Functional impact of silencing the Helicoverpa armigera sex-peptide receptor on female reproductive behaviour.

Female Helicoverpa armigera sex pheromone production is under the control of pheromone biosynthesis-activating neuropeptide (PBAN). After mating, females undergo suppression of sex pheromone production and enhanced oviposition as a result of the transfer of male-derived seminal peptides. In a previous study we identified a putative H. armigera sex-peptide receptor (HeaSP-R) and demonstrated a significant up-regulation in gene expression levels of this receptor in brains and pheromone glands of mated females, thereby implicating a regulatory role for sex peptide in the reproductive behaviour of H. armigera. In the present study, we show that virgin females injected with Drosophila melanogaster SP (DrmSP), in addition to inhibition of pheromone production, also exhibited a suppression of calling behaviour and a significant reduction in the gene expression levels of the PBAN-receptor. In addition, RNA interference (RNAi) silencing of the HeaSP-R expression by 50-60% prevented DrmSP-suppression of pheromone production and calling behaviour. Moreover, mated, silenced females failed to increase their oviposition rates as is normally observed in mated females, and their behaviour did not differ from that of virgin females. However, sex pheromone production by mated, silenced females remained low, comparable to mated, normal females, thereby indicating the probable involvement of additional factors in the suppression of sex pheromone production after mating.

Female sex pheromone suppression and the fate of sex-peptide-like peptides in mated moths of Helicoverpa armigera.

Insect males produce accessory gland (MAG) factors that are transferred in the seminal fluid to females during copulation, and elicit changes in the mated female's behavior and physiology. Our previous studies showed that the injection of synthetic Drosophila melanogaster sex-peptide (DrmSP) into virgin females of the moth Helicoverpa armigera causes a significant inhibition of pheromone production. In this and other moth species, pheromone production, correlated with female receptivity, is under neuroendocrine control due to the circadian release of the neuropeptide PBAN. In this study, we show that PBAN, present in the hemolymph during the scotophase in females, is drastically reduced after mating. We also identify 4 DrmSP-like HPLC peaks (Peaks A, S1, S2, and B) in MAGs, with increasing levels of DrmSP immunoreactivity during the scotophase, when compared to their levels observed during the photophase. In H. armigera MAGs, a significant reduction in the pheromonostatic peak (Peak B) was already evident after 15 min of copulation, and depletion of an additional peak (Peak S2) was evident after complete mating. Peak A is also detected in female brains, increasing significantly 1 h after mating, at which time inhibition of pheromone biosynthesis also occurs. However, changes corresponding to the other MAG peaks were not detected in mated female tissues.

Tomato fruit size, maturity and alpha-tomatine content influence the performance of larvae of potato tuber moth Phthorimaea operculella (Lepidoptera: Gelechiidae).

Various physical and chemical properties of host plants influence insect larval performance and subsequent adult fitness. Tomato plants are relatively new hosts to the potato tuber moth, Phthorimaea operculella (Zeller), with the fruit being its preferred feeding site. However, it is unclear how the biochemical and physical properties of tomato fruits relate to potato tuber moth performance. Significant amounts of alpha-tomatine were detected in maturing green and ripening fruits of cherry (cv. Ceres) and processing (cv. Serio) types of tomatoes whereas none was detected in a fresh market variety (cv. Marglobe), at comparable stages. alpha-Tomatine is negatively and significantly correlated with development rate (head capsule size) of larvae reared in the fruits of the cherry and processing type tomatoes. Generally, survival, growth and development were significantly superior for larvae reared in the ripening fruits of the fresh market cultivar. At this stage, the fruits of this cultivar are also the largest. Based on these results it is concluded that fruit alpha-tomatine content, as well as fruit size and maturity, all affect performance of P. operculella larvae in the fruits of cultivated tomatoes.

Presence and titer of methyl palmitate in the Medfly (Ceratitis capitata) during reproductive maturation.

