Post-diapause Development Research Articles

A gene catalogue for post-diapause development of an anhydrobiotic arthropod Artemia franciscana

BackgroundDiapause is a reversible state of developmental suspension and found among diverse taxa, from plants to animals, including marsupials and some other mammals. Although previous work has accumulated ample data, the molecular mechanism underlying diapause and reactivation from it remain elusive.ResultsUsing Artemia franciscana, a model organism to study the development of post-diapause embryos in Arthropod, we sequenced random clones up to a total of 28,039 ESTs from four cDNA libraries made from dehydrated cysts and three time points after rehydration/reactivation, which were assembled into 8,018 unigene clusters. We identified 324 differentially-expressed genes (DEGs, P < 0.05) based on pairwise comparisons of the four cDNA libraries. We identified a group of genes that are involved in an anti-water-deficit system, including proteases, protease inhibitors, heat shock proteins, and several novel members of the late embryogenesis abundant (LEA) protein family. In addition, we classified most of the up-regulated genes after cyst reactivation into metabolism, biosynthesis, transcription, and translation, and this result is consistent with the rapid development of the embryo. Some of the specific expressions of DEGs were confirmed experimentally based on quantitative real-time PCR.ConclusionWe found that the first 5-hour period after rehydration is most important for embryonic reactivation of Artemia. As the total number of expressed genes increases significantly, the majority of DEGs were also identified in this period, including a group of water-deficient-induced genes. A group of genes with similar functions have been described in plant seeds; for instance, one of the novel LEA members shares ~70% amino-acid identity with an Arabidopsis EM (embryonic abundant) protein, the closest animal relative to plant LEA families identified thus far. Our findings also suggested that not only nutrition, but also mRNAs are produced and stored during cyst formation to support rapid development after reactivation.

Diapause and prolonged development in the embryo and their ecological significance in two cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata

The seasonal timing mechanism of egg hatching was examined in two cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata, with different but overlapping geographical distributions. These species lay eggs in summer, and nymphs hatch in the summer of the following year after egg durations of 10–12 months. When eggs were maintained at 25 °C from oviposition, both the species entered embryonic diapause within 60 days irrespective of photoperiod, but at different developmental stages between the two species. The optimal temperature for diapause development was approximately 15 °C in both the species. The development rate for postdiapause morphogenesis increased linearly with temperature in the range of 20–27.5 °C in C. facialis, and of 15–25 °C in G. nigrofuscata. The lower development threshold and the sum of effective temperatures were computed as 14.3 °C and 715.3 day-degrees in C. facialis and 12.1 °C and 566.6 day-degrees in G. nigrofuscata, respectively. The hatching dates predicted by these large thermal constants accorded with the hatching dates observed in the field, i.e., late June and mid-July in G. nigrofuscata and C. facialis, respectively. Therefore, the high thermal requirements for postdiapause development compel the cicadas to hatch in summer.

ArHsp22, a developmentally regulated small heat shock protein produced in diapause‐destined Artemia embryos, is stress inducible in adults

Diapause embryos of the crustacean Artemia franciscana exhibit extreme stress tolerance, a property thought to involve molecular chaperones known as small heat shock proteins. To further explore this idea, the structure, function and synthesis of ArHsp22, an Artemia small heat shock protein, were characterized. ArHsp22 contains amino-terminal WXDPF motifs, an alpha-crystallin domain with a highly conserved arginine, and a carboxy-terminal I/VXI/V motif, all typical of small heat shock proteins. ArHsp22 formed large oligomers and exhibited molecular chaperone activity in vitro, protecting citrate synthase and insulin from denaturation. Quantitative PCR and immunoprobing of western blots revealed that ArHsp22 synthesis is restricted to diapause-destined Artemia embryos and that the protein is degraded during post-diapause development. ArHsp22 was observed in cyst nuclei, a location shared by p26 but not ArHsp21, which are two other diapause-specific Artemia small heat shock proteins. ArHsp22 production was enhanced by thermal stress, but only in adults, thus representing the first crustacean small heat shock protein whose synthesis is known to be both developmentally regulated and stress inducible. The results demonstrate that expression of the gene for ArHsp22 is modulated by multiple cues that vary with life history stage. Such findings are of importance in understanding diapause maintenance in Artemia embryos and the survival of adult animals experiencing environmental insult.

