Trial and error: New insights into recombinant expression of membrane-bound insect cytochromes P450 in Escherichia coli systems.

Insect cytochromes P450 (CYP450s) are key enzymes responsible for a wide array of oxidative transformations of both endogenous and exogenous substrates. However, there is currently no a universal guideline established for heterologous expression of membrane-bound CYP450s, which hampers their downstream biochemical and structural studies. In this study, we conducted large-scale screening of protein overexpression in Escherichia coli using 71 insect CYP450 sequences and optimized the expression of a difficult-to-express CYP450 (CYP6HX3) using eight different optimizations, including selection of host strains and expression vectors, alternative of leader signal peptides, and N-terminal modifications. We confirmed that 1) Only insect CYP450s belonging to the CYP347 family could be expressed with N-terminal fusion of ompA2+ signal peptide in E. coli expression system. 2) E. coli Lemo 21 (DE3) effectively improved the expression of CYP6HX3 in the plasma membrane. 3) A brick-red appearance occurred frequently in the expressed thallus or membrane proteins, but this phenomenon could not necessarily indicate successful overexpression of target CYP450s. These findings provide new insights into the recombinant expression of insect CYP450s in E. coli systems and will facilitate the theoretical approaches for functional expression and production of eukaryotic CYP450s.

Heat stress-induced oviposition behavioral change correlates with sperm damage in the pine sawyer beetle, Monochamus alternatus.

BACKGROUND: Global climate change is causing an increase in extreme high temperatures (EHTs), which subject insects to unprecedented stress. Behavior plasticity in response to EHTs, particularly oviposition behavior, is important for the persistence and outbreak of insect populations. Investigating the plasticity of oviposition behavior and its underlying mechanisms has theoretical importance to pest management, but knowledge gaps still remain. RESULTS: Herein, we characterized the reproductive traits of Monochamus alternatus, a dominant insect vector of the destructive pine wilt disease, including oviposition behavioral patterns, fecundity, offspring fitness and sperm viability, under simulated heatwave conditions in the laboratory. The results showed that (i) EHTs induced a novel oviposition behavior, whereby females deposited multiple eggs into a single groove rather than laying one egg per groove under normal condition; (ii) EHTs exerted stage- and sex-specific effects on fecundity, offspring fitness and sperm viability; and (iii) there was a significant correlation between frequency of the novel oviposition strategy and sperm viability. CONCLUSION: We hypothesized that this beetle pest has the ability to flexibly shift towards a low-cost oviposition strategy to counteract the fitness costs caused by heat stress. Taken together, these findings provide a theoretical foundation for personalized pest management strategies in the context of climate change. © 2024 Society of Chemical Industry.

A gut-isolated Enterococcus strain (HcM7) triggers the expression of antimicrobial peptides that aid resistance to nucleopolyhedrovirus infection of Hyphantria cunea larvae.

BACKGROUND: Commensal microorganisms are widely distributed in insect gut tissues and play important roles in host nutrition, metabolism, reproductive regulation, and especially immune functioning and tolerance to pathogens. Consequently, gut microbiota represent a promising resource for the development of microbial-based products for pest control and management. However, the interactions among host immunity, entomopathogen infections, and gut microbiota remain poorly understood for many arthropod pests. RESULTS: We previously isolated an Enterococcus strain (HcM7) from Hyphantria cunea larvae guts that increased the survival rates of larvae challenged with nucleopolyhedrovirus (NPV). Here, we further investigated whether this Enterococcus strain stimulates a protective immune response against NPV proliferation. Infection bioassays demonstrated that re-introduction of the HcM7 strain to germfree larvae preactivated the expression of several antimicrobial peptides (particularly H. cunea gloverin 1, HcGlv1), resulting in the significant repression of virus replication in host guts and hemolymph, and consequently improved host survivorship after NPV infection. Furthermore, silencing of the HcGlv1 gene by RNA interference markedly enhanced the deleterious effects of NPV infection, revealing a role of this gut symbiont-induced gene in host defenses against pathogenic infections. CONCLUSION: These results show that some gut microorganisms can stimulate host immune systems, thereby contributing to resistance to entomopathogens. Furthermore, HcM7, as a functional symbiotic bacteria of H. cunea larvae, may be a potential target for increasing the effectiveness of biocontrol agents against this devastating pest. © 2023 Society of Chemical Industry.

Isolation and characterization of gut bacteria associated with the degradation of host-specific terpenoids in Pagiophloeus tsushimanus (Coleoptera: Curculionidae) larvae.

