Photoperiod-dependent adult diapause within a geographical cline in the multivoltine bruchid Bruchidius dorsalis

Abstract The bruchid beetle Bruchidius dorsalis Fahraeus (Coleoptera: Bruchidae) has been known to undergo larval diapause during the final instar under short photoperiods ( Kurota & Shimada, 2001 ). This species has a multivoltine life cycle and the overwintering stages show a geographical variation across Japan ( Kurota & Shimada, 2002 ). In cooler areas, overwintering occurs during the final instar, whereas in warmer climates overwintering can occur during several developmental stages: non‐diapausing young instars, diapausing instars, and adults. In this study, we investigated the adult reproductive diapause in three populations from different geographical regions to clarify the role of geographical variation on overwintering strategies. We found that: (1) B. dorsalis entered reproductive diapause in addition to larval diapause under short photoperiods, (2) diapause propensity was higher and the critical photoperiod was longer in populations from cooler regions, and (3) the sensitive photoperiod range was the first 5 days after emergence. Predictions of the overwintering stage, derived from critical photoperiods, were consistent with actual overwintering stages observed in each population. The geographical variation in diapause induction is likely to reflect the adaptive overwintering strategy in each local environment.     

Geographical variation in photoperiodic induction of larval diapause in the bruchid beetle, Bruchidius dorsalis: polymorphism in overwintering stages

The bruchid beetle, Bruchidius dorsalis Fahraeus (Coleoptera: Bruchidae), has a multivoltine life cycle and shows geographical variation of overwintering stages in Japan. Our previous study found that B. dorsalis enters larval diapause in the final instar under short photoperiods. In cooler areas, we observed that most individuals overwinter in the final larval stage in diapause, whereas beetles at different developmental stages (non‐diapausing young instars, diapausing instars, and adults) were overwintering in warmer areas. In this study, we investigated geographical variation in the photoperiodic response for induction of larval diapause at 20 °C (three populations) and 24 °C (two populations) to clarify the overwintering strategy of B. dorsalis. We observed that (1) diapause incidence at 20 °C changed sharply from ca. 100% to 0% with a change in photoperiod in all the populations, (2) critical photoperiod was longer at 20 °C in populations from cooler areas, and (3) critical photoperiod at 24 °C was shorter than at 20 °C and a fraction of the larvae did not enter diapause, even under short photoperiods. Overwintering stages estimated from these results were consistent with those actually observed in the field. This study indicates that the geographical variation of overwintering stages is likely to reflect adaptive diapause induction in each local environment.     

Cold tolerance in diapausing and non-diapausing stages of the flesh fly, Sarcophaga crassipalpis

ABSTRACT. Supercooling points (SCP) and low temperature tolerance were determined for larval, pupal and adult stages of Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae). No stage tolerates tissue-freezing. Ontogenetic changes in SCP profiles are similar for comparable developmental stages of diapause and non-diapause groups. Feeding larvae have SCPs near -7°C which decrease to -11°C in the postfeeding wandering phase of the final larval instar. The lowest SCPs are recorded for pupae at -23°C. The capacity to survive at -17°C varies with age of the diapausing pupae: 10-day-old pupae are less cold tolerant than pupae that have been in diapause for 45–80 days. Although the SCP of non-diapausing pupae is as low as in diapausing pupae, non-diapausing pupae are extremely sensitive to low temperature exposure and do not survive to adult eclosion when exposed to -17°C for as little as 20 min. The use of hexane to break pupal diapause has no effect on SCPs or low temperature tolerance.     

