Modulatory effects of bombyxin on ecdysteroidogenesis in Bombyx mori prothoracic glands

In the present study, we investigated the modulatory effects of ecdysteroidogenesis of prothoracic glands (PGs) by bombyxin, an endogenous insulin-like peptide in the silkworm, Bombyx mori. The results showed that bombyxin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. Moreover, the injection of bombyxin into day 4-last instar larvae increased ecdysteroidogenesis 24 h after the injection, indicating its possible in vivo function. Phosphorylation of the insulin receptor and Akt, and the target of rapamycin (TOR) signaling were stimulated by bombyxin, and stimulation of Akt phosphorylation and TOR signaling appeared to be dependent on phosphatidylinositol 3-kinase (PI3K). Bombyxin inhibited the phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK), and the inhibition appeared to be PI3K-independent. Bombyxin-stimulated ecdysteroidogenesis was blocked by either an inhibitor of PI3K (LY294002) or a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR), indicating involvement of the PI3K/Akt and AMPK signaling pathway. Bombyxin did not stimulate extracellular signal-regulated kinase (ERK) signaling of PGs. Bombyxin, but not prothoracicotropic hormone (PTTH) stimulated cell viability of PGs. In addition, bombyxin treatment also affected mRNA expression levels of insulin receptor, Akt, AMPKα, -β, and -γ in time-dependent manners. These results suggest that bombyxin modulates ecdysteroidogenesis in B. mori PGs during development.     

Prothoracicotropic hormone of Rhodnius prolixus: partial characterization and rhythmic release of neuropeptides related to Bombyx PTTH and bombyxin

Summary

The brain-retrocerebral complex (br-complex) of Rhodnius prolixus was found both to contain and release neuropeptides related to Bombyx mori PTTH and bombyxin. A > 10 kDa peptide fraction obtained from extracts and incubation media of br-complex exhibited high steroidogenic activity on Rhodnius prothoracic glands and reacted with a Bombyx PTTH antibody on dot blots. The release from the Br-complex of this immunoreactive peptide fraction showed a daily rhythm: high release during the night and little on no release during the day. On Western blots, a single 68 kDa peptide in the > 10 kDa peptide fraction was recognized by the Bombyx PTTH antibody and was also released rhythmically during a day. This peptide was reduced to a doublet of about 17 kDa that retained immunoreactivity. Double immunoprecipitation of the > 10 kDa peptide fraction from brain media using the Bombyx PTTH antibody and agarose-bound secondary antibody removed the steroidogenic activity in this fraction; it also removed the 68 kDa peptide on Western blots. A bombyxin antiserum recognized a 3–5 kDa peptide in a <10 kDa peptide fraction; this peptide fraction was also released with a daily rhythm but possessed weak steroidogenic activity. The natural PTTH of Rhodnius, therefore, appears to be a 68 kDa peptide, possibly composed of several 17 kDa subunits, that is related to Bombyx PTTH.     

Regulation of capacitative Ca2+ entry by prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca2+ influx in Fura-2/AM loaded steroidogenic cells (prothoracic glands; PGs) of the silkworm, Bombyx mori showed that application of the neuropeptide prothoracicotropic hormone (PTTH) can increase the intracellular [Ca2+]i. This PTTH-mediated Ca2+ influx in PG cells had kinetic patterns and pharmacological characteristics similar to those induced by thapsigargin. Namely, it produced increases in intracellular Ca2+ levels only in the presence of extracellular Ca2+, it was blocked by Gd3+ and 2-Aminoethoxydiphenylborate (2-APB), and it was unaffected by several toxins or compounds that block voltage-activated Ca2+ channels. Moreover, the PTTH-stimulated increase of Ca2+ levels was eliminated in the presence of heparin (an IP3 receptor blocker), and by TMB-8 which also blocked any PTTH-dependent increase of ecdysteroid secretion. The PTTH-mediated increase of Ca2+ levels was not affected by the non-hydrolysable GDP analogue, GDPbetaS, an indication that a G protein is not downstream of the PTTH receptor. These results argue strongly in favor of gating by the PTTH receptor of capacitative Ca2+ entry (CCE) channels (or store-operated Ca2+ channels (SOCs)) by a mechanism that does not involve any G proteins but requires the presence of functional IP3 receptors. Because the ability of PTTH to stimulate the [Ca2+]i levels of PG cells was completely mimicked by thapsigargin and exhibited a pharmacological profile similar to CCE mechanisms, we believe that PTTH directly regulates a CCE pathway in PG cells thereby activating a plethora of downstream regulators responsible for ecdysteroid secretion by the PGs of Bombyx mori.     

