To understand the role of Juvenile Hormone (JH) on changes in the integument of Omphisa fuscidentalis Hampson (Lepidoptera: Pyralidae) during larval–pupal development, the expression patterns of transcripts of the O. fuscidentalis JH binding protein gene (OfJHBP) are determined in the integument of diapausing larvae. Quantitative real-time polymerase chain reaction (qRT-PCR) studies show that the relative levels OfJHBP mRNA in the integument do not differ significantly in different segments of the body. Application of the JH analogue (JHA) methoprene to diapausing larvae results in a relatively low expression of OfJHBP mRNA from days 0 to 8 after JHA application and reaches maximal levels in the pupal stage. Changes in OfJHBP expression after 20-hydroxyecdysone (20E) injection also show low levels of OfJHBP mRNA from days 0 to 8 and show a peak of expression in the pupal stage. Incubation of the integument in vitro in the presence of JHA (3.22 µm) induces high levels of OfJHBP expression within 120 min, whereas incubation with 20E (2.08 µm) induces gene expression at 150 min. To study the cooperative effect on OfJHBP expression of these two hormones, dose–response experiments are performed. Larval integument is maintained in the presence of 0.32 µm JHA or 2.08 µm 20E or a combination of both hormones in vitro for 120 min. Induction of OfJHBP expression by JHA and 20E in combination is significantly higher than that of either hormone alone. The results impact not only on our understanding of how methoprene (and hence JH) terminates larval diapause through an increase in ecdysteroid titre in the haemolymph, but also on how JH acts directly on the integument to stimulate the expression of a JH binding protein gene.

The collembolan Cryptopygus antarcticus Willem is potentially exposed to habitat salinities equal to (or greater than) sea water, as a result of sea spray, drying of littoral habitats, dispersal or temporary entrapment on the surface of sea water, or exposure to localized salt deposits from dense vertebrate populations on terrestrial habitats. To test the impact of this exposure on C. antarcticus, the tolerance of the collembolan to being placed on the surface of sea water and solutions of higher salt concentrations is investigated. The effects of acclimation to exposure to liquids of different salinities [44, 100 and 200 parts per thousand (ppt) sea salt] on cold and heat tolerance, as well as thermal activity thresholds, are also explored. Cryptopygus antarcticus shows > 75% survival after 10 days of exposure to both sea water and 100-ppt salt, whereas it exhibits significantly lower survival after 5 days (60% survival) and 10 days (40%) of exposure to a 200-ppt solution. Body water content also decreases after exposure to all salinities, and particularly to the 200-ppt solution, in which > 50% of body water is lost after 10 days. Acclimation results in greater cold tolerance, although heat tolerance at 33, 35 and 37 °C is either unaltered or reduced. The thermal activity thresholds of C. antarcticus at both high and low temperatures are also negatively affected by saline exposure. The data demonstrate the capacity of C. antarcticus to tolerate periods of exposure to saline conditions, and also show that this exposure can enhance cross-tolerance to low temperatures. The present study also demonstrates that salinity-associated stress at moderately low and high temperatures narrows the thermal range of activity, thus reducing the ability of collembolans to forage, develop and reproduce. © 2013 The Royal Entomological Society

Quantities of ecdysteroid are compared in the haemolymph and ovaries of the blowfly Protophormia terraenovae Robineau-Desvoidy (Diptera: Calliphoridae) under reproductive (LD 18 : 6 h at 25 °C) and diapause (LD 12 : 12 h at 20 °C) conditions. The effects of ablation of the pars intercerebralis or ovaries on ecdysteroid quantities and of ablation of the pars intercerebralis on yolk protein expression are examined. Under reproductive conditions, the levels of ecdysteroid in vitellogenic females are high, although the levels in previtellogenic females and females with mature ovaries are low. Under diapause conditions, there are low quantities of ecdysteroid in both the haemolymph and ovaries. Ecdysteroid titres in the haemolymph are not significantly affected by the removal of the ovaries, suggesting that tissues other than the ovaries are also involved in the production of ecdysteroids. Reproductive females in which the pars intercerebralis of the brain is experimentally ablated have ecdysteroid levels that are not significantly different from sham-operated or intact females. However, yolk protein expression in the fat body is suppressed after removal of the pars intercerebralis. These results suggest that the suppression of ecdysteroid levels in the haemolymph and ovaries is associated with reproductive diapause, and that the pars intercerebralis could play a role in yolk protein synthesis without mediating ecdysteroid production.

The ‘choice’ of whether to enter diapause or to develop directly has profound effects on the life histories of insects, and may thus have cascading consequences such as seasonal morphs and other less obvious forms of seasonal plasticity. Present knowledge of the control of diapause and seasonal morphs at the physiological and molecular levels is briefly reviewed. Examples, mainly derived from personal research (primarily on butterflies), are given as a starting point with the aim of outlining areas of research that are still poorly understood. These include: the role of the direction of change in photoperiod; the role of factors such as temperature and diet in modifying the photoperiodic responses; and the role of sex, parental effects and sex linkage on photoperiodic control. More generally, there is still a limited understanding of how external cues and physiological pathways regulating various traits are interconnected via gene action to form a co-adapted complete phenotype that is adaptive in the wild despite environmental fluctuation and change.

