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Infect Immun. Jul 2004; 72(7): 4114–4126.
PMCID: PMC427405

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.

The maintenance of mosquito-borne disease transmission cycles is dependent upon the compatibility, as dictated by the genomes, of both the pathogen and the host. The decreasing efficacy of traditional methods for vector-borne disease control has provided the impetus to explore host-pathogen interactions at the molecular level with the aim of designing novel control methods (13). Innate immune responsiveness is of particular interest in such explorations, because extensive research efforts have demonstrated that vector mosquito species and mosquito cell lines produce robust humoral and cellular immune responses against invading pathogens.

Experimental evidence for a pivotal role of mosquito hemocytes (blood cells) as initiators and mediators of mosquito innate immune responses has recently become available. But because mosquito hemocytes are difficult to collect from mosquitoes and cannot be cultured or even maintained for significant amounts of time in vitro, they have received little research attention. Hemocytes phagocytose and melanize large numbers of bacteria, fungi, and malaria parasites (15, 24, 25, 28, 29). Several subpopulations of mosquito hemocytes have been characterized, and immunocytochemical and cytochemical assays have demonstrated that these cells are differentially responsive to invading pathogens (24, 27). For example, cell types are distinguished by ultrastructural characteristics, phagocytic capacity, or the presence and activity of enzymes involved in the melanin biosynthetic pathway (24, 28, 29, 36). It is hypothesized that hemocytes also participate in pattern recognition and mediate the production of immune peptides from the fat body (6).

In conjunction with the structural characterization of hemocytes done in our laboratory, we undertook a high-throughput molecular approach to gene discovery and characterization with the aim of gaining insight into vector-pathogen interactions mediated by hemocytes. Directional cDNA libraries and expressed sequence tags (ESTs) were generated from the perfusate (which contains the hemocytes) of the mosquitoes Aedes aegypti and Armigeres subalbatus. Aedes aegypti is a well-studied model mosquito for studies of Plasmodium and filarial worm vector competence, as well as a principal vector for yellow fever and dengue viruses. A genome sequencing project has been proposed for this species (37). Armigeres subalbatus is a model organism for studies of inherent resistance to filarial worms, which cause lymphatic filariasis in 120 million people annually (7). Material was collected from mosquitoes that were immune response activated by inoculation with the bacteria Escherichia coli and Micrococcus luteus. These species of bacteria have been used extensively to study insect innate immunity, particularly immune peptide production (35, 41). It has been shown that each bacterium elicits a distinct effector arm of the mosquito innate immune response; following inoculation of either Aedes aegypti or Armigeres subalbatus, the primary response to E. coli is phagocytosis, and that to M. luteus is melanization (28, 29). Most recently, it was shown that melanization and/or phagocytosis is elicited by a variety of bacterial species, independent of the Gram type (30).

Sequences from each mosquito species were assembled into contigs to reduce redundancy. Based on sequence similarity, consensus sequences for EST clusters were annotated using a controlled vocabulary in the web-based interface called A Systematic Annotation Package for Community Analysis of Genomes (ASAP) (21). Each cluster set was compared to the Drosophila melanogaster transcriptome and proteome sequences, the GenPept database, predicted peptides from the Anopheles gambiae genome sequence, the Pfam protein families database, and the other cluster set by use of BLAST analyses. Annotators manually analyzed these data and, whenever possible, attached information to the sequences, including a predicted gene product, protein domains, and indication of biological function, by use of a controlled vocabulary. Each entry is supported by the evidence used to make the annotation and typically includes intact hyperlinks to relevant databases. Consensus sequences from EST clusters and their annotations were submitted to GenBank. However, the data in ASAP are subject to change because the ASAP platform readily supports the addition of new sequence data, sequence comparisons, and experimental data. To view and download sequences and annotations, readers are invited to enter the ASAP website at https://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm.

