Growth and mitogenic effects of arylphorin in vivo and in vitro.

In insects, developmental responses are organ- and tissue-specific. In previous studies of insect midgut cells in primary tissue cultures, growth-promoting and differentiation factors were identified from the growth media, hemolymph, and fat body. Recently, it was determined that the mitogenic effect of a Manduca sexta fat body extract on midgut stem cells of Heliothis virescens was due to the presence of monomeric alpha-arylphorin. Here we report that in primary midgut cell cultures, this same arylphorin stimulates stem cell proliferation in the lepidopterans M. sexta and Spodoptera littoralis, and in the beetle Leptinotarsa decemlineata. Studies using S. littoralis cells confirm that the mitogenic effect is due to free alpha-arylphorin subunits. In addition, feeding artificial diets containing arylphorin increased the growth rates of several insect species. When tested against continuous cell lines, including some with midgut and fat body origins, arylphorin had no effect; however, a cell line derived from Lymantria dispar fat body grew more rapidly in medium containing a chymotryptic digest of arylphorin.

Timing and ecdysteroid regulation of the molt in last instar greenhouse whiteflies (Trialeurodes vaporariorum).

A system of markers has been devised to track the development of 3rd and 4th instar/pharate adult greenhouse whiteflies. Instars were identified based on measurements of body width and body length. Depending upon the host plant, the product of the two measurements was exceptionally useful in distinguishing between instars. Body depth was used to divide the 3rd instar into eight stages and body depth and color and appearance of the developing adult eye were used to divide the 4th instar/pharate adult into nine stages. Under conditions of L:D 16:8 and a temperature of 26+/-2 degrees C, the body depth of 3rd instars reared on greenbean increased from 0.025 (stage 1) to 0.2mm (stage 8) and the instar duration was approximately 3 days. The body depth of 4th instars increased from approximately 0.1+/-0.02 (Stage 1) to 0.3+/-0.03mm (Stage 5) and then remained constant or decreased slightly during adult development. Ecdysteroid titers peaked at approximately 120fg/&mgr;g protein during Stages 3 through 6 of the 4th instar. Based on an external examination of developing 4th instars and the fluctuations in ecdysteroid titer, it appears that adult development is initiated in Stage 4 or 5 4th instars. Results from histological studies support this view. In Stage 4 nymphs, a subtle change was observed in the corneagenous cells of the eye. However, most Stage 4 4th instars possessed wing development characteristic of earlier, immature stages. In all Stage 5 insects, wing development had been initiated and the corneagenous cells had become quite distinct. In Stage 6 whiteflies, the wing buds were deeply folded and by Stage 7, spines were observed on the new cuticle, indicating that the adult cuticle was well-formed by this stage. Our study is the first to investigate the timing and regulation of the molt, to monitor ecdysteroid titers in precisely staged 4th instar whiteflies and to examine the internal anatomical changes associated with metamorphosis in these tiny homopteran insects.

Testis ecdysiotropin, an insect gonadotropin that induces synthesis of ecdysteroid.

Testes of lepidoptera synthesized ecdysteroid in a somewhat different temporal pattern than the prothoracic glands that release ecdysteroid to the hemolymph. Brain extracts from Heliothis virescens and Lymantria dispar induced testes to synthesize ecdysteroid, but did not affect prothoracic glands. The testis ecdysiotropin (LTE) was isolated from L. dispar pupal brains by a series of high-pressure chromatography steps. Its sequence was Ile-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH, of molecular mass 2,473 Daltons. The predominant signaling pathway for LTE was via G(i) protein, IP3, diacylglycerol and PKC; a modulating pathway, apparently mediated by an angiotensin II-like peptide, was controlled via G(s) protein, cAMP, and PKA. Testis ecdysteroid caused isolated testis sheaths to also synthesize a growth factor that induced development of the male genital tract. The growth factor appeared to be a glycoprotein similar to vertebrate alpha-1-glycoprotein. A polyclonal antibody to LTE indicated LTE-like peptide in L. dispar brain medial neurosecretory cells, the suboesophageal, and other ganglia, and also in its target organ, the testis sheath. LTE immunoreactivity was also seen in testis sheaths of Rhodnius prolixus. LTE-like immunoactivity was also detected in developing optic lobes, antennae, frontal ganglia, and elongating spermatids of developing L. dispar pupae. This may indicate that LTE has a role in development as well as stimulation of testis ecdysteroid synthesis. Published 2001 Wiley-Liss, Inc.

