Farnesoic Acid O-methyltransferase Research Articles

Effects of Different Levels of Antarctic Krill Oil on the Ovarian Development of Macrobrachium rosenbergii

Antarctic krill oil has been proven to be able to promote the ovarian development of crustaceans, but its optimal application dose and potential regulatory mechanism in Macrobrachium rosenbergii are still unclear. In this study, five isonitrogenous and isolipidic diets with gradient additions of Antarctic krill oil (0%, 1.5%, 3%, 4.5%, and 6%) were served exposed to 8 weeks of feeding. The results show that 3–4.5% Antarctic krill oil supplementation significantly increases the weight gain rate and specific growth rate of M. rosenbergii (p < 0.05). In addition, 3–4.5% Antarctic krill oil supplementation significantly increased the content of hemolymph vitellogenin (VTG) and the levels of reproductive hormones, including methyl farnesoate (MF), estradiol (E2), and progesterone (P4) (p < 0.05). The differences in ovarian index, oocyte volume, yolk granule deposition in oocytes, and the transcription levels of VTG genes in hepatopancreas and ovarian tissues demonstrated that the addition of Antarctic krill oil significantly promoted ovarian development and vitellogenesis, especially at the 4.5% addition level. In terms of molecular signaling, this study confirms that the retinol metabolic signaling pathway, MF signaling pathway, steroid hormone signaling pathway, and ecdysone signaling pathway, along with their specific molecules, such as Farnesoic acid-O-methyltransferase (FAMeT), retinoid x receptor (RXR), ecdysone receptor (EcR), and estrogen-related receptor (ERR), are involved in the regulation of the ovarian development of M. rosenbergii by adding Antarctic krill oil at appropriate doses. The findings indicate that the supplementation of 4.5% Antarctic krill oil in the diet is optimal for stimulating the secretion of reproductive hormones in female M. rosenbergii, thereby promoting vitellogenesis and ovarian development.

Molecular signature of the ontogenic development of the prawn Macrobrachium tenellum

The prawn Macrobrachium tenellum shows aquaculture potential due to its well-defined reproductive cycle linked to female nutritional requirements. Significant changes occur in egg composition during the 16 to 17-day embryo development. Understanding the ontogenic proteins is crucial for developmental insights and controlled reproduction. We employed free-label quantitative proteomics to analyze egg peptides at the initial and final stages of wild females. Using the emPAI protocol and Proteome Discoverer 2.0, we identified 89 differentially expressed proteins in M. tenellum eggs. Of these, 27 were exclusive to early-stage development and three to late-stage. Abundant proteins included Vitellogenin, glyceraldehyde-3-phosphate dehydrogenase, histone 4, beta-actin, and hemocyanin. Gene Ontology analysis revealed 518 terms across molecular functions, biological processes, and cellular components using the GoRetriever tool of AgBase and the CateGOrizer tool of the Animal Genome Research Program. Carbohydrate metabolism was significant in early-stage development, with glyceraldehyde-3-phosphate dehydrogenase being the second most abundant protein. Proteins involved in ATP synthesis and cytoplasmic proteins associated with catalytic and binding activities related to primary metabolism were also detected. Our study elucidates the role of Vitellogenin in lipid transport activity and its potential involvement in the juvenile hormone feedback pathway. This pathway includes farnesoic acid O-methyltransferase and juvenile hormone epoxide oxidase, regulating protein biosynthesis, molt cycles (including chitinase activity), and potentially influencing controlled reproduction. Our proteomic analysis provides insights into the molecular mechanisms driving Ontogenic development in Macrobrachium tenellum, with implications for controlled reproduction strategies and advancements in aquaculture practices.

Target gene selection for RNAi-based biopesticides against the hawthorn spider mite, Amphitetranychus viennensis (Acari: Tetranychidae).

