Effects of host （Boettcherisca peregrina） copper exposure on development, reproduction and vitellogenesis of the ectoparasitic wasp, Nasonia vitripennis

Effects of copper （Cu） accumulation by the flesh fly Boettcherisca peregrina （R.- D.） （Diptera： Sarcophagidae） on the ectoparasitic wasp Nasonia vitripennis （Walker） （Hymenoptera： Pteromalidae） were investigated experimentally by exposing host larvae to contaminated diets with final Cu concentrations of 400μg/g and 800μg/g diet fresh weight （DFW）, respectively. Results showed that Cu can be transferred along food chains to secondary consumers （parasitoids） in small amounts, resulting in negative effects on parasitoid growth and development （body weight and developmental duration） as well as fecundity （number of offspring per female）. Copper exposure also inhibited vitellogenesis ofparasitoids from Cu-contaminated host pupae. It is suggested that the decreased fecundity and inhibition of vitellogenesis ofN. vitripennis resulted from poor host nutritional state rather than from direct effects of Cu stress.

Effects of "Candidatus Liberibacter solanacearum"(haplotype B)on Bactericera cockerelli fitness and vitellogenesis

"Candidatus Liberibacter solanacearum"(Lso)are phloem-restricted and unculturable Gram-negative bacteria.Presently five haplotypes have been identified worldwide;but only haplotypes A and B are associated with the vector Bactericera cockerelli(Sulc.)in the Americas.Previous studies showed that Lso-infection reduces B.cockerelli reproductive output and that Lso haplotype B is more pathogenic than Lso haplotype A.To understand the interaction of Lso haplotype B and B.cockerelli,the fitness of Lso-free and Lso B-infected insects,and the expression of vitellogenin(5cKg7-like),a gene involved directly in the insect reproduction were analyzed.Statistical differences in the number of eggs oviposited,and the total number of progeny nymphs and adults were found among crosses of insects with or without Lso.Significant differences in sex proportions were found between Lso B-infected and Lso-free crosses:a higher proportion of Fi adult females were obtained from Lso B-infected mothers.A significant reduction of BcVgl-like was observed in crosses performed with Lso B-infected females compared to the Lso-free insects.In female cohorts of different age,a significant reduction of BcVgl-like expression was measured in 7-d-old Lso B-infected females(virgin and mated)compared with 7-d-old Lso-free females(virgin and mated),respectively.The reduction of BcVgl-like transcript was associated with a lower number of developing oocytes observed in female5s reproductive systems.Overall,this study represents the first step to understand the interaction of Lso B with B.cockerelli,highlighting the effect of Lso B infection on egg production,BcVgl-like expression,and oocyte development.

Expression and localization of vitellogenin genes(VTG)and receptor(VGR)in the gonad development of silver pomfret Pampus argenteus

Vitellogenesis is the main event of oocyte growth in oviparous animals,which is mainly manifested by the accumulation of vitellogenin(VTG).The accumulation of vitellogenin depends mainly on the absorption of exogenous vitellogenin,which enters oocyte through endocytosis mediated by its receptor(VGR).We investigated the expression and localization of VTG and VGR during gonad development of Pampus argenteus.The qPCR results show that vtgs were not expressed in male fish,but in the ovary and liver of female fish;the expression levels went up at first and then down.The expression levels of vgr in the testis were low and only 1%-3%of that in ovary.ELISA results show that during the ovarian development of P.argenteus,VTG in liver,serum,and ovary all showed a trend from increasing to decreasing.However,VTG in liver peaked in StageⅣ,and in serum and ovary peaked in Stage V,reflecting changes in the characteristics of VTG in the liver(synthesis),blood(transport),and ovaries(accumulation).During gonad development,VGR in the ovaries first increased and then decreased,reaching a peak in Stage V,in contrast to vgr mRNA expression.The VGR content in the testis was extremely low and stable,consistent with vgr mRNA.Immunohistochemistry results show that the location and intensity of VTG and VGR positive signals were synchronized with the changes of their protein content,which revealed that VTG was mainly synthesized in the liver cytoplasm,secreted into the blood,and transported to ovary in StageⅢ.VGR is highly expressed in oocytes in StageⅡ.In StageⅢ,a large amount of VTG reaches the ovary,when VGR begins to translate and is subsequently transported to the plasma membrane of the oocyte.Therefore,the positive signal of VGR was stronger near the plasma membrane of oocytes in StagesⅠandⅡ.By using qPCR,ELISA,and immunohistochemistry,the synthesis,transport,and accumulation of vitellogenin were elucidated and the mechanism of its endocytosis on egg membrane mediated by VTG during the development of P.argenteus was revealed preliminarily.

