A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.     

Repeated cycles of chilling and warming effectively terminate prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis (Coleoptera: Curculionidae) requires one or more years to complete its life cycle, owing to prolonged larval diapause. To compare the effects of temperature cycles and total periods of chilling on the termination of prolonged diapause, larvae were subjected to different chilling (5 degrees C) and warming (20 degrees C) cycles ranging from 30 to 720 days, and all cycles were repeated until the sum of chilling and warming periods reached 720 days. The prolonged diapause of C. sikkimensis was more effectively terminated by repeated cycles of chilling and warming than by prolonging the continuous chilling period. However, extremely short temperature cycles were not highly effective in enhancing diapause termination, even when such cycles were repeated many times. To examine the role of warming periods on diapause termination, diapause larvae were subjected to a sequence of chilling (120 days at 5 degrees C) and warming (240 days at 20 degrees C) with a warming period (0-120 days at 20 degrees C) inserted in the chilling period. Diapause larvae that were not reactivated in the first chilling period required exposure to a certain period of warming before they were able to complete diapause development in the subsequent chilling. Thus, C. sikkimensis appears to spread its reactivation times over several years in response to seasonal temperature cycles.     

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, ¹³C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were -helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no -helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.     

Termination of diapause in the Cinnabar moth (Tyria jacobaeae) by chilling

The effect of chilling time on the termination of pupal diapause was studied in the Cinnabar moth, Tyria jacobaeae L., Lepidoptera: Arctiidae. Chilling followed by a temperature of 20°C was effective in terminating diapause. Chilling for 120 days immediately after pupation yielded the highest percentage emergence and resulted in the shortest total pupal development time (162 days). A more natural treatment, a warm period before cold treatment, did not result in a higher percentage of emergence or in a shorter total pupal development time.During hibernation this species experiences two successive processes: diapause development during the first three to four months of cold, followed by a period of quiescence. Application of high temperatures in this latter period immediately starts morphogenesis. The impact of a relative humidity of 70% during the pupal stage for complete wing unfolding is demonstrated. The underlying mechanism of diapause is discussed.

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Résumé L'effet sur la levée de la diapause de différents régimes de température infligés aux chrysalides est étudié chez Tyria jacobaeae L. (Lepidoptera: Arctiidae). Une mise au froid suivie de hautes températures lève efficacement la diapause. Un refroidissement pendant 120 jours immédiatement après la nymphose entraîne le plus grand pourcentage d'éclosions et la durée totale du développement des chrysalides la plus courte (162.3 j). Un traitement plus naturel, une période chaude avant la période froide, n'entraîne pas un plus grand pourcentage d'éclosions ni une durée plus courte de développement.Il est proposé que durant l'hibernation cette espèce passe par deux processus successifs: un diapause durant les premiers trois ou quatre mois de froid, suivie d'une période de quiescence. Le transfert à des hautes températures pendant cette derpériode déclanche immédiatement la morphogénèse. L'influence d'un taux élevé d'humidité relative durant le stade chrysalide sur le déploiement complet des ailes est démontré. Le méchanisme de diapause sous-jacent est discuté.

     

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly,Delia antiqua

The influence of pupal diapause on adult eclosion rhythm of Delia antiqua was investigated. When non-diapause and diapause pupae were exposed to various photoperiods at 15, 20 and 25 °C, both of them emerged as adults close to the light-on time, but the phase of eclosion varied with photoperiod and temperature. Moreover, there was a significant difference in the eclosion time between non-diapause and diapause pupae; the eclosion peak of diapause pupae was earlier than that of non-diapause pupae. When non-diapause and diapause pupae were transferred to constant darkness (DD) after having experienced LD 12:12 at 15, 20 and 25 °C, both showed circadian rhythmicity in eclosion. Although the free-running period (τ) decreased slightly as temperature increased in both non-diapause and diapause pupae, the latter tended to show shorter τ than the former. This observation suggests that the observed difference in eclosion time in LD cycles between non-diapause and diapause pupae is due to differences in τ.     

High temperature and hexane break pupal diapause in the flesh fly, Sarcophaga crassipalpis, by activating ERK/MAPK

Pupal diapause in the flesh fly, Sarcophaga crassipalpis, can be terminated by exposure to high temperatures or, artificially, with a topical application of organic solvents. To analyze the molecular mechanisms involved in diapause termination we explored the possibility that the mitogen-activated protein kinases (MAPK) are involved in this response. Levels of phospho-ERK increased within 10 min after hexane application. Extracellular signal-regulated kinase (ERK) was also activated when pupae were transferred from 20 to 25 degrees C, thus suggesting that ERK activation is a likely component of the signal transduction pathway used to initiate development in response to diapause-terminating signals. 20-Hydroxyecdysone and cyclic GMP terminate diapause in this fly, and the juvenile hormone analog methoprene shortens the diapause, but none of these agents activated ERK. ERK was readily activated in isolated abdomens treated with hexane, thus we conclude that ERK is directly activated by the hexane treatment. ERK activation was evident in the brain, epidermis, midgut and fat body, but not in the ventral nerve mass or ring gland, thus suggesting that ERK does not act directly on the ring gland to promote ecdysteroid synthesis but exerts its effect through stimulation of the brain.     