Recently, RNA interference (RNAi)-based biopesticide, a species-specific pest control alternative, has been deregulated and commercialized in the US and Canada. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is a major pest for rosaceous plants, which has been controlled primarily by synthetic pesticides. To address the emerging resistance issues in A. viennensis, we initiated a project to develop RNAi-based biopesticides. In this study, we (i) developed a dietary RNAi system for A. viennensis using leaf disc, (ii) assessed the suitability of multiple control genes to distinguish sequence-specific silencing from non-specific effects within this RNAi system, and (iii) screened for the target gene candidates. As a result, β-Glucuronidase (GUS), an enzyme derived from E. coli and a broadly used reporter for plants is the appropriate control for A. viennensis RNAi, while green fluorescent protein (GFP), is not suitable due to its significantly higher mortality than the other controls. For target gene screening, suppression was confirmed for all the candidates, including two housekeeping genes (Vacuolar-type H + -ATPase subunit A (V-ATPase A) and Glyceraldehyde 3-phosphate dehydrogenase, (GAPDH)), and three genes associated with development (ATP-dependent RNA Helicase DDX3Y (Belle), CREB-binding protein (CBP), and Farnesoic acid O-methyltransferase (FaMet)). Knocking down of V-ATPase A resulted in the highest mortality (~ 90%) and reduced fecundity (over 90%) than other candidates. As for the genes associated with development, suppression of Belle and CBP, led to approximately 65% mortality, as well as 86% and 40% reduction in fecundity, respectively. Silencing of FaMet, however, had negligible biological impacts on A. viennensis. The combined efforts not only establish an effective dsRNA delivery method, but also provide potential target genes for RNAi-based biopesticides against A. viennensis, a devastating invasive pest for fruit trees and woody ornamental plants throughout Asia and Europe. © 2023 Society of Chemical Industry.

Sesquiterpenoid pathway in the mandibular organ of Penaeus monodon: Cloning, expression, characterization of PmJHAMT and its alteration response to eyestalk ablation

Methyl farnesoate (MF), a crustacean equivalent of juvenile hormone (JH) of insects, is known to be produced from the mandibular organ (MO). This study reports transcriptome analysis of Penaeus monodon MO and identifies putative genes encoding enzymes in the sesquiterpenoid pathway. A total of 44,490,420 clean reads were obtained and utilized for subsequent analysis. De novo assembly created 31,201 transcripts and 31,167 unigenes. To archive the functional annotation, all unigenes were annotated with KOG, KEGG, and GO. Putative genes encoding enzymes and regulatory proteins involved in the sesquiterpenoid pathway were obtained from the MO transcriptome data based on the conserved domains and sequence homology. They included S-adenosylmethionine synthetase, farnesyl pyrophosphate synthase, short chain dependent dehydrogenase/reductase (SDR), NAD(P) + -dependent aldehyde dehydrogenase, S-adenosylmethionine-dependent methyltransferases or juvenile hormone acid-O-methyl transferase (JHAMT), farnesoic acid O-methyl transferase (FAMeT), juvenile hormone binding protein, cytochrome C/P-450 family 15 (CRYP15A1)/methylfarnesoate epoxidase (MFE), juvenile hormone epoxide hydrolase (JHEH), and juvenile hormone esterase (JHE). We first identified and characterized JHAMT orthologs inP. monodon(PmJHAMT). The complete cDNA sequence ofPmJHAMTconsisted of 1,221 nt encoded 271 amino acids with a conserved S-adenosyl methionine (SAM) binding domain. Phylogenetic analysis clusteredPmJHAMTinto the group JHAMT with the same clade of the crabPortunus trituberculausJHAMT. Moreover, the predicted three-dimensional structure of PmJHAMT showed remarkable similarity with the recent crystal structure ofthe Bombyx moriJHAMT homodimer. RT–PCR analysis revealed that PmJHAMT was exclusively expressed in MO and initially expressed at stage 3 postlarvae. In situ hybridization with a specific probe to PmJHAMT validated the specific expression of this gene in MO cells. Finally, we evaluated the regulation of MO by eyestalk inhibitory peptides. Diminishing MO inhibitory hormone through unilateral eyestalk ablation resulted in a significantly higher expression ofPmJHAMTin MO by quantitative PCR. This result indicated that the eyestalk inhibitory hormone inhibited MF synthesis byPmJHAMTgene suppression in the MO. This finding provides insight into the crustacean sesquiterpenoid pathway and improves our understanding of crustacean endocrinology.

Cloning, expression analysis and RNAi of farnesoic acid O-methylransferase gene from Neocaridina denticulata sinensis.