Artificial diets for rearing the Colorado potato beetle, Leptinotarsa decemlineata.

Colorado potato beetles have been reared successfully through 12 generations on artificial diets containing either 2.5% potato leaf powder or 2.5% lettuce leaf powder/0.75% potato leaf powder. For all but one of the treatment groups, the mean duration of each of the four larval stages was between 0.8 and 1.5 days longer than the durations exhibited by control beetles that had been fed on potato leaves. Maximum weights of prepupae, newly emerged adults and day 5-9 adults were approximately 78, 80 and 82%, respectively, of the weights for comparable stages of control beetles. Mean percent mortality for 1(st) instars was two to six times higher for artificial diet-fed CPBs than for leaf-fed beetles. However, since pupal mortality was four times higher for control beetles than for beetles reared on artificial diet, mean percent total mortality (newly hatched through the 9 day old adult) was equivalent for leaf-fed beetles and for later generations of potato and Lettuce+Potato diet-fed CPBs. Hemolymph ecdysteroid levels and fluctuations in mature 4th instar larvae and prepupae were similar in controls and experimental groups. Number of hatchlings produced per adult pair per day (fertility) was approximately eight times greater in control beetles than in later generations of artificial diet-fed beetles, primarily because fewer egg masses were laid per day, percent hatch was lower and cannibalism of eggs was higher in these latter groups. Interestingly, the mean percent hatch, although only 68% of the control value, was 1.5 times greater for beetles reared on diet containing lettuce-leaf powder, and a small percentage of potato leaf powder, than on diet containing only potato leaf powder. Percent hatch was equal for beetles fed on diet containing only lettuce-leaf powder and those fed on potato leaves. Finally, it is noteworthy that the quality of eggs, as judged by the ability of the wasp parasitoid, Edovum puttleri, to parasitize and develop in the eggs, was similar for eggs produced by control beetles and for those produced by beetles fed on potato and Lettuce+Potato diets. The diets and rearing system described here will be useful for providing beetles on a year-round basis for experiments designed to evaluate the effects of potential insect control agents, to investigate the mechanism(s) by which insects become resistant to control agents and for other applied and fundamental studies related to the control of this serious pest. The use of lettuce leaf powder in place of most of the potato leaf powder is especially advantageous because of the much reduced cost and greater availability of lettuce as compared to potato leaves.

Aedes aegypti TMOF modulates ecdysteroid production by prothoracic glands of the gypsy moth, Lymantria dispar.

Trypsin modulating oostatic factor (TMOF) is a decapeptide that inhibits the biosynthesis of trypsin-like enzymes in the midgut of several insect species and, as such, serves as a dipteran oostatic hormone. In vitro incubation of lepidopteran prothoracic glands with Aedes aegypti TMOF revealed that this decapeptide, in the presence of brain extract, modulates ecdysteroid production. The modulatory effect was highly dependent on both the concentration of TMOF and brain extract. Typically, TMOF was stimulatory in the presence of lower concentrations of Lymantria dispar brain extract (0.01 and 0. 025 brain equivalent), and either neutral or inhibitory at higher concentrations (0.25, 0.5, and 1.0 brain equivalent) of extract. In the presence of European corn borer (Ostrinia nubilalis) brain extract, TMOF also exhibited modulatory effects, effects that again were dependent on the concentrations of both brain extract and TMOF present in the incubation medium. At 1.5 brain equivalents, TMOF was inhibitory at all but the highest concentration tested (5x10(-6) M), at 1.0 brain equivalent, TMOF was stimulatory at 10(-6) M and at 0. 5 brain equivalents, TMOF did not significantly affect PTG synthesis of ecdysteroids. Results suggest the presence of a modulatory peptide(s), which fine tunes the synthesis and release of ecdysteroids by PTGs in accordance with the insect's developmental/physiological requirements.

Ecdysteroid and free amino acid content of eggs of the Colorado potato beetle, Leptinotarsa decemlineata.

