Per- and polyfluoroalkyl substance (PFAS) mixtures induce gut microbiota dysbiosis and metabolic disruption in silkworm (Bombyx mori L.).

Mixed legacy and emerging per- and polyfluoroalkyl substances (PFASs) are commonly found in soil and dust; however, the potential toxicity of PFAS mixtures (mPFASs) in insects is unknown. Using 16S rRNA gene sequencing and transcriptome sequencing (RNA-Seq), we evaluated the adverse effects of mPFASs on silkworms, a typical lepidopteran insect. After exposure to mPFASs, the silkworm midgut was enriched with high levels of PFASs, which induced histopathological changes. The composition of the midgut microbiota was significantly affected by mPFAS exposure, and functional predictions revealed significant disruption of some metabolic pathways. RNA-seq analysis revealed that mPFASs significantly changed the transcription profiles. Functional enrichment analysis of the differentially expressed genes also revealed that biological processes related to metabolic pathways and the digestive system were significantly affected, similar to the results of the gut microbiota analysis, suggesting that mPFAS exposure had an adverse effect on the metabolic function of silkworms and may further affect their normal growth. Finally, the significant correlation between abundance changes in the gut microbiota and metabolism/digestion-related genes further highlighted the role of the gut microbiota in mPFAS-related processes affecting the metabolic functions of silkworms. To our knowledge, this study is the first to evaluate the toxic effects of mPFASs in insects and provide basic data for further PFAS toxicity investigations in insects and comprehensive ecological risk assessments of mPFASs.

Isolation and genomic characterization of a cypovirus from Clanis bilineata.

Clanis bilineata Walker, soybean hawkmoth, belongs to the subfamily Ambulicinae (Sphingidae, Lepidoptera) and is an edible insect that usually grows on soybean leaves. In this study, we isolated a new cypovirus from naturally diseased Clanis bilineata larvae (named CbCPV), scanned its structure, sequenced its genome, and studied its phylogenetic relationship to other cypoviruses. Microscopy showed that CbCPV polyhedral occlusion bodies were about 1.878 µm on average and contained many virions in the ultrathin sections. The complete genome sequence of CbCPV is 22,812 bp comprising 10 segmented double-stranded RNAs. Apart from segment 1 containing one open reading frame (ORF) and one sub-ORF, the other nine segments all contain one open reading frame and encoded one putative protein. The non-coding regions contained conserved sequences at 5' termini (AGUCAAA) and 3' termini (AGC), except segment 4 containing a different 5' termini (AUGUUUA). The whole sequence of the polyhedrin gene in CbCPV contained 892 nucleotides, encoding a protein of 246 amino acids. Based on amino acid sequences of polyhedrin or RNA dependent RNA polymerase (RdRp), the phylogenetic analysis indicated that CbCPV was closely related to DnCPV-23. The putative function of all segments differed from each other, but the most closely related species of segments were DnCPV-23 with 98.2-99.8% nucleotide identity. Overall, the evidence of morphology, protein analysis and nucleic acids (genomic pattern) showed that CbCPV is a new isolate in the cypovirus-23 type and can be termed Clanis bilineata cypovirus type 23 (CbCPV-23).

Discovery of mosquitocides from fungal extracts through a high-throughput cytotoxicity-screening approach.

BACKGROUND: Mosquitoes transmit a variety of diseases. Due to widespread insecticide resistance, new effective pesticides are urgently needed. Entomopathogenic fungi are widely utilized to control pest insects in agriculture. We hypothesized that certain fungal metabolites may be effective insecticides against mosquitoes. METHODS: A high-throughput cytotoxicity-based screening approach was developed to search for insecticidal compounds in our newly established global fungal extract library. We first determined cell survival rates after adding various fungal extracts. Candidate insecticides were further analyzed using traditional larval and adult survival bioassays. RESULTS: Twelve ethyl acetate extracts from a total of 192 fungal extracts displayed > 85% inhibition of cabbage looper ovary cell proliferation. Ten of these 12 candidates were confirmed to be toxic to Anopheles gambiae Sua5B cell line, and six showed > 85% inhibition of Anopheles mosquito cell growth. Further bioassays determined a LC50, the lethal concentration that kills 50% of larval or adult mosquitoes, of 122 µg/mL and 1.7 µg/mosquito, respectively, after 24 h for extract 76F6 from Penicillium toxicarium. CONCLUSIONS: We established a high-throughput MTT-based cytotoxicity screening approach for the discovery of new mosquitocides from fungal extracts. We discovered a candidate extract from P. toxicarium that exhibited high toxicity to mosquito larvae and adults, and thus were able to demonstrate the value of our recently developed approach. The active fungal extracts discovered here are ideal candidates for further development as mosquitocides.