EST clusters from mosquito hemocytes not only represent diverse cellular processes but also include numerous gene products related to immunity, many of which parallel those employed in vertebrate defense responses. These data constitute the first high-throughput, comprehensive analysis of immunity genes from these two mosquito species and are supplemented with corresponding information from the Anopheles gambiae genome sequencing project. The sequences and their annotations should prove to be an important resource for comparative genomic studies of mosquito species and for the prediction of gene products from and annotation of mosquito genome sequences. Additionally, because of the source of the library, these data provide valuable insight into potential transcript localization and abundance for the represented gene products.

MATERIALS AND METHODS

Mosquito maintenance.

Aedes aegypti (Liverpool-strain) larvae were hatched from dry oviposition papers in deoxygenated, deionized water. Armigeres subalbatus larvae were hatched in oviposition dishes and transferred to enamel rearing pans. Approximately 300 Aedes aegypti or 250 Armigeres subalbatus larvae per pan were placed in deionized water. Female pupae were mechanically separated, and 100 Aedes aegypti or 80 Armigeres subalbatus pupae were placed into 0.473-liter ice cream cartons covered with fine-mesh marquisette. Adults were maintained as previously described for Culex pipiens adults and used for experimentation within 3 days of eclosion (3).

Bacterial inoculations.

E. coli K-12 and M. luteus were used for bacterial inoculations as described previously (42). Cold-immobilized mosquitoes were held in place with a vacuum saddle, and a stainless steel probe that had been dipped into a bacterial pellet was inserted into the cervical membrane. The mosquitoes were returned to the ice cream cartons and placed in our insectary.

Tissue collection.

A volume displacement technique was used to collect hemocytes. A tear was made above the penultimate abdominal segment of the cold-immobilized mosquitoes, which were then placed on a vacuum saddle. A pulled glass capillary needle, attached to a syringe containing an RNA extraction solution, was inserted into the cervical membrane between the head and the thorax. The RNA extraction solution was injected, and the perfusate was immediately placed on dry ice and then stored at −80°C. For each mosquito, the perfusion takes only seconds. Hemocyte material was collected within minutes (<1 h) and at 3, 6, 12, and 24 h postinoculation.

cDNA library construction and EST production.

RNA extractions were performed by the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method (11). A directional cDNA library was generated from each mosquito species. RNA was electrophoresed to confirm its integrity, and then RNA from all time points was pooled. Total RNA was used as the template for constructing libraries by use of the Long-Distance PCR protocol of the SMART cDNA library construction kit (Clontech, Palo Alto, Calif.). Total RNA, as opposed to poly(A)-selected RNA, was used for the library construction because initial attempts to isolate selected RNA were unsuccessful, even when perfusate from approximately 500 mosquitoes was used.

Visual assessments of the quality of the libraries demonstrated that more than 80% of the phage out of 4 × 106 to 5 × 106 independent clones were recombinant. At the University of Wisconsin—Madison, sequences from recombinant plaques were generated from the resulting primary library as previously described (3). Sequencing efforts were primarily directed toward randomly picked clones, with the exception of a single batch of sequences generated at the University of Wisconsin—Madison. These clones were selected based on size such that approximately half of the sequences were more than 500 bp in length and half were more than 1,000 bp in length. PCR products in the desired size range, electrophoresed and stained with ethidium bromide, were chosen for sequencing. At National Yang-Ming University, primary and secondary libraries were first converted from phage to plasmids by use of the excision protocol provided by the manufacturer (Clontech).

Sequence compilation.

Raw sequences obtained from the University of Wisconsin—Madison included sequences from the vector and primer used in library construction. Those obtained from National Yang-Ming University were subjected to Phred analysis for base calling, and then the vector sequence was trimmed by using Vector Strips (EMBOSS). The sequences were assembled to reduce redundancy by using Seqman (DNASTAR Inc., Madison, Wis.) set to trim sequence ends, scan for pTriplEx vector, and optimize sequence assembly order. A minimum match length of 18 nucleotides was used in vector-searching parameters. End trimming was executed by using the quality low-stringency parameters. All other assembly parameters were default settings. Low-stringency end-trimming parameters were used in order to keep sequences of lower quality in the assembly because these sequences could yield hits in BLAST analyses. Because of the parameters used, each contig was manually examined following assembly. Any vector sequence not recognized by the defined parameters was removed. Contigs containing what appeared to be different genes were manually split into appropriate separate contigs. Contigs containing fewer than 50 bp were removed.

