An integrative approach to untangling species delimitation in the Cataglyphis bicolor desert ant complex in Israel.

Although extensive research has been carried out on the desert ants in the genus Cataglyphis in recent years, some of the specific intra- and interspecific relationships remain elusive. The present study disentangles the phylogenetic relationships among the C. bicolor complex in Israel using an integrative approach based on genetic markers, morphometric measurements, and chemical analyses (cuticular hydrocarbons). Several species delimitation approaches based on four nuclear, two mitochondrial, and eleven microsatellite markers, as well as 16 body measurements and 56 chemical variables, were employed to deciphering the occurrence of cryptic species in our data set. Our findings support the occurrence of at least four distinct species in the C. bicolor group in Israel, one of which may be a complex of three more recent species. The findings confirm the distinctiveness of C. isis and C. holgerseni. They attest the presence of a recently discovered species, C. israelensis, in the central mountain ridge and the occurrence of another clade distributed from the Negev to the Mediterranean coast, comprising the species C. niger, C. savignyi, and C. drusus. Although these three species are separated on the basis of mtDNA, this subgrouping was not supported by any of the nuclear sequence markers nor by the microsatellite analysis. This genetic structure may thus either reflect a possible recent speciation, or a geographical structuring of a single species. Overall, using these different sources of evidence we locate our samples within a global phylogeny of the bicolor group and discuss the processes that underlie speciation in this group.

Precocene-I inhibits juvenile hormone biosynthesis, ovarian activation, aggression and alters sterility signal production in bumble bee (Bombus terrestris) workers.

Juvenile hormone (JH) is an important regulator of development and physiology in insects. While in many insect species, including bumble bees, JH functions as gonadotropin in adults, in some highly eusocial insects its role has shifted to regulate social behavior including division of labor, dominance and aggression. Studying JH functions across social insect species is important for understanding the evolution of sociality; however, these studies have been limited because of the inability to reduce JH levels without surgically removing its glandular source, the corpora allata. Precocene is known to inhibit JH biosynthesis in several non-social insects, but has been poorly studied in social insects. Here, we tested whether precocene-I can effectively reduce JH levels in Bombus terrestris workers, and examined its effects on their physiology and behavior. Precocene-I treatment of three-worker groups decreased JH titer and ovarian activation, irrespective of the bees' dominance rank within the group, and was remedied by JH replacement therapy. Precocene-I also decreased aggressiveness and increased ester-sterility signal production; these changes were rank-dependent, and affected mainly the most reproductive and the least aggressive workers, respectively, and could not be remedied by JH replacement therapy. These results clearly confirm the role of JH as a gonadotropin and mediator of aggression in B. terrestris, and indicate that JH effects are associated with worker dominance rank. The ability to chemically reduce JH titer provides us with a non-intrusive method to probe the evolutionary changes associated with JH and the hormonal mechanisms that are associated with reproduction and behavior in social insects.

Variation in the level of aggression, chemical and genetic distance among three supercolonies of the Argentine ant in Europe.

In their invasive ranges, Argentine ant populations often form one geographically vast supercolony, genetically and chemically uniform within which there is no intraspecific aggression. Here we present regional patterns of intraspecific aggression, cuticular hydrocarbons (CHCs) and population genetics of 18 nesting sites across Corsica and the French mainland. Aggression tests confirm the presence of a third European supercolony, the Corsican supercolony, which exhibits moderate to high levels of aggression, depending on nesting sites, with the Main supercolony, and invariably high levels of aggression with the Catalonian supercolony. The chemical analyses corroborated the behavioural data, with workers of the Corsican supercolony showing moderate differences in CHCs compared to workers of the European Main supercolony and strong differences compared to workers of the Catalonian supercolony. Interestingly, there were also clear genetic differences between workers of the Catalonian supercolony and the two other supercolonies at both nuclear and mitochondrial markers, but only very weak genetic differentiation between nesting sites of the Corsican and Main supercolonies (F(ST) = 0.06). A detailed comparison of the genetic composition of supercolonies also revealed that, if one of the last two supercolonies derived from the other, it is the Main supercolony that derived from the Corsican supercolony rather than the reverse. Overall, these findings highlight the importance of conducting more qualitative and quantitative analyses of the level of aggression between supercolonies, which has to be correlated with genetic and chemical data.