In order to identify components of the Colorado potato beetle (CPB) egg that may be required by Edovum puttleri, a parasitic wasp that parasitizes the CPB egg, to complete development, ecdysteroid and free amino acid content of CPB eggs were analyzed by reversed phase high pressure liquid chromatography followed by radioimmunoassay to identify ecdysteroids. Ecdysteroid titers were relatively low (<300 pg/egg) through day 2 post-oviposition and then increased sharply, reaching concentrations >2,500 pg/egg on day 3 post-oviposition. Ecdysone (E), 20-hydroxyecdysone (20E), and polar conjugates of E were prominent ecdysteroids present in eggs sampled on days 0 and 1 post-ecdysis, and E, 20E, three peaks containing more polar ecdysteroids (metabolic inactivation products), and polar conjugates of E were present in eggs sampled on day 2. Thus, at a time when parasitization of CPB eggs by E. puttleri is relatively high (0-48 h), physiologically-active ecdysteroids (20E and perhaps E are physiologically active) are present at concentrations between 50 and 200 pg/egg. Ecdysone and 20E reached their highest levels in day-3 eggs, indicating that ecdysteroid may direct physiological processes associated with the completion of CPB embryonic development. In day-4 eggs, the concentration of E and 20E fall dramatically and polar metabolites of E and/or 20E are now responsible for the high ecdysteroid content of the eggs. Interestingly, conjugates of E decrease to relatively low levels in day-3 eggs and are absent in day-4 eggs. Therefore, it is likely that the increase in E in day-3 eggs is due, in part, to the breakdown of polar conjugates of E. Nine amino acids were present in significant quantities in eggs sampled at various times between 0 and 48 h post-oviposition. These include histidine, glutamine, proline, asparagine, serine, glutamic acid, threonine, lysine, and tyrosine. The first three amino acids were present at concentrations that were approximately 2 to 6 times greater than the concentrations of the last six amino acids. Amounts of most of the free amino acids varied with the age of the eggs from which the extract was prepared, but in general, there was no correlation between the levels at times of maximum parasitization (0 and 30 h) and the levels at the less favored times of parasitization (16 and 48 h). This information should facilitate the development of diets for both parasites and predators of pest species of beetles. Arch. Insect Biochem. Physiol. 44:172-182, 2000. Published 2000 Wiley-Liss, Inc.

Ecdysteroid levels/profiles of the parasitoid wasp, Diapetimorpha introita, reared on its host, Spodoptera frugiperda and on an artificial diet.

Diapetimorpha introita is an ichneumonid ectoparasitoid of the fall armyworm, Spodoptera frugiperda. Since it has been reported that D. introita wasps reared on an artificial diet exhibit a significantly lower percentage of adult eclosion and fecundity than host-reared wasps, this study was undertaken to elucidate the factors responsible for the reduced viability observed in diet-reared wasps. A system of markers has been devised to track the development (from the initiation of cocooning through adult eclosion) of D. introita. Although wasps reared on artificial diet developed more slowly than did those reared on host pupae, both diet- and host-reared wasps passed through the same stages of development - the eyes enlarged and moved backward, the gut was purged and upon ecdysis the exarate pupa emerged. The thorax was the first to darken, followed by the head and then the abdomen. Pharate pupal formation occurred before gut purge. Two peaks of hemolymph ecdysteroids were observed, one in wasps in which gut purge was almost complete and the second in day-2 exarate pupae. Ecdysone and 20-hydroxyecdysone were the major ecdysteroids present in hemolymph sampled at these times. Small quantities of 20,26-dihydroxyecdysone, polar ecdysteroids and/or possibly 26-hydroxyecdysone were also present. In six stages of development, hemolymph ecdysteroid titers were significantly higher in host-reared than in diet-reared wasps (Eye 1, Eye 2, Gut Purge 2, Pharate Pupa, Head/Thorax Dark, and Abdomen Dark). Relatively high percentages of mortality were observed in diet-reared wasps in four of these stages and in two others which occurred in close proximity to one of the stages, the Abdomen Dark stage. Thus, insufficient ecdysteroid in the hemolymph may be responsible, in part, for the relatively high percentage of mortality that occurred in wasps reared on an artificial diet.

Two polypeptide factors that promote differentiation of insect midgut stem cells in vitro.

