Molecular mechanism of diapause in insect (I)

The embryonic diapause of the silkworn,Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expreon is a direct response to the environmend stimulus, such as high incubation temperature. The cDNA from the embryonic stage wa cloned and sequenm analysis showed the cDNA encoding DH. Expmion patterns of the DH gene in embryonic stage are different ar incubation temperatures 15°C and 25°C, suggesting that the incubation tempcreturt as an environmental signal is kept within the body to control the DH gene expmion at the pupal stage, so that the embryonic diapause of next generation can be determined.     

Effect of temperature on the termination of diapause in the univoltine almond seed wasp Eurytoma amygdali

Diapausing larvae of Eurytoma amygdali Enderlein (Hymenoptera, Eurytomidae) were collected in early August and late September. They were subjected to various photoperiod and temperature regimens for up to 20 weeks, then kept at L16:D8 and 19 °C for another 14 to 26 weeks for diapause to be terminated and pupation to take place. Photoperiod did not affect diapause completion. It was confirmed that the two morphologically distinct diapause stages have different temperature requirements for their completion. The first diapause stage was completed synchronously at temperatures between 16 and 19 °C. A higher temperature of 26 °C delayed diapause development. The second stage required lower temperatures between 4 and 10 °C. Spontaneous termination of diapause was observed at constant 19 °C. When applied to the first diapause stage for 20 weeks, low temperatures made the larvae refractory to subsequent intermediate temperatures. The first stage was thus maintained until a higher temperature of 26 °C made the larvae regain their ability to respond to the intermediate temperatures and complete this stage. Larvae grown in Retsou almonds had a higher diapause intensity than larvae grown in Truoito almonds. The results suggest that, in nature, the high temperatures of late summer and early autumn are likely to maintain the first diapause stage. Subsequently, the less warm temperatures of autumn allow the completion of the first stage by late autumn, and the low temperatures of late autumn and of winter allow the completion of the second diapause stage by mid winter.     

Diapause hormone directly stimulates the prothoracic glands of diapause larvae under juvenile hormone regulation in the bamboo borer,Omphisa fuscidentalis Hampson

Larval diapause in many lepidopteran insects is induced and maintained by high juvenile hormone (JH). In the case of the bamboo borer, Omphisa fuscidentalis, the effect of JH is the opposite: The application of juvenile hormone analog (JHA: S‐methoprene) terminates larval diapause, unlike in other insect species. Here, we analyzed the expression of JH‐receptor Met, DH‐PBAN, and Kr‐h1 in the subesophageal ganglion (SG) from October to April using semi‐quantitative polymerase chain reaction (PCR). The results show that OfMet and OfDH‐PBAN messenger RNA in the SG are mainly expressed during the larval diapause stage, while OfKr‐h1 increases during the pupal stage. Using tissue culture techniques and an enzyme‐linked immunosorbent assay (ELISA), diapause hormone (DH) was found to induce ecdysteroidogenesis in the culture medium of the prothoracic gland (PG) after incubation for 30 min with 25 ng and 50 ng of DH. Thus, DH is a novel stimulator for the PG. We identified a DHR homolog in the bamboo borer and confirmed that it is expressed in the PG. In addition, for in vitro experiments, DH increased the expression levels of OfDHR, OfEcR‐A, and ecdysone‐inducible genes in the PG. These results demonstrate that DH can function as a prothoracicotropic factor, and this function of DH might be through of DHR expressed on PG cells. Consequently, DH is one of the key factors in larval diapause break which is triggered by JH in the bamboo borer, O. fuscidentalis.     

