Further evidence that diapause in the gypsy moth,Lymantria dispar,is regulated by ecdysteroids: a comparison of diapause and nondiapause strains

A nondiapause strain of the gypsy moth offers an additional tool for evaluating the regulation of diapause in this species. Patterns of protein expression in the gut and gut enzyme activity distinguished the two strains. Synthesis of a 55kDa gut protein, previously linked to diapause, began 14days after oviposition in both the diapause (D) and nondiapause (ND) strains. Though synthesis of this protein persisted in the D strain, its synthesis decreased after day 18 in the ND strain. In the D strain, activity of the proteolytic enzymes (trypsin, chymotrypsin, elastase, aminopeptidase) and esterase remained low, while activity of all of these enzymes increased dramatically in the ND strain 18-20days after oviposition. By contrast, alkaline phosphatase (ALP) activity was high in both strains 15-17days after oviposition, activity remained high in the D strain but in the ND strain activity then decreased. Patterns of ALP zymograms were similar in the two strains on day 15, but later a band of high mobility appeared only in the D strain. When 20-hydroxyecdysone was added to hanging drop cultures containing ND pharate larvae 15days after oviposition, the larvae assumed the characteristics of diapause larvae: the 55kDa gut protein was synthesized, the ALP zymogram revealed the characteristic diapause pattern, and they failed to ingest culture medium. The fact that 20-hydroxyecdysone could elicit these responses in ND individuals further supports previous results indicating that ecdysteroids promote the induction and maintenance of the pharate larval diapause in this species.     

A Role for Ecdysteroids in the Induction and Maintenance of the Pharate First Instar Diapause of the Gypsy Moth,Lymantria dispar

Several lines of evidence suggest a novel regulatory mechanism for diapause regulation in the gypsy moth. We propose that ecdysteroids play a role in the induction and maintenance of the pharate first instar larval diapause in this species. A 55 kDa gut protein that is indicative of diapause is expressed in intact and neck-ligated pharate larvae but is not expressed when a ligature is placed posterior to the prothorax, site of the prothoracic gland. Guts cultured in vitro for 12 h cease to synthesize the 55 kDa protein, but synthesis of the protein resumes if the culture medium is enriched with a prothorax extract from pharate larvae or a prothoracic gland extract from fifth instar larvae. Injection of 20-hydroxyecdysone or the ecdysteroid agonist, RH-5992, into isolated abdomens stimulates synthesis of the diapause-specific 55 kDa protein, suggesting that the essential factor from the prothorax is an ecdysteroid. KK-42, an imidazole derivative known to inhibit ecdysteroid biosynthesis, averts diapause when applied to prediapausing pharate first instar larvae, but this effect can be countered by application of 20-hydroxyecdysone or RH-5992, i.e. KK-42 treated pharate larvae that are exposed to an ecdysteroid or RH-5992 readily enter diapause. A chilling period (120 days at 5 degrees C) is normally adequate to prompt an immediate termination of diapause when pharate larvae are transferred to 25 degrees C, but if such larvae are held in hanging drop cultures with ecdysteroids they fail to terminate diapause. Together, these results suggest that ecdysteroids are essential for the induction and maintenance of diapause and imply that a drop in the ecdysteroid titer is essential for diapause termination. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth,Lymantria dispar

During the embryonic (pharate first instar) diapause of the gypsy moth, Lymantria dispar, a 55 kDa protein is highly up-regulated in the gut. We now identify that protein as hemolin, an immune protein in the immunoglobulin superfamily. We isolated a gypsy moth hemolin cDNA and demonstrated a high degree of similarity with hemolins from three other moth species. Hemolin mRNA levels increased at the time of diapause initiation and remained high throughout the mandatory period of chilling required to terminate diapause in this species, and then dropped in late diapause. This mRNA pattern reflects the pattern of protein synthesis. These results suggest that hemolin is developmentally up-regulated in the gut during diapause. Diapause in this species can be prevented using KK-42, an imidazole derivative known to inhibit ecdysteroid biosynthesis, and gypsy moths treated in this manner failed to elevate hemolin mRNA. Conversely, this diapause appears to be initiated and maintained by the steroid hormone, 20-hydroxyecdysone, and the addition of 20-hydroxyecdysone to the culture medium elevated hemolin mRNA in the gut. Our results thus indicate a role for 20-hydroxyecdysone in the elevation of hemolin mRNA during diapause. Presumably, hemolin functions to protect the gypsy moth from microbial infection during its long, overwintering diapause.     

