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. Insect Biochem. Physiol. 34:313–328, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Diflubenzuron-Induced alterations during in vitro development of Tenebrio molitor pupal integument

The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20-hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (? 10 μg/ml) resulted in epicuticle deposition. These observations confirm in vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells. In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after in vivo DFB treatment in Tenebrio pupae.     

The ecdysteroids from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity.

Six naturally occurring C27 ecdysteroids were isolated and identified from the tobacco hornworm during pupal-adult development five days after peak titer of molting hormone activity. In order of decreasing quantities the hormones were: 20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, 20hydroxyecdysone, 3-epi-20,26-dihydroxyecdysone, 3-epi-ecdysone, and ecdysone. 20-Hydroxyecdysone, in an earlier study, was the major molting hormone present at peak titer during pupal-adult development. The major ecdysteroid present during embryonic development in this insect, 26-hydroxyecdysone, was not detected. The copresence of all six of these ecdysteroids from a single developmental stage of an insect provides information on the metabolic interrelationships that exist among these steroids and on their possible function(s) in insects. The 3alpha-ecdysteroids were far less active than the 3 beta-epimers in the house fly assay. The significance of epimerization is discussed.     

An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells

The addition of physiological concentrations of the arthropod molting hormone 20-hydroxyecdysone results in the cessation of cell division in the Kc cell line of Drosophila melanogaster. Fluorometric mononitoring of the cell cycle reveals that treatment of the cells with hormone for 12 hr causes a G2 arrest. The dose-response curves are in agreement with those obtained for other hormonal effects in both the Kc line and the intact animal. In the continual presence of hormone, cells remain G2-arrested for approximately 100 hr, resuming division by 120 hr. Cells which have responded once to ecdysteroids and subsequently reentered the cell cycle are insensitive to hormonal restimulation. This lack of response has been correlated with, and is probably due to, the loss of ecdysteroid receptors in stimulated cells.     

The ecdysteroids from young embryonated eggs of the tobacco hornworm

26-Hydroxyecdysone, which is the major free recoverable ecdysteroid of older age groups of embryonated eggs of the tobacco hornworm was also the major component in 4- to 18-hour-old embryonated eggs. The other 3β-ecdysteroids, ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxy-ecdysone, were also present and accounted for an the molting hormone activity; 26-hydroxyecdysone was devoid of molting hormone activity in the house fly assay. 20-Hydroxyecdysone was a minor component, which confirms the earlier observations that the main metabolic route for ecdysteroids during embryonic development is that leading to 26-hydroxy-ecdysone, whereas formation of 20-hydroxyecdysone is a minor pathway. A new 3α-ecdysteroid, 3-epi-26-hydroxyecdysone, also devoid of molting hormone activity, was the second major ecdysteroid isolated from the eggs. 3-Epi-20,26-dihydroxyecdysone was detected in very minute amounts. In additon to the six 3β-and 3α-ecdysteroids there were at least an equivalent number of unknown ecdysteroids an of which lacked molting hormone activity. Their physical properties including chromatographic behavior are discussed.     

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.     

The establishment of cell lines from imaginal wing discs of Spodoptera frugiperda and Plodia interpunctella

Continuous cell cultures were established from imaginal wing discs of 2 Lepidoptera, Spodoptera frugiperda and Plodia interpunctella. The S. frugiperda line (IAL-SFD1) grows as multicellular vesicles and responds morphologically and biochemically to the insect hormone, 20-hydroxyecdysone. In contrast, the P. interpunctella cells (IAL-PID2) grow as attached monolayers of small spindle-shaped cells and do not appear to have specific responses to 20-hydroxyecdysone, although growth rates are slowed in these cells upon exposure to the hormone.     