Geographic variation in embryonic diapause,cold‐hardiness and life cycles in the migratory locust Locusta migratoria (Orthoptera: Acrididae) in China

To investigate geographic adaptation of the migratory locust Locusta migratoria in China, locusts were collected from six localities, ranging from 47.4°N to 19.2°N. Using offspring from the various populations, we compared embryonic diapause, reproductive traits, cold‐hardiness and adult body size. The incidence of embryonic diapause was influenced by the genetic makeup, parental photoperiod, and incubation temperature of the eggs. The northern strain (47.4°N) produced diapause eggs under all photoperiodic conditions, whereas the other strains produced a higher proportion of diapause eggs when exposed to a short photoperiod. The incubation temperature greatly influenced diapause induction. At a low temperature, all eggs entered diapause, even some of those from a tropical strain (19.2°N) in which no diapause was induced at high temperatures. Photoperiodic changes during the parental generation affected the incidence of embryonic diapause. Diapause intensity decreased with decreasing original latitude. Cold hardiness was compared by exposing eggs in diapause to either ?10 or ?20°C for various periods; the northern strain was more cold‐hardy than the southern strain, although some eggs in the tropical strain were probably not in a state of diapause. Adult body size and head width showed a complicated pattern of variation along the latitudinal gradient, whereas egg pod size (egg pod width and egg number) and hatchling weight tended to decrease with decreasing latitude. These results reveal that L. migratoria has adapted to local environments and that the latitudinal gradient appears to play an important role in shaping L. migratoria life cycle and development.     

Silkworm strains that can be clearly destined towards either embryonic diapause or direct development by adjusting a single ambient parameter during the preceding generation

In the silkworm, Bombyx mori (Lepidoptera: Bombycidae), embryonic diapause is under maternal control and the decision between diapause and direct development in bivoltine strains depends on environmental factors such as temperature and photoperiod experienced by the preceding generation. We reared ten bivoltine silkworm strains (c10, g32, k06, n25, p21, p22, p24, p44, p50 and p63) under various thermal and light conditions and examined the incidence of embryonic diapause in the next generation to identify strains in which the incidence of diapause can be controlled within the range of 0 to 100% by adjusting a single ambient parameter. Some strains were clearly destined towards either diapause or direct development. The diapause incidence in the c10, p22 and p50 strains was controlled by temperature during the egg stage (0% at 18°C and 100% at 25°C), that in the p50 strain was also determined to be dependent on illumination during the egg stage (0% under continuous darkness and 100% under continuous illumination), and photoperiod during the larval stage regulated diapause in p44 and p50 (0% and 100% under long‐day and short‐day photoperiod, respectively), when all other external parameters remained constant under each experimental condition. These diapause‐controllable silkworm strains might serve as model systems for studies of insect diapause, especially for the differential screening of related factors.     

Interactive effects of photoperiod and temperature on diapause induction and termination in the yellow-spotted longicorn beetle, Psacothea hilaris

Abstract. The interactive effects of temperature (20 °C or 25 °C) and photoperiod (LD 12 : 12 h or LD 15 : 9 h) on diapause induction and termination are investigated in the west‐Japan type yellow‐spotted longicorn beetle, Psacothea hilaris (Pascoe) (Coleoptera: Cerambycidae). Larval diapause of P. hilaris is induced under three diapause‐inducing conditions (20 °C–SD, 20 °C–LD and 25 °C–SD), and the diapause larvae are transferred to one of four conditions (20 °C–SD, 20 °C–LD, 25 °C–SD or 25 °C–LD) for observation of pupation, which indicates termination of diapause. The intensity of diapause induced under the three conditions increases in the order 20 °C–SD < 25 °C–SD < 20 °C–LD, when assessed by the time course of pupation after the transfer. On the other hand, the effectiveness of the temperature–photoperiod combinations to terminate diapause is in the order 25 °C–SD (ineffective) < < 20 °C–LD < 25 °C–LD < 20 °C–SD. Among the temperatures (5, 10, 15 and 20 °C) examined, 15 °C is the most effective in terminating diapause under the short day; diapause in most larvae appears to have been completed in 15 days.     

