Chemical Heterogeneity in Inbred European Population of the Invasive Hornet Vespa velutina nigrithorax.

Invasive social insect populations that have been introduced to a new environment through a limited number of introduction events generally exhibit reduced variability in their chemical signatures (cuticular hydrocarbons) compared to native populations of the same species. The reduced variability in these major recognition cues could be caused by a reduction of genetic diversity due to a genetic bottleneck. This hypothesis was tested in an inbred European population of the invasive hornet Vespa velutina nigrithorax. Our results show that, in spite of the limited amount of genetic diversity present in the European population, the chemical signatures of individuals were highly heterogeneous according to their caste, sex, and colony origin. In queens, some specific saturated and unsaturated hydrocarbons were identified. These results suggest that epigenetic and/or environmental factors could play a role in modifying cuticular hydrocarbon profiles in this introduced hornet population despite the observed reduction of genetic diversity.

Varroa destructor changes its cuticular hydrocarbons to mimic new hosts.

Varroa destructor (Vd) is a honeybee ectoparasite. Its original host is the Asian honeybee, Apis cerana, but it has also become a severe, global threat to the European honeybee, Apis mellifera. Previous studies have shown that Varroa can mimic a host's cuticular hydrocarbons (HC), enabling the parasite to escape the hygienic behaviour of the host honeybees. By transferring mites between the two honeybee species, we further demonstrate that Vd is able to mimic the cuticular HC of a novel host species when artificially transferred to this new host. Mites originally from A. cerana are more efficient than mites from A. mellifera in mimicking HC of both A. cerana and A. mellifera. This remarkable adaptability may explain their relatively recent host-shift from A. cerana to A. mellifera.

Relationship between invasion success and colony breeding structure in a subterranean termite.

Factors promoting the establishment and colonization success of introduced populations in new environments constitute an important issue in biological invasions. In this context, the respective role of pre-adaptation and evolutionary changes during the invasion process is a key question that requires particular attention. This study compared the colony breeding structure (i.e. number and relatedness among reproductives within colonies) in native and introduced populations of the subterranean pest termite, Reticulitermes flavipes. We generated and analysed a data set of both microsatellite and mtDNA loci on termite samples collected in three introduced populations, one in France and two in Chile, and in the putative source population of French and Chilean infestations that has recently been identified in New Orleans, LA. We also provided a synthesis combining our results with those of previous studies to obtain a global picture of the variation in breeding structure in this species. Whereas most native US populations are mainly composed of colonies headed by monogamous pairs of primary reproductives, all introduced populations exhibit a particular colony breeding structure that is characterized by hundreds of inbreeding reproductives (neotenics) and by a propensity of colonies to fuse, a pattern shared uniquely with the population of New Orleans. These characteristics are comparable to those of many invasive ants and are discussed to play an important role during the invasion process. Our finding that the New Orleans population exhibits the same breeding structure as its related introduced populations suggests that this native population is pre-adapted to invade new ranges.

Endocrine control of cuticular hydrocarbon profiles during worker-to-soldier differentiation in the termite Reticulitermes flavipes.

The social organization of termites, unlike that of other social insects, is characterized by a highly plastic caste system. With the exception of the alates, all other individuals in a colony remain at an immature stage of development. Workers in particular remain developmentally flexible; they can switch castes to become soldiers or neotenics. Juvenile hormone (JH) is known to play a key role in turning workers into soldiers. In this study, we analyzed differences in cuticular hydrocarbon (CHC) profiles among castes, paying particular attention to the transition of workers to soldiers, in the subterranean termite species Reticulitermes flavipes. CHCs have a fundamental function in social insects as they serve as cues in inter- and intraspecific recognition. We showed that (1) the CHC profiles of the different castes (workers, soldiers, nymphs and neotenics) are different and (2) when workers were experimentally exposed to a JH analog and thus induced to become soldiers, their CHC profiles were modified before and after the worker-presoldier molt and before and after the presoldier-soldier molt.

Global genetic analysis reveals the putative native source of the invasive termite, Reticulitermes flavipes, in France.

Biological invasions are recognized as a major threat to both natural and managed ecosystems. Phylogeographic and population genetic analyses can provide information about the geographical origins and patterns of introduction and explain the causes and mechanisms by which introduced species have become successful invaders. Reticulitermes flavipes is a North American subterranean termite that has been introduced into several areas, including France where introduced populations have become invasive. To identify likely source populations in the USA and to compare the genetic diversity of both native and introduced populations, an extensive molecular genetic study was undertaken using the COII region of mtDNA and 15 microsatellite loci. Our results showed that native northern US populations appeared well differentiated from those of the southern part of the US range. Phylogenetic analysis of both mitochondrial and nuclear markers showed that French populations probably originated from southeastern US populations, and more specifically from Louisiana. All of the mtDNA haplotypes shared between the United States and France were found in Louisiana. Compared to native populations in Louisiana, French populations show lower genetic diversity at both mtDNA and microsatellite markers. These findings are discussed along with the invasion routes of R. flavipes as well as the possible mechanisms by which French populations have evolved after their introduction.

