Comparative genomic analysis of ABC transporter genes in Tenebrio molitor and four other tenebrionid beetles (Coleoptera: Tenebrionidea).

ATP-binding cassette (ABC) transporters, one of the largest transmembrane protein families, transport a diverse number of substate across membranes. Details of their diverse physiological functions have not been established. Here, we identified 87 ABC transporter genes in the genomes of Tenebrio molitor along with those from Asbolus verrucosus (104), Hycleus cichorii (65), and Hycleus phaleratus (80). Combining these genes (336 in total) with genes reported in Tribolium castaneum (73), we analyzed the phylogeny of ABC transporter genes in all five Tenebrionids. They are assigned into eight subfamilies (ABCA-H). In comparison to other species, the ABCC subfamily in this group of beetles appears expanded. The expression profiles of the T. molitor genes at different life stages and in various tissues were also investigated using transcriptomic analysis. Most of them display developmental specific expression patterns, suggesting to us their possible roles in development. Most of them are highly expressed in detoxification-related tissues including gut and Malpighian tubule, from which we infer their roles in insecticide resistance. We detected specific or abundant expressions of many ABC transporter genes in various tissues such as salivary gland, ovary, testis, and antenna. This new information helps generate new hypotheses on their biological significance within tissues.

Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor.

Serine protease inhibitors (SPIs) act in diverse biological processes in insects such as immunity, development, and digestion by preventing the unwanted proteolysis. So far, the repertoire of genes encoding SPIs has been identified from few insect species. In this study, 62 SPI genes were identified from the genome of the yellow mealworm, Tenebrio molitor. According to their modes of action, they were classified into three families, serpin (26), canonical SPI (31), and α-macroglobulins (A2M) (5). These SPIs feature eight domains including serpin, Kazal, TIL, Kunitz, WAP, Antistasin, pacifastin, and A2M. In total, 39 SPIs contain a single SPI domain, while the others encode at least two inhibitor units. Based on the amino acids in the cleaved reactive sites, the abilities of these SPIs to inhibit trypsin, chymotrypsin, or elastase-like enzymes are predicted. The expression profiling based on the RNA-seq data showed that these genes displayed stage-specific expression patterns during development, suggesting to us their significance in development. Some of the SPI genes were exclusively expressed in particular tissues such as hemocyte, fat body, gut, ovary, and testis, which may be involved in biological processes specific to the indicated tissues. These findings provide necessary information for further investigation of insect SPIs.

Genomic and transcriptomic analyses of chitin metabolism enzymes in Tenebrio molitor.

Chitin is of great importance in the cuticle and inner cuticular linings of insects. Chitin synthases (CHSs), chitin deacetylases (CDAs), chitinases (CHTs), and ß-N-acetylhexosaminidases (HEXs) are important enzymes required for chitin metabolism, and play essential roles in development and metamorphosis. Although chitin metabolism genes have been well characterized in limited insects, the information in the yellow mealworm, Tenebrio molitor, a model insect, is presently still unavailable. With the help of bioinformatics, we identified 54 genes that encode putative chitin metabolism enzymes, including 2 CHSs, 10 CDAs, 32 CHTs, and 10 HEXs in the genome of T. molitor. All these genes have the conserved domains and motifs of their corresponding protein family. Phylogenetic analyses indicated that CHS genes were divided into two groups. CDA genes were clustered into five groups. CHT genes were phylogenetically grouped into 11 clades, among which 1 in the endo-ß-N-acetylglucosaminidases group and the others were classified in the glycoside hydrolase family 18 groups. HEX genes were assorted into six groups. Developmental and tissue-specific expression profiling indicated that the identified chitin metabolism genes showed dynamical expression patterns concurrent with specific instar during molting period, suggesting their significant roles in molting and development. They were predominantly expressed in different tissues or body parts, implying their functional specialization and diversity. The results provide important information for further clarifying their biological functions using the yellow mealworm as an ideal experimental insect.

Identification and expression profiling of serine protease-related genes in Tenebrio molitor.

