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 degrees 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 degrees 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 degrees 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 degrees 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 degrees C. Transferring the prepupae to 25 degrees 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 degrees C but decreased to very low levels after being transferred to 25 degrees 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 degrees C and increased slightly in the post-diapausing pupae. Transferring prepupae stored at 4 to 25 degrees 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.     

Post-embryonic functions of HSP90 in Tribolium castaneum include the regulation of compound eye development

Heat shock protein 90 (HSP90) belongs to a family of conserved chaperons with multiple roles in stress adaptation and development, including spermatogenesis, oogenesis and embryogenesis in insects. In the red flour beetle, Tribolium castaneum, we found that HSP90 is transiently upregulated during larval development, in prepupae, in female pupae and in adults, suggesting multiple post-embryonic roles. We found that silencing HSP90 expression by RNA interference was lethal within 10 days at all developmental stages. Titration experiments revealed that larvae were more susceptible than pupae or beetles. Interestingly, HSP90 silencing in final instar larvae resulted in abnormal pupal phenotypes lacking compound eyes and exhibiting prepupal features, suggesting developmental arrest at the prepupal stage. Our results suggest that HSP90 functions can be expanded beyond the known ones in insect embryogenesis to include roles in post-embryonic development such as the regulation of compound eye development.     

Relationships between cold hardiness and diapause, and between glycerol and free amino acid contents in overwintering larvae of the oriental corn borer, Ostrinia furnacalis

To elucidate the relationship between diapause and cold hardiness in the oriental corn borer, Ostrinia furnacalis, the levels of various substances, cold hardiness and respiration were measured in diapausing and post-diapausing overwintering larvae. Under field conditions, diapause terminated between November and January, although O(2) consumption, measured at 20 degrees C in the laboratory, remained at a high level from October to January. Glycerol content was low during October and November but greatly increased during December and January. Serine was the most abundant of the free amino acids, and its concentrations were especially high during October and November, while the concentration of alanine increased in December and January. Under laboratory conditions, glycerol levels were low in diapausing larvae, and in post-diapausing larvae that were acclimated at either high temperatures or under anaerobic conditions, while they were high in post-diapausing larvae kept under aerobic, low temperature conditions. The survival rate (cold hardiness) was strongly correlated with glycerol content but not with serine or alanine levels. These results suggest that O. furnacalis has a highly developed cold hardiness mechanism in which termination of diapause enables the larvae to increase glycerol levels when the temperature decreases.     

Expression profiling of a diapause-specific peptide (DSP) of the leaf beetle Gastrophysa atrocyanea and silencing of DSP by double-strand RNA

A diapause-specific peptide (DSP) composed of 41 amino acid residues including 6 cysteines, has been isolated from diapausing adults of the leaf beetle Gastrophysa atrocyanea. In this study, DSP was found to be expressed primarily in diapausing adults and to a minor extent in pupae, but not in eggs, larvae, or post-diapausing adults. DSP was not induced by bacterial or fungal challenge. DSP-less adults were generated by the injection of double-stranded RNA (dsRNA) corresponding to the dsp gene into pre-diapausing adults. Gene silencing induced by dsRNA was found to be a useful tool for the analysis of DSP in diapausing adults. DSP-less adults showed similar burrowing behavior and oxygen consumption as control insects suggesting that DSP is not essential for the normal onset and maintenance of diapause.     

高温条件下棉铃虫化蛹率、夏滞育率和蛹重的变化

在预蛹期,高温处理能诱导棉铃虫蛹进入夏滞育。本实验着重就33～39℃的变温下滞育蛹和未滞育蛹的失重动态进行了对比研究,同时以常温（27℃）下蛹作为参照。研究发现： 在33～39℃的变温条件下,棉铃虫化蛹率显著低于其在常温下的化蛹率,且所化蛹中有63.2%的雄性和10.9%的雌性进入高温夏滞育,其中高温滞育蛹和未滞育蛹分别都轻于正常发育蛹。化蛹后第2日至第5日期间,高温滞育蛹失重量显著低于高温未滞育蛹和正常发育蛹的失重量,分别为3.62、13.30和5.49 mg;蛹期总失重量结果与化蛹后第2～5日间蛹失重量趋势一致,高温滞育蛹、未滞育蛹和正常发育蛹失重量分别为15.60、49.35和26.30 mg。蛹失重动态研究发现高温滞育蛹在夏滞育期间其失重曲线平缓,显著低于高温未滞育蛹和正常发育蛹;高温滞育蛹滞育解除后,其失重曲线与正常发育蛹的失重趋势基本一致。结果表明,棉铃虫夏滞育蛹能通过维持低的代谢水平来度过不利环境,具有一定的生态适应意义。     

