Identification of steroid hormone signaling pathway in insect cell differentiation.

To dissect the steroid hormone signaling pathway involved in insect cell morphological differentiation, we extended the application of the double-stranded RNA-mediated interference (dsRNAi) method to the epidermal IAL-PID2 cell line from Plodia interpunctella Lepidoptera. We first demonstrated that dsRNA was capable of efficiently blocking the steroid hormone 20-hydroxyecdysone (20E) inducibility of proteins that belong to the nuclear receptor superfamily, including the ecdysone receptor (EcR), its partner Ultraspiracle (USP), the insect homolog of the vertebrate retinoid X receptor and the HR3 transcription factor. We then showed that inhibiting the 20E induction of EcR, USP or HR3 proteins prevented the increased synthesis of beta tubulin and consequently the morphological transformation of cells. Thanks to this functional approach, we have shown, for the first time, the participation of EcR, USP and HR3 in a 20E signaling pathway that directs morphological differentiation in insect cells by regulating beta tubulin expression.

Cell cycle profiles of EcR, USP, HR3 and B cyclin mRNAs associated to 20E-induced G2 arrest of Plodia interpunctella imaginal wing cells.

Using the IAL-PID2 cell line established from pupally committed imaginal wing discs of Plodia interpunctella, we have investigated the dynamics of cellular and molecular events involved in the G2/M arrest. We have first cloned a cDNA sequence named PIUSP-2 that likely encodes a homologue of the Ultraspiracle-2 isoform of Manduca sexta. When the IAL-PID2 cells were exposed to a 8 h 20E treatment applied at different times of the cell cycle, an optimal period of sensitivity of cells to 20E, in inducing G2 arrest, was determined at the S/G2 transition. Using cDNA probes specifically designed from Plodia B cyclin (PcycB), ecdysone receptor B1-isoform (PIEcR-B1) and HR3 transcription factor (PHR3), we provide evidence that the 20E-induced G2 arrest was correlated to a high induction of PHR3, PIEcR-B1, PIUSP-2 mRNAs at the S/G2 transition and a decrease in PcycB mRNA level at the end of G2 phase.

Synchronization of Plodia interpunctella lepidopteran cells and effects of 20-hydroxyecdysone.

We have investigated the molecular and cellular mechanisms involved in the control of insect cell cycle by 20-hydroxyecdysone (20E) using the IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. We first defined conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, in order to synchronize the IAL-PID2 cells in their division cycle. A high degree of synchrony was reached when cells were exposed to two consecutive hydroxyurea treatments at 1 mm for 36 h spaced 16 h apart. Under these conditions, flow cytometry analysis demonstrated that 20E at 10(-6) m induced an inhibition of cell growth by an arrest of 90% of the cells in G2/M phase. Using cDNA probes specifically designed from E75 and HR3 nuclear receptors of Plodia interpunctella, we showed that PiE75 and PHR3 were highly induced by 20E through S and G2 phases with maximal enhancement just before the G2/M arrest of cells. These findings suggest that PiE75 and PHR3 could be involved in a 20E-induced genetic cascade leading to G2/M arrest.

Periodic expression of an ecdysteroid-induced nuclear receptor in a lepidopteran cell line (IAL-PID2).

A set of DNA primers was designed within the DNA-binding domain of the Manduca hormone receptor 3 (MHR3) cDNA. These primers were used in RT-PCR to isolate a 204 bp cDNA fragment from IAL-PID2 cells exposed to 10(-6) M 20-hydroxyecdysone (20E) for 12 h. The amino acid sequence deduced from the cDNA fragment presented 100% identity with the zinc finger domain of Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Choristoneura hormone receptor 3 (CHR3). This cDNA fragment was used as a probe on total RNA from IAL-PID2 cells exposed to 20E and hybridized to mRNA, the size of which was close to 4.5 kb and named Plodia hormone receptor 3 (PHR3). Kinetics of induction of PHR3 mRNA were similar to that of HR3 genes but varied according to the position of cells in their cell cycle. The non-steroidal ecdysone agonist, RH-5992 induced the expression of PHR3 at lower concentrations than 20E. From sequence similarity, mRNA size, 20E and RH-5992 inducibilities, we conclude that PHR3 transcript could encode a Plodia hormone receptor 3 involved in the genetic signalling cascade of 20E. Thanks to its periodic expression, this putative orphan nuclear receptor could serve as a suitable cellular marker for studying changes of epidermal cell sensitivity to 20E during the cell cycle.

