Jeremy F. Fuchs

Description of the Transcriptomes of Immune Response-Activated Hemocytes from the Mosquito Vectors Aedes aegypti and Armigeres subalbatus

ABSTRACT Mosquito-borne diseases, including dengue, malaria, and lymphatic filariasis, exact a devastating toll on global health and economics, killing or debilitating millions every year (54). Mosquito innate immune responses are at the forefront of concerted research efforts aimed at defining potential target genes that could be manipulated to engineer pathogen resistance in vector populations. We aimed to describe the pivotal role that circulating blood cells (called hemocytes) play in immunity by generating a total of 11,952 Aedes aegypti and 12,790 Armigeres subalbatus expressed sequence tag (EST) sequences from immune response-activated hemocyte libraries. These ESTs collapsed into 2,686 and 2,107 EST clusters, respectively. The clusters were used to adapt the web-based interface for annotating bacterial genomes called A Systematic Annotation Package for Community Analysis of Genomes (ASAP) for analysis of ESTs. Each cluster was categorically characterized and annotated in ASAP based on sequence similarity to five sequence databases. The sequence data and annotations can be viewed in ASAP at https://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm . The data presented here represent the results of the first high-throughput in vivo analysis of the transcriptome of immunocytes from an invertebrate. Among the sequences are those for numerous immunity-related genes, many of which parallel those employed in vertebrate innate immunity, that have never been described for these mosquitoes.

Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers.

Mosquitoes vector pathogens. One aspect that has been overlooked in mosquito–pathogen relationships is the effect of host age on immune competence. Here, we show that there is age‐associated mortality following immune challenge with Escherichia coli. This mortality correlates with a decrease in haemocyte numbers (blood cells) and a decreased ability to kill E. coli. Although the number of haemocytes decreases, the available haemocytes retain their phagocytic ability regardless of age, and we estimate that individual granulocytes can phagocytose approximately 1500 E. coli. Moreover, transcription profiles for cecropin, defensin and gambicin in E. coli challenged mosquitoes do not change with age, indicating that the increased susceptibility is not attributed to fewer humoral antimicrobial peptides. These results suggest that a contributing factor for the age‐associated mortality is the decrease in circulating haemocytes, which reduces the overall phagocytic capacity of mosquitoes. To our knowledge, this is the first report detailing an age‐associated decline in the immunological capabilities of mosquitoes following challenge with an infectious agent. These data also call for caution in the analysis and interpretation of experimental results when mosquito age has not been closely monitored. Lastly, a model for haemocyte function is presented.

Reassessing the role of defensin in the innate immune response of the mosquito, Aedes aegypti.

Defensin is the predominant inducible immune peptide in Aedes aegypti. In spite of its activity against Gram‐positive bacteria in vitro, defensin expression is detected in mosquitoes inoculated with Gram‐positive or negative bacteria, or with filarial worms. Defensin transcription and expression are dependent upon bacterial dose; however, translation is inconsistent with transcription because peptide is detectable only in mosquitoes inoculated with large doses. In vitro translation assays provide further evidence for post‐transcriptional regulation of defensin. Clearance assays show that a majority of bacteria are cleared before defensin is detected. In gene silencing experiments, no significant difference in mortality was observed between defensin‐deficient and control mosquitoes after bacteria inoculation. These studies suggest that defensin may have an alternative function in mosquito immunity.

A novel lectin with a fibrinogen-like domain and its potential involvement in the innate immune response of Armigeres subalbatus against bacteria

Mosquitoes have an efficient cellular innate immune response that includes phagocytosis of microbial pathogens and encapsulation of metozoan parasites. In this study, we describe a novel lectin in the mosquito, Armigeres subalbatus (aslectin or AL‐1). The 1.27 kb cDNA clone for the AL‐1 gene (AL‐1) encodes a 279 deduced amino acid sequence that contains a C‐terminal fibrinogen‐like domain. AL‐1 is transcribed in all life stages. AL‐1 mainly exists in the haemolymph of adult female mosquitoes, and is upregulated following both Escherichia coli and Micrococcus luteus challenge. AL‐1 specifically recognizes N‐acetyl‐d‐glucosamine and is able to bind both E. coli and M. luteus. These results suggest that AL‐1 might function as a pattern recognition receptor in the immune response in Ar. subalbatus.