Methyl farnesoate (MF) is an essential endocrine hormone in crustaceans, which can promote the occurrence of crustaceans molting, control morphogenesis, affect gonad development, and regulate the stress stimulation to the external environment. The farnesoic acid O-methyltransferase (FAMeT) is a key rate-limiting enzyme in MF synthesis, catalyzing the conversion of farnesoic acid (FA) into MF. Neocaridina denticulata sinensis [Decapoda] is a suitable animal model for studying crustaceans because it can reproduce many times under artificial control and has a short reproductive cycle. According to its transcriptomic and genomic information, the full-length cDNA sequence of FAMeT from N. denticulata sinensis (NdFAMeT) was cloned and the characterization of its deduced amino acid sequence was also analyzed. The relative expression of NdFAMeT in different tissues was determined. The NdFAMeT protein was recombinantly expressed in E. coli and its enzyme activity was determined. After gene knockdown by RNAi technology, the protein activity of shrimp was decreased and the individual phenotype was also observed.

Molecular evidence for farnesoic acid O-methyltransferase (FAMeT) involved in the biosynthesis of vitellogenin in the Chinese mitten crab Eriocheir sinensis

Sesquiterpenoid methyl farnesoate (MF), a crustacean equivalent of insect juvenile hormone (JH III), has essential functions in regulating physiological processes in crustaceans, including reproduction and vitellogenesis. Farnesoic acid O-methyltransferase (FAMeT) is a key rate-limiting enzyme catalyzing the conversion of farnesoic acid (FA) to JH/MF in insects and crustaceans. In this study, a full-length cDNA of EsFAMeT from Eriocheir sinensis was isolated and characterized. The deduced EsFAMeT amino acid sequence indicated there were two conserved Methyltransf-FA domains characteristic of FAMeT family proteins. With use of sequence alignment analysis procedures, there was an indication that FAMeT proteins are highly conserved among crustaceans and FAMeT is more closely related to crustacean FAMeT than to insect FAMeT. Results from quantitative real-time PCR analysis revealed there was ubiquitous EsFAMeT in all tissues examined, with greater abundances of mRNA transcripts in the ovary. The transcription of EsFAMeT indicated there were stage-specific patterns in the hepatopancreas and ovary during ovarian development, with the greatest abundance during ovarian development Stages II and III, respectively. To investigate functions of EsFAMeT in vitellogenin biosynthesis in E. sinensis, RNA interference-mediated gene knockdown was used in vitro and in vivo. Injection of EsFAMeT dsRNA resulted in a marked decrease in EsVg (encoding vitellogenin) transcripts in the ovary and hepatopancreas both in vitro and in vivo. Results from the present study indicated EsFAMeT is involved in vitellogenin biosynthesis in the ovary and hepatopancreas of E. sinensis, providing a new resource to study modulatory effects of the FAMeT family of enzymes in crustacean reproduction.

Linolenic acid improves growth performance and immune status of Penaeus vannamei infected by Enterocytozoon hepatopenaei

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) leads to significant growth retardation of Penaeus vannamei and is increasing the economic losses in the shrimp aquaculture. Our previous metabolomic results showed that 24 metabolites including linolenic acid (LNA) significantly decreased in EHP-infected shrimp compared with healthy groups. This study aims to evaluate whether LNA can improve the growth performance and immune status of EHP-infected shrimp. The EHP-infected shrimp was fed with dietary LNA (0, 1.2, 2.4 and 4.8 g/kg diet) for 30 days. The results showed that compared to the other groups, the body length and weight of shrimp in groups of 2.4 g LNA/kg diet were significantly increased. The copy numbers of EHP were significantly reduced in group of 1.2, 2.4 and 4.8 LNA g/kg diet compared to without LNA group. Further study showed that when shrimp were fed with 2.4 g LNA/kg diet, growth-related genes (farnesoic acid O-methyltransferase and ecdysteroid-regulated protein) were down-regulated, but juvenile hormone esterase-like carboxylesterase 1 up-regulated, suggesting that LNA could improve the growth ability of EHP-infected shrimp. More importantly, three immune genes (peritrophin-44-like protein, lysozyme, and cathepsin C) in shrimp were significantly up-regulated after fed with addition of LNA. In addition, the enzyme activity of superoxide dismutase and catalase in the hepatopancreas of P. vannamei was obviously increased after fed with different concentrations of LNA. Overall, the appropriate LNA content in the feed (2.4 g/kg diet) can improve the growth of P. vannamei and enhance its non-specific immunity and antioxidant activity. These findings indicate that LNA could be used as a potential metabolic modulator for controlling the HMP caused by EHP, and shed some light on host metabolic prevention to infectious diseases.