Biosynthesis and Accumulation of Formic Acid in the Poison Gland of the Carpenter Ant Camponotus pennsylvanicus.

Formic acid synthesis in the poison gland of Camponotus pennsylvanicus is closely related to the C-1 metabolism of the glandular cells. Serine, glycine, and histidine are potential C-l donors to formic acid by several tetrahydrofolate intermediates. Formic acid is accumulated by its transfer to an insulated reservoir, so that the ant avoids the acid's cytotoxicity. This combination of biochemical and morphological features provides an autodefensive mechanism. Possible factors that regulate the biosynthesis of formic acid in the poison gland apparatus are discussed.

Natural Polyesters: Dufour's Gland Macrocyclic Lactones Form Brood Cell Laminesters in Colletes Bees.

Bees in the genus Colletes make their brood cells in the ground and coat them with a highly resistant, waterproof, transparent membrane. This membrane is a polyester constructed mainly from 18-hydroxyoctadecanoic acid and 20-hydroxy-eicosanoic acid, which are stored as their corresponding lactones in the Dufour's gland of the bee. When lining the cells, the bee secretes its glandular content, and the membrane is apparently a product of polycondensation reaction of its contents. This appears to be the first report of a naturally occurring linear polyester. The term laminester (lamina approximately layer + ester) for this class of compounds is proposed.

Anthophora Bees: Unusual Glycerides from Maternal Dufour's Glands Serve as Larval Food and Cell Lining.

The Dufour's gland of Anthophora abrupta, a solitary bee, secretes a complex mixture of liquid triglycerides containing one long-chain and two shortchain fatty acids. This is applied inside the earthen brood cells and added to the provision, where it is converted, perhaps by enzymes from the bee's saliva or gut, to solid diglycerides that are later eaten by the bee larvae. This use of Dufour's gland secretion as food and its nutritive function are reminiscent of the royal jelly secreted by honey bees.

Proteins in a nonvenomous defensive secretion: biosynthetic significance.

In common with many arthropods, the true bug, Leptoglossus phyllopus, when disturbed, emits a two-phase secretion that consists of an organic phase and an aqueous phase. The organic phase is a mixture of highly reactive low-molecular-weight compounds, analogous to those produced by other arthropods, and is deterrent to many kinds of predators. The aqueous phase, heretofore ignored in most analyses of arthropod defensive secretions, contains proteins. Even though the secretion is not injected, the proteins enzymatically catalyze the derivation of the most reactive components within the impermeable cuticular storage reservoir and, thus, constitute part of the defensive system that appears to be commonly used by arthropods producing irritating chemicals.

Stock-specific chemical brood signals are induced by Varroa and Deformed Wing Virus, and elicit hygienic response in the honey bee.

The health of the honey bee Apis mellifera is challenged by the ectoparasitic mite Varroa destructor, and the numerous harmful pathogens it vectors. Existing pesticide-based Varroa controls are not sustainable. In contrast, one promising approach for improved honey bee health is the breeding of hygienic bees, capable of detecting and removing brood that is parasitized or diseased. In three experiments we find evidence to support the hypothesis that stock-specific chemical brood signals are induced by Varroa and Deformed Wing Virus, and elicit hygienic response in the honey bee. By collecting, analyzing, and running bioassays involving mite-infested and control brood extracts from three honey bee breeding stocks we: 1) found evidence that a transferrable chemical signal for hygienic behavior is present in Varroa-infested brood extracts, 2) identified ten stock-specific hydrocarbons as candidates of hygienic signaling, and 3) found that two of these hydrocarbons linked to Varroa and DWV were also elevated in brood targeted for hygienic behavior. These findings expand our understanding of honey bee chemical communication, and facilitate the development of improved hygienic selection tools to breed honey bees with greater resistance to Varroa and associated pathogens.