Factors affecting carbohydrate and free amino acid content in overwintering larvae of Enosima leucotaeniella

Amounts of several metabolites were measured in overwintering larvae of Enosima leucotaeniella acclimated to temperatures between -5 and 15 degrees C for 30days. In the diapausing stage, cold hardiness, as shown by the survival rate, began rising below 15 degrees C. Glycogen content decreased as the temperature decreased from 10 to 0 degrees C. Trehalose content rose as the temperature decreased from 15 to 5 degrees C, but remained unchanged as the temperature decreased from 5 and 0 degrees C. Twenty-eight free amino acids were detected in the haemolymph; levels of proline, glutamine and glutamic acid increased at high temperatures, but alanine increased at low temperatures, especially as temperature decreased from 5 to 0 degrees C. Lipid content was unchanged by the different acclimation temperatures. The effects of temperature, diapause and aerobic conditions on the levels of carbohydrates and amino acids in overwintering larvae were analyzed. Alanine levels rose at low temperature only when the larvae were in the diapausing stage. The level of trehalose rose at low temperature in both the diapausing and post-diapausing stages, although it was higher at aerobic conditions in the post-diapausing stage. These results suggest that efficient trehalose synthesis occurs under the combination of low temperature and aerobic conditions of the post-diapausing stage, so that cold hardiness in overwintering E. leucotaeniella larvae may rise to a high level in winter.     

Differential expression of two HSP70 transcripts in response to cold shock,thermoperiod, and adult diapause in the Colorado potato beetle

Partial clones for two members of Leptinotarsa decemlineata inducible 70kDa heat shock protein family (LdHSP70A and B) were developed using RT-PCR. LdHSP70A, but not LdHSP70B, was upregulated during adult diapause. The ability of L. decemlineata to express these two genes in response to subzero temperatures depended on the thermal history of the beetles. Chilling diapausing beetles increased the rate at which both LdHSP70A and B were expressed following a cold shock at -10 degrees C. Following cold shock at -10 degrees C, LdHSP70B expression peaked after 3h at 15 degrees C for chilled diapausing individuals, decreasing to near background levels by the sixth hour. In contrast, nonchilled diapausing beetles expressed their highest level of LdHSP70B only after 6h at 15 degrees C. Diapausing beetles exposed to a thermoperiod with a mean temperature of either 0 or -2.5 degrees C expressed significantly higher levels of both LdHSP70A and B than beetles exposed to constant 0 or -2.5 degrees C. These results demonstrate that the expression of LdHSP70A and B is differentially regulated in response to diapause and environmental conditioning.     

HSP70 and Genomic Stability

The 70 kDa heat shock proteins (HSP70) were initially identified by their elevated expression following hyperthermic cell stress, however, these highly conserved proteins also protect critical cellular functions from a wider range of important environmental and physiological stresses. At least one result of HSP70 expression is inhibition of stress induced caspase activation as well as downstream events in the apoptotic cell death pathway. HSP70 have been reported upregulated in tumor cells, selective inhibition of such proteins might be valuable approach to treat cancer. A recent study revealed that cells with inactivated HSP70 displayed telomere instability and high frequency of spontaneous chromosomal aberrations, indicating a possible role for HSP70 proteins in the maintenance of genomic stability.     

HSP70 and genomic stability

The 70 kDa heat shock proteins (HSP70s) were initially identified by their elevated expression following hyperthermic cell stress, however, these highly conserved proteins also protect critical cellular functions from a wider range of important environmental and physiological stresses. At least one result of HSP70 expression is inhibition of stress induced caspase activation as well as downstream events in the apoptotic cell death pathway. HSP70 have been reported upregulated in tumor cells, selective inhibition of such proteins might be valuable approach to treat cancer. A recent study revealed that cells with inactivated HSP70 displayed telomere instability and high frequency of spontaneous chromosomal aberrations, indicating a possible role for HSP70 proteins in the maintenance of genomic stability.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

Induction and termination of prepupal summer diapause in Pseudopidorus fasciata (Lepidoptera: Zygaenidae)