Prothoracicotropic hormone induces tyrosine phosphorylation in prothoracic glands of the silkworm,Bombyx mori

In the present study, we investigated the tyrosine phosphorylation of Bombyx mori prothoracic glands using phosphotyrosine‐specific antibodies and Western blot analysis. Results showed that prothoracicotropic hormone (PTTH) stimulates a rapid increase in tyrosine phosphorylation of at least 2 proteins in prothoracic glands, one of which was identified as extracellular signal‐regulated kinase (ERK). The phosphorylation of another 120‐kDa protein showed dose‐ and time‐dependent stimulation by PTTH in vitro. In vitro activation of tyrosine phosphorylation was also verified by in vivo experiments: injection of PTTH into day‐6 last‐instar larvae greatly increased tyrosine phosphorylation. Treatment of prothoracic glands with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, also resulted in tyrosine phosphorylation of several proteins and increased ecdysteroidogenesis. The PTTH‐stimulated phosphorylation of the 120‐kDa protein was markedly attenuated by genistein, a broad‐spectrum tyrosine kinase inhibitor, but not by HNMPA‐(AM)₃, a specific inhibitor of insulin receptor tyrosine kinase. PP2, a more‐selective inhibitor of the Src‐family tyrosine kinases, partially inhibited PTTH‐stimulated tyrosine phosphorylation, but not ecdysteroidogenesis. This result implies the possibility that in addition to ERK, the phosphorylation of the 120‐kDa protein, which is not Src‐family tyrosine kinase, is likely also involved in PTTH‐stimulated ecdysteroidogenesis in B. mori. © 2010 Wiley Periodicals, Inc.     

Isolation and purification of the diuretic hormone from Rhodnius prolixus

Extracts of Rhodnius prolixus mesothoracic ganglionic masses were subjected to column chromatographic separation procedures. The eluates were assayed biologically for diuretic hormone activity.Gel filtration through columns of polyacrylamide gel was found to separate soluble diuretic activity into two distinct zones. Poor recovery was obtained with respect to the activity of the crude extract. No diuretic activity was found to co-chromatograph with a sample of 5-hydroxytryptamine. When samples of haemolymph from freshly fed Rhodnius were chromatographed under similar conditions only the second zone (low molecular weight) was found to have diuretic activity. It is this low molecular weight zone which would more reasonably qualify as the physiologically active diuretic hormone. Likewise, the diuretic activity released from isolated mesothoracic ganglionic masses by K⁺-rich Ringer's solution showed only low molecular weight activity after chromatography.     

Involvement of calcium, inositol-1,4,5 trisphosphate and diacylglycerol in the prothoracicotropic hormone-stimulated ecdysteroid synthesis and secretion in the prothoracic glands of Bombyx mori

The objective of this study was to determine which intracellular second messenger systems are activated by prothoracicotropic hormone in the prothoracic glands (PGs) of Bombyx mori. Recombinant prothoracicotropic hormone (rPTTH) could stimulate ecdysteroid synthesis and secretion from day 6 PGs of the 5th instar of Bombyx mori within 30 min of in vitro incubation. However, rPTTH did not stimulate any increases in the glandular content of inositol 1,4,5-trisphosphate and cAMP during this short incubation period. Extracellular Ca2+ influenced the basal and rPTTH-stimulated ecdysteroid synthesis and release in a dose-dependent manner. The L-type Ca2+ channel antagonist, nitrendipine, inhibited the rPTTH-stimulated ecdysteroid synthesis and secretion (IC50-28 microM). The phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50-19 microM). The protein kinase C inhibitor, chelerythrine chloride, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50-14 microM). The protein kinase C activator, phorbol-12-myristate 13-acetate (PMA), could stimulate basal ecdysteroid synthesis and secretion (EC50-1 microM) and its inactive alpha-isomer (4 alpha-PMA) was ineffective. The combined results suggest that the PTTH-stimulated ecdysteroid synthesis and release in the PGs of Bombyx is dependent on extracellular Ca2+ and the bifurcating second messenger signalling cascade of inositol 1,4,5-triphosphate and diacylglycerol.     