European field collections and Canadian releases ofCeranthia samarensis (Dipt.: Tachinidae), a parasitoid of the gypsy moth

A programme to collect, import and release into Canada the gypsy moth parasitoid,Ceranthia samarensis (Diptera: Tachinidae) is described. The parasitoid's potential for biological control in Canada is also discussed. The parasitoid was collected in Europe by exposing experimental gypsy moth larvae in areas where local gypsy moth populations were at low densities. Following field exposure, the host larvae were returned to the laboratory and parasitoids reared from them. This technique has shown thatC. samarensis is the suffers 7–16% hyperparasitism. From 83–90% of theC. samarensis typically enter diapause as pharate adults within the puparia. Laboratory tests of post-exposure host rearing conditions indicate that constant temperatures disrupt the normal parasitoid diapause and that this effect can not be offset by use of either static long or short photoperiods or natural daylengths. Shipping and cold-storage procedures for puparia are described. Post-storage time to emergence of adultC. samarensis decreased with longer cold storage periods and with higher post-storage incubation temperatures. Emergence requires 112 degree-days above a threshold of 8°C after a period of at least 8 months cold storage. Releases of adultC. samarensis into field cages at four locations in southern Ontario are documented. While dissection of host larvae from the field cages has failed so far to demonstrate evidence of parasitism, we remain hopeful that some establishment of the parasitoid has occurred.      

Effect of KK-42 on growth,development, molting,and metamorphosis of the european corn borer,Ostrinia nubilalis (Hübner)

KK-42 (1-benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl]imidazole), administered by feeding, delayed the growth and development of nondiapause-bound and diapause-bound Ostrinia nubilalis larvae and increased the length of the instar. At doses of 80–240 ppm, 62–100% of nondiapause-bound fourth instars precociously pupated or remained as fourth instars, while 52–100% of diapause-bound fourth instars did not molt to the fifth instar. Injection of these nondiapause- and diapause-bound KK-42-fed fourth instars with ecdysone elicited a molt and resulted in the production of larval-pupal intermediates. When mature fourth instar controls were similarly injected, they molted into normal fifth instars. These results support the view that KK-42 delays/inhibits ecdysteroid production. Both eupyrene and apyrene spermiogenesis were prematurely initiated in nondiapause-bound fourth instars that were fed on medium containing 160 ppm KK-42. Fenoxycarb, a potent juvenile hormone mimic, rescued nondiapause-bound fourth instars from precocious pupation. All fenoxycarbtreated larvae either molted to the fifth instar or remained as fourth instars and eventually died. These results support the view that treatment with KK-42 inhibits JH production. When KK-42 treatment was begun in the third instar, a considerable number of nondiapause-bound and some diapause-bound third instars precociously molted to the fifth instar. There was a correlation between weight and the incidence of precocious molting in that third instars destined to skip the fourth instar attained a weight, as pharate fifth instars, of two to three times more than pharate fourth instar controls. Similarly, fourth instars that were destined to undergo precocious pupation attained a weight, as pharate pupae, that was approximately two times more than pharate fifth instar controls. More potent analogues of KK-42 may prove useful in controlling populations of 0. nubilalis by interfering with their growth, development, and metamorphosis. © 1995 Witey-Liss, Inc.

1 This article is a US Government work and, as such, is, in the public domain in the United States of America.

     

Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species

The imidazole derivative KK-42 is a synthetic insect growth regulator known previously to be capable of averting embryonic diapause in several Lepidoptera, but whether it also affects diapauses occurring in other developmental stages remains unknown. In the present study, we examined the effect of KK-42 on pupal diapause in two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and in one species of Diptera, the flesh fly Sarcophaga crassipalpis. In A. pernyi, KK-42 delayed pupal diapause termination under the long day conditions that normally break diapause in this species. Likewise, in H. zea, KK-42 delayed termination of pupal diapause, a diapause that, in this species, is normally broken by high temperature. KK-42-treated pupae of these two species eventually terminated diapause and successfully emerged as adults, but the timing of diapause termination was significantly delayed. KK-42 also significantly increased the incidence of pupal diapause in H. zea and S. crassipalpis when administered to larvae that were environmentally programmed for diapause, but it was not capable of inducing pupal diapause in H. zea if larvae were reared under environmental conditions that do not normally evoke the diapause response. Experiments with H. zea showed that the effect of KK-42 on pupal diapause was dose- and stage-dependent, but not temperature-dependent. Results presented here are consistent with a link between KK-42 and the ecdysteroid signaling pathway that regulates pupal diapause.     