Isolated stem cells from the midguts of Manduca sexta and Heliothis virescens can be induced to differentiate in vitro by either of two polypeptide factors. One of the peptides was isolated from culture medium conditioned by differentiating mixed midgut cells; we used high performance liquid chromatographic separation and Edman degradation of the most prominent active peak. It is a polypeptide with 30 amino acid residues (3,244 Da), with the sequence HVGKTPIVGQPSIPGGPVRLCPGRIRYFKI, and is identical to the C-terminal peptide of bovine fetuin. A portion of this molecule (HVGKTPIVGQPSIPGGPVRLCPGRIR) was synthesized and was found to be very active in inducing differentiation of H. virescens midgut stem cells. It was designated Midgut Differentiation Factor 1 (MDF1). Proteolysis of bovine fetuin with chymotrypsin allowed isolation of a pentamer, Midgut Differentiation Factor 2 (MDF2) with the sequence HRAHY corresponding to a portion of the fetuin molecule near MDF1. Synthetic MDF2 was also biologically active in midgut stem cell bioassays. Dose response curves indicate activity in physiological ranges from 10(-14) to 10(-9) M for MDF1 and 10(-15) to 10(-5) M for MDF2.

Manipulation of fifth-instar host (Manduca sexta) ecdysteroid levels by the parasitoid wasp Cotesia congregata.

Although 5th (last) instar parasitized Manduca sexta larvae undergo developmental arrest and do not wander, they exhibit a small hemolymph ecdysteroid peak (300-400pg/&mgr;l) which begins one day prior to the parasitoid's molt to the 3rd (last) instar and concomitant emergence from the host. Ecdysteroids present in this peak were 20-hydroxyecdysone, 20,26-dihydroxyecdysone and one or more very polar ecdysteroids, as well as small amounts of 26-hydroxyecdysone and ecdysone. In parasitized day-1 and -2 5th instars ligated just behind the 1st abdominal proleg, hemolymph ecdysteroid levels increased in both anterior and posterior portions (100-500pg/&mgr;l), while in unparasitized larvae, hormone levels only increased in the anterior portion (100-350pg/&mgr;l). Thus, the ecdysteroid peak observed in host 5th instars was probably produced, at least in part, by the parasitoids. It may serve to promote Cotesia congregata's molt from the second to the third instar and/or to facilitate parasitoid emergence from the host. In parasitized day-1 and -2 5th instars ligated between the last thoracic and 1st abdominal segments, hemolymph ecdysteroid titers reached much higher levels (500-3500pg/&mgr;l) in the anterior portion (no parasitoids present) than in the posterior portion (150-450pg/&mgr;l). Therefore, it appears that the parasitoid's regulation of hemolymph ecdysteroid titers occurs at two levels. First, parasitization neutralizes the host's ability to maintain its normal hemolymph ecdysteroid levels. Second, in a separate action, the parasitoid manipulates the ecdysteroid-producing machinery so that hemolymph levels are maintained at the 200-400pg/&mgr;l characteristic of day 3-4 hosts. This is the first report of a parasitoid's ability to interfere with the normal inhibitory mechanisms which prevent prothoracic gland production of ecdysteroid at inappropriate periods of insect growth and development.

Effects of parasitization by Cotesia congregata on the brain-prothoracic gland axis of its host, Manduca sexta.

The ability of prothoracic glands (PTGs) from parasitized and unparasitized Manduca sexta 5th-instars to respond to ecdysiotropic extracts prepared from day-5 5th instar brains was compared. An in vitro bioassay revealed that PTGs from parasitized animals were much less responsive to brain PTTH than glands from unparasitized larvae. However, when incubated in Grace's medium in the absence of brain extract, glands from day-3 and -4 hosts remained active for a much longer period of time than did those dissected from their unparasitized counterparts. Rather than exhibiting reduced (basal) levels of synthesis after the 3rd hour of incubation, glands from these parasitized larvae continued to synthesize/release ecdysteroid into the medium at relatively high rates. The timing of this enhanced secretory activity is coincident with the ecdysteroid peak that occurs just prior to and during wasp emergence. Following parasite emergence, gland activity decreased, and by the third day after emergence, was reduced to low levels. Results suggest that the requirement for PTTH to stimulate ecdysteroid production has been bypassed, i.e. that the parasite has uncoupled the normal mechanisms that permit brain regulation of PTG activity. The ability of brains from parasitized M. sexta to stimulate PTGs from unparasitized day-2 5th instars was also examined. Dose-response analyses performed for the first 7 days of the 5th instar showed that on a per brain basis ecdysiotropic activity in brains from parasitized and unparasitized animals was similar. However, when differences in brain size were considered, ecdysiotropic activity appeared to be more concentrated in brains from day-7 parasitized larvae than in brains from similarly aged unparasitized larvae. Analysis of the size distribution of the ecdysiotropic activity in brains from parasitized larvae revealed a unique form that was larger than the 29kDa standard. This suggests that parasitization may inhibit neuropeptide processing, particularly during the final stages preceding emergence of the wasps from the host. Thus, both an inhibition of prothoracicotropic hormone processing and the inability to respond to this neurohormone may contribute to the developmental arrest characteristic of parasitized 5th instars.

