Brain modulation of Dufour’s gland ester biosynthesis in vitro in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Caste-specific pheromone biosynthesis is a prerequisite for reproductive skew in the honeybee. Nonetheless, this process is not hardwired but plastic, in that egg-laying workers produce a queen-like pheromone. Studies with Dufour’s gland pheromone revealed that, in vivo, workers’ gland biosynthesis matches the social status of the worker, i.e., sterile workers showed a worker-like pattern whereas fertile workers showed a queen-like pattern (production of the queen-specific esters). However, when incubated in vitro, the gland spontaneously exhibits the queen-like pattern, irrespective of its original worker type, prompting the notion that ester production in workers is under inhibitory control that is queen-dependent. We tested this hypothesis by exposing queen or worker Dufour’s glands in vitro to brain extracts of queens, queenright (sterile) workers and males. Unexpectedly, worker brain extracts activated the queen-like esters biosynthesis in workers’ Dufour’s gland. This stimulation was gender-specific; queen or worker brains demonstrated a stimulatory activity, but male brains did not. Queen gland could not be further stimulated. Bioassays with heated and filtered extracts indicate that the stimulatory brain factor is below 3,000 Da. We suggest that pheromone production in Dufour’s gland is under dual, negative–positive control. Under queenright conditions, the inhibitor is released and blocks ester biosynthesis, whereas under queenless conditions, the activator is released, activating ester biosynthesis in the gland. This is consistent with the hypothesis that queenright workers are unequivocally recognized as non-fertile, whereas queenless workers try to become “false queens” as part of the reproductive competition.     

Conservation of Bio synthetic pheromone pathways in honeybees Apis

Social insects use complex chemical communication systems to govern many aspects of their life. We studied chemical changes in Dufours gland secretions associated with ovary development in several genotypes of honeybees. We found that C28–C38 esters were associated only with cavity nesting honeybee queens, while the alcohol eicosenol was associated only with their non-laying workers. In contrast, both egg-laying anarchistic workers and all parasitic Cape workers from queenright colonies showed the typical queen pattern (i.e. esters present and eicosenol absent), while egg-laying wild-type and anarchistic workers in queenless colonies showed an intermediate pattern, producing both esters and eicosenol but at intermediate levels. Furthermore, neither esters nor eicosenol were found in aerial nesting honeybee species. Both esters and eicosenol are biosynthetically similar compounds since both are recognizable products of fatty acid biosynthesis. Therefore, we propose that in honeybees the biosynthesis of esters and eicosenol in the Dufours gland is caste-regulated and this pathway has been conserved over evolutionary time.     

Fever in honeybee colonies

 Honeybees, Apis spp., maintain elevated temperatures inside their nests to accelerate brood development and to facilitate defense against predators. We present an additional defensive function of elevating nest temperature: honeybees generate a brood-comb fever in response to colonial infection by the heat-sensitive pathogen Ascosphaera apis. This response occurs before larvae are killed, suggesting that either honeybee workers detect the infection before symptoms are visible, or that larvae communicate the ingestion of the pathogen. This response is a striking example of convergent evolution between this "superorganism" and other fever-producing animals. Received: 2 September 1999 / Accepted in revised form: 28 February 2000     

Brood comb as a humidity buffer in honeybee nests

Adverse environmental conditions can be evaded, tolerated or modified in order for an organism to survive. During their development, some insect larvae spin cocoons which, in addition to protecting their occupants against predators, modify microclimatic conditions, thus facilitating thermoregulation or reducing evaporative water loss. Silk cocoons are spun by honeybee (Apis mellifera) larvae and subsequently incorporated into the cell walls of the wax combs in which they develop. The accumulation of this hygroscopic silk in the thousands of cells used for brood rearing may significantly affect nest homeostasis by buffering humidity fluctuations. This study investigates the extent to which the comb may influence homeostasis by quantifying the hygroscopic capacity of the cocoons spun by honeybee larvae. When comb containing cocoons was placed at high humidity, it absorbed 11% of its own mass in water within 4 days. Newly drawn comb composed of hydrophobic wax and devoid of cocoons absorbed only 3% of its own mass. Therefore, the accumulation of cocoons in the comb may increase brood survivorship by maintaining a high and stable humidity in the cells.     