Rhythmic autocrine activity in cultured insect epidermal cells.

It is now well established that ecdysteroids can be produced in insects in the absence of prothoracic glands. In this respect, it has been shown that cells in culture can produce ecdysteroids. Our aims were: (1) to determine whether ecdysteroid target cells of epidermal origin could also be the source of ecdysteroids; (2) to monitor more accurately the kinetics of ecdysteroid production; and (3) to check for possible relationships between this synthetic activity and dynamics of cell division. An insect cell line (IAL-PID2) established from imaginal discs of the Indian meal moth, Plodia interpunctella, with wild-type sensitivity to ecdysteroids was used in our study. Our results showed that the Plodia cell line exhibited autocrine activity. When division of IAL-PID2 cells was synchronized, a rhythmic production of ecdysteroids was observed. However, further experiments indicated that this rhythmicity could be cell autonomous. This led us to anticipate the existence of two cell subpopulations that would be able to produce ecdysteroids rhythmically, a minor one that would be cell cycle serum-independent population, and a major population that would need serum growth factors to proliferate and produce ecdysteroids. Qualitative study of the ecdysteroid content of the media clearly showed that ecdysone was the major immunoreactive product. Taken together, our findings clearly show that an insect cell line of epidermal origin is capable of rhythmic autocrine production of ecdysteroids. These results support the hypothesis that alternate sites for ecdysteroid production in vivo may exist and could play a role in local regulation of development. We now plan to determine the cellular basis of this rhythmic autocrine activity and to confirm the existence of growth factor-autonomous cells in the culture as well as the potent role played by ecdysteroids in the cross-talk between various cell subpopulations.

Activity of insulin growth factors and shrimp neurosecretory organ extracts on a lepidopteran cell line.

Ecdysteroids, or molting hormones, have been proven to be key differentiation regulators for epidermal cells in the postembryonic development of arthropods. Regulators of cell proliferation, however, remain largely unknown. To date, no diffusible insect peptidic growth factors have been characterized. Molecules structurally related to insulin have been discovered in insects, as in other eucaryotes. We developed in vitro tests for the preliminary characterization of potential growth factors in arthropods by adapting the procedures designed to detect such factors in vertebrates to an insect cell line (IAL-PID2) established from imaginal discs of the Indian meal moth. We verified the ability of these tests to measure the proliferation of IAL-PID2 cells. We tested mammalian insulin and insulin-like growth factors (IGF-I, IGF-II). Following an arrest of cell proliferation by serum deprivation, IGF-I and IGF-II caused partial resumption of the cell cycle, evidenced by DNA synthesis. In contrast, the addition of 20-hydroxyecdysone arrested the proliferation of the IAL-PID2 cells. The cell line was then used in a test for functional characterization of potential growth factors originating from the penaeid shrimp, Penaeus vannamei. Crude extracts of neurosecretory and nervous tissues, eyestalks, and ventral neural chain compensated for serum deprivation and stimulated completion of mitosis. Arch.

Comparison of ecdysteroid concentration in different morphs of aphids.

The amounts of ecdysteroids were determined in different morphs of holocyclic monoecious aphids (Acyrthosiphon pisum Harris, Dysaphis devecta Walk., Lachnus roboris L., Schizolachnus pineti F.) and holocyclic heteroecious aphids (Aphis sambuci L., Rhopalosiphum padi L.) by means enzyme immunoassay. Among the parthenogenetic morphs (fundatrices, virginoparae, and oviparae), the fundatrices have consistently higher amounts of ecdysteroids than those of other morphs of the same species. Alate and apterous virginoparae showed slight differences in their ecdysteroid titer both in heteroecious and monoecious aphids. The migrant morphs (i.e., alate fundatrices and gynoparae) have the lowest amounts of ecdysteroids within a species. With the exception of D. devecta, the oviparae of both heteroecious and monoecious aphids have the second greatest amounts of ecdysteroids among the morphs living on same host plant. Polyphenism, dispersal behavior, and fecundity in connection with weight, ovariole number, and ecdysteroid concentration of different morphs of aphids are discussed.