Effects of Parasitism by the Braconid Wasp Cotesia congregata on Metabolic Rate in Host Larvae of the Tobacco Hornworm, Manduca sexta.

We examined growth rates, gas exchange patterns and energy metabolism of tobacco hornworm (Manduca sexta) larvae parasitized by the braconid wasp Cotesia congragata. Larvae parasitized at the beginning of the fourth-instar had reduced growth compared to unparasitized larvae of the same age and short-term differences in metabolism (measured as rates of CO(2) production, Vdot; CO(2)) were apparent almost immediately after wasp oviposition. However, over the growth period between parasitization and the last part of the fifth-instar, there was no significant difference between parasitized and unparasitized hosts as seen in the relationship between mass and Vdot; CO(2). One day prior to parasitoid emergence, host larvae stopped eating, ceased spontaneous locomotor activity and showed a dramatic decline in metabolism. The 60% decline of Vdot; CO(2) at this time is consistent with lack of specific dynamic action because the animals were not feeding. Gas exchange became highly cyclical on the day of parasitoid emergence, but the cause and significance of this phenomenon, which disappeared by the third day following emergence, are not clear. This pattern of cycling was not induced by starving nonparasitized larvae for 6days, nor by immobilizing nonparasitized larvae with tetrodotoxin. Ecdysteroid levels in the host's hemolymph significantly increased on the day when parasitoids completed their L2-L3 molt and began emerging, but not during the wasps' L1-L2 molt which occurred a few days earlier. Contrary to our initial expectation that hemolymph ecdysteroid titers might be linked to alterations in the host's metabolic rate, we observed no such correlation.

Conversion of 3-dehydroecdysone by a ketoreductase in post-diapause, pre-hatch eggs of the gypsy moth Lymantria dispar.

The prothoracic glands (PGs) of Lymantria dispar (day-5 female, last-stage larvae) produce both ecdysone and an ecdysteroid which has the same retention time on reverse-phase liquid chromatography (RPLC) as a known standard of 3-dehydroecdysone. The latter ecdysteroid can be converted by a heat-labile factor in extracts of post-diapause, pre-hatch L. dispar eggs to an ecdysteroid which has the same retention time on RPLC as ecdysone. Purified 3-dehydroecdysone, similarly treated with egg extract, also gives the same retention time on RPLC as ecdysone. Taken together, these data suggest that, like Manduca sexta, a major product of the PGs in L. dispar is 3-dehydroecdysone. Furthermore, these data suggest that L. dispar eggs, which contain mature embryos, possess ecdysteroid ketoreductase activity capable of converting 3-dehydroecdysone to ecdysone. This is the first report of ecdysteroid ketoreductase activity in embryonated eggs.

The pharate adult clasper as a tool for measuring chitin synthesis and for identifying new chitin synthesis inhibitors.

A rapid, reliable, repeatable bioassay for measuring chitin synthesis is described. It utilizes the clasper from male pharate adult European corn borers and measures the incorporation of [14C]N-acetylglucosamine. Chitin synthesis is maximum in claspers taken from animals 5 and 6 days postpupation. The system is very sensitive to inhibition by the phenylbenzoyl ureas and polyoxins and should be useful for identifying potential inhibitory agents.

Ammonium sulfate activation of phosphodiesterase in homogenates of larvae of the european corn borer.

European corn borer phosphodiesterase is highly activated by (NH4)2SO4 and moderately activated by NH4C1 (pH 7.6, 33 degrees). Vertebrate and crayfish diesterases, on the other hand, are inhibited by (NH4)2SO4. It is likely that (NH4)2SO4 causes some configurational change in the European corn borer phosphodiesterase molecule which results in the exposure of more active sites and hence greater enzyme activity. In in vitro tests caffeine (0.008 M) and theophylline (0.008 M) inhibit phosphodiesterase more effectively in European corn borer larvae than in crayfish, ovine, bovine, or rat tissue.