Effects of photoperiod and temperature on the development and diapause of the bark beetle Ips typographus

Abstract:  Diapause was induced in a Central European population of Ips typographus grown at 20°C when the day length decreased below 16 h [50% diapause incidence occurred in the 14.7:9.3 h L:D (light:dark) regime]. The non-diapausing adults fed on days 2–6 and 10–14 after the ecdysis and swarmed after the second feeding bout with chorionated eggs in the ovaries and sperm in the spermiducts. Neither gonads nor the flight muscles matured and no swarming occurred in the diapausing adults. The development from egg to adult took about 34 days in both 18:6 h (no diapause) and 12:12 h L:D (diapause) regimes, but it was extended by up to 30% without diapause induction when only larvae or pupae were exposed to L:D 12:12 h. Diapause was induced in insects reared at L:D 12:12 h through the last larval and the pupal instars and/or in the adult stage. Temperature ≥ 23°C prevented diapause induction at L:D 12:12 h but diapause occurred at L:D 14:10 h associated with 26:6°C thermoperiod. The effect of thermoperiods on the developmental rate requires further research. Exposure of the non-diapausing adults to 5°C for several days blocked feeding and evoked a diapause-like state, whereas diapausing adults fed and their gonads slowly developed at this temperature. Diapausing adults exposed in forest to low night temperatures and transferred in October to 20°C readily reproduced at 18:6, but not 12:12 h L:D photoperiods. After 2-months at 5°C and darkness, they became insensitive to the photoperiod, matured and most of them also swarmed at 20°C in the 12:12 h L:D regime. In a Scandinavian population, diapause occurred at 18:6 h L:D and was terminated either by exposure to 5°C or by very long photoperiod (L:D 20:4 h) combined with high temperature (23°C).     

Termination and induction of diapause in the gypsy moth larval parasitoid, Apanteles melanoscelus

Diapause in Apanteles melanoscelus can be terminated by exposure of the diapausing last instar larvae within their cocoons to 5°C for a period of 8 or more weeks. Photoperiod has no consistent influence upon diapause termination, but is of paramount importance for diapause induction. At less than 16 hr light per day virtually all larvae diapause, and at 18 hr and above very few larvae diapause. By exposing different larval stages to different photoperiods it was found that older larvae were most sensitive to the light-dark cycle. It was also noted that cocoons of diapausing larvae are larger than those of non-diapausing larvae.     

Effects of changing photoperiods in the life cycle regulation of the comma butterfly, Polygonia c-album (Nymphalidae)

Abstract. 1. Regulation of adult reproductive diapause and seasonal polyphenism was studied in two populations of the comma butterfly, Polygonia c-album (L.) (Nymphalidae), from Stockholm, Sweden, and Oxford, England.
2. In the univoltine Stockholm population short and long constant day-lengths (in the range 12–22 h) at 20°C were ineffective in averting the production of the dark, generally diapausing, morph, but the non-diapausing light morph could be produced if daylengths were increased during larval development. The procedure was especially effective with increases from 12 to 22 h, but the tendency was the same with a more realistic increase, from 18 to 20 h.
3. In the partially bivoltine English population a critical constant day-length for morph and diapause induction was found between 12 and 18 h. Decreasing daylengths above the critical daylength early or late in larval development resulted in production of the diapausing morph.
4. These results suggest a system for environmental control where day-lengths which increase throughout the larval period indicate an early date before summer solstice, meaning that there is sufficient time for a second generation of offspring to reach the hibernating adult stage before winter. The ecological significance of the results is discussed.     

A small HSP gene is not responsible for diapause and cold tolerance acquisition in Chilo suppressalis

Abstract:  The cDNA sequence of a small heat shock protein ( hsp19.7 ) was cloned and sequenced from the rice stem borer, Chilo suppressalis Walker. The cDNA encoded a protein of 177 amino acids with a calculated molecular weight of 19.7 kDa. The deduced amino acid sequence showed the highest identity of 90% to Bombyx mori hsp19.9 . Expression levels of hsp19.7 were similar between diapausing and non-diapausing larvae. In non-diapausing larvae, but not in diapausing ones, hsp19.7 expression was upregulated by cold acclimation. Involvement of hsp19.7 in larval diapause and cold tolerance in C. suppressalis is discussed.     