Evaluation of the Function of a Type I Peritrophic Matrix as a Physical Barrier for Midgut Epithelium Invasion by Mosquito-Borne Pathogens in Aedes aegypti

In addition to modulating blood meal digestion and protecting the midgut epithelial cells from mechanical and chemical damage, a biological function attributed to the mosquito type I peritrophic matrix (PM) is preventing or reducing pathogen invasion, especially from Plasmodium spp. Previously, we demonstrated that chitin is an essential component of the PM and is synthesized de novo in response to blood feeding in Aedes aegypti. Therefore, knocking down chitin synthase expression by RNA interference severely disrupts formation of the PM. Utilizing this artificial manipulation, we determined that the absence of the PM has no effect on the development of Brugia pahangi or on the dissemination of dengue virus. However, infectivity of Plasmodium gallinaceum is lower, as measured by oocyst intensity, when the PM is absent. Our findings also suggest that the PM seems to localize proteolytic enzymes along the periphery of the blood bolus during the first 24 hours after blood feeding. Finally, the absence of the PM does not affect reproductive fitness, as measured by the number and viability of eggs oviposited.

Replication of Flock House Virus in Three Genera of Medically Important Insects

Abstract Flock House Virus (family Nodaviridae, genus Alphanodavirus, FHV) was originally isolated from grass grubs Costelytra zealandica (White) (Coleoptera: Scarabaeidae) in New Zealand and belongs to a family of divided genome, plus-sense RNA insect viruses. FHV replicates in insects, a nematode, plants, and yeast. We previously reported replication of FHV in four genera of mosquitoes and expression of green fluorescent protein in Aedes aegypti (L.) produced by an FHV-based vector. We report here that FHV multiplies vigorously in vivo in the malaria vectors Anopheles gambiae Giles and An. stephensi Liston and in vitro in a cell line derived from An. gambiae. In addition, FHV multiplies extensively in two other medically important insects, the tsetse fly, Glossina morsitans morsitans Westwood, and the reduviid bug Rhodnius prolixus Stal, extending its host range to four orders of insects (Coleoptera, Lepidoptera, Diptera, and Hemiptera). The virus disseminates in all the major tissues of the insects studied. Anopheles and Glossina show mortality when FHV is injected at a dose above 104 plaque-forming units (pfu) or the virus accumulates to titer above 108 pfu. A lower dose (103 pfu) promotes more extensive virus multiplication and reduces mortality to <10%. No adverse effects are observed in Ae. aegypti, Culex pipiens pipiens L., and Armigeres subalbatus (Coquillett), when injected with a dose of up to 107 pfu. Mosquitoes orally fed with FHV exhibited slower virus growth rate with lower mortality. Our results indicate that FHV has uniquely broad insect host range and that the virus can be used to study virus host interactions in a variety of medically important insects.

Mosquito transcriptome changes and filarial worm resistance in Armigeres subalbatus

BackgroundArmigeres subalbatus is a natural vector of the filarial worm Brugia pahangi, but it rapidly and proficiently kills Brugia malayi microfilariae by melanotic encapsulation. Because B. malayi and B. pahangi are morphologically and biologically similar, the Armigeres-Brugia system serves as a valuable model for studying the resistance mechanisms in mosquito vectors. We have initiated transcriptome profiling studies in Ar. subalbatus to identify molecular components involved in B. malayi refractoriness.ResultsThese initial studies assessed the transcriptional response of Ar. subalbatus to B. malayi at 1, 3, 6, 12, 24, 48, and 72 hrs after an infective blood feed. In this investigation, we initiated the first holistic study conducted on the anti-filarial worm immune response in order to effectively explore the functional roles of immune-response genes following a natural exposure to the parasite. Studies assessing the transcriptional response revealed the involvement of unknown and conserved unknowns, cytoskeletal and structural components, and stress and immune responsive factors. The data show that the anti-filarial worm immune response by Ar. subalbatus to be a highly complex, tissue-specific process involving varied effector responses working in concert with blood cell-mediated melanization.ConclusionThis initial study provides a foundation and direction for future studies, which will more fully dissect the nature of the anti-filarial worm immune response in this mosquito-parasite system. The study also argues for continued studies with RNA generated from both hemocytes and whole bodies to fully expound the nature of the anti-filarial worm immune response.