柞蚕小热休克蛋白20.1的基因鉴定及免疫功能研究

热休克蛋白(HSP)是一类广泛存在于各类生物中的具有分子伴侣功能的蛋白质。近年来研究发现HSP与机体许多功能如免疫、凋亡、衰老等密切相关。柞蚕(Antheraea pernyi)小热休克蛋白20.1(Aphsp20.1)基因的开放读码框长度为534bp,编码178个氨基酸。序列比对结果表明,柞蚕小热休克蛋白20.1属于HSP20家族。组织定量显示这Aphsp20.1在中肠和脂肪体分布较高。此外用大肠杆菌Escherichia coli及Micrococcus luteus病原微生物注射入5龄3天柞蚕幼虫后,发现Aphsp20.1的基因表达菌明显上调。另外体外抑菌试验结果发现纯化后的蛋白也具有一定的抑菌作用。该研究表明ApHSP20.1在柞蚕的免疫功能中具有重要作用,该研究不仅为我们进一步了解更加复杂的高等生物天然免疫反应提供一些相关的研究线索;而且对柞蚕天然免疫的研究有利于更好地理解昆虫自身的免疫系统,为保护益虫防治害虫提供重要依据。     

Regulation of gonad development and respiratory metabolism associated with food availability and reproductive diapause in the rice bug Leptocorisa chinensis

Food has an influence on many life history traits related to dormancy in insects. In our previous study with the rice bug Leptocorisa chinensis (Dallas) (Hemiptera: Alydidae), diapausing females transferred to conditions physically favorable for promoting the gonad development required food intake to resume gonad development, whereas males did not. These differences in response to food between males and females lead to two questions: (1) Was diapause in the starved females completed? (2) Were the starved males that resumed gonad development at the same physiological status as fed males? We tested these questions with two physiological indicators: gonad status and respiratory rate. Results indicate that starved females are able to complete diapause, but show depressed respiration relative to well-fed insects in diapause. Similar to females, starved males that resumed postdiapause gonad development also had depressed respiratory rate, and hence physiological status is presumed to be different between starved and fed individuals. In this study, it was also found that the photoperiodic signal is storable, whereas the food signal acts directly. The adaptive significance of regulation of gonad development and respiratory metabolism in relation to phenology of suitable host plant and reproductive strategy is discussed in both sexes.     

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly, Delia antiqua: the change of rhythmicity

When pupae of Delia antiqua were transferred to constant darkness (DD) from light-dark (LD) cycles or constant light (LL), the sensitivity to light of the circadian clock controlling eclosion increased with age. The daily rhythm of eclosion appeared in both non-diapause and diapause pupae only when this transfer was made during late pharate adult development. When transferred from LL to DD in the early pupal stage, the adult eclosion was weakly rhythmic in non-diapause pupae but arrhythmic in diapause pupae. However, the sensitivity of the circadian clock to temperature cycles or steps was higher in diapause pupae than in non-diapause pupae; in the transfer to a constant 20 degrees C from a thermoperiod of 25 degrees C (12 h)/20 degrees C (12 h) on day 10 after pupation or from chilling (7.5 degrees C) in DD, the adult eclosion from diapause pupae was rhythmic but that from non-diapause pupae arrhythmic. In a transfer to 20 degrees C from the thermoperiod after the initiation of eclosion, rhythmicity was observed in both types of pupae. The larval stage was insensitive to the effect of LD cycle initiating the eclosion rhythm. In D. antiqua pupae in the soil under natural conditions, therefore, the thermoperiod in the late pupal stage would be the most important 'Zeitgeber' for the determination of eclosion timing.     

The effect of chronic chlorpromazine administration on monoamine levels in various regions of rat brain

The neuroleptic drug, chlorpromazine (CPZ) has been shown to exert its antipsychotic effect by blocking post synaptic dopamine receptors. However, its effect on steady state levels of monoamines is still in discrepancy. In the present study, CPZ (4 mg/kg body weight) was administered intraperitoneally to adult Wistar rats chronically for 75 days and the levels of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) were assayed in various brain regions by high performance liquid chromatography (HPLC). After the experimental period body and brain weights were not statistically different from controls. NE and 5-HT levels were increased only in hippocampus by 15% (p<0.01) and 16% (p<0.01) respectively. DA levels were consistently increased in cortex by 39% (p<0.001), striatum-accumbens by 18% (p<0.01), hippocampus by 27% (p<0.01), hypothalamus by 34% (p<0.001), cerebellum by 36% (p<0.001) and brainstem by 40% (p<0.001) in CPZ treated rats compared to controls. The results suggest that chronic CPZ administration increases DA levels in almost all regions of brain and reflect the ability of CPZ to preferentially interfere with synaptic transmission mediated by DA in brain. It also suggests that this increase in DA might be responsible for certain side effects seen in patients after chronic CPZ treatment.     