Effects of two insect growth regulators on ecdysteroid production in Aedes aegypti (Diptera: Culicidae).

Diflubenzuron and OMS 2017 are insect growth regulators that affect larval to adult development in Aedes aegypti (L.) by altering ecdysis. When larvae were exposed to sublethal concentrations, surviving adults express reduced reproductive potential. In mosquitoes, ecdysteroids are important in larval and adult ovarian development. We applied 30% emergence reduction concentrations (EI30) of OMS 2017 and diflubenzuron to 4th-instar Ae. aegypti to determine if changes in ecdysteroid production may explain these physiological effects. Ecdysteroid concentrations were measured in the larvae, pupae, and adults after treatment with both IGRs. After treatment with OMS 2017, the 1st peak of ecdysteroid production in larvae was totally inhibited, but after pupation, ecdysteroid concentrations were the same as in untreated controls. In diflubenzuron-treated larvae, the second peak of production was delayed and the ecdysteroid concentrations of the larvae, pupae, and adults were depressed slightly when compared with untreated controls. The production of ecdysteroids by the ovaries was not altered by sublethal larval treatment with both IGRs. Although OMS 2017 and diflubenzuron belong to the same chemical family, their mode of action apparently is different.

Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin.

During postembryonic development of insects, molting cycles affect epidermal cells with alternate periods of proliferation and differentiation. Cells of the cell line established from imaginal discs of the Indian meal moth (IAL-PID2) differentiate under the action of the molting hormone, 20-hydroxyecdysone, in a manner that is meaningful in terms of the development of the tissue from which they were derived. In particular, the hormone caused an accumulation of the cells in the G2 phase of their cycle and induced the formation of epithelial-like aggregates and the synthesis of specific proteoglycans. Recent discovery of members of the insulin superfamily in insects and the role of growth factors played by this family of molecules in vertebrates led us to check for their potential effects on IAL-PID2 cell cycle regulation. On the one hand, our results showed that insulin was involved in partial resumption of the cell cycle after an arrest caused by serum deprivation, but that other growth factors present in fetal calf serum were needed for full completion of mitosis. On the other hand, the cytostatic effect of 20-hydroxyecdysone was reversible, and, prior exposure of the cells to the hormone allowed the cells to complete one cell cycle in serum-free medium. These results suggest that the production of autocrine growth factors induced by ecdysteroids could circumvent the absence of serum. This cell culture model provides potential for further study of interactions between ecdysteroids and growth factor homologs during differentiation of insect epidermal cells.

Levels of ecdysteroid-like material in adults of Nippostrongylus brasiliensis during the intestinal phase.

Total ecdysteroid-like immunoreactive material was assayed and quantified in adults of the parasitic nematode N. brasiliensis during the intestinal phase in the rat in order to detect possible physiological fluctuations in titre. Worms of the same sex isolated from one rat were pooled in order to quantify ecdysteroids using enzyme immunoassay (EIA). The concentration of ecdysteroids fluctuated during adult life according to the sex and the age of the parasite. Important differences of levels of ecdysteroid-like compounds between the two sexes of parasites were noted particularly at 128 and 168 h post-infection. The peak at 128 h, present in female, but not in male worms, corresponds to the time of egg-laying. Following HPLC-EIA analysis, the presence of ecdysone, 20-hydroxyecdysone and apolar ecdysteroid-like material was ascertained during this peak. The physiological role and the possible origin of ecdysteroids in this nematode are discussed.

Ovarian development and correlated changes in hemolymphatic ecdysteroid levels in two spiders, Coelotes terrestris and Tegenaria domestica (Araneae, Agelenidae).

Oocyte development during the first vitellogenic cycle of Coelotes terrestris and Tegenaria domestica is described. Under the present conditions, this development took about 40 days during which the oocytes went through six stages of maturation. For the first time presence of ecdysteroids is reported in adult females of spiders. Hemolymphatic ecdysteroid peaks were observed in both species at the transition between previtellogenesis and vitellogenesis. 20-Hydroxyecdysone and ecdysone were present in slightly different ratios in C. terrestris as well as T. domestica. These data largely agree with current views of ovarian development in Arthropods.

