticulitermes lucifugus</Emphasis> (Isoptera,Rhinotermitidae)

Summary The subterranean termite, Reticulitermes lucifugus (Rossi), is found throughout Italy. The purpose of this study was to assess variations among colonies collected from the north to south of Italy. A multidisciplinary approach was used including behavioral tests, cuticular hydrocarbon analysis and partial sequencing of mitochondrial DNA. Results showed that Italian R. lucifugus populations were moderately aggressive and that aggressive behavior was unrelated to intercolonial geographic distance. Analysis of cuticular hydrocarbons demonstrated no qualitative variations between colonies, but quantitative differences were found according to caste and colony. Alkene content tended to decrease from north to south. Sequencing of mtDNA indicated kinship between two Tuscan populations and R. l. corsicus. This finding is suggestive of transtyrrenian distribution of the Corsican subspecies. Moreover, distance between haplotypes appears to be associated with intercolonial geographic distance.Received 25 March 2003; revised 3 October 2003; accepted 10 November 2003.     

Workers and queens of the European hornetVespa crabro L. have colony-specific cuticular hydrocarbon profiles (Hymenoptera: Vespidae)

Summary Cuticular hydrocarbons were extracted from sixty individual workers from six colonies ofVespa crabro L. and analyzed by combined gas-chromatography/mass spectrometry. Discriminant analysis of the cuticular hydrocarbon profiles of workers and queens showed that the wasps could be grouped by colony and by caste. Stepwise discriminant analysis selected the components which were weighted most heavily in these analyses. Different combinations of cuticular hydrocarbons were important in grouping workers by colony, queens and workers by colony, and workers and queens by caste.     

Comparative study of the cuticular hydrocarbon composition of Melipona bicolor Lepeletier, 1836 (Hymenoptera, Meliponini) workers and queens

In social insects, cuticular hydrocarbons are involved in species, kin, caste and nestmate recognition. Gas chromatography and mass spectrometry were used to compare the cuticular hydrocarbon composition of workers, males and queens of Melipona bicolor. The cuticular hydrocarbon composition of this species was found to consist mainly of C23, C25:1, C25, C27:1, C27, C29:1 and C29, which are already present in imagoes that have not yet abandoned the brood cell. This composition varied quantitatively and qualitatively between and within the castes and sexes. The newly emerged workers and young queens (virgins) had similar cuticular hydrocarbon profiles, which were different from those of the males. When the females start executing their tasks in the colony, the cuticular hydrocarbon profile differences appear. The workers have less variety, while the queens conserve or increase the number of cuticular hydrocarbon compounds. The queens have more abdominal tegumentary glands than the workers, which apparently are the source of the new cuticular compounds.     

Rank integration in dominance hierarchies of host colonies by the paper wasp social parasite Polistes sulcifer (Hymenoptera, Vespidae)

In multiple-foundress nests of the wasp Polistes dominulus, dominance hierarchies are established among foundresses, and only the dominant (=alpha) individual lays eggs. The alpha female can be distinguished from subordinate females and workers on the basis of the proportions of some hydrocarbons present on the cuticle, suggesting that chemical signaling of her reproductive status could occur. P. dominulus is also the host species of the obligate social parasite Polistes sulcifer. After aggressively usurping host colonies and behaviorally replacing the host alpha female, parasites are characterized by a change in the proportions of their cuticular hydrocarbons to match that of the host cuticular profile at both species and colony levels. In the current study, we demonstrate that P. sulcifer queens also modify their cuticular hydrocarbon proportions after usurpation to match that of the host alpha female. Parasite females, therefore, acquire the dominant rank in host colonies both reproductively and chemically by mimicking the typical alpha profile of the host. Parasite females were not able to fully inhibit ovary development in host foundresses, and 10 days after usurpation, parasites, alpha and beta foundresses show similar chemical profiles and ovarian development.     

Cuticular hydrocarbons in a termite: phenotypes and a neighbour–stranger effect

Abstract The composition of cuticular hydrocarbons of different colonies of the fungus-growing termite Macrotermes falciger shows considerable intercolonial variation. Ordination, as well as cluster analyses, separate profiles into three distinct chemical phenotypes. Behavioural tests with major workers reveal no alarm behaviour or mortality in pairings of workers from the same colony but a full range from no alarm to overt aggression, with associated death, when individuals were paired from different colonies. The level of mortality increases with differences in the composition of cuticular hydrocarbons between colonies. However, no mortality occurs in pairings of individuals from neighbouring colonies belonging to different phenotypes. The data thus provide evidence for a 'neighbour–stranger' effect (so-called 'dear-enemy' phenomenon) in termites.     

Development changes of cuticular hydrocarbons in Chrysomya rufifacies larvae: potential for determining larval age

Age determination is the basis of determining the postmortem interval using necrophagous fly larvae. To explore the potential of using cuticular hydrocarbons for determining the ages of fly larvae, changes of cuticular hydrocarbons in developing larvae of Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) were investigated using gas chromatography with flame-ionization detection and gas chromatography-mass spectrometry. This study showed that the larvae produced cuticular hydrocarbons typical of insects. Most of the hydrocarbons identified were alkanes with the carbon chain length of 21-31, plus six kinds of alkenes. The hydrocarbon composition of the larvae correlated with age. The statistical results showed that simple peak ratios of n-C29 divided by another eight selected peaks increased significantly with age; their relationships with age could be modelled using exponential or power functions with R(2) close to or > 0.80. These results suggest that cuticular hydrocarbon composition is a useful indicator for determining the age of larval C. rufifacies, especially for post-feeding larvae, which are difficult to differentiate by morphology.     

Variation in the male pheromones and mating success of wild caught Drosophila melanogaster

Drosophila melanogaster males express two primary cuticular hydrocarbons (male-predominant hydrocarbons). These act as sex pheromones by influencing female receptivity to mating. The relative quantities of these hydrocarbons vary widely among natural populations and can contribute to variation in mating success. We tested four isofemale lines collected from a wild population to assess the effect of intrapopulation variation in male-predominant hydrocarbons on mating success. The receptivity of laboratory females to males of the four wild-caught lines varied significantly, but not consistently in the direction predicted by variation in male-predominant hydrocarbons. Receptivity of the wild-caught females to laboratory males also varied significantly, but females from lines with male-predominant hydrocarbon profiles closer to a more cosmopolitan one did not show a correspondingly strong mating bias toward a cosmopolitan male. Among wild-caught lines, the male-specific ejaculatory bulb lipid, cis-vaccenyl acetate, varied more than two-fold, but was not associated with variation in male mating success. We observed a strong inverse relationship between the receptivity of wild-caught females and the mating success of males from their own lines, when tested with laboratory flies of the opposite sex.     

Nestmate recognition and temporal modulation in the patterns of cuticular hydrocarbons in natural colonies of japanese carpenter antCamponotus japonicus mayr (hymenoptera: formicidae)

Camponotus japonicus workers can discriminate nestmates from alien individuals. In the field, freeze-killed alien workers received significantly more attacks than nestmate carcasses. Gas chromatography (GC) analysis showed that the compositions of cuticular hydrocarbons of foraging workers from different colonies were the same, but the relative proportions of some compounds were colony-specific. These compounds are thus likely to function as colonial signatures. Characterization of the cuticular hydrocarbons by GC for 2 natural colonies at an interval of about 30 days over 4 months revealed that the patterns of cuticular hydrocarbon of foraging workers were not fixed but changed with time. The significant temporal modulation in term of proportions occurred in 5 of the 6 compounds that seemed to be the potential colonial signatures. The biological significance of temporal modulation in colonial signature is also discussed.