Inheritance of the photoperiodic response controlling imaginal summer diapause in the cabbage beetle, Colaphellus bowringi

Adults of the cabbage beetle Colaphellus bowringi display a summer diapause in response to the exposure of their larvae to long photoperiods. In the present study, the inheritance of the photoperiodic response controlling summer diapause in C. bowringi by crossing a high diapause strain (D strain) with a laboratory selected nondiapause strain (N strain) was investigated under different photoperiods at 22, 25 and 28 °C. The beetles in both reciprocal crosses and backcrosses showed a clear short-day response for the induction of diapause at all temperatures, similar to that of the D strain, suggesting that photoperiodic response of this beetle is heritable. The diapause incidences in the progeny from all the crosses under LD 15:9 or LD 12:12 at 25 °C suggest that genetic and genetic-environmental interactions are involved in diapause induction. The incidence of diapauses in F₁ progeny was significantly lower than that in the D × D strain but significantly higher than that in the N × N strain, indicating that the diapause capability is inherited in an incomplete dominant manner. The incidence of diapause was greater among the offspring of hybrid females when those females had a D strain mother or grandmother rather than a N strain mother or grandmother, indicating that maternal effects on diapause induction are stronger than paternal effects. The laboratory selected nondiapause strain also showed a short-day photoperiodic response at a low temperature of 22 °C, indicating that the photoperiodic photoreceptor and photoperiodic clock still function in the nondiapause strain.     

Geographic differences on accumulation of sugars and polyols in locust eggs in response to cold acclimation

The accumulation of low molecular weight sugars and polyols is one of major mechanisms hypothesized to increase cold tolerance in overwintering insects. But little is known about whether these sugars and polyols are involved in geographic variation of cold tolerance. In this study, we investigated accumulation patterns of eight low molecular weight sugars and polyols of eggs in tropical and temperate populations of the migratory locust, which exhibits between-population variation in cold tolerance, in response to cold acclimation (5, 0 and −5 °C). Excluding erythritol, the other seven carbohydrates were identified as possible cryoprotectants in locust eggs. Basal maximal and minimal concentrations were 45 μg/g wet weight for trehalose and 0.59 μg/g wet weight for glycerol. Most sugars and polyols were elevated after a −5 °C exposure. In a tropical population, fructose, glucose, sorbitol and myo-inositol were significantly accumulated by low temperature treatments, but glycerol was not. In the temperate population, glycerol, glucose, mannitol, sorbitol, myo-inositol were significantly accumulated but trehalose did not increase. Our results suggest different accumulation patterns of these carbohydrates of locust eggs between tropical and temperate populations and highlighted possible roles for them in geographic variation of cold tolerance in the migratory locust.     

Quantitative short-day photoperiodic response in larval development and its adaptive significance in an adult-overwintering cerambycid beetle, Phytoecia rufiventris

The chrysanthemum longicorn beetle, Phytoecia rufiventris, overwinters in the adult stage and reproduces in spring. Larvae of this beetle develop during summer inside a host stem or root. In the present study, photoperiodic control of larval development and its adaptive significance were examined in this beetle using an artificial diet. Larvae showed a short-day photoperiodic response at 25 °C with a critical day length of around 14 h; larvae reared under short-day conditions pupated, whereas those reared under long-day conditions entered summer diapause with some supernumerary molts and did not pupate. A similar response was found at 30 °C, but with a shorter critical day length. Below the critical day length, a shorter day length corresponded to a shorter larval period. Larvae transferred from long-day conditions to various photoperiods showed a similar quantitative response. Field rearing of larvae starting at various times of year showed that pupation occurs within a relatively short period in early autumn. Field rearing of pupae and adults at various times indicated that only pupation in early autumn results in a high survival rate until winter. Earlier or later pupation led to a low survival rate due to death before overwintering in the adult and pupal stages, respectively. Thus, in P. rufiventris, timing of pupation regulated by the quantitative short-day photoperiodic response is vital for survival. Relatively lower developmental threshold in the pupal stage supports this hypothesis.     