In insects, serine proteases and serine protease homologs (SPs/SPHs) are involved in a variety of physiological processes including digestion, development, and immunity. Here, we identified 112 SP and 88 SPH genes in the genome of the yellow mealworm, Tenebrio molitor. Based on the features of domain structure, they were divided into "S" group containing single Tryp-SPc or Tryp-SPHc domain, "C" group containing 1-4 CLIP domain (CLIPA-D) and "M" group containing the CBD, CUB, EGF, Fz, Gd, LDLa, PAN, SEA, SR, Sushi, and TSP domains, and have 115, 48, and 37 gene members, respectively. According to the active sites in the catalytic triad, the putative trypsin, chymotrypsin, or elastase-like enzyme specificity of the identified SPs/SPHs were predicted. Phylogenetic and genomic location analyses revealed that gene duplication exists in the large amount of SPs/SPHs. Gene expression profiling using RNA-seq data along with real time reverse transcription-polymerase chain reaction analysis showed that most SP/SPH genes display life stage specific expression patterns, indicating their important roles in development. Many SP/SPH genes are specifically or highly expressed in the gut, salivary gland, fat body, hemocyte, ovary, and testis, suggesting that they participate in digestion, immunity, and reproduction. The findings lay the foundation for further functional characterization of SPs/SPHs in T. molitor.

Genome-wide and expression-profiling analyses of the cytochrome P450 genes in Tenebrionidea.

Cytochrome P450 monooxygenases (CYPs) are present in almost all areas of the tree of life. As one of the largest and most diverse superfamilies of multifunctional enzymes, they play important roles in the metabolism of xenobiotics and biosynthesis of endogenous compounds, shaping the success of insects. In this study, the CYPome (an omics term for all the CYP genes in a genome) diversification was examined in the four Tenebrionidea species through genome-wide analysis. A total of 483 CYP genes were identified, of which 103, 157, 122, and 101 were respectively deciphered from the genomes of Tebebrio molitor, Asbolus verucosus, Hycleus cichorii and Hycleus phaleratus. These CYPs were classified into four major clans (mitochondrial, CYP2, CYP3, and CYP4), and clans CYP3 and CYP4 are most diverse. Phylogenetic analysis showed that most CYPs of these Tenebrionidea beetles from each clan had a very close 1:1 orthology to each other, suggesting that they originate closely and have evolutionally conserved function. Expression analysis at different developmental stages and in various tissues showed the life stage-, gut-, salivary gland-, fat body-, Malpighian tubule-, antennae-, ovary- and testis-specific expression patterns of T. molitor CYP genes, implying their various potential roles in development, detoxification, immune response, digestion, olfaction, and reproduction. Our studies provide a platform to understand the evolution of Tenebrionidea CYP gene superfamily, and a basis for further functional investigation of the T. molitor CYPs involved in various biological processes.

Comparative genomic analysis of carboxylesterase genes in Tenebrio molitor and other four tenebrionids.

Carboxylesterases (COEs) have various functions in wide taxons of organisms. In insects, COEs are important enzymes involved in the hydrolysis of a variety of ester-containing xenobiotics, neural signal transmission, pheromone degradation, and reproductive development. Understanding the diversity of COEs is basic to illustrate their functions. In this study, we identified 53, 105, 37, and 39 COEs from the genomes of Tenebrio molitor, Asbolus verucosus, Hycleus cichorii, and H. phaleratus in the superfamily of Tenebrionidea, respectively. Phylogenetic analysis showed that 234 COEs from these four species and those reported in Tribolium castaneum (63) could be divided into 12 clades and three major classes. The α-esterases significantly expanded in T. molitor, A. verucosus, and T. castaneum compared to dipteran and hymenopteran insects. In T. molitor, most COEs showed tissue and stage-specific but not a sex-biased expression. Our results provide insights into the diversity and evolutionary characteristics of COEs in tenebrionids, and lay a foundation for the functional characterization of COEs in the yellow mealworm.

The Wnt gene family in Tenebrio molitor and other coleopterans.

The Wnt gene family is involved in a wide range of developmental processes. Despite its significance, the evolution and function of Wnt genes remain largely unclear. Here, an exhaustive survey of Wnt genes was conducted in Tenebrio molitor and 17 other beetle genomes. A total of 146 Wnt genes were identified, creating a comprehensive coleopteran Wnt gene catalog. Comparative genomics indicates that dynamic evolutionary patterns of Wnt gene loss and duplication occurred in Coleoptera, leading to the diverse Wnt gene repertoire in various beetles. A striking loss of particular Wnt gene subfamilies occurs in Coleoptera. Remarkably, Wnt gene duplication was discovered for the first time in insects. Further analysis of Wnt gene expression in T. molitor indicates that each Wnt gene, including the duplicated ones, has a unique spatial or temporal expression pattern. The current study provides valuable insight into the evolution and functional validation of Wnt genes in Coleoptera.

