Towards disrupting Varroa -honey bee chemosensing: A focus on a Niemann-Pick type C2 transcript.

We focused our study on the 12 recently identified putative odorant carrier proteins in the ectoparasitic mite, Varroa destructor. Here we show, via an exclusion of the chemosensory appendages (forelegs and gnathosoma) that transcripts of five of the 12 genes were significantly lower, suggesting that they are likely involved in carrying host volatiles. Specifically, three transcripts were found to be foreleg-specific while the other two transcripts were expressed in both the forelegs and gnathosoma. We focused on one of the highly expressed and foreleg-specific transcript Vd40090, which encodes a Niemann-Pick disease protein type C2 (NPC2) protein. Effects of dsRNA-mediated silencing of Vd40090 were first measured by quantifying the transcript levels of genes that encode other putative odorant carrier proteins as well as reproduction related proteins. In addition, the impact of silencing on mites behaviour and survival was tested. Silencing of Vd40090 effectively disrupted Varroa host selection, acceptance and feeding and significantly impaired the expression of genes that regulate its reproduction in brood cells, resulting in reduced reproduction and survival.

Varroa chemosensory proteins: some are conserved across Arthropoda but others are arachnid specific.

The tight synchronization between the life cycle of the obligatory parasitic mite Varroa destructor (Varroa) and its host, the honeybee, is mediated by honeybee chemical stimuli. These stimuli are mainly perceived by a pit organ located on the distal part of the mite's foreleg. In the present study, we searched for Varroa chemosensory molecular components by comparing transcriptomic and proteomic profiles between forelegs from different physiological stages, and rear legs. In general, a comparative transcriptomic analysis showed a clear separation of the expression profiles between the rear legs and the three groups of forelegs (phoretic, reproductive and tray-collected mites). Most of the differentially expressed transcripts and proteins in the mite's foreleg were previously uncharacterized. Using a conserved domain approach, we identified 45 transcripts with known chemosensory domains belonging to seven chemosensory protein families, of which 14 were significantly upregulated in the mite's forelegs when compared to rear legs. These are soluble and membrane bound proteins, including the somewhat ignored receptors of degenerin/epithelial Na+ channels and transient receptor potentials. Phylogenetic clustering and expression profiles of the putative chemosensory proteins suggest their role in chemosensation and shed light on the evolution of these proteins in Chelicerata.

Identification and gene-silencing of a putative odorant receptor transcription factor in Varroa destructor: possible role in olfaction.

The ectoparasitic mite Varroa destructor is one of the major threats to apiculture. Using a behavioural choice bioassay, we determined that phoretic mites were more successful in reaching a bee than reproductive mites, suggesting an energy trade-off between reproduction and host selection. We used both chemo-ecological and molecular strategies to identify the regulation of the olfactory machinery of Varroa and its association with reproduction. We focused on transcription regulation. Using primers designed to the conserved DNA binding region of transcription factors, we identified a gene transcript in V. destructor homologous to the pheromone receptor transcription factor (PRTF) gene of Pediculus humanus corporis. Quantitative PCR (qPCR) revealed that this PRTF-like gene transcript is expressed in the forelegs at higher levels than in the body devoid of forelegs. Subsequent comparative qPCR analysis showed that transcript expression was significantly higher in the phoretic as compared to the reproductive stage. Electrophysiological and behavioural studies revealed a reduction in the sensitivity of PRTF RNA interference-silenced mites to bee headspace, consistent with a reduction in the mites' ability to reach a host. In addition, vitellogenin expression was stimulated in PRTF-silenced mites to similar levels as found in reproductive mites. These data shed light upon the regulatory mechanism of host chemosensing in V. destructor.

Quantitative GC analysis of secondary alcohol pheromones: determination of release rate of red palm weevil, Rhynchophorus ferrugineus, pheromone from lures.

Aliphatic secondary alcohols are components of several aggregation pheromones of important beetle and weevil pests. Some of these pheromones are used frequently for the monitoring and mass trapping of the relevant insects. We encountered severe difficulties in direct GC quantitative analysis of these compounds. Therefore, we developed a simple GC analysis of secondary alcohols convening them to trifluoroacetyl derivatives and using secondary alcohol acetates as internal standards. This method was applied for the quantitative analysis of several secondary alcohols, including the aggregation pheromone components of the almond bark beetle and the red palm weevil. The release rate of the latter pheromone from commercial lures was also determined.