Effects of temperature during chilling and pre-chilling periods on diapause and post-diapause development in a katydid, Eobiana engelhardti subtropica

In Eobiana engelhardti subtropica, early laid eggs reach the diapause stage in early autumn. For long periods before winter, the eggs are exposed to temperatures higher than their theoretical lower threshold for development. In contrast, late-laid eggs cannot reach their diapause stage before winter. Our study showed that E. e. subtropica copes with these difficulties via the thermal response involving embryonic diapause. In this katydid, the almost fully developed embryo undergoes an obligatory diapause. When diapause eggs were maintained at a temperature of 20 degrees C or higher, diapause persisted for a long time. Diapause was effectively terminated by temperatures ranging from 1 to 11 degrees C, and hatching occurred successfully at temperatures from 11 to 15 degrees C. In addition to the chilling temperature, pre-chilling temperature modified diapause intensity and hatching time. Diapause eggs hatched earlier after chilling when the pre-chilling temperature was lower, within a range of 14.5-25 degrees C. Thus, the low-temperature requirement for diapause termination prevents early laid eggs from untimely hatching in autumn, and low temperatures before and during winter decrease diapause intensity and shorten the hatching time in the following spring. When eggs were chilled before diapause, they tolerated chilling and averted diapause. Thus, even if eggs encounter low temperatures before diapause, they can hatch in the following spring.     

Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are encoded by a single mRNA in the suboesophegeal ganglion (SG) and are responsible for induction of embryonic diapause in Bombyx mori and sex pheromone biosynthesis in lepidopteran insects. PBAN cDNA analyses revealed that the DH-like peptide is present in several species that have a pupal diapause. However, the function of the DH-like peptide remains unknown. In the present study, we cloned the cDNA encoding DH-PBAN in Helicoverpa armigera utilizing the rapid amplification of the cDNA ends method. The nucleotide se quence analysis revealed that the longest open reading frame of this cDNA encodes a 194-amino acid precursor protein that con tains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which have a common C-terminal pentapeptide motif FXPR/KL ( X=G, T, S). A homology search showed that H. armigera DH-like and PBAN are highly homologous to those from other insects. Northern blot analysis demonstrated a single message RNA corresponding to the size of Har-DH-PBAN cDNA from pupal SG with significantly higher expression in the SG of nondiapause pupae than diapausing pupae. Western blot analysis showed DH-like peptide expression from SG of both males and females. When DH-like peptide was injected into nondiapause larvae and pupae, it did not induce diapause, but rather efficiently broke pupal diapause in H. armigera. The ED(50) of DH to terminate pupal diapause is 20 pmol/pupae. The other four FXPRLamide neuropeptides from the DH-PBAN polyprotein precursor have cross activity for diapause termination. These observations therefore suggest a potential role for these FXPRL family peptides in promoting continuous development in several noctuid species. The high expression of this gene in pharate adults and adults indicates that the FXPRL family peptides may have multiple physiological functions.     

Stress-related proteins compared in diapause and in activated, anoxic encysted embryos of the animal extremophile, Artemia franciscana

Previous work indicated similarities between diapause and anoxic quiescence in encysted embryos (cysts) of the brine shrimp Artemia franciscana. That possibility was examined further in the present study through an immunochemical study of the following stress-related proteins in low speed supernatants and pellets: hsc70, artemin, p26, hsp21, LEA Group 1 protein and p8. Changes in the amounts and locations of these proteins occurred during the initial period after release of diapause cysts from females, and after activated (diapause-terminated) cysts were made anoxic. However, with the passage of incubation time the patterns seen in both kinds of cysts were more similar than different, lending further support to the possibility that activated anoxic embryos retain many of the mechanisms operative in the previous diapause condition.     

