Brain hormone carrier haemocytes in the moth Monema flavescens

The movement of neurosecretory substances released from the neurosecretory B cell in the pars intercerebralis to the haemolymph was examined with the progress of the termination of diapause in the slug moth pharate pupa, Monema flavescens.The injection of precipitates in the haemolymph of the pharate pupa just before the termination of diapause into diapausing pharate pupae reduced the numbers of days required for them to pupate. In the precipitates, seven types of haemocytes were present. The number of haemocytes, especially the granular cell, increased just before the termination of diapause. AF and CHP positive substances not detected in the haemocytes of diapausing pharate pupae appeared in the granular cells just before the termination of diapause. The period also coincided well with the releasing period of the neurosecretory B cell. Histological examination showed that granular haemocytes gathered around the pars intercerebralis at this period and exchange of neurosecretory substances occurred between granular haemocytes and neurosecretory B cells. Then granular haemocytes migrated to the region of the prothoracic gland. From digestion tests of the neurosecretory substances with rabbit serum and from the implantation tests of the neuroendocrine system, the substances detected in both the neurosecretory B cell and the granular haemocytes seemed to be the same. The dye injection caused a delay in larval-pupal ecdysis emergence. Droplets of black ink are incorporated into the granular haemocytes. This seems to be caused by blocking of the transport of neurosecretory substances released from cytoplasmic processes of the neurosecretory B cell.From these experiments, it is suggested that neurosecretory substances of the prothoracotropic hormone are transported to the prothoracic gland, along with granular haemocytes, after being released directly from the neurosecretory B cell to the haemolymph.     

The induction of an injury reaction in cultured haemocytes from saturniid pupae

The plasmatocytes of diapausing saturniid pupae are round or spindle-shaped cells floating free in the haemolymph. Upon injury to the pupa, these haemocytes become amoeboid and adhesive. A technique is described for the isolation and short-term culture of pupal haemocytes in their inactive state and for their conversion in vitro into the active, ‘injured’ form. The activation of ‘uninjured’ haemocytes was stimulated by fragments of epidermal tissue or by plasma from previously activated blood samples. A fraction capable of stimulating the activation of haemocytes was partially purified from both plasma and epidermal tissue and has been called haemokinin. Haemokinin from either source has a molecular weight in the range of 50,000. The intensity of the haemocyte injury reaction in vitro changes systematically during diapause; the nature and significance of the changes are discussed.     

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.     

Diapause and development in the tobacco budworm,Heliothis virescens: A comparison of haemolymph ecdysteroid titres

The signal to induce diapause in H. virescens comes early in development (prior to the third instar in most insects), but the signal to break diapause can come shortly after entrance into diapause at pupation. Haemolymph ecdysteroid titres in both diapause-bound and non-diapause-bound Heliothis virescens larvae were similar in the first two thirds of the last-larval instar, when similar changes in morphology and behaviour occurred. However, the number of stepwise increases in titre and the timing of the steps was different in the two groups of larvae. Haemolymph ecdysteroid titres in the last third of the instar were approx, five times higher in non-diapause than in diapause-bound larvae. In diapausing pupae, haemolymph ecdysteroid titres dropped to levels found in larvae which had completed two thirds of the last instar. When diapausing pupae were warmed to break diapause, haemolymph ecdysteroid titres rose again. However, 2 of the 4 high ecdysteroid levels detected in pupae developing after diapause break were considerably lower than those detected for non-diapause pupae.     

Comparative studies of oxidative enzyme systems in epidermis and fat body of diapausing and non-diapausing silkmoths

The activities of four oxidative enzyme systems, including NADH oxidase, succinate-cytochrome c reductase, NADH-cytochrome c reductase, and cytochrome c oxidase, were compared in mitochondrial-microsomal preparations from wing epidermis and fat body of diapausing Samia cynthia pupae, presumptively non-diapausing S. cynthia ricini pupae which were caused to diapause by removal of the brain, and non-diapausing S. cynthia ricini during the pupal and pharate adult period. In diapausing pupae the activities of all enzyme systems were low and presented a profile similar to that previously reported for the Cecropia silkmoth. By contrast, in non-diapausing individuals the activities showed substantially higher levels, and an essentially unchanging pattern from just after the larval-pupal ecdysis through most of adult development. These events are functionally correlated with the patterns of biosynthetic activity in diapausing and non-diapausing silkmoths and are discussed in relation to the endocrine control of diapause and development.     

