Non-diapause Eggs Research Articles

Paralytic Peptide Binding Protein (PP-BP) Gene Expression During Egg Diapause and Its Multi-Gene Organization in the Silkworm Bombyx mori

Paralytic peptide binding proteins (PP-BP) are 30KP proteins that show similarity to ENF binding proteins. The ENF-BP act as active regulators of ENF peptides. ENF peptides are multifunctional insect cytokines. The comparison of gene expression in diapause induced and non-diapause eggs at different time intervals after oviposition showed an upregulation of PP at 18h as well as PP-BP at 12 and 18h after oviposition along with few other genes. The current study has been taken up to investigate the role of PP as well as PP-BP in diapause induction in polyvoltine silkworms and to study the multigene organization of PP-BP in the Bombyx mori genome. The tissue specific expression analysis revealed that, PP-BP is highly expressed in fat body followed by egg and brain while no expression was observed in midgut. The expression levels of PP and PP-BP in diapause and non-diapause eggs from 0h to 48h after oviposition, validated through realtime PCR revealed that PP is highly expressed at 18 and 24h while PP-BP expression is higher at 12 and 18h time intervals suggesting their possible role in diapause induction. The whole genome survey of the PP-BP paralogous sequences revealed a total of 46 B. mori PP-BP homologs that are classified into 3 categories viz., ENF-BP, Typical 30KPs and serine/threonine rich 30KPs. These paralogous sequences are distributed on chromosomes 7, 20, 22 and 24, all 30KP and S/T rich 30KP proteins are present in the same locus of chromosome 20.

Influence of Photoperiod on Hydrogen Peroxide Metabolism during Diapause Induction in Cotesia vestalis (Haliday) (Hymenoptera: Braconidae)

Abstract Hydrogen peroxide is a toxic metabolite of oxidative metabolism, but may also serve as a broad spectrum signaling molecule controlling a variety of essential physiological processes. Photoperiod influences diapause beyond the maternal generation in Cotesia vestalis and we speculated that intracellular hydrogen peroxide was a possible signal mediating this generational effect. To investigate the signaling functions of hydrogen peroxide in diapause induction in C. vestalis, hydrogen peroxide and activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase were measured at diapause, and in maternal non-diapausing prepupa, adult, and offspring egg stages form the cultures reared at 25°C under the photoperiods of 8∶16, 12∶12, and 16∶8 (L∶D) h. The hydrogen peroxide contents and activities of superoxide dismutase and peroxidase in maternal non-diapausing prepupae and adults declined as the light period was reduced, whereas catalase activity increased significantly. Compared to d...

The role of gene flow asymmetry along an environmental gradient in constraining local adaptation and range expansion

Theoretically, asymmetric gene flow along an environmental gradient can limit species range expansion by keeping peripheral populations from locally adapting. However, few empirical studies have examined this potentially fundamental evolutionary mechanism. We address this possibility in the cricket Allonemobius socius, which exist along a season-length gradient where the probability of producing a single generation per year (univoltinism) increases with latitude. As the probability of univoltinism increases northwards, populations are expected to hedge their bets by producing a greater proportion of diapause eggs when exposed to a mild diapause cue. However, gene flow from southern populations may disrupt local adaptation in the north by reducing the proportion of diapause eggs (expected to be 100% in pure univoltine environments). This may limit range expansion along the northern periphery where A.socius compete with A.fasciatus, a sister species that exhibits an invariant diapause-only egg-laying strategy. To assess the potential for range limitation, we examined diapause incidence (the proportion of diapause eggs produced under diapause conditions), gene flow symmetry and population structure across nine A.socius populations. We found that gene flow was asymmetric and biased northwards towards the periphery. Furthermore, peripheral populations that inhabited pure univoltine environments produced numerous nondiapause eggs (a southern, bivoltine diapause phenotype), which we assume to be a suboptimal phenotype. These patterns suggest that asymmetric gene flow along the gradient constrains adaptation in peripheral populations, potentially constraining species range expansion.