Role of SfJHAMT and SfFAMeT in the reproductive regulation of Sogatella furcifera and its expression under insecticide stress

The isoprene branching pathway is a unique downstream synthesis pathway of juvenile hormone (JH) in arthropods, which plays an important role in the growth, development, and reproduction of insects. Juvenile hormone acid O-methyltransferase (JHAMT) and farnesoic acid O-methyltransferase (FAMeT) are two key proteins that are regulated in the isoprene branching pathway. Based on the available transcriptomic and genomic data of Sogatella furcifera, full-length cDNAs of SfJHAMT and SfFAMeT were identified. In vitro injection of dsRNA targeted to silence SfJHAMT and SfFAMeT inhibited the fecundity, ovarian development, and transcription levels of SfKr-h1 and SfVg significantly. Of note, The transcription levels of SfJHAMT and SfFAMeT are regulated mutually; i.e., silencing of SfJHAMT causes an increase in the SfFAMeT transcription level and vice versa, and the negative effect of simultaneous silencing on reproduction is greater. The results revealed a coordinated effect of SfJHAMT and SfFAMeT on the reproductive capabilities of S. furcifera. Furthermore, a JH analog (methoprene) partially rescued the negative effect of simultaneous silencing by SfJHAMT and SfFAMeT on reproduction. In addition, the expression profile analysis after insecticide stress showed that triazophos (LC25) can induce the transcription of SfMet and SfKr-h1 to promote JH signal transduction, which affects the transcription of SfVg and ultimately promotes the reproduction of S. furcifera. The results of the present study lay a foundation to further explain the isoprene branch pathway function in insect reproduction and can open up new avenues for sustainable pest control while expanding the current understanding of molecular mechanisms through which insecticides stimulate reproduction and lead to pest resurgence.

Integrative Proteomic and MicroRNA Analysis: Insights Into Mechanisms of Eyestalk Ablation-Induced Ovarian Maturation in the Swimming Crab Portunus trituberculatus.

Eyestalk ablation is the most common method to induce ovarian maturation in decapod crustacean aquaculture, but it jeopardizes broodstock survival and larvae production. It is important to understand the molecular basis underlying the maturation triggered by ablation and thereby develop an alternative measure for maturation manipulation. In this study, we investigate alterations of ovarian proteome and miRNA profile after ablation in a commercially important marine crab Portunus trituberculatus. Quantitative proteomic analysis using iTRAQ reveals that 163 proteins are differentially expressed following ablation, and modulation of methyl farnesoate metabolism and activation of calcium signaling may play important roles in the ovarian maturation induced by ablation. miRNA expression profiling identifies 31 miRNAs that show statistically significant changes. Integration of miRNA and proteome expression data with miRNA target prediction algorithms generates a potential regulatory network consisting of 26 miRNAs and 30 proteins linked by 71 possible functional associations. The miRNA-protein network analysis suggests that miRNAs are involved in promoting ovarian maturation by controlling expression of proteins related to methyl farnesoate synthesis, calcium signals, and energy metabolism. Experimental validation and temporal expression analysis indicate multiple miRNAs can act synergistically to regulate expression of Farnesoic acid O-methyltransferase and Calmodulin. Our findings provide new insights for elucidating the mechanisms underlying eyestalk ablation-induced ovarian maturation and could be useful for devising an alternative technique for manipulating reproduction in P. trituberculatus and other decapods.

Gammarid exposure to pyriproxyfen and/or cadmium: what effects on the methylfarnesoate signalling pathway?