Carotenoids of the pupae of the Large White butterfly (Pieris brassicae) and the Small White butterfly (Pieris rapae)

The light cues received by the larvae of Pieris brassicae which determine diapause can also influence the carotenoid distribution (and hence the colour) in the epidermis and cuticle of the diapausing pupae. Irrespective of background or light cues received during the "sensitive period" of the pharate pupa, these diapausing pupae are coloured green. They then contain more than double the concentration of carotenoids in their epidermis than the non-diapausing pupae. This green colour can be somewhat modified by switching the full grown larvae to long day regimes immediately after feeding ceases.
The Large White and Small White butterfly each has a characteristic carotenoid storage pattern, which can be demonstrated by feeding the larvae on portions of the same cabbage leaves. The braconid parasite Apanteles glomeratus mirrors the carotenoids in its host.     

Toxicity of some insecticides to the haemocytes of giant honeybee,Apis dorsata F. under laboratory conditions

Quantitative studies concerning total and differential haemocyte counts and abnormalities were performed under laboratory conditions for larvae, pupae and adults collected from a wild Apis dorsata colony. Haemolymph samples were observed immediately, thirty and sixty minutes after field recommended concentration exposure of five different insecticides. Total haemocyte counts were significantly higher for larvae and pupae but less for adult bees, however, differential haemocyte counts insignificantly different. Exposure of insecticides showed variable response for tested insecticides with immediate increased change with ethofenprox, diafenthiuron and imidacloprid but decreased for all tested insecticides after sixty minutes. For differential haemocyte counts, plasmatocytes and granulocytes increased with exposure of insecticides. Immune response of haemocytes against insecticides showed different degrees of abnormalities like agglutination, denucleation and cell shape distortion. Such studies may help in possible identification of insect defense mechanisms against their exposure to external hazards for instance insecticide exposure.     

Cycles of protein synthesis during pupal diapause in the flesh fly,Sarcophaga crassipalpis

Protein synthesis is cyclic during pupal diapause in Sarcophaga crassipalpis. These cycles are in phase with infradian MO₂ cycles, which have a periodicity of about 4 days at 25°C. Mean incorporation of [³⁵S]methionine by diapausing pupae was 5.4% during the 2 days of highest MO₂ but dropped to 1.7% during the 2 days of low MO₂. Diapausing pupae treated with a juvenile hormone analog prior to pupariation had a constant high MO₂ similar to peak values observed in untreated pupae, and such pupae consistently incorporated [³⁵S]methionine at a high rate (7.7%). [³⁵S]Methionine incorporation by nondiapausing pupae and pharate adults was eightfold higher than the peak rates observed during diapause. Autoradiography of in vivo labeled proteins indicated quantitative and qualitative changes in the synthesis of proteins by diapausing pupae during different phases of the MO₂ cycle. Brains from diapausing pupae labeled in vitro showed higher incorporation at the peak of the MO₂ cycle than at the nadir of the cycle, but no such differences were detected for integument, fat body, or fat body supernatant. Theses differences in tissue response indicate that control of protein synthesis during diapause is not cell autonomous, but is a function of the metabolism of the intact organism.     

