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G3 (Bethesda, Md.) May 2024While CRISPR-Cas9 technology has demonstrated remarkable promise as a gene editing tool, its application in certain insects, such as the jewel wasp, Nasonia vitripennis,...
While CRISPR-Cas9 technology has demonstrated remarkable promise as a gene editing tool, its application in certain insects, such as the jewel wasp, Nasonia vitripennis, has been hindered by a lack of a tractable method for reagent delivery. Direct Parental-CRISPR (DIPA-CRISPR) recently emerged as a facile way to induce gene lesions because it involves adult injection with commercially available Cas9-sgRNA with no helper reagent. However, DIPA-CRISPR has so far been tested in only a few insects. Here, we have assessed the viability of DIPA-CRISPR in N. vitripennis by targeting two eye-pigmentation genes, cinnabar and vermilion, which function in the ommochrome pathway. Successful generation of lesions in both genes demonstrated the functionality of DIPA-CRISPR in N. vitripennis and its potential application to other genes, thereby expanding the range of insects suitable for this method. We varied two parameters, Cas9-sgRNA concentration and injection volume, to determine optimal injection conditions. We found that the larger injection volume coupled with either higher or lower concentration was needed for consistent mutation production. However, DIPA-CRISPR yields an overall low mutation rate in N. vitripennis when compared to other tested insects, a characteristic that may be attributed to a proportionally low vitellogenic import efficiency in the jewel wasp. We discuss different factors that may be considered in determining when DIPA-CRISPR may be preferable over other reagent delivery methods.
PubMed: 38734969
DOI: 10.1093/g3journal/jkae095 -
Insect Molecular Biology May 2024
PubMed: 38716739
DOI: 10.1111/imb.12920 -
BioRxiv : the Preprint Server For... Apr 2024The molecular mechanisms that govern the metabolic commitment to reproduction, which often occurs at the expense of somatic reserves, remain poorly understood. We...
The molecular mechanisms that govern the metabolic commitment to reproduction, which often occurs at the expense of somatic reserves, remain poorly understood. We identified the F-box protein FBXL-5 as a negative regulator of maternal provisioning of vitellogenin lipoproteins, which mediate the transfer of intestinal lipids to the germline. Mutations in partially suppress the vitellogenesis defects observed in the heterochronic mutants and both of which ectopically express at the adult developmental stage. FBXL-5 functions in the intestine to negatively regulate expression of the vitellogenin genes; and consistently, intestine-specific over-expression of FBXL-5 is sufficient to inhibit vitellogenesis, restrict lipid accumulation, and shorten lifespan. Our epistasis analyses suggest that functions in concert with , a cullin gene, and the Skp1-related gene to regulate vitellogenesis. Additionally, acts genetically upstream of , which encodes the core mTORC2 protein Rictor, to govern vitellogenesis. Together, our results reveal an unexpected role for a SCF ubiquitin-ligase complex in controlling intestinal lipid homeostasis by engaging mTORC2 signaling.
PubMed: 38712300
DOI: 10.1101/2024.04.18.590113 -
General and Comparative Endocrinology Aug 2024The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for...
The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for characterization. Ventral lobe extract (VLE) from the pituitary contains homologous FSH and LH, and it stimulated the cAMP signaling of FSHR and LHR dose-dependently. Two transcript variants of LHR (LHR-L with exon 10 and LHR-S without) were identified, and LHR-S was the dominant form with higher basal cAMP activity without VLE stimulation. Among various developmental stages of follicles, FSHR expression was mainly associated with the pre-vitellogenic and early white follicles. When follicles were recruited into vitellogenesis, the expression of FSHR decreased while of LHR was upregulated reciprocally, suggesting that LHR may also be responsible for the control of vitellogenesis in chondrichthyans. The expression of LHR-L was upregulated among maturing follicles before ovulation, indicating LHR-L could have a specific role in receiving the LH surge signal for final maturation. Plasma LH-like activity was transiently increased prior to the progesterone (P4)-surge and testosterone-drop at the beginning of P4-phase, supporting a pituitary control of follicle-maturation via LH signaling in chondrichthyans. The expression of follicular LHR was downregulated during the P4-phase when LH-like activity was high, indicating that the LH-dependent downregulation of LHR is conserved in chondrichthyans as it is in other vertebrate lineages. (213 words).