Due to expected changes in climate, it is predicted that disease-carrying mosquitoes will expand their geographical range, resulting in increased use of insect growth regulators (IGRs) to face their proliferation. Among IGRs, pyriproxyfen (PXF) is widely used and has been shown to prevent larvae from developing into adults, rendering them unable to reproduce. However, because of the similarity of crustacean and insect endocrine systems, PXF could also impact aquatic crustaceans. In addition, when spreading in the environment, PXF is found in a mixture with other pollutants such as metallic trace elements, which could alter its effect. Consequently, the present work was devoted to analysing the effects of PXF on the methylfarnesoate (MF) hormonal pathway of the freshwater amphipod Gammarus pulex, as well as its combined binary effects with cadmium (Cd), by measuring MF concentration, as well as the relative transcriptional expression of the farnesoic acid O-methyltransferase (FAMeT) (enzyme limiting the MF production), the methoprene-tolerant receptor (Met), and the broad-complex (BrC) as a transcription factor. Results revealed that single exposures to PXF or Cd have mainly overexpressed FAMeT, Met, and BrC but did not significantly affect MF concentration. Conversely, the mixture exposures seemed to suppress these effects and even achieve antagonistic effects. This work confirmed that PXF single exposure could impact non-target organisms such as amphipods through changes in hormonal pathways of MF. In the same way, it highlighted that Cd could also impair the endocrine system of exposed organisms. However, antagonistic effects have been observed in exposure to mixtures, suggesting different long-term consequences on the growth of amphipods under realistic exposure conditions.

Characterization and expression profile of farnesoic acid O-methyltransferase gene from Indian white shrimp, Penaeus indicus.

Methyl farnesoate (MF), a sesquiterpenoid synthesized in the mandibular organ, regulates many physiological processes in crustaceans including growth and reproduction. In the present study, farnesoic acid O-methyltransferase (FAMeT), the key enzyme responsible for final step conversion of farnesoic acid (FA) to methyl farnesoate (MF), was cloned and characterized from the nervous tissues of Penaeus indicus. Multiple sequence alignment, prediction of conserved domain regions, phosphorylation sites identification and phylogenetic analysis indicated that putative FAMeT fragment from P. indicus (PiFAMeT), shares a high degree of sequence identity to FAMeT proteins isolated from other crustaceans species. Quantitative real-time PCR analysis revealed ubiquitous expression of PiFAMeT in all the tissues examined, with comparative higher mRNA levels in nervous tissue and ovary. Additionally, the levels of PiFAMeT also showed gradual increase of expression correlating with the advancement in ovarian maturation. Further to support their role in promoting ovarian development, serotonin treatment (5HT, 50 μg/g body weight) was given to eyestalk intact and unilaterally eyestalk ablated females which resulted in significant increase in PiFAMeT transcript levels at day 7 and day 14. The relatively higher levels of PiFAMeT, reflecting higher levels of MF, suggest a role during secondary vitellogenesis thereby regulating ovarian development in P. indicus. Further research is required to understand the synergistic interaction of MF pathways with serotonergic and other regulatory pathways in regulating ovarian maturation in penaeid shrimps.

Elucidation of the role of farnesoic acid O-methyltransferase (FAMeT) in the giant freshwater prawn, Macrobrachium rosenbergii: Possible functional correlation with ecdysteroid signaling

Farnesoic acid O-methyltransferase (FAMeT) is a key enzyme involved in catalyzing the conversion of farnesoic acid (FA) to methylfarnesoate (MF) in the mandibular organ (MO) of crustaceans. In this study, a full-length cDNA encoding a 278-amino-acid FAMeT protein (MrFAMeT) was characterized from the giant freshwater prawn, Macrobrachium rosenbergii. Bioinformatics analysis revealed a high degree of conservation of FAMeT among crustaceans and a close phylogenetic relationship between MrFAMeT and that of Scylla paramamosain. The prokaryotic expressed MrFAMeT could catalyze the conversion of FA to MF in a radiochemical assay. Expression analysis by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that MrFAMeT mRNA was highly expressed in the muscle and the hepatopancreas of both females and males. During the molt cycle and the ovarian development, the mRNA expressions of MrFAMeT displayed stage-specific patterns in the muscle of both sexes and the female hepatopancreas, and the highest expressions were detected at intermolt and ovarian development stage V. Double stranded RNA (dsRNA)-mediated RNA interference (RNAi) of MrFAMeT increased expressions of myostatin in the muscle of both sexes and reduced expressions of vitellogenin (Vg) in the female hepatopancreas. Furthermore, both in the muscle and the hepatopancreas, silence of MrFAMeT downregulated the expression of ecydone receptor gene (MrEcR) and silence of MrEcR decreased the expression of MrFAMeT as well. Results in our study indicate that MrFAMeT is involved in prawn muscle growth and female vitellogenin biosynthesis and its function may be closely related with the ecdysteroid signaling.