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT7 genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.

Genome-wide characterization and transcriptomic analyses of neuropeptides and their receptors in an endoparasitoid wasp, Pteromalus puparum.

In insects, neuropeptides constitute a group of signaling molecules that act in regulation of multiple physiological and behavioral processes by binding to their corresponding receptors. On the basis of the bioinformatic approaches, we screened the genomic and transcriptomic data of the parasitoid wasp, Pteromalus puparum, and annotated 36 neuropeptide precursor genes and 33 neuropeptide receptor genes. Compared to the number of precursor genes in Bombyx mori (Lepidoptera), Chilo suppressalis (Lepidoptera), Drosophila melanogaster (Diptera), Nilaparvata lugens (Hemiptera), Apis mellifera (Hymenoptera), and Tribolium castaneum (Coleoptera), P. puparum (Hymenoptera) has the lowest number of neuropeptide precursor genes. This lower number may relate to its parasitic life cycle. Transcriptomic data of embryos, larvae, pupae, adults, venom glands, salivary glands, ovaries, and the remaining carcass revealed stage-, sex-, and tissue-specific expression patterns of the neuropeptides, and their receptors. These data provided basic information about the identity and expression profiles of neuropeptides and their receptors that are required to functionally address their biological significance in an endoparasitoid wasp.

Genomic and transcriptomic analyses of glutathione S-transferases in an endoparasitoid wasp, Pteromalus puparum.

Pteromalus puparum is a gregarious pupal endoparasitoid with a wide host range. It deposits eggs into pierid and papilionid butterfly pupae. Glutathione S-transferases (GSTs) are a family of multifunctional detoxification enzymes that act in xenobiotic metabolism in insects. Insect genome projects have facilitated identification and characterization of GST family members. We identified 20 putative GSTs in the P. puparum genome, including 19 cytosolic and one microsomal. Phylogenetic analysis showed that P. puparum GSTs are clustered into Hymenoptera-specific branches. Transcriptomic data of embryos, larvae, female pupae, male pupae, female adults, male adults, venom glands, carcass, salivary glands, and ovaries revealed stage-, sex-, and tissue-specific expression patterns of GSTs in P. puparum. This is the most comprehensive study of genome-wide identification, characterization, and expression profiling of GST family in hymenopterans. Our results provide valuable information for understanding the metabolic adaptation of this wasp.

Identification of a C-type lectin possessing both antibacterial and antiviral activities from red swamp crayfish.

C-type lectins are important immune molecules that participate in crustacean defense response. The present work reports a novel C-type lectin (PcLec6) from the red swamp crayfish Procambarus clarkii. PcLec6 encodes a single-peptide protein of 385 amino acids, which include a C-type lectin domain (CTLD) and a serine-rich region. PcLec6 expression in lymph organ and gills was up-regulated after bacterial challenge by Vibrio alginolyticus or white spot syndrome virus (WSSV). Recombinant full-length PcLec6 or its CTLD proteins were used for the functional analyses. Results showed that these two proteins had the capacity to bind to carbohydrates and bacteria. Both the full-length PcLec6 and CTLD facilitated the bacterial clearance, but only full-length PcLec6 protected crayfish from WSSV infection. Furthermore, PcLec6 regulated the expression of ALF genes. These results suggest that PcLec6 is involved in the innate immune response of crayfish against both bacterial and viral pathogens.

Characterization of a double WAP domain-containing protein from the red swamp crayfish Procambarus clarkii.

Crustaceans express multiple whey acidic protein (WAP) domain containing proteins which are components of host immunity. In the present study, a new double WAP domain containing protein was identified from red swamp crayfish Procambarus clarkii, designated Pc-DWD. The ORF is 387 bp, encoding 128 amino acids consisting of signal peptide of 18 residues, and two tandem WAP domains of 38 and 44 residues. Multiple alignment indicates the presence of conserved motifs in both WAP domains, and phylogenetic analysis shows that Pc-DWD is a new member of the type-IV crustin family. Pc-DWD transcripts were found most abundantly in hemocytes, gills, intestine and heart, and induced by Vibrio anguillarum, Staphylococcus aureus and white spot syndrome virus challenge. RNAi knockdown of Pc-DWD expression led to increased expression of white spot syndrome virus genes and increased crayfish mortality after virus infection. Recombinant Pc-DWD exhibited strong protease inhibitory activity towards commercial subtilicin A and protease K. Pc-DWD inhibited the crude proteases from V. anguillarum and S. aureus cultures and from the crayfish tissue extracts. We infer that Pc-DWD acts in crayfish bacterial and viral immunity.