Factors affecting the natural duration of diapause and post-diapause development in the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae)

Sesamia nonagrioides responds to photoperiod for diapause termination at high temperature with a Type II response curve, and only photoperiods longer than 12:12 terminate diapause. However, these photoperiods never occur in the field when diapausing larvae are competent to terminate diapause. Under a temperature similar to the natural field temperatures diapause terminates spontaneously in approximately 4 months, which ensures that the larvae reach the middle of winter without pupation. S. nonagrioides larvae pupate after going through a specific number of light–dark cycles or days: the required day number (RDN) for diapause completion. This RDN could be modulated by temperature but more research is necessary to clarify this point. In post-diapause development, when a suitable temperature threshold is considered, the absolute accumulation of heat is more important than whether the temperature received is fluctuating or constant. The temperature threshold for diapause and post-diapause development was lower than the temperature threshold of larvae in continuous development. This is important for adjusting phenological models in S. nonagrioides and in other species, and may explain why in many cases adults appear in the field when the supposed temperature threshold for development has not been attained.

ArHsp21, a developmentally regulated small heat-shock protein synthesized in diapausing embryos of Artemia franciscana

Embryos of the crustacean, Artemia franciscana, undergo alternative developmental pathways, producing either larvae or encysted embryos (cysts). The cysts enter diapause, characterized by exceptionally high resistance to environmental stress, a condition thought to involve the sHSP (small heat-shock protein), p26. Subtractive hybridization has revealed another sHSP, termed ArHsp21, in diapause-destined Artemia embryos. ArHsp21 shares sequence similarity with p26 and sHSPs from other organisms, especially in the alpha-crystallin domain. ArHsp21 is the product of a single gene and its synthesis occurred exclusively in diapause-destined embryos. Specifically, ArHsp21 mRNA appeared 2 days post-fertilization, followed 1 day later by the protein, and then increased until embryo release at day 5. No ArHsp21 protein was detected in embryos developing directly into larvae, although there was a small amount of mRNA at 3 days post-fertilization. The protein was degraded during post-diapause development and had disappeared completely from second instar larvae. ArHsp21 formed large oligomers in encysted embryos and transformed bacteria. When purified from bacteria, ArHsp21 functioned as a molecular chaperone in vitro, preventing heat-induced aggregation of citrate synthase and reduction-driven denaturation of insulin. Sequence characteristics, synthesis patterns and functional properties demonstrate clearly that ArHsp21 is an sHSP able to chaperone other proteins and contribute to stress tolerance during diapause. As such, ArHsp21 would augment p26 chaperone activity and it may also possess novel activities that benefit Artemia embryos exposed to stress.

Post-diapause development and multimodal oviposition activity of arrowhead scale, Unaspis yanonensis (Homoptera: Diaspididae) and its modeling

The longevity and fecundity of overwintered Unaspis yanonensis Kuwana (Homoptera: Diaspididae) were examined at constant temperature 24°C, and its periodical oviposition activity was modeled. Adult females collected in early November showed abnormal post-diapause development; prolonged pre-oviposition period, not producing progenies and much lower fecundity. All females collected from early December through early March showed normal post-diapause development and had <20 d of pre-oviposition period and <80 d of longevity. Overwintered U. yanonensis showed a periodical oviposition activity with a maximum 4 times of oviposition cycle during their life time, and the pattern was flexibly fitted to a 4-peak Gaussian function. The average peak timings (parameter a) occurred at 5.5, 25.2, 41.6, and 56.4 d for the 1st, 2nd, 3rd, and 4th peak from the start of oviposition, respectively. The height of the peaks (parameter b) gradually decreased from 14.9 for the 1st peak to 5.0 for the 4th peak. The parameter values of the 4-peak equation were successfully converted to degree-days unit with a lower threshold temperature 13°C for field validation. The model predicted the 1st oviposition peak of overwintered U. yanonensis very well (<282 DD from 1 January), while showed large discrepancy with actual data in the later peak period. A 3-modal oviposition curve that includes the individual variations of oviposition activity of U. yanonensis females with removing the 4th cycle showed a better shape fitting ability in the later peak period. Further, application and improvement of the multi-peak model were discussed.