Contact versus feeding intoxication by fipronil in Reticulitermes termites (Isoptera: Rhinotermitidae): laboratory evaluation of toxicity, uptake, clearance, and transfer among individuals.

The susceptibility to fipronil of U.S. and French populations of Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) was evaluated in two types of laboratory bioassays: contact intoxication with 0.01-1 ppm treated sand and feeding intoxication with 0.1-10 ppm treated filter paper. Contact intoxication with 0.01 ppm fipronil caused 60% mortality after 55 and 64 h in the French and U.S. populations, respectively, whereas in the 5 ppm feeding assay 70 and 60% mortality was observed after 7 d in the French and U.S. populations, respectively. We evaluated the uptake, clearance, and transfer of fipronil among workers of French R. flavipes using [14C] fipronil in contact (0.01 ppm) and feeding (3 ppm) bioassays. Fipronil amounts were measured on their cuticle and in their bodies. Maximal uptake was observed in the contact assay. A significant uptake of fipronil occurred in the feeding assay. Transfer from exposed donors to unexposed recipients occurred within 24 h. Frequent horizontal transfer resulted in a significant uptake in recipients, particularly when donor fipronil acquisition was by feeding. Donors transferred approximately 46% of the toxicant to recipients. Social behaviors such as contact and grooming, together with internalization of the biocide, may be components of the horizontal transfer process and contribute to the efficacy of fipronil in the field.

Unicoloniality in Reticulitermes urbis: a novel feature in a potentially invasive termite species.

Social insects are among the world's most successful species at invading of new habitats. A good example of this invasive ability is Reticulitermes (Rhinotermitidae), a prominent group of subterranean termites. As a result of human intervention, i.e. transportation and creation of urban heat islands, Reticulitermes have been able to invade and thrive in cities located in areas where the natural habitat is normally too cold for colonization. They commonly infest man-made structures where they can cause extensive damage.This study was designed to evaluate the invasiveness of Reticulitermes urbis that was probably introduced in France from the Balkans. Invasive potential was assessed on the basis of features typical to invasive social insects, i.e. unicoloniality, low intraspecific aggression, high level of polygyny and colony reproduction by budding. The opportunity to study establishment and spreading processes arose after extensive sampling of an imported Reticulitermes urbis population was performed over the entire city of Domène, France (Rhône-Alpes region).For the first time, genetic analysis showed that the termites belonged to a single 'genetic entity' forming a vast colony covering about seven hectares. The colony was structured as an extended family with separate reproductive centres. We speculate that termites were introduced in a single location from which they gradually budded throughout the old town. Based on the absence of aggression among different nests within the colony, we defined this 'genetic entity' as a supercolony.

The breeding system and population structure of the termite Reticulitermes grassei in Southwestern France.

We assessed colony and population structure in three French populations of the termite Reticulitermes grassei using eight polymorphic microsatellite loci. Although most colonies contained the offspring of multiple, highly related replacement reproductives (complex families), some contained the offspring of a single pair of reproductives (simple families), and the proportion of such colonies varied across populations. Populations also showed variability in the numbers of reproductives within complex families; the F-statistics of these families in one population were consistent with having upwards of 100 replacement reproductives, while in another population these colonies contained fewer than 10 pairs of reproductives. Colony boundaries in all populations were well defined, in spite of reports of a widespread breakdown in nestmate recognition and unicolonial populations of R. grassei from these regions in France. A second unexpected finding was a lack of significant isolation by distance among colonies within populations, indicating that colony reproduction by budding was rare or absent. The lack of this form of colony reproduction even within populations where it is expected to be common suggested that the propensity for colony budding may not be as common as suggested by the literature.

Dynamics of chemical mimicry in the social parasite wasp Polistes semenowi (Hymenoptera: Vespidae).