Antennal thermo- and hygrosensitive sensilla in Antheraea pernyi (Lepidoptera,Saturniidae): ultrastructure and immunohistochemical localization of Na+,K+-ATPase

Summary In an attempt to identify and localize the components of voltage sources involved in sensory transduction in insect sensilla, the thermo-/hygrosensitive sensilla of the moth Antheraea pernyi were probed with a polyclonal antiserum against Na⁺,K⁺-ATPase in cryofixed and freeze-substituted preparations. The antiserum recognized epitopes on the cytoplasmic membranes of the dendritic inner segments and somata of the sensory cells and also on the cytoplasmic membranes of glial cells surrounding the initial axon segments. The findings support the current concept that ion pumps in the cytoplasmic membranes of the dendritic inner segments and somata of the sensory cells contribute to the maintenance of the resting potential of the sensory cells and to the driving forces generating the receptor currents in response to stimulation of the sensillum. Morphological features and immunohistochemical characteristics of the region of the initial axon segment are also discussed with respect to the initiation of action potentials in these sensilla.     

Cerebral ischemia: Changes in brain choline,acetylcholine, and other monoamines as related to energy metabolism

The relationship of cerebral neurotransmitters acetylcholine (ACh), noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5HT) to the energy state of the brain was examined in mice at various times following complete ischemia produced by decapitation, in gerbils submitted to transient global ischemia (10 min bilateral carotid artery occlusion, 5 or 30 min recirculation), and in rats 24 hr after irreversible microembolism. Ischemia caused significant reductions in brain monoamine concentrations. The alterations in NA, DA, and 5HT levels persisted during recirculation and were unrelated to energy restoration. They were accompanied by an increase in the concentrations of related metabolites, suggesting that synthesis was unable to compensate for the release of the transmitters at early post-ischemic time periods. As described for the catecholamines and 5HT, ischemia resulted in a significant decrease in ACh level, but recirculation was associated with a rapid increase in ACh concentration. Impaired synthesis and/or increased release of ACh can be responsible for the decrease in ACh concentration during ischemia. Early post-ischemic elevation of ACh may be related to the large increase in brain choline brought about by ischemia.     

Social rank and brain levels of monoamines and monoamine metabolites in Arctic charr,Salvelinus alpinus (L.)

Summary Dominance hierarchy was determined in 5 groups of juvenile Arctic charr (Salvelinus alpinus), each group consisting of 4 fish. Telencephalon and brain stem (remaining parts of the brain) were analyzed with regard to their content of monoamines and monoamine metabolites. No significant differences were observed in the concentrations of norepinephrine (NE), dopamine (DA), or serotonin (5-hydroxytryptamine, 5-HT) between fish with different social rank. However, the concentration of 5-hydroxyindoleacetic acid (5-HIAA), the principle metabolite of 5-HT, was significantly higher in subordinate fish, and a significant inverse linear correlation was found between 5-HIAA concentration and social rank (as measured by dominance index) in the brain stem. In the telencephalon the dominant fish had a significantly higher level of homovanillic acid (HVA), a major DA metabolite. These findings indicate a greater serotonergic activity, possibly associated with increased stress, as well as a lower dopaminergic activity, possibly associated with reduced aggression, in subordinate charr. The differences between dominant and subordinate fish could either be caused by social interactions or reflect innate individual differences in monoamine utilization, predisposing individuals for dominant or subordinate positions in the dominance hierarchy.Abbreviations DA dopamine - DI dominance index - NE norepinephrine - 5-HT serotonin (5-hydroxytryptamine) - 5-HIAA 5-hydroxyindoleacetic acid - 5-HTOH 5-hydroxytryptophol - HVA homovanillic acid     

Differential expression, phosphorylation of COX subunit 1 and COX activity during diapause phase in the cotton bollworm, Helicoverpa armigera

Helicoverpa armigera (Lepidoptera, Noctuidae) is an important agricultural pest with a pupal diapause. Cytochrome c oxidase (COX) is a key speed-limited enzyme of oxidative phosphorylation in mitochondria for ATP production. A differentially expressed cDNA fragment encoding COX subunit 1 (cox1) was cloned by differential display-PCR from the pupal brain at diapause termination with an injection of ecdysone. We then obtained the full length of H. armigera cox1 (Hea-cox1) cDNA which has an open reading frame of 1530 nucleotides encoding a putative protein of 510 amino acid residues, with CGA as a start codon. To evaluate the response to different energy demands during pupal development and at diapause termination, we assessed the expression of Hea-cox1 mRNA and protein, COX activity and its phosphorylation. The results show that Hea-cox1 expression at the mRNA and protein levels is associated with COX activity, and high levels of Hea-cox1 expression and COX activity are present in nondiapause pupae, suggesting that low energy metabolism provided by oxidative phosphorylation in mitochondria in diapause individuals is necessary. After diapause is broken by injection of 20-hydroxyecdysone, expression of Hea-cox1 mRNA and protein increases gradually and COX activity increases significantly. Furthermore, Hea-cox1 phosphorylation is closely correlated with COX activity, suggesting that reversible protein phosphorylation may play a key role in insect diapause by suppressing the rate of energy production.