Regulation of gonad development and respiratory metabolism associated with food availability and reproductive diapause in the rice bug Leptocorisa chinensis

Food has an influence on many life history traits related to dormancy in insects. In our previous study with the rice bug Leptocorisa chinensis (Dallas) (Hemiptera: Alydidae), diapausing females transferred to conditions physically favorable for promoting the gonad development required food intake to resume gonad development, whereas males did not. These differences in response to food between males and females lead to two questions: (1) Was diapause in the starved females completed? (2) Were the starved males that resumed gonad development at the same physiological status as fed males? We tested these questions with two physiological indicators: gonad status and respiratory rate. Results indicate that starved females are able to complete diapause, but show depressed respiration relative to well-fed insects in diapause. Similar to females, starved males that resumed postdiapause gonad development also had depressed respiratory rate, and hence physiological status is presumed to be different between starved and fed individuals. In this study, it was also found that the photoperiodic signal is storable, whereas the food signal acts directly. The adaptive significance of regulation of gonad development and respiratory metabolism in relation to phenology of suitable host plant and reproductive strategy is discussed in both sexes.

Host plant and latitude‐related diapause variation in Rhagoletis pomonella: a test for multifaceted life history adaptation on different stages of diapause development

Variation in the overwintering pupal diapause of Rhagoletis pomonella appears to adapt sympatric populations of the fly to seasonal differences in the fruiting times of their host plants, generating ecological reproductive isolation. Here, we investigate what aspects of diapause development are differentially affected (1) by comparing the propensities of apple vs. hawthorn-infesting host races of R. pomonella to forgo an initially deep diapause and directly develop into adults, and (2) by determining the chronological order that R. pomonella races and sibling species break diapause and eclose when reared under standardized environmental conditions. The results imply that factors affecting initial diapause depth (and/or differential mortality during the prewintering period) and those determining the timing of diapause termination or rates of post-diapause development are both under differential selection and are to some degree genetically uncoupled in flies. The modular nature of diapause life history adaptation in Rhagoletis suggests that phenology may involve multiple genetic changes and represent a stronger ecological barrier separating phytophagous specialists than is generally appreciated.

Sexual differences in the crucial environmental factors for the timing of postdiapause development in the rice bug Leptocorisa chinensis

Although the regulation of reproductive diapause by environmental factors has been extensively studied in a variety of insect orders, sexual differences in the regulation of diapause are still poorly understood. We examined the effects of environmental factors including photoperiod, temperature and food on the regulation of reproductive diapause in both sexes of the rice bug Leptocorisa chinensis (Dallas) (Hemiptera: Alydidae) in the laboratory and under natural conditions. There was no significant difference in critical daylength (CDL) for diapause induction between the sexes. The CDL corresponded to the daylength in which development of the gonads was suppressed in half the insects caught in the field. Both sexes enter diapause at the same time in successive seasons. In diapause insects, on the other hand, there were sexual differences not only in retention of responsiveness to photoperiod and temperature but also in the food requirement, and these sexual differences lead to different timing of the resumption of reproductive development after overwintering.

Antioxidant defense in mitochondria during diapause and postdiapause development of European corn borer (Ostrinia nubilalis, Hubn.)

Antioxidant enzymes (CAT, catalase; GPx, selenium nondependent glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; DHAR, dehydroascorbate reductase) were determined in the mitochondria of diapausing and non-diapausing larvae and pupae of both diapausing and non-diapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). CAT, GST, and DHAR activity in mitochondria of diapausing larvae were reduced compared to non-diapausing larvae. Pupae of diapaused-larvae possessed lower GST, but higher DHAR activities compared to pupae of non-diapaused individuals. Comparison between larvae and pupae revealed lower GPx activity in the mitochondria of pupae. CAT activity in the mitochondria of pupae was higher compared to diapausing larvae, but lower than in non-diapausing ones. Correlation and canonical discriminant analyses revealed different antioxidant enzyme compositions for a particular stage and developmental pattern. Our results show that antioxidant enzymes have a similar role in the regulation of energetics in mitochondria as that in diapause and metamorphosis.