Topics: Animals; Receptors, LH; Female; Receptors, FSH; Luteinizing Hormone; Follicle Stimulating Hormone; Fishes; Ovarian Follicle
PubMed: 38685391
DOI: 10.1016/j.ygcen.2024.114542 -
Vavilovskii Zhurnal Genetiki I Selektsii Apr 2024Juvenile hormone plays a "status quo" role in Drosophila melanogaster larvae, preventing the untimely metamorphosis, and performs a gonadotropic function in imagoes,...
Fertility differences between two wild-type Drosophila melanogaster lines correlate with differences in the expression of the Jheh1 gene, which codes for an enzyme degrading juvenile hormone.
Juvenile hormone plays a "status quo" role in Drosophila melanogaster larvae, preventing the untimely metamorphosis, and performs a gonadotropic function in imagoes, ensuring the ovaries' preparedness for vitellogenesis. The decreased level of juvenile hormone results in reproductive disorders in D. melanogaster females including a delay in the oviposition onset and a fertility decrease. Another factor that can affect the insect reproduction is an infection with the maternally inherited symbiotic α-proteobacterium Wolbachia. The present study is devoted to the analysis of the expression of two juvenile hormone metabolism genes encoding enzymes of its synthesis and degradation, juvenile hormone acid O-methyltransferase ( jhamt) and juvenile hormone epoxide hydrase (Jheh1), respectively, in four wild-type D. melanogaster lines, two of them being infected with Wolbachia. Lines w153 and Bi90 were both derived from an individual wild-caught females infected with Wolbachia, while lines w153T and Bi90T were derived from them by tetracycline treatment and are free of infection. Line Bi90 is known to be infected with the Wolbachia strain wMel, and line w153, with the Wolbachia strain wMelPlus belonging to the wMelCS genotype. It was found that infection with either Wolbachia strain does not affect the expression of the studied genes. At the same time, it was shown that the w153 and w153T lines differ from the Bi90 and Bi90T lines by an increased level of the Jheh1 gene expression and do not differ in the jhamt gene expression level. Analysis of the fertility of these four lines showed that it does not depend on Wolbachia infection either, but differs between lines with different nuclear genotypes: in w153 and w153T, it is significantly lower than in lines Bi90 and Bi90T. The data obtained allow us to reasonably propose that the inter-line D. melanogaster polymorphism in the metabolism of the juvenile hormone is determined by its degradation (not by its synthesis) and correlates with the fertility level.
PubMed: 38680182
DOI: 10.18699/vjgb-24-22 -
Marine Biotechnology (New York, N.Y.) Jun 2024The vitellogenin present in the bloodstream undergoes internalization into developing oocytes through the vitellogenin receptor (VgR), a process mediated by...
The vitellogenin present in the bloodstream undergoes internalization into developing oocytes through the vitellogenin receptor (VgR), a process mediated by receptor-mediated endocytosis. VgR plays a crucial role in facilitating the accumulation of vitellogenin and the maturation of oocytes. In this study, we characterized a Tachypleus tridentatus vitellogenin receptor (TtVgR) gene from the tri-spine horseshoe crab, revealing a length of 1956 bp and encoding 652 amino acid residues with 12 exons. TtVgR has a molecular weight of 64.26 kDa and an isoelectric point of 5.95. Predictions indicate 85 phosphorylation sites and 7 glycosylation sites within TtVgR. Transcriptional analysis demonstrated specific expression of TtVgR in the ovary and yellow connective tissue. TtVgR was identified and distributed in the plasma membrane of oocytes. The siRNA-mediated TtVgR knockdown significantly reduced the transcriptional activity of TtVgR. This depletion induced excessive ROS production, resulting in DNA damage in ovarian primary cells. TUNEL and flow cytometry analyses confirmed ovarian cell apoptosis following TtVgR knockdown, indicating DNA damage in ovarian primary cells. These findings underscore the importance of TtVgR in ovarian cell development, suggesting its potential involvement in vitellogenesis and oocyte maturation. This knowledge may inform innovative breeding strategies and contribute to the sustainable management and conservation of the tri-spine horseshoe crab.