Molecular basis for insecticide-enhanced thermotolerance in the brown planthopper Nilaparvata lugens Stål (Hemiptera:Delphacidae).

Climate change is likely to have marked ecological effects on terrestrial ecosystems, including the activities of insect pests. Most attention has focused on the increasing geographical ranges of pests; however, if extrinsic factors enhance their thermotolerance, populations may express increased voltinism and longer daily and annual activity periods. These changes in pest populations have the potential for severe consequences, including increased crop losses and decreased food security at the global level. The brown planthopper (BPH) Nilaparvata lugens Stål (Hemiptera: Delphacidae) is a serious pest of rice crops in temperate and tropical regions of Asia. It is often present in rice microclimates at temperatures close to its maximum thermotolerance. Recent BPH outbreaks in tropical Asia are considered to be associated with excess use of pesticides and increasing temperature. This study tested whether exposure to sublethal concentrations of triazophos (tzp), an insecticide widely used in Asian rice production, enhances thermotolerance of BPH. Tzp exposure (40 ppm at 40 °C) significantly decreased mortality (from 94% in controls to 50% at 48 h post-treatment) and increased lethal mean time (LT50 ) of adults by 17.2 h. To investigate the underlying molecular mechanism of this tzp-enhanced thermotolerance, we selected Hsp70 and Arginine kinase (Argk) for detailed study. Transcripts encoding both proteins in third-instar nymphs and brachypterous adult females were up-regulated, compared with controls, after exposure to tzp. RNAi silencing of both genes demonstrated that Hsp70 and Argk are essential for survival and tzp-increased thermotolerance. We propose that tzp induces thermotolerance in BPHs by increasing the expression of genes that act in cell protection mechanisms. The significance of our proposal relates to the importance of understanding the influence of sublethal concentrations of insecticides on pest biology. In addition to its influence on thermotolerance, tzp also enhances BPH reproduction. We infer that exposure to a pesticide stressor can produce cross-tolerance, that is, increased tolerance to one stressor also increases tolerance to other stressors, including temperature. Aside from needing a better understanding of these effects in nature and in other pest/cropping systems, we suggest that pest management programmes can be improved with better understanding of the influences of stressors, including increased environmental temperatures and sublethal concentrations of insecticides, on pest biology.

Integrated transcriptome and proteome unveiled distinct toxicological effects of long-term cadmium pollution on the silk glands of Pardosa pseudoannulata.

Cadmium (Cd) induces severe soil pollution worldwide and exerts adverse effects on paddy field arthropods. Spiders grant a novel perspective to assess the Cd-induced toxicity, yet the impacts of long-term Cd stress on spider silk glands and its underlying mechanism remain elusive. The study showed that Cd stress enervated the antioxidant system in the spider Pardosa pseudoannulata, manifested as the decreases of glutathione peroxidase and peroxidase, and the increase of malonaldehyde (p < 0.05). In addition, a total of 1459 differentially expressed genes (DEGs) and 404 differentially expressed proteins (DEPs) were obtained from the silk glands' transcriptome and proteome. DEGs and DEPs encoding spidroin (e.g., tubuliform spidroin and ampullate spidroin) and amino acids metabolism (e.g., alanine, proline, and glycine) were distinctively down-regulated. Further enrichment analysis verified that Cd stress could inhibit amino acid metabolism via the down-regulation of several key enzymes, including glutathione synthase, methylthioadenosine phosphorylase, S-adenosylmethionine synthetase, etc. In addition, the hedgehog signaling pathway regulating cellular growth and development was down-regulated under Cd stress. A protein-protein interaction network showed that long-term Cd stress could inhibit some key biological processes in the silk glands, including peptide biosynthetic process and cytoskeleton part. Collectively, this comprehensive study established an effective animal detection model for evaluating Cd-induced toxicity, presented key biomarkers for further validation, and provided novel insights to investigate the molecular mechanisms of spiders to Cd pollution.

Bt rice expressing Cry1Ab does not stimulate an outbreak of its non-target herbivore, Nilaparvata lugens.