Effect of Cold Exposure and Photoperiod on Diapause Termination of the Predatory MiteEuseius finlandicus(Acari: Phytoseiidae)

The effect of cold storage and photoperiod on diapause termination of the predatory mite Euseius finlandicus was studied under laboratory conditions. Females in diapause that developed under a short-day photoperiod of L:D 8:16 h and 20°C were exposed for 1–13 wk to 5°C and subsequently maintained under L:D 8:16 h and 20°C, after which the percentage of females terminating diapause was recorded. After 1–4 wk at 5°C, the number of days required for diapause termination in 50% of females (TA50) was similar to that of the control mites maintained continuously at L:D 8:16 h and 20°C. This indicates that cold is not a prerequisite for the completion of diapause development. Furthermore, when mites were maintained for longer periods (7, 10, or 13 wk) at 5°C, TA50 was significantly longer than in the control mites. This may indicate an adverse effect of cold storage on the completion of diapause development or on postdiapause development. However, the longer exposures to cold resulted in synchronous diapause termination. When diapause females were maintained under a series of different photoperiods and 20°C without previous cold exposure, diapause was terminated rapidly under the photoperiods of L:D 16:8, 14:10, and 13:11 h. In contrast, under the shorter-day photoperiods of LD 8:16, 10:14, and 12:12 h, diapause was maintained and eventually ended spontaneously after a long period of time. These results may be useful for the long-term storage of this predatory mite, which could be required for use in biological control programs.

Effect of Cold Exposure and Photoperiod on Diapause Termination of the Predatory Mite &lt;I&gt;Euseius finlandicus&lt;/I&gt; (Acari: Phytoseiidae)

The effect of cold storage and photoperiod on diapause termination of the predatory mite Euseius finlandicus was studied under laboratory conditions. Females in diapause that developed under a short-day photoperiod of L:D 8:16 h and 20°C were exposed for 1–13 wk to 5°C and subsequently maintained under L:D 8:16 h and 20°C, after which the percentage of females terminating diapause was recorded. After 1–4 wk at 5°C, the number of days required for diapause termination in 50% of females (TA50) was similar to that of the control mites maintained continuously at L:D 8:16 h and 20°C. This indicates that cold is not a prerequisite for the completion of diapause development. Furthermore, when mites were maintained for longer periods (7, 10, or 13 wk) at 5°C, TA50 was significantly longer than in the control mites. This may indicate an adverse effect of cold storage on the completion of diapause development or on postdiapause development. However, the longer exposures to cold resulted in synchronous diapause termination. When diapause females were maintained under a series of different photoperiods and 20°C without previous cold exposure, diapause was terminated rapidly under the photoperiods of L:D 16:8, 14:10, and 13:11 h. In contrast, under the shorter-day photoperiods of LD 8:16, 10:14, and 12:12 h, diapause was maintained and eventually ended spontaneously after a long period of time. These results may be useful for the long-term storage of this predatory mite, which could be required for use in biological control programs.

Thermal history influences diapause development in the solitary bee Megachile rotundata

Respiration rate, time to pupation and the expression patterns of selected genes were examined during the diapause to post-diapause transition in the alfalfa leafcutting bee, Megachile rotundata held at constant 4 °C in winter storage. Respiration quotients were at or below 0.7 from December to May and then increased to over 0.8 in June and July. The time required for prepupae to reach the pupal stage following transfer to 29 °C decreased from 23 days in December to 10 days in July. HSP70 was expressed at a consistently high level in all the diapausing prepupae stored at 4 °C. In contrast, we demonstrated previously that HSP70 expression in diapausing prepupae maintained under field conditions began decreasing after December and was expressed at trace levels in the June samples. Transferring prepupae stored at 4 to 25 °C at monthly intervals from December to July induced a significant decrease in HSP70. Levels of HSC70 showed no changes during the transition to post-diapause development in prepupae maintained at 4 °C. Transferring the prepupae to 25 °C during the April–June time interval elicited an increase in HSC70 expression. HSP90 was expressed at a consistent level in prepupae stored at 4 °C but decreased to very low levels after being transferred to 25 °C in December–February prepupae: no decrease was noted in the April–July prepupae. Actin was expressed at trace levels in the diapausing prepupae maintained at 4 °C and increased slightly in the post-diapausing pupae. Transferring prepupae stored at 4 to 25 °C at monthly intervals from December to July induced an increase in actin expression. These results indicate that the level of gene expression for selected genes in diapausing and post-diapause bees is highly influenced by their thermal history.

