Male sexual enhancement after methoprene treatment in Anastrepha fraterculus (Diptera: Tephritidae): A sustained response that does not fade away after sexual maturation.

The juvenile hormone (JH) of insects triggers physiological changes related to reproduction in adults of both sexes. Methoprene is a sesquiterpene with some effects that are analogous to those of JH. Treatments with methoprene accelerate sexual maturation in males of the South American fruit fly Anastrepha fraterculus, giving young males a mating advantage over non-treated males of the same age. Here, we evaluated the effects of methoprene treatment on A. fraterculus males after the sexual maturation phase and tested whether this compound provides a long-term mating advantage. Moreover, we took the first step to unravel the mechanisms that underlie male sexual enhancement. We treated males 1day or 8days after adult emergence and compared mate choice between recently matured (young) females and females that had been mature for ca. 10days (aged females). We also addressed methoprene treatment effects on male sexual signalling. We found that methoprene treatment enhanced male sexual competitiveness even after the sexual maturation phase, and the effect did not decrease until males were older than 20days. However, when methoprene treatment was carried out close to sexual maturity, the mating enhancement was no longer observed, suggesting a non-immediate effect and excluding the possibility that methoprene acts as a pheromonal compound. Young and aged females tended to mate more frequently with treated-males. This might indicate that in a context of sexual selection, the potential benefits associated with reproductive success would be similar for females of both ages. Treated males released larger amounts of pheromonal compounds than non-treated males, but their courtship behaviour was not altered to the same extent, suggesting that methoprene treatment may accelerate differently the components of male courtship. We discuss potential benefits of using methoprene to increase the efficiency of the sterile insect technique, which is an environmentally safe method to control this important South American fruit pest.

Three Halloween genes from the Varroa mite, Varroa destructor (Anderson & Trueman) and their expression during reproduction.

The ecdysteroid biosynthetic pathway involves sequential enzymatic hydroxylations by a group of enzymes collectively known as Halloween gene proteins. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), were identified in varroa mites and sequenced. Phylogenetic analyses of predicted amino acid sequences for Halloween orthologues showed that the acarine orthologues were distantly associated with insect and crustacean clades indicating that acarine genes had more ancestral characters. The lack of orthologues or pseudogenes for remaining genes suggests these pathway elements had not evolved in ancestral arthropods. Vdspo transcript levels were highest in gut tissues, while Vddib transcript levels were highest in ovary-lyrate organs. In contrast, Vdshd transcript levels were lower overall but present in both gut and ovary-lyrate organs. All three transcripts were present in eggs removed from gravid female mites. A brood cell invasion assay was developed for acquiring synchronously staged mites. Mites within 4 h of entering a brood cell had transcript levels of all three that were not significantly different from mites on adult bees. These analyses suggest that varroa mites may be capable of modifying 7-dehydro-cholesterol precursor and hydroxylations of other steroid precursors, but whether the mites directly produce ecdysteroid precursors and products remains undetermined.

Methoprene application and diet protein supplementation to male melon fly, Bactrocera cucurbitae, modifies female remating behavior.

Methoprene (an analogue of juvenile hormone) application and feeding on a protein diet is known to enhance male melon fly, Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), mating success. In this study, we investigated the effect of these treatments on male B. cucurbitae's ability to inhibit female remating. While 14-d-old females were fed on protein diet, 6-d-old males were exposed to one of the following treatments: (i) topical application of methoprene and fed on a protein diet; (ii) no methoprene but fed on a protein diet; (iii) methoprene and sugar-fed only; and (iv) sugar-fed, 14-d-old males acted as controls. Treatments had no effect on a male's ability to depress the female remating receptivity in comparison to the control. Females mated with protein-deprived males showed higher remating receptivity than females first mated with protein-fed males. Methoprene and protein diet interaction had a positive effect on male mating success during the first and second mating of females. Significantly more females first mated with sugar-fed males remated with protein-fed males and females first mated with methoprene treated and protein-fed males were more likely to remate with similarly treated males. Females mating latency (time to start mating) was significantly shorter with protein-fed males, and mating duration was significantly longer with protein-fed males compared with protein-deprived males. These results are discussed in the context of methoprene and/or dietary protein as prerelease treatment of sterile males in area-wide control of melon fly integrating the sterile insect technique (SIT).