Population genetic structure, worker reproduction and thelytokous parthenogenesis in the desert ant Cataglyphis sabulosa.

In social Hymenoptera, within-colony relatedness is usually high due to the haplodiploid sex-determining system. However, factors such as the presence of multiple reproductive queens (polygyny), multiple queen matings (polyandry) or worker reproduction result in decreased relatedness among workers and the brood they rear, and consequently dilute their inclusive fitness benefits from helping. Here, we investigated population genetic structure, mating system, worker reproduction and parthenogenesis in the desert ant Cataglyphis sabulosa. Analysis of worker genotypes showed that colonies are headed by a single queen, mated with 1-5 males. The inbreeding coefficient within colonies and the levels of relatedness between the queens and their mates were positive, indicating that mating occurs between related individuals. Moreover, the mates of a queen are on average related and contribute equally to worker production. Our analyses also indicate that colonies are genetically differentiated and form a population exhibiting no isolation-by-distance pattern, consistent with the independent foundation of new colonies (that is, without the help of workers). Finally, both ovarian dissections and genetic data on the parentage of males show that workers do not reproduce in queenright colonies; however, they lay both haploid (arrhenotokous males) and diploid (thelytokous females) eggs in queenless colonies. In contrast to the congeneric species C. cursor, where new queens are produced by thelytokous parthenogenesis, female sexuals of C. sabulosa result from classical sexual reproduction.

Biosynthesis of formic acid by the poison glands of formicine ants.

The biosynthesis of formic acid in the poison glands of formicine ants is closely related to the C-1 metabolism of the glandular cells. Experiments utilizing radiolabeled amino acids revealed that serine is a major precursor, contributing both its alpha and beta carbons to formic acids. 5,10[14C]methylene H4folate and 5,10[14C]methenyl H4folate also serve as precursors of formic acid in the poison gland, suggesting that they are intermediates in the pathway. Furthermore, these H4folate derivates were isolated from poison glands following incubation with [3-14C]serine and proved radioactive. The glandular cells are also exceptionally rich in the enzymes responsible for these reactions, supporting the proposed pathway. Although this pathway has been established in various organisms, the uniqueness of the poison gland system is that it accumulates formic acid to large extent, yet avoids its cytotoxicity. This is made possible by a combination of the biochemical characteristics of the pathway and the special morphological features of the poison gland.

Aggressive reproductive competition among hopelessly queenless honeybee workers triggered by pheromone signaling.

In the honeybee, Apis mellifera, the queen monopolizes reproduction, while the sterile workers cooperate harmoniously in nest maintenance. However, under queenless (QL) conditions, cooperation collapses and reproductive competition among workers ensues. This is mediated through aggression and worker oviposition, as well as shifts in pheromones, from worker to queen-like composition. Many studies suggest a dichotomy between conflict resolution through aggression or through pheromonal signaling. In this paper, we demonstrate that both phenomena comprise essential components of reproductive competition and that pheromone signaling actually triggers the onset of aggression. We kept workers as QL groups until first aggression was observed and subsequently determined the contestants' reproductive status and content of the mandibular (MG) and Dufour's glands (DG). In groups in which aggression occurred early, the attacked bee had consistently more queen-like pheromone in both the MG and DG, although both contestants had undeveloped ovaries. In groups with late aggression, the attacked bee had consistently larger oocytes and more queen-like pheromone in the DG, but not the MG. We suggest that at early stages of competition, the MG secretion is utilized to establish dominance and that the DG provides an honest fertility signal. We further argue that it is the higher amount of DG pheromone that triggers aggression.

Function of secretion of mandibular gland of male in territorial behavior ofXylocopa sulcatipes (Hymenoptera: Anthophoridae).