Influence of the braconid Glyptapanteles liparidis on the juvenile hormone titer of its larval host,the gypsy moth,Lymantria dispar

The increase in the juvenile hormone (JH) III titer in the hemolymph of Lymantria dispar larvae that were parasitized by the endoparasitoid braconid, Glyptapanteles liparidis, during the host's premolt to third instar, coincided with the molt of the parasitoid larvae to the second instar between day 5 and 7 of the fourth host instar. It reached a maximum mean value of 89 pmol/ml on day 7 of the fifth instar while it remained below 1 pmol/ml in unparasitized larvae. Only newly molted fifth instar hosts showed a low JH III titer similar to that of the unparasitized larvae. JH II, which is the predominant JH homologue in unparasitized gypsy moth larvae, also increased relative to controls in the last two samples (days 7 and 9) from parasitized fourth and fifth instars. Compared to unparasitized larvae, a generally reduced activity of JH esterase (JHE) was found in parasitized larvae throughout both larval stages. The reduction in enzyme activity at the beginning and at the end of each instar, when the JHE activity in unparasitized larvae was high, may be in part responsible for the increased JH II and JH III titers in parasitized larvae. Ester hydrolysis was the only pathway of JH metabolism in the hemolymph of unparasitized and parasitized gypsy moth larvae as detected by chromatographic assays. © 1996 Wiley-Liss, Inc.     

Hostplant,larval age,and feeding behavior influence midgut pH in the gypsy moth (Lymantria dispar)

Summary The midgut pH of late instar gypsy moth (Lymantria dispar L.) larvae is strongly alkaline, and varies with diet, larval stadium, and time since feeding. Midgut pH rises with time since feeding, and does so more quickly, reaching greater maximum values, on some diets than others. Leaf tissues of 23 tree species resist increases in alkalinity differentially; this trait and differing initial leaf pH may explain the impact of diet on gut pH. Third instar larvae may have gut conditions favorable for tannin-protein binding shortly after ingesting certain foods, but with time midgut alkalinity becomes great enough to dissociate tannin-protein complexes. Older instars rarely exhibit gut pHs low enough to permit tannin activity. Alkaline gut conditions may explain the gypsy moth's ability to feed on many tanniniferous plant species, especially in later instars. Consequences for pathogen effectiveness are discussed.     

Juvenile hormone esterases in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

Haemolymph levels of juvenile hormone esterase, 1-naphthyl acetate esterase, and juvenile hormone were measured in synchronously staged diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis. Juvenile hormone esterase levels were monitored using juvenile hormone I as a substrate while juvenile hormone titres were measured with the Galleria bioassay. Haemolymph of nondiapause larvae showed two peaks of juvenile hormone hydrolytic activity: one near the end of the feeding phase and a smaller one just prior to pupal ecdysis. These peaks of enzyme activity correlated well with the low levels of haemolymph juvenile hormone. Juvenile hormone titres were high early in the stadium then showed a second peak during the prepupal stage coinciding with low esterase activity. Diapause haemolymph had peak juvenile hormone esterase activity nearly 4 times the nondiapause level, reaching a peak near the end of the feeding phase. Diapause-destined larvae retained high juvenile hormone titres even during the rise of the high esterase levels. 1-naphthyl acetate esterase levels did not correlate with the juvenile hormone esterase levels in either the diapause or nondiapause haemolymph. High levels of 1-naphthyl acetate esterase activity were associated with moulting periods.     

Induction of Non-diapause Eggs by Imidazole Derivative KK-42 in the Diapause Type of Bombyx mori Silkworm

The injection of an imidazole compound, KK-42, into fifth instar larvae of a silkworm (Bombyx mori, Daizo strain), which had been destined to produce diapause eggs, induced the moths to lay non-diapause eggs. The critical period for KK-42 injection in the induction of non-diapause eggs was 24 h to 72 h after the fourth ecdysis. Topical application of KK-42 to 48 h-old fifth instar larvae also induced non-diapause eggs.     

Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris

At 25 degrees C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at approximately 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of approximately 1.1 ng/ml was maintained even 30-60 days into the instar, when they were in diapause. Ecdysteroid titers remained approximately 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers.     

Diapause development in Bombyx eggs in relation to ‘esterase A’ activity

Diapause is broken by hydrochloric acid treatment and also terminated by long chilling of eggs in the silkworm, Bombyx mori. One of esterases in silkworm eggs named ‘esterase A’ is closely related to diapause of this insect.Hydrochloric acid treatment of diapause eggs induced a prompt elevation of esterase A activity. The elevation was observed within 30 min after treatment. This HCl treatment effectively stimulated the eggs to hatch. This indicates that the increase of esterase A activity is correlated with an active resumption of morphogenesis.The question was examined of whether chilling also increases esterase A activity or not. It was found that chilling also caused an increase of esterase A activity. This increase occurred before the re-appearance of glycogen in eggs, which indicates the termination of diapause in this insect. In fact, the establishment of hatchability in chilled eggs was observed after esterase A activity has reached the maximum level. Thus the increase of esterase A activity could be regarded as associated with the termination of diapause per se but not with the subsequent process of post-diapause development. This change in esterase A activity was observed only in chilled diapause eggs and was not observed in diapause eggs without chilling and non-diapause eggs.These results suggest that the increase of esterase A activity during chilling may be a kind of activity that occurs during the diapause stage in preparation for resumption of morphogenesis or diapause development.     