Sequence similarity searches.

In order to predict the gene products and associated biological functions represented in mosquito hemocyte ESTs, sequences were compared to those of the GenBank nonredundant database, the D. melanogaster and Anopheles gambiae genomes, and the reciprocal mosquito EST cluster sets created during this project. A FASTA file generated with Seqman (DNASTAR Inc.) was used for BLAST analyses. Arguments were used to produce the output in HTML format, and an expectation (e) value threshold was set at 0.01. Nucleotide sequences were translated and compared to the most recent version of the GenPept protein database (available at ftp://ftp.ncbi.nih.gov/genbank/) by blastx analysis using stand-alone BLAST software available at ftp://ftp.ncbi.nih.gov/blast/executables/. Both a standard BLAST (blastn) and a translated BLAST (blastx) were used to compare our sequences to the D. melanogaster genome (release 3.1) available through FlyBase at ftp://flybase.org/flybase-data/ (19). A blastx analysis also was done for 16,112 known and novel predicted peptides from Anopheles gambiae downloaded from Ensembl at ftp://ftp.ensembl.org/pub/current_mosquito/data/ (version 14.2, updated on 2 June 2003). Lastly, reciprocal BLAST (tblastx) analyses were done (with the BLAST parameters described above) for the two mosquito species to define orthologous sequences (i.e., for each species, a database was created against which sequences from the opposing species were compared). In order to qualify as orthologs, similarity between sequences had to be observed in reciprocal BLAST analyses and then those similar sequences had to have equivalent hits in the GenPept and Drosophila databases. Hyperlinks to orthologous sequences were then built into the database.

To obtain information about protein motifs, domains, and families, clusters were compared to the Pfam database of alignments and hidden Markov models (5). Sequences were translated in six reading frames by using a program written in Perl code. The translations were subjected to analysis against the Pfam_ls (version 9.0) library global alignment models (downloaded from http://pfam.wustl.edu) by using profile hidden Markov models software (hmmer-2.2g.bin.dos-cygwin) from the HMMER site at Washington University (http://hmmer.wustl.edu/). Annotations were made, by use of a controlled vocabulary, for Pfam hits with positive scores and expectation (E) values of less than 1.

Sequence annotation.

Each EST cluster and its corresponding sequence similarity data were uploaded into ASAP, a relational database and web interface that was designed to facilitate the annotation of enterobacterial genomes. By using this platform, a community of annotators analyzed and attached information to the sequence data; that information was reviewed by a curator before being made available for public viewing. During the course of this project, ASAP has been adapted to accommodate EST data. Each cluster is identified by two unique numbers for two feature types: “EST_cluster” and “source.” The source feature contains qualifiers that define the origin of the sequence, such as organism and library information. The EST_cluster feature type encompasses the sequence data, multiple sequence similarity searches, and annotations. Under the EST_cluster category in ASAP, annotators assessed sequence alignments and followed intact hyperlinks to the National Center for Biotechnology Information, the Wellcome Trust Sanger Institute (Ensembl and Pfam), FlyBase, and orthologous sequences within ASAP. For each cluster sequence, a predicted gene product and/or function was noted in the ASAP annotation tables. Supporting evidence for each annotation, including products and notes, is typically in the form of a hyperlink to a database. Annotations can be viewed in ASAP by following links to sequences for the mosquito species of interest that lead to the “Query Genome Annotations” page. Within this interface, users can search for genes of interest by entering an ASAP feature identification number (as listed in Table Table5,5, for example) or by querying with a keyword.