Methionine‐rich storage protein gene in the rice stem borer, Chilo suppressalis, is expressed during diapause in response to cold acclimation

Gene expressions of acclimatized and non-acclimatized diapausing larvae were examined in Chilo suppressalis using a subtraction technique. A gene encoding a methionine-rich storage protein, CsSP1, was cloned and its complete cDNA sequence was determined. Potentially, CsSP1 encoded a 758-amino acid protein, with a calculated molecular weight of 88.8 kDa. The expression level of CsSP1 was higher in nondiapausing larvae than in diapausing ones. The CsSP1 expression was up-regulated in diapausing larvae when the temperature of cold acclimation was shifted to 5 degrees C. The up-regulated level was maintained at 40 days after incubation at 5 degrees C. In nondiapausing larvae, CsSP1 expression was down-regulated when the temperature was below developmental zero. Involvement of CsSP1 in diapause, cold tolerance acquisition and postdiapause development in C. suppressalis is discussed.

Characterization of the microtubule proteome during post-diapause development of Artemia franciscana

The microtubule proteome encompasses tubulin and a diverse group of proteins which associate with tubulin upon microtubule formation. These proteins either determine microtubule organization and function or their activity is influenced by microtubule association. To characterize the microtubule proteome in Artemia franciscana, tubulin assembly was induced with taxol in vitro after 0 and 12 h of post-diapause development. Proteins obtained by extraction of microtubules with 0.5 M NaCl were electrophoresed in two-dimensional gels and analyzed by mass spectrometry. Fifty-five proteins were identified with 10 of these occurring at both developmental stages, and multiple isoforms were observed for some proteins of the Artemia proteome. Their functions include roles in membrane transport, metabolism, chaperoning and protein synthesis, thus reflecting physiological properties of encysted Artemia such as stress resistance and the ability to rapidly initiate post-diapause development. For example, chaperones may protect tubulin during encystment and facilitate folding in metabolically active embryos. Additionally, the interaction of metabolic enzymes with microtubules funnels reaction intermediates, potentially enhancing efficiency within biochemical processes. This study represents the first systematic characterization of a crustacean microtubule proteome. Although it is difficult to be certain that all protein associations documented herein occur in vivo, the results suggest how protein–protein interactions contribute to cytoplasmic organization while implying how Artemia embryos resist stress and remain capable of development once diapause terminates.

Six Years after the Commercial Introduction of Bt Maize in Spain: Field Evaluation, Impact and Future Prospects

We carried out a 6-year-field evaluation to assess potential hazards of growing Compa, a transgenic Bt maize variety based on the transformation event CG 00256-176. Two categories of hazards were investigated: the potential of the target corn borer Sesamia nonagrioides to evolve resistance to Bt maize and effects on non-target organisms. In order to address the first hazard, dispersal capacity of the corn borer was measured and our results indicated that larvae move to plants other than those onto which the female oviposited - even to plants in adjacent rows - in remarkable numbers and they do so mostly at a mature age, suggesting that mixing Bt and non-Bt seeds in the same field would not be a very useful deployment strategy to delay/prevent resistance. In addition, adults move among fields to mate and males may do so for up to 400 m. Three different aspects of potential non-target effects were investigated: sub-lethal effects on the target S. nonagrioides, effects on non-target maize pests, and effects on maize-dwelling predators. Larvae collected in Bt fields at later growth stages, in which event 176 Bt maize expresses Bt toxin at sub-lethal concentrations, had longer diapause and post-diapause development than larvae collected in non-Bt fields, a feature that might lead to a certain isolation between populations in both type of fields and accelerate Bt resistance evolution. Transgenic maize did not have a negative impact on non-target pests in the field; more aphids and leafhoppers but similar numbers of cutworms and wireworms were counted in Bt versus non-Bt fields; in any case differences in damage or yield were recorded. We observed no difference in the numbers of the most relevant predators in fields containing transgenic or no transgenic maize.

The effect of pasture improvement and burning on Orthoptera populations of Culm grasslands in northwest Devon, UK

Very little research has been conducted into the impact of grassland management on insect populations of Culm grasslands located in northwest Devon, UK. This paper presents the effects of agricultural improvement and burning on Orthoptera populations of these wet, heathy pastures at Dunsdon National Nature Reserve. The results from this 2-year study should aid conservation management of this important UK habitat. Orthoptera populations and habitat variables of improved (drained and cropped from 1840-1875) and unimproved pastures, were monitored in 2002 and 2003. There was no significant difference in Orthoptera abundance between pasture cropped from 1840-1875 and unimproved grassland, suggesting that mobile grasshopper species such as the meadow grasshopper Chorthippus parallelus, recolonised the improved swards from surrounding pastures, after a lengthy period of arable cropping in the 1800s. Winter burning led to higher numbers of Orthoptera in the post-burn year, perhaps due to the reduced sward height and biomass on the burnt swards, making them more favorable for postdiapause development.