Epidermal collagenase activity and its induction by 20-hydroxyecdysone in the fiddler crab Uca pugilator

The epidermal collagenase activity and its induction by 20-hydroxyecdysone in Uca pugilator were investigated.Zymographic electrophoresis showed four bands of collagenae activity,16,19,22 and 29 kDa in molecular weight,with the former two accounting for 60% and 36%,respectively,of the total coUagenase activity. The collagenase activity varies during the molting cycle. Among the molt stages tested,Premoh Stage Do exhibited the highest epidermal coUagenase activity for both the 16 and 19 kDa isoenzymes and,as the molt stage proceeded,the enzymatic activity of these two isoenzymes decreased,with the lowest activity for both found in Premoh Stage D3-4. Injection of 20-hydroxyecdysone significantly induced the activity of the 16 kDa collagenase in the epidermis of Uca pugilator,suggesting that the activity of this isoenzyme is under molting hormone control.Although 20-hydroxyecdysone injection did not result in a statistically significant increase in the activity of the 19 kDa isocnzyme,a tendency of the induction was nonetheless demonstrated. This is the first report on epidermal collagenase activity and its induction by the molting hormone in a crustacean.     

Morphological and cytochemical studies of the effects of ecdysteroids in a lepidopteran cell line (IAL-PID2)

Summary The Indian meal-moth cell line, IAL-PID2, established from larval wing discs was examined from the 250th to the 300th passages. The cultured cells retain various structural and functional qualities of epidermal cells. Under hormone-free conditions PID2 cells grow as a monolayer of round or spindle-shaped cells. They appear as weakly active epidermal cells. The endoplasmic reticulum and Golgi apparatus are poorly developed and secretory activity is reduced. Culture conditions resulted in considerable cellular expansions, abundance of storage products (glycogen, lipids), and hypertrophy of the lysosomal system. The PID2 cell line retains the ability to respond to ecdysteroids; 20-hydroxyecdysone treatment (2×10^-6 M) triggered morphogenetic and secretory processes. Cells formed pseudoepithelial aggregates interconnected and linked by desmosome-like structures. The hormone-stimulated cells are involved in the biosynthesis of N-acetyl-D-glucosamine-rich glycoproteins. The glycosylation sites were located, by use of WGA-gold particles, on cellular expansions and all along the plasma membrane. The possible significance of these glycoproteins is discussed.     

Entomogenous fungus Nomuraea rileyi inhibits host insect molting by C22-oxidizing inactivation of hemolymph ecdysteroids

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae.     

Hormonal Control of Molting in Decapod Crustacea

The involvement of the molting hormone, 20-hydroxyecdysone,in the mediation of molting in decapod crustaceans is brieflyreviewed. Aspects of the secretion and metabolism of its precursor,ecdysone, are discussed. Experiments are described that demonstratethe presence of a molt-inhibiting hormone (MIH) in the sinusglands of juvenile lobsters (Homarus americanus). Assays forMIH include measurement of the molt interval and radioimmunoassayof circulating titers of ecdysteroids in eyestalk-ablated lobsters.This latter assay indicates that sinus gland extracts significantlydecrease the concentration of circulating ecdysteroids 24 hrafter injection. Data are also presented on the circulatingtiters of ecdysteroids during multiple molt cycles of lobstersfollowing eyestalk ablation. These data indicate that theremust be another factor that ultimately regulates the circulatinglevels of the molting hormone.     

Identification of the molting hormone of the sweet potato (Bemisia tabaci) and greenhouse (Trialeurodes vaporariorum) whitefly