Low-temperature threshold for egg survival of a post-diapause and non-diapause European aedine strain, Aedes albopictus (Diptera: Culicidae)

BackgroundThe interplay between global warming and invasive arthropods in temperate zones is of utmost interest in terms of the potential expansions of vector-borne diseases. Up to now, investigations on the recent establishment of mosquito vectors have focused on temperatures during their phases of activity. However, cold temperatures may also act as a strong ecological constraint. Projected changes in winter climate indicate an increase of mean minimum temperatures of the coldest quarter, less frequent days with frost and a shorter frost-season in Europe at the end of the century. Nevertheless, single cold extremes are also expected to persist under warming scenarios, which have a strong impact on reproduction success.MethodsHere, the temperature constraints of European Aedes albopictus eggs, which had passed through a diapause, compared to non-diapausing eggs were examined systematically under controlled laboratory conditions. Additionally, one tropical strain of Ae. albopictus and of Ae. aegypti was used in the comparison.ResultsThe lower temperature threshold tolerated by the European eggs of Ae. albopictus which have undergone a diapause, was -10°C for long term exposures (12 and 24h) and -12°C for 1h exposure. Non-diapausing eggs of European Ae. albopictus were found to hatch after a -7°C cold treatment (8, 12 and 24h exposure). Both tropical aedine species only tolerated the long term treatment at -2°C. Neither Ae. albopictus nor Ae. aegypti eggs hatched after being exposed to -15°C. Survival was mainly influenced by temperature (F = 329.2, df = 1, p < 0.001), whereas the duration of the cold treatment only significantly influenced the hatching response at the thermal limits of survival (F = 5.6, df = 1, p = 0.031) but not at 0°C (F = 0.1, df = 1, p = 0.730). Hatching success after the cold treatment was significantly increased in European eggs, which have undergone a diapause compared to non-diapausing eggs (F = 14.7, df = 3, p < 0.001). These results illustrate rapid adaptation.ConclusionsHere, low temperature thresholds for aedine mosquito egg survival were detected. The compilation of risk maps for temperate regions can substantially be improved by considering areas where an establishment of a vector population is unlikely due to winter conditions.

Diapause specific gene expression in the eggs of multivoltine silkworm Bombyx mori, identified by suppressive subtractive hybridization

Molecular mechanism controlling egg diapause remains obscure in silkworm, Bombyx mori. An attempt is made to decipher various molecular events occurring during embryonic diapause in multivoltine silkworm, B. mori. Using suppressive subtractive hybridization (SSH), 186 cDNA clones isolated from both diapause and nondiapause eggs were sequenced. Of the sequenced clones, 29 matched with silkbase entries and these identified putative genes were classified into six functional groups such as regulatory, food utilization, stress response, metabolic, ribosomal and transposable elements. Among these genes, twelve belonged to regulatory group while, one taste receptor type 2 member 117 gene was related to food utilization. One heat shock cognate 70 kDa protein and 3 of the ubiquitin family were identified under stress response category. Similarly, four genes were identified as metabolic genes, 3 belonging to chitin family and one propanediol utilization protein. Of the seven genes identified in ribosomal groups, most of them were 60s ribosomal protein subunits. However, one negative regulation of transcription gene identified was a transposable element. The qPCR analysis confirmed the expression of 21 of the above genes, wherein, 6 were upregulated during diapause, 12 during non-diapause, while, 3 remained unchanged.

Developmental Changes in the Localization of Protein Kinase CK2 in Non-Diapause and Diapause Eggs of the Silkworm,Bombyx mori

To analyze the role of protein kinase CK2 (CK2) during early embryogenesis in non-diapause and diapause of the silkworm, the distribution and localization of Bombyx mori CK2 (BmCK2) were investigated by an immunohistochemical technique using antibodies against the α- and β-subunits of BmCK2. Both were localized in blastoderm cells of non-diapause and diapause eggs until 24 h after oviposition. More than 24 h after oviposition, however, the distribution of BmCK2 was different in non-diapause and diapause eggs. In non-diapause eggs, BmCK2 was mainly localized in yolk cells. In contrast, in diapause eggs, the localization was mainly observed in germ-band cells. Furthermore, we confirmed that the RNA helicase-like protein that was localized together with BmCK2 in non-diapause eggs was phosphorylated by BmCK2 in vitro. These data suggest that the role of BmCK2 is different in non-diapause and diapause eggs.

Metabolic rate in diapause and nondiapause brown locust eggs correlated with embryonic development

Insects use dormancy to survive adverse conditions. Brown locust Locustana pardalina (Walk.) eggs offer a convenient model to study dormancy (diapause and quiescence), which contributes to their survival under arid conditions. The metabolic rates of developing nondiapause, diapause and quiescent eggs are compared in the present study using closed‐system respirometry. The embryo becomes committed to continue development and hatch or to enter diapause 6 days after the eggs are placed on moist soil. The metabolic rate of nondiapause eggs increases exponentially until hatching, whereas that of diapause eggs is low and stable. The metabolic rate of diapause laboratory eggs (1.9 ± 0.6 µL CO2 mg−1 h−1) is significantly higher than that of field eggs (0.5 ± 0.3 µL CO2 mg−1 h−1), although the ranges of metabolic rate overlap and the embryos are all in late anatrepsis. The metabolic rate of quiescent eggs is similar to that of diapause eggs but decreases with time. Low metabolic rates during arrested development allow eggs to persist over long periods before hatching.