Consequences of reproductive barriers for genealogical discordance in the European corn borer

Speciation involves the origin of trait differences that limit or prevent gene exchange and ultimately results in daughter populations that form monophyletic or exclusive genetic groups. However, for recently diverged populations or species between which reproductive isolation is often incomplete, gene genealogies will be discordant, and most regions of the genome will display nonexclusive genealogical patterns. In these situations, genome regions for which one or both species are exclusive groups may mark the footprint of recent selective sweeps. Alternatively, such regions may include or be closely linked to "speciation genes," genes involved in reproductive isolation. Therefore, comparisons of gene genealogies allow inferences about the genetic architectures of both reproductive isolation and adaptation. Contrasting genealogical relationships in sexually isolated pheromone strains of the European corn borer moth (Ostrinia nubilalis) demonstrate the relevance of this approach. Genealogies for five gene regions are discordant, and only one molecular marker, the sex-linked gene Tpi, has evidence for pheromone strain exclusivity. Tpi maps to a position on the sex chromosome that is indistinguishable from a major factor (Pdd) affecting differences in postdiapause development time. The major factor (Resp) determining male behavioral response to pheromone is also sex-linked, but maps 20-30 cM away. Exclusivity at Tpi may be a consequence of these linkage relationships because evidence from phenotypic variation in natural populations implicates both Pdd and Resp as candidates for genes involved in recent sweeps and/or reproductive isolation between strains.

Effects of temperature during chilling and pre-chilling periods on diapause and post-diapause development in a katydid, Eobiana engelhardti subtropica

In Eobiana engelhardti subtropica, early laid eggs reach the diapause stage in early autumn. For long periods before winter, the eggs are exposed to temperatures higher than their theoretical lower threshold for development. In contrast, late-laid eggs cannot reach their diapause stage before winter. Our study showed that E. e. subtropica copes with these difficulties via the thermal response involving embryonic diapause. In this katydid, the almost fully developed embryo undergoes an obligatory diapause. When diapause eggs were maintained at a temperature of 20 °C or higher, diapause persisted for a long time. Diapause was effectively terminated by temperatures ranging from 1 to 11 °C, and hatching occurred successfully at temperatures from 11 to 15 °C. In addition to the chilling temperature, pre-chilling temperature modified diapause intensity and hatching time. Diapause eggs hatched earlier after chilling when the pre-chilling temperature was lower, within a range of 14.5–25 °C. Thus, the low-temperature requirement for diapause termination prevents early laid eggs from untimely hatching in autumn, and low temperatures before and during winter decrease diapause intensity and shorten the hatching time in the following spring. When eggs were chilled before diapause, they tolerated chilling and averted diapause. Thus, even if eggs encounter low temperatures before diapause, they can hatch in the following spring.

Gene expression of heat-shock proteins ( Hsp23, Hsp70 and Hsp90) during and after larval diapause in the blow fly Lucilia sericata

Genes encoding heat-shock protein 23 ( Hsp23), Hsp70 and Hsp90 were cloned from Lucilia sericata to examine whether their expression is related to the regulation of its larval diapause. The level of all three Hsps mRNAs was consistently low irrespective of diapause status. These results indicate that expression of Hsp23, Hsp70 and Hsp90 is not regulated in response to diapause, in contrast to the flesh fly Sarcophaga crassipalpis that shows upregulation of Hsp23 and Hsp70 and downregulation of Hsp90 during its pupal diapause. On the other hand, Hsp90 transcripts were regulated developmentally in nondiapause larvae of L. sericata, i.e., they were at low levels after cessation of feeding but were considerably upregulated a day before pupariation, suggesting that Hsp90 is involved in a developmental process that occurs between the cessation of feeding and pupariation. When diapause larvae were transferred to all of the conditions that terminate diapause and allow postdiapause development, Hsp90 transcripts were promptly upregulated. These results indicate that Hsp90 may serve as an early marker to predict diapause termination in this species.