Chemical cues are so important in the recognition mechanism of social insects that most social parasites (which rely on hosts to rear their brood) have been documented as overcoming the mechanism by which colony residents recognize non-nestmates, by mimicking the odour of the usurped colony. We simulated in the laboratory the process by which the obligate social parasite, Polistes semenowi, invades nests of the host species, Polistes dominulus, in the field and analysed the epicuticular lipid layer before and after host nest usurpation. The experiment documents that P. semenowi social parasites have an epicuticular hydrocarbon pattern which is very similar to that of their host but, after entering host colonies, parasites mimic the odour of the colonies they invade, to the point that they perfectly match the hydrocarbon profile peculiar to the colony they entered. However, both before and after host nest invasion, parasites show a tendency to possess diluted recognition cues with respect to their hosts.

Phylogeography of two European Reticulitermes (Isoptera) species: the Iberian refugium.

Chemical, i.e. cuticular hydrocarbons, and molecular data were used to probe the phylogeography of Reticulitermes termites collected from various parts of France, Spain and Portugal. Phylogenetic relationships were inferred from sequences of the internal transcribed spacer (ITS2) of nuclear ribosomal RNA genes as well as from two partial mitochondrial DNA segments, the cytochrome oxidase II gene and a sequence combining the tRNA-Leu gene and fragments of the NADH dehydrogenase I and ribosomal 16S genes. Two species, namely, R. grassei and R. banyulensis, were identified based on an analysis of cuticular hydrocarbons and the identification was confirmed by ITS2 haplotyping. However, phylogeny based on the analysis of mitochondrial DNA was not completely in agreement with the conclusions drawn from the chemical and nuclear data. An analysis of 56 R. grassei colonies revealed intraspecific differentiation into two major lineages with distinct geographical ranges. Whereas analysis of cuticular hydrocarbons showed that R. banyulensis was chemically distinct from R. grassei, analysis of mitochondrial DNA showed its close kinship with the R. grassei lineage occurring in southern Spain. This kinship could be explained by their evolution from a common polymorphic ancestor species in this ice age refugium.

Concealing identity and mimicking hosts: a dual chemical strategy for a single social parasite? (Polistes atrimandibularis, Hymenoptera: Vespidae).

Polistes atrimandibularis is a species of obligate social wasp parasite, which introduces into nests of the host species P. biglumis, where it rears its own brood relying on the workers of the host species. Wasps of the host species discriminate between nestmates and non-nestmates by means of their epicuticular hydrocarbons. To overcome the host recognition systems, parasites have to modify their epicuticular recognition cues. Compared to individuals of the host species, parasites have 3-4 times less epicuticular hydrocarbons, although they share with their hosts the habitat and even the nests. We hypothesize that a dilution of the recognition cues in social parasites could have a facilitating role in entering host nests, in integrating within their host colony and in sneaking into secondary colonies to steal larvae and pupae.

Modifications of the cuticular hydrocarbon profile of Apis mellifera worker bees in the presence of the ectoparasitic mite Varroa jacobsoni in brood cells.

Varroa jacobsoni is an ectoparasite of Apis mellifera which invades brood cells, on 8-day-old larvae several hours before cell capping. Reproduction of the parasite takes place in the capped brood cells during the nymphose of the bee. Cuticular hydrocarbons of unparasitized bees and of bees parasitized by Varroa jacobsoni were extracted and analysed by gas chromatography (GC) coupled with mass spectrometry (GC-MS). Three developmental stages of worker honey bees were studied: larvae, pupae and emergent adults. The comparison between unparasitized and parasitized hosts was performed with Principal Components Analysis coupled with a multivariate variance analysis. The cuticular hydrocarbon profiles of honey bees were qualitatively similar, for the 3 developmental stages and regardless of the presence of Varroa in the cells. Nevertheless, comparison of the relative proportions of hydrocarbons showed that the cuticular profiles of pupae and emergent adults parasitized by 1 mite and of larvae parasitized by 2 mites were significantly different from the corresponding unparasitized individuals. Such modifications could be regarded (i) as a cause of the multi-infestation in larvae during invasion of brood and (ii) as a consequence of stress and/or removal of proteins contained in the haemolymph of the host during its development.

Effect of age and sex on the production of internal and external hydrocarbons and pheromones in the housefly, Musca domestica.

