-
Frontiers in Physiology 2023CRISPR/Cas9 is a gene-editing technology which could specifically cleave dsDNA and induce target gene mutation. CRISPR/Cas9 has been widely used in gene functional...
CRISPR/Cas9 is a gene-editing technology which could specifically cleave dsDNA and induce target gene mutation. CRISPR/Cas9 has been widely used in gene functional studies in many fields, such as medicine, biology, and agriculture due to its simple design, low cost, and high efficiency. Although it has been well developed in model fish and freshwater fish for gene function analysis, it is still novel in the studies dealing with economic crustacean species. In this study, we established a CRISPR/Cas9 system based on microinjection for , an important economic crustacean aquaculture species. The () gene and the () gene were selected as the targeted genes for mutation. Two sgRNAs were designed for and gene editing, respectively. For sg--1, the gastrula survival ratio was 8.69%, and the final hatching ratio was 4.83%. The blastula mutant ratio was 10%, and the hatching individual mutant ratio was 30%. For sg--2, the gastrula survival ratio was 5.85%, and the final hatching ratio was 3.89%. The blastula mutant ratio was 16.67%, and no mutant sequences were detected in hatching individuals. For sg--1, the gastrula survival ratio was 6.25%, and the final hatching ratio was 2.34%. The blastula mutant ratio was 10.00%, and the hatching individual mutant ratio was 66.67%. For sg--2, the gastrula survival ratio was 6.00%, and the final hatching ratio was 2.67%. No mutant sequence was detected in both blastula stage and hatching individuals. There were no significant morphological changes observed in the group. Two deformed types were detected in sg--1-injected embryos. An evident developmental delay of the compound eye was detected in sg1-H1 in the zoea stage. The compound eyes of the sg1-H2 embryo could not form well-defined spheres, and the whole compound eye appeared to diffuse at the end of the late zoea stage. The establishment of a gene-editing platform based on CRISPR/Cas9 will not only provide an efficient and convenient method for gene function analysis but also provide a powerful tool for molecular-assisted breeding of .
PubMed: 37035655
DOI: 10.3389/fphys.2023.1141359 -
PLoS Pathogens Dec 2023Microsporidia are a group of obligate intracellular parasites that infect almost all animals, causing serious human diseases and major economic losses to the farming...
Microsporidia are a group of obligate intracellular parasites that infect almost all animals, causing serious human diseases and major economic losses to the farming industry. Nosema bombycis is a typical microsporidium that infects multiple lepidopteran insects via fecal-oral and transovarial transmission (TOT); however, the underlying TOT processes and mechanisms remain unknown. Here, we characterized the TOT process and identified key factors enabling N. bombycis to invade the ovariole and oocyte of silkworm Bombyx mori. We found that the parasites commenced with TOT at the early pupal stage when ovarioles penetrated the ovary wall and were exposed to the hemolymph. Subsequently, the parasites in hemolymph and hemolymph cells firstly infiltrated the ovariole sheath, from where they invaded the oocyte via two routes: (I) infecting follicular cells, thereby penetrating oocytes after proliferation, and (II) infecting nurse cells, thus entering oocytes following replication. In follicle and nurse cells, the parasites restructured and built large vacuoles to deliver themselves into the oocyte. In the whole process, the parasites were coated with B. mori vitellogenin (BmVg) on their surfaces. To investigate the BmVg effects on TOT, we suppressed its expression and found a dramatic decrease of pathogen load in both ovarioles and eggs, suggesting that BmVg plays a crucial role in the TOT. Thereby, we identified the BmVg domains and parasite spore wall proteins (SWPs) mediating the interaction, and demonstrated that the von Willebrand domain (VWD) interacted with SWP12, SWP26 and SWP30, and the unknown function domain (DUF1943) bound with the SWP30. When disrupting these interactions, we found significant reductions of the pathogen load in both ovarioles and eggs, suggesting that the interplays between BmVg and SWPs were vital for the TOT. In conclusion, our study has elucidated key aspects about the microsporidian TOT and revealed the key factors for understanding the molecular mechanisms underlying this transmission.
Topics: Animals; Humans; Vitellogenins; Spores, Fungal; Nosema; Bombyx
PubMed: 38060601
DOI: 10.1371/journal.ppat.1011859 -
Frontiers in Physiology 2019Vitellogenin (Vg) is precursor of vitellin. Here, we identified a () and two -likes (-like1 and -like2) in the brown planthopper, . Phylogenetic analyses showed that...
