Basic pattern of fluctuation in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm,Bombyx mori

General features of the changes in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm Bombyx mori were analyzed by comparing the patterns of the titer changes between different races and between silkworms reared under different environmental conditions. In common to all types of the silkworms tested, we observed low PTTH titers during the phagoperiod of the final instar, a small rise in PTTH titer on the day before wandering, two middle-sized peaks of the titer at the wandering and prepupal stages, high PTTH titers during early pupal-adult development, and a gradual titer increase shortly before adult eclosion. Increases in hemolymph PTTH titer were closely correlated with increases in ecdysteroid titers and with subsequent occurrences of morphological and behavioral changes characteristic of the initiation or progression of metamorphosis. The timing of the increase in hemolymph PTTH titer on the day of wandering was photoperiodically controlled, but that timing at the later stages seemed not to be influenced by the light-dark cycle.     

Roles of ecdysteroid and juvenile hormone in vitellogenesis in an endoparasitic wasp, Pteromalus puparum (Hymenoptera: Pteromalidae)

To elucidate the endocrine regulation of vitellogenesis in an endoparastic wasp (Pteromalus puparum), the titers of ecdysteroid and juvenile hormone (JH) from the whole bodies are measured using the method of radioimmunoassay and GC-MS, and compared with the levels of vitellogenin (Vg) mRNA in the fat bodies, hemolymph Vg and ovarian vitellin (Vt), respectively. The results show that the ecdysteroid titer and fat body Vg mRNA level have a similar dynamics tendency, and the peak titer is at adult eclosion. The titer of JH III and ovarian Vt also have a similar dynamics tendency, and the peak titer is at 48 h after eclosion. The profiles of hemolymph Vg, Vg mRNA in fat bodies and ovarian Vt, are also measured in the wasps after treated with different amounts of 20-hydroxyecdysone (20HE) or JH III in female pupa and adults. The results show that 20HE stimulates Vg synthesis in the fat bodies and its release into the hemolymph, and that JH III only accelerates Vg sequestration in the oocytes. Decapitation, which is believed to terminate synthesis of JH in insects, can not inhibit vitellogenesis and oocyte maturation in P. puparum. Furthermore, Vg gene is expressed with a lower titer of JH and depressed by a higher titer of JH III. These studies suggest that ecdysteroids play a role in Vg synthesis and believed to be the dominant hormones in regulation of vitellogenesis in P. puparum, and JHs are not the essential factors to female reproduction in this wasp.     

Stage-specific regulation of juvenile hormone biosynthesis by ecdysteroid in Bombyx mori

In the penultimate (4th) instar larvae of Bombyx mori, juvenile hormone (JH) synthesis by corpora allata (CA) fluctuates. When diet containing 20-hydroxyecdysone (20E) was fed, JH synthetic activity of the CA was first stimulated as the ecdysteroid titer increased, then suppressed slightly by the higher molting concentration of ecdysteroids (>250 ng/ml). The overall JH biosynthetic activity was modulated by the expression of JH biosynthetic enzymes in the CA: primarily JH acid O-methyltransferase (JHAMT), isopentenyl diphosphate isomerase, and farnesyl diphosphate synthase 1. After the last (5th) larval ecdysis, the artificially increased high ecdysteroid level due to the 20E diet activated JH synthesis by the CA, which required intact nervous connections with the brain. A factor(s) from the 20E-activated brain controls mainly JHAMT and HMG Co-A reductase expression to stimulate the JH synthesis. In the normal last instar larvae, the ecdysteroid titer declines so that these activation mechanisms are absent; therefore the decline of the ecdysteroid titer after the final larval ecdysis is one of the factors which induces the cessation of the JH synthesis by CA.     

Endocrine changes associated with the starvation-induced premature metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris

Under 25 degrees C and a long day photoperiod, about half of the fourth instar Psacothea hilaris larvae molt to the fifth instar on day 13 and pupate about 18 days later; the rest pupate without a further larval molt with a mean fourth instar period of 24 days. However, starvation of fourth instar larvae exceeding a threshold weight induces premature pupation, resulting in the formation of small but morphologically normal adults. To clarify the endocrine basis for this premature pupation, hemolymph juvenile hormone (JH) and ecdysteroid titers were quantified during the fed and the starved periods. Normally fed fourth instar larvae exhibited two populations with regard to JH and ecdysteroid titers, one having JH titers ranging from 1.2 to 2.1 ng/ml through to day 13, the other, similarly high titers in the early part of the instar but low titers reaching 0.1 ng/ml on day 13. One population had ecdysteroid titers with a peak of 43 ng/ml on day 10, coinciding well with the period when some larvae normally molt to the fifth instar (day 13), the other, a small peak of 14 ng/ml on day 14 and a large peak of 70 ng/ml on day 17 coinciding well with the period just before the prepupa stage. When fourth instar larvae were starved after 4 days of feeding, JH titers decreased sharply in the next 24h and never recovered, and a small but significant increase (to 21 ng/ml) in ecdysteroid titers was observed on day 6, followed by a large peak of 63 ng/ml on day 11. Altogether, these results suggest that starvation induces a rapid decline in the JH titer, and this cues the early occurrence of a small ecdysteroid peak that commits larvae to early metamorphosis.     