In order to identify the whitefly molting hormone, whole body extracts of mature 4th instar and newly formed pharate adult Bemisia tabaci (Biotype B) and Trialeurodes vaporariorum were prepared and subjected to reverse phase high performance liquid chromatography (RPHPLC). Ecdysteroid content of fractions was determined by enzymeimmunoassay (EIA). The only detectable ecdysteroids that were present in significant amounts in whitefly extracts were ecdysone and 20-hydroxyecdysone. The concentrations of 20-hydroxyecdysone in B. tabaci and T. vaporariorum extracts, respectively, were 40 and 15 times greater than the concentrations of ecdysone. The identity of the two ecdysteroids was confirmed by normal phase high performance liquid chromatography (NPHPLC). When ecdysteroid content of RPHPLC fractions was assayed by radioimmunoassay (RIA), small amounts of polar ecdysteroids were also detected indicating that these ecdysteroids have a very low affinity for the antiserum used in the EIA. Ecdysteroid at 10.4 mM administered by feeding stimulated 2nd instar whitefly nymphs to molt. Based on our results, it appears that 20-hydroxyecdysone is the whitefly molting hormone.     

Insulin signaling is involved in hematopoietic regulation in an insect hematopoietic organ

Only a few extracellular hematopoietic factors have been identified in insects. We previously developed an in vitro culture system for the larval hematopoietic organ (HPO) of the silkworm Bombyx mori, and found that cell proliferation is linked to hemocyte discharge from the HPO. In this study, we tested hematopoietic activity of bombyxin, a peptide in the insulin family. When silkworm HPO was cultured with synthetic bombyxin-II, the number of discharged hemocytes increased in a dose-dependent manner, indicating that bombyxin promoted cell proliferation in the HPO. However, a neutralization experiment using anti-bombyxin-II antibody revealed that bombyxin is not the primary effector in larval plasma. Similarly, bovine insulin showed hematopoietic activity. Addition of molting hormone, 20-hydroxyecdysone, circumstantially enhanced the hematopoietic activity of bombyxin and insulin. Bombyxin and insulin induced phosphorylation of different sets of proteins in the HPO, suggesting that their signaling pathways are different.     

Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: an in vitro study

In relation with the digestive cycle, the digestive gland cells of bivalve molluscs undergo a sequence of cytological changes which is controlled by external and internal effectors such as putative gastrointestinal hormones and growth differentiation factors. A tissue dissociation method was developed to investigate the in vitro effect of the vertebrate growth and differentiation factors: insulin, insulin growth factor I (IGF-I), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the digestive gland cells of the scallop Pecten maximus. All these vertebrate peptides induced a dose-dependent increased incorporation of ³H-leucine and ¹⁴C-uridine in whole digestive gland cell suspensions. However, after Percoll density gradient purification of the digestive cells, only stem and undifferentiated enriched cell fractions were responsive to the different peptides. In addition, insulin and IGF-I, but not EGF and bFGF, stimulated ³H-leucine incorporation in control dispersed mantle edge cells. These results suggest that insulin-related peptides could work as general growth promoting factors in molluscs. On the other hand, EGF and bFGF, or at least their molluscan counterparts, may be efficient growth differentiation factors in the regenerative processes occurring in the digestive gland of molluscs. Accepted: 26 September 1997     

Growth of an ovarian cell line of Galleria mellonella and its response to immune-inducing factors

Antibacterial proteins are produced in the reproductive tracts of some insect species. The advent of a pupal ovarian cell line of the lepidopteran Galleria mellonella offered an opportunity for exploring the use of ovarian tissue culture to induce antimicrobial proteins in lieu of the larvae. The ovarian cell growth rates and cell yields were maximized by adjusting Grace's medium to pH 6.5, adding 15% (v/v) qualified heat-inactivated fetal calf serum, and lowering the sucrose concentration to 9.3 g/L. Five cell forms and biochemical profiles of the collective cell types were analyzed throughout the culture growth cycle. The final modified culture medium did not affect morphogenesis, whereas it increased the culture growth rate by 50% and the final cell yield threefold. The molting and immunoprotein-inducing hormone, 20-hydroxyecdysone, increased culture growth rate and altered the levels of cell types A and D. Neither 20-hydroxyecdysone nor the larval immunizing agents, apolipophorin-III or Bacillus subtilis, in combination or alone, induced antibacterial activity. The bacterium did induce immunity in both larval and adult stages.