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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.

Phosphohexomutases reversibly catalyse the transfer of the phosphate group of a glycosyl phosphate between the C6 and C1 positions, and uridine diphosphate (UDP)-hexose pyrophosphorylases catalyse the synthesis of UDP-hexose from uridine triphosphate (UTP) and hexose-1-phosphate. Both enzyme families are essential for nucleoside diphosphate hexose biosynthesis and are therefore critical for various physiological functions in the midgut of mosquitoes after a blood meal. We cloned and sequenced three phosphohexomutase and two UDP-hexose pyrophosphorylase cDNAs from Aedes aegypti. The products of the cDNAs were expressed and substrate specificities were examined. Herein we describe Ae. aegypti phosphoglucomutase 1, phosphoglucomutase 2, phosphoacetylglucosamine mutase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase. Transcripts of the genes expressing the enzymes are constitutively present in all life stages and blood-feeding does not seem to influence transcript abundance.

Chorion melanization is a vital biochemical event for the survival of mosquito eggs in the environment. This study describes the identification and molecular characterization of a prophenoloxidase (proPO) involved in chorion melanization in Aedes aegypti by various biochemical and molecular techniques. Results revealed that transcription of the chorion proPO occurs only in adults, blood feeding greatly stimulated its transcription and haemocytes are responsible for its transcription. Our study provides a solid basis for suggesting an essential role of the isolated proPO in chorion melanization during chorion hardening and also raises fundamental questions regarding its transportation and distribution in the chorion. This study should serve as a useful reference towards functional elucidation of other proPOs in mosquitoes.

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.

Aedes aegyptiglutamine synthetase (GS) is expressed constitutively at various developmental stages and its relative mRNA abundance increases in the midgut following blood feeding in support of the biosynthesis of chitin, a component of the peritrophic matrix. To understand the regulation of GS expression better, GS-luciferase reporter fusion genes were constructed and analysed in transiently transfected C6/36 cells. These studies have identified three GS regions: GS-A, -B and -C (C1, C2) that are required for efficient transcription. The crucial regulatory DNA sequence is located within 140 nucleotides of the GS-C region in the first exon. GS-B region between -209 and +4 contains a negative modulator that represses transcription of the GS-C promoter, but the 5'-GS-A region, between -476 and -282, can negate the transcription inhibition of GS-B and promote GS transcription of the GS-C promoter. Electrophoretic mobility shift assays showed that nuclear proteins for GS-A, GS-B and GS-C1 are present in the C6/36 cells, and therefore that GS-A, GS-B and GS-C1 indeed possess regulatory function. By contrast, nuclear proteins isolated from both cultured cells and midgut tissues bound to GS-C2, suggesting that GS-C2 plays an important role in GS transcription and that GS-C2 is regulated by several different and redundant transcription factors to achieve constitutive expression in a wide variety of tissues.

Glucosamine:fructose-6-phosphate aminotransferase (GFAT) catalyses the formation of glucosamine 6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. The final product of the hexosamine pathway, UDP-N-acetyl glucosamine, is an active precursor of numerous macromolecules containing amino sugars, including chitin in fungi and arthropods. Chitin is one of the essential components of insect cuticle and peritrophic matrix. The peritrophic matrix is produced in the midgut of mosquitoes in response to bloodfeeding, and may affect vector competence by serving as a physical barrier to pathogens. It is hypothesized that GFAT plays a regulatory role in biosynthesis of chitin and peritrophic matrix formation in insects. We cloned and sequenced the GFAT gene (AeGfat-1) and its 5' regulatory region from Aedes aegypti. There is no intron in AeGfat-1 and there are two potential transcription start sites. AeGfat-1 cDNA is 3.4 kb in length and its putative translation product is 75.4 kDa. The amino acid sequence of GFAT is highly conserved in lower and higher eukaryotes, as well as in bacteria. AeGfat-1 message is constitutively expressed but is gradually up-regulated in the midgut after bloodfeeding. The putative regulatory region of the gene contains the ecdysone response element, E74, and Broad complex motifs, similar to what is found in the glutamine synthetase gene in Ae. aegypti. Results suggest that Ae. aegypti GFAT-1 may have a regulatory role in chitin biosynthesis and peritrophic matrix formation, and probably is under the regulation of ecdysteroids.