Effects of neuropeptides on feeding initiation in larvae of the silkworm, Bombyx mori

In insects, especially phytophagous insects, feeding behavior occurs at a regular frequency. Although a number of physiological studies have revealed various causal factors leading to feeding behavior in insects, little has been demonstrated regarding the regulatory mechanisms underlying insect feeding behavior. To confirm the presence of an endocrinological regulatory mechanism in feeding behavior, we tested the effects of several biologically active peptides on silkworm, Bombyx mori larvae feeding behaviors. To evaluate the effects of the biologically active peptides, we measured the period of latency to the first bite following sample injection into starved Bombyx larvae. Of the chemically synthesized peptides tested, myosuppressin exhibited a prolonged latency, indicating that myosuppressin is a possible inhibitory peptide in Bombyx larvae. In contrast, injections of tachykinin and short neuropeptide F, which are members of the structurally related RF-amide peptide family, had a shorter latency period, indicating that these two peptides are possible stimulatory peptides. In addition, the present study suggests that this bioassay will be advantageous for screening for peptides that regulate insect feeding behavior.     

Bombykol receptors in the silkworm moth and the fruit fly

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the “empty neuron” system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor.     

The heterodimeric ecdysteroid receptor complex in the brown shrimp Crangon crangon: EcR and RXR isoform characteristics and sensitivity towards the marine pollutant tributyltin

Decapod crustaceans are characterized by multiple ecdysteroid receptor (EcR) and retinoid-X-receptor (RXR) isoforms, which likely exhibit variant dimerization and transactivation interactions. In the brown shrimp C. crangon we cloned C-terminally truncated CrcEcR and CrcRXR isoforms and isoforms exhibiting deletions within the hinge region. For the former, in silico modeling of the CrcEcR indicated that, where the conserved helices H10 and H11 of the ligand-binding domain (LBD) are missing, an alternative C-terminal α-helix repairs the ligand-binding pocket (LBP). The truncated CrcRXR isoforms lack a major part of the LBD (H4-H12), thereby compromising ligand binding and dimerization. Through an in vitro ecdysteroid responsive reporter assay, we showed that these natural receptor variations do not impair receptor functioning but probably alter the receptor dimerization preferences. By the same in vitro assay, using full-length CrcEcR and CrcRXR, the effect of tributyltin (TBT) on ecdysteroid-induced transactivation was evaluated. The transactivation by 10 nM PonA was reduced with 64% by 20 nM TBT. In silico modeling confirmed that TBT fits in the full-length CrcRXR-LBD. Furthermore, semi-quantitative PCR indicated altered expression of CrcEcR and CrcRXR isoforms after in vivo acute exposure to TBT, especially in the ovaries.     

Isolation and expression of the retinoid X receptor from last instar nymphs and adult females of the soft tick Ornithodoros moubata (Acari: Argasidae)

Retinoid X receptors (RXR) exist broadly from invertebrates to vertebrates, and play essential roles in physiological processes of these organisms. In arthropods, RXRs form a complex with the ecdysteroid receptor (EcR) and ecdysteroids to mediate the regulation of ecdysis and reproduction. Compared to EcR, RXR and its homologue ultraspiracle (USP) are much less well understood. Therefore, we identified RXR of the soft tick Ornithodoros moubata (OmRXR) and used real-time PCR to examine the expression of OmRXR. This is the first report of RXR from a soft tick. OmRXR showed higher homology to hard tick, crustacean and vertebrate RXRs than insect RXRs and USPs. OmRXR expression was observed during molting in the last instar nymphs coinciding with EcR expression and increases in ecdysteroid titers. Tick vitellogenesis normally occurs soon after engorgement and OmRXR expression coinciding with EcR expression and ecdysteroid titers in engorged females occurred before vitellogenin (Vg) synthesis and egg maturation. The ecdysteroid/EcR/RXR complex appears to be important in the regulation of molting and vitellogenesis of soft ticks.     