How species-specific is the phoretic relationship between the broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae), and its insect hosts?

Broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae), is a serious plant pest in tropical and subtropical regions. Phoretic associations between broad mite and two genera of whiteflies (Insecta: Homoptera: Aleyrodidae). namely Bemisia and Trialeurodes, have been reported from different parts of the world. Our purpose was to determine the specificity of the association between the mite and its phoretic hosts. Two host plants, potatoes and cucumbers, were used to study these relationships in the laboratory. Insects frozen for 24 h were used as potential phoretic hosts in all experiments. Attachment levels were monitored by counting the number of mites attached to each insect. All tests were conducted for 4-6 h. as attachment to Bemisia tabaci on potato shoots leveled off after 4 h, increasing only marginally after 8 h. Attachment levels to thrips (the western flower thrips, Frankliniella occidentalis) and to allate aphids (Myzus persicae) was negligible relative to the attachment to B. tabaci. Broad mite also attached to the whiteflies Dialeurodes citri, Aleyrodes singularis and Trialeurodes lauri, the latter being the least attractive species (including B. tabaci). Washing B. tabaci with pentane greatly reduced attachment. Using cucumbers as hosts did not substantially change the general trend, but attachment levels were lower. The phoretic relationship between broad mite and its insect hosts appears to be specific to whiteflies, with some whiteflies having a higher potential as phoretic hosts.

Task-related chemical analysis of labial gland volatile secretion in worker honeybees (Apis mellifera ligustica).

Chemical analyses revealed that the labial gland complex of worker honeybees possesses a series of hydrocarbons dominated by odd-numbered carbon chain alkanes along with minor amounts of alkenes and branched alkanes. Foragers contained significantly more secretion than nurse bees. Experiments with bees from colonies induced to have a division of labor independent of age revealed that the differences in the amount of secretion were task, but not age dependent.

Segregation of colony odor in the desert ant Cataglyphis niger.

There are two separate, and presumably opposing, processes that affect colony odor in the desert ant Cataglyphis niger: (1) biosynthesis and turnover of these chemicals by individual ants, and (2) homogenization of colony odor through exchange of cues. The first increases signal variability; the latter decreases it. The impact of these factors was tested by splitting colonies and monitoring the profile changes occurring in the postpharyngeal glands (PPG) and cuticular hydrocarbons. From each of two polygynous nests four daughter colonies were formed, three monogynous and one queenless. Thereafter, 10 ants from each were randomly selected each month, for three successive months, for analyses of their PPG and cuticular hydrocarbons. From two colonies we also obtained ants from a known matriline. Over time, there was a shift in hydrocarbon profiles of both the PPG and cuticular washes in each of the tested colonies. Moreover, by subjecting selected hydrocarbon constituents to a discriminant analyses based on their relative proportions, all of the daughter colonies (queenright and queenless) were distinguishable from each other and from their respective mother colonies. In each of the queenright daughter colonies, the queen profile was indiscriminable from that of the workers and often was in the center of the group. Full sisters were clearly distinguishable from their nestmates, emphasizing the genetic versus environmental processes that govern colony odor. The effect of time was always superior to the separation effect in contributing to odor segregation. Comparison of the Mahalanobis distances indicated that the shift in hydrocarbon seems to proceed along parallel lines rather than in divergence. However, there was no overt aggression between ants that originated from the different subgroups in dyadic encounters. It appears that in this species a three-month separation pe riod is not sufficient to change the hydrocarbon profile beyond the recognition threshold.

Camponotus fellah colony integration: worker individuality necessitates frequent hydrocarbon exchanges.

Our aim was to test the existence of Gestalt colony odour in Camponotus fellah. We isolated individual workers to prevent trophallaxis, allogrooming and body contact. After 20 days, the cuticular hydrocarbon profile of the isolated ants diverged from that of the parent colony. Moreover, each isolated individual had its own specific blend. This procedure showed that after about 20 days of isolation there was a turnover of the colony odour, revealing the genetically expressed hydrocarbon profile of each individual. It also showed that the cuticular hydrocarbon profile is polymorphic, and that its homogeneity within a colony is maintained by frequent exchanges of hydrocarbons between workers. Behavioural observations of resident workers, in their nest, towards nestmates reintroduced after isolation indicated that a short isolation period (3-5 days), which induced a minor change in hydrocarbon profile, provoked frequent trophallactic solicitations. These were likely to permit the isolated ants to readjust their hydrocarbon profile to that of the ants in the mother colony. Longer isolation periods (20-40 days) induced a greater change in hydrocarbon profile and made the residents intolerant towards their introduced nestmates. Therefore, our results clearly support the existence of a Gestalt colony odour in C. fellah. They also show that since individual hydrocarbon production is dynamic, workers are obliged to exchange hydrocarbons continually (mainly by trophallaxis) in order to be in the Gestalt, and properly integrate into the colony. Copyright 2000 The Association for the Study of Animal Behaviour.