Vitellogenin (Vg) is precursor of vitellin. Here, we identified a () and two -likes (-like1 and -like2) in the brown planthopper, . Phylogenetic analyses showed that NlVg-like1 and NlVg-like2 are not clustered with the conventional insect Vgs associated with vitellogenesis. Temporo-spatial expression analyses showed that the and -like2 transcript levels increased significantly 24 h after emergence and were primarily expressed in female adults. However, -like1 was expressed during all stages, and in both genders. Tissue-specific analyses showed that all three genes were most highly expressed in the fat body. The injection of double-stranded RNA targeting showed that is essential not only for oocyte development but also for nymph development. The knockdown of -like1 in female adults resulted in failure to hatch or death before eggshell emergence in 18% of offspring embryos, suggesting that -like1 plays an important role during late embryogenesis. Approximately 65% of eggs laid by females that were treated with double-stranded RNA targeting -like2 failed to hatch, indicating that -like2 plays a role in nutrition absorption during oocyte, or embryonic development. Our results illustrate the structural and functional differences among the and -like genes and provide potential targets for RNA-interference-based insect pest management strategies.
PubMed: 31620015
DOI: 10.3389/fphys.2019.01181 -
PloS One 2016Vitellogenin (Vg) plays vital role in oocytes and embryo development in insects. Vg is synthesized in the fat body, moves through haemolymph and accumulates in oocytes....
Vitellogenin (Vg) plays vital role in oocytes and embryo development in insects. Vg is synthesized in the fat body, moves through haemolymph and accumulates in oocytes. Vitellogenin receptors (VgR) present on the surface of oocytes, are responsible for Vg transportation from haemolymph to oocytes. Here, we cloned and characterized these genes from Bemisia tabaci Asia1 (BtA1) species. The cloned BtA1Vg and BtA1VgR genes consisted of 6,330 and 5,430 bp long open reading frames, which encoded 2,109 and 1,809 amino acid (AA) residues long protein. The BtA1Vg protein comprised LPD_N, DUF1943 and VWFD domains, typical R/KXXR/K, DGXR and GL/ICG motifs, and polyserine tracts. BtA1VgR protein contained 12 LDLa, 10 LDLb and 7 EGF domains, and a trans-membrane and cytoplasmic region at C-terminus. Phylogenetic analyses indicated evolutionary association of BtA1Vg and BtA1VgR with the homologous proteins from various insect species. Silencing of BtA1VgR by siRNA did not affect the transcript level of BtA1Vg. However, BtA1Vg protein accumulation in oocytes was directly influenced with the expression level of BtA1VgR. Further, BtA1VgR silencing caused significant mortality and reduced fecundity in adult whiteflies. The results established the role of BtA1VgR in transportation of BtA1Vg in oocytes. Further, these proteins are essential for fecundity, and therefore these can be potential RNAi targets for insect control in crop plants.
Topics: Amino Acid Sequence; Animals; Data Mining; Diptera; Egg Proteins; Phylogeny; Receptors, Cell Surface; Sequence Homology, Amino Acid; Vitellogenins
PubMed: 27159161
DOI: 10.1371/journal.pone.0155306 -
Insects Mar 2020The relationship between reproductive diapause and the genes related to vitellogenin (Vg) and its receptor (VgR) in insectoid ovarian development is still unclear....
The relationship between reproductive diapause and the genes related to vitellogenin (Vg) and its receptor (VgR) in insectoid ovarian development is still unclear. Accordingly, in the present study, we used hematoxylin and eosin staining to study the ovarian structure in the predatory mite Neoseiulus barkeri, a species that shows promise as a biological pest control agent. Staining revealed the presence of oocytes on ovary surfaces, and the oocytes were deposited as yolk granules through the intake of Vg and other nutrients with the development of the ovary. Development of the ovary stopped at the oocyte stage in diapausing adult mites, and this stage presented the same characteristics as the first day of adulthood in non-diapause female adults, where oocytes with nutrient cells, but no yolk granules are observed. In order to further explore the effects of the Vg gene and its receptor on reproduction, the sequences of the N. barkeri vitellogenin genes NbVg1, NbVg2, NbVg3, and NbVgR were analyzed using bioinformatics, and the expression levels of the NbVgs and the VgR at different developmental stages were determined by quantitative polymerase chain reaction (qPCR). The results showed that the NbVgs and NbVgR have complete domains and that the positions of many conservative regions and conservative motif are consistent. The expression levels of the NbVgs and NbVgR were highest in the ovipositional period, followed by those in the preovipositional period. The expression levels of the NbVgs and the VgR in non-diapause female adult mites were significantly higher than those in reproductive diapause female adult mites.