Insect intestinal bacteria play an important role in resisting defensive substances of host plants. Pagiophloeus tsushimanus (Coleoptera: Curculionidae) feeds exclusively on camphor trees (Cinnamomum camphora, Laurales: Lauraceae) in China, causing substantial economic and ecological losses. It is unclear how the larvae of P. tsushimanus outcome the main secondary metabolites of C. camphora such as D-camphor, eucalyptol, and linalool. In this study, we isolated terpenoid-degrading bacteria from the gut of P. tsushimanus larvae by using selective culture medium. Maximum likelihood phylogenetic analyses were performed with 16S rDNA sequences to identify the bacteria, and results showed ten strains belonged to four genera, including Pseudomonas, Enterobacter, Serratia, and Corynebacterium. Then, gas chromatography was employed to determine the degradability of D-camphor, eucalyptol, and linalool by the isolated strains, results showed that Z5 strain (i.e., Corynebacterium variabile, Actinomycetales: Corynebacteriaceae), F1 strain (i.e., Pseudomonas aeruginosa, Pseudomonadales: Pseudomonaceae), and A3 strain (i.e., Serratia marcescens, Enterobacterales: Enterobacteriaceae) had the highest degradation rates of D-camphor, linalool, and eucalyptol, respectively. The intestinal bacteria were capable of terpenoid degradation in vitro, which suggested that these gut bacteria associated with P. tsushimanus play an important role in overcoming host plant secondary metabolite defense, thereby facilitating the host specialization of this pest.

Tolerance to dietary linalool primarily involves co-expression of cytochrome P450s and cuticular proteins in Pagiophloeus tsushimanus (Coleoptera: Curculionidae) larvae using SMRT sequencing and RNA-seq.

BACKGROUND: Pagiophloeus tsushimanus (Coleoptera: Curculionidae), an emerging forest pest exclusively infesting camphor trees, has recently caused severe ecological and economic damage in localized areas in China. Its population outbreak depends largely on the capacity to overcome the pressure of terpenoid-derived metabolites (e.g. linalool) from camphor trees. At present, the molecular basis of physiological adaptation of P. tsushimanus to dietary linalool is poorly understood, and there is no available reference genome or transcriptome. RESULTS: Herein, we constructed the transcriptome profiling of P. tsushimanus larvae reared on linalool-infused diets using RNA sequencing and single-molecule real-time sequencing. A total of 20,325 high-quality full-length transcripts were identified as a reference transcriptome, of which 14,492 protein-coding transcripts including 130 transcription factors (TFs), and 5561 long non-coding RNAs (lncRNAs) were detected. Also, 30 alternative splicing events and 8049 simple sequence repeats were captured. Gene ontology enrichment of differential expressed transcripts revealed that overall up-regulation of both cytochrome P450s (CYP450s) and cuticular proteins (CPs), was the primary response characteristic against dietary linalool. Other physiological effects possibly caused by linalool exposure, such as increase in Reactive Oxygen Species (ROS) and hormetic stimulation, were compensated by a handful of induced genes encoding antioxidases, heat shock proteins (HSPs), juvenile hormone (JH) epoxide hydrolases, and digestive enzymes. Additionally, based on co-expression networks analysis, a diverse array of hub lncRNAs and TFs co-expressed with CYP450s and CPs were screened as the potential gene regulators. Temporal expression of candidate transcripts determined by quantitative real-time PCR also indicated a cooperative relationship between the inductions of CYP450s and CPs upon exposure to linalool. CONCLUSIONS: Our present study provides an important transcriptome resource of P. tsushimanus, and lays a valuable foundation for understanding how this specialist pest copes with chemical challenges in its specific host environments.

A heat shock 70kDa protein MaltHSP70-2 contributes to thermal resistance in Monochamus alternatus (Coleoptera: Cerambycidae): quantification, localization, and functional analysis.

BACKGROUND: Heat Shock Proteins 70 (HSP70s) in insects act on a diverse range of substrates to assist with overcoming extreme high temperatures. MaltHSP70-2, a member of HSP70s, has been characterized to involve in the thermotolerance of Monochamus alternatus in vitro, while quantification and localization of MaltHSP70-2 in various tissues and its functional analysis in vivo remain unclear. RESULTS: In this study, temporal expression of MaltHSP70-2 indicated a long-last inductive effect on MaltHSP70-2 expression maintained 48 hours after heat shock. MaltHSP70-2 showed a global response to heat exposure which occurring in various tissues of both males and females. Particularly in the reproductive tissues, we further performed the quantification and localization of MaltHSP70-2 protein using Western Blot and Immunohistochemistry, suggesting that enriched MaltHSP70-2 in the testis (specifically in the primary spermatocyte) must be indispensable to protect the reproductive activities (e.g., spermatogenesis) against high temperatures. Furthermore, silencing MaltHSP70-2 markedly influenced the expression of other HSP genes and thermotolerance of adults in bioassays, which implied a possible interaction of MaltHSP70-2 with other HSP genes and its role in thermal resistance of M. alternatus adults. CONCLUSIONS: These findings shed new insights into thermo-resistant mechanism of M. alternatus to cope with global warming from the perspective of HSP70s functions.