TABLE 5.
Abundant and immunity-related EST clusters observed from A. aegypti and A. subalbatus immune response-activated hemocyte libraries

Within ASAP, products were defined by using the “/product” qualifier, and additional information was annotated within “/note” qualifiers that are compatible with legal GenBank vocabulary. Whenever possible, products were annotated according to D. melanogaster (FlyBase) because the genome is well characterized and annotated. Products with homology to sequences in other databases that have no attributed function were annotated as “conserved unknown.” Those that did not yield convincing hits in any database were annotated as “unknown.” Annotations were preceded by additional qualifiers to categorize the quality of BLAST alignments to existing described gene products. The “questionable:” qualifier was used if the alignment was poor (i.e., low score, high e value, or short alignment), or “putative:” was used if the annotated product was likely according to the alignment (i.e., score close to 100 or low e value). ESTs without a preceding qualifier aligned very well to the sequence referred to in the annotation (i.e., high score, low e value, or alignment along the majority of the sequence length).

A controlled vocabulary was used to insert additional information related to the biological function of proposed gene products within the “/note” category. Broad categories of interest were defined, some of which reflect previously published mosquito genomics information, including cytoskeletal, antioxidant-related, stress response, detoxification, apoptosis-related, nuclear regulation, signal transduction, iron metabolism, and immunity-related categories. Within the immunity-related category, clusters were further described as being serine proteases or serpins or antimicrobial peptides or as being related to melanization, pattern recognition, antioxidant formation, or signal transduction; any of these can be used as a keyword search term in ASAP.

Pfam annotations were also added as notes for EST clusters with hits to the Pfam database that scored greater than 1 and had E values of less than 0.01. In order to provide preliminary information about the abundance of transcripts for a specific product, a note was added to describe the number of sequences that were assembled to produce each EST cluster, and an additional note (“/homolog”) was created to support the annotation of sequences with homology (threshold set as the sum of identical and positive residues equal to approximately 120 or more) to Anopheles gambiae. This feature appears in the GenBank flat files as a note with the corresponding Ensembl identifier embedded in its content.

Sequence annotations were ongoing while we continued to collect the additional sequences that make up version 2 of our mosquito databases. ASAP provides a mechanism for tracking features across versions of a sequencing project. Should we choose to collect more EST data, ASAP provides the facility to integrate new data with existing data while tracking the history of features in all versions. We have already found it necessary to continuously revise our annotations to address the lack of this functionality in other database resources for ongoing sequencing projects.

Nucleotide sequence accession numbers.

The sequences and annotations presented in this paper have been submitted to GenBank under accession numbers AY431103 to AY433788 (Aedes aegypti) and AY439334 toAY441440 (Armigeres subalbatus). More detailed information is available at https://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm.

RESULTS AND DISCUSSION

Sequence compilation.

Raw sequences, generated from multiple rounds of sequencing, were assembled to construct EST clusters that were uploaded into ASAP. A total of 11,952 Aedes aegypti and 12,790 Armigeres subalbatus sequences yielded 2,686 and 2,107 EST clusters, respectively. Of the clusters, 66% of those from Aedes aegypti and 62% of those from Armigeres subalbatus are composed of a single sequence (singletons). The hemocyte libraries were not normalized, so this yield is not surprising. Those clusters that are not singletons are composed of an average of 9.4 Aedes aegypti or 12.3 Armigeres subalbatus sequences; because these clusters may reflect transcriptionally up-regulated genes, the number of ESTs that were assembled in each cluster has been incorporated into the annotations and is presented here for genes of interest. Summaries of sequence assembly results and cluster composition data are presented in Tables Tables11 and and22.

TABLE 1.
Summary of EST assembly data from the mosquito species A. aegypti and A. subalbatus
TABLE 2.
Composition of EST clusters from the mosquito species A. aegypti and A. subalbatus

Sequence annotation.