Hydrocarbon site of synthesis and circulation in the desert ant Cataglyphis niger.

Chemical analyses revealed that in Cataglyphis niger both the hemolymph and the crop contain the same hydrocarbons that are found in the postpharyngeal gland (PPG). On the cuticle, on the other hand, alkanes, and in particular nonacosane, were more abundant than in the PPG. Studies of their biosynthesis in vivo, using intact ants, revealed the presence of newly synthesized hydrocarbons in both the PPG and the crop. In decapitated ants (in the absence of the PPG), however, the crop did not contain any newly synthesized hydrocarbons, indicating the PPG as the major source of crop hydrocarbons. The fat body, as demonstrated by in vitro studies, is the major tissue that biosynthesizes hydrocarbons. The PPG failed to do so, but showed good de novo biosynthesis of other lipid constituents. The large amount of hydrocarbons in the crop suggests that the alimentary canal may serve as an outlet for the overflow of PPG hydrocarbons, or as a route for the directed clearance of hydrocarbons from the PPG.These results confirm and enlarge the model proposed for hydrocarbon circulation in C. niger. They are synthesized by the fat body, released to the hemolymph and transported to the cuticle and the PPG. The PPG hydrocarbons are applied to the cuticle by self-grooming, but can also be cleared via the alimentary canal. Partial emptying of the PPG may facilitate the admixing of recognition cues that the ant may acquire from nestmates by trophallaxis. The reason for the dissimilarity in hydrocarbon composition between the PPG and the cuticle is not yet clear; it may be due to secretions from additional glands, or reflect deviant hydrocarbon transport mechanisms between the PPG and the cuticle.

Plasticity in caste-related exocrine secretion biosynthesis in the honey bee (Apis mellifera).

Plasticity of Dufour's gland secretion in the honey bee is correlated with the individual's plasticity. Queens and queenless (QL) egg-laying workers possess a bouquet of esters and hydrocarbons, whereas queenright (QR) workers produce exclusively hydrocarbons. The effects of social environment (QR vs. QL conditions) and possible physiological constraints on the gland were studied by following the biosynthesis of these classes of compounds in vivo and in vitro. Biosynthesis in vivo followed the prediction based on glandular chemistry. Queens and QL egg-laying workers, but not QR workers or QL foragers, showed incorporation of sodium acetate into both hydrocarbons and esters. In contrast, the in vitro studies revealed that, in addition to queens and QL egg-laying workers, QR nurses retained their ability to produce the queen characteristic esters. Although there was some ester production in foragers, it occurred to a lesser extent. It is possible that the glands in the older foragers undergo irreversible changes. The in vitro incubation also revealed a temporal activation of ester biosynthesis in QR workers. In these glands alcohols, corresponding to the alcohol moiety of the esters, predominated in short-term incubations but decreased as the amount of newly synthesized esters increased. In contrast, queens and QL egg-laying workers showed predominant incorporation into esters from the onset of incubation. Thus, expression within the workers' Dufour's gland is regulated. In the presence of a queen, ester production is inhibited. Once the queen is removed the physiologically unconstrained gland starts to biosynthesize the queen-specific esters after a certain lag needed for the build-up of precursors and the enzymatic machinery.

Octopamine reverses the isolation-induced increase in trophallaxis in the carpenter ant Camponotus fellah.