Genomic organization and reproductive regulation of a large lipid transfer protein in the varroa mite, Varroa destructor (Anderson & Trueman).

The complete genomic region and corresponding transcript of the most abundant protein in phoretic varroa mites, Varroa destructor (Anderson & Trueman), were sequenced and have homology with acarine hemelipoglycoproteins and the large lipid transfer protein (LLTP) super family. The genomic sequence of VdLLTP included 14 introns and the mature transcript coded for a predicted polypeptide of 1575 amino acid residues. VdLLTP shared a minimum of 25% sequence identity with acarine LLTPs. Phylogenetic assessment showed VdLLTP was most closely related to Metaseiulus occidentalis vitellogenin and LLTP proteins of ticks; however, no heme binding by VdLLTP was detected. Analysis of lipids associated with VdLLTP showed that it was a carrier for free and esterified C12 -C22 fatty acids from triglycerides, diacylglycerides and monoacylglycerides. Additionally, cholesterol and ß-sitosterol were found as cholesterol esters linked to common fatty acids. Transcript levels of VdLLTP were 42 and 310 times higher in phoretic female mites when compared with males and quiescent deutonymphs, respectively. Coincident with initiation of the reproductive phase, VdLLTP transcript levels declined to a third of those in phoretic female mites. VdLLTP functions as an important lipid transporter and should provide a significant RNA interference target for assessing the control of varroa mites.

Variable induction of vitellogenin genes in the varroa mite, Varroa destructor (Anderson & Trueman), by the honeybee, Apis mellifera L, host and its environment.

Transcript levels of vitellogenins (Vgs) in the varroa mite, Varroa destructor (Anderson & Trueman), were variably induced by interactions between the developing honeybee, Apis mellifera L, as a food source and the capped honeybee cell environment. Transcripts for two Vgs of varroa mites were sequenced and putative Vg protein products characterized. Sequence analysis of VdVg1 and VdVg2 proteins showed that each had greater similarity with Vg1 and Vg2 proteins from ticks, respectively, than between themselves and were grouped separately by phylogenetic analyses. This suggests there was a duplication of the ancestral acarine Vg gene prior to the divergence of the mites and ticks. Low levels of transcript were detected in immature mites, males and phoretic females. Following cell invasion by phoretic females, VdVg1 and VdVg2 transcript levels were up-regulated after cell capping to a maximum at the time of partial cocoon formation by the honeybee. During oviposition the two transcripts were differentially expressed with higher levels of VdVg2 being observed. A bioassay based on assessing the transcript levels was established. Increases in VdVg1 and VdVg2 transcripts were induced experimentally in phoretic females when they were placed inside a cell containing an early metamorphosing last instar bee but not when exposed to the metamorphosing bee alone. The variable response of Vg expression to the food source as well as environmental cues within the capped cell demonstrates that perturbation of host-parasite interactions may provide avenues to disrupt the reproductive cycle of the varroa mites and prevent varroasis.

Precocious sexual signalling and mating in Anastrepha fraterculus (Diptera: Tephritidae) sterile males achieved through juvenile hormone treatment and protein supplements.

Sexual maturation of Anastrepha fraterculus is a long process. Methoprene (a mimic of juvenile hormone) considerably reduces the time for sexual maturation in males. However, in other Anastrepha species, this effect depends on protein intake at the adult stage. Here, we evaluated the mating competitiveness of sterile laboratory males and females that were treated with methoprene (either the pupal or adult stage) and were kept under different regimes of adult food, which varied in the protein source and the sugar:protein ratio. Experiments were carried out under semi-natural conditions, where laboratory flies competed over copulations with sexually mature wild flies. Sterile, methoprene-treated males that reached sexual maturity earlier (six days old), displayed the same lekking behaviour, attractiveness to females and mating competitiveness as mature wild males. This effect depended on protein intake. Diets containing sugar and hydrolyzed yeast allowed sterile males to compete with wild males (even at a low concentration of protein), while brewer´s yeast failed to do so even at a higher concentration. Sugar only fed males were unable to achieve significant numbers of copulations. Methoprene did not increase the readiness to mate of six-day-old sterile females. Long pre-copulatory periods create an additional cost to the management of fruit fly pests through the sterile insect technique (SIT). Our findings suggest that methoprene treatment will increase SIT effectiveness against A. fraterculus when coupled with a diet fortified with protein. Additionally, methoprene acts as a physiological sexing method, allowing the release of mature males and immature females and hence increasing SIT efficiency.