The role of tyramine and octopamine in the regulation of reproduction in queenless worker honeybees

In honeybees, workers under queenless condition compete for reproduction and establish reproductive dominance hierarchy. Ovary activation is generally accompanied by the expression of queen-like pheromones. Biogenic amines (BAs), in particular dopamine, are believed to be involved in this process by regulating ovarian development. However, the role of BAs in establishing reproductive dominance or their effect on queen-like pheromone production was not investigated. Here, we explored the effect of octopamine (OA) and tyramine (TA) oral treatments on the propensity of treated bees to become reproductively dominant and produce queen-like pheromones in Dufour’s and mandibular glands. One bee in a pair was treated with either OA or TA while the other was fed sugar solution. TA was found to enhance ovary development and the production of esters in the Dufour’s gland and 9HDA (queen component) in the mandibular glands, thus facilitating worker reproductive dominance. OA, on the other hand, did not enhance ovarian development or ester production, but increased the production of 10HDA (worker major component) in the mandibular glands of their sugar-paired mates. OA is known to induce foraging behavior by workers, while increased production of 10HDA characterizes nursing workers. Therefore, we suggest that TA induces reproductive division of labor, while OA treatment results in caste differentiation of workers to foragers and nurses.     

Pheromone-modulated behavioral suites influence colony growth in the honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

The success of a species depends on its ability to assess its environment and to decide accordingly which behaviors are most appropriate. Many animal species, from bacteria to mammals, are able to communicate using interspecies chemicals called pheromones. In addition to exerting physiological effects on individuals, for social species, pheromones communicate group social structure. Communication of social structure is important to social insects for the allocation of its working members into coordinated suites of behaviors. We tested effects of long-term treatment with brood pheromone on suites of honey bee brood rearing and foraging behaviors. Pheromone-treated colonies reared significantly greater brood areas and more adults than controls, while amounts of stored pollen and honey remained statistically similar. Brood pheromone increased the number of pollen foragers and the pollen load weights they returned. It appeared that the pheromone-induced increase in pollen intake was directly canalized into more brood rearing. A two-way pheromone priming effect was observed, such that some workers from the same age cohorts showed an increased and extended capacity to rear larvae, while others were recruited at significantly younger ages into pollen-specific foraging. Brood pheromone affected suites of nursing and foraging behaviors allocating worker and pollen resources associated with an important fitness trait, colony growth.     

Sensory reception of the primer pheromone ethyl oleate

Social work force distribution in honeybee colonies critically depends on subtle adjustments of an age-related polyethism. Pheromones play a crucial role in adjusting physiological and behavioral maturation of nurse bees to foragers. In addition to primer effects of brood pheromone and queen mandibular pheromone—both were shown to influence onset of foraging—direct worker–worker interactions influence adult behavioral maturation. These interactions were narrowed down to the primer pheromone ethyl oleate, which is present at high concentrations in foragers, almost absent in young bees and was shown to delay the onset of foraging. Based on chemical analyses, physiological recordings from the antenna (electroantennograms) and the antennal lobe (calcium imaging), and behavioral assays (associative conditioning of the proboscis extension response), we present evidence that ethyl oleate is most abundant on the cuticle, received by olfactory receptors on the antenna, processed in glomeruli of the antennal lobe, and learned in olfactory centers of the brain. The results are highly suggestive that the primer pheromone ethyl oleate is transmitted and perceived between individuals via olfaction at close range.     

Temperature and parasitism by <Emphasis Type="BoldItalic">Asobara tabida</Emphasis> (Hymenoptera: Braconidae) influence larval pupation behaviour in two <Emphasis Type="BoldItalic">Drosophila</Emphasis> species

In holometabolous insects, pupation site selection behaviour has large consequences for survival. Here, we investigated the combined effects of temperature and parasitism by the parasitoid Asobara tabida on larval pupation behaviour in two of its main Drosophila sp. hosts differing in their climate origin. We found that larvae of Drosophila melanogaster—a species with a (sub)tropical origin—placed at 25°C pupated higher in rearing jars than those placed at 15°C. The opposite pattern was observed for Drosophila subobscura larvae—a species from temperate regions—which pupated lower, i.e. on or near the substrate at 25°C, than those placed at 15°C. When placed at 25°C, parasitized larvae of both species pupated closer to the substrate than unparasitized ones. Moreover, the Drosophila larvae that had been exposed and probably stung by A. tabida, but were not parasitized, pupated lower than the control unparasitized larvae. These results provide new insights of host behaviour manipulation by A. tabida larvae.     