Social deprivation is an unusual situation for ants that normally maintain continuous contact with their nestmates. When a worker was experimentally isolated for 5 days and then reunited with a nestmate, she engaged in prolonged trophallaxis. It is suggested that trophallaxis allows her to restore a social bond with her nestmates and to re-integrate into the colony, particularly via the exchange of colony-specific hydrocarbons. Octopamine reduced trophallaxis in these workers as well as hydrocarbon transfer between nestmates, but not hydrocarbon biosynthesis. Administration of serotonin to such 5-day-isolated ants had no effect on the percentage of trophallaxis. Administration of phentolamine alone, an octopamine antagonist, had no effect, but when co-administrated with octopamine it reduced the effect of octopamine alone and restored trophallaxis to control levels. Moreover, the observed effect of octopamine was not due to a non-specific effect on locomotor activity. Therefore, we hypothesise that octopamine mediates behaviour patterns linked to social bonding, such as trophallaxis. On the basis of an analogy with the role of norepinephrine in vertebrates, we suggest that the levels of octopamine in the brain of socially deprived ants may decrease, together with a concomitant increase in their urge to perform trophallaxis and to experience social contacts. Octopamine administration may reduce this social deprivation effect, and octopamine could therefore be regarded as being partly responsible for the social cohesion between nestmates in ant colonies.

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.

Spatial and temporal distribution of pheromone biosynthesis-activating neuropeptide in Helicoverpa (Heliothis) armigera using RIA and in vitro bioassay.

A [3H]-PBAN (pheromone biosynthesis-activating neuropeptide) analog was synthesized, and binding of the radioligand to a specific PBAN-antiserum was achieved. The inhibition of binding of the radioligand by unlabeled PBAN, several PBAN analogs, and other competitors was studied and a specific radio-immunoassay was developed. Using this radioimmunoassay we found PBAN-like immunoreactivity in methanol extracts of hemolymph and neural tissues from females. Higher levels of PBAN-like immunoreactivity in extracts of brain-suboesophageal ganglion complexes, corpora cardiaca, thoracic ganglia, and abdominal ganglia were observed during the 4-5th h scotophase when compared to the PBAN-like immunoactivity levels during the 6-11th h photophase. On the other hand, the concentrations of PBAN-like immunoreactivity, in the terminal abdominal ganglion were higher during the photophase relative to minimal levels observed during the scotophase, indicating an accumulation before the onset of pheromone production. These differences in concentrations of PBAN were also reflected in the stimulation of in vitro pheromone glands, whereby significant stimulations were obtained by scotophase and photophase brain extracts, scotophase thoracic ganglia extracts, and photophase terminal abdominal ganglia extracts. No detectable levels of PBAN were found in hemolymph extracts during the sampling periods.

Cyclic AMP mediation of the hormonal stimulation of 14C-acetate incorporation by Heliothis armigera pheromone glands in vitro.

Evidence is presented for hormone-controlled adenosine 3',5'-cyclic monophosphate (cAMP)-mediated incorporation of 14C-acetate by in vitro pheromone gland preparations of Heliothis armigera. Basal intracellular cAMP concentrations of in vitro pheromone glands increase 28-fold in the presence of methanolic brain-complex extracts after 5 min of incubation. This intracellular cAMP increase shows a dose-dependent response. Studies on isolated pheromone gland preparations show that 8-bromo-cAMP added to the incubation media stimulates acetate incorporation thereby mimicking the hormonal response. This effect was duplicated by forskolin and isobutyl-methylxanthine. In addition, forskolin exhibits a dose-dependent stimulatory effect on the incorporation of 14C-acetate by such isolated in vitro pheromone glands. High-pressure liquid chromatographic analysis of the radioactive products, extracted by hexane from pheromone glands and media, revealed a stimulation by forskolin of a product exhibiting the same retention time as (Z)-11-hexadecenal, the main pheromone component of H. armigera. In addition, thin-layer chromatographic analysis revealed co-elution with (Z)-11-hexadecenal on stimulation by 8-bromo-cAMP.

Influence of diel rhythm and brain hormone on pheromone production in two lepidopteran species.

Heliothis armigera andSpodoptera littoralis females produced sex pheromone during their scotophase. Maximum levels of pheromone in Heliothisarmigera were extracted from 2- to 3-day-old females during their 4.5-hr scotophase. On ligation between the head and the thorax of both species, normal pheromone production was inhibited. Homogenates of brains (consisting of brains, subesophageal ganglia, corpora cardiaca, and corpora allata) from both species induced a normal level of pheromone production by ligated females. Moreover, inS. littoralis, female brain hormone induced the production of the two components (Z)-9- and (Z, E)-9,11-tetradecenyl acetate tested by gas chromatography. Partial separation of brain homogenates, using low pressure C18-silica reversed-phase columns, produced a stable and active hormone source that gave a dose-dependent response. This hormone was found in brain extracts of both male and femaleH. armigera andS. littoralis females. In addition,S. littoralis induced pheromone production byH. armigera ligated females.