Thomas A. Rocheleau

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.

Cloning, sequencing and functional expression of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti

A degenerate PCR strategy was used to isolate a fragment of the acetylcholinesterase gene (Ace) homolog from Aedes aegypti and screen for a cDNA clone containing the complete open reading frame of the gene. The predicted amino acid sequence of the Aedes gene shares 64% identify with Ace from Drosophila and 87% identity with the acetylcholinesterase gene from another mosquito species Anopheles stephensi. High levels of expression of the Aedes gene were achieved by infection of Sf21 cells with a recombinant baculovirus containing the Aedes Ace cDNA. The catalytic properties and sensitivity of the recombinant enzyme to insecticide inhibition are described and discussed in relation to the role of insensitive AChE in conferring resistance to organophosphorus and carbamate insecticides.

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.

Expression of a Drosophila GABA receptor in a baculovirus insect cell system : functional expression of insecticide susceptible and resistant GABA receptors from the cyclodiene resistance gene Rdl

Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed. Spodoptera frugiperda (Sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). Western blotting of cell membrane extracts and immunoprecipitation experiments with an anti‐Rdl antiserum recognized a protein of the expected size of ~65 kDa. Whole cell patch clamp analysis of cells infected with either splice form revealed functional expression of GABA gated chloride ion channels which were blocked by application of 1 μM picrotoxinin. Following replacement of alanine 302 with a serine, a mutation associated with resistance to picrotoxinin and cyclodiene insecticides, mutant channels showed similar levels of insensitivity to picrotoxinin (~ 100‐fold) as those observed in recordings from cultured Drosophila neurons. The significance of the expression of an insect GABA receptor in an insect cell line and the similarity of the results from these functional expression studies to recordings from cultured neurons is discussed.

A potential role for phenylalanine hydroxylase in mosquito immune responses.

In mosquitoes the melanotic encapsulation immune response is an important resistance mechanism against filarial worms and malaria parasites. The rate limiting substrate for melanin production is tyrosine that is hydroxylated by phenoloxidase (PO) to produce 3, 4-dihydroxyphenylalanine. The single pathway for endogenous production of tyrosine is by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH). In this study we describe a potential role for PAH in melanotic immune responses in the yellow fever mosquito, Aedes aegypti. A 1.6 kb A. aegypti PAH cDNA, encoding a 51 kDa protein, was isolated and subsequently expressed in an Escherichia coli expression system. In developing mosquitoes, PAH transcript is present in all stages and it is differentially expressed in adult tissues. Following an immune-challenge with Dirofilaria immitis microfilariae (mf) or bacteria, PAH transcript is up-regulated in hemocytes. Likewise, western analysis of hemocytes collected from immune-activated mosquitoes show an increase in gene product over control samples. Like PO, ultrastructure observations provide verification that PAH is located in oenocytoid and granulocyte hemocytes. Our results offer the first data that suggest PAH is used in mosquito melanin synthesis and defense responses.

Heat shock protein 27 stimulates recovery of RNA and protein synthesis following a heat shock

Constitutive expression of human hsp27 resulted in a 100‐fold increase in survival to a single lethal heat shock in CHO cells without effecting the development of thermotolerance. A possible mechanism for the thermoprotective function of hsp27 may be increased recovery of protein synthesis and RNA synthesis following a heat shock. A lethal heat shock (44°C, 30 min) results in a 90% reduction in the rate of protein synthesis in non‐tolerant cells. Control transfected cells recovered protein synthesis to a pre‐heat shock rate 10 h after the heat shock; while cell lines that constitutively express human hsp27 recovered 6 h after the heat shock. Thermotolerant cells had a 50% reduction in protein synthesis, which recovered within 7 h following the heat shock. The same lethal heat shock (44°C, 30 min) reduced RNA synthesis by 60% in the transfected cell lines, with the controls recovering in 7 h; while the hsp27 expressing cell lines recovered within 5 h. Thermotolerant cells had a 40% reduction in RNA synthesis and were able to recover within 4 h. The enhanced ability of hsp27 to facilitate recovery of protein synthesis and RNA synthesis following a heat shock may provide the cell with a survival advantage. J. Cell. Biochem. 66:153–164, 1997.

Drosophila Cyclodiene Resistance Gene Shows Conserved Genomic Organization with Vertebrate γ‐Aminobutyric AcidA Receptors

Abstract: Genomic clones from the Rdl locus of Drosophila, whose mutant phenotype is resistant to cyclodiene insecticides and picrotoxin, were characterized by restriction mapping and partial sequencing to determine intron/exon structure. The coding region of the gene comprises nine identified exons and spans >25 kb of genomic DNA. The structure of the Drosophila Rdl receptor subunit was compared with those of vertebrate γ‐aminobutyric acid subtype A (GABAA) receptors and nicotinic acetylcholine receptors (nAChRs). The first six introns in Rdl show positions similar to those in vertebrate GABAA receptors, whereas the last two differ. It is interesting that the last intron appears to be in a position similar to that in nAChRs. These results are examined in relation to the proposal, based on amino acid identities, that Rdl codes for a novel class of GABAA receptor subunit more closely related to glycine receptors, and the possible place ofRdl in the lineage of the receptor superfamily is discussed.

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.