In this study we describe the purification and molecular cloning of a dopachrome conversion enzyme (DCE) from the yellow fever mosquito, Aedes aegypti. DCE catalyzes the conversion of L-dopachrome to 5,6-dihydroxyindole in the melanization pathway. Melanin biosynthesis is involved with crucial protective phenomena in mosquitoes, including egg chorion and cuticular tanning, wound healing, and the melanotic encapsulation immune response. The enzyme was purified to homogeneity by various chromatographic techniques from A. aegypti larvae and has a relative molecular mass of 51 kDa as-revealed by SDS-PAGE analysis. Physiochemical analysis of DCE revealed a pH optimum of 7.5-8.0 and substrate activity for L-dopachrome and aminochromes generated from dopa methyl ester, alpha-methyl dopa and dopamine. Trypsin digestion of the isolated DCE and subsequent reverse-phase separation resulted in the isolation of several polypeptide fragments, from which two partial internal amino acid sequences were obtained by Edman degradation. PCR amplification, using a degenerate primer based on one internal amino acid sequence and an oligo-dT primer, produced a 650 bp DNA fragment. Subsequent screening of an A. aegypti pupal cDNA library resulted in the isolation of a 1.6 kb clone containing coding sequence for both internal DCE amino acid sequences, thereby confirming the identity of the isolated gene product (pAaDce1) as DCE. Northern analysis revealed the constitutive expression of DCE message in developmental stages and adults, with the majority of transcript localized in the fat body and ovaries of adult females. AaDce1 mRNA increased in abundance above constitutive levels in adult females when a melanotic encapsulation immune response was initiated by the intrathoracic inoculation of Dirofilaria immitis microfilariae.

The peritrophic matrix (PM) is the first natural barrier a mosquito-borne parasite faces when ingested with a blood meal; consequently, understanding the biology of PM formation could provide novel transmission control strategies. Because the PM is composed of chitin (a molecule of repeating units of N-acetyl glucosamine), glycoproteins and glucose, characterizing the regulation of enzymes involved in chitin production should provide information concerning factors that influence PM formation. We previously have shown that glutamine synthetase (GS) provides the glutamine needed in the initial steps of chitin biosynthesis in the yellow fever mosquito, Aedes aegypti. In the present study we show that GS is encoded by a single 4.5 kb gene, designated mGS, containing three exons and two introns. Multiple transcripts are generated from mGS presumably by differential splicing of the introns. Sequences of two cDNAs encoding GS are identical at the protein level, but differ in their 5'-untranslated regions. GS message is constitutively expressed in all developmental stages and in most tissues, with an increase in GS transcription observed in midgut and fat body tissues of female mosquitoes following a blood meal. Transcripts are localized to the apical side of the mosquito midgut epithelium and data suggest that mGS transcription is regulated by an Oct-1 transcription factor.

The functions of insect peroxidases include detoxification, stabilization of extracellular matrices, and possible involvement in insect immunity. The current study describes the isolation of a peroxidase gene, AePox, and its cDNA from the mosquito, Aedes aegypti. AePox codes for a protein that is homologous to various heme-peroxidases from vertebrates and invertebrates, with highest identity to Drosophila melanogaster peroxidase (62%). Sequence comparison identified several functionally and structurally conserved domains in the mosquito peroxidase, including a heme environment, a calcium binding site, and five possible disulfide bridges. These results imply that AePOX may likely have a similar structure and catalytic mechanism as those described for the mammalian myeloperoxidase superfamily. Expression studies demonstrate that AePox is transcribed in mosquito larvae and pupae, but not in adults, in ovaries, or in early embryos. However, AePOX protein is present in all mosquito stages and possibly has a maturation process that is similar to that of human myeloperoxidase. Unlike most human peroxidases, the AePox gene contains a TATA box and an ecdysone response element (EcRE).