Males of the carpenter bee,Xylocopa sulcatipes, establish and defend territories which they mark with the secretions of their mandibular glands. Chemical analysis of the glandular blend revealed that it is composed of guaiacol,p-cresol, and vanillin. Territorial males recognize other intruding males either by sight or by the odor emitted from their mandibular glands. They chase the intruder out of the territory immediately, but will not do so if the visitor is a female. If a female, however, is scented with the reconstituted synthetic pheromonal blend and introduced into a territory of a male, it is treated as a male and chased out of the territory. It is suggested that the blend has a dual function: Guaiacol seems to be the aggressiveness elicitor, while vanillin is the territorial marker and female attractant and therefore could be considered a sex pheromone.

Are queen Bombus terrestris giant workers or are workers dwarf queens? Solving the 'chicken and egg' problem in a bumblebee species.

In the social bee, Bombus terrestris, the two castes differ in size and physiology, but not in any other morphological and anatomical aspects. The size differences between the castes are the result of longer instar duration in prospective queen larvae. It appears that queen larvae are programmed to have a higher molting weight at the end of the 2nd, 3rd and 4th instars. Calculation of the growth ratio, the ratio between the logarithm of molting weight at two successive instars, revealed that queen larvae have a linear growth ratio over the entire larval development as predicted by Dyar's rule. In the worker larvae, in contrast, linearity of the growth ratio breaks after the second instar, resulting in larval molting at lower weights than expected by Dyar's rule. We therefore suggest that workers' development is abnormally shortened, either by parental manipulation or by adopting a different growth plan in response to the queen's signal.

Hydrocarbon dynamics within and between nestmates inCataglyphis niger (Hymenoptera: Formicidae).

The objective of the present study was to evaluate the interrelationship between the cuticular and postpharyngeal glands' hydrocarbons, both in the individual ant and during its interaction with nestmates. In vivo radiochemical assays were employed to monitor the de novo hydrocarbon biosynthesis from acetate in the ant's body. The newly synthesized hydrocarbons appeared first internally and after 24 hr they accumulated in the postpharyngeal gland and on the cuticular surface. Blocking the possibility of external transfer of hydrocarbons between cuticle and postpharyngeal gland led to a significant decrease of labeled hydrocarbons in the postpharyngeal gland. In addition, during encounters between labeled and unlabeled ants, newly synthesized hydrocarbons were transferred, mainly via trophallaxis, but also by allo-grooming and physical contact. In view of these results, we propose as a model for their dynamics that hydrocarbons are synthesized in tissues associated with the integument. Through self-grooming, there is a constant exchange of hydrocarbons between the cuticular surface and the postpharyngeal gland. Furthermore, in encounters between nestmates, hydrocarbons are exchanged among them mostly by trophallaxis, with the mediation of the postpharyngeal gland. Thus, this gland acts as a pool for mixing colonial hydrocarbons and may serve to attain a unified colony odor.

Species, individual and kin specific blends in Dufour's gland secretions of halictine bees : Chemical evidence.

The compositions of Dufour's gland secretions of four sympatric halictine bee species were chemically studied by combined gas chromatography-mass spectrometry. The secretion is composed of C16-C24 macrocyclic lactones, isopentenyl esters, and hydrocarbons. Comparative analyses have revealed that the blends of compounds are species specific. Studies of individual glands belonging to bees of the speciesEvylaeus malachurum have demonstrated that each bee exhibits a specific blend in its Dufour's gland. Nestmate bees (considered as sister bees), however, are more similar to each other than nonrelated bees. The relative importance of species specificity, individual specificity, and similarities between kins are discussed.

Exocrine glands ofPolyrhachis simplex: Chemistry and function.

The mandibular glands of the Israeli weaver ant,Polyrhachis simplex, contain a mixture of 4-heptanone, 6-methyl-5-hepten-2-one and 6-methyl-5-hepten-2-ol;its Dufour's gland secretion consists mainly ofn-tridecane. The significance of these glandular secretions in the biology of the weaver ant is discussed.

Identification of new components from anal glands ofTapinoma simrothi pheonicium.

In addition to 2-methyl-4-heptanone, 6-methyl-5-hepten-2-one, and iridodials, the anal gland exudate of the dolichoderine ant,Tapinoma simrothi, contains 4-heptanone and 4-hydroxy-4-methyl-2-pentanone. The secretion functions as an alarm pheromone.