In this study, the non-target effects of Bt rice "KMD2" expressing a Cry1Ab protein on the performance of the brown planthopper (BPH), Nilaparvata lugens, over multiple generations were evaluated under laboratory and field conditions. In the laboratory, BPH was reared to observe the impact of the Bt rice as compared to its parental non-Bt cultivar Xiushui 11, while the population dynamics and oviposition performance of BPH were investigated in the field. The survival of BPH nymphs fed Bt and non-Bt rice did not differ significantly. The nymph developmental duration of BPH was significantly delayed by the Bt rice by comparison with the non-Bt rice for the 1st and 2nd but not the 4th generation. Most importantly, the fecundity of BPH on the Bt rice was significantly decreased in every generation when compared with the non-Bt rice. In the field investigations, the population density of BPH nymphs was significantly lower in the Bt rice field. However, the temporal pattern of population dynamics of BPH adults was similar between the Bt and non-Bt rice, presumably due to migratory interference of the adults. In the Bt rice field, the percentage of tillers with eggs and the number of eggs per tiller were also significantly lower from tillering to mature stage. Additionally, Cry1Ab protein could not be detected in guts from single BPH adults. In general, our results suggest that the Bt rice "KMD2" could not stimulate an outbreak of BPH.

[Evaluation of tuberculin skin test conversion and interferon-gamma release assay in diagnosis of tuberculosis infection].

OBJECTIVE: To compare PPD conversion with γ-interferon release assay (IGRA) in determining the newly Mycobacterium tuberculosis (MTB) infection during a TB outbreak. METHODS: The 505 subjects exposed to a TB outbreak were divided into 3 groups based upon the induration diameters of PPD before the outbreak. The changes of PPD induration diameters were observed in different groups, and correlation between PPD conversion or IGRA and exposure levels were analyzed by logistic regression. RESULTS: In subjects with the highest exposure level, the increase in induration of PPD was (12 ± 5) mm, (7 ± 4) mm, and (5 ± 3) mm respectively among the previous PPD < 5 mm, 5 - < 10 mm, and ≥ 10 mm groups, the difference being significant among groups, χ(2) = 43.12, P < 0.01. Using logistic regression analysis, PPD conversion was related to the exposure level (OR = 4.70, P < 0.05) only in the PPD < 5 mm group, while IGRA positivity was closely related to the exposure levels in all 3 groups (OR values were 2.16 - 3.60, P < 0.05). In the high exposure group, the subjects with IGRA positivity combined with PPD conversion rate was 26.1% (31/119), and the subjects with IGRA positivity but no PPD conversion rate was 39.5% (47/119), while in the low exposure group the results were 6.5% (12/185) and 16.8% (31/185) respectively, the difference being significant (χ(2) = 22.82 and 19.63, P < 0.01). After 1 year of follow-up, the reversion rate of the subjects with IGRA positivity was 48.1% (91/189), while the mean increase value of PPD was (1.1 ± 4.0) mm. CONCLUSION: IGRA is superior to PPD conversion in the diagnosis of tuberculosis infection, and more valuable in the detection of newly infected cases.

Integrated transcriptomics and proteomics provide new insights into the cadmium-induced ovarian toxicity on Pardosa pseudoannulata.

Cadmium (Cd) pollution is intractable heavy metal pollution in the farmland ecosystem, posing a life-threatening challenge to the paddy field organisms. Spiders are riveting animal biomarkers for evaluating Cd-induced toxicity, yet the effects of long-term Cd toxicity on spider reproductive function and its underlying mechanism remain unclear. In the present study, we found that Cd exposure impaired the antioxidant enzyme system in the wolf spider Pardosa pseudoannulata and decreased the concentration of four antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase, and peroxidase) (p < 0.05). The content of vitellogenin and the number of hatched spiderlings were also dramatically reduced under Cd stress (p < 0.05), indicating that Cd stress could vitiate the fecundity of P. pseudoannulata. Moreover, a total of 10,511 differentially expressed genes (DEGs) and 391 proteins (DEPs) were yielded from the ovarian transcriptome and proteome, and a mass of genes and proteins involved in protein processing in endoplasmic reticulum (ER) were significantly down-regulated. DEGs and DEPs directly encoding the antioxidant enzyme system and/or vitellogenesis were also distinctively down-regulated. In addition, we illustrated that the PI3K-AKT signaling pathway might play a crucial role in regulating protein synthesis, cell cycle, growth, differentiation and survival in P. pseudoannulata. The effects of protein processing in ER and PI3K-AKT pathways could further trigger transcriptional factor Forkhead shackling the protein synthesis and cell growth process. Collectively, this integrated analysis identified the Cd-induced reproductive toxicity on P. pseudoannulata and provided multifaceted insights to investigate the molecular mechanisms of spiders to Cd pollution.

