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1. A Tale of Two Transcriptomic Responses in Agricultural Pests via Host Defenses and Viral Replication

   https://doi.org/10.3390/ijms22073568  

   Pantha, Pramod; Chalivendra, Subbaiah; Oh, Dong-Ha; Elderd, Bret D.; Dassanayake, Maheshi (April 2021, International Journal of Molecular Sciences)

   Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus that causes systemic infections in many arthropod pests. The specific molecular processes underlying the biocidal activity of AcMNPV on its insect hosts are largely unknown. We describe the transcriptional responses in two major pests, Spodoptera frugiperda (fall armyworm) and Trichoplusia ni (cabbage looper), to determine the host–pathogen responses during systemic infection, concurrently with the viral response to the host. We assembled species-specific transcriptomes of the hemolymph to identify host transcriptional responses during systemic infection and assessed the viral transcript abundance in infected hemolymph from both species. We found transcriptional suppression of chitin metabolism and tracheal development in infected hosts. Synergistic transcriptional support was observed to suggest suppression of immune responses and induction of oxidative stress indicating disease progression in the host. The entire AcMNPV core genome was expressed in the infected host hemolymph with a proportional high abundance detected for viral transcripts associated with replication, structure, and movement. Interestingly, several of the host genes that were targeted by AcMNPV as revealed by our study are also targets of chemical insecticides currently used commercially to control arthropod pests. Our results reveal an extensive overlap between biological processes represented by transcriptional responses in both hosts, as well as convergence on highly abundant viral genes expressed in the two hosts, providing an overview of the host–pathogen transcriptomic landscape during systemic infection. 

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2. A single-cell atlas of the Culex tarsalis midgut during West Nile virus infection

   https://doi.org/10.1371/journal.ppat.1012855  

   Fitzmeyer, Emily A; Dutt, Taru S; Pinaud, Silvain; Graham, Barb; Gallichotte, Emily N; Hill, Jessica L; Campbell, Corey L; Ogg, Hunter; Howick, Virginia; Lawniczak, Mara_K N; et al (January 2025, PLOS Pathogens)

   Coffey, Lark L (Ed.)

   The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and virus infection dynamics are scarce, and inCulex tarsalis—an extremely efficient vector of West Nile virus (WNV)—nonexistent. We performed single-cell RNA sequencing onCx. tarsalismidguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprising 8 distinct cell types, consistent with existing descriptions ofDrosophilaandAedes aegyptimidgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells (EE), suggesting enhanced replication in this population. In contrast, proliferating intestinal stem cells (ISC) had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in infection control. ISCs were also found to have a strong transcriptional response to WNV infection; genes involved in ribosome structure and biogenesis, and translation were significantly downregulated in WNV-infected ISC populations. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g.,AGO2,R2D2, etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA load in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish aCx. tarsalismidgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level. 

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3. Transcriptional responses of the Trichoplusia ni midgut to oral infection by the baculovirus Autographa californica Multiple Nucleopolyhedrovirus

   https://doi.org/10.1128/JVI.00353-19  

   Shrestha, Anita; Bao, Kan; Chen, Wenbo; Wang, Ping; Fei, Zhangjun; Blissard, Gary W. (May 2019, Journal of Virology)