Involvement of 4E-BP phosphorylation in embryonic development of the silkworm, Bombyx mori

Phosphorylation of the translational repressor 4E-binding protein (4E-BP) plays a critical role in regulating the overall translation levels in cells. In the present study, we investigated 4E-BP phosphorylation of Bombyx mori eggs by an immunoblot analysis of a conserved phospho-specific antibody to 4E-BP and demonstrated its role during embryonic development. When HCl treatment was applied to diapause-destined eggs at 20h after oviposition, a dramatic increase in the phosphorylation of 4E-BP occurred 5min after treatment with HCl, and high phosphorylation levels were maintained throughout embryonic stage in HCl-treated eggs compared to those in diapause (control) eggs. When HCl treatment was applied to diapause eggs on day 10 after oviposition, no dramatic activation in 4E-BP phosphorylation occurred, indicating stage-specific effects of HCl treatment. In both non-diapause eggs and eggs whose diapause had been terminated by chilling of diapausing eggs at 5°C for 70 days and then were transferred to 25°C, high phosphorylation levels of 4E-BP were also detected. Moreover, 4E-BP phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of rapamycin, a specific inhibitor of mammalian target of rapamycin (TOR) signaling, and LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, but not the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, dose-dependently inhibited 4E-BP phosphorylation in dechorionated eggs, indicating that PI3K/TOR signaling is an upstream signaling event involved in 4E-BP phosphorylation. Examination of 4E-BP gene expression levels showed no differences between treatments with HCl and water in the first hour after treatment, indicating that changes in phosphorylation of 4E-BP upon HCl treatment are mainly regulated at the post-transcriptional level. In addition, MAPK pathways and glycogen synthase kinase (GSK)-3β phosphorylation were not significantly affected in the first hour after HCl treatment. These results demonstrate that the rapid phosphorylation of 4E-BP is an early signaling event in embryonic development in the eggs whose diapause initiation was prevented by HCl treatment, thus being involved in the embryonic development of B. mori.

Variation in Cold Hardiness of Nondiapausing Eggs of Nine Aedes (Stegomyia) Species (Diptera: Culicidae) From Eastern Asia and Pacific Islands Ranging From the Tropics to the Cool-Temperate Zone

Variation in cold hardiness was studied in the laboratory for the nondiapausing eggs of nine Aedes (Stegomyia) species from eastern Asia and Pacific islands, ranging from the tropics to the cool-temperate zone. Species included were Aedes aegypti (L.), Aedes albopictus (Skuse), Aedes flavopictus Yamada, Aedes galloisi Yamada, Aedes hensilli Farner, Aedes palauensis Bohart, Aedes paullusi Stone and Farner, Aedes riversi Bohart and Ingram, and Aedes scutellaris (Walker). For species with distributions covering two or three climatic zones, at least one population was included from each zone, except for Ae. aegypti (subtropical populations were unavailable). Some eggs of four species confined to the tropical zone survived short exposure to freezing temperature (> or = -5 degrees C) when humidity was high. There were inter- and intraspecies variations in cold hardiness of nondiapausing eggs. Cold hardiness in each climatic zone differed in that populations of species with more northerly distributions were more cold-hardy than species with southerly distributions. In Ae. albopictus and Ae. flavopictus that cover three climatic zones, populations from cooler regions were more cold-hardy than populations from warmer regions. A possibility of southern species without diapausing eggs to colonize temperate eastern Asia was discussed.