Endogenous and induced monooxygenase activity in gypsy moth larvae feeding on natural and artificial diets

NADPH oxidase activity was measured in third to sixth instar gypsy moth larvae fed oak or pine foliage. Activity levels ranged from 400 to 1,900 pmol NADPH oxidized/min/mg microsomal protein, but enzyme activity was not correlated with host plant ingested. Similarly, activity levels in larvae fed diets containing inducers, such as the terpenoid α-pinene or pentamethylbenzene, ranged from 700 to 1,500 pmol NADPH oxidized/min/mg protein, levels that were comparable to those measured for larvae fed control diets. O-demethylase activity in older instar gypsy moth larvae fed pine averaged 109 pmol p-nitrophenol/min/mg protein, and activity levels in those fed diet containing α-pinene ranged from 22 to 55 pmol/min/mg protein. Although statistically significant, these induced O-demethylase levels are well below those observed for Heliothis zea larvae. Our findings indicate that monooxygenases play a minor, if any, role in the ability of later instar gypsy moth larvae to develop successfully on pine foliage.     

Controlled ecdysteroid accumulation in eggs of the silkworm,Bombyx mori,by an imidazole compound (KK-42), and embryogenesis in these eggs

An imidazole compound (KK-42), a potent inhibitor of ecdysone synthesis, was applied to the female pharate adult of the silkworm, Bombyx mori, to control ecdysteroid accumulation in developing ovaries and mature eggs. KK-42 applied on day 2 or later completely suppressed an increase in ecdysteroid content in developing ovaries. The inhibitory action of KK-42 was restricted to vitellogenic follicles, i.e., those in which active ecdysteroid synthesis is occurring. Ecdysteroid content in the mature eggs of moths remained at the level accumulated in ovaries before KK-42 application. Thus, KK-42 was shown to be a novel agent to suppress the ecdysteroid accumulation in eggs. Eggs containing different amounts of ecdysteroids showed different levels of embryonic development. About 80% of the eggs which contained less than 10 ng free ecdysteroids/g eggs were not fertilized. More than 80% of the eggs containing less than 40 ng/g eggs of free ecdysteroids initiated embryogenesis but failed to hatch. Larvae hatched from almost all eggs which accumulated free ecdysteroids of more than 150 ng/g. Thus, maternal ecdysteroids appear to be required at different titers for fertilization, embryogenesis, and hatching of the silkworm larvae. © 1994 Wiley-Liss, Inc.     

Control of diapause in codling moth larvae

Diapause in a New Zealand strain of codling moth (Cydia pomonella Linnaeus [Lepidoptera: Olethreutidae]) was induced in larvae by photoperiods of 15 h or less. Once diapause had been initiated, it could not be terminated by any combination of conditions tested for at least 20 days after cocooning. In diapausing larvae a low rate of pupation occurred at 25 °C under a long day (18 h) photoperiod. A high rate of pupation was achieved under a long day regime when larvae were decocooned, and provided with apple as nourishment. Diapause could be terminated predictably in 94–100% of larvae by 1) conditioning at 15 °C and constant darkness for periods of 40–100 days, then 2) chilling at 2±2 °C and constant darkness for 20–50 days followed by 3) any post-chill condition periods at 25 °C, 18 h photoperiod. Complete diapause termination was achieved when 100 days conditioning was followed by 30 days or 50 days post-chill period. Under these conditions, 76% termination occurred in the post-chill period after 10 days, and 93% after 25 days.To terminate diapause in codling moth larvae, we recommend that a 100 days conditioning followed by 30 days chilling and 50 days post chilling periods be used.     

Haemocytes of the pink bollworm, Pectinophora gossypiella, during larval development and diapause

Seven types of haemocytes were observed in the last larval instar of the pink bollworm, Pectinophora gossypiella (Saunders): prohaemocytes, plasmatocytes, granular haemocytes, spherule cells, adipohaemocytes, oenocytoids, and podocytes. Total and differential haemocyte counts made from diapausing and non-diapausing larvae showed that during diapause there was a significant reduction in the numbers of all haemocyte types. Upon termination of diapause, the haemocyte level increased. There were no significant differences in the level of haemocytes in the pharate pupae that developed from diapause or non-diapause type larvae, except in the case of adipohaemocytes, which were three times as prevalent in pharate pupae from diapausing larvae. Functional aspects of various types of haemocytes are discussed, and it is suggested that the lower haemocyte level observed during diapause is the result of lower metabolic activity.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.