Diapause induction, maintenance and termination in the rice stem borer Chilo suppressalis (Walker)

The rice stem borer, Chilo suppressalis, enters facultative diapause as fully grown larvae in response to short-day conditions during the autumn. Our results showed that the critical night length for diapause induction in C. suppressalis was between 10 h 22 min and 10 h 45 min at 22, 25 and 28 °C, 11 h 18 min at 31 °C, and between 10 h 5 min and 10 h 20 min under field conditions (average temperature ranged from 27.2 to 30.7 °C). The diapause incidence declined in ultra-long nights (18-22 h scotophases) and DD, and increased in ultra-short nights (2-6 h scotophases) and LL. Moreover, we found that the third instar was the stage most sensitive to the photoperiod, and night length played an essential role in the initiation of diapause. Night-interruption experiments with a 1-h light pulse at LD 12:12 (light 12:dark 12) exhibited two troughs of diapause inhibition, with one occurring in early scotophase and the other in late scotophase. Field observations for six years showed that most larvae entered winter diapause in August in response to declining day lengths, despite the high temperatures prevailing during August. By periodically transferring the field-collected overwintering larvae to different photoperiods and temperatures, the results showed that photoperiod had a significant influence on diapause development during the early phase of diapause, while high temperature significantly accelerated the termination of larval diapause.     

Geographic variation in diapause induction and termination of the cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

Overwintering diapause in Helicoverpa armigera, a multivoltine species, is controlled by response to photoperiod and temperature. Photoperiodic responses from 5 different geographical populations showed that the variation in critical photoperiod for diapause induction was positively related to the latitudinal origin of the populations at 20, 22 and 25 °C. Diapause response to photoperiod and temperature was quite different between northern and southern populations, being highly sensitive to photoperiod in northern populations and temperature dependence in southern populations. Diapause pupae from southern population showed a significantly shorter diapause duration than from northern-most populations when they were cultured at 20, 22, 25, 28 and 31 °C; by contrast, overwintering pupae from southern populations emerged significantly later than from northern populations when they were maintained in natural conditions, showing a clinal latitudinal variation in diapause termination. Diapause-inducing temperature had a significant effect on diapause duration, but with a significant difference between southern and northern populations. The higher rearing temperature of 22 °C evoked a more intense diapause than did 20 °C in northern populations; but a less intense diapause in southern population. Cold exposure (chilling) is not necessary to break the pupal diapause. The higher the temperature, the quicker the diapause terminated. Response of diapause termination to chilling showed that northern populations were more sensitive to chilling than southern population.     

Thermal response and reversibility of prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis undergoes prolonged larval diapause that is terminated by chilling and warming cycles. To examine the effects of warming temperatures and their duration on diapause termination, we exposed diapause larvae that had not been reactivated after chilling at 5 °C to 20 or 25 °C and chilled them again before incubation at 20 °C. With increasing warming duration at 20 °C, diapause termination after chilling increased and shorter chilling durations became effective. In contrast, few or no larvae warmed at 25 °C terminated diapause after chilling, irrespective of the warming duration. To investigate the effect of warming temperature on diapause intensity, larvae with diapause weakened by initial incubation at 20 °C after the first chilling were subsequently incubated at 15, 20, or 25 °C, then chilled at 5 °C before incubation at 20 °C. Diapause termination increased significantly after the larvae were treated at 15 or 20 °C but decreased significantly after they were treated at 25 °C. The intensification of prolonged diapause at 25 °C was reversed when the larvae were transferred to 20 °C. Diapause intensity in C. sikkimensis therefore decreases at 20 °C, increases at 25 °C, and can be reversed by alternately exposing diapause larvae to 20 and 25 °C. In C. sikkimensis, prolonged diapause does not always proceed in one direction, and its intensity fluctuates in response to ambient temperature conditions.     

The role of photoperiod and temperature in determination of summer and winter diapause in the cabbage beetle,Colaphellus bowringi (Coleoptera: Chrysomelidae)

The cabbage beetle, Colaphellus bowringi, is a short-day species undergoing an imaginal summer and winter diapause. Its photoperiodic response highly depends on temperature. All adults entered diapause at ≤ 20 °C regardless of photoperiods. High temperatures strongly weakened the diapause-inducing effects of long daylengths. The diapause-averting influence of short daylengths was expressed only at high temperatures (above 20 °C). This indicates that the beetle has a cryptic ability to reproduce in summer. In fact, summer and winter diapause were induced principally by relatively low temperatures in the field, whereas photoperiod had less influence on diapause induction. The critical daylength for the autumnal population was between 12 h and 13 h. By transferring from a long day to a short day or vice versa at different times after hatching, it was shown that the sensitive stage with regard to photoperiod was the larva, whereas a long day was photoperiodically more potent than a short day. The sensitive stage to temperature encompassed the larval, pupal and adult stages. This different response pattern serves to ensure that the beetle enters summer and winter diapause in time. The selections for non-diapause trait under laboratory (at 25 °C) and natural conditions (at >24 °C) showed that the beetle could lose its sensitivity to photoperiod very rapidly.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae)

We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 °C under a 60 ± 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C and 25 °C under a 60 ± 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 °C, and 12.2, 48.8 and 39.0% at 25 °C, respectively. On the other hand, when mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C, 18 °C and 25 °C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 °C, 14.5, 26.9 and 58.6% at 18 °C, and 8.3, 21.2 and 70.5% at 25 °C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues.     