The epicuticular and internal waxes of male and female houseflies were examined by capillary gas chromatography-mass spectrometry at closely timed intervals from emergence until day-6 of adulthood. New components identified included tricosan-10-one, 9,10-epoxyheptacosane, heptacosen-12-one, a series of odd-carbon numbered dienes from C31 to C39, several positional isomers of monoenes including (Z)-9- and 7-pentacosene and a number of methyl- and dimethylalkanes. (Z)-9-tricosene appears in internal lipids prior to appearing on the surface of the insect, suggesting that it is transported in the hemolymph to its site of deposition on the epicuticle. The large increases in the amount of (Z)-9-tricosene in females from day-2 until day-6 is compensated for by a concomitant decrease in (Z)-9-heptacosene. The C23 epoxide and ketone only appear in females after the production of (Z)-9-tricosene is induced, and are only abundant in epicuticular waxes, suggesting they are formed after (Z)-9-tricosene is transported to the cells which are involved in taking them to the surface of the insect. Mathematical analysis indicated that the time shift between internal production and external accumulation in females is more than 24 h. The divergence between male and female lipid production occurs at an early stage, when insects are less than one day old.

Tissue distribution and lipophorin transport of hydrocarbons and sex pheromones in the house fly, Musca domestica.

We investigated the relationship between epicuticular and internal hydrocarbons in the adult house fly, Musca domestica and the distribution of hydrocarbons, including the female sex pheromone component, (Z)-9-tricosene, in tissues. Internal hydrocarbons increased dramatically in relation to sexual maturation and were found in the hemolymph, ovaries, digestive tract, and fat body. (Z)-9-Tricosene comprised a relatively large fraction of the hydrocarbons in the female carcass and hemolymph, and less so in other tissues, while other hydrocarbons were represented in greater amounts in the ovaries than in other tissues. It therefore appears that certain hydrocarbons were selectively provisioned to certain tissues such as the ovaries, from which pheromone was relatively excluded. Both KBr gradient ultracentrifugation and specific immunoprecipitation indicated that > 90% of hemolymph hydrocarbons were associated with a high-density lipophorin (density = 1.09 g ml(-1)), composed of two apoproteins under denaturing conditions, apolipophorin I (approximately 240 kD) and apolipophorin II (approximately 85 kD). Our results support a predicted model (Chino, 1985) that lipophorin is involved in the transport of sex pheromone in M. domestica. In addition to delivering hydrocarbons and sex pheromones to the cuticular surface, we suggest that lipophorin may play an important role in an active mechanism that selectively deposits certain subsets of hydrocarbons at specific tissues.

Modification of morphological characters and cuticular compounds in worker ants Leptothorax nylanderi induced by endoparasites Anomotaenia brevis.

Anomotaenia brevis (Cestoda, Cyclophyllidea) induces major changes in the morphological characters of the host ant, Leptothorax nylanderi (Hymenoptera, Formicidae): alteration of pigmentation, lowering of adult mean size, reduction of legs, eyes and head, enlargement of petiole. The presence of parasites in adult ants also modifies the quantity of cuticular compounds but not their quality. The parasite induces some changes in the synthesis/release of 13 cuticular hydrocarbons. The higher the number of parasites within a worker, the larger the quantitative changes in four cuticular hydrocarbons in comparison with normal ants. Such modifications (morphology, chemistry, behaviour) may explain the partial intolerance exerted by normal workers against the parasitized ants.

Differential adsorption of allospecific hydrocarbons by the cuticles of two termite species, Reticulitermes santonensis and R. lucifugus grassei, living in a mixed colony. Passive transfer by contact.

When members of the two termite species Reticulitermes santonensis and Reticulitermes lucifugus grassei were placed together, each species acquired some of the allospecific cuticular products. When living individuals of each of these two species were placed together, their cuticular hydrocarbon profiles changed very quickly, since it was within the first two hours of cohabitation that they differed most from those of the corresponding control individuals. After the first two hours, the profiles of the R. santonensis individuals continued to change only very little if at all, whereas 24h later, the process of change continued in the R. lucifugus grassei individuals until their profiles resembled those of the mixed R. santonensis individuals more than their own original profiles. The profiles of the R. l. grassei individuals therefore underwent a greater change than those of the R. santonensis individuals during the period of cohabitation. The fact that similar results were obtained when dead members of these two species were placed together suggests that this difference in the adsorption of allospecific hydrocarbon by the cuticles of the members of the two species cannot be attributable to any behavioural differences, but to differences in physico-chemical composition of the cuticles between the two species.

Sex recognition inDiglyphus isaea walker (Hymenoptera: Eulophidae): Role of an uncommon family of behaviorally active compounds.