The relative amounts of methyl palmitate (MP) during the first 10 days post-eclosion were determined in whole-body extracts of adult female Ceratitis capitata by SIM monitoring of the 74 m/z fragment. MP peaks in receptive 3-day-old virgin females coincide with previously reported production of Juvenile Hormone (JH) by the corpus allatum (CA). Mating in the Medfly induces female non-receptivity. Indirect evidence suggests that the mevalonate pathway to sesquiterpene biosynthesis is underdeveloped in newly eclosed females. We propose that the pathway leading to synthesis of JH is markedly diverted in non-receptive virgin females to fatty acid synthesis, and partly so-in non-receptive mated females, leading to production of palmitic acid, presumably methylated thereafter. MP is depressed and remains marginal thereafter for the 7 days examined in the virgin female but goes through an apparent second cycle in the mated female. This contrasts with the consistent increase of allatal biosynthesis of MP of virgin and mated females previously reported and suggests additional control mechanisms in vivo. During the period of reduced receptivity following the first mating a second apparent peak of MP is observed. MP is a metabolic default metabolite of reproductively immature females whose putative role in reproductive physiology remains to be defined.

Methyl palmitate: a novel product of the Medfly (Ceratitis capitata) corpus allatum.

The corpus allatum (CA) of adult female Ceratitis capitata produces methyl palmitate (MP) in vitro, in addition to JHB(3) and JH III. Biosynthesized MP migrates on TLC and co-elutes from RP-18 HPLC with synthetic MP. Its identity is verified herein by GCMS. MP production is up-regulated twofold by mevastatin, an inhibitor of mevalonic acid-dependent isoprene biosynthesis. Fosmidomycin, an inhibitor of mevalonic acid-independent isoprene synthesis in graminaceous plants, up-regulates MP synthesis by about fourfold. However, it does not depress JHB(3) biosynthesis concurrently. This suggests that the initial enzyme(s) in the conversion of 1-deoxy-xylulose 5-phosphate to isoprene is presumably present in C. capitata, but is inhibited by fosmidomycin, and this inhibition diverts precursors to MP synthesis. Phytol, an acyclic diterpene, might be suppressing isoprene biosynthesis by CA, thereby resulting in a fourfold increase in the MP biosynthesis. Linolenic acid is an end-product and its presence in incubation media up-regulates MP biosynthesis by twofold, presumably due to the feedback diversion to biosynthesis of C(16:0) and its methyl ester. Biosynthesis of MP is markedly depressed after mating, while otherwise maintained at significantly higher levels in virgin females. MP biosynthesis is significantly reduced in virgin females by direct axonal control but is less consistent after mating.

Inhibition of pheromone biosynthesis in Helicoverpa armigera by pheromonostatic peptides.

Male insect accessory glands contain factors that are transferred during mating to the female, some inducing post-mating behavior, including the cessation of pheromone production, non-receptivity and the initiation of oviposition. One such factor is the Drosophila melanogaster sex-peptide (DrmSP). A pheromone suppression peptide, termed HezPSP, was identified in the moth Helicoverpa zea, isolated by HPLC and the active peak sequenced, but the activity of the synthesized peptide has not been reported to date. HezPSP bears no sequence homology to DrmSP. However, both peptides contain a disulfide bridge separated by an equal number, but dissimilar, amino acids. We herein report on the pheromonostatic activity of HezPSP partial peptides in the moth Helicoverpa armigera.

Biosynthetic maturation of the corpus allatum of the female adult medfly, Ceratitis capitata, and its putative control.