Decapitation or starvation raise haemolymph ecdysteroid titres in the ovoviviparous female cockroach, Blaberus craniifer Burm.

1. Decapitating newly emerged Blaberus craniifer females near the prothorax severs connections between the suboesophageal and prothoracic ganglia, thus depriving them of the neuroendocrine cephalic complex (including brain and suboesophageal ganglion) and the anterior end of prothoracic glands (PGs). 2. As demonstrated by enzyme immunoassay (EIA), headless females have higher levels of ecdysteroids (ECDs) in haemolymph than starved or fed females, indicating that the neuroendocrine cephalic complex influences circulating ECD levels. 3. The time course of hormonal peaks in decapitated females resembles that in starved females during the first post-ecdysial week, suggesting that some as yet unknown regulating mechanism of ECD production lies outside the head. 4. It is suggested that: (a) The PGs are sites for ECDs production in the early post-imaginal period, (b) the prothoracic and suboesophageal ganglia (linked by nerves to PGs) regulate PGs activity, possibly via neural inputs.

A protein from Daudi cell line conditioned medium induces ovarian dysgenesis in Drosophila melanogaster.

From a medium in which Daudi cells had been grown, we isolated by HPLC a protein that caused ovarian abnormalities in adult females of Drosophila melanogaster when injected into preblastoderm embryos. This protein, whose apparent M(r) is between 30,000 and 50,000, was found to be a moderately polar compound which is heat stable and whose activity is destroyed by acidification. The protein is characteristic of medium conditioned from Daudi cells.

Insect tissue culture systems: models for study of hormonal control of development.

The regulation of growth and development of insects is under endocrine control and involves both juvenile hormones and ecdysteroids. Neuropeptides are master regulators which control the secretion of these hormones. Most experiments in insect endocrinology have been conducted in vivo, but tissue culture methodology is playing an increasing role due to the great interest in simpler model systems for the study of complex processes that occur in vivo. The availability of appropriate media has allowed the culture of a variety of insect organs and cell lines of defined origin which have kept certain properties of the parent tissues. Tissue culture approaches have been useful for studying hormonal control of morphogenetic processes. Cell lines are particularly suited to the study of hormonally regulated mechanisms of macromolecular biosynthesis and gene expression. Thus, the value of in vitro analysis in studies of regulation of hormone production is now recognized. Results obtained from tissue culture allow more precise definition of the hormonal requirements of insect cells and tissues for growth and differentiation and might make possible the discovery of new growth regulators.

Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

Relationships among hormonal changes, cuticular hydrocarbons, and attractiveness during the first gonadotropic cycle of the female Calliphora vomitoria (Diptera).

Attractiveness in adult females of Calliphora vomitoria is correlated with ovarian development and there is a marked increase during the previtellogenic and vitellogenic periods. The development of attractiveness may result from the combined actions of ecdysteroids and juvenile hormone. A rise in total hydrocarbons parallels the first increase in levels of these hormones during the previtellogenic stage. Cuticular hydrocarbons subsequently fall, along with the disappearance of hemolymphatic ecdysteroids, and then rise again during the vitellogenic phase of JH production. Increasing and decreasing of some cuticular hydrocarbons, some hydrocarbons implicated in the attractiveness, are correlated with variation of the titer of these hormones, especially JH III.

Ecdysteroids in the shrimp Palaemon serratus: relations with molt cycle.

Ecdysteroids were studied during the molt cycle of the shrimp Palaemon serratus using radioimmunoassay and HPLC. Continuous presence of ecdysteroids was observed in whole extracts, with two hormonal peaks: a minor peak during stage B and a major one during stage D2. The ecdysteroid titers were generally higher in females than in males. The ecdysteroid peak at stage B was more pronounced in the integument (cuticle and epidermis) than in other tissues and, at this time, 20-hydroxyecdysone was the predominant form. The observed 20-hydroxyecdysone peaks in stages B and D2 are correlatable to the previously reported rise in integumental carbonic anhydrase activity.

