Ontogenetic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis

Developmental patterns of low-temperature tolerance and glycerol production were determined for larval, pupal and adult stages of the flesh fly Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae). Both diapause and non-diapause-destined flies were reared at relatively high temperatures, 20° or 25°C, prior to testing. Cold tolerance was greatest for diapause pupae aged 12–35 days after pupariation. Among non-diapause-destined flies, pupae exhibited a greater level of low temperature tolerance than larvae or adults. Although diapause pupae were more tolerant than non-diapause pupae maximal cold tolerance was not attained in either group until 10 days after pupariation. Non-diapause-destined feeding and wandering larvae had higher glycerol levels than larvae destined for diapause. During the first 6 weeks after pupariation glycerol titres increased steadily in diapause pupae. Rapid loss of glycerol is associated with the termination of pupal diapause.     

Molecular mechanism of diapause in insect (I) --Expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori

The embryonic diapause of the silkworm, 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 expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.     

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.     

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.     

Regulation of heat shock proteins in the apple maggot Rhagoletis pomonella during hot summer days and overwintering diapause

Abstract Developing larvae of the apple maggot Rhagoletis pomonella are frequently exposed to summertime apple temperatures that exceed 40 °C and, during their overwintering diapause, pupae are exposed to sub‐zero soil temperatures for prolonged periods. To investigate the potential involvement of heat shock proteins (Hsps) in response to these environmental extremes, the genes encoding Hsp70 and Hsp90 in R. pomonella are cloned and expression monitored during larval feeding within the apple and during overwintering pupal diapause. Larvae reared in the laboratory at constant temperatures of 25, 28 or 35 °C express Hsp90 but very little Hsp70. Larvae do not survive rearing at 40 °C. The temperature cycles to which larvae were exposed inside apples in the field, ranging 16–46.9 °C over a 24‐h period, elicit strong Hsp70 and Hsp90 expression, which begins at mid‐day and reaches a peak in late afternoon, coinciding with peak air and apple temperatures. Heat shock proteins are also expressed strongly by pupae during their overwintering diapause. Hsp70 is not expressed in nondiapausing pupae but is highly expressed throughout diapause. Hsp90 is constitutively expressed in both diapausing and nondiapausing pupae. Rhagoletis pomonella thus strongly expresses its Hsps during pupal diapause, presumably as a protection against low temperature injury, and during larval development to cope with natural temperature cycles prevailing in late summer.     

Selection of reference genes for tissue/organ samples on day 3 fifth‐instar larvae in silkworm,Bombyx mori

The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth‐instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori.     

Effects of transgenic overexpression of diapause hormone and diapause hormone receptor genes on non-diapause silkworm

Diapause is a state of developmental arrest that is most often observed in arthropods, especially insects. The domesticated silkworm, Bombyx mori, is a typical insect that enters diapause at an early embryonic stage. Previous studies have revealed that the diapause hormone (DH) signaling molecules, especially the core members DH and DH receptor 1 (DHR1), are crucial for the determination of embryonic diapause in diapause silkworm strains. However, whether they function in non-diapause silkworm strains remains largely unknown. Here, we generated two transgenic lines overexpressing DH or DHR1 genes in a non-diapause silkworm strain, Nistari. Our results showed that developmental expression patterns of DH and DHR1 are quite similar in transgenic silkworms: both genes are highly expressed in the mid to late stages of pupae and are most highly expressed in day-6 pupae but are expressed at very low levels in other developmental stages. Moreover, the overexpression of DH or DHR1 can affect the expression of diapause-related genes but is not sufficient to induce embryonic diapause in their offspring. This study provides new insights into the function of DH and DHR1 in a non-diapause silkworm strain.     

Water and carbohydrate levels at different developmental stages and dynamics in hibernating pupae of Pieris melete (Lepidoptera: Pieridae)

For insight into the physiological indicators of diapause in Pieris melete, water and carbohydrate (glycogen and trehalose) levels were measured under both natural and laboratory conditions. The highest water content (3.71–3.79 mg/mg dry weight) was found in larvae and developing pupae, which was substantially higher than in diapausing pupae (2.59 mg/mg dry weight). Water content was almost stable during diapause, except for individuals approaching diapause termination (3.43–3.58 mg/mg dry weight). The total carbohydrate level was significantly higher in pre‐pupae (47.41 μg/mg) compared to larvae (22.80 μg/mg) and developing pupae (21.48 μg/mg). The highest level of trehalose was detected in winter diapausing pupae, and no trehalose was found in larvae or developing pupae. Levels of glycogen were highest in pre‐pupae and lowest in diapausing pupae. Levels of total carbohydrate decreased as diapause proceeded, and no significant changes were found in trehalose levels for diapausing pupae under natural conditions or treated for 60–90 days at 5°C. Pupae treated at 20°C for 60–90 days had significantly lower levels of trehalose than those treated for 30 days. Glycogen content was relatively stable at 5°C, but increased after treatment under natural conditions and 20°C for more than 60 days. These results suggest that the dynamics of water and carbohydrate levels are potential physiological diapause indicators, which show metabolic differences between trehalose and glycogen during diapause development.     