The seasonal life cycle of the zygaenid moth, Pseudopidorus fasciata is complicated by two different developmental arrests: a winter diapause as a fourth larval instar and a summer diapause as a prepupa in a cocoon. Both larval diapause induction and termination are under photoperiodic control. Short days induce larval diapause with a critical daylength of 13.5h and long days terminate diapause with a critical daylength of 14h. In the present study photoperiodic control of summer diapause was investigated in Pseudopidorus fasciata. Under long photoperiods ranging from LD 14:10 to LD 18:6, only part of the population entered summer diapause, the rest continued to develop. The lowest number of prepupae entered diapause at LD 14:10, followed by LD 16:8 and LD 17:7. The highest incidence of diapause occurred with photoperiods of LD 15:9 and LD 18:6. By transferring the diapausing prepupae induced by various long photoperiods (LD 14:10, LD 15:9, LD 16:8, LD 17:7, LD 18:6) to LD 13:11, 25 degrees C, the duration of diapause induced by LD 14:10 was significantly shorter than those induced by longer photoperiods. By keeping aestivating prepupae induced by LD 15:9, 28 degrees C or by natural conditions at short photoperiods (LD 11:13 and LD 13:11) and at a long photoperiod (LD 15:9), the duration of diapause at LD 15:9 was more than twice as long as than those at LD 11:13 and LD 13:11. Moreover, adult emergence was highly dispersed with a high mortality at LD 15:9 but was synchronized with low mortality at LD 11:13 and LD 13:11. When the naturally induced aestivating prepupae were kept under natural conditions, the early aestivating prepupae formed in May exhibited a long duration of diapause (mean 126 days), whereas the later-aestivating prepupae formed in July exhibited a short duration of diapause (mean 69 days). These results indicate that aestivating prepupae require short or shortening photoperiod to terminate their diapause successfully. By transferring naturally induced aestivating prepupae to 25, 28 and 30 degrees C, the duration of diapause at the high temperature of 30 degrees C was significantly longer than those at 25 and 28 degrees C, suggesting that high temperature during summer also plays an important role in the maintenance of summer diapause in Pseudopidorus fasciata. All results reveal that summer diapause can serve as a "bet hedging" against unpredictable risks due to fluctuating environments or as a feedback mechanism to synchronize the period of autumn emergence.     

Glycerol accumulation in developmentally arrested pupae of Papilio machaon obtained by brain removal

Brains were removed from non-diapausing pupae of Papilio machaon within 24 hr after pupation. The developmentally arrested pupae were examined for glycerol metabolism by comparison with naturally diapausing pupae. The decerebrate non-diapausing pupae accumulated appreciable amounts of glycerol (13.0 mg/ml haemolymph) during exposure to 5°C for 2 months. However, this amount was considerably lower than that accumulated in the naturally diapausing pupae (24.5 mg/ml) under the same conditions. A difference was also observed in the initial level of the glycogen in the fat body. The naturally diapausing pupae contained about 105 mg/g glycogen, while the decerebrate non-diapausing pupae contained only about 70 mg/g, one day after pupation.     

Cellular localization of HSP23 during Drosophila development and following subsequent heat shock

The low-molecular-weight heat-shock protein HSP23 is synthesized in the absence of heat shock during Drosophila development. Here, I present a quantitative analysis of this phenomenon and describe the cellular localization of this protein during normal development and after a subsequent heat shock. HSP23 is first detected in the late third instar larvae and continues to accumulate reaching a maximum level in late pupae. In a 1-week-old adult, HSP23 can no longer be detected. Following lysis of whole pupae, HSP23 is found in the soluble lysate fraction in a form which sediments between 10 and 20 S. Exposure of larvae, pupae, and the adult fly to heat stress (37 degrees C) results in an increased amount of HSP23 which, however, is recovered in an insoluble particulate form following insect lysis. During recovery from heat shock, HSP23 is again found in the soluble 10- to 20-S lysate fraction. In pupae which are exposed to a severe heat stress (41 degrees C) HSP23 remains in the pellet fraction after the heat stress and no pupae are able to emerge as adult flies. However, when pupae are first exposed to a mild heat-shock treatment prior to the 41 degrees C stress, the thermotolerance process is induced and HSP23 is again rapidly found in the soluble lysate fraction during the recovery from heat shock. These observations suggest a possible correlation between the survival of pupae after heat shock and the recovery of HSP23 in the soluble lysate fraction as 10- to 20-S structures after the heat shock.     

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.     

TERMINATION OF PUPAL DIAPAUSE IN THE BOLLWORM HELICOVERPA ARMIGERA BY PRECOCENE II

Abstract Precocene II terminated pupal diapause in the bollworm Helicoverpa armigera as 20-hydroxyecdysterone did, whereas juvenile hormone analog ZR-515, cyclic adenosine monophosphate (CAMP), and 5-hydroxytryptamine (5-HT) did not. The results indicate that precocene II affects diapausing pupae in the similar way as what was found in the prepupae of the aphid parasitoids, Aphidius matricariae Haliday and Praon volucre Haliday (Hymenoptera: Aphidiidae). It is suggested that precocene II may affect different kinds of termination of diapause in insects.