The biosynthesis of melanotic materials is an important process in the life of a mosquito. Melanin production is critical for many diverse processes such as egg chorion tanning, cuticular sclerotization, and melanotic encapsulation of metazoan parasites. Prophenoloxidase plays a critical role in this biochemical cascade. Two cDNAs, one full length and one partial clone, and two genomic clones encoding prophenoloxidase (pro-PO) were isolated from the yellow fever mosquito, Aedes aegypti. The full-length cDNA, pAaProPO1, is 2286 bp long with a 2055 bp open reading frame encoding a 685 amino acid protein that shares 89% identity with Armigeres subalbatus pro-PO. It contains two putative copper binding domains (amino acids 197-243 and 346-423) that are homologous to other insect pro-POs. AaProPO1 messenger RNA (mRNA) was detected by reverse transcription polymerase chain reaction (RT-PCR) only from third-stage larvae and not in adult mosquitoes after blood feeding, during the melanotic encapsulation of Dirofilaria immitis microfilariae or following exposure to bacteria. A 750 bp fragment of the second cDNA (pAaProPO2) was cloned using RT-PCR from mRNA obtained from 14-day postovipostional eggs. AaProPO2 mRNA was not found in any other life stages, and may be in low abundance or transiently expressed. AaProPO2 and AaProPO1 each contain three introns that are 60, 68 and 58 bp and 61, 69 and 59 bp long, respectively, and the intron sequences of these two genes are not similar.

Sindbis virus expression vectors have been used successfully to express and silence genes of interest in vivo in several mosquito species, including Aedes aegypti, Ae. albopictus, Ae. triseriatus,Culex pipiens, Armigeres subalbatus and Anopheles gambiae. Here we describe the expression of an endogenous gene, defensin, in Ae. aegypti using the orally infectious Sindbis virus, MRE/3'2J expression vector. We optimized conditions to infect mosquito larvae per os using C6/36Ae. albopictus cells infected with the recombinant virus to maximize virus infection and expression of defensin. Infection with the parental Sindbis virus (MRE/3'2J) did not induce defensin expression. Mosquito larvae infected by ingestion of recombinant Sindbis virus-infected C6/36 cells expressed defensin when they emerged as adults. Defensin expression was observed by western analysis or indirect fluorescent assay in all developmental stages of mosquitoes infected with MRE/3'2J virus that contained the defensin insert. The multiplicity of infection of C6/36 cells and the quantity of infected cells consumed by larvae played an important role in defensin expression. Parental viruses, missing the defensin insert, and/or other defective interfering virus may have contributed to these observations.

Characterization of the enzymes involved in the chitin biosynthetic pathway in mosquitoes is critical due to the importance of chitin in the formation of the peritrophic matrix [PM] and its potential impact on vector competence. Chitin is the homopolymer of the amino sugar N-acetyl-D glucosamine [GlcNAc]. The final step of incorporation of GlcNAc into the chitin polymer is catalyzed by the enzyme chitin synthase [CS]. CS is a membrane bound enzyme, but the mechanism of its action in the biosynthesis of the PM is not understood. We have isolated and sequenced a CS-encoding cDNA clone from the mosquito Aedes aegypti, compared its sequence with CS from other organisms and studied its RNA expression. The cDNA is 3.5 kb in length with an open reading frame of 2.6 kb that encodes a protein of 865 amino acids with a predicted molecular mass of 99.5 kDa. The putative translation product shares 90% similarity to two CS proteins from Caenorhabditis elegans and 50% similarity to Saccharomyces cerevisiae in the catalytic domain of CS enzymes. Data suggest that CS is a single copy gene. RT-PCR analysis shows CS message in whole non-blood-fed females, whole blood-fed females, non-blood-fed midguts and in midguts dissected at different time points post-blood-feeding. In situ hybridization studies of midgut samples revealed that CS mRNA increases following a bloodmeal and is localized to the periphery of the epithelial cells facing the midgut lumen.

The RING finger is a zinc-binding domain that is found in proteins from viruses, plants and animals. Here we report the characterization and tissue-specific expression of a mosquito gonadal protein gene, mgp, from the mosquito, Aedes aegypti. The putative gene product, MGP, contains two RING fingers, a B-box, and a hydrophobic core. These mosquito MGP structural motifs are highly conserved in proteins found in mouse and nematode. Northern blot analysis and in situ hybridization demonstrated the presence of multiple mgp RNA transcripts in male and female reproductive tissues. Expression of mgp in the ovary is constitutive, but an increase in message was observed in the ovaries of female mosquitoes previously exposed to a blood meal. These results suggest that MGP is a protein that might play a role(s) in mosquito gametogenesis.