Ring gland and prothoracic gland sensitivity to interspecific prothoracicotropic hormone extracts

Summary Using the techniques of intraspecific in vitro activation of prothoracic glands and ring glands by serial dilutions of prothoracicotropic hormone (PTTH) extracts from pupalManduca sexta (Lepidoptera) and larvalSarcophaga bullata (Diptera), a dose-response of activation was observed for both species. In both species maximum activation was at 0.5 brain equivalents while the number of brain equivalents necessary for half maximal stimulation (ED₅₀) was 0.20 forManduca and 0.15 forSarcophaga. When prothoracic glands or ring glands were challenged with interspecific PTTH extracts from a stage different from that of the gland donor, no dose-response of gland activation was observed. However, whenM. sexta larval prothoracic glands were challenged byS. bullata larval PTTH extract, activation was observed. The dose-response profile fell midway between the dose-response curves obtained for the intraspecific assays. Thus, PTTH extract from one insect has the ability to activate the prothoracic glands of an insect representing another order.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

S6 phosphorylation results from prothoracicotropic hormone stimulation of insect prothoracic glands: A role for S6 kinase

The insect prothoracic glands are the source of steroidal molting hormone precursors and the glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH). Previous work from this laboratory revealed that PTTH acts via a cascade including Ca²⁺/calmodulin activation of adenylate cyclase, protein kinase A, and the subsequent phosphorylation of a 34 kDa protein (p34) hypothesized, but not proven, to be the 56 protein of the 40S ribosomal subunit. The jmmunosuppressive macrolide, rapamycin, is a potent inhibitor of cell proliferation, a signal transduction blocker, and also prevents ribosomal S6 phosphorylation in mammalian systems. We demonstrate here that rapamycin inhibited PTTH-stimulated ecdysteroidogenesis in vitro by the prothoracic glands of the tobacco hornworm, Manduca sexta, with half-maximal inhibition at a concentration of about 5 nM. At concentrations above 5 nM, there was a 75% inhibition of ecdysteroid biosynthesis. Similar results, were observed with the calcium ionophore (A23187), a known stimulator of ecdysteroidogenesis. Most importantly, the inhibition of ecdysteroid biosynthesis was accompanied by the specific inhibition of the phosphorylation of p34, indicating that p34 indeed is ribosomal protein S6. In vivo assays revealed that injection of rapamycin into day 6 fifth instar larvae resulted in a decreased hemolymph ecdysteroid titer and a dose-dependent delay in molting and metamor-phosis. When S6 kinase (S6K) activity was examined using rapamycin-treated prothoracic glands as the enzyme source and a synthetic peptide (S6-21) or a 40S ribosomal subunit fraction from Manduca tissues as substrate, the date revealed that rapamycin inhibited S6K activity. The composite data suggest that rapamycin inhibits a signal transduction element leading to p34 phosphorylation that is necessary for PTTH-stimulated ecdysteroidogenesis in this insect endocrine gland, and lend further support to the concept that p34 is S6. © 1994 Wiley-Liss, Inc.     

The brain neurosecretory cells of the moth Samia cynthia ricini: Immunohistochemical localization and developmental changes of the Samia homologues of the Bombyx prothoracicotropic hormone and bombyxin

We produced mouse antisera against synthetic peptides corresponding to the sequences of the Samia cynthia ricini homologues of the Bombyx mori PTTH and bombyxin. Immunohistochemical analyses of the Samia cephalic neuroendocrine system using these antisera were performed to identify the neurosecretory cells (NSC) containing the PTTH and bombyxin homologues and to examine the developmental changes in their amounts in the NSC. The results show that the PTTH and bombyxin homologues are produced by two pairs of dorsolateral and 16 pairs of dorsomedial NSC of Samia brain, respectively, and both are transported to, and released from, the corpora allata. No clear-cut correlation was found between the fluctuation in the amount of immunoreactive substances in the brain NSC and the endocrinologically anticipated timings of PTTH secretion. From Samia brain extract, two forms of PTTH activity (∼30 kDa and ∼5 kDa) were resolved through Sephadex gel filtration. The ∼30 kDa and ∼5 kDa PTTH seem to represent the PTTH and bombyxin homologues, respectively. We discuss that the ∼30 kDa PTTH homologue is the true PTTH of Samia .     