Activity of gut alkaline phosphatase,proteases and esterase in relation to diapause of pharate first instar larvae of the gypsy moth,Lymantria dispar

Two distinctly different patterns of gut enzyme activity were noted in relation to diapause in pharate first instar larvae of the gypsy moth, Lymantria dispar. Trypsin, chymotrypsin, elastase, aminopeptidase and esterase activities were low at the initiation of diapause and through the period of chilling needed to terminate diapause. At the completion of a 150 day chilling period, activity of each of these enzymes quickly increased when the pharate larvae were transferred to 25°C. By contrast, activity of alkaline phosphatase (ALP) increased rapidly at the onset of diapause, remained elevated throughout diapause, increased again during postdiapause, and then dropped at the time of hatching. In addition, zymogram patterns of ALP activity differed qualitatively in relation to diapause: several bands were detectable during the pre- and postdiapause periods, but only one band, a band of high mobility, was visible during diapause. The ALP isozyme present in diapausing pharate larvae had a pH optimum of 10.6. Diapause in the gypsy moth can be averted by application of an imidazole derivative, KK-42, and pharate larvae treated with KK-42 showed elevated protease and esterase activity, low ALP activity, and expressed ALP isozymes with low mobility. Thus the overall patterns of gut enzyme activity and the ALP zymogram in KK-42 treated individuals were similar to those observed in untreated individuals at the termination of diapause. Our results suggest a unique pattern of enzyme activity in the gut that is regulated by the diapause program. Arch. Insect Biochem. Physiol. 37:197–205, 1998. © 1998 Wiley-Liss, Inc.     

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.     

高温条件下棉铃虫化蛹率、夏滞育率和蛹重的变化

在预蛹期,高温处理能诱导棉铃虫蛹进入夏滞育。本实验着重就33～39℃的变温下滞育蛹和未滞育蛹的失重动态进行了对比研究,同时以常温（27℃）下蛹作为参照。研究发现： 在33～39℃的变温条件下,棉铃虫化蛹率显著低于其在常温下的化蛹率,且所化蛹中有63.2%的雄性和10.9%的雌性进入高温夏滞育,其中高温滞育蛹和未滞育蛹分别都轻于正常发育蛹。化蛹后第2日至第5日期间,高温滞育蛹失重量显著低于高温未滞育蛹和正常发育蛹的失重量,分别为3.62、13.30和5.49 mg;蛹期总失重量结果与化蛹后第2～5日间蛹失重量趋势一致,高温滞育蛹、未滞育蛹和正常发育蛹失重量分别为15.60、49.35和26.30 mg。蛹失重动态研究发现高温滞育蛹在夏滞育期间其失重曲线平缓,显著低于高温未滞育蛹和正常发育蛹;高温滞育蛹滞育解除后,其失重曲线与正常发育蛹的失重趋势基本一致。结果表明,棉铃虫夏滞育蛹能通过维持低的代谢水平来度过不利环境,具有一定的生态适应意义。     

Wax secretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta

Scanning electron microscopy of the epicuticle of the tobacco hornworm, Manduca sexta, showed that pupae in diapause possess thicker wax layers than non-diapausing pupae. Extractions of the epicuticle with CHCl₃ established that diapausing pupae secreted over three times as much wax as non-diapausing pupae, and that the total period of wax secretion in diapausing pupae was two to three times longer than that of non-diapausing pupae. Apparently, the extra thickness of the wax layer of diapausing pupae protects the insect from desiccation during diapause. The deposition of additional wax may result from hormonal changes accompanying entry into diapause.     

Cold-hardiness: a component of the diapause syndrome in pupae of the flesh flies, Sarcophaga crassipalpis and S. bullata

ABSTRACT. Diapausing pupae of Sarcophaga crassipalpis Macquart and S. bullata Parker reared at 20 or 25C readily survive exposure to - 10C for at least 25 days. In contrast, non-diapausing pupae produced by a variety of means are consistently intolerant of the low temperature. Non-diapausing pupae are not immediately killed by exposure to -10C: pupae exposed to the low temperature for up to 3 days proceed with pharate adult development but ultimately die before adult eclosion. Unlike many temperate zone insects, diapausing flesh fly pupae do not require a period of chilling for induction of cold-hardiness, and the attribute of cold-hardiness cannot be separated from other features of the diapause syndrome. Some cold-hardiness is already acquired during the third larval instar: diapause-destined larvae exposed to -10C are more successful in pupariating than non-diapause-destined larvae of the same age.     

Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is a medically important invasive species whose geographic distribution has expanded dramatically during the past 20 years, and one of the key elements of its success is its capacity to survive long distance transport as a diapausing pharate first instar larva, encased within the chorion of the egg. We report that pharate larvae entering diapause are larger and contain 30% more lipid than their nondiapausing counterparts. To improve our understanding of the molecular regulation of lipid metabolism during diapause, we assessed the relative mRNA abundance of 21 genes using qRT-PCR. Elevated expression of lipid storage droplet protein 2 during embryonic development likely contributes to the higher amounts of lipid we noted in diapausing individuals. The conservation of lipids during diapause is reflected in downregulation of genes involved in lipid catabolism, including lipase 2, lipase 3, lipase 4, acyl-CoA dehydrogenase 4, and isovaleryl-CoA dehydrogenase. Two genes involved in fatty acid synthesis and modification, Δ(9)-desaturase, and fatty acyl-CoA elongase, were both upregulated in diapausing pharate larvae, suggesting roles for their gene products in generating unsaturated fatty acids to enhance membrane fluidity at low temperatures and generating precursors to the surface hydrocarbons needed to resist desiccation, respectively. Together, the results point to substantial distinctions in lipid metabolism within the embryo as a consequence of the diapause program, and these differences occur both before the actual onset of diapause as well as during the diapause state.     

Neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta

The frontal ganglion of the tobacco hornworm, Manduca sexta (L.), was found to contain two neurosecretory (NS) cells (max dia = 40–45 μm). The cytoplasmic inclusions of the NS cells were stained purple with paraldehyde fuchsin, and marked fluctuations in amounts of NS material in the perikarya were observed, depending upon the developmental status of the insect. The perikarya of NS cells in the frontal ganglia of starved larvae and diapause pupae contained large accumulations of NS material, whereas feeding larvae and developing pharate adults showed relatively low amounts of neurosecretion. Electron microscopy revealed large accumulations of NS granules (dia = 80–240 nm) in the frontal ganglia of diapause pupae, but only slight accumulations of granules were seen in the NS cells of developing larvae and pharate adults.It was concluded that axonal transport and release but not synthesis is shut down during starvation and diapause, leading to accumulation of NS material in the perikarya. It is also suggested that the failure of many investigators to differentiate NS cells in the frontal ganglion of various insects may have been due to the selection of very active stages when the amount of available NS material was too low to be visualized by conventional staining techniques.     

Effects of transgenic Bt cotton on overwintering characteristics and survival of Helicoverpa armigera

The effects of transgenic Bt cotton on the overwintering generation of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), are unknown. We hypothesized that a Bt cotton diet may adversely affect fitness of this generation and examined fresh weight, lipids, glycogens, low-molecular-weight sugars and SCPs (supercooling points) of pupae, as well as survival of larvae, diapausing pupae and adult emergence in comparison with controls. Field and laboratory experiments showed that larvae fed on Bt cotton had a decreased pupation rate, and fewer entered diapause and emerged as adults compared with larvae fed non-Bt cotton. Furthermore, larvae fed Bt cotton had reduced pupal weight, glycogen content and trehalose levels both in diapausing and in non-diapausing pupae, and only diapausing pupae had an increased SCP compared to controls. The SCPs of diapausing pupae reared on Bt cotton were significantly higher than those reared on non-Bt cotton. The trehalose levels of diapausing pupae reared on Bt cotton were significantly lower than those of larvae reared on non-Bt cotton. Thus, these results suggest that a Bt cotton diet weakens the preparedness of cotton bollworm for overwintering and reduces survival of the overwintering generation, which will in turn reduce the density of the first generation in the following year. Effects of transgenic Bt cotton on the overwintering generation of cotton bollworm appear to have significantly contributed to the suppression of cotton bollworm observed throughout northern China in the past decade.     