The major juvenile hormone (JH) homolog synthesized in vitro by the adult female Medfly (Ceratitis capitata) corpus allatum (CA) is JHB(3), with JH-III the minor homolog. Methyl-incorporation in vitro in post-eclosion virgin females is age-dependent. Basal activity occurs during the first four days post-eclosion and increases significantly thereafter, peaking at five days. Biosynthetic maturation of the mated female CA is delayed by one day and reduced considerably. The delayed response may be due to direct cerebral or neural inhibition. Synthetic Drosophila melanogaster sex peptide depresses JH biosynthesis by the Medfly female CA in vitro. Male-derived accessory gland peptides of the Medfly are transferred to the female during mating and a Medfly SP-analog may be responsible for down-regulation of JH synthesis by the CA in mated Medfly females. Mevinolin, an inhibitor of the mevalonate pathway, significantly reduces the biosynthesis of JHB(3), while farnesoic acid, a proximate precursor of JHIII, significantly stimulates the biosynthesis of both JHB(3) and JHIII in vitro.

Population structure of the pestiferous moth Helicoverpa armigera in the eastern Mediterranean using RAPD analysis.

The genetic structure of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), was studied in the eastern Mediterranean. Moths were sampled in six locations (five in Israel, and one in Turkey) and their genetic relationship was analysed using RAPD-PCR. Three 10-oligonucleotide primers revealed 84 presumptive polymorphic loci that were used to estimate population structure. Results reveal low level of genetic distances among Israeli and Turkish populations. The estimated values of FST and theta for the eastern Mediterranean populations were very low across all populations, indicating a high level of gene flow. Four distinct RAPD-product profile types were defined, and found in all Israeli and Turkish populations. Although no isolation by geographical distance was detected, topographical barriers may play a role in such isolation.

Common functional elements of Drosophila melanogaster seminal peptides involved in reproduction of Drosophila melanogaster and Helicoverpa armigera females.

Sex peptide (SP) and Ductus ejaculatorius peptide (Dup) 99B are synthesized in the retrogonadal complex of adult male Drosophila melanogaster, and are transferred in the male seminal fluid to the female genital tract during mating. They have been sequenced and shown to exhibit a high degree of homology in the C-terminal region. Both affect subsequent mating and oviposition by female D. melanogaster. SP also increases in vitro juvenile hormone (JH) biosynthesis in excised corpora allata (CA) of D. melanogaster and Helicoverpa armigera. We herein report that the partial C-terminal peptides SP(8-36) and SP(21-36) of D. melanogaster, and the truncated N-terminal SP(6-20) do not stimulate JH biosynthesis in vitro in CA of both species. Both of these C-terminal peptides reduce JH-III biosynthesis significantly. Dup99B, with no appreciable homology to SP in the N-terminal region, similarly lacks an effect on JH production by H. armigera CA. In contrast, the N-terminal peptides - SP(1-11) and SP(1-22) - do significantly activate JH biosynthesis of both species in vitro. We conclude that the first five N-terminal amino acid residues at the least, are essential for allatal stimulation in these disparate insect species. We have previously shown that the full-length SP(1-36) depresses pheromone biosynthesis in H. armigera in vivo and in vitro. We now show that full-length Dup99B and the C-terminal partial sequence SP(8-36) at low concentrations strongly depress (in the range of 90% inhibition) PBAN-stimulated pheromone biosynthesis of H. armigera. In addition, the N-terminal peptide SP(1-22), the shorter N-terminal peptide SP(1-11) and the truncated N-terminal SP(6-20) strongly inhibit pheromone biosynthesis at higher concentrations.

Effect of temperature and photoperiod on juvenile hormone biosynthesis and sexual maturation in the cotton bollworm, Helicoverpa armigera: implications for life history traits.

The Israeli population of the cotton bollworm, Helicoverpa armigera (Hübner), undergoes a short-day, low-temperature pupal diapause and is also suspected of being a seasonal migrant in the eastern Mediterranean region. H. armigera were reared in the laboratory under several constant temperature and photoperiodic combinations which simulate average conditions encountered in the spring, summer, early-autumn and late-autumn in Israel. Juvenile hormone (JH) biosynthesis, the onset of calling behavior, sex pheromone production and ovarian development were examined in virgin female moths subsequent to eclosion. Allatal maturation, defined as acquisition of competence to synthesize JH, was significantly delayed in moths reared under simulated spring conditions. This was probably the cause for the observed delay in ovarian development and the onset of calling behavior, and to the reduction in sex pheromone biosynthesis. The delay in female sexual maturation, commonly associated with migratory flight, is consistent with presumptive pre-reproductive migration in H. armigera.