Dynamics of the “Popcorn” Wolbachia Infection in Outbred Aedes aegypti Informs Prospects for Mosquito Vector Control

Forty percent of the world's population is at risk of contracting dengue virus, which produces dengue fever with a potentially fatal hemorrhagic form. The wMelPop Wolbachia infection of Drosophila melanogaster reduces life span and interferes with viral transmission when introduced into the mosquito Aedes aegypti, the primary vector of dengue virus. Wolbachia has been proposed as an agent for preventing transmission of dengue virus. Population invasion by Wolbachia depends on levels of cytoplasmic incompatibility, fitness effects, and maternal transmission. Here we characterized these traits in an outbred genetic background of a potential target population of Ae. aegypti using two crossing schemes. Cytoplasmic incompatibility was strong in this background, and the maternal transmission rate of Wolbachia was high. The infection substantially reduced longevity of infected adult females, regardless of whether adults came from larvae cultured under high or low levels of nutrition or density. The infection reduced the viability of diapausing and nondiapausing eggs. Viability was particularly low when eggs were laid by older females and when diapausing eggs had been stored for a few weeks. The infection affected mosquito larval development time and adult body size under different larval nutrition levels and densities. The results were used to assess the potential for wMelPop-CLA to invade natural populations of Ae. aegypti and to develop recommendations for the maintenance of fitness in infected mosquitoes that need to compete against field insects.

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-NH(2) (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.

Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause

Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8kDa proteins function to block permeability of O2 during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis.

Electron-microscope investigation on the early developmental stages of diapause and non-diapause eggs in the silkworm, Bombyx mori L.

Electron-microscope investigation on the early developmental stages of diapause and non-diapause eggs in the silkworm, Bombyx mori L.

Changes in RNA/DNA ratios during the embryogenesis of non-diapause silkworm eggs

Changes in RNA/DNA ratios during the embryogenesis of non-diapause silkworm eggs

Induction of non-diapause egg by dark and low temperature incubation in local variety of the silkworm, Bombyx mori

Induction of non-diapause egg by dark and low temperature incubation in local variety of the silkworm, Bombyx mori

Some notes of the pigmented and non-diapausing egg strain, Bombyx mori

Some notes of the pigmented and non-diapausing egg strain, Bombyx mori

Genetical studies of the pigmented and non-diapausing egg mutant in Bombyx mori

Genetical studies of the pigmented and non-diapausing egg mutant in Bombyx mori

Further Studies on the Alteration of Egg Diapause in Bombyx mori by Ouabain-Diapause Egg Production in Non-diapause Egg Producers

Further Studies on the Alteration of Egg Diapause in Bombyx mori by Ouabain-Diapause Egg Production in Non-diapause Egg Producers

Metabolism of hydrogen peroxide between diapause and non‐diapause eggs of the silkworm, Bombyx Mori during chilling at 5°C

When diapause and non-diapause eggs of the same bivoltine strain of Bombyx mori were chilled at 5 degrees C for more than 30 days, the hatchability of diapause eggs increased while that of non-diapause eggs decreased, respectively. To investigate the relationship between effects of chilling on the hatchability and the metabolism of hydrogen peroxide (H2O2), content of H2O2 and activities of superoxide dismutase (SOD), xanthine oxidase (XO), and catalase (CAT) between diapause and non-diapause eggs were determined during the chilling at 5 degrees C. The significant enhancement of H2O2 occurred prior to the quick increase of the hatchability in diapause eggs and coincided with the quick decline of the hatchability in non-diapause eggs, respectively. Diapause eggs contained significantly higher H2O2 and XO activity and lower CAT activity compared to non-diapause eggs. Our results showed that there were significant differences in the metabolism of H2O2 between diapause and non-diapause eggs during chilling and that significant enhancement of H2O2 may be involved in the diapause termination of diapause eggs and the cell damage of non-diapause eggs.

The molecular physiology of increased egg desiccation resistance during diapause in the invasive mosquito,Aedes albopictus

Photoperiodic diapause is a crucial adaptation to seasonal environmental variation in a wide range of arthropods, but relatively little is known regarding the molecular basis of this important trait. In temperate populations of the mosquito Aedes albopictus, exposure to short-day (SD) lengths causes the female to produce diapause eggs. Tropical populations do not undergo a photoperiodic diapause. We identified a fatty acyl coA elongase transcript that is more abundant under SD versus long-day (LD) photoperiods in mature oocyte tissue of replicate temperate, but not tropical, A. albopictus populations. Fatty acyl CoA elongases are involved in the synthesis of long chain fatty acids (hydrocarbon precursors). Diapause eggs from a temperate population had one-third more surface hydrocarbons and one-half the water loss rates of non-diapause eggs. Eggs from a tropical population reared under SD and LD photoperiods did not differ in surface hydrocarbon abundance or water loss rates. In both a temperate and tropical population, composition of hydrocarbon chain lengths did not differ between eggs from SD versus LD conditions. These results implicate the expression of fatty acyl coA elongase and changes in quantity, but not composition, of egg surface hydrocarbons as important components of increased desiccation resistance during diapause in A. albopictus.