二化螟滞育幼虫的蛋白和核酸含量以及保护酶活性的变化

为了阐明二化螟Chilo supprressalis滞育幼虫的分子特征及滞育期间保护酶活性的变化规律, 本研究应用Trizol法、 总量DNA提取法和蛋白定量试剂盒, 测定了在长光周期16L∶8D和25℃下发育的非滞育老熟幼虫、 在短光周期12L∶12D 和25℃下诱导滞育51 d的幼虫（称为滞育0个月）、 滞育1, 2和3个月幼虫的核酸含量和总蛋白含量; 同时应用试剂盒测定了老熟幼虫、 滞育0, 1和2个月的二化螟幼虫5种保护酶（POD, CAT, SOD, LDH和ATP酶）的活性。结果表明： 滞育幼虫的总RNA含量显著低于非滞育的老熟幼虫（P<0.05）, 而滞育1, 2和3个月的幼虫之间没有显著差异（P≥0.05）。老熟幼虫的总DNA含量显著高于滞育幼虫（P<0.05）。老熟幼虫的RNA/DNA比值较低, 滞育幼虫的RNA/DNA比值较高, RNA/DNA比值随着滞育时间的推移呈现出先上升后下降的趋势。滞育期大于1个月的幼虫中蛋白含量均显著高于非滞育的老熟幼虫（P<0.05）, 而滞育1, 2和3个月的幼虫之间没有显著差异（P≥0.05）。二化螟幼虫体内5种保护酶活性随发育阶段不同而存在差异。滞育幼虫中POD, CAT和SOD的活性随滞育时间延长而逐渐增强, 滞育2个月幼虫中的活性最高, 而非滞育老熟幼虫中的活性最低; LDH和ATP酶的活性则相反, 非滞育老熟幼虫中的活性最高, 滞育2个月幼虫中的活性最低。这些结果说明, 总RNA和DNA含量降低、 RNA/DNA比值先升后降、 总蛋白含量升高以及保护酶活性的变化是二化螟幼虫滞育过程中的主要生理特征。     

A trade-off between diapause duration and fitness in female parasitoids

Abstract 1. Diapause allows insects to cope with adverse weather conditions but also poses substantial fitness costs, e.g. through reduced survival of the diapausing stage or sublethal effects in the following season.
2. The energetic costs of diapause in females of the parasitoid Asobara tabida Nees (Hymenoptera: Braconidae) were assessed using experimental manipulation of diapause duration.
3. An increase in diapause length not only led to higher mortality among diapausing pupae but also caused a significant decrease in egg load, fat reserves, and dry weight of the emerging adult females.
4. Only larvae with sufficient resources were able to survive the entire diapause period and there was a trade-off between the metabolic costs of diapause and adult fitness components.
5. In contrast, the size (tibia length) of emerging females increased with increasing diapause duration. It is proposed that this may be due to a genetic correlation between larval dry weight and adult size, so that selection for heavier larvae would result in larger adults even when resources are limited.
6. Ultimately, the evolution of diapause duration and its consequences for adult performance will depend on the costs of overwintering and the ecological conditions in the habitat.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

大斑芫菁滞育幼虫在滞育不同阶段体内糖类和醇类含量的变化

在以卵滞育的昆虫中昆虫滞育时的生理代谢特点已经得到了大量研究。本文对以末龄幼虫（5龄）滞育的大斑芫菁Mylabris phalerate（Pallas）在不同滞育阶段体内糖类和醇类代谢的特征进行了研究。结果表明： 滞育个体血淋巴中的海藻糖含量高于非滞育个体,且随滞育时间的加大逐渐升高,滞育5个月时达到最大值,为5.61 μmol/mL。糖原的含量随滞育的进程逐渐减少,滞育初期（0.5个月）为0.72 mg/mL,到滞育末期（5个月）时仅为0.1 mg/mL。滞育个体脂肪体中的海藻糖含量都高于非滞育个体,滞育1个月时为非滞育个体的3倍,至滞育末期时达非滞育个体的5倍,为2.5 μmol/g脂肪体。糖原含量总体变化趋势是随滞育时间的加大逐渐减少,滞育早期和中期都高于非滞育个体。在滞育过程中血淋巴积累的小分子多元醇主要为甘油,其次是山梨醇;而在脂肪体中主要为甘油,其次是甘露醇,少量积累山梨醇：表明大斑芫菁滞育幼虫主要积累的是海藻糖和一些小分子多元醇。滞育幼虫在准备滞育时储备了大量糖原,这些糖原可能为滞育期间海藻糖、山梨醇和甘油的代谢提供了原料。     

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.     