滞育和非滞育棉铃虫血淋巴类固醇蜕皮素含量变化的比较

采用放射免疫分析法对不同时期的注定滞育和非滞育棉铃虫的血淋巴中的类固醇蜕皮素的含量进行了测定,发现在预蛹期间,注定滞育的棉铃虫的类固醇蜕皮素含量高于非滞育的棉铃虫,化蛹后,注定滞育的棉铃虫的类固醇蜕皮素含量则迅速降到极低的水平,明显低于非滞育棉铃虫。用20-羟基蜕皮素处理不同时期的滞育蛹,均能打破滞育。由此可见,类固醇蜕皮素含量的降低或缺乏乃是导致棉铃虫滞育的关键因子之一。     

Difference in nutrient accumulation patterns between diapause-destined and non-diapause-destined larvae of Hyphantria cunea

Nutrient accumulation is crucial in insect diapause preparation because insufficient nutrient accumulation can shorten the diapause period, interfere with diapause development completion, and decrease the probability of surviving the overwintering period. The amounts of lipids and carbohydrates stored in diapausing pupae of Hyphantria cunea (Drury) (Lepidoptera: Erebidae, Arctiinae) are greater than those in the non-diapausing pupae. In this study, we tested the hypothesis that diapause-destined (DD) and non-diapause-destined (NDD) larvae of H. cunea have different nutrient accumulation patterns in penultimate and final instars. The body mass, as well as lipid, carbohydrate, and soluble protein contents, and the efficiency of converting digested food and ingested food into body matter were greater in the DD penultimate and final instars than in the NDD penultimate and final instars. Larger amounts of lipids, carbohydrates, and proteins were absorbed by DD penultimate and final instars and the final instar development period in the DD larvae was prolonged relative to NDD larvae. The activities of fatty acid synthase and glycogen synthase of DD penultimate and final instars were significantly higher than those of NDD larvae. These results suggest that the changes in nutrient accumulation patterns between DD and NDD larvae occur in penultimate and final instars, and that the DD larvae increase their nutrient accumulation during diapause preparation by the combined effect of extending their final-instar development period and improving their digestive efficiency; they increase their lipid and carbohydrate stores by increasing the activities of fatty acid synthase and glycogen synthase in the fat body.     

Expression of metabolic enzyme genes and heat-shock protein genes during embryonic development in diapause and non-diapause egg of multivoltine silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

The expression of metabolic enzyme genes and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm Bombyx mori was quantified by semi-quantitative RT-PCR. The trehalase gene (Tre) was expressed in non-diapause eggs up-to nine days, while in diapause eggs was not up regulated. The glycogen phosphorylase gene (GPase) was expressed in non-diapause eggs, whereas in diapause eggs a high level was observed in early stage, but down regulated in later stage. The phosphofructokinase gene (PFK) and sorbitol dehyrogenase-2 gene (SDH-2) expression was fluctuated in non-diapause eggs, whereas in diapause eggs these were expressed only at early stage and not observed in later stage. The glucose-6-phosphate dehydrogenase gene (G6P-DH) in non-diapause eggs was highly expressed during the differentiation phase and decreased in the organogenesis phase. In contrast to this, expression in diapause eggs was of low level during differentiation phase and of high level observed in the organogenesis phase. In the tissues, PFK and SDH-2 were selectively expressed in cuticle and midgut, whereas Tre expression was high in midgut and ovary of larvae incubated at 15°C. The Hsp (20.4, 20.8, 40, 70, and 90) were expressed in both diapause and non-diapause eggs. Their expression was, however, selective in tissues with Hsp20.4 in midgut and ovary, Hsp40 in head, Hsp70 in cuticle and Hsp90 in ovary and head in high amounts at 15°C. These results suggest that the metabolic enzyme genes studied except Hsp play a major role during embryogenesis of diapause and non-diapause silkworm.     

Relationship between photoperiod and secretion of the diapause hormone during larval stages of the silkworm, Bombyx mori L., reared on an artificial diet

The suboesophageal ganglion of the silkworm, Bombyx mori synthesizes sufficient diapause hormone to produce diapause eggs, regardless of the photoperiodic conditions experienced during the larval stages. When larvae destined to produce non-diapause eggs are implanted with the brain-suboesophageal ganglion complex from larvae which have been reared under short-day conditions, the resulting adults lay diapause eggs. The larvae receiving the complex from larvae reared under long-day conditions gave rise to adults which did not produce any diapause eggs. The brains from pupae which have been reared under long-day conditions show an activity inhibiting the secretion of diapause hormone by the suboesophageal ganglion. The mechanism through which the brain controls the secretion of diapause hormone from the suboesophageal ganglion can be modified by photoperiodic conditions during the larval stages.     