Density-dependent physiological phase in insects.

Insects respond to crowding in a variety of ways that are usually exemplified by rapid changes in behavior and culminate in enduring long-term morphological and/or chromatic responses. A common feature of both short-term and long-term effects is that they are graded, dependent not only on density but also on the duration and on phase history of the maternal generation. Because of their exoskeletons, which are persistent for the duration of each instar and endure throughout adult life, overt changes in morphology or coloration are restricted to the molting period and shortly afterward, when cuticular hardening and pigmentation are expressed. Changes in internal organs or metabolism elicited by population density, being independent of integumental constraints, are not restricted to the molting period, but the temporal difference between internal and external responses is not of fundamental significance. Intraspecific responses to the presence of sibling insects are of apparent ecological significance and often involve directional movement and/or migration. They are mediated via the sensory system, involve signal transduction, and elicit downstream biochemical and physiological changes.

Drosophila melanogaster sex peptide stimulates juvenile hormone synthesis and depresses sex pheromone production in Helicoverpa armigera.

Previous studies demonstrate that virgin female adult Helicoverpa armigera (Lepidoptera: Noctuidae) moths exhibit calling behaviour and produce sex pheromone in scotophase from the day after emergence, and that mating turns off both of these pre-mating activities. In the fruit fly Drosophila melanogaster, a product of the male accessory glands, termed sex peptide (SP), has been identified as being responsible for suppressing female receptivity after transfer to the female genital tract during mating. Juvenile hormone (JH) production is activated in the D. melanogaster corpus allatum (CA) by SP in vitro. We herein demonstrate cross-reactivity of D. melanogaster SP in the H. armigera moth: JH production in photophase virgin female moth CA in vitro is directly activated in a dose-dependent manner by synthetic D. melanogaster SP, and concurrently inhibits pheromone biosynthesis activating neuropeptide (PBAN)-activated pheromone production by isolated pheromone glands of virgin females. Control peptides (locust adipokinetic hormone, AKH-I, and human corticotropin, ACTH) do not inhibit in vitro pheromone biosynthesis. Moreover, SP injected into virgin H. armigera females, decapitated 24 h after eclosion, or into scotophase virgin females, suppresses pheromone production. In the light of these results, we hypothesize the presumptive existence of a SP-like factor among the peptides transmitted to female H. armigera during copulation, inducing an increased level of JH production and depressing the levels of pheromone produced thereafter.

Sex-peptide activates juvenile hormone biosynthesis in the Drosophila melanogaster corpus allatum.

Mating elicits two well-defined reactions in sexually matured females of many insects: reduction of receptivity and increased oviposition. These post-mating responses have been shown to be induced by factors synthesized in the reproductive tract of the adult male and transferred in the seminal fluid to the female during copulation. One of these factors, named sex-peptide (SP), has been identified in Drosophila melanogaster. Using an in vitro radiochemical assay, we show that synthetic sex-peptide considerably activates juvenile hormone III-bisepoxide (JHB3) synthesis in corpus allatum (CA) excised from Days 3 and 4 post-eclosion virgin females. Base levels are significantly lower at emergence (Day 0) than on subsequent days, and only weak stimulation is obtained on Day 1, while none is obtained on Day 2, where maximal basal synthesis occurs. The CA of mated females cannot be stimulated further for at least 7 days, but regain responsiveness by Day 10 after mating. Synthesis of JHB3 stimulated by SP in vitro persists for at least 4 h after removal of the peptide. Development of responsiveness of the CA to SP in vitro is compared with development of the post-mating reactions of sex-peptide injected virgin females. Our results suggest that the CA is a direct target for SP in vivo and that sexual maturity is established separately for the two post-mating reactions.