Dopa decarboxylase converts L-dopa to dopamine, a precursor molecule for diverse biological activities in insects including neurotransmission and a variety of tanning reactions required for development, reproduction and defence against parasites. Herein, we report the cloning and sequencing of the Aedes aegypti Ddc gene, including 2.1 kb of the upstream promoter region. The transcribed region of the gene spans more than 16 kb and contains five exons. In situ hybridization localizes the blood-meal-induced ovarian transcription of this gene to the follicular epithelial cells surrounding individual oocytes. Ovary tissue transcription of Ddc is increased in response to injection of 20-hydroxyecdysone to levels equal to those observed for blood-fed controls, however coinjection with the translational inhibitor cycloheximide negates the effect, indicating an indirect regulatory role for this hormone. Clusters of putative ecdysone-responsive elements and zinc-finger binding domains for the products of Broad-Complex gene family are identified in the 5'-promoter region. These elements are discussed in the context of common insect Ddc regulatory mechanisms.

An antimicrobial peptide belonging to the cecropin family was isolated from the hemolymph of bacteria-challenged adult Aedes aegypti. This new peptide, named cecropin A, was purified to homogeneity and fully characterized after cDNA cloning. The 34-residue A. aegypti cecropin A is different from the majority of reported insect cecropins in that it is devoid of a tryptophan residue and C-terminal amidation. The importance of these two structural features on the activity spectrum was investigated using a chemically synthesized peptide. A comparison of the antimicrobial activity spectrum of A. aegypti and Drosophila cecropin A showed a lower activity for the mosquito molecule. A. aegypti cecropin mRNA expression was not detected by Northern blot or reverse transcription-polymerase chain reaction analysis in any immature stage of the mosquito, nor in naïve adults, but it was observed in challenged adults 6 h after bacteria inoculation, and it continued over 7-10 days.

Oxygen consumption of single mosquitoes was measured using a differential pressure transducer (DPT) connected to two small chambers. A mosquito was placed in the experimental chamber (P1) and was separated from NaOH by 4 cm2 of marquisette mesh. The reference chamber (P2) contained the same amount of NaOH and the marquisette mesh but without a mosquito. When these two chambers were sealed, removed O2 from P1 resulted in a pressure decrease with respect to P2. This pressure differential was transduced into an output voltage that was directly proportional to the amount of O2 consumed by the mosquito. An array of eight DPTs was interfaced with an IBM 486 PC using an ADAC 5500MF analog to digital converter and software from ADAC (Direct View) to automate the recording procedure. We determined that our apparatus was sensitive enough to detect subtle differences in O2 consumption in mosquitoes subjected to different physiologic conditions.

Aedes albopictus was introduced into the United States in used tires in 1985. Its successful colonization of the upper Midwest has potential to alter the current epidemiology of bunyaviruses that circulate in the region. It is permissive for the replication of several arboviruses, including La Crosse (LACV) and Jamestown Canyon (JCV) bunyaviruses. In this study, we demonstrate the ability of LACV and JCV to coinfect Ae. albopictus mosquitoes and to form all six possible reassortant genotypes. All reassortant viruses infect Ae. albopictus orally and can be transmitted to suckling mice. All reassortants are neurovirulent in mice. However, reassortant viruses carrying the LACV M segment in the foreign genetic background of JCV are more neuroinvasive than JCV, or any other reassortant genotype. In addition, these reassortants can replicate in gerbils and infect Ae. triseriatus, characteristics of LACV, but not JCV. Because Ae. albopictus is spreading into new geographic areas and feeds on a variety of mammals, including humans, it has the potential to transmit new, emerging bunyaviruses in nature.

Aedes aegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10 days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 microM at 24 h post inoculation. cDNAs encoding the preprodefensins of three previously described mature Ae. aegypti defensins were amplified by PCR, cloned and sequenced. Genomic clones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter- and intra-isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty-eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty-three amino acids, a prodefensin region of thirty-six amino acids, and the mature protein domain of forty amino acids. Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B. The defensin gene mapped to chromosome 3, between two known loci, blt and LF168.