In vitro and in vivo stimulation of extracellular signal-regulated kinase (ERK) by the prothoracicotropic hormone in prothoracic gland cells and its developmental regulation in the silkworm, Bombyx mori

In this study, we investigated activation of the extracellular signal-regulated kinase (ERK) by the prothoracicotropic hormone (PTTH) in prothoracic gland cells of the silkworm, Bombyx mori. The results showed that the PTTH stimulated ERK phosphorylation as this depends on time and dose and ecdysteroidogenic activity. The ERK phosphorylation inhibitors, PD 98059 and U0126, blocked both basal and PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis. In addition, activation of glandular ERK phosphorylation by the PTTH appeared to be developmentally regulated with the refractoriness of gland cells to the PTTH occurring during the latter stages of both the fourth and last larval instars. Moreover, in vitro activation of ERK phosphorylation of prothoracic glands by the PTTH was also verified by in vivo experiments: injection of the PTTH into day 6 last instar larvae greatly increased the activity of glandular ERK phosphorylation and ecdysteroidogenesis. These results suggest that development-specific changes in ERK phosphorylation may play a role in PTTH stimulation of ecdysteroidogenesis.     

The fine structure of developing type 'B' dermal glands in Rhodnius prolixus

The fine structure of the developing type 'B' dermal gland, the more common type in fifth instar larvae of Rhodnius, is described. Each gland consists of three cells responsible for secretion of the duct, saccule and secretion product or precursors, and a fourth capsule cell. The cuticular linings of the duct and saccule are secreted and shed synchronously with the cuticle of the body surface. Mechanisms of secretion and discharge are suggested.     

Insulin stimulates ecdysteroidogenesis by prothoracic glands in the silkworm,Bombyx mori

It is generally accepted that the prothoracicotropic hormone (PTTH) is the stimulator of ecdysteroidogenesis by prothoracic glands in larval insects. In the present study, we investigated activation of ecdysteroidogenesis by bovine insulin in prothoracic glands of the silkworm, Bombyx mori. The results showed that the insulin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. In addition, insulin also stimulated both DNA synthesis and viability of prothoracic glands. Insulin-stimulated ecdysteroidogenesis was blocked by either LY294002 or wortmannin, indicating involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Activation of ecdysteroidogenesis by insulin appeared to be developmentally regulated. Moreover, in vitro activation of ecdysteroidogenesis of prothoracic glands by insulin was also verified by in vivo experiments: injection of insulin into day 6 last instar larvae greatly increased both hemolymph ecdysteroid levels and ecdysteroidogenesis 24 h after the injection, indicating its possible in vivo function. Phosphorylation of Akt and the insulin receptor was stimulated by insulin, and stimulation of Akt phosphorylation appeared to be PI3K-dependent and developmentally regulated. Insulin did not stimulate extracellular signal-regulated kinase (ERK) signaling of the prothoracic glands. These results suggest that in silkworm prothoracic glands, in addition to the PTTH and an autocrine factor, ecdysteroidogenesis is also stimulated by insulin during development.     

Autocrine activation of ecdysteroidogenesis in the prothoracic glands of the silkworm, Bombyx mori

Ecdysteroidogenesis in the prothoracic glands is activated by the neuropeptide, prothoracicotropic hormone (PTTH). The present study demonstrates autocrine activation of ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori. Using both a long-term in vitro organ culture system and an ecdysteroid radioimmunoassay, it was found that either decreasing the incubation volume, from 100 to 5 microl, or increasing the number of glands incubated per drop (50 microl) from 1 to 5 significantly increased ecdysteroid secretion. Prothoracic gland-conditioned medium was used to clarify the autocrine factor. The results showed that activation of ecdysteroidogenesis by the prothoracic gland-conditioned medium appeared to be dose dependent and a dramatic increase in ecdysteroid secretion was observed after 6h of incubation in the conditioned medium. Moreover, it appeared that autocrine activation occurred when glands were incubated in large volumes of incubation medium and during a short incubation period, indicating that the factor may exert its action in situ at some specific developmental stages. This tropic factor was further characterized, and it was found that the factor seemed to be heat-stable, with a molecular weight estimated to be between 1000 and 3000 Da. Injection of the concentrated putative autocrine factor into day 5 last instar larvae greatly increased ecdysteroidogenic activity of the prothoracic glands compared to those injected with saline, indicating the possible in vivo function of the present factor.     