Transcriptional responses of Daphnis nerii larval midgut to oral infection by Daphnis nerii cypovirus-23.

BACKGROUND: Daphnis nerii cypovirus-23 (DnCPV-23) is a new type of cypovirus and has a lethal effect on the oleander hawk moth, Daphnis nerii which feeds on leave of Oleander and Catharanthus et al. After DnCPV-23 infection, the change of Daphnis nerii responses has not been reported. METHODS: To better understand the pathogenic mechanism of DnCPV-23 infection, 3rd-instar Daphnis nerii larvae were orally infected with DnCPV-23 occlusion bodies and the transcriptional responses of the Daphnis nerii midgut were analyzed 72 h post-infection using RNA-seq. RESULTS: The results showed that 1979 differentially expressed Daphnis nerii transcripts in the infected midgut had been identified. KEGG analysis showed that protein digestion and absorption, Toll and Imd signaling pathway were down-regulated. Based on the result, we speculated that food digestion and absorption in insect midgut might be impaired after virus infection. In addition, the down-regulation of the immune response may make D. nerii more susceptible to bacterial infections. Glycerophospholipid metabolism and xenobiotics metabolism were up-regulated. These two types of pathways may affect the viral replication and xenobiotic detoxification of insect, respectively. CONCLUSION: These results may facilitate a better understanding of the changes in Daphnis nerii metabolism during cypovirus infection and serve as a basis for future research on the molecular mechanism of DnCPV-23 invasion.

Alleviating chronic ER stress by p38-Ire1-Xbp1 pathway and insulin-associated autophagy in C. elegans neurons.

ER stress occurs in many physiological and pathological conditions. However, how chronic ER stress is alleviated in specific cells in an intact organism is an outstanding question. Here, overexpressing the gap junction protein UNC-9 (Uncoordinated) in C. elegans neurons triggers the Ire1-Xbp1-mediated stress response in an age-dependent and cell-autonomous manner. The p38 MAPK PMK-3 regulates the chronic stress through IRE-1 phosphorylation. Overexpressing gap junction protein also activates autophagy. The insulin pathway functions through autophagy, but not the transcription of genes encoding ER chaperones, to counteract the p38-Ire1-Xbp1-mediated stress response. Together, these results reveal an intricate cellular regulatory network in response to chronic stress in a subset of cells in multicellular organism.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.

[Isolation and Identification of a Novel Cypovirus from Daphnis nerii].

In order to develop a novel effective biological insecticide for controlling oleander hawk moth, a new pathogen was isolated from naturally diseased Daphnis nerii. Based on scanning electron microscopy, full-length amplification of cDNAs (FLAC), and phylogenetic analysis of genome segments 2and 10,the virus was identified as a new type of cypovirus (Da phnis nerii cypovirus [DnCPV]). Electrophoresis analysis showed that DnCPV had a genome comprising 10double-stranded RNA (dsRNA) segments, ranging from 892 to 4160bp.Using FLAC, the cDNAs from the 10 dsRNA segments of the new CPV were cloned and genome segments 2and 10 were sequenced. Sequencing results showed that segment 2 encoded RNA-dependent-RNA-polymerases (RdRps) and segment 10 encoded polyhedrin. These two segments shared conserved terminal sequences of AGUCAAA and AGC at the 5'and 3'ends,respectively.These conserved terminal sequences were not consistent with any of the known CPV types.Phylogenetic analysis of the RdRp and polyhedrin indicated that this CPV was more closely related to CPV type 19 and type 5than other CPV types. Based on the unique conserved terminal sequences and the electrophoresis pattern of the new virus, we tentatively named it DnCPV Nanchang isolate: DnCPV-NC.

Identification of the chitin-binding proteins from the larval proteins of silkworm, Bombyx mori.

The silkworm is a model organism for Lepidoptera. Its cuticle is composed mainly of chitin and proteins, which plays essential roles in multiple physiological functions. The binding of proteins to chitin plays an important role for cuticle formation. In this research, a chitin-binding assay followed by a proteomics analysis was carried out using the proteins extracted from the 5th instar larval cuticles. As results, twenty-two proteins were identified including nine cuticular proteins, two lysozyme precursors, two proteins with chitin-binding-type 2 domains, and other proteins. A cuticular protein with the RR-1 consensus, BmorCPR56, and a silkworm Tweedle protein, BmorCPT1, were detected in the chitin-binding fraction for the first time and their chitin-binding activities were further confirmed in vitro. The results of this research increase our understanding of the structure of the silkworm larval cuticle.