We report construction of a comparative linkage map for the mosquito (Culex pipiens) based on restriction fragment length polymorphisms (RFLPs) using cDNA clones from Aedes aegypti as probes to Southern blots of Cx. pipiens genomic DNA. Seventy-one cDNA clones were screened for hybridization and genetic diversity among three Cx. pipiens strains. Fifty-two of 71 cDNA clones, isolated from and previously mapped in Ae. aegypti (73.2%), were hybridized under high-stringency conditions with Cx. pipiens genomic DNA. Thirty-four clones (47.9%) reflected strain-specific polymorphisms. The map consists of 21 cDNA markers that identify 22 loci covering 165.8 cM. The loci mapped in Cx. pipiens cover 7.1 cM on chromosome 1, 80.4 cM on chromosome 2, and 78.3 cM on chromosome 3. Linkage relationships of the RFLP markers for chromosome 1 in Cx. pipiens are the same as chromosome 1 in Ae. aegypti, indicating that chromosome 1 is highly conserved between the two species. The comparative RFLP linkage maps for chromosomes 2 and 3 in Cx. pipiens and Ae. aegypti reflect apparent whole-arm translocations. RFLP markers for chromosome 2 in Ae. aegypti identified homologous loci on one arm of chromosomes 2 and 3 in Cx. pipiens, and RFLP markers for chromosome 3 in Ae. aegypti identified homologous loci on the opposite arms of chromosomes 2 and 3 in Cx. pipiens.

Glutamine synthetase (GS) in the mosquito, Aedes aegypti, is induced in the midgut following a blood meal. Mosquito GS message is detected as soon as 1 h post-blood feeding and remains stable for 18 h. Using a PCR product encoding mosquito GS, a lambda gt10 adult female mosquito cDNA library was screened. A cDNA clone, pCl5A2, encoding the full translation product of mosquito GS was isolated and sequence analyses performed. Mosquito GS cDNA is 2.5 kb in length and its putative translation product shares all the conserved regions characteristic of the GS gene family, including the presumed ATP biding site. Glutamine synthetase activity in the mosquito midgut is highest at 18 h post-blood feeding. Activity can be detected over a broad pH range, from 6.0 to 7.5. Unlike other cellular GS enzymes, mosquito GS is not active in the presence of ATP. Very low dosages (0.05 mM) of L-methionine S-sulfoximine are sufficient to partially inhibit mosquito GS activity. Inhibition of GS disrupts the normal formation of the midgut peritrophic matrix, suggesting that GS enzyme might be involved in the initial pathway of chitin synthesis. The unique expression pattern and inducible nature of the mosquito GS gene make it an interesting candidate for studying promoter function. Additionally, the blood meal activation of the GS gene makes this a potentially valuable tool in mosquito transformation studies.

One of the causative agents of lympahtic filariasis is the nematode parasite Brugia malayi that requires a competent mosquito vector for its development and transmission. Armigeres subalbatus mosquitoes rapidly destroy invading B. malayi microfilariae via a defense response known as melanotic encapsulation. We have constructed a genetic linkage map for this mosquito species using RFLP markers from Aedes aegypti. This heterologous approach was possible because of the conserved nature of the coding sequences used as markers and provided an experimental framework to evaluate the hypothesis that linkage and gene order are conserved between these mosquito species. Of the 56 Ae. aegypti markers tested, 77% hybridize to genomic DNA digests of Ar. subalbatus under stringent conditions, with 53% of these demonstrating strain-specific polymorphisms. Twenty-six Ae. aegypti markers have been mapped using an F2- segregating Ar. subalbatus population derived from a cross of strains originating in Japan and Malaysia. Linear order of these marker loci is highly conserved between the two species. Only 1 of these markers, LF92, was not linked in the manner predicted by the Ae. aegypti map. In addition, the autosomal sex-determination locus that occurs in linkage group 1 in Ae. aegypti resides in group 3 in Ar. subalbatus. The Ar. subalbatus map provides a basic genetic context that can be utilized in further genetic studies to clarify the genetic basis of parasite resistance in this mosquito and is a necessary precursor to the identification of genome regions that carry genes that determine the encapsulation phenotype. [The composite map and sequence database information for Ae. aegypti markers can be retrieved directly from the Ae. aegypti Genome Database through the World Wide Web: http://klab.agsci.colostate.edu.]