EST clusters were compared to the D. melanogaster transcriptome and proteome sequences, the GenPept database, predicted peptides from the Anopheles gambiae genome sequence, and to one another via BLAST analyses. The number of sequences that yielded hits in these databases is summarized in Table Table3,3, as is the number of products annotated according to those databases. A brief summary of the results from the sequence annotations appears in Table Table4.4. Although the libraries were constructed from total RNA, fewer than 10% of the EST clusters encode ribosomal genes or gene products. Over 50% of the sequences from both mosquito species were annotated as “unknown” or “conserved unknown,” meaning that they have no similarity to sequences in other databases (unknown) or are similar to those of gene products with unknown function from other organisms.

TABLE 3.
Results from multiple sequence similarity searches of A. aegypti and A. subalbatus EST clusters against five publicly available databases and number of annotations made using evidence from each database
TABLE 4.
Sequence annotations in general categories of interest

In the interest of brevity, we have restricted the discussion and tables in this paper primarily to clusters that were annotated with confidence to known gene products, with a particular emphasis on immunity-related products. The evolutionary conserved nature of innate immune responsiveness to parasites makes this information pertinent to the communities of research groups interested in the biology of vector-pathogen-vertebrate host relationships (2, 31). The text will not be replete with references. Readers interested in particular EST clusters are encouraged to visit ASAP at https://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm, where the evidence for each annotation provides hyperlinks to GenBank, FlyBase, Ensembl, and the Pfam database and, therefore, links to relevant literature.

The libraries from which the ESTs were generated probably contain clones from three sources: hemocytes, the bacteria used to activate the mosquitoes' immune responses, and the fat body. Although the source libraries were not generated from poly(A)-selected RNA, only four Aedes aegypti clusters (composed of 33 sequences) and two Armigeres subalbatus clusters (composed of 5 sequences) were annotated as bacterial contaminants, perhaps owing to the fact that these libraries were not normalized and therefore did not select for less abundant sequences. Fat body contamination is inherent in the perfusion process employed to collect hemocytes (27). The inevitable presence of ESTs from the fat body in these data sets does not diminish their value, because this tissue is essential for immune responsiveness (26, 52). Molecules that are likely produced in the fat body are noted in the text.

Immunity-related gene products.

Hemocytes function in pattern recognition, phagocytosis, melanization, and signaling cascades that initiate varied cytotoxic effector responses, and they are the central element mediating systemic mosquito innate immune responses (6). As a framework for examining hemocyte sequences, we defined the following as subcategories of immunity relatedness: general (no subcategory), pattern recognition molecules, Toll signaling pathway related, antimicrobial peptides (AMPs), melanization related, antioxidant related, serine proteases, and serpins, all of which are summarized in Table Table5.5. Using these criteria, 169 Aedes aegypti EST clusters were identified (based on sequence similarity alone) as immunity related, as were 103 from Armigeres subalbatus. These numbers do not reflect a differential analysis of transcription between mosquito species. This is, however, a substantial number of genes, considering that only 38 were identified in a previous study of 2,380 clone clusters from a normalized Anopheles gambiae hemocyte-like cell line library (16), and it underscores the importance of in vivo evaluations of these cells. In comparison, the genomes of Anopheles gambiae and D. melanogaster contain 242 and 185 immunity-related genes, respectively, from 18 families (12). Of those families, 16 are represented in this study (and are indicated in Table Table5),5), with only STATs and galectins not being represented.

Abundant sequences.

Eleven EST clusters in Aedes aegypti and 16 in Armigeres subalbatus were categorized as abundant because they are composed of more than 100 sequences (Table (Table5).5). Transcriptional up-regulation of these genes will have to be confirmed in further assays; however, transcript abundance alone is a valuable and important measure used to identify genes of interest in EST projects (see reference 43). We speculate that the sequences in this category are illustrative of the concerted investment that hemocytes make in immune responsiveness, employing multiple effector arms to combat pathogens, including phagocytosis via cytoskeletal rearrangements, apoptosis, and the production of effector molecules and serine proteases. One intriguing example is an abundantly represented sensory appendage protein with similarity to odorant or pheromone binding proteins that was noted among the Aedes aegypti EST clusters. Two such molecules recently have been reported as having a role in tissue remodeling and are inducible by viral and bacterial infection in Drosophila (49).