Juvenile hormone and diapause in the Mediterranean corn borer,Sesamia nonagrioides

Juvenile hormone content of Sesamia nonagrioides larvae reared under different environmental conditions that induce diapause was studied using a bioassay on newly-emerged Tribolium confusum pupae. Two analytical methods were also used to measure JHIII content. Extracts from larvae that developed under different conditions of photoperiod and temperature caused different effects in T. confusum pupae. This can be related to different diapause intensities. Extracts from diapausing larvae produced a higher juvenilizing effect than the expected, considering their JHIII titer calculated by the chromatographic analysis. This indicates that hormones other than JHIII must be present in extracts of diapausing larvae of S. nonagrioides. The analytical procedures confirm that diapausing larvae have a higher JHIII titer in the haemolymph than non-diapausing larvae. This shows that JH is involved in the maintenance of diapause in this species.     

Hormonal control of seasonal morph determination in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

Seasonal morphs (spring and sumer forms) of Papilio xuthus L. are determined coincidentally with diapause and non-diapause in pupae by larval exposure to short days and long days respectively. The neuroendocrine principle underlying seasonal-morph determination was studied using surgical operations in P. xuthus.When recipient 0-day old or chilled diapause pupae were joined to donor 0-day old non-diapause pupae, the recipients developed into summer or intermediate morphs. When the same kinds of recipients used above were joined to 0-day old or chilled diapause pupae, there were no significant effects on the adult morph. In contrast, recipient non-diapause pupae all developed into summer morphs, regardless of groups of the type of donors.Furthermore, when the brain of 5th-instar larvae, pharate pupae or pupae, predetermined to be diapause, was transplanted into the abdomen of 0-day old, 30-day old or chilled diapause pupae, the recipients developed into summer or intermediate morphs.The results indicate that the brain of non-diapause pupae secretes a humoral factor producing the summer morph. In non-diapause pupae, the factor may be secreted at about the stage of larval-pupal ecdysis coincidentally with that of prothoracicotropic hormone.     

Role of antioxidant defense during different stages of preadult life cycle in European corn borer (Ostrinia nubilalis, Hubn.): Diapause and metamorphosis

Antioxidant enzymes, total glutathione (GSH), and ascorbic acid (ASA) were determined in whole body homogenates of nondiapausing larvae, diapausing larvae during the diapausing period (October, December, and February), and in pupae emerged from both diapausing and nondiapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). The activities of catalase, selenium nondependent glutathione peroxidase (GPx), and glutathione-S-transferase (GST), as well as the content of GSH and ASA, were found to vary throughout the larval diapause. Compared to diapausing larvae, nondiapausing larvae were higher in levels of catalase, GPx, GST, and dehydroascorbate reductase (DHAR) activity. GSH content was also increased. However, nondiapausing larvae contained less ASA than diapausing ones. Pupae had higher GPx and GST activity and an increased ASA content compared to larvae. The pupae emerged from nondiapausing larvae had higher GST, glutathione reductase (GR), and DHAR activities, but lower GPx activity and ASA content than those emerged from diapausing larvae. Correlation analysis revealed differences in the way the antioxidant level is equilibrated for a particular stage and developmental pattern. The results suggest that cellular antioxidants are involved in both the protection of cells and the regulation of redox levels during the pre-adult stages of Ostrinia nubilalis. Arch. Insect Biochem. Physiol. 55:79-89, 2004.     

Diapause phenomena in non-diapausing last instar larvae,pupae, and pharate adults of the Colorado beetle

From the first day of the last (fourth) larval instar no trace of juvenile hormone (JH) can be detected in the haemolymph by Galleria bioassay. Three specific diapause proteins, which are also found in diapausing adults, appear in the haemolymph. These proteins disappear towards the end of the pupal stage. Study of the ultrastructure of the fat body revealed the formation from lysosomes of proteinaceous bodies which are also characteristic for adult diapause. The behaviour of last instar larvae and pupae resembles that of prediapausing and diapausing adults respectively. Injection of synthetic JH delays the appearance of the diapause proteins in the haemolymph and of proteinaceous bodies in the fat body for 2 to 3 days. The absence of JH seems to trigger off these diapause phenomena.