Insects have evolved a number of physiological mechanisms for coping with the detrimental effects of low temperature. As autumn progresses, insects use environmental signals such as shortening day lengths and gradually decreasing temperatures to trigger seasonal cold-hardening adaptations. These mechanisms include dramatic changes in biochemistry, cell function and gene expression that permit improved cell function and viability at low temperature. Insects are also capable of enhancing cold tolerance on a much shorter time scale, in a process called rapid cold-hardening (RCH). Rapid cold-hardening allows insects to improve cold tolerance almost instantaneously (i.e. within minutes to hours) to cope with sudden cold snaps and regularly-occurring diurnal drops in temperature. Initially, it was assumed that RCH would share many of the same basic mechanisms as seasonal cold-hardening, albeit on a shorter time scale. Although there is some evidence supporting this, recent work has called into question some of the original hypotheses concerning the mechanisms of RCH. Also, some mechanisms important for seasonal cold-hardening, such as up-regulation of stress proteins, are unlikely to function at the temperatures and time scales at which RCH occurs. In the present review, the current understanding of the physiological mechanisms governing both seasonal cold-hardening and RCH are summarized. A synthesis of the current literature suggests that these two forms of cold-hardening may be more mechanistically distinct than originally anticipated.

Aphids are major crop pests and show a high level of phenotypic plasticity. They display a seasonal, photoperiodically-controlled polyphenism during their life cycle. In spring and summer, they reproduce efficiently by parthenogenesis. At the end of summer, parthenogenetic individuals detect the transition from short nights to long nights, which initiates the production of males and oviparous females within their offspring. These are the morphs associated with the autumn season. Deciphering the physiological and molecular events associated with this switch in reproductive mode in response to photoperiodic conditions is thus of key interest for understanding and explaining the remarkable capacity of aphids to adapt to fluctuations in their environment. The present review aims to compile earlier physiological studies, focussing on the neuroendocrine control of seasonal photoperiodism, as well as a series of large-scale transcriptomic approaches made possible by the recent development of genomic resources for the model aphid species: the pea aphid Acyrthosiphon pisum. These analyses identify genetic programmes putatively involved in the control of the initial steps of detection and transduction of the photoperiodic signal, as well as in the regulation of the switch between asexual and sexual oogenesis within embryonic ovaries. The contribution of small RNAs pathways (and especially microRNAs) in the post-transcriptional control of gene expression, as well as the role of epigenetic mechanisms in the regulation of genome expression associated with the photoperiodic response, is also summarized.

Nondiapause pupae of Papilio machaon L. exhibit pupal colour diphenism comprising green–yellow and brown–white types. To understand the regulatory mechanism underlying the control of pupal colouration in P. machaon, the effect of environmental cues on diapause and nondiapause pupal colouration is investigated. When larvae reared under short-day and long-day conditions are allowed to pupate in sites with a smooth surface and a yellow background colour, all diapause pupae exhibit a brown–white type and 89.5% of nondiapause pupae exhibit a green–yellow type, respectively. With rough-surface pupation sites, all diapause pupae exhibit brown–white and intermediate types, whereas a large proportion of nondiapause pupae exhibit brown–white and intermediate types, although some exhibit a green–yellow type. When extracts prepared from the head-thoracic and thoracic-abdominal regions of larval central nervous systems are injected into the ligated abdomens of P. machaon short-day pharate pupae, all recipients exhibit a brown–white colouration. Furthermore, when each extract is injected into the ligated abdomen of Papilio xuthus L. short-day pharate pupae with orange-pupa-inducing factor activity, recipients injected with the head-thoracic extract exhibit the brown type, whereas those injected with the thoracic-abdominal extract exhibit an orange colour. The results indicate that the response to the environmental cues of pupation site in P. machaon changes according to the photoperiodic conditions experienced during larval stages, and that at least two hormonal factors producing brown–white pupae are located in the larval central nervous system, with the secretion of these factors being regulated by the recognition of environmental cues in long-day larvae.

The lifecycle of Paratlanticus ussuriensis Uvarov can extend for longer than 1 year (plurennial) as a result of prolonged diapause. Eggs can overwinter by entering a facultative initial diapause in the early embryonic stage within the egg, followed by further overwintering by entering an obligatory final diapause at the fully developed embryonic stage. The initial diapause is temperature-dependent and initiated by continuous incubation at 20 °C but not at 30 °C. To understand the mechanism of initial diapause at the molecular level, cDNAs of small heat shock protein genes (shsp), hsp90, as well as three hsp70 genes (hsp70a, hsp70b and hsp70c), are determined and the transcript levels of the five hsp genes are compared in eggs incubated at 20 or 30 °C for 60 days after oviposition using real-time reverse transcriptase-polymerase chain reaction analysis. At 30 °C, transcript levels of shsp, hsp70a, hsp70b and hsp90 increase in eggs with age. However, hsp70c levels uniquely peak only in 25-day-old eggs, corresponding to the time of initial diapause. At 20 °C, distinct levels of shsp, hsp70a and hsp90 appear in some stages and fluctuate throughout the 60-day observation period, although both hsp70b and hsp70c are undetectable. These results suggest that each hsp may play a specific role in relation to initial diapause. In addition, up-regulation of hsp70c may be associated with the mechanism of embryonic activation for averting initial diapause in warmer conditions.