Evaluating light attraction to increase trap efficiency for Tribolium castaneum (Coleoptera: Tenebrionidae).

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major coleopteran pest in flour mills and storage facilities. An aggregation pheromone has been identified for this pest; however, the pheromone is of limited value for population monitoring. To develop more efficient methods to monitor this pest, experiments were conducted to determine whether light functioned as an attractant for the red flour beetle. Light-emitting diodes (LEDs) of various wavelengths were examined as light sources because they produce bright, narrow light spectra. A comparison of responses to light spectra across the visible and UV regions of the electromagnetic spectrum indicated that the beetle was most attracted to near UV LED at a 390 nm dominant wavelength. The use of LEDs in competitive laboratory experiments resulted in a 20% capture of released beetles, compared with a 1% capture with the aggregation pheromone alone. Even more beetles were captured with a combination of LEDs and commercially available chemical lures in traps. LEDs can easily be added onto existing trap designs or new traps can be designed to take full advantage of positive phototaxis.

Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon fly Bactrocera cucurbitae: effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast.

The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments known to enhance mating success in the male melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), supporting their use in mass rearing protocols for sterile males in the context of sterile insect technique (SIT) programmes. The objective of the present laboratory study was to investigate the effect of methoprene application and diet supplementation with hydrolyzed yeast (protein) on the turnover of body lipids and protein to confirm the feasibility of their application in melon fly SIT mass-rearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males were exposed to one of the following treatments: (1) topical application of methoprene and access to diet including protein (M+P+); (2) only diet including protein (M-P+); (3) only methoprene (M+P-) and (4) untreated, only sugar-fed, control males (M-P-). Total body carbon (TBC) and total body nitrogen (TBN) of flies were measured at regular intervals from emergence to 35 days of age for each of the different treatments. Nitrogen assimilation and turnover in the flies were measured using stable isotope ((15)N) dilution techniques. Hydrolyzed yeast incorporation into the diet significantly increased male body weight, TBC and TBN as compared to sugar-fed males. Females had significantly higher body weight, TBC and TBN as compared to all males. TBC and TBN showed age-dependent changes, increasing until the age of sexual maturity and decreasing afterwards in both sexes. Methoprene treatment did not significantly affect TBC or TBN. The progressive increase with age of TBC suggests that lipogenesis occurs in adult male B. cucurbitae, as is the case in other tephritids. Stable isotope dilution was shown to be an effective method for determining N uptake in B. cucurbitae. This technique was used to show that sugar-fed males rely solely on larval N reserves and that the N uptake rate in males with access to diet including hydrolyzed yeast was higher shortly after emergence and then stabilized. The implications of the results for SIT applications are discussed.

Identification of methyl farnesoate from in vitro culture of the retrocerebral complex of adult females of the moth, Heliothis virescens (Lepidoptera: Noctuidae) and its conversion to juvenile hormone III.

Gas chromatographic-mass spectral analysis of extracts obtained from in vitro culture of isolated retrocerebral complexes obtained from adult females of the moth Heliothis virescens resulted in identification of methyl farnesoate as well as juvenile hormone III (JH III) but not JH III acid. Inhibition of JH biosynthesis by incubation of tissue in synthetic Manduca sexta allatostatin (Manse-AST, pGlu-Val-Arg-Phe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-COOH) reduced production of these chemicals to negligible levels. However, incubation of tissue in the presence of Manse-AST plus farnesol resulted in production of significant amounts of both methyl farnesoate and JH III. Tissue incubated in the presence of Manse-AST plus methyl farnesoate produced only JH III. The results indicated that methyl farnesoate is naturally produced by the corpora allata of adult females of Heliothis virescens. However, tissue incubated in the presence of Manse-AST plus JH III acid also produced JH III in amounts equivalent to that produced by tissue incubated with methyl farnesoate. Thus, both methyl farnesoate and JH III acid could serve as a precursor for biosynthesis of JH III.

Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta.

Retrocerebral complexes (RCs) were isolated from adult females of the moths Heliothis virescens and Manduca sexta. Different homologs of juvenile hormone (JH) produced by the isolated RCs were identified and amounts measured by capillary gas chromatography-chemical ionization (isobutane)-mass spectroscopy. Only JH I, II and III were identified. Incubation of RCs from both species in media containing acetate, but no propionate, induced production of approximately equal amounts of JH II and JH III, but the amount of JH I present was very low in all samples. Incubation of RCs with synthetic Manduca sexta allatotropin stimulated significant increases in production of all three homologs but increases in JH I and JH II were greater than those for JH III. The effect of allatotropin was mimicked by addition of propionate to the medium, which indicated that allatotropin increased supply of acetyl- and propionyl-CoA precursors. Incubation of tissue from H. virescens females during the first 24 h after eclosion with synthetic Manduca sexta allatostatin did not reduce production of JH. However, incubation of tissue from 3-day-old females with allatostatin significantly reduced production of JH. Similarly, incubation of tissue from H. virescens females during the first 24 h after eclosion with both allatotropin and allatostatin did not increase JH over the amount present in extracts from tissue incubated without the neuropeptides, indicating that allatostatin negated the action of allatotropin. Incubation of tissue from H. virescens females with allatostatin plus farnesol or JH III acid resulted in significant production of JH III, but neither JH I nor JH II was detected. These findings indicated that allatostatin acts prior to formation of the sesquiterpene alcohol precursors of JH.

Identification and action of juvenile hormone III from sexually mature alate females of the red imported fire ant, Solenopsis invicta.

Analysis of extracts of hemolymph obtained from sexually mature alate females of Solenopsis invicta from monogyne colonies resulted in identification of juvenile hormone III (JH III). The average amount of JH III was 0.32+/-0.04 pmol/&mgr;molof hemolymph. Topical application of 0.038 pmol of JH III was sufficient to stimulate alates to shed their wings in the presence of the queen. The time in which alates were induced to dealate decreased linearly with increasing concentrations of JH III from 0.038 to 3.8 pmol. However, higher JH III concentrations deviated from linearity and did not reach dealation times comparable with those that occur after mating flights. Thus, it appears that the mechanism of dealation that occurs when female alates are out of the influence of their queen is different from the one associated with mating flights. Application of 0.42 &mgr;mol of precocene II inhibited dealation of alates in queenless colonies. However, this inhibition was reversed after applying 38 pmol JH III to precocene-treated alates. The sizes of corpora allata (CA) from sexuals treated with JH III did not differ from those of controls. However, the sizes of CA were reduced in alates treated with precocene II. The results indicated that JH was important to dealation.

Enzymatic decomposition of elicitors of plant volatiles in Heliothis virescens and Helicoverpa zea.

Feeding by larvae of Heliothis virescens induces cotton, corn and tobacco plants to release blends of volatile organic compounds that differ in constituent proportions from blends released when Helicoverpa zea larvae feed on the same plant species. The same elicitors (and analogs) of plant biosynthesis and release of volatiles, originally identified in oral secretions of Spodoptera exigua larvae, were also found in oral secretions of H. virescens and H. zea. However, relative amounts of these compounds, particularly N-(17-hydroxylinolenoyl)-L-glutamine (volicitin), 17-hydroxylinolenic acid, and N-linolenoyl-L-glutamine, varied among batches of oral secretions, more so in H. virescens than in H. zea. This variation was due to cleavage of the amide bond of the fatty acid-amino acid conjugates by an enzyme, or enzymes, originating in the midgut. The enzymatic activity in guts of H. virescens was significantly greater than that found in guts of H. zea. Furthermore, H. zea frass contains N-linolenoyl-L-glutamine in more than 0.1% wet weight, while this conjugate comprises only 0.003% wet weight in H. virescens frass. These results indicated that physiological differences between these two species affect the proportions of volicitin and its analogs in the caterpillars. Whether this causes different proportions of volatiles to be released by plants damaged by each caterpillar species is yet to be determined.