Topics: Animals; Female; Ovary; RNA, Small Interfering; Receptors, Cell Surface; Gene Knockdown Techniques; Horseshoe Crabs; Apoptosis; Oocytes; Amino Acid Sequence; Reactive Oxygen Species; Phylogeny; Egg Proteins
PubMed: 38676851
DOI: 10.1007/s10126-024-10319-7 -
Biology of Reproduction Apr 2024In oviparous animals, egg yolk is largely derived from vitellogenin, which is taken up from the maternal circulation by the growing oocytes via the vitellogenin...
In oviparous animals, egg yolk is largely derived from vitellogenin, which is taken up from the maternal circulation by the growing oocytes via the vitellogenin receptor. Recently, a novel member of the lipoprotein receptor superfamily termed low-density lipoprotein receptor-related protein 13 was identified and proposed as a candidate of vitellogenin receptor in oviparous animals. However, the roles of low-density lipoprotein receptor-related protein 13 in vitellogenesis are still poorly defined. Here, we investigated the expression, vitellogenin-binding properties, and function of low-density lipoprotein receptor-related protein 13 in zebrafish. Two different lrp13 genes termed lrp13a and lrp13b were found in zebrafish. Reverse transcription polymerase chain reaction and quantitative polymerase chain reaction revealed both lrp13s to be predominantly expressed in zebrafish ovary, and in situ hybridization detected both lrp13s transcripts in the ooplasm of early stage oocytes. Two yeast hybrid studies showed that among eight vitellogenins of zebrafish, Vtg1, 2, and 3 bind to Lrp13a, while Vtg1, 2, and 5 bind to Lrp13b. We created zebrafish lrp13a and lrp13b mutant lines using CRISPR/Cas9. Knockout of lrp13a leads to a male-biased sex ratio and decreased diameter of embryo yolk, while knockout of lrp13b and double knockout of lrp13a and lrp13b leads to the delay of vitellogenesis, followed by follicular atresia. These phenotypes of mutants can be explained by the disruption of vitellogenesis in the absence of Lrp13s. Taken together, our results indicate that both Lrp13a and Lrp13b can serve as vitellogenin receptors in zebrafish among other vitellogenin receptors that are not yet described.
PubMed: 38660750
DOI: 10.1093/biolre/ioae040 -
Development (Cambridge, England) Oct 2024Ecdysone-induced protein 93 (E93), known as the 'adult-specifier' transcription factor in insects, triggers metamorphosis in both hemimetabolous and holometabolous...
Ecdysone-induced protein 93 (E93), known as the 'adult-specifier' transcription factor in insects, triggers metamorphosis in both hemimetabolous and holometabolous insects. Although E93 is conserved in ametabolous insects, its spatiotemporal expression and physiological function remain poorly understood. In this study, we first discover that, in the ametabolous firebrat Thermobia domestica, the previtellogenic ovary exhibits cyclically high E93 expression, and E93 mRNA is broadly distributed in previtellogenic ovarioles. E93 homozygous mutant females of T. domestica exhibit severe fecundity deficiency due to impaired previtellogenic development of the ovarian follicles, likely because E93 induces the expression of genes involved in ECM (extracellular matrix)-receptor interactions during previtellogenesis. Moreover, we reveal that in the hemimetabolous cockroach Blattella germanica, E93 similarly promotes previtellogenic ovarian development. In addition, E93 is also essential for vitellogenesis that is necessary to guarantee ovarian maturation and promotes the vitellogenesis-previtellogenesis switch in the fat body of adult female cockroaches. Our findings deepen the understanding of the roles of E93 in controlling reproduction in insects, and of E93 expression and functional evolution, which are proposed to have made crucial contributions to the origin of insect metamorphosis.