Dopamine is a key factor for the induction of egg diapause of the silkworm, Bombyx mori.

The silkworm, Bombyx mori, enters diapause in the early embryonic stage. Embryonic diapause is induced by incubating eggs of the maternal generation at high temperature (diapause type), whereas incubation at low temperature results in non-diapausing progeny (non-diapause type). Measurement of catecholamine concentrations in haemolymph and brain-subesophageal ganglia (Br-SGs) showed that only dopamine concentrations in both tissues are consistently higher in diapause-type than non-diapause-type larvae and pupae. In particular, the difference in dopamine concentrations in both tissues increases around pupal ecdysis. During the early pupal stage, Dopa decarboxylase activities and mRNA concentrations in Br-SGs were also much higher in diapause-type than non-diapause-type insects. Elevation of dopamine levels induced by feeding Dopa to penultimate-instar and last-instar larvae, and by injecting Dopa or dopamine into pupae 2 days after pupation made the non-diapause-destined insects lay diapause-destined eggs at 59% and approximately 70% frequencies, respectively. Furthermore, injection of Dopa or dopamine elevated mRNA levels of the diapause hormone in the Br-SGs of non-diapause-type pupae 1 day after injection. Incubation of Br-SGs isolated from non-diapause-type day-2 pupae with Dopa or dopamine also stimulated the expression of diapause hormone mRNA. These data indicate that environmental stimuli during embryonic development increase dopamine levels in both hemolymph and Br-SGs from the larval stage to early pupal stage, which results in laying of diapause-destined eggs by female adults through enhanced expression of the diapause hormone gene.     

Effects of putrescine on diapause induction and intensity,and post‐diapause development of Helicoverpa armigera

Effects of putrescine on diapause induction and intensity, and post‐diapause development in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) from Wuhan, Hubei, China were studied in the laboratory at different temperatures and photoperiod conditions. The results showed a generally higher incidence of diapause in putrescine‐feeding groups. Putrescine feeding distinctly restricted the development of non‐diapause‐destined and diapause‐destined larvae, except at 0.01% putrescine concentration, which accelerated the development of larvae at 21 °C. Putrescine had no significant effect on the duration of diapause, although it inhibited the post‐diapause development of pupae. Photoperiod played a different role in diapause induction and post‐diapause development between 21 and 23 °C. Putrescine played a definite role in diapause induction and intensity, and post‐diapause development in H. armigera, and a significant interaction between putrescine, photophase, and temperature is to be expected.     

Inhibition of Spermiogenesis of Eupyrene Spermatozoa by Elevated,Sublethal Temperatures During Pupal-Adult Development of the Bertha Armyworm,Mamestra con figurata Wlk. (Lepidoptera,Noctuidae)

Summary

Eupyrene spermiogenesis and spermatozoa production in Mamestra configurata Walker were inhibited by temperatures >24° C during a “critical period” of about 7 days during the first half of post-diapause pupal-adult development. The critical period coincided with the appearance of early-stage eupyrene spermatids. If temperatures >24° C occurred during the critical period, eupyrene spermatozoa production was reduced, the result of irreversible autolysis of the early- and mid-stage eupyrene spermatids. Each day of exposure to 25 or 27.5° C during the critical period reduced the number of eupyrene cysts with spermatozoa on average by 12 to 14%. There were no observable effects on eupyrene spermiogenesis and spermatozoa production of exposing post-diapausing pupae to 25 or 27.5° C before or after the critical period. Apyrene spermatogenesis was not affected by 25 or 27.5° C.

Exposure of pupae to 25°or 27.5° C during diapause had no apparent effect on eupyrene spermiogenesis and spermatozoa production. Meiosis usually was not detected among the eupyrene cysts with lalje primary spermatocytes until after diapause, indicating that meiosis is suppressed during diapause. By providing a meiotic block, diapause governs the time of onset of the temperature-sensitive critical period of spermiogenesis. In nature, male pupae of M. configurata in diapause are protected from sterilizing temperatures in the soil during August and post-diapause developing pupae pass through the critical period of spermiogenesis in spring (April to June) when soil temperatures throughout the range of M. configurata in Canada are well below 25° C. The distribution of M. configurata in the northern region of the temperate zone may in part be attributable to the sensitivity of spermatogenesis to relatively mild temperatures.     

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.