Wing polyphenism in aphids represents an outstanding example of adaptive phenotypic plasticity. During summer, parthenogenic mother aphids alter the developmental fate of their embryos to produce wingless or winged adult forms in response to high population density (i.e. crowded conditions). Although this maternal effect is well known, the mechanisms underlying transgenerational winged-morph determination remain largely unresolved. In the present study, the effects of different high-density treatment durations are tested on the vetch aphid Megoura crassicauda Mordvilko aiming to investigate how and when the density signals detected by mothers are transmitted to embryos. The duration of density treatment shows additive effects on both the number of crowded females producing winged aphids (winged-producers) and the number of winged progeny. In addition, even when high-density treatment is stopped, the production of winged offspring continues for several days and depends on the duration of treatment. The results indicate that mother aphids retain high-density signals for a period after removal of the stimulus. Furthermore, observations of the progeny sequence (i.e. the order in which the offspring are born) and the embryonic stages developing in the mothers reveal that high-density information may affect embryonic fate at the late embryonic stage immediately before cuticle formation.

The role of the circadian clock gene Clock in the circadian rhythm and the photoperiodic regulation of reproductive diapause in the bean bug Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) is investigated. Clock RNA interference (RNAi) disrupts the circadian rhythm in an alternating deposition of polarized and nonpolarized cuticle layers in the endocuticle, and produces only a polarized layer. This indicates that Clock is a core component of the circadian clock and that it acts as a positive element in activating the transcription of downstream genes. In addition, Clock RNAi suppresses ovarian development irrespective of day-length conditions, which indicates that Clock is involved in the photoperiodic response. The observed phenotypes in Clock RNAi insects, the production of a single polarized layer in the endocuticle and an arrested ovarian development irrespective of photoperiod are the same as those induced by RNAi of cycle, a positive element in the circadian clock. However, the phenotypes induced by RNAi of negative elements period and mammalian-type cryptochrome differ from those induced by the positive elements. Considering the current data together with previously published findings, it is concluded that the circadian clock, comprising the circadian clock genes, is involved in the photoperiodic response of R. pedestris.

The cricket Modicogryllus siamensis Chopard shows photoperiod-dependent changes in the duration of nymphal development: nymphs become adult within 60 days after hatching, undergoing seven moults under long-day conditions, whereas, under short-day conditions, nymphal development takes much longer (approximately 180 days) with an increased number of moults. Because removal of the compound eyes alters this photoperiodic response, the eyes may be involved in light detection during the photoperiodic response. The role of opsins, expressed in the compound eye, is examined in the present study with reference to the photoperiodic response. Molecular cloning identifies cDNAs of three opsins, opsin-Ultra Violet (Ms'op-UV), opsin-Blue (Ms'op-B) and opsin-Long Wave (Ms'op-LW), and in situ hybridization reveals that the opsin genes are expressed in specific regions of the compound eye in a gene-specific manner. RNA interference (RNAi) technology using the opsin genes results in a partial disruption in the long-day responses; most of the treated crickets showed eight or more moults and up to 23.5% show a prolonged nymphal period that is typical of short-day responses. Under short-day conditions, op-UV RNAi crickets show earlier adult development, whereas no distinct alterations are observed in op-B and op-LW RNAi insects. The results suggest that the opsin genes may play differential roles in the photoperiodic response in the cricket and that the results can be at least partially explained in terms of the external coincidence model of photoperiodic time measurement.

Diapause has long been recognized as a crucial ecological adaptation to spatio-temporal environmental variation. More recently, rapid evolution of the diapause response has been implicated in response to contemporary global warming and during the range expansion of invasive species. Although the molecular regulation of diapause remains largely unresolved, rapidly emerging next-generation sequencing (NGS) technologies provide exciting opportunities for addressing this longstanding question. In the present study, a new assembly from life-history stages relevant to diapause in the Asian tiger mosquito Aedes albopictus (Skuse) is reported, along with unique methods for the analysis of NGS data and transcriptome assembly. A digital normalization procedure that significantly reduces the computational resources required for transcriptome assembly is evaluated. Additionally, a method for protein reference-based and genomic reference-based merged assembly of 454 and Illumina reads is described. Finally, a gene ontology analysis is presented, which creates a platform for identifying the physiological processes associated with diapause. Taken together, these methods provide valuable tools for analyzing the transcriptional underpinnings of many complex phenotypes, including diapause, and provide a basis for determining the molecular regulation of diapause in Ae. albopictus.