Cold tolerance of the harlequin ladybird Harmonia axyridis in Europe

As an essential aspect of its invasive character in Europe, this study examined the cold hardiness of the harlequin ladybird Harmonia axyridis. This was done for field-collected populations in Belgium overwintering either in an unheated indoor or an outdoor hibernaculum. The supercooling point, lower lethal temperature and lower lethal time at 0 and −5 °C were determined. Possible seasonal changes were taken into account by monitoring the populations during each winter month. The supercooling point and lower lethal temperature remained relatively constant for the overwintering populations in the outdoor hibernaculum, ranging from −17.5 to −16.5 °C and −17.1 to −16.3 °C, respectively. In contrast, the supercooling point and lower lethal temperature of the population overwintering indoors clearly increased as the winter progressed, from −18.5 to −13.2 °C and −16.7 to −14.1 °C, respectively. A proportion of the individuals overwintering indoors could thus encounter problems surviving the winter due to premature activation at times when food is not available. The lower lethal time of field populations at 0 and −5 °C varied from 18 to 24 weeks and from 12 to 22 weeks, respectively. Morph type and sex had no influence on the cold hardiness of the overwintering adults. In addition, all cold tolerance parameters differed greatly between the laboratory population and field populations, implying that cold tolerance research based solely on laboratory populations may not be representative of field situations. We conclude from this study that the strong cold hardiness of H. axyridis in Europe may enable the species to establish in large parts of the continent.     

Effect of temperature regime on diapause intensity in an adult-wintering Hymenopteran with obligate diapause

Osmia lignaria is a solitary bee that over-winters as a fully eclosed, cocooned, unfed adult. Our objective is to understand the effect of wintering temperature on diapause maintenance and termination in this species. We measure respiration rates and weight loss in individuals exposed to various wintering temperatures (0, 4, 7, 22 °C, outdoors) and durations (28, 84, 140, 196, 252 days). We use time to emerge and respiration response (respiration rate measured at 22 °C) as indicators of diapause intensity. Adults spontaneously lower their respiration rates to ∼0.1 ml/g h within 1 month after adult eclosion, indicating obligatory diapause. Non-wintered individuals maintain low respiration rates, but lose weight rapidly and die by mid-winter. In wintered adults, two phases can be distinguished. First, respiration response undergoes a rapid increase and then reaches a plateau. This phase is similar in bees wintered at 0, 4 and 7 °C. In the second phase, respiration response undergoes an exponential increase, which is more pronounced at the warmer temperatures. Composite exponential functions provide a good fit to the observed respiration patterns. Adults whose respiration response has reached 0.45 ml/g h emerge promptly when exposed to 20 °C, indicating diapause completion. Individuals wintered for short periods do not reach such respiration levels. When exposed to 20 °C these individuals lower their metabolic rate, and their emergence time is extended. The relationship between respiration rates and emergence time follows a negative exponential function. We propose two alternative models of diapause termination to interpret these results.     

Diapause termination, post-diapause development and reproduction in the beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae)

Effects of photoperiod and cold exposure on diapause termination, post-diapause development and reproduction in Loxostege sticticalis were examined. Larvae were reared at diapause inducing condition (22 °C, L:D 12:12) consistently or transferred to long day photoperiod (L:D 16:8) and darkness (L:D 0:24) respectively, after entering into diapause. Diapause was terminated in approximately 40% of the larvae after 36 days, and no significant differences were observed between photoperiods, suggesting larval diapause was terminated spontaneously without being induced by photoperiods. Cold exposure significantly hastened diapause termination. The diapause termination incidence increased significantly with peaks of 98% at both 5 °C and 0 °C exposure for 30 days, as compared to 42% in controls not exposed to cold, while the mortality and number of days required for diapause termination decreased dramatically. The optimal low temperature exposure periods under 5 °C or 0 °C were 20 days and 30 days, showing a higher termination incidence and shorter time for diapause termination. This suggests that the low temperatures in winter play an important role in diapause termination under natural conditions. The threshold temperatures for post-diapause development in prepupae and pupae were 9.13 °C and 10.60 °C respectively, with corresponding accumulations of 125 and 200 degree-days. Adults that experienced larval diapause significantly delayed their first oviposition, oviposition period was prolonged, and the lifetime number of eggs laid was decreased, however both males and females have significantly longer longevity. The field validation of diapause termination, the degree-days model, and the relationship between diapause and migration in L. sticticalis were also discussed.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.     