Larval diapause of the European corn borer, Ostrinia nubilalis: Further experiments examining its hormonal control

The possible involvement of juvenile hormone (JH) in controlling the mature larval diapause of the European corn borer, Ostrinia nubilalis, was examined using biological and chemical assays for JH titres, topical applications of JH mimic, and injections of 20-hydroxy-ecdysone. Bioassays of extracts of larval haemolymph showed that (1) 4th instar pre-diapausing larvae had a higher JH titre (ca. 1450 Galleria Units (GU)/ml) than equivalent non-diapausing larvae (ca. 340 GU/ml), and that (2) 5th instar pre-diapausing larvae contained a JH titre of ca. 320 GU/ml, which declined to ca. 90 GU/ml in newly-diapaused larvae. Chemical assasys carried out on extracts of whole larvae showed that early diapausing larvae contained an extremely low titre of JH. In addition, the application of JH mimic or 20-hydroxy-ecdysone or both agents to diapausing larvae failed to reveal the presence of a functional JH titre during diapause. The application of JH mimic to early 5th instar non-diapausing larvae produced moribund larval-pupal intermediates rather than supernumerary larvae. Our results, therefore, suggest that although JH may control some phases of diapause induction, it is not involved in maintaining diapause.     

The roles of temperature and diapause in the life history of a temperate-zone dragonfly: Argia vivida (Odonata: Coenagrionidae)

Abstract. 1. The life cycle of Argia vivida Hagen generally took longer to complete in the field than was predicted on the basis of the thermal sum accumulated in laboratory rearing.
2. The prediction of a bivoltine life-cycle from geothermal sites with either a constant annual temperature of 26°C or thermal range of 11–31°C was not borne out because the intervention of short-day induced developmental delays in later larval instars extended the life cycle to 1 year.
3. This diapause, which synchronizes adult emergence with favourable summer temperatures, was also present in larvae from sites with annual temperature ranges of 0–33°C and 5–20°C.
4. At these colder sites completion of the life cycle takes 2 and 3 years respectively and dragonflies must be in cold-resistant stages during the winter. A long-day diapause, principally affecting late-instar larvae below a certain size during the summer, achieves this.
5. Large diurnal temperature fluctuations at the 0–33°C site markedly increase the useful thermal energy available to larvae for growth over that predicted by the thermal sum equation.
6. The interaction between the effects of temperatures favourable for growth and day-length-governed diapause, synchronize the emergence of the low-temperature sensitive adult stage of this tropical dragonfly with northern-latitude summers at a variety of habitats.     

Diapause induction in Trichogramma embryophagum Htg. (Hym., Trichogrammatidae): the dynamics of thermosensitivity

It is known that the low temperature is the most important factor inducing the pre-pupal diapause in Trichogramma species. The position of the thermosensitive period over the life cycle and temporal variation of the degree of responsiveness were investigated in T. embryophagum Htg. by transferring pre-imaginal stages between 'neutral' temperature of 15°C and 'diapause-inducing' temperature of 10°C. Our experiments showed that 6 days long exposure at 10°C significantly increased the percentage of diapausing pre-pupae when started during rather large part of development: from embryo up to early pre-pupa. The highest thermosensitivity was recorded during the embryo and the larval stages, with some decrease during the hatching period. Treatments with shorter cold exposures (2–3 days) gave similar results. Even 24 h long exposure at 10°C increased the percentage of diapausing pre-pupae when applied during egg or early larval stage. Being started at the same stage of development, longer cold exposures caused stronger increase in the percentage of diapausing individuals. The experiments did not reveal any significant daily changes in thermosensitivity: at 12 : 12 h light : dark, larvae subjected to the low temperature during six photophases showed practically the same percentage of diapausing individuals as those subjected to the low temperature during six scotophases, and as those subjected to the 3 days long uninterrupted cold exposure. Hence, in natural conditions even occasional short-term cold periods could be accumulated.     