The sexual behavior of the ectoparasitoidDiglyphus isaea is described. Recognition of the female by the male occurs at close range. Males initiate courtship behavior in the presence of a living female regardless of age, as well as in the presence of a female killed by freezing. Courtship behavior is not observed in the presence of a dead female washed with organic solvents but could be elicited using a lure covered with a female organic extract. These findings demonstrate that each sex develops a specific chemical signature that can be dissolved in hexane and transferred to a lure. Analysis of organic extracts by gas chromatography revealed chemical dimorphism between the two sexes. Gas chromatography coupled with mass spectrometry showed that the main components in females were esters of medium-chain fatty acids and long-chain 11-alcohols. There were few hydrocarbons. Female esters, which were present in only small proportions in males, were recovered in the nonhydrocarbon fraction obtained after fractionation of the total extract on a silica-filled microcolumn as a mixture containing 11-heneicosyl, 11-docosyl, 11-tricosyl, 11-tetracosyl, and 11-pentacosyl octanoate, and 11-docosyl, 11-tricosyl, 11-tetracosyl, and 11-pentacosyl decanoate. These results demonstrate that there is a specific gender-related chemical signature.

Role of plant volatiles in the search for a host by parasitoidDiglyphus isaea (Hymenoptera: Eulophidae).

Diglyphus isaea Walker is a larval ectoparasitoid used in biological pest control against the American serpentine leaf minerLiriomyza trifolii Burgess. We studied the parasitoid's host searching behavior, using olfactometric methods. Our data show that the parasitoids locate host larvae (a leafmining dipteran) on the basis of volatile signals released by the plant-host complex. FemaleD. isaea are strongly attracted to the odors arising from damaged bean plants, whereas they show practically no response to intact plants. The results of our chemical analyses showed that about 15 components were present, two of which,cis-3-hexen-1-ol and 4-hydroxy-4-methyl-2-pentanone, were present in significantly larger quantities in the leaf extracts from mined or damaged bean plants than in those from healthy plants. The damage inflicted by the host larvae on these plants triggers the release of larger amounts of these substances, which probably lead the parasites to their hosts. The compounds thus act as synomones.

Mechanism underlying cuticular hydrocarbon homogeneity in the antCamponotus vagus (SCOP.) (Hymenoptera: Formicidae): Role of postpharyngeal glands.

The aim of the present study was to investigate the regulation and the homogeneity of the chemical signature between members ofCamponotus vagus after experimentally changing the cuticular chemical signature by topically applying hydrocarbons. Topical application of pentane (solvent) to the cuticle of isolated workers led to a significant decrease in the quantities of the cuticular hydrocarbons measured within 3 hr, followed by an increase within the following 3 hr and a period of relative stability from 9 hr to 14 days. On the other hand, after topical application to isolated workers ofn-tetracosane, a hydrocarbon existing only in trace quantity in this species, the quantity of this hydrocarbon measured over time in the epicuticular wax tended to level out at about 14 days after treatment. In contrast, topically applied (Z)-9-tricosene, an unsaturated hydrocarbon not normally synthesized by this species, decreased dramatically within a few hours and had completely disappeared within 14 days. (Z)-9-Tricosene applied to one member of a group was present in the postpharyngeal glands of the other members from 30 min to seven days of cohabitation. The highest levels were recorded in all six workers in each group after one day. GC-MS analyses showed that (Z)-9-tricosene was present in the cuticles of some untreated workers only after four and seven days of cohabitation with a treated worker. These data suggest: (1) that the deposited (Z)-9-tricosene decreased very quickly on the cuticle of the treated worker, although the total amount was spread over the cuticle and postpharyngeal gland and (2) that it was absorbed by the nontreated workers via the postpharyngeal glands during licking or grooming activities and reincorporated into the cuticle at four and seven days. When the treated worker was separated from the other ants by a wire mesh, (Z)-9-tricosene was detected neither in the cuticle nor in the postpharyngeal gland of nontreated workers.

Cuticular hydrocarbons wherebyMessor barbarus ant workers putatively discriminate between monogynous and polygynous colonies. Are workers labeled by queens?

The results of laboratory experiments carried out with both monogynous and artificially polygynousMessor barbarus ant colonies (which under natural conditions are always monogynous) have shown that the workers belonging to monogynous colonies were able to discriminate between intruders from other monogynous colonies and those from polygynous (di- and trigynous) ones. What mechanisms are involved in this discriminatory ability? Since differences in the relative proportions of the hydrocarbons they carry are known to convey complex messages that are used for recognition purposes, it was proposed here to investigate whether there existed any differences in the proportions of the various hydrocarbons carried by the diverse categories of intruders tested in our experiments. It emerged that one set of hydrocarbons, which were usually present in rather small proportions and included all the families that constitute this species' chemical signature (n-alkanes, mono-, di-, and trimethylalkanes), was characteristically associated with workers from monogynous colonies. Another set of hydrocarbons, which included some of the above components, mostly in larger relative proportions, can be said to have characterized the digynous and trigynous colonies.