Restriction fragment length polymorphism (RFLP) markers were used to evaluate Aedes aegypti genome structure and genetic variability within and between substrains selected for different levels of refractoriness to the malaria parasite, Plasmodium gallinaceum. The MOYO-R substrain was previously selected for complete refractoriness and the MOYO-IS substrain for intermediate susceptibility from the Moyo-In-Dry (MOYO) strain by selective inbreeding (F = 0.5). Eighteen mapped RFLP markers were used to provide coverage of the mosquito genome. The two substrains showed reduced genetic diversity compared with the MOYO strain, including significant reductions in mean heterozygosity, number of alleles per locus, and proportion of polymorphic loci. Genetic differentiation between the two substrains was statistically significant, as reflected by differences in allele frequencies. Significant pairwise linkage disequillbrium among the RFLP loci was detected in all three strains, most evidently in the MOYO strain. This is surprising because the RFLP loci examined are separated by large map distances, and therefore linkage disequilibrium should decay to zero after many generations of laboratory culture. Our hypothesis to explain this phenomena is that lack of recombination, or low recombination rates in some regions of the A. aegypti genome, is a result of chromosome inversions. Finally, we used graphical genotyping, wherein whole genome genotypic information for individual mosquitoes is represented in a simple graphic format, to illustrate genome structure and allelic variation within and among the mosquito strains. Our analysis revealed an apparent chromosomal deletion on chromosome 3 for some individuals in the MOYO strain and MOYO-IS substrain.

The effect of host immune activation on the development of Brugia malayi in one susceptible and four refractory strains of Aedes aegypti and in Armigeres subalbatus was assessed. A. aegypti that were immune activated by the injection of saline or bacteria 24 hr before feeding on a B. malayi-infected gerbil had significantly reduced prevalences and mean intensities of infection from those of naive controls when exposed to bloodmeals with low (105 mf/20 microliters) and medium (160 mf/20 microliters) microfilaremias. At a higher microfilaremia (237 mf/20 microliters) there were no significant differences in mean intensities, suggesting that the number of parasites ingested may affect the host's ability to mount an effective defense response. Because the major immune proteins in A. aegypti are defensins, we did Northern analyses of fat body RNA 8 hr after immune activation or bloodfeeding. All mosquitoes demonstrated rapid transcriptional activity for defensins following immune activation by intrathoracic inoculation with either saline or bacteria. However, no strain of A. aegypti, susceptible or refractory to B. malayi, nor Ar. subalbatus produced defensin transcripts after bloodfeeding on an uninfected or a B. malayi-infected gerbil. These data suggest that inducible immune proteins of mosquitoes can reduce the prevalence and mean intensity of infections with ingested parasites, but these proteins are not expressed routinely after parasite ingestion and midgut penetration and probably do not contribute to existing refractory mechanisms. Immune proteins such as defensins, however, represent potential candidates to genetically engineer mosquitoes for resistance to filarial worms.

Dopa decarboxylase (DDC) functions in insect catecholamine biochemistry to produce materials essential for cross-linking reactions that result in tanning and/or melanization, include tanning of the mosquito egg chorion and encapsulation of parasites. We have cloned Ddc from the mosquito, Aedes aegypti, and studied its expression in response to blood-feeding, which initiates events necessary for egg maturation in mosquitoes. The Ae. aegypti Ddc cDNA was isolated via heterologous screening using a clone from Drosophila melanogaster. A resulting 1.87 kilobase (kb) clone was sequenced to reveal an open reading frame of 1464 bp, as well as 5'- and 3'-untranslated segments. The inferred amino acid sequence of this clone shares 81% identity with the published Drosophila Ddc cDNA, including complete identity with twenty-four contiguous amino acids encompassing the pyridoxal-5-phosphate cofactor binding domain. Analysis of an F2 intercross population derived from a parental cross between two Ae. aegypti strains (Hamburg and Moyo-In-Dry) allowed us to map Ddc to a locus on linkage group 2. Expression studies demonstrated the presence of a 2.1 kb message, the majority of which occurs in the ovaries where Ddc-specific mRNA is up-regulated in response to ingestion of a blood meal. The potential for egg-tanning in anautogenous mosquitoes as a model for understanding specific genetic events in the regulation of catecholamine metabolism is addressed.