Topics: Animals; Female; Reproduction; Metamorphosis, Biological; Ovary; Gene Expression Regulation, Developmental; Vitellogenesis; Insect Proteins; Transcription Factors; Drosophila Proteins
PubMed: 38646855
DOI: 10.1242/dev.202518 -
Experimental & Applied Acarology May 2024Blood feeding and digestion are vital physiological activities essential for the survival and reproduction of ticks. Chemical acaricides viz., ivermectin, amitraz and...
Blood feeding and digestion are vital physiological activities essential for the survival and reproduction of ticks. Chemical acaricides viz., ivermectin, amitraz and fipronil, are known to act on the central nervous system, resulting in the mortality of ticks. The present study is focused on the effect of these acaricides on the midgut and gut enzymes of Rhipicephalus microplus. The ultra-thin sections of midgut of ivermectin-treated ticks showed irregular basal membrane and ruptured digestive vesicles. Amitraz treatment resulted in a notable decrease in digestive cells with pleats in the basal membrane, while fipronil-exposed ticks exhibited reduced digestive cells, loss of cellular integrity, and disintegration of the basal membrane and muscle layer. The gut tissue homogenate of ivermectin and fipronil treated ticks showed a significant reduction of cathepsin D level, 76.54 ± 3.20 μg/mL and 92.67 ± 3.72 μg/mL, respectively, as compared to the control group (150.0 ± 3.80 μg/mL). The leucine aminopeptidase level (4.27 ± 0.08 units/mL) was significantly decreased in the ivermectin treated ticks compared to other treatment groups. The acid phosphatase activity (29.16 ± 0.67 μmole/min/L) was reduced in the ivermectin treated group whereas, increased activity was observed in the fipronil and amitraz treated groups. All the treatment groups revealed increased alkaline phosphatase levels (17.47-26.72 μmole/min/L). The present finding suggests that in addition to the established mechanism of action of the tested acaricides on the nervous system, the alterations in the cellular profile of digestive cells and enzymes possibly affect the blood digestion process and thus the synthesis of vital proteins which are essential for vitellogenesis, and egg production in ticks.
Topics: Animals; Rhipicephalus; Ivermectin; Pyrazoles; Toluidines; Acaricides; Female; Epithelium; Gastrointestinal Tract
PubMed: 38642307
DOI: 10.1007/s10493-024-00913-8 -
Frontiers in Insect Science 2024In insects and other animals, nutrition-mediated behaviors are modulated by communication between the brain and peripheral systems, a process that relies heavily on the... (Review)
Review
In insects and other animals, nutrition-mediated behaviors are modulated by communication between the brain and peripheral systems, a process that relies heavily on the insulin/insulin-like growth factor signaling pathway (IIS). Previous studies have focused on the mechanistic and physiological functions of insulin-like peptides (ILPs) in critical developmental and adult milestones like pupation or vitellogenesis. Less work has detailed the mechanisms connecting ILPs to adult nutrient-mediated behaviors related to survival and reproductive success. Here we briefly review the range of behaviors linked to IIS in insects, from conserved regulation of feeding behavior to evolutionarily derived polyphenisms. Where possible, we incorporate information from and other model species to describe molecular and neural mechanisms that connect nutritional status to behavioral expression via IIS. We identify knowledge gaps which include the diverse functional roles of peripheral ILPs, how ILPs modulate neural function and behavior across the lifespan, and the lack of detailed mechanistic research in a broad range of taxa. Addressing these gaps would enable a better understanding of the evolution of this conserved and widely deployed tool kit pathway.
PubMed: 38638680
DOI: 10.3389/finsc.2024.1360320