Transcriptome Changes in Culex quinquefasciatus (Diptera: Culicidae) Salivary Glands During West Nile Virus Infection

ABSTRACT Persistent West Nile virus (WNV) infection in the mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is associated with pathological changes in the salivary glands, including apoptotic cell death and a corresponding reduction in virus transmission over time. The vector host response to WNV infection and the molecular basis of WNV pathogenesis in Cx. quinquefasciatus was investigated using oligonucleotide microarrays designed to detect differences in the salivary gland transcriptome between WNV-infected mosquitoes and uninfected controls. Transcripts with increased abundance in infected salivary glands included those related to immunity, transcription, protein transport and degradation, amino acid and nucleotide metabolism, signal transduction, and cellular detoxification. Microarray-based analysis detected a decrease in transcript levels of a Culex inhibitor of apoptosis gene (IAP-1) and a decrease in abundance of 11 transcripts encoding salivary gland proteins, Transcript levels for an endonuclease, a proline-rich mucin, and several D7 protein family members also decreased. Transcripts with the greatest change in abundance during infection had either no similarity to sequences found in GenBank, VectorBase, and FlyBase, or were similar to sequences with uncharacterized protein products. These transcripts represent exciting targets for future analysis. Results from this study suggest that WNV infection influences transcriptional changes in an invertebrate host target tissue that may confer an advantage to the replicating virus, induce a host defense response, and alter the composition of vector saliva. The ramifications of these changes are discussed in terms of mosquito vector competence and WNV pathogenesis.

Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti

Pathogens that infect and/or are transmitted by mosquitoes typically are exposed to the body cavity, and to haemocytes circulating therein, during development or dissemination. Aedes aegypti haemocytes produce a range of immune response‐related gene products, and an endpoint response of phagocytosis and/or melanization that is temporally and structurally distinct for the invading pathogen. Expressed sequence tags were generated from haemocyte libraries and then used to design oligonucleotide microarrays. Arrays were screened with haemocyte material collected 1‐, 8‐ and 24‐h post‐inoculation with Escherichia coli or Micrococcus luteus bacteria. Data from these studies support the discovery of novel immune response‐activated genes, provide an expanded understanding of antimicrobial peptide biology and highlight the coordination of immune factors that leads to an endpoint response.

Mosquito Transcriptome Profiles and Filarial Worm Susceptibility in Armigeres subalbatus

Background Armigeres subalbatus is a natural vector of the filarial worm Brugia pahangi, but it kills Brugia malayi microfilariae by melanotic encapsulation. Because B. malayi and B. pahangi are morphologically and biologically similar, comparing Ar. subalbatus-B. pahangi susceptibility and Ar. subalbatus-B. malayi refractoriness could provide significant insight into recognition mechanisms required to mount an effective anti-filarial worm immune response in the mosquito, as well as provide considerable detail into the molecular components involved in vector competence. Previously, we assessed the transcriptional response of Ar. subalbatus to B. malayi, and now we report transcriptome profiling studies of Ar. subalbatus in relation to filarial worm infection to provide information on the molecular components involved in B. pahangi susceptibility. Methodology/Principal Findings Utilizing microarrays, comparisons were made between mosquitoes exposed to B. pahangi, B. malayi, and uninfected bloodmeals. The time course chosen facilitated an examination of key events in the development of the parasite, beginning with the very start of filarial worm infection and spanning to well after parasites had developed to the infective stage in the mosquito. At 1, 3, 6, 12, 24 h post infection and 2–3, 5–6, 8–9, and 13–14 days post challenge there were 31, 75, 113, 76, 54, 5, 3, 13, and 2 detectable transcripts, respectively, with significant differences in transcript abundance (increase or decrease) as a result of parasite development. Conclusions/Significance Herein, we demonstrate that filarial worm susceptibility in a laboratory strain of the natural vector Ar. subalbatus involves many factors of both known and unknown function that most likely are associated with filarial worm penetration through the midgut, invasion into thoracic muscle cells, and maintenance of homeostasis in the hemolymph environment. The data show that there are distinct and separate transcriptional patterns associated with filarial worm susceptibility as compared to refractoriness, and that an infection response in Ar. subalbatus can differ significantly from that observed in Ae. aegypti, a common laboratory model.