Relationships between body weight of overwintering larvae and supercooling capacity; diapause intensity and post-diapause reproductive potential in Chilo suppressalis Walker

The rice stem borer, Chilo suppressalis Walker, overwinters in China as a larva in facultative diapause. The instars and body weights of overwintering larvae vary widely. In this paper, the relationships between body weight and supercooling capacity, diapause intensity and post-diapause reproductive potential of overwintering larvae collected in late-stage rice field were examined. There was a significant positive correlation between body weight, instar, and head capsule width, thus the overwintering larvae were divided into five groups based on body weight (I, up to 35.0 mg; II, 35.1-57.0 mg; III, 57.1-79.0 mg; IV, 79.1-101.0 mg; and V, over 101.1 mg) for further analysis. The body water content of the lighter group (I) was significantly higher than that of the heavier groups (IV-V). However, the mean supercooling point decreased with an increase of the mean larval body weight in five groups; mean supercooling point of group I was significantly lower than that of group V, except in January 2009. After transfer of overwintering larvae to 15, 20 and 25 °C on different dates, smaller individuals pupated slightly faster than larger ones at the same temperature, suggesting that diapause was less intense in smaller overwintering larvae. On 19 March 2009 there was a strong positive correlation between larval body weight and the weight of 3 day-old pupae, and the number of eggs carried by 2 day-old adult females at 15, 20 and 25 °C. The average number of eggs carried by 2 day-old adult females differed significantly among different groups. The average number of eggs carried by 2 day-old adult females in group V was significantly greater than those of other groups, and that of group I was significantly lower than those of other groups, suggesting that post-diapause reproductive potential was determined, to a certain extent, by body weight of the overwintering larvae.     

Photoperiodism of diapause induction in Thyrassia penangae (Lepidoptera: Zygaenidae)

Thyrassia penangae enters winter diapause as a prepupa in a cocoon. Photoperiodism of diapause induction was systematically investigated in this moth. The photoperiodic response curves under 24-h light-dark cycles showed that this insect is a typical long-day species. The critical daylength was 13 h 30 min at 25 °C, 13 h at 30 °C and 12 h 20 min at 28 °C. Transferring experiments from a short day (LD 12:12) to a long day (LD 15:9) or vice versa indicated that photoperiodic sensitivity mainly occurs during the larval period. In experiments using non-24-h light-dark cycles, when the length of photophase exceeded the critical daylength (13.5 h), was diapause inhibited effectively, even when the length of scotophase exceeded the critical nightlength (10.5 h). Only when a long scotophase was combined with a short photophase, diapause was induced effectively. This result suggests that daylength measurement is more important than nightlength measurement in T. penangae. Night interruption experiments under 24-h light-dark cycles exhibited two points of apparent light sensitivity, but the photosensitive position was highly influenced by temperature and the length of scotophase. Nanda-Hamner experiments failed to reveal the involvement of a circadian system in this photoperiodic time measurement. All light-dark cycles from LD 12:12 to LD 12:72 resulted in a short day response, and all cycles from LD 14:4 to LD 14:72 resulted in a long day response, suggesting that photoperiodic time measurement in this moth is performed by a day-interval timer or an hourglass-like clock.     

The physiological response of reef corals to diel fluctuations in seawater temperature

An opportunity to explore the effects of fluctuating temperatures on tropical scleractinian corals arose when diurnal warming (as large as 4.7 °C) was detected over the rich coral communities found within the back reef of Moorea, French Polynesia. In April and May 2007, experiments were completed to determine the effects of fluctuating temperature on Pocillopora meandrina and Porites rus, and consecutive trials were used to expose them for 13 days to 26 °C, 28 °C (ambient conditions), 30 °C, or a fluctuating treatment ranging from 26 to 30 °C over 24 h. The multivariate response was assessed using maximum dark-adapted quantum yield of PSII (F_V/F_M), Symbiodinium density, chlorophyll-a content, and calcification. In trial 1, multivariate physiology of both species was significantly affected by treatments, with the fluctuating temperature resulting in a 17-45% decline in Symbiodinium density (relative to the ambient) matching that occurring at a constant 30 °C; F_V/F_M, chlorophyll-a content, and calcification, did not differ between the fluctuating and the steady treatments. In contrast, in trial 2 that utilized corals collected two weeks after those used in trial 1, the corals were unaffected by the treatments, likely due to an environment × trial interaction caused by seasonal declines in Symbiodinium density. Together, these results demonstrate that short transgressions to ecologically relevant high and low temperatures can elicit a potentially detrimental response equivalent to that occurring upon exposure to a constant high temperature. The dissimilar responses among dependent variables and consecutive trials underscore the importance of temporal replication and multivariate approaches in coral ecophysiology. It is likely that recent history has a stronger effect on the response of corals to treatments than is currently recognized.     