Inter- and intra-population variations in diapause attribute of the two-spotted spider mite,Tetranychus urticae Koch,in Japan

Populations of the two-spotted spider mite, Tetranychus urticae Koch collected from various localities and from various host plants in Japan showed wide variations in diapause attribute. Diapause percentages at 18°C/9L15D varied from nearly 100% in the north to 0% in the south-west. At intermediate latitudes the mites showed wide inter-population variations. Populations on herbaceous hosts in vinyl- or glass-houses gave significantly lower incidence of diapause than those on roses and deciduous fruit trees. Presence of winter hosts and better host quality under protected environments seemed to favour non-diapausing mites. The temperature threshold for diapause expression also varied widely among local populations. Northern populations consistently had higher and less variable thresholds than populations at intermediate latitudes with thresholds between 15 and 18°C. Inbred lines derived from a population in Kyoto exhibited a wide variation in diapause percentage at 18°C. These results show that diapause in T. urticae is a quantitative threshold trait and that populations in central Japan consist of a variety of genotypes with different diapause traits. This might provide a genetic source for adaptation to local and temporal variations in environmental conditions.     

Geographic variation in photoperiodic diapause induction and diapause intensity in Sericinus montelus (Lepidoptera: Papilionidae)

Abstract Due to the risk of extinction and ornamental value of the swallowtail butterfly, Sericinus montelus Gray (Lepidoptera: Papilionidae) in China, knowledge about local adaptations is important for the conservation and economical utilization of the species. In the present study, photoperiodic diapause induction and diapause intensity of S. montelus populations from Jiamusi (46°37′N), Beijing (40°15′N), Zibo (36°48′N), Fangxian (32°36′N), Wuhan (30°33′N) and Huaihua (27°33′N) were characterized at 25°C. Logistic regression analysis revealed a significant population × hours of light interaction, confirming that photoperiodic responses varied among populations. The critical photoperiod was positively correlated with latitude and increased toward the north at a rate of about 1 h for each 6.67 degrees of latitude. Survival analyses indicated that survival time of diapausing pupae before adult eclosion differed significantly among populations at 25°C and 16 : 8 L : D h. The mean duration of pupal diapause was also positively correlated with latitude. Our study reveals geographic variation in the critical photoperiod for diapause induction and in diapause intensity of S. montelus. These results provide useful information for our general understanding about seasonal adaptation in insects and may also be used to predict how geographic populations respond to climate warming.     

Maternal induction of larval diapause and its sensitive stage in the blow fly Lucilia sericata (Meigen) (Diptera: Calliphoridae)

Lucilia sericata has a facultative diapause in the third larval instar after cessation of feeding. Induction of the diapause is influenced by the photoperiod and temperature conditions experienced by insects in the parental generation as well as those experienced by the larvae themselves. The sensitive stage of the parental generation for induction of diapause was examined using diapause‐averting conditions of 16 h light : 8 h darkness (LD 16:8) at 25°C and diapause‐inducing conditions of LD 12:12 at 20°C. The incidence of diapause in the progeny was predominantly determined by the conditions experienced by the parents in the adult stage. Moreover, the results of reciprocal crosses showed that only the mother's experience is involved in the induction of diapause in the progeny.     

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

Abstract To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time‐consuming to maintain non‐diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15°C‐IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.     

Effects of thermoperiods on diapause induction in the cabbage beetle, Colaphellus bowringi (Coleoptera: Chrysomelidae)

Abstract. The effects of thermoperiods on diapause induction in continuous darkness or under a 12 : 12 h LD photoperiod were investigated in the cabbage beetle, Colaphellus bowringi Baly, a typical short‐day species. The diapause response curves both at different constant temperatures and at the thermocycle of format CT x: (24 ? x) h (16 : 28 °C) under continuously dark rearing conditions showed that the incidence of diapause depended mainly on whether or not the mean temperature was ≤20 °C or >20 °C. If the mean temperature was ≤20 °C, all individuals entered diapause; if >20 °C, the incidence of diapause declined gradually with increasing mean temperatures. The thermocycle (CT 12 : 12 h) with a series of different cryophases (8–22 °C) and thermophases (24–32 °C) under continuous darkness demonstrated a cryophase response threshold temperature of approximately 19 °C and a thermophase response threshold temperature of approximately 31 °C. Thermoperiodic amplitude (temperature difference between cryophase and thermophase) was shown to have a significant influence on diapause induction at the mean temperatures of 22, 23 and 24 °C, but not at ≥25 °C. Thermoperiodic responses under LD 12 : 12 h clearly showed that the incidence of diapause was influenced strongly by the photophase temperature. The thermoperiod under LD 12 : 12 h induced a much lower incidence of diapause than the thermoperiod with the same temperature in continuous darkness. The ecological significance of thermoperiodic induction of diapause in this species is discussed.     