Products that are necessary for basic cellular function also were noted in this category. The three large protein complexes (NADH dehydrogenase, cytochrome reductase, and cytochrome c oxidase) involved in the respiratory chain, in addition to ATPase (ATP synthase), are represented. These enzyme systems likely serve to provide requisite energy for various cellular processes, or their up-regulation may be indicative of impending apoptosis, as has been observed in various vertebrate cells (9). Notably, four highly abundant EST clusters could not be identified by comparison to sequence databases. It is interesting to speculate that orthologous EST clusters that were assembled from disparate numbers of sequences, or abundantly represented EST clusters that do not have an orthologous sequence, are associated with observed experimental differences in immune responsiveness between these two mosquito species (7, 28).

Pattern recognition.

Recognition of pathogen-associated molecular patterns initiates innate immune responsiveness in both vertebrates and invertebrates (44). The cascade of events begins when pathogen-associated molecular patterns are bound by pattern recognition receptors such as peptidoglycan recognition proteins (see reference 12). These and a number of additional molecules that likely function in pattern recognition, including scavenger receptors, lectins, gram-negative binding proteins, and products containing fibrinogen domains, were identified in hemocyte ESTs of both mosquito species (Table (Table5).5). Four ESTs from Aedes aegypti are similar to thioester-containing proteins shown to be immune responsive with complement-like opsonin properties (38, 40). Three EST clusters were identified as having high similarity (score, 116; e = 7.1e-32) to the Pfam model MD-2-related lipid-recognition domain (E1_DerP2_DerF2), a lipid-binding protein essential for mammalian recognition of lipopolysaccharide in cooperation with Toll-like receptor 4 (34).

Phagocytosis.

Phagocytosis is a primary innate immune response and a mediator of subsequent responses in adaptive immunity. Like vertebrates, insects utilize subpopulations of professional phagocytic cells, sometimes referred to as macrophage-like, in response to invading pathogens. Evidence for phagocytic events is manifested in the large number of signal transduction, cytoskeletal rearrangement, and apoptotic elements identified (23) (Table (Table5).5). In agreement with the results from ultrastructural studies of Aedes aegypti and Armigeres subalbatus hemocytes (28, 29), the sequences presented here emphasize the importance of phagocytosis in the mosquito immune response.

Signal transduction.

Considering the extensive cell-cell communication and collaborative interaction mandated by phagocytic activity, it is not surprising to see numerous signal transduction elements and pathways represented in ESTs from immune response-activated mosquito hemocytes. Emphasis has been placed on the Toll and immune deficiency (imd) signaling pathways as elicitors of antimicrobial responses (reviewed in reference 32), but the importance of alternative signaling pathways in insect cell phagocytosis and immunity is also becoming clear (8, 33). A report concerning Anopheles gambiae showed that Anopheles gambiae STAT is translocated to the nucleus in response to a bacterial challenge (1). Drosophila Janus kinase kinase hopscotch (hop) gain-of-function mutants constitutively express the complement-like protein Tep-1 (38). Molecules related to signaling pathways including Toll and imd are presented in Table Table55 and include numerous Rho family GTPases that may be involved in actin reorganization during phagocytosis (23).

Cytoskeletal elements.

Phagocytic cells in Aedes aegypti and Armigeres subalbatus, called granulocytes, have the capacity to engulf hundreds of bacteria, which undoubtedly requires extensive cytoskeletal remodeling (23, 29). In support of this observation, 31 EST clusters were identified as cytoskeletal elements, including actin, actin-binding and polymerizing factors, and alpha- and beta-tubulins (Table (Table5).5). Noteworthy among these is gelsolin, an actin-severing molecule represented by 148 sequences in a single EST from Armigeres subalbatus.

Cytotoxic molecule production and antioxidant-related immune response.