Peroxidase activity is detectable in Aedes aegypti ovaries, containing developing eggs, at 24 h following blood feeding, and peak peroxidase activity is reached at 36-48 h after the blood-meal. Peroxidase is associated with the chorion layer in mature eggs and the majority of the enzyme is released from the chorion layer by treating the isolated chorion fraction with SDS/urea. Analysis of the SDS/urea solubilized chorion proteins using SDS-PAGE with tropolone/H2O2 or dopa staining verified the presence of both peroxidase and phenol oxidase in the released chorion proteins. The molecular weight of chorion peroxidase is about 61,000 Da as determined by SDS-PAGE analysis. Incubation of the solubilized chorion proteins with tyrosine and H2O2 produces dityrosine, and hyrolysis of hardened egg chorion results in the detection of dityrosine and trityrosine in the chorion hydrolysate. Data suggest that chorion peroxidase is involved in the hardening of the mosquito egg chorion by catalyzing the formation of ditryrosine through tyrosine residues on structural proteins. The overall hardening of the A. aegypti egg chorion includes both peroxidase-mediated chorion protein crosslinking through dityrosine formation and phenol oxidase-catalyzed chorion melanization.

Because intensity of infection was significantly increased in a substrain of Aedes aegypti selected for susceptibility to the filarial worm, Brugia malayi, experiments were designed to assess numbers of microfilariae (mf) ingested and midgut penetration by mf in this susceptible substrain as compared to a refractory substrain selected from the same parental stock. Refractory mosquitoes ingested significantly fewer mf than susceptible mosquitoes and significantly fewer numbers of mf penetrated through refractory midguts as compared to susceptible midguts. In 16.7% of the refractory midguts, no mf were able to penetrate the midgut and in three refractory mosquitoes over 250 mf were ingested, but no mf penetrated the midgut. These results indicate that permissiveness of the midgut for penetration by microfilariae can determine not only parasite intensity, but also prevalence of infection. The genetic basis for ingestion of mf and midgut penetration was assessed using restriction fragment length polymorphism markers and quantitative trait loci (QTL) mapping. This mapping identified a QTL on chromosome 2, idb[2,LF181] (idb, intensity determinant for Brugia), that seems to influence ingestion ability. This QTL is linked to a previously identified QTL for susceptibility to B. malayi, fsb[2,LF98], as well as to loci for susceptibility to the malaria parasite, Plasmodium gallinaceum, and yellow fever virus. These results suggest that this region of chromosome 2 contains one or more genes that influence susceptibility of A. aegypti to several mosquito-transmitted pathogens.

The mechanisms by which hemocytes mediate a mosquito's defense response to parasites or pathogens are not well understood. This is due in part to difficulty in collecting intact mosquito hemocytes for experiments and to a lack of reagents, such as antibodies. Our objectives were to collect adult Aedes aegypti hemocytes under conditions suitable for immunofluorescence microscopy, and to test whether monoclonal antibodies, generated against larval Manduca sexta hemocytes, bind adult Ae. aegypti hemocytes. We present immunofluorescence micrographs of M. sexta and Ae. aegypti hemocytes stained by six monoclonal antibodies. Two antibodies, MS11 and MS32, immunolocalized to hemocyte nuclei in both species. On Western blots, these antibodies generate one signal at approximately 40 kDa and four others between 10 and 25 kDa. Immunofluorescence staining patterns of the other four antibodies were more complex. That these antibodies bind hemocytes from both species suggests significant molecular similarities exist between hemocytes from evolutionarily divergent species.

The injection of Escherichia coli and Micrococcus luteus into the hemocoel of Aedes aegypti induces a potent antibacterial activity in the hemolymph. We have purified and fully characterized three 40-residue antibacterial peptides from the hemolymph of bacteria-challenged