Molecular cloning,developmental expression and tissue distribution of diapause hormone and pheromone biosynthesis activating neuropeptide in the bamboo borer Omphisa fuscidentalis

Diapause, an arrested period of post‐embryonic development in insects, is under the control of hormonal interactions. In the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae), larvae remain in diapause for as long as 9 months during the dry season, from September to the following June, although the factors that regulate larval diapause are poorly understood. The present study describes the cloning and expression analysis of the diapause hormone and pheromone biosynthesis activating neuropeptide (DH‐PBAN) precursor of O. fuscidentalis (Ompfu‐DH‐PBAN cDNA), aiming to reveal how it may be involved regulating larval diapause in this species in combination with environmental factors. The open reading frame (ORF) of the cDNA encodes a 199‐amino acid precursor protein that contains DH, PBAN and three other neuropeptides, all of which share a conservative C‐terminal pentapeptide motif FXPR/KL (X = G, T or S). The Ompfu‐DH‐PBAN is highly similar (74%) to the DH‐PBAN of the legume pod borer (Maruca vitrata). A quantitative real‐time polymerase chain reaction reveals that Ompfu‐DH‐PBAN mRNA is expressed only in neural tissues and that expression is highest in the suboesophageal ganglion. In addition, the expression level of Ompfu‐DH‐PBAN mRNA in the suboesophageal ganglion is consistently high during the fifth larval instar, increasing moderately in early diapause before reaching a peak during late diapause. After pupation, expression of the Ompfu‐DH‐PBAN precursor decreases to a low level. In addition to endocrine factors, the results demonstrate that photoperiod increases the expression level of Ompfu‐DH‐PBAN mRNA in larval diapause. These results also suggest that the expression of the Ompfu‐DH‐PBAN gene correlates with larval diapause development and may be activated by photoperiod in O. fuscidentalis.     

Effects of photoperiod and temperature on diapause induction and termination in the swallowtail,Sericinus montelus

Abstract Sericinus montelus overwinters as diapausing pupae. In the present study, the effects of photoperiod and temperature on diapause induction and termination of diapause are investigated. The results obtained demonstrate that high temperature can reverse the effect of short day‐lengths on diapause induction. Under an LD 12 : 12 h photoperiod, all pupae enter diapause at 15, 20 and 25 °C, whereas all pupae develop without diapause at 35 °C. No pupae enter diapause under an LD 14 : 10 h photoperiod when the temperature is above 20 °C. Photoperiodic response curves obtained at 25 and 30 °C indicate that S. montelus is a long‐day species and the critical day‐length is approximately 13 h at 25 °C. At 25 °C, the duration of diapause is shortest when the diapausing pupae are maintained under an LD 16 : 8 h photoperiod and increases under LD 14 : 10 h and LD 12 : 12 h photoperiods. Under an LD 16 : 8 h photoperiod, the duration of diapause is shortest when the diapausing pupae are maintained at 25 °C, followed by 20 and 30 °C, and then at 15 °C. These results suggest that a moderate temperature favours diapause development under a diapause‐averting photoperiod in this species. The duration of diapause induced by an LD 12 : 12 h photoperiod is significantly longer at 25 °C than those at 15, 20 and 30 °C, and is shortest at 15 °C. At 25 °C, the duration of diapause induced by LD 6 : 18, LD 12 : 12 and LD 13 : 11 h photoperiods is similar and longer than 90 days. Thus, the diapause‐inducing conditions may affect diapause intensity and a photoperiod close to the critical day‐length has significant influence on diapause intensity in S. montelus.     