Proteomic and metabolomic responses in hepatopancreas of whiteleg shrimp Litopenaeus vannamei infected by microsporidian Enterocytozoon hepatopenaei

Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. HPM is not normally associated with shrimp mortality, but is associated with significant growth retardation. In this study, the responses induced by EHP were investigated in hepatopancreas of shrimp Litopenaeus vannamei using proteomics and metabolomics. Among differential proteins identified, several (e.g., peritrophin-44-like protein, alpha2 macroglobulin isoform 2, prophenoloxidase-activating enzymes, ferritin, Rab11A and cathepsin C) were related to pathogen infection and host immunity. Other proteomic biomarkers (i.e., farnesoic acid o-methyltransferase, juvenile hormone esterase-like carboxylesterase 1 and ecdysteroid-regulated protein) resulted in a growth hormone disorder that prevented the shrimp from molting. Both proteomic KEGG pathway (e.g., “Glycolysis/gluconeogenesis” and “Glyoxylate and dicarboxylate metabolism”) and metabolomic KEGG pathway (e.g., “Galactose metabolism” and “Biosynthesis of unsaturated fatty acids”) data indicated that energy metabolism pathway was down-regulated in the hepatopancreas when infected by EHP. More importantly, the changes of hormone regulation and energy metabolism could provide much-needed insight into the underlying mechanisms of stunted growth in shrimp after EHP infection. Altogether, this study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of microsporidial infection in the shrimp L. vannamei.

Relative expression of three key genes involved in the hormonal cycle of the freshwater amphipod, Gammarus pulex.

Ecdysteroids and sesquiterpenoids are the two groups of hormones controlling molt and growth processes in amphipods, and are mostly represented by ecdysone (i.e., 20-hydroxyecdysone) and methyl farnesoate (MF), respectively. The endocrine system responsible for their syntheses is located in the cephalothorax and is composed of three main organs: the X-organ/sinus gland complex (XO), the Y-organ (YO) and the mandibular organ. Ecdysone synthesis is negatively controlled by the molt-inhibiting hormone (MIH) produced by XO, and its signal is mediated through the ecdysteroid receptor (EcR); whereas the MF production is limited by the farnesoic acid O-methyltransferase enzyme (FAMeT). As little is known on MIH, EcR, and FAMeT in amphipods, this study focused on the evaluation of the expression variations of the gene of these three proteins in Gammarus pulex, during the embryonic development and the molt cycle of females. Results highlighted the presence of ecr and famet genes from the first stages of the embryogenesis, suggesting key roles in the embryo development. The mih gene was only measured from Stage 3 of embryogenesis, probably related to the gastrulation and the cephalothorax development. Regardless of the gene, a strong overexpression was also measured at the hatch time. Besides, differential expression levels of mih, ecr, and famet genes through the molt cycle were observed. The highest expressions of the three genes were measured at the premolt stage, confirming key roles of MIH and EcR in the ecdysteroid pathways, and also suggesting the involvement of the FAMeT enzyme during the ecdysis in G. pulex.

Transcriptomic analysis of interactions between Hyphantria cunea larvae and nucleopolyhedrovirus

