The effects of glyphosate exposure on gene transcription and immune function of the silkworm, Bombyx mori.

Glyphosate is a widely used herbicide and crop desiccant. However, whether its extensive use has any effect on the species diversity of nontarget organisms is still unclear. In this study, we used the silkworm, Bombyx mori, as the research subject, and performed RNA sequencing to analyze the transcriptional profile of silkworm midgut after exposure to glyphosate at 2975.20 mg/L (a concentration commonly used at mulberry fields). A total of 125 significantly differentially expressed genes (DEGs) were detected in the midgut of glyphosate-exposed silkworm (q < 0.05), of which 53 were upregulated and 72 were downregulated. Gene ontology enrichment analysis showed that the DEGs were mainly enriched in biological process, cellular component, and molecular function. Kyoto encyclopedia of genes and genomes analysis showed that the differential genes were mainly related to oxidative stress, nutrient metabolism, and immune defense pathways, including oxidative stress-related Cat and Jafrac1, nutrient metabolism-related Fatp and Scpx, and immune-related CYP6AN2, UGT40B4, CTL11, serpin-2, and so forth. Experimental verification showed that glyphosate exposure led to a 4.35-fold increase in the mortality of silkworm after Beauveria bassiana infection, which might be caused by the decreased PO (phenoloxidase) activity and impaired immunity. These results provide evidence for the potential effects of residue glyphosate on the physiological functions of silkworm, and also provide a reference for the biosafety evaluation of glyphosate.

A selection marker-free method for gene deletion and editing in baculovirus genomes.

Here, we develop a simple, efficient, bacmid-based, selection marker-free method for gene deletion and editing in baculovirus genomes. Specifically, based on pFastbac1, a donor plasmid with long left and right homology arms but without a reporter was constructed for disrupting ie1, an essential baculovirus gene. Instead of ligating with a plasmid, the homology arms were introduced to the polyhedrin locus of BmNPV bacmid using the BmNPV bac-to-bac expression system. Two viruses generated from the modified bacmid and unmodified BmNPV bacmid were then used to co-infect BmN cells in order that recombination takes place at the ie1 locus between them. Finally, without multiple rounds of purification, total cellular DNA was isolated, transformed into Cacl2-treated competent DH10B cells, and then blue colonies were selected for PCR screening. Remarkably, the proportion of blue colonies containing ie1-disrupted bacmid was found to be around 7 %. Moreover, using primers flanking the homology arms further confirmed that all these positive recombinants were double crossovers. These findings indicate that our method is also capable of gene modification if inverse PCR or seamless cloning is used to construct the donor plasmid and sequencing is employed to select positive colonies.

Effects of glyphosate on the growth, development, and physiological functions of silkworm, Bombyx mori.

Glyphosate is an herbicide widely used worldwide, but whether it is safe to nontarget organisms is controversial. In this study, the lepidopteran model insect silkworm was used to investigate the effects of glyphosate residues. The LC50 (72 h) of glyphosate on silkworm was determined to be 14875.98 mg/L, and after exposure to glyphosate at 2975.20 mg/L (a concentration comparable to that used for weed control in mulberry fields), silkworm growth was inhibited by 9.00%, total cocoon weight was lowered by 10.53%, feed digestibility was decreased by 7.56%, and the activities of alpha-amylase and trypsin were reduced by 10.41% and 21.32%, respectively. Pathological analysis revealed that glyphosate exposure led to significantly damaged midgut, along with thinner basal layer, shedding microvilli, blurred cytoplasmic membrane, and appearance of vacuoles. Exposure to glyphosate also led to accumulation of peroxides in the intestinal tissue; the messenger RNA transcription of SOD, Cu/Zn-SOD, and Mn-SOD was all significantly upregulated by glyphosate treatment for 24 h, while CAT transcription was increased at 24, 48, and 72 h. The activity of SOD was increased significantly at 24 h, while significant activity changes were observed for CAT at 72 and 96 h. These results indicated that exposure to glyphosate caused oxidative stress in the midgut of silkworm and affected the midgut's physiological function. This study provides important insights in evaluating the impact of glyphosate residues in the environment on nontarget organisms.

A novel system for the generation of baculoviruses mutant for an essential gene.

BACKGROUND: Currently, to delete an essential gene from a baculovirus genome, a cell line stably expressing the gene to be knocked-out should be first generated, which is time-consuming. Alternatively, essential genes can be deleted in E. coli using the λ Red recombination system, which requires an electroporation system. Here, based on homologous recombination in insect cells, we develop an alternative efficient system that requires neither generation of a cell line nor an electroporation system. METHODS AND RESULTS: Using puc19-based inverse PCR, a transfer vector for deleting BmNPV orf92 (Bm92, an essential gene) was efficiently constructed. A copy of Bm92 was introduced into the polyhedrin locus of BmNPV bacmid. The transfer vector was then co-transfected into BmN cell with the modified bacmid to enable homologous recombination at the Bm92 locus. An agarose-free approach was developed for the purification of Bm92-disrupted bacmid viruses in insect cells. Subsequently, BmN cells were co-infected with purified Bm92-disrupted bacmid viruses and unmodified bacmid viruses to allow recombination at the Tn7 insertion site between the two viruses. Finally, bacmid DNA extracted from BmN cells was transformed into chemically-treated competent DH10B cells, and blue colonies containing Bm92-disrupted bacmid were selected using PCR. CONCLUSIONS: For its efficiency and convenience, the system has great potential to be used for the generation of baculovirus knockout mutants.