Reactive oxygen intermediates function ubiquitously in phagocytic cells as cytotoxic effector molecules. Evidence for both reactive oxygen species production and restorative chemistry following an oxidative burst are evident in hemocyte EST clusters (Table (Table5).5). Reductases, catalase, and peroxidases remove excess reactive oxygen intermediates to return cells to homeostasis following oxidative bursts.

Apoptosis-related immunity.

Apoptosis and phagocytic immune responses are intimately linked so that organisms can achieve homeostasis before and after an immune challenge (47). As do many immune response elements, this phenomenon transcends phyla (20, 31). The sequence of the Anopheles gambiae genome revealed both pro- and antiapoptotic regulators (12). To further demonstrate that mosquitoes employ these gene products in response to bacteria, ESTs from immune response-challenged mosquito hemocytes include 19 Aedes aegypti and 22 Armigeres subalbatus apoptosis-related molecules (Table (Table55).

Melanotic encapsulation.

Melanin biosynthesis is a hemocyte-mediated immune response that involves a complex yet well-characterized (6) cascade of reactions beginning with tyrosine and ending with the polymerization of a capsule that surrounds an invading parasite (55), eventually killing the parasite (10, 45). This is an important mechanism for mosquito resistance to eukaryotic parasites, including malaria parasites and filarial worms (7, 14). Ultrastructural studies demonstrate that this response is also rapidly deployed against bacteria (24, 25, 28-30). In Aedes aegypti and Armigeres subalbatus, enzymes involved in melanization, including prophenoloxidase and phenylalanine hydroxylase, are produced primarily by oenocytoids (27, 28, 36). These enzymes, as well as dopachrome conversion enzyme and multiple sequences with similarity to prophenoloxidase activating factors, are represented in EST clusters from both species (Table (Table55).

AMPs.

The AMPs—effector molecules that exhibit in vitro activities against bacteria, fungi, and protozoa—are considered a primary defense element in mosquito innate immunity. Transcriptional up-regulation of AMPs has been correlated not only with responses to bacteria and fungi but also with various stages of Plasmodium infection in Anopheles gambiae (17, 48). A number of novel and previously reported defensins, cecropins, and lysozymes, as well as gambicin, are represented in EST clusters from immune response-challenged hemocytes. The production of AMPs occurs predominantly in the fat body but has also been reported to occur in various tissues and in immune-responsive mosquito cell lines (see references 18 and 26). The extent to which the immune peptides discussed herein are produced by the fat body or hemocytes will need to be resolved with cytochemical assays.

Previously reported immune peptides from Aedes aegypti include three isoforms of defensin and a single isoform of cecropin (41). Immune peptide sequences from Armigeres subalbatus have not been reported. A number of sequences encoding immune peptides are represented in the EST clusters produced for the present project, several of which were compiled from a large number of sequences. For example, 3 of the 11 Aedes aegypti EST clusters that are composed of more than 100 sequences encode defensins; likewise, 1 of the 16 ESTs from Armigeres subalbatus that is most abundantly represented is a defensin. Another Armigeres subalbatus EST encoding lysozyme is abundantly represented with 123 sequences (Tables (Tables55 and and6).6). Further sequence analysis was done to resolve the type and number of representatives of each immune peptide for both mosquito species.

TABLE 6.
AMPs observed in immune response-activated A. aegypti and A. subalbatus EST clusters

Among the EST clusters from both mosquito species, four isoforms of defensin were observed. An Armigeres subalbatus sequence shows similarity to defensin genes but lacks the conserved cysteine residues necessary for the characteristic structure of the molecule. Five different cecropin isoforms were observed. Isoform N, not previously described, exhibits a unique amino acid sequence. Three isoforms of lysozyme were also among the sequences reported here, as was gambicin, a newly described immune peptide from Anopheles gambiae that has broad activity against bacteria, fungi, and Plasmodium parasites (53). Gambicin was not found in our BLAST analysis of the Anopheles gambiae predicted peptide sequences; however, the sequence was identified in a more recent version (version 17.2a.1) of the genome. Two Aedes aegypti sequences, categorized as questionable, have weak similarity to diptericin and holotricin. A tabular description of these sequences and this nomenclature is presented in Table Table66.