GROWTH, REPRODUCTION & POPULATION STRUCTURE OF THE FRESHWATER CRAB SINOPOTAMON YANG TSEKIENSE BOTT, 1967, FROM ZHEJIANG, CHINA

Monthly investigatiom were made on the population of Chinese freshwater crab, Sinopotamon yangteskiense Bott, 1967 from April, 1984 to Match, 1985. The data on 4413 specimens show that the growth was affected mainly by temperature. During the April to November growth period, the crabs’ major development occured from June through October. One year was required for a fine white oocyte to develop into a mature egg. The reproduction period was June-October. Females bearing eggs were taken from June-August, and crabs with young were found from July-October. The females reproduced once a year but could for more than one year. The number of eggs carried by a female varied greatly according to the size of the crab, ranging from 30 to 100 eggs. New-bom crabs become mature after 1-2 yeats. The sex ratio was approximately 1:1 in the overall population. However, the larger crabs are predominantly male. The age distribution of S. yangtsekinese was estimated from size frequency histograms. There were more adult

Molecular characterization of yolk proteins in the female crab Neptunus pelagicus(A.Milne-Edwards,1861) from the Mediterranean Sea of Alexandria,Egypt

This study aimed to characterize the morphological changes in the ovary of the female crab Neptunus pelagicus and to identify specific fractions of vitelloginin and vitelline molecules during primary and secondary vitellogenesis.Samples of the blue crab were collected from the Mediterranean Sea of Alexandria monthly during 2017.Ovaries and oocytes in primary and secondary vitellogenesis were detached and treated for histological test.Native polyacrylamide gel electrophoresis(PAGE)Bis-Tris Gels was applied to identify vitelloginin(VN)and vitelline(VL)molecules.Protein Analyses were done by PAGE-SDS.The initial degenerate primers were built regarding the conserved amino acid domains of the yolk proteins.Primary and secondary vitellogeneses consisted of 8 phases.Lipoprotein fraction with molecular weight 550 kDa was identified in the hemolymph in secondary vitellogenesis.Two protein fractions(VLI&VLII)were identified in secondary vitellogenic oocytes.The electrophoresis performed with extract of stage I oocyte showed two protein fractions with molecular weights 550 kDa and 460 kDa.In stage II and III oocyte,4 subunits were presented of 180,195,140 and 120 kDa in VLI and 2 subunits with molecular weight of 110 kDa and 95 kDa in VLII.Another two fractions in stage V oocyte presented with molecular weights of 380 kDa and 360 kDa.Western blot analysis proved that both fractions were of four major polypeptide subunits with molecular weight of 180,125,90 and 85 kDa in each of the two VLs.The hybridization signal obtained by the Northern blot was detected in the hepatopancreas during ovarian cycle and in the ovary during secondary vitellogenesis.The result of the reverse transcription-polymerase chain reaction(RT-PCR)analysis showed that the mRNA that encodes the C-terminal region of the VN cDNA was found in the ovary in secondary vitellogenesis and in the hepatopancreas.