Imbalance of intestinal microbial homeostasis caused by acetamiprid is detrimental to resistance to pathogenic bacteria in Bombyx mori.

The neonicotinoid insecticide acetamiprid is widely applied for pest control in agriculture production, and its exposure often results in adverse effects on a non-target insect, Bombyx mori. However, only few studies have investigated the effects of exposure to sublethal doses of neonicotinoid insecticides on gut microbiota and susceptibility to pathogenic bacteria. In this study, we aimed to explore the possible mechanisms underlying the acetamiprid-induced compositional changes in gut microbiota of silkworm and reduced host resistance against detrimental microbes. This study indicated that sublethal dose of acetamiprid activated the dual oxidase-reactive oxygen species (Duox-ROS) system and induced ROS accumulation, leading to dysregulation of intestinal immune signaling pathways. The evenness and structure of bacterial community were altered. Moreover, after 96 h of exposure to sublethal dose of acetamiprid, several bacteria, such as Pseudomonas sp (Biotype A, DOP-1a, XW34) and Staphylococcus sp (RCB1054, RCB314, X302), invaded the silkworm hemolymph. The survival rate and bodyweight of the acetamiprid treated silkworm larvae inoculated with Enterobacter cloacae (E. cloacae) were significantly lower than the acetamiprid treatment group, suggesting that acetamiprid reduced silkworm resistance against pathogens. These findings indicated that acetamiprid disturbed gut microbial homeostasis of Bombyx mori, resulting in changes in gut microbial community and susceptibility to detrimental microbes.

Low concentration acetamiprid-induced oxidative stress hinders the growth and development of silkworm posterior silk glands.

Acetamiprid is a new type of nicotinic insecticide that is widely used in pest control. Its environmental residues may cause silkworm cocooning disorder. In this study, silkworms that received continuous feeding of low concentration acetamiprid (0.15 mg/L) showed significantly decreased silk gland index and cocooning rate. Gene expression profiling of posterior silk glands (PSGs) revealed that the differentially expressed genes were significantly enriched in oxidative stress-related signal pathways with significant up-regulation. The contents of both H2O2 and MDA were increased, along with significantly elevated SOD and CAT activities, all of which reached maximal values at 48 h when H2O2 and MDA's contents were 10.46 and 7.98 nmol/mgprot, respectively, and SOD and CAT activities were 5.51 U/mgprot and 33.48 U/gprot, respectively. The transcription levels of antioxidant enzyme-related genes SOD, Mn-SOD, CuZn-SOD, CAT, TPX and GPX were all up-regulated, indicating that exposure to low concentration acetamiprid led to antioxidant response in silkworm PSG. The key genes in the FoxO/CncC/Keap1 signaling pathway that regulates antioxidant enzyme activity, FoxO, CncC, Keap1, NQO1, HO-1 and sMaf were all up-regulated during the whole process of treatment, with maximal values being reached at 72 h with 2.91, 1.46, 1.82, 2.52, 2.32 and 4.01 times of increases, respectively. These results demonstrate that exposure to low concentration acetamiprid causes oxidative stress in silkworm PSG, which may be the cause of cocooning disorder in silkworm. Our study provides a reference for the safety evaluation of environmental residues of acetamiprid on non-target insects.

Study on the effect of graphene oxide (GO) feeding on silk properties based on segmented precise measurement.

Silk is widely used in the biomedical field (e.g., surgical sutures) for its excellent mechanical properties and biocompatibility. The properties of silk can be further enhanced by a multitude of methods, including nano particle feeding, which is convenient and green. Generally, the filament length of a silkworm cocoon ranges from 1300 to 1700 m. Despite the fact that the filament size, a key factor affecting the mechanical properties of silk, varies along the length, evaluation of strengthened silk by segment and the specific distribution along the length has not been reported. Therefore, in the present study, we fed silkworms with graphene oxide-sprayed mulberry leaves and evaluated the silk properties segment by segment. The silk's strength and elongation were significantly enhanced, with more α-helical/random coils and thicker mesophase regions. Specifically, the silk from 2‰ GO-treated group had higher strength in the first 60% of the length, whereas the silk from 1‰ GO-treated group was stronger in the last 40% of the length. Notably, the silk from 1‰ GO-treated group had the highest strength and Young's modulus in the last 20% of the length, indicating that this segment is more suitable for use as a surgical suture. Our findings demonstrate that different silk segments offer a great range of desirable assets, and the feasibility to select a specific segment with the desired properties for a specific application.