PubMed: 32225063
DOI: 10.3390/insects11040203 -
International Journal of Molecular... Oct 2022The fall armyworm is a highly polyphagous invasive pest. The strong reproductive capacity is an important factor in the rapid colonization and expansion of...
The fall armyworm is a highly polyphagous invasive pest. The strong reproductive capacity is an important factor in the rapid colonization and expansion of Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in insect reproduction. As the precursor of vitellin (Vn), Vg provides essential nutrition for embryonic development, and VgR mediates the uptake of Vg by oocytes. In this context, we cloned and characterized these two genes of ( and ) and evaluated their expression profiles in different developmental stages and tissues. The RNA interference experiment was used to investigate their function in vitellogenesis. The ORF values of and were 5250 and 5445 bp, encoding 1749 and 1815 amino acid residues, respectively. The qRT-PCR results revealed that both and were highly expressed in female adults; was specifically expressed in the fat body, whereas was highly expressed in the ovary. In addition, the depletion of either or hindered oocyte maturation and ovarian development, leading to a significant decrease in fecundity. The present study reveals the importance of and in the vitellogenesis of , laying a theoretical foundation for the development of pollution-free pest control strategies with and as new targets.
Topics: Amino Acids; Animals; Female; Spodoptera; Vitellins; Vitellogenesis; Vitellogenins
PubMed: 36233286
DOI: 10.3390/ijms231911972 -
Cells Feb 2023Insect immunity is assorted into humoral and cellular immune reactions. Humoral reactions involve the regulated production of anti-microbial peptides, which directly... (Review)
Review
Insect immunity is assorted into humoral and cellular immune reactions. Humoral reactions involve the regulated production of anti-microbial peptides, which directly kill microbial invaders at the membrane and intracellular levels. In cellular immune reactions, millions of hemocytes are mobilized to sites of infection and replaced by hematopoiesis at a high biological cost after the immune defense. Here, we considered that the high biological costs of maintaining and replacing hemocytes would be a better investment if hemocytes carried out meaningful biological actions unrelated to cellular immunity. This idea allows us to treat a set of 10 hemocyte actions that are not directly involved in immunity, some of which, so far, are known only in . These include (1) their actions in molting and development, (2) in surviving severe hypoxia, (3) producing phenoloxidase precursor and its actions beyond immunity, (4) producing vitellogenin in a leafhopper, (5) recognition and responses to cancer in , (6) non-immune actions in , (7) clearing apoptotic cells during development of the central nervous system, (8) developing hematopoietic niches in , (9) synthesis and transport of a lipoprotein, and (10) hemocyte roles in iron transport. We propose that the biological significance of hemocytes extends considerably beyond immunity.
Topics: Animals; Drosophila melanogaster; Hemocytes; Insecta; Drosophila; Immunity, Cellular
PubMed: 36831266
DOI: 10.3390/cells12040599 -
Frontiers in Physiology 2019While much effort has been put into understanding vitellogenesis in insects and other organisms, much less is known of this process in ticks. There are several steps... (Review)
Review
While much effort has been put into understanding vitellogenesis in insects and other organisms, much less is known of this process in ticks. There are several steps that facilitate yolk formation in developing oocytes of which the vitellogenin receptor (VgR) is a key component. The tick VgR binds vitellogenin (Vg) circulating in the hemolymph to initiate receptor-mediated endocytosis and its transformation into vitellin (Vn). The conversion of Vg into Vn, the final form of the yolk protein, occurs inside oocytes of the female tick ovary. Vn is critical to tick embryos since it serves as the nutritional source for their development, survival, and reproduction. Recent studies also suggest that pathogenic microbes, i.e., spp., that rely on ticks for propagation and dissemination likely "hitchhike" onto Vg molecules as they enter developing oocytes through the VgR. Suppressing VgR messenger RNA synthesis via RNA interference (RNAi) completely blocked spp. transmission into developing tick oocytes, thereby inhibiting vertical transmission of these pathogenic microbes from female to eggs. To date, VgRs from only four tick species, , , , and , have been fully sequenced and characterized. In contrast, many more VgRs have been described in various insect species. VgR is a critical component in egg formation and maturation that can serve as a precise target for tick control. However, additional research will help identify unique residues within the receptor that are specific to ticks or other arthropod disease vectors while avoiding cross-reactivity with non-target species. Detailed knowledge of the molecular structure and functional role of tick VgRs will enable development of novel vaccines to control ticks and tick-borne diseases.