Effects of diapause and cold-acclimation on the avoidance of freezing injury in fat body tissue of the rice stem borer, Chilo suppressalis Walker

Overwintering freeze-tolerant larvae of Chilo suppressalis can survive at -25 degrees C, but non-diapausing larvae cannot. We reported earlier that to prevent intracellular freezing, which causes death in overwintering larvae of the Saigoku ecotype distributed in southwestern Japan, water leaves and glycerol enters fat body cells through water channels during freezing. However, it is still unclear how diapause and low-temperature exposure are related to the acquisition of freeze tolerance. We compared the extent of tissue damage, accumulation of glycerol, and transport of glycerol and water in fat body tissues between cold-acclimated and non-acclimated non-diapausing and diapausing larvae. The tissue from cold-acclimated diapausing larvae could survive only when frozen in Grace's insect medium with 0.25 M glycerol at -20 degrees C. The protection provided by glycerol was offset by mercuric chloride, which is a water-channel inhibitor. Fat body tissue isolated from non-acclimated diapausing larvae was injured by freezing even though glycerol was added to the medium, but the level of freezing injury was significantly lower than in non-diapausing larvae. Radiotracer assays in cold-acclimated diapausing larvae showed that during freezing, water left the cells into the medium and glycerol entered the cells from the medium at the same time. Therefore, in Saigoku ecotype larvae of the rice stem borer, both diapause and cold-acclimation are essential to accumulate glycerol and activate aquaporin for the avoidance of freezing injury.     

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.     

Feeding,reproductive and locomotor activities in diapausing and non-diapausing adults ofDrosophila

Feeding, reproductive and locomotor activities of fourDrosophila species were studied under short and long daylengths at 15°C. A short daylength induced firm reproductive diapause in experimental strains ofD. subauraria andD. triauraria from northern Japan, but very shallow diapause in those ofD. lutescens andD. rufa from southern Japan. A subtropical strain ofD. triauraria had no diapause. The influence of diapause on feeding activity was detected only in aged (> 12 day old) females; that is, the feeding activity was lower in diapausing females than in non-diapausing ones. Females that do not produce eggs would not require so much energy. On the other hand, young adults of the study species exhibited a high feeding activity and rapidly increased bodyweight irrespective of sex and the diapause state. They would need nutrition to build up their adult body. In males, the feeding activity decreased with age irrespective of the diapause state. Males would not require so much energy for reproductive activity. Diapausing males became heavier than non-diapausing males, perhaps because they accumulated triacylglycerols in fat bodies. However, female bodyweight did not differ by the diapause state, perhaps because diapausing females accumulated triacylglycerols and reproducing females had eggs in their ovaries. InD. triauraria, diapausing individuals exhibited somewhat lower locomotor activity than non-diapausing ones.     

Physiology of diapause and cold hardiness in overwintering pupae of the apple leaf miner Phyllonorycter ringoniella in Japan

Abstract The apple leaf miner Phyllonorycter ringoniella (Matsumura) (Lepidoptera: Gracillariidae) overwinters as a diapausing pupa. The diapause rate reaches 100% in early October. Diapause intensity decreases gradually from early October and diapause terminates in early February. The fresh body weight of diapausing pupae is 1.6 times that of non-diapausing pupae. The main cryoprotectant in P. ringoniella pupae is trehalose. Three stages are distinguishable as indicated by the correlations between diapause intensity, levels of cold hardiness and the trehalose content: diapause induction occurred in October, diapause development from November to December, and post-diapause quiescence from January to April. During diapause induction, the pupae accumulate low levels of trehalose and do not survive exposure to −15 °C. During diapause development, the pupae gradually accumulate more trehalose and show some ability to survive exposure to −15 °C, but not to −20 °C. During post-diapause quiescence, the pupae accumulate relatively more trehalose and cold hardiness fully develops, but decreases quickly in April. The trehalose content in pupae sampled in December is unaffected by acclimation temperatures in the range 0–30 °C, but decreases in pupae sampled in March after acclimation at temperatures from 5 to 15 °C. These results suggest that overwintering pupae of P. ringoniella have the ability to accumulate trehalose and develop a high level of cold hardiness during diapause development.