1-Tridecene—male-produced sex pheromone of the tenebrionid beetle <Emphasis Type="Italic">Parastizopus transgariepinus</Emphasis>

Males of the genus Parastizopus (Coleoptera: Tenebrionidae) exhibit a special pheromone-emitting behaviour. They do a headstand, expose the aedeagus and remain in this posture for a few seconds. The pheromone emitted by P. transgariepinus was collected by solid-phase micro-extraction (100 microm polydimethylsiloxane fibre) and identified as 1-tridecene by gas chromatography/mass spectrometry. Presumably, this compound originates from the aedeagal gland, a special feature in Parastizopus, as 1-tridecene is the main compound in the gland reservoirs (23.6+/-3.8%), accompanied by various less volatile fatty acid esters (25.2+/-2.0%) and hydrocarbons (51.2+/-5.7%). 1-Tridecene is also part of the pygidial defensive secretion of both sexes, together with other 1-alkenes, monoterpene hydrocarbons and 1,4-benzoquinones, but as none of these other compounds was detected during calling, the pygidial gland could be ruled out as pheromone source. Extracts of the aedeagal gland reservoirs and the pygidial defensive secretion contained comparable amounts of 1-tridecene, 1.24+/-0.41 and 1.88+/-0.54 microg/male, respectively. Chemo-orientation experiments using a servosphere showed that 1 microg of 1-tridecene was attractive to females but not to males.     

Anatomical localization and stereoisomeric composition of <Emphasis Type="Italic">Tribolium castaneum</Emphasis> aggregation pheromones

We report that the abdominal epidermis and associated tissues are the predominant sources of male-produced pheromones in the red flour beetle, Tribolium castaneum and, for the first time, describe the stereoisomeric composition of the natural blend of isomers of the aggregation pheromone 4,8-dimethyldecanal (DMD) in this important pest species. Quantitative analyses via gas chromatography–mass spectrometry showed that the average amount of DMD released daily by single feeding males of T. castaneum was 878 ± 72 ng (SE). Analysis of different body parts identified the abdominal epidermis as the major source of aggregation pheromone; the thorax was a minor source, while no DMD was detectable in the head. No internal organs or obvious male-specific glands were associated with pheromone deposition. Complete separation of all four stereoisomers of DMD was achieved following oxidation to the corresponding acid, derivatization with (1R, 2R)- and (1S, 2S)-2-(anthracene-2,3-dicarboximido)cyclohexanol to diastereomeric esters, and their separation on reversed-phase high-performance liquid chromatography at −54°C. Analysis of the hexane eluate from Porapak-Q-collected volatiles from feeding males revealed the presence of all four isomers (4R,8R)/(4R,8S)/(4S,8R)/(4S,8S) at a ratio of approximately 4:4:1:1. A walking orientation bioassay in a wind tunnel with various blends of the four synthetic isomers further indicated that the attractive potency of the reconstituted natural blend of 4:4:1:1 was equivalent to that of the natural pheromone and greater than that of the 1:1 blend of (4R,8R)/(4R,8S) used in commercial lures.     

Caps and gaps: a computer model for studies on brood incubation strategies in honeybees (<Emphasis Type="Italic">Apis mellifera carnica</Emphasis>)

In addition to heat production on the comb surface, honeybee workers frequently visit open cells (“gaps”) that are scattered throughout the sealed brood area, and enter them to incubate adjacent brood cells. We examined the efficiency of this heating strategy under different environmental conditions and for gap proportions from 0 to 50%. For gap proportions from 4 to 10%, which are common to healthy colonies, we find a significant reduction in the incubation time per brood cell to maintain the correct temperature. The savings make up 18 to 37% of the time, which would be required for this task in completely sealed brood areas without any gaps. For unnatural high proportions of gaps (>20%), which may be the result of inbreeding or indicate a poor condition of the colony, brood nest thermoregulation becomes less efficient, and the incubation time per brood cell has to increase to maintain breeding temperature. Although the presence of gaps is not essential to maintain an optimal brood nest temperature, a small number of gaps make heating more economical by reducing the time and energy that must be spent on this vital task. As the benefit depends on the availability, spatial distribution and usage of gaps by the bees, further studies need to show the extent to which these results apply to real colonies. M. Fehler and M. Kleinhenz contributed equally to this work.     