The brown shrimp (Crangon crangon L.) ecdysteroid receptor complex: Cloning, structural modeling of the ligand-binding domain and functional expression in an EcR-deficient Drosophila cell line

cDNAs encoding ecdysteroid receptor (EcR) and retinoid X receptor (RXR) were cloned and sequenced from brown shrimp Crangon crangon (Crustacea: Decapoda), a common faunal species and commercially important in the North-West European coastal waters. A 3D model of the ligand-binding domain (LBD) of EcR was created and docking of ponasterone A (PonA) was simulated in silico. Finally, we report the transfection of expression plasmids for these receptors in the mutant Drosophila L57-3-11 cell line. Through an ecdysteroid responsive reporter assay we clearly prove the functionality of shrimp ecdysteroid receptor in the transfected L57-3-11 cell line. Our results indicate that the Drosophila L57-3-11 cell line and in silico LBD modeling can be used to study the function of crustacean ecdysteroid receptors and be applied to assess endocrine disrupting effects on non-target crustacean species.     

Juvenile hormone synthesis as related to egg development in neotenic reproductives of the termite Reticulitermes flavipes, with observations on urates in the fat body

The relationship between juvenile hormone (JH) synthesis and egg development, which is well documented in cockroaches, is much less studied in their close relatives, the termites. In this study of neotenic reproductives of the subterranean termite Reticulitermes flavipes, in vitro rates of juvenile hormone (JH) synthesis by corpora allata (CA) are related to vitellogenic egg development and the size of CA. The first study compared brachypterous and apterous neotenics in their first cycle of egg development and a second study compared physogastric and non-physogastric brachypterous and apterous neotenics. In both studies, rates of JH synthesis correlated with the size of CA as indicated by their length. Unlike the cockroach in which all basal oocytes are in the same stage of development, those in termites are in various stages. In brachypterous and apterous in the first cycle of egg development, CA with high rates of JH synthesis were from females with early vitellogenic basal oocytes, whereas CA with low rates of JH synthesis were from females with either pre-vitellogenic or mature basal oocytes. This pattern of JH synthesis is similar to the cycle of JH synthesis correlated with oocyte development in several cockroach species. In later oocyte maturations, CA from physogastric apterous females with ovaries containing mature, as well as growing oocytes, showed a wide range of JH production; the CA with the highest rates of JH synthesis were from females with the highest proportion of early vitellogenic oocytes suggesting that both mature and early vitellogenic oocytes interact to regulate JH synthesis. Rates of JH synthesis were related to the number of vitellogenic ovarioles. Physogastric brachypterous neotenics, compared to the other classes of neotenic females, had CA with 2- to 4-fold higher rates of JH synthesis and ovaries with 2.5- to 8-fold greater number of vitellogenic ovarioles. However, both physogastric brachypterous and apterous neotenics had more vitellogenic basal oocytes and less urate in their fat bodies than the respective non-physogastric neotenics. These results demonstrate the similarities and differences between the classes of neotenic termites and between reproductive females in cockroaches and termites.     

Effects of retinoids and juvenoids on moult and on phenoloxidase activity in the blood-sucking insect Rhodnius prolixus

Retinoic acid and insect juvenile hormone (JH) are structurally related terpenoids which are widespread in nature and are involved in many biological events such as morphogenesis, embryogenesis and cellular differentiation. Here, we investigated the effects of the retinoids 9-cis retinoic acid (9cisRA), all trans retinol (atROH), all trans retinoic acid (atRA) and the juvenoids methoprene (Met) and JH injection on moult and on phenoloxidase activity in the blood-sucking insect Rhodnius prolixus. Overall, we observed that injection of retinoids or juvenoids (120 pmols) in the hemocoel of 4th instar nymphs reduced the percentage of insects which appeared normal in morphology upon moult. Noteworthy, insects exposed to 9cisRA or JH underwent profound morphological changes upon moult, generating abnormal 5th instar nymphs and also markedly increased the death of insects during the moulting process. In addition, reduction in the percentage of insects that moult without any morphological alteration, induced by retinoids or juvenoids treatment, was negatively correlated with insects that both display abnormal moult and those that die during moult. Hemolymphatic phenoloxidase activity in adult male insects injected with 9cisRA, Met and JH were significantly reduced after a bacterial challenge. Together, these results indicate that not only juvenoids but also retinoids play an important role on morphogenesis and on immune response in R. prolixus, suggesting that the molecular mechanisms involved in these events recognize the terpenoid backbone as an important structural determinant in insects.     