Differences in pulmonary nodular consolidation and pulmonary cavity among drug-sensitive, rifampicin-resistant and multi-drug resistant tuberculosis patients: a computerized tomography study with history length matched cases.

Background: There have been concerns that literature described radiological feature differences between drug-sensitive pulmonary tuberculosis (DS-PTB) and multidrug-resistant (MDR)-PTB were confounded by that MDR-PTB cases tend to have a longer history. Using history length matched DS-PTB and MDR-PTB cases from a well-defined urban region in Dalian, we retrospectively analysed the CT feature differences of these paired cases with a focus on pulmonary nodular (PN) consolidation and pulmonary cavity (PC). Methods: There were 33 consecutive MDR-PTB cases [inclusive of rifampicin-resistant (RR) cases, 27 males and 6 females, mean age: 49.2 years], with 19 cases had a history of <1 month and 8 and 6 cases had a history of 1-6 and >6 months respectively. To pair the MDR-PTB cases with history length, matched 33 cases of DS-PTB patients (21 males and 12 females, mean age: 56.5 years) were included. All patients were new PTB without HIV infection. The first CT exams prior to treatment were analysed. Results: Compared with DS cases, MDR cases had a much higher prevalence of PN (75.76% vs. 45.45%) and a higher number of PN per positive case for PN (6.2 vs.1.53). For the cases >1 month history, MDR-PTB had a higher number of PC per positive case than that of DS-PTB cases (7.18 vs. 2.36). To differentiate DS-PTB from MDR-PTB, receiver operating characteristic (ROC) analysis showed a cutoff PN number of ≥3 had 48.5% sensitivity and 93.9% specificity, and a cutoff PC number of ≥4 had 39.4% sensitivity and 84.9% specificity. The lung field distribution of all lesions tended to be wider for MDR-PTB cases. MDR-PTB cases appeared to be associated with a faster progression in the absence of treatment. Conclusions: MDR-TB is likely intrinsically more invasive than DS-TB. Multiple PN and Multiple PC are promising signs for the suspicion of MDR-PTB on chest imaging.

DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyx mori.

Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression. However, the mechanisms by which DNA methylation regulates these processes in insects remain unclear. Here, we studied the impacts of DNA methylation on early embryonic development in Bombyx mori. Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5' region of protein metabolism-related genes. The transcription factor gene zinc finger protein 615 ( ZnF615) was methylated by DNA methyltransferase 1 (Dnmt1) to be up-regulated and bind to protein metabolism-related genes. Dnmt1 RNA interference (RNAi) revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615. The same sites in the ZnF615 gene were methylated in ovaries and embryos. Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout. Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo. Thus, Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.

Comparative analysis unveils the cadmium-induced reproductive toxicity on the testes of Pardosa pseudoannulata.

Cadmium (Cd) pollution is one of the most serious heavy metal pollutions in the world, which has been demonstrated to cause different toxicities to living organisms. Cd has been widely suggested to cause reproductive toxicity to vertebrates, yet its reproductive toxicity to invertebrates is not comprehensive. In this study, the wolf spider Pardosa pseudoannulata was used as a bioindicator to evaluate the male reproductive toxicity of invertebrates under Cd stress. Cd stress had no effect on the color, size and length of testis. However, Cd significantly increased the contents of catalase, glutathione peroxidase and malondialdehyde, the antioxidants in the testis of P. pseudoannulata. Then we analyzed the transcriptome of testis exposed to Cd, and identified a total of 4739 differentially expressed genes (DEGs) compared to control, with 2368 up-regulated and 2371 down-regulated. The enrichment analysis showed that Cd stress could affect spermatogenesis, sperm motility, post-embryonic development, oxidative phosphorylation and metabolism and synthesis of male reproductive components. At the same time, the protein-protein interaction network was constructed with the generated DEGs. Combined with the enrichment analysis of key modules, it revealed that Cd stress could further affect the metabolic process in testis. In general, the analysis of testicular damage and transcriptome under Cd stress can provide a novel insight into the reproductive toxicity of Cd on rice filed arthropods and offer a reference for the protection of rice filed spiders under Cd pollution.