Hyphantria cunea is a destructive forest pest. To utilise H. cunea nucleopolyhedrovirus (HcNPV) for biological control, understanding insect-virus interactions is essential. Four cDNA libraries were constructed from H. cunea larvae (two HcNPV-infected and two uninfected groups) and 76 004 expressed genes were obtained by next-generation sequencing. Compared with controls, 272 differentially expressed genes (DEGs) were identified in infected groups, including 162 up-regulated and 110 down-regulated genes. Transcription levels of 20 random DEGs were consistent with the Solexa expression profiles obtained by quantitative real-time PCR. DEGs associated with innate immunity were grouped into several categories, including pattern recognition proteins, heat-shock proteins, UDP-glycosyltransferases, cytochrome P450s, antimicrobial peptides and hormonal signalling proteins. Interestingly, up-regulated host genes included farnesoic acid O-methyltransferase, two juvenile hormone (JH) binding proteins, and a circadian clock-controlled protein related to JH regulation. Pathway enrichment analysis indicates that mitogen-activated protein kinase (MAPK) signalling pathways, key candidate genes and important biological pathways may be associated with molecular modification in H. cunea larvae in response to virus stress. These findings provide insight for future research on the molecular mechanisms of HcNPV invasion and anti-HcNPV mechanisms in H. cunea. A better understanding of gene regulation following HcNPV invasion could help to develop the virus as a bio-insecticide. © 2018 Society of Chemical Industry.

Identification of target genes for RNAi-mediated control of the Twospotted Spider Mite

RNA interference (RNAi) is being developed for the management of pests that destroy crops. The twospotted Spider Mite (TSSM), Tetranychus urticae is a worldwide pest due to its unique physiological and behavioral characteristics including extraordinary ability to detoxify a wide range of pesticides and feed on many host plants. In this study, we conducted experiments to identify target genes that could be used for the development of RNAi-based methods to control TSSM. Leaf disc feeding assays revealed that knockdown in the expression genes coding for proteins involved in the biosynthesis and action of juvenile hormone (JH) and action of ecdysteroids [Methoprene-tolerant (Met), retinoid X receptor β, farnesoic acid O-methyltransferase, and CREB-binding protein] caused 35–56% mortality. Transgenic tobacco plants expressing hairpin dsRNA targeting Met gene were generated and tested. About 48% mortality was observed in TSSM raised on transgenic tobacco plants expressing dsMet. These studies not only broaden our knowledge on understanding hormone action in TSSM but also identified target genes that could be used in RNAi-mediated control of TSSM.

Cloning, characterization, and expression profile of an insect farnesoic acid O-methyltransferase orthologue from the mud crab Scylla paramamosain Estampador, 1950 (Brachyura: Portunidae): putative relationship with methyl farnesoate

Cloning, characterization, and expression profile of an insect farnesoic acid O-methyltransferase orthologue from the mud crab Scylla paramamosain Estampador, 1950 (Brachyura: Portunidae): putative relationship with methyl farnesoate

Proteomic response of Macrobrachium rosenbergii hepatopancreas exposed to chlordecone: Identification of endocrine disruption biomarkers?

The present work is the first study investigating the impacts of chlordecone, an organochlorine insecticide, on the proteome of the decapod crustacean Macrobrachium rosenbergii, by gel-free proteomic analysis. The hepatopancreas protein expression variations were analysed in organisms exposed to three environmental relevant concentrations of chlordecone (i.e. 0.2, 2 and 20µg/L). Results revealed that 62 proteins were significantly up- or down-regulated in exposed prawns compared to controls. Most of these proteins are involved in important physiological processes such as ion transport, defense mechanisms and immune system, cytoskeleton dynamics, or protein synthesis and degradation. Moreover, it appears that 6% of the deregulated protein are involved in the endocrine system and in the hormonal control of reproduction or development processes of M. rosenbergii (e.g. vitellogenin, farnesoic acid o-methyltransferase). These results indicate that chlordecone is potentially an endocrine disruptor compound for decapods, as already observed in vertebrates. These protein modifications could lead to disruptions of M. rosenbergii growth and reproduction, and therefore of the fitness population on the long-term. Besides, these disrupted proteins could be suggested as biomarkers of exposure for endocrine disruptions in invertebrates. However, further investigations are needed to complete understanding of action mechanisms of chlordecone on proteome and endocrine system of crustaceans.