The clear energetic investment made by these mosquitoes to produce redundant AMPs in abundance, when the capacity of hemocytes is likely sufficient to clear an infection (16, 28), suggests that these molecules might have additional roles in insect innate immunity beyond cytotoxicity (4). Signaling and chemotaxis may be functions of mosquito AMPs, as is the case in vertebrate immunity (50). An alternative view is that redundant AMPs in the Anopheles gambiae genome have alternate antimicrobial specificities (12).

Stress response.

Bacterial infection and the robust cellular and humoral responses elicited by that infection undoubtedly serve as stressors for infected mosquitoes. Numerous stress-responsive molecules were detected among these ESTs, including heat shock proteins and ubiquitin-63 (Table (Table5).5). These data implicate a link between the innate immune and stress responses in mosquitoes and support a previous assessment of altered immune capacity in environmentally stressed Anopheles gambiae (51).

Iron metabolism.

Iron transport and storage proteins have diverse roles in insect physiology. Of interest for innate immunity is the host's ability to sequester iron to hinder pathogen survival. ESTs from both species contain multiple molecules related to iron metabolism, including iron-sulfur cluster proteins, ferredoxin, and cysteine desulfhydrase (Table (Table5).5). Several reports have described up-regulation of transferrins in insects or insect cells challenged with bacteria (reviewed in reference 46), and the transferrin molecule is represented by three EST clusters in immune response-activated hemocytes. In contrast, ferritin, an iron storage protein, is represented by five clusters that contain numerous sequences. Two ferritin light chain-like clusters contain 92 Aedes aegypti sequences, suggesting the importance of this molecule in the immune response; whether this apparent transcriptional abundance is inducible will need to be examined further, because ferritin modulation has not previously been demonstrated in association with innate immune responses in insects (39).

Serine proteases and serpins.

Diverse functions in insect innate immunity, including hemolymph coagulation, AMP production, and melanotic encapsulation, are modulated by various serine proteases and serpins (12, 22). The importance of these molecules is illustrated by the number of immunity-related serine proteases identified and their abundance (Table (Table5).5). One serine protease in Armigeres subalbatus is represented by 107 sequences, and its Aedes aegypti ortholog contains 35 sequences.

Mosquito-specific EST clusters.

Following the addition of Anopheles gambiae BLAST results to Aedes aegypti and Armigeres subalbatus sequences in ASAP, 102 EST clusters that had no similarity to sequences in nonmosquito databases were noted (data not shown). These intriguing sequences, especially those that are represented multiple times, may represent mosquito-specific gene products related to immune responsiveness and merit further investigation.

Future prospects.

The data presented here represent the first high-throughput analysis of the transcriptome of immunocytes from an invertebrate and provide a road map for future investigations that aim to elucidate interactions between the mosquito host and the pathogens it transmits. Host-parasite interactions represent coevolved adaptations of considerable complexity, and the compatibility of these relationships is dependent on the relative capacities of the host to recognize and respond to a foreign object and of the parasite to weaken or inhibit the immune system of the host. These dynamic interactions are unquestionably manifested in the transcriptomes of immune response-activated mosquito hemocytes. In anticipation of conducting in-depth studies of the dynamics of the hemocyte transcriptome in response to bacteria, malaria, and filarial worm parasites, microarrays are being generated. Having well-characterized ESTs provided a unique capacity to strategically design oligonucleotide-based microarrays.

Acknowledgments

This work was supported by NIH grants AI19769, AI053772, and AI46032 (to B. M. Christensen), National Science Council of Taiwan grant NSC 91-3112-B-010-001 (to C.-C. Chen), and NIAID graduate fellowship AI07414-11 (to L. C. Bartholomay).

Notes

Editor: W. A. Petri, Jr.

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