Complete mitochondrial genome of Spilosoma lubricipedum (Noctuoidea: Erebidae) and implications for phylogeny of noctuid insects.

Mitochondrial genomes (mitogenomes) have been widely used for studies on phylogenetic relationships and molecular evolutionary biology. Here, the complete mitogenome sequence of Spilosoma lubricipedum (Noctuoidea: Erebidae: Arctiinae) was determined (total length 15,375 bp) and phylogenetic analyses S. lubricipedum were inferred from available noctuid sequence data. The mitogenome of S. lubricipedum was found to be highly A + T-biased (81.39%) and exhibited negative AT- and GC-skews. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for cox1 with CGA. All tRNAs exhibited typical clover-leaf secondary structures, except for trnS1. The gene order of the S. lubricipedum mitogenome was trnM-trnI-trnQ-nad2. The A + T-rich region of S. lubricipedum contained several conservative features common to noctuid insects. Phylogenetic analysis within Noctuoidea was carried out based on mitochondrial data. Results showed that S. lubricipedum belonged to Erebidae and the Noctuoidea insects could be divided into five well-supported families (Notodontidae + (Erebidae + (Nolidae + (Euteliidae + Noctuidae)))).

Phylogenetic relationships of Limacodidae and insights into the higher phylogeny of Lepidoptera.

To determine the systematic status of family Limacodidae within Lepidoptera, the complete mitochondrial genome (mitogenome) of Thosea sinensis (Lepidoptera: Zygaenoidea: Limacodidae) was sequenced. The genome is 15,544 base pairs (bp), including 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and an AT-rich region. These characteristics are similar to of other lepidopterans. The gene order of T. sinensis is identical to that of Ditrysia lepidopterans. The nucleotide composition of the T. sinensis mitochondrial genome is highly biased toward A + T nucleotides (81.1%) and exhibits negative AT and GC skew. All the other 13 PCGs except cox1 are initiated by ATN codons. All tRNA genes are folded into the typical cloverleaf secondary structure, except for trnS1, which lacked the dihydrouridine (DHU) stem. There are 20 intergenic spacer regions ranging from 1 to 56 bp in length, and two gene overlap regions throughout the entire genome. The AT-rich region includes the ATAGA motif, followed by a 19-bp poly T stretch, a microsatellite-like (AT)10, and a poly-A element. Analysis of phylogenetic relationships indicated that T. sinensis belongs to the Limacodidae, and the monophyly of each lepidopteran family was well supported.

Species-specific size expansion and molecular evolution of the oleosins in angiosperms.

Oleosins are hydrophobic plant proteins thought to be important for the formation of oil bodies, which supply energy for seed germination and subsequent seedling growth. To better understand the evolutionary history and diversity of the oleosin gene family in plants, especially angiosperms, we systematically investigated the molecular evolution of this family using eight representative angiosperm species. A total of 73 oleosin members were identified, with six members in each of four monocot species and a greater but variable number in the four eudicots. A phylogenetic analysis revealed that the angiosperm oleosin genes belonged to three monophyletic lineages. Species-specific gene duplications, caused mainly by segmental duplication, led to the great expansion of oleosin genes and occurred frequently in eudicots after the monocot-eudicot divergence. Functional divergence analyses indicate that significant amino acid site-specific selective constraints acted on the different clades of oleosins. Adaptive evolution analyses demonstrate that oleosin genes were subject to strong purifying selection after their species-specific duplications and that rapid evolution occurred with a high degree of evolutionary dynamics in the pollen-specific oleosin genes. In conclusion, this study serves as a foundation for genome-wide analyses of the oleosins. These findings provide insight into the function and evolution of this gene family in angiosperms and pave the way for studies in other plants.

A novel way to purify recombinant baculoviruses by using bacmid.

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.

Expression of Bombyx mori nucleopolyhedrovirus ORF76 in permissive and non-permissive cell lines by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Open reading frame 76 of Bombyx mori nucleopolyhedrovirus (BmNPV), designated as Bm76, is a gene whose function is completely unknown. With EGFP fused to the 3' terminal of Bm76 as the reporter gene and BmNPV bacmid as the expression vector, a recombinant bacmid was successfully constructed expressing Bm76-EGFP fusion protein under the control of polyhedrin promoter in Bombyx mori cells (Bm cells), BmNPV's permissive cell line, laying the foundation for rescue experiment of Bm76 deletion mutant. Moreover, the supernatant from Bm cells transfected with the recombinant bacmid was used to infect Trichoplusia Ni cells (Tn cells), BmNPV's non-permissive cell line. Unexpectedly, the expression of Bm76-EGFP fusion protein in some Tn cells was detected, implying that viral DNA was replicated in these cells. The causes are being studied for the inability of BmNPV to produce enough viable budded viruses in Tn cells despite of viral DNA replication.