   Baculoviruses are large dsDNA viruses that are virulent pathogens of certain insect species. In a natural host, Trichoplusia ni, infection by the model baculovirus Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) begins when the occluded form of the virus disassembles in the midgut and virions infect midgut epithelial cells to establish the primary phase of the infection. To better understand the primary phase of the AcMNPV infection cycle, newly molted 5 th instar T. ni larvae were orally infected with AcMNPV occlusion bodies and transcriptional responses of the T. ni midgut were analyzed at various times from 0-72 hours post infection, using RNA-Seq analysis and a T. ni reference genome. The numbers of differentially expressed host genes increased as the infection progressed, and we identified a total of 3,372 differentially expressed T. ni transcripts in the AcMNPV-infected midgut. Genes encoding orthologs of HMG176, atlastin, and CPH43 were among the most dramatically upregulated in response to AcMNPV infection. A number of cytochrome P450 genes were downregulated in response to infection. We also identified the effects of AcMNPV infection on a large variety of genes associated with innate immunity. This analysis provides an abundance of new and detailed information on host responses to baculovirus infection during the primary phase of the infection in the midgut, and will be important for understanding how baculoviruses establish productive infections in the organism. IMPORTANCE Baculoviruses are virulent pathogens of a number of important insect pest species. In the host Trichoplusia ni , infection begins in the midgut when infectious virions of the occulsion derived virus (ODV) phenotype enter and subsequently replicate in cells of the midgut epithelium. A second virion phenotype (budded virus or BV) is produced there and BV mediates systemic infection of the animal. Most prior detailed studies of baculovirus infections have focused on BV infections of cultured cells. In this study, we examined the transcriptional responses of the T. ni midgut to infection by ODV of the baculovirus AcMNPV, and identified a variety of host genes that respond dramatically to viral infection. Understanding transcriptional responses of the host midgut to viral infection is critically important for understanding the biphasic infection in the animal as a whole. 

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4. Global Analysis of Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Gene Expression in the Midgut of the Lepidopteran Host Trichoplusia ni

   https://doi.org/10.1128/JVI.01277-18  

   Shrestha, Anita; Bao, Kan; Chen, Yun-Ru; Chen, Wenbo; Wang, Ping; Fei, Zhangjun; Blissard, Gary W.; Shisler, Joanna L. (December 2018, Journal of Virology)

   ABSTRACT The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ∼156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes ( p6.9 , orf76 , orf75 , pp31 , Ac-bro , odv-e25 , and odv-ec27 ) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies ( polh and p10 ) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production ( fp-25k ), acceleration of systemic infection ( v-fgf ), and enhancement of viral movement ( arif-1/orf20 ). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut. IMPORTANCE Baculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion-derived virus (ODV) virions and a secondary phase in which other tissues are infected by budded-virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut in Trichoplusia ni and compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to (i) overcome or avoid host defenses in the gut and (ii) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection. 

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5. Culex pipiens and Culex restuans egg rafts harbor diverse bacterial communities compared to their midgut tissues

   https://doi.org/10.1186/s13071-020-04408-4  

   Juma, Elijah O.; Kim, Chang-Hyun; Dunlap, Christopher; Allan, Brian F.; Stone, Chris M. (December 2020, Parasites & Vectors)

   null (Ed.)

   Abstract Background The bacterial communities associated with mosquito eggs are an essential component of the mosquito microbiota, yet there are few studies characterizing and comparing the microbiota of mosquito eggs to other host tissues. Methods We sampled gravid female Culex pipiens L. and Culex restuans Theobald from the field, allowed them to oviposit in the laboratory, and characterized the bacterial communities associated with their egg rafts and midguts for comparison through MiSeq sequencing of the 16S rRNA gene. Results Bacterial richness was higher in egg rafts than in midguts for both species, and higher in Cx pipiens than Cx. restuans . The midgut samples of Cx. pipiens and Cx. restuans were dominated by Providencia. Culex pipiens and Cx. restuans egg rafts samples were dominated by Ralstonia and Novosphingobium , respectively. NMDS ordination based on Bray-Curtis distance matrix revealed that egg-raft samples, or midgut tissues harbored similar bacterial communities regardless of the mosquito species. Within each mosquito species, there was a distinct clustering of bacterial communities between egg raft and midgut tissues. Conclusion These findings expand the list of described bacterial communities associated with Cx. pipiens and Cx. restuan s and the additional characterization of the egg raft bacterial communities facilitates comparative analysis of mosquito host tissues, providing a basis for future studies seeking to understand any functional role of the bacterial communities in mosquito biology. 

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