Comparative Transcriptome Analysis Reveals Molecular Insights in Overwintering Monochamus alternatus (Coleoptera: Cerambycidae).

Monochamus alternatus, the dominant vector of Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae), has caused immense damage to forest resources. In China, this vector was native to the southern regions but has spread northward recently. To adapt to more challenging environments in the northern winter, M. alternatus has evolved an intricate strategy for overwintering, which remains largely unknown. Herein, we compared the transcriptome data of the overwintering and non-overwintering larvae of M. alternatus larvae to investigate the molecular mechanisms in overwintering. A total of 53.10 GB clean bases and 28, 245 unigenes were obtained by RNA-seq. Analysis of 2597 upregulated and 2429 downregulated unigenes, as well as the enrichment of DEGs showed that many genes and pathways were jointly involved in the overwintering period. Besides, the accuracy of the RNA-seq data was tested by using qPCR experiment involving 13 selected genes. The results revealed that the overwintering process relied largely on the energy allocation trade-off. Specifically, overwintering M. alternatus inhibited energy-intensive activities, such as growth and molting, detoxification, and trehalose transport, and the reserved energy was skewed towards the synthesis of antifreeze compounds and immune response to cope with the deleterious effects of winter.

Insights into chemosensory genes of Pagiophloeus tsushimanus adults using transcriptome and qRT-PCR analysis.

Pagiophloeus tsushimanus is a new, destructive, and monophagous weevil pest that thrives on Cinnamomum camphora, found in Shanghai. The functions of chemosensory genes involved in the host location and intraspecific communication of P. tsushimanus remain unknown. The male-female transcriptomes of P. tsushimanus adults were assembled using Illumina sequencing, and we focused on all chemosensory genes in transcriptomes. In general, 58,088 unigenes with a mean length of 1018.19 bp were obtained. In total, 39 odorant binding proteins (OBPs), 10 chemosensory proteins (CSPs), 22 olfactory receptors (ORs), 16 gustatory receptors (GRs), eight ionotropic receptors (IRs), and five sensory neuron membrane proteins (SNMPs) were identified. PtsuOBPs comprised four subfamilies (20 Minus-C, one Plus-C, two Dimer, and 15 Classic). Both PtsuOBPs and PtsuCSPs contained a highly conserved sequence motif of cysteine residues. PtsuORs including one olfactory receptor co-receptors (Ptsu/Orco) comprised seven predicted transmembrane domains. Phylogenetic analysis revealed that PtsuOBPs, PtsuCSPs, and PtsuORs in P. tsushimanus exhibited low homology compared to other insect species. The results of tissue- and sex-specific expression patterns indicated that PtsuOBPs and PtsuORs were highly abundant in the antennae; whereas, PtsuCSPs were not only highly abundant in antennae, but also abdominal apexes, wings, and legs. In conclusion, these results enrich the gene database of P. tsushimanus, which may serve as a basis for identifying novel targets to disrupt olfactory key genes and may provide a reverse validation method to identify attractants for formulating potential eco-friendly control strategies for this pest.

Characterization, expression profiling, and thermal tolerance analysis of heat shock protein 70 in pine sawyer beetle, Monochamus alternatus hope (Coleoptera: Cerambycidae).

Monochamus alternatus Hope (Coleoptera: Cerambycidae) warrants attention as a dominant transmission vector of the pinewood nematode, and it exhibits tolerance to high temperature. Heat shock protein 70 (HSP70) family members, including inducible HSP70 and heat shock cognate protein 70 (HSC70), are major contributors to the molecular chaperone networks of insects under heat stress. In this regard, we specifically cloned and characterized three MaltHSP70s and three MaltHSC70s. Bioinformatics analysis on the deduced amino acid sequences showed these genes, having close genetic relationships with HSP70s of Coleopteran species, collectively shared conserved signature structures and ATPase domains. Subcellular localization prediction revealed the HSP70s of M. alternatus were located not only in the cytoplasm and endoplasmic reticulum but also in the nucleus and mitochondria. The transcript levels of MaltHSP70s and MaltHSC70s in each state were significantly upregulated by exposure to 35-50°C for early 3 h, while MaltHSP70s reached a peak after exposure to 45°C for 2-3 h in contrast to less-upregulated MaltHSC70s. In terms of MaltHSP70s, the expression threshold in females was lower than that in males. Also, both fat bodies and Malpighian tubules were the tissues most sensitive to heat stress in M. alternatus larvae. Lastly, the ATPase activity of recombinant MaltHSP70-2 in vitro remained stable at 25-40°C, and this recombinant availably enhanced the thermotolerance of Escherichia coli. Overall, our findings unraveled HSP70s might be the intrinsic mediators of the strong heat tolerance of M. alternatus due to their stabilized structure and bioactivity.