PubMed: 31178755
DOI: 10.3389/fphys.2019.00618 -
Environmental Science and Pollution... Oct 2016Chlordecone is a persistent organochlorine pesticide widely used in Guadeloupe (French West Indies) to control the banana weevil Cosmopolites sordidus. Although it was...
Chlordecone is a persistent organochlorine pesticide widely used in Guadeloupe (French West Indies) to control the banana weevil Cosmopolites sordidus. Although it was previously highlighted that chlordecone may affect the reproduction and growth of vertebrate species, little information is available on the chlordecone effects in invertebrates. The present study investigated the effects of chlordecone on a hormone and a protein having key roles in reproduction and growth of the decapod crustacean Macrobrachium rosenbergii, by measuring the 20-hydroxyecdysone concentration, vitellogenin, and vitellogenin receptor gene expression, as well as the bioconcentration of chlordecone in exposed prawns. First, the results revealed that chlordecone was accumulated in M. rosenbergii. Then, it was found that Vg and VgR gene expression were increased in male and female M. rosenbergii exposed to chlordecone for 90 and 240 days, while the 20-hydroxyecdysone concentrations were decreased. This work suggests that chlordecone accumulates in prawn tissues and could affect key molecules involved in the reproduction and the growth of the invertebrate M. rosenbergii. However, many questions remain unresolved regarding the impacts of chlordecone on growth and reproduction and the signaling pathways responsible for these effects, as well as the potential role of confounding factors present in in situ studies.
Topics: Animals; Arthropod Proteins; Chlordecone; Ecdysterone; Egg Proteins; Endocrine Disruptors; Female; Gene Expression; Insecticides; Male; Palaemonidae; Receptors, Cell Surface; Vitellogenins; Water Pollutants, Chemical
PubMed: 27470247
DOI: 10.1007/s11356-016-7273-1 -
Microbiology Spectrum Aug 2022Liberibacter solanacearum (CLso) haplotype D, transmitted by the carrot psyllid , is a major constraint for carrot production in Israel. Unveiling the molecular...
Liberibacter solanacearum (CLso) haplotype D, transmitted by the carrot psyllid , is a major constraint for carrot production in Israel. Unveiling the molecular interactions between the psyllid vector and CLso can facilitate the development of nonchemical approaches for controlling the disease caused by CLso. Bacterial surface proteins are often known to be involved in adhesion and virulence; however, interactions of CLso with carrot psyllid proteins that have a role in the transmission process has remained unexplored. In this study, we used CLso outer membrane protein (OmpA) and flagellin as baits to screen for psyllid interacting proteins in a yeast two-hybrid system assay. We identified psyllid vitellogenin (Vg) to interact with both OmpA and flagellin of CLso. As Vg and autophagy are often tightly linked, we also studied the expression of autophagy-related genes to further elucidate this interaction. We used the juvenile hormone (JH-III) to induce the expression of Vg, thapsigargin for suppressing autophagy, and rapamycin for inducing autophagy. The results revealed that Vg negatively regulates autophagy. Induced Vg expression significantly suppressed autophagy-related gene expression and the levels of CLso significantly increased, resulting in a significant mortality of the insect. Although the specific role of Vg remains obscure, the findings presented here identify Vg as an important component in the insect immune responses against CLso and may help in understanding the initial molecular response in the vector against Liberibacter. Pathogen transmission by vectors involves multiple levels of interactions, and for the transmission of liberibacter species by psyllid vectors, much of these interactions are yet to be explored. Liberibacter solanacearum (CLso) haplotype D inflicts severe economic losses to the carrot industry. Understanding the specific interactions at different stages of infection is hence fundamental and could lead to the development of better management strategies to disrupt the transmission of the bacteria to new host plants. Here, we show that two liberibacter membrane proteins interact with psyllid vitellogenin and also induce autophagy. Altering vitellogenin expression directly influences autophagy and CLso abundance in the psyllid vector. Although the exact mechanism underlying this interaction remains unclear, this study highlights the importance of immune responses in the transmission of this disease agent.
Topics: Animals; Autophagy; Flagellin; Hemiptera; Liberibacter; Plant Diseases; Rhizobiaceae; Vitellogenins
PubMed: 35863005
DOI: 10.1128/spectrum.01577-22