Cyclitols protect glutamine synthetase and malate dehydrogenase against heat induced deactivation and thermal denaturation

The accumulation of cyclitols in plants is a widespread response that provides protection against various environmental stresses. The capacity of myo-Inositol, pinitol, quercitol, and other compatible solutes (i.e., sorbitol, proline, and glycinebetaine) to protect proteins against thermally induced denaturation and deactivation was examined. Enzymatic activity measurements of l-glutamine synthetase from Escherichia coli and Hordeum vulgare showed that the presence of cyclitols during heat treatment resulted in a significantly higher percentage of residual activity. CD spectroscopy experiments were used to study thermal stabilities of protein secondary structures upon the addition of myo-Inositol, pinitol, and glucose. 0.4 M myo-Inositol was observed to raise the melting temperature (T_m) of GS from E. coli by 3.9 °C and MDH from pig heart by 3.4 °C, respectively. Pinitol showed an increase in T_m of MDH by 3.8 °C, whereas glucose was not effective. Our results show a great potential of stabilizing proteins by the addition of cyclitols.     

Metabolic responses of sand fiddler crabs, Uca pugilator, in northwest Florida to seasonal temperature change

Metabolic responses of sand fiddler crab, Uca pugilator, populations in northwest Florida are greatly influenced by seasonal temperature fluctuations. Crabs acclimated at 20 °C and immediately transferred to either 14 or 26 °C produced an acute metabolic response with respective temperature quotient (Q₁₀) values of 3.46 and 3.91. Crabs acclimated at 10 and 20 °C exhibited a Q₁₀ of 2.62 indicating a partial compensation response. A brumation (reverse) response (Q₁₀ value of 20.11) was observed for acclimated crabs between 5 and 10 °C. Brumation is advantageous during winter when food supplies are scarce and crabs must survive extensive periods of inactivity.     

Functional response of Tetrastichus gallerucae (Hym: Eulophidae) to different densities of the elm leaf beetle eggs,Xanthogaleruca luteola (Col: Chrysomelidae)

The elm leaf beetle is often considered as much of an indoor nuisance as a garden pest since large numbers of adults migrate into homes seeking a protected place to overwinter. Outdoors, both adults and larvae feed on the emerging leaves of virtually all species of elm trees, leaving skeleton zed foliage in their wake. Repeated severe infestations can markedly weaken a host tree, making it susceptible to other insects and diseases. The elm leaf beetle does not transmit the well-known Dutch elm disease, which is carried by another insect than elm bark beetle. The functional response of Tetrastichus gallerucae bees, that is the most important natural enemy of egg elm beetle Xanthogallerucae luteola, has been detected in Kerman province. The experiment was carried out in the growth room under conditions of 25 ± 1°C, 60 ± 5 RH and 16L:8D. Every female bee was exposed for 24 hours at densities of 2, 4, 6, 8, 10, 12, 16, 20, 30 and 40 eggs of elm leaf beetle. Every density had 10 repetitions. The results showed that T. gallerucae has the second type of functional response. The searching efficiency and the handling time of T. gallerucae was 2.63 and 0.114 on the eggs of elm leaf beetle and 2.193 and 0.112 on the eggs of hostess with diluted honey.     

Cloning of cDNAs encoding sorbitol dehydrogenase-2a and b, enzymatic characterization, and up-regulated expression of the genes in Bombyx mori diapause eggs exposed to 5 °C

We previously cloned a cDNA for sorbitol dehydrogenase (SDH1) from Bombyx mori. In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and SDH2b). The amino acid sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to B. mori SDH1. The SDH2ab and SDH1genes were located in tandem within about 40 kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5 °C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25 °C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the SDH activity seen in diapause eggs exposed to 5 °C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in Escherichia coli and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4∼9.0, and the Km value for sorbitol was 12.6 mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified SDH from B. mori diapause-terminated eggs exposed to 5 °C for 300 days which showed higher SDH activity using two-step affinity chromatography. The highly purified SDH showed a higher Km value (125 mM) for sorbitol, being similar to the value (136 mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no SDH genes other than SDH1 and 2ab are present in B. mori genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased SDH activity seen in diapause eggs after acclimation to 5 °C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original SDH gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and SDH2b genes.