Pheromonal contest between honeybee workers (Apis mellifera capensis)

 Queenless workers of the Cape honeybee (Apis mellifera capensis) can develop into reproductives termed pseudoqueens. Although they morphologically remain workers they become physiologically queenlike, produce offspring, and secrete mandibular gland pheromones similar to those of true queens. However, after queen loss only very few workers gain pseudoqueen status. A strong intracolonial selection governs which workers start oviposition and which remain sterile. The “queen substance”, 9-keto-2(E)-decenoic acid (9-ODA), the dominant compound of the queen's mandibular gland pheromones, suppresses the secretion of queenlike mandibular gland pheromones in workers. It may act as an important signal in pseudoqueen selection. By analysing the mandibular gland pheromones of workers kept in pairs, we found that A. m. capensis workers compete to produce the strongest queen-like signal. Received: 2 July 2000 / Accepted in revised form: 28 July 2000     

Chemical compounds of the foraging recruitment pheromone in bumblebees

When the frenzied and irregular food-recruitment dances of bumblebees were first discovered, it was thought that they might represent an evolutionary prototype to the honeybee waggle dance. It later emerged that the primary function of the bumblebee dance was the distribution of an alerting pheromone. Here, we identify the chemical compounds of the bumblebee recruitment pheromone and their behaviour effects. The presence of two monoterpenes and one sesquiterpene (eucalyptol, ocimene and farnesol) in the nest airspace and in the tergal glands increases strongly during foraging. Of these, eucalyptol has the strongest recruitment effect when a bee nest is experimentally exposed to it. Since honeybees use terpenes for marking food sources rather than recruiting foragers inside the nest, this suggests independent evolutionary roots of food recruitment in these two groups of bees.     

Sex pheromone chemistry and field trapping studies of the elm spanworm Ennomos subsignaria (Hübner) (Lepidoptera:Geometridae)

The elm spanworm, Ennomos subsignaria (Hübner), occurs throughout Canada and the eastern United States and can be a destructive forest pest on a wide range of deciduous trees. Gas chromatography/mass spectrometry (GC/MS) and coupled gas chromatographic–electroantennographic detection (GC/EAD) analysis of pheromone gland extracts, in combination with chemical synthesis and field trapping studies have identified (2S, 3R)-2-((Z)-oct-2′-enyl)-3-nonyl oxirane (hereafter Z6-9S, 10R-epoxy-19:H) as the female-produced sex pheromone. Significantly more male moths were captured between 1–100 μg loadings of this compound on red rubber septa in sticky traps compared to blank (unbaited) traps; catches then declined at higher dosages (500–1000 μg). The other isomeric enantiomer, (2R, 3S)-2-[(Z)-oct-2′-enyl]-3-nonyl oxirane (Z6-9R, 10S-epoxy-19:H), at a 10-μg dosage did not elicit trap capture. The likely precursor to the active epoxide, (Z, Z)-6, 9-nonadecadiene ((Z, Z)-6, 9-19:H), identified in virgin female sex pheromone glands, did not elicit trap capture either, and inhibited trap capture when combined with the active epoxide. Racemic 2-((Z)-oct-2′-enyl)-3-nonyl oxirane showed no significant difference in trap capture compared with Z6-9S, 10R-epoxy-19:H, indicating that the opposite enantiomer was not antagonistic. The addition of the EAD-active diene epoxide enantiomers (2S, 3R)-2-[(Z, Z)-octa-2′, 5′-dienyl]-3-nonyl oxirane or (2R, 3S)-2-[(Z, Z)-octa-2′, 5′-dienyl]-3-nonyl oxirane in admixture with Z6-9S, 10R-epoxy-19:H (at 10% of the latter) did not enhance or decrease trap capture compared to Z6-9S, 10R-epoxy-19:H oxirane alone, so they are not likely pheromone components. This pheromone, impregnated in rubber septa at less than 100-μg dosage, can now be used as a trap bait to develop detection and monitoring strategies for this insect.     