Ecdysteroids during the third larval instar in 1(3)ecd-1ts, a temperature-sensitive mutant of Drosophila melanogaster.

The temperature-sensitive 1(3)ecd-1ts mutation (A. Garen, L. Kauvar, and J.A. Lepesant (1977). Proc. Natl. Acad. Sci USA 74, 5099-5103.) has been used in several laboratories to obtain Drosophila larvae deprived of moulting hormone. The development of mutants and controls during the third larval instar at permissive (20 degrees C) and restrictive temperatures (29 degrees C) was compared. Pupariation was inhibited when larvae were shifted to the restrictive temperature immediately at the second moult. The permanent larvae obtained remained active, did not leave the food, and reached a maximum weight superior to the weight of controls. Ecdysteroids were studied during the third larval instar by HPLC analysis and radioimmunoassays. A careful synchronization of the larvae at the second moult enabled the confirmation that at least one ecdysteroid peak occurs during the third larval instar, prior to the wandering stage in controls (20 or 29 degrees C). Ecdysone was then the predominant moulting hormone, whereas 20-hydroxyecdysone was the main ecdysteroid at the time of pupariation. Low levels of ecdysteroid were measured in mutant larvae shifted to 29 degrees C immediately at the second moult but larvae completely deprived of immunoreactive material were never observed. Nearly normal levels of ecdysteroids appeared at 27.5 degrees C. Feeding ecd-1 larvae maintained at restrictive temperature on 20-hydroxyecdysone-yeast mixture for 16 hr triggered abortive pupariation. Ecdysteroid levels were measured after the return of the larvae to the standard medium; normal levels were restored 24 hr later. The mutant ecd-1 appears to present interesting opportunities for the detailed study of the hormonal induction of a developmental process during the third larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytophysiological correlations between prothoracic gland activity and hemolymph ecdysteroid concentrations in Rhodnius prolixus during the fifth larval instar: further studies in normal and decapitated larvae.

Hemolymph ecdysteroid titers in fifth instar larvae of Rhodnius prolixus were determined by radioimmunoassay, and their prothoracic glands were excised and examined by electron microscopy. During the last larval instar, the titer of ecdysteroid increased between the head-critical period until Day 13, at which time the peak titer was 3100 pg 20-hydroxyecdysone equivalents/microliter. The activation of secretory cells at the time of the second period of prothoracicotropic hormone release was correlated with the development of major cellular organelles. The smooth endoplasmic reticulum first appeared at the head-critical period and then proliferated in close relation to the increase in ecdysteroid titer until Day 13, after which time it disappeared. Mitochondria expand and develop tubular cristae. They are closely associated with the smooth endoplasmic reticulum. When insects were decapitated, hemolymph ecdysteroid titer remained below 10 pg/microliter and the prothoracic gland cells failed to develop smooth endoplasmic reticulum. We conclude that in the prothoracic gland cells as well as other steroidogenic tissues the smooth endoplasmic reticulum in association with mitochondria is involved in ecdysone biosynthesis.

Activity of corpora allata, endocrine balance and reproduction in female Labidura riparia (Dermaptera).

The reproductive activity of Labidura riparia females involves, after a 5-day maturation stage, a regular alternation of ovarian cycles and egg-care stages averaging 10 days each. Vitellogenesis is characterized by an increase in the size of the corpora allata (CA) where structured SER bodies appear, and by a rise of juvenile hormone (JH III) content in the hemolymph which is followed by an increase in the level of ecdysteroids. During the egg-care periods, the CA are inactive; structured bodies generate autophagic vacuoles, the titer of JHs and later that of ecdysteroids in the hemolymph decreases and remains stationary. Ovariectomy causes hypertrophy and hyperactivity of the CA for about two months. Subsequently, the titer of JH decreases and old females may display parental behaviour; the level of ecdysteroids falls and remains unchanged. After cauterization of the pars intercerebralis (PI) of the protocerebrum, the ovarian activity stops, the ovary shrinks, the JHs rapidly disappear but ecdysteroids remain at the same or even higher levels than those of normal females of the same age. On the basis of these data, we postulate the existence of a center located in the PI, inhibiting the production of ecdysteroids, and of a stimulating center located outside this area. The PI also exhibits an allatotropic function.