Diuretic hormone in adult Rhodnius prolixus: total store and speed of release

Adult Rhodnius take smaller blood meals than do fifth stage larvae and excrete less fluid afterwards. The total amount of diuretic hormone and active precursors that can be extracted from their nervous system is also smaller. However, more diuretic hormone is released in adult than in fifth stage insects when the hormone release sites are depolarized for short periods with potassium-rich solutions. This shows that the adult can release diuretic hormone at a higher rate than can the fifth stage larva, a feature that can be correlated with the larger volume of haemo-lymph which the adult insect has to load with hormone in order to promote diuresis. In vivo , the concentration of diuretic hormone in the haemo-lymph of adult and fifth stage insects rises at the same rate. Detectable amounts of hormone are found in samples of haemolymph from insects which have been feeding for periods as short as 15 s.     

The effect of the persistence of juvenile hormone mimics on their activity on Rhodnius prolixus

The apparent juvenile hormone (JH) activity of methyl 12-homo-juvenate on fed fifth instar Rhodnius prolixus is nearly twelve times greater if the compound is applied topically over a period of 7 days (serial application) rather than as a single dose 1 day after feeding. Of five other JH mimics tested, four were more active by serial application, although none showed so great an increase in activity as methyl 12-homojuvenate; only farnesyl methyl ether was more active by single application. It is considered that the generally greater activity shown in the serial application tests is because less JH mimic is wasted by degradation and/or excretion. Therefore the serial application test provides a more accurate estimate of inna e JH activity than the single application test.     

Thermosensation and the TRPV channel in Rhodnius prolixus

The thermal sense of triatomine bugs, vectors of Chagas disease, is unique among insects. Not only do these bugs exhibit the highest sensitivity to heat known in any animal up to date, but they can also perceive the infrared radiation emitted by the body of their warm-blooded hosts. The sensory basis of this capacity has just started to be unravelled. To shed additional light on our understanding of thermosensation, we initiated an analysis of the genetic basis of the thermal sense in Rhodnius prolixus. We tested the hypothesis that a TRPV (transient receptor potential vanilloid) channel receptor is involved in the evaluation of heat in this species. Two different approaches were adopted. Initially, we analysed the expression of a TRPV candidate for this function, i.e., RproIav, in different tissues. Subsequently, we tested the effects of capsaicin and capsazepine, two molecules known to interact with mammal TRPV1, using three different behavioural protocols for evaluating thermal responses: (1) proboscis extension response (PER), (2) thermopreference in a temperature gradient and (3) spatial learning in an operant conditioning context. Bioinformatic analyses confirmed that the characteristic features typical of the TRPV channel subfamily are found in the RproIav protein sequence. Molecular analysis showed that RproIav is expressed in R. prolixus, not only in the antennae, but also in other body structures bearing sensory organs. Behavioural experiments consistently revealed that capsaicin treated insects are less responsive to heat stimuli and prefer lower temperatures than non-treated insects, and that they fail to orient in space. Conversely, capsazepine induces the opposite behaviours. The latter data suggest that triatomine thermoreception is based on the activation of a TRP channel, with a similar mechanism to that described for mammal TRPV1. The expression of RproIav in diverse sensory structures suggests that this receptor channel is potentially involved in bug thermoreception. This constitutes solid evidence that thermosensation could be based on the activation of TRP receptors that are expressed in different tissues in R. prolixus. Whether RproIav channel is a potential target for the compounds tested and whether it mediates the observed effects on behaviour still deserves to be confirmed by further research.     

The development of responsiveness to juvenile hormone in the follicle cells of Rhodnius prolixus

During the previtellogenic phase of the development of the follicle in Rhodnius, the JH-sensitive Na/K ATPase in the membranes of the follicle cells displays display a rapid increase in activity, so that the level in vitellogenic follicle cells is about six times that in early previtellogenic cells. The sharp increase in activity during the development of previtellogenic cells is abolished in an allatectomized female. Topical application of JH I early in development restores the level in previtellogenic cells and produces a nearly normal level in vitellogenic cells. Treatment with JH I later in the cycle fails to yield an increase in the activity of the JH-sensitive Na/K ATPase in the previtellogenic cell and leads to only partial restoration of the activity in vitellogenic follicles. These effects of allatectomy and early and late hormone replacement are duplicated by effects on the maximal binding capacity for [³H]JH I of the membranes of follicle cells. These results are interpreted in the light of “activation”, the JH-mediated process by which follicles acquire the competence to respond to JH by becoming patent.