Characterization of a native whitefly vitellogenin gene cDNA and its expression pattern compared with two invasive whitefly cryptic species

supported by grants from the National Basic Research Program of China （2014CB138404）;the China National Science Fund for Innovative Research Groups of Biological Control （31321063）;the National Basic Research Program of China （973 Program, 2009CB119203）;the National Natural Science Foundation for Young Scientists in China （31101674）

Hexamerin and allergen are required for female reproduction in the American cockroach,Periplaneta americana

Reproduction is of great importance for the continuation of the species.In insects,the fat body is the major tissue for nutrient storage and involved in vitellogenesis,which is essential for female reproduction.Here,2 proteins,hexamerin and allergen,were separated from the fat bodies of adult female American cockroaches(Periplaneta americana)and identified as storage proteins,encoding for 733 amino acids with molecular weight of 87.88 kDa and 686 amino acids with molecular weight of 82.18 kDa,respectively.The encoding genes of these 2 storage proteins are mainly expressed in the fat body.RNA interference-mediated knockdown of Hexamerin and Allergen in the early stage of the first reproductive cycle in females suppressed vitellogenesis and ovarian maturation,indicating that these storage proteins are involved in controlling reproduction.Importantly,the expression of Hexamerin and Allergen was repressed by knockdown of the juvenile hormone(JH)receptor gene Met and the primary response gene Kr-h1,and was induced by methoprene,a JH analog,in both in vivo and in vitro experiments.Altogether,we have determined that hexamerin and allergen are identified as storage proteins and play an important role in promoting female reproduction in the American cockroach.The expression of their encoding genes is induced by JH signaling.Our data reveal a novel mechanism by which hexamerin and allergen are necessary for JH-stimulated female reproduction.

Rho 1 participates in parasitoid wasp eggs maturation and host cellular immunity inhibition

Endoparasitoid wasps introduce venom into their host insects during the egglaying stage.Venom proteins play various roles in the host physiology,development,immunity,and behavior manipulation and regulation.In this study,we identified a venom protein,MmRhol,a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone,egg production,and cocoons formation in the female wasps.We demonstrated that MmRho1 entered the cotton bollworm's(host)hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining.Furthermore,wasp MmRhol interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2-hybrid and glutathione s-transferase pull-down.In conclusion,this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses.

Delivery methods for CRISPR/Cas9 gene editing in crustaceans

In this mini-review,we provide an up-to-date overview of the delivery methods that have been used for CRISPR/Cas9 genomic editing in crustacean species.With embryonic microinjection as the main workforce for delivering CRISPR/Cas9 reagents,biologists working with crustacean species have to tackle the technical challenges involved in microinjection.We use examples of three Crustacean species(the branchiopod Daphnia,amphipod Parhyale hawaiensis,and decapod Exopalaemon carinicauda)to provide a technical guide for embryonic microinjection.Moreover,we outline two potentially useful new techniques for delivering CRISPR/Cas9 components into crustaceans,i.e.,Receptor-Mediated Ovary Transduction of Cargo(ReMOT Control)and electroporation.

Production of the yolk protein precursor vitellogenin is mediated by target of rapamycin (TOR) in the soft tick Ornithodoros moubata (Acari: Argasidae)

Initiation of vitellogenesis by blood feeding is essential for egg maturation in ticks.Nutrients derived from the blood meal are utilized by female ticks to synthesize the yolk protein precursor vitellogenin(Vg).Engorged Ornithodoros moubata ticks can synthesize Vg whether mated or virgin,thus O.moubata is an excellent model for studying the relative roles of blood feeding and mating in tick vitellogenesis.Injection of rapamycin into engorged O.moubata resulted in a reduction of ovarian growth and yolk accumulation in the oocytes of mated females.OmVg expression in the midgut and fat body and protein concentrations in the hemolymph significantly decreased in mated ticks after injection with rapamycin,indicating that inhibition of the nutrient-sensing target of rapamycin(TOR)pathway disrupts egg maturation at the levels of Vg expression and synthesis.These results suggest that the TOR-signaling pathway induces vitellogenesis in response to nutritional stimulation after a blood meal in O.moubata and is functionally independent of the mating-induced pathway.