Rare royal families in honeybees, Apis mellifera

The queen is the dominant female in the honeybee colony, Apis mellifera, and controls reproduction. Queen larvae are selected by the workers and are fed a special diet (royal jelly), which determines caste. Because queens mate with many males a large number of subfamilies coexist in the colony. As a consequence, there is a considerable potential for conflict among the subfamilies over queen rearing. Here we show that honeybee queens are not reared at random but are preferentially reared from rare “royal” subfamilies, which have extremely low frequencies in the colony's worker force but a high frequency in the queens reared.     

The ladybird<Emphasis Type="Italic"> Thalassa saginata</Emphasis>, an obligatory myrmecophile of<Emphasis Type="Italic"> Dolichoderus bidens</Emphasis> ant colonies

The larvae and pupae of the ladybird Thalassa saginata develop inside colonies of the dolichoderine ant Dolichoderus bidens. This association is the first specific and obligatory relationship recorded between ants and ladybirds. The ants provide shelter and protection to the larvae but the diet of the latter remains unclear. The integration of T. saginata larvae into the ant colonies is achieved by mimicking the cuticular patterns of the ants brood. Moreover, the larvae secrete substances from their hairs and anal gland that are likely to enhance their attractiveness.     

Male-produced anti-sex pheromone in a plant bug

In plant bugs (Miridae), females produce sex pheromones in the metathoracic scent gland, which in most other true bugs (Heteroptera) is responsible for chemical defense. The possibility that the metathoracic gland secretion of male plant bugs plays a role other than defense has been largely overlooked. Here we show that in a pine-inhabiting mirid, Phytocoris difficilis Knight, hexyl butyrate and (E)-2-hexenyl butyrate are abundantly produced only in males; we demonstrate that these metathoracic gland compounds elicit strong antennal responses in conspecific males, and that these butyrates totally interrupt attraction of males to the female-produced sex pheromone. Our results suggest that in at least some plant bugs the male metathoracic scent gland esters have a natural communicative function as anti-sex pheromones, probably to interrupt further mating attempts by other males.     

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.     

Can Dufour��s gland compounds honestly signal fertility in the primitively eusocial wasp Ropalidia marginata?

Unlike queens of typical primitively eusocial species, Ropalidia marginata queens are docile and non-interactive, and hence cannot be using dominance to maintain their status. It appears that the queen maintains reproductive monopoly through a pheromone, of which the Dufour's gland is at least one source. Here, we reconfirm earlier results showing that queens and workers can be correctly classified on a discriminant function using the compositions of their respective Dufour's glands, and also demonstrate consistent queen-worker differences based on categories of compounds and on single compounds also in some cases. Since the queen pheromone is expected to be an honest signal of the fecundity of a queen, we investigate the correlation of Dufour's gland compounds with ovarian activation of queens. Our study shows that Dufour's gland compounds in R. marginata correlate with the state of ovarian activation of queens, suggesting that such compounds may portray the fecundity of a queen, and may indeed function as honest signals of fertility.     

Effects of clay turbidity and density of pikeperch (Sander lucioperca) larvae on predation by perch (Perca fluviatilis)

Increased turbidity reduces visibility in the water column, which can negatively affect vision-oriented fish and their ability to detect prey. Young fish could consequently benefit from high turbidity levels that can provide a protective cover, reducing predation pressure. Perch (Perca fluviatilis) are commonly found in littoral zones of temperate lakes and coastal areas of the Baltic Sea. Pikeperch (Sander lucioperca) spawn in these areas, so perch is a potential predator for pikeperch larvae. We conducted laboratory experiments to test the predation of perch on pikeperch larvae at different turbidity levels (5–85 nephelometric turbidity units), densities of pikeperch larvae (2–21 individuals l⁻¹) and volumes of water (10–45l). The logistic regression showed that the probability of larvae eaten depended significantly on turbidity and volume of water in the bags, while density of larvae was not significant. However, because container size is known to affect predation, the data was divided into two groups based on water volume (10–20 and 25–45l) to reduce the effects of container size. In either group, probability of predation did not significantly depend on volume, whereas turbidity was significant in both groups, while density was significant in larger water volumes. Thus, high turbidity impaired perch predation and protected pikeperch larvae from perch predation. Because density of larvae was also a significant factor affecting predation of perch, the dispersal of pikeperch larvae from spawning areas should also increase the survival of larvae.