Factors affecting behavioral phase transition in the desert locust,Schistocerca gregaria (Forskål) (Orthoptera: Acrididae).

The behavior of the desert locust,Schistocerca gregaria (Forskål) (Orthoptera: Acrididae), is adjusted rapidly to population density and is a phase characteristic. We used discriminant analysis to quantify the extent of phase transition from the solitary to the gregarious phase and accurately classify the phase status on the basis of two decisive behavioral parameters: nymphal activity and social interaction. Fecal extracts. examined by olfactometry, attract solitarious nymphs but do not contribute to behavioral phase transition. Neither do visual stimuli alone. Short-range olfaction of airborne volatiles slightly affects behavioral phase transition. Antennectomy abolishes density response. Cuticular lipid extract, containing presumptive contact pheromones, does not attract nymphs, but does significantly affect behavioral phase transition.

Pathway and regulation of JHIII-Bisepoxide biosynthesis in adult Drosophila melanogaster corpus allatum.

Adult female Drosophila melanogaster corpus allatum (CA) synthesize JHB3 from endogenous and exogenous precursors in vitro. We present evidence supporting the thesis that biosynthesis proceeds from precursor FA via initial epoxidation and terminal methylation on the basis of the following: (1) Methyl farnesoate is not epoxidized to JHIII or JHB3; (2) Authentic JHIII is not epoxidized to JHB3; and (3) FABE is markedly metabolized to JHB3. Cerebral allatostatic factors act at some stage subsequent to FA and this precursor is not normally rate-limiting. Additionally, neural inhibition from the brain acts at some biosynthetic step prior to FA.

The kinetics of nutrient incorporation into body tissues of gilthead seabream (Sparus aurata) females and the subsequent effects on egg composition and egg quality.

The interaction between essential dietary components and changes in tissue nutrient reserves, egg quality and egg composition, were studied from 60 d before and during the spawning of Sparus aurata broodstock. Fish were given isonitrogenous (550 g/kg dry weight) and isolipidic (100 g/kg dry weight) diets, based on protein and lipid extracts of squid meal. Diets differed in the levels of n-6 (10-30 mg/g dry weight) and n-3 (0-10 mg/g dry weight) essential fatty acids. The effects of these diets on biochemical and fatty acid composition of body tissues, and the subsequent effects on egg composition and egg viability were measured. Dietary essential fatty acids were mostly incorporated into the liver, ovaries, digestive tract and associated adipose tissues. The lipid composition of these tissues reached an equilibrium with dietary lipid composition within 15 d of feeding on any given diet. Muscle and gill cartilage tissues did not show any significant changes in their biochemical and fatty acid composition, even after 60 d feeding. Egg viability decreased significantly within 10 d of feeding the broodstock with a diet deficient in n-3 highly unsaturated fatty acids (n-3 HUFA). The levels of n-3 HUFA in both polar and neutral fractions of egg lipid were directly correlated with their levels in the broodstock diet. When the total amount of egg n-3 HUFA dropped below 17 mg/g dry weight, egg viability and larvae hatching rate decreased by 53% and 47% respectively. These results suggest that the biochemical composition of organs involved in S. aurata reproduction are highly sensitive to the nutritional value of the diet, which affects egg and larval quality rapidly.

Maturation and responsiveness to extracts of corpora allata from male Locusta migratoria containing allatotropic factors.