Hormonal coordination of molting and female reproduction by ecdysteroids in the mole crab Emerita asiatica (Milne Edwards)

The measurement of hemolymph ecdysteroids using radioimmunoassay (RIA) indicates a biphasic increase during intermolt and premolt stages of the mole crab Emerita asiatica. The gradual rise during intermolt stage corresponds to vitellogenic activities in the ovary; whereas a distinctive premolt peak is characteristic of molting crustaceans. Injection experiments with 20-hydroxyecdysone (20E) during different molt cycle stages revealed the onset of precocious premolt changes, as determined by the epidermal retraction and setal development. Injection of 20E augmented protein synthesis in the ovary, hepatopancreas and integumentary tissues. Quantification of ecdysteroids in different developmental stages of ovary also indicated a progressive increase of ovarian ecdysteroids. Interestingly, the ovarian ecdysteroids after reaching a peak at C3 stage, start declining drastically to reach the lowest level at D(3-4) stage. This decline in the ovarian ecdysteroids is inversely related to rising hemolymph ecdysteroids during these active premolt stages. The hatching of the embryos, attached to the pleopods of the ovigerous females also occurs under a high titer of hemolymph ecdysteroids. In support, 20E injection at C3 stage crabs indicated a significant reduction in time duration of pleopodal embryonic development leading to hatching of zoea larvae. Understandably, the augmented hemolymph ecdysteroid titer helps in the synchronization of embryo hatching and the premolt changes, as occurring under the normal premolt conditions.     

PolV(PolIVb) function in RNA-directed DNA methylation requires the conserved active site and an additional plant-specific subunit

Two forms of a plant-specific RNA polymerase (Pol), PolIV(PolIVa) and PolV(PolIVb), currently defined by their respective largest subunits [NRPD1(NRPD1a) and NRPE1(NRPD1b)], have been implicated in the production and activity of 24-nt small RNAs (sRNAs) in RNA-directed DNA methylation (RdDM). Prevailing models support the view that PolIV(PolIVa) plays an upstream role in RdDM by producing the 24-nt sRNAs, whereas PolV(PolIVb) would act downstream at a structural rather than an enzymatic level to reinforce sRNA production by PolIV(PolIVa) and mediate DNA methylation. However, the composition and mechanism of action of PolIV(PolIVa)/PolV(PolIVb) remain unclear. In this work, we have identified a plant-specific PolV(PolIVb) subunit, NRPE5a, homologous to NRPB5a, a common subunit shared by PolI-III and shown here to be present in PolIV(PolIVa). Our results confirm the combinatorial diversity of PolIV(PolIVa)/PolV(PolIVb) subunit composition and indicate that these plant-specific Pols are eukaryotic-type polymerases. Moreover, we show that nrpe5a-1 mutation differentially impacts sRNAs accumulation at various PolIV(PolIVa)/PolV(PolIVb)-dependent loci, indicating a target-specific requirement for NRPE5a in the process of PolV(PolIVb)-dependent gene silencing. We then describe that the triad aspartate motif present in the catalytic center of PolV(PolIVb) is required for recapitulation of all activities associated with this Pol complex in RdDM, suggesting that RNA polymerization is important for PolV(PolIVb) to perform its regulatory functions.     

Natural variability and response interpretation of fecundity, vertebrate-like sex-steroid levels and energy status in the New Zealand mudsnail Potamopyrgus antipodarum (Gray)

Potamopyrgus antipodarum is a promising test organism that is often used in ecotoxicology, both in laboratory and field exposures. As no data are available on the physiological variation range of its life-traits and the biomarkers it uses, we studied the variation of fecundity, steroid levels and energy reserves over the course of a year in a field population. The reproductive cycle was described and showed seasonal activity during summer and autumn. Steroid levels (17β-estradiol and testosterone) varied significantly during the year and were correlated with the reproductive cycle, which suggested a potential role for sex-steroids in P. antipodarum reproduction. Energy status also showed seasonal variations. Triglycerides (TG) seemed to be the main energy lipid, whereas cholesterol appeared to be mostly used as a structural lipid. Proteins were also involved in the reproductive cycle, but only when TG were not sufficient to support the reproductive strain, similar to cholesterol. Glycogen seemed to be used as an early reserve. Threshold values under which no reproduction occurred were defined in starved snails. We proposed a range of variation in the measured parameters, allowing for a better understanding and interpretation of their levels during laboratory or in situ exposures. The data suggest that the variability of fecundity in snails has not been fully appreciated in literature.