Seasonal variation in embryonic diapause of the striped ground cricket, Allonemobius fasciatus

ABSTRACT. The incidence and intensity of embryonic diapause in the striped ground cricket, Allonemobius fasciatus DeGeer (Gryllidae), is influenced by the date of oviposition and the incubation temperature. At 20C, embryos develop normally until the end of the appendage-formation stage when diapause occurs. The intensity of 'winter' diapause at this stage is greatest in eggs laid early in the season. Incubation at higher temperature induces a different and earlier 'summer' diapause. The higher the temperature the earlier the stage at which this developmental suppression is imposed. At 27 and 30C, some individuals develop without any interruption and hatch within 20 days. The proportion of these fast-developing eggs is low in summer but increases towards the end of the laying season. In nature, this species is univoltine and precocious hatching before winter is unlikely.
When embryos diapausing at an early stage at 27C are transferred to low temperatures, they resume development and enter winter diapause. The time required to reach the winter-diapause stage after transfer is inversely related to the temperature (30-30C). At 30C, sunmer diapause is neither induced nor maintained.     

Immunoreactive intensity of FXPRL amide neuropeptides in response to environmental conditions in the silkworm, Bombyx mori

In the silkworm Bombyx mori, the diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is a neuropeptide gene that encodes a polypeptide precursor consisting in five Phe-X-Pro-Arg-Leu-NH₂ (FXPRL) amide (FXPRLa) neuropeptides; DH (diapause hormone), PBAN (pheromone-biosynthesis-activating neuropeptide) and α-, β- and γ-SGNPs (subesophageal ganglion neuropeptides). These neuropeptides are synthesized in DH-PBAN-producing neurosecretory cells contained within three neuromeres, four mandibular cells, six maxillary cells, two labial cells (SLb) and four lateral cells of the subesophageal ganglion. DH is solely responsible, among the FXPRLa peptide family, for embryonic diapause. Functional differentiation has been previously suggested to occur at each neuromere, with the SLb cells releasing DH through brain innervation in order to induce embryonic diapause. We have investigated the immunoreactive intensity of DH in the SLb when thermal (25°C or 15°C) and light (continuous illumination or darkness) conditions are altered and following brain surgery that induces diapause or non-diapause eggs in the progeny. We have also examined the immunoreactivity of the other FXPRLa peptides by using anti-β-SGNP and anti-PBAN antibodies. Pupal SLb somata immunoreactivities seem to be affected by both thermal and light conditions during embryogenesis. Thus, we have been able to identify a close correlation between the immunoreactive intensity of neuropeptides and environmental conditions relating to the determination of embryonic diapause in B. mori.     

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.     

Effect of photoperiod on the development and diapause of the green lacewing Chrysopa pallens (Neuroptera: Chrysopidae)

To investigate the physiology of Chrysopa pallens, the effect of photoperiod on diapause and development was examined in a Japanese population (33.4°N). The response stage for diapause of C. pallens was considered to be the prepupal stage. The critical photoperiod for diapause induction at 20.0°C was between 13 h light : 11 h dark (LD 13:11) and LD 14:10. The larval developmental period was affected by photoperiod: larvae in diapause took longer to complete their development. This difference of larval developmental period in relation to photoperiod was considered to be an adjustment of larval diapause timing.     

Mode of inheritance of face fly diapause and its correlation with other developmental traits

Abstract.

• 1 Adult face flies, Musca autumnalis De Geer, respond to short photoperiods by entering diapause. Diapause responses to different photoperiods at a mean temperature of 17°C varied among seven laboratory face fly strains. Old strains lost much of their diapause responses.
• 2 To analyse the nature of diapause inheritance, hybrids and their back-crosses were made between two strains that showed the greatest difference in diapause introduction at 10:14 h(L:D), and 17°C.
• 3 Responses of hybrids to the short photoperiod were intermediate to those of their parents. Joint scaling tests showed that diapause did not fit an additive model and the lack of fit was attributed to incomplete dominance. No cytoplasmic effect was detected, but diapause was partially sex linked.
• 4 Diapause heritabilities were 47% and 30% in the broad and narrow senses, respectively.
• 5 Estimates of the minimum number of diapause genes suggested a small number.
• 6 Developmental rates of egg to adult, ovarian follicle growth, and pterin accumulation were inversely correlated with diapause propensities, suggesting pleiotropy or linkage.