Corpora allata (CA) from male Locusta migratoria were tested for their ability to synthesize juvenile hormone (JH) and to respond to stimulating brain/corpora cardiaca (CC)-extracts under in vitro conditions. We found that a preincubation of the CA of both sexes at 4 degrees C for 24 h lowers their basal rate of synthesis and retains their responsiveness to allatotropic factors. Male CA can be stimulated by brain/CC extracts as well as female CA. JH biosynthesis stimulating factors are also present in male brain/CC extracts. Thus such extracts from male locusts can be used for the isolation of locust allatotropin. Furthermore male locust CA are appropriate for bioassaying such allatotropic factors.

Is There Extraovarian Synthesis of Vitellogenin in Penaeid Shrimp?

Extraovarian synthesis of vitellogenin (Vg), has been reported for several crustaceans, mainly in the subepidermal adipose tissue (SAT) or the hepatopancreas (HEP). The precise site(s) of Vg synthesis in penaeid shrimp is hitherto unknown and was investigated in a large local species Penaeus semisulcatus de Haan. Protein synthesis was determined in SAT and HEP tissue pieces incubated in vitro. Incubations were at 25{deg}C for eight hours in an oxygen enriched atmosphere, under sterile conditions in a physiological medium, containing 14C-leucine. At the end of the incubation period, tissue homogenates and medium samples were analyzed for de novo protein synthesis. Total protein synthesis was determined by trichloroacetic acid precipitation. Specific vitellin (Vt) synthesis was determined by radioimmunoprecipitation with a polyclonal Vt-specific antiserum. Characterization of other de novo synthesized proteins was carried out by fluorography from polyacrylamide gels. Subepidermal adipose tissues removed from females at all stages of ovarian development did not synthesize Vt-specific proteins, in spite of the fact that total protein synthesis levels were high. The major protein synthesized de novo in the SAT of males and females is a protein with an identical electrophoretic mobility as hemocyanin in polyacrylamide gels. In vitro protein synthesis in HEP tissues was low compared to SAT or ovary systems. Vt-specific de novo synthesized protein was identified in HEP's from early vitellogenic females, but constituted less than 15% of total protein synthesis. We have previously shown that ovarian tissues from vitellogenic females incubated in vitro exhibited high levels of protein synthesis, an average of 38% of which is Vt-specific (Browdy et al., 1990, J. Exp. Zool. 255:205-215). The calculated Vt synthesis rates in ovaries were up to 23 times higher than in HEP. We conclude that the extraovarian contribution to vitellogenesis in P. semisulcatus is low.

Regulation of juvenile hormone synthesis in wild-type and apterous mutant Drosophila.

Juvenile hormone (JH) is a major regulator of insect development and reproduction and its titer is determined largely by central nervous system regulation of JH synthesis by the corpora allata. To establish the basis for a molecular genetic dissection of the neuroendocrine system responsible for modulating JH titer, a radiochemical assay was utilized to examine JH synthesis in vitro by the isolated corpus allatum as well as the regulation of this synthesis by brain extracts of wild-type and apterous mutant Drosophila melanogaster females during reproductive maturation. JH production by glands of wild-type females increases in parallel with the progress of ovarian maturation, the major product of the adult corpus allatum being juvenile hormone 3 bis-epoxide (JHB3). Gland activity appears to be regulated by both the availability of JH precursors and the level of terminal oxidase(s) in the JH biosynthetic pathway. The brain contains an allatostatic factor, that is transmitted to the glands via nervous connections. Allatostatin production in the brain appears to be positively regulated by JHB3. Adult corpora allata from the mutants ap4 and ap56f synthesize very low levels of JH; additionally, brains of ap56f homozygotes lack allatostatic activity.

Identification of a diuretic hormone of Locusta migratoria.

We have isolated a peptide from brains and corpora cardiaca of Locusta migratoria which is immunologically related to the diuretic hormone of Manduca sexta. We determined its structure as a 46 amino acid linear peptide with 43-50% identity to the M. sexta hormone. Moreover, we showed that the new peptide functions as a diuretic hormone in L. migratoria, stimulating urine production by Malpighian tubules and elevating levels of cAMP in tubules.