Transcriptomic information from Pacific white shrimp (Litopenaeus vannamei) ovary and eyestalk, and expression patterns for genes putatively involved in the reproductive process

The increased use of massive sequencing technologies has enabled the identification of several genes known to be involved in different mechanisms associated with reproduction that so far have only been studied in vertebrates and other model invertebrate species. In order to further investigate the genes involved in Litopenaeus vannamei reproduction, cDNA and SSH libraries derived from female eyestalk and gonad were produced, allowing the identification of expressed sequences tags (ESTs) that potentially have a role in the regulation of gonadal maturation. In the present study, different transcripts involved in reproduction were identified and a number of them were characterized as full-length. These transcripts were evaluated in males and females in order to establish their tissue expression profiles during developmental stages (juvenile, subadult and adult), and in the case of females, their possible association with gonad maturation was assessed through expression analysis of vitellogenin. The results indicated that the expression of vitellogenin receptor (vtgr) and minichromosome maintenance (mcm) family members in the female gonad suggest an important role during previtellogenesis. Additionally, the expression profiles of genes such as famet, igfbp and gpcr in brain tissues suggest an interaction between the insulin/insulin-like growth factor signaling pathway (IIS) and methyl farnesoate (MF) biosynthesis for control of reproduction. Furthermore, the specific expression pattern of farnesoic acid O-methyltransferase suggests that final synthesis of MF is carried out in different target tissues, where it is regulated by esterase enzymes under a tissue-specific hormonal control. Finally, the presence of a vertebrate type steroid receptor in hepatopancreas and intestine besides being highly expressed in female gonads, suggest a role of that receptor during sexual maturation.

Expression of catechol O-methyltransferase during ovarian development and association between its SNP and reproduction-related parameters of the giant tiger shrimp Penaeus monodon

Catechol O-methyltransferase cDNA and gene of the giant tiger shrimp Penaeus monodon (PmCOMT) were characterized. Genomic sequence of PmCOMT spanned 1466bp in length containing 3 exons (194, 111 and 361bp) and 2 introns (143 and 147bp). The full-length cDNA of PmCOMT was 1176bp in length with an open reading frame (ORF) of 666bp corresponding to a polypeptide of 221 amino acids. PmCOMT mRNA was not differentially expressed during ovarian development in wild intact P. monodon adults (P>0.05). Eyestalk ablation resulted in up-regulation of PmCOMT in stages II (vitellogenic) and IV (mature) relative to stage I (previtellogenic) ovaries (P<0.05). In domesticated shrimp, its expression was reduced in 18-month-old adults compared with 6-month-old juveniles and 14-month-old adults (P<0.05). In situ hybridization indicated that PmCOMT mRNA was clearly localized in ooplasm of previtellogenic oocytes but not in oogonia and other oocytes stages. Recombinant PmCOMT protein and anti-rPmCOMT polyclonal antibody were successfully produced. The PmCOMT protein was localized in ooplasm of previtellogenic oocytes. It was temporally found in both ooplasm and nucleus of vitellogenic oocytes and subsequently in cortical rods (Crs) and the Cr membrane of mature oocytes. In addition, single nucleotide polymorphism (SNP) in PmCOMT of domesticated P. monodon (14-month-old shrimp, N=64) was identified by single-strand conformational polymorphism (SSCP) and PCR-direct sequencing. Two SSCP patterns (A and B) corresponding to G/Cc.305+g.84 and C/Cc.305+g.84 SNP genotypes were found. Shrimp carrying the former genotype (OW=0.92±0.37g and GSI=1.06±0.39%, N=31) had a greater average ovarian weight and GSI value than those carrying the latter genotype (OW=0.75±0.30g and GSI=0.86±0.32%, N=33) (P<0.05). Taken the information together, PmCOMT should play the functional role during ovarian development of P. monodon. Statement of relevanceReduced reproductive performance of the giant tiger shrimp Penaeus monodon in captivity prevents the potential application of domestication and selective breeding program for genetic improvement of this economically important species. Understanding molecular mechanisms controlling the development and maturation of ovaries/oocytes is essential for finding an approach to promote the reproductive maturation of P. monodon without the use of eyestalk ablation. Two kinds of O-methyltransferase (OMT) transcripts; farnesoic acid O-methyltransferase (FAMeT) and catechol O-methyltransferase (COMT) have been characterized in penaeid species. P. monodon FAMeT was differentially expressed during ovarian development and its SNPs showed association with ovarian weight of domesticated adults. Similarly, the expression profile of P. monodon COMT was examined and eyestalk ablation affected its expression level during vitellogenic and mature stages. Relationships between a SNP of P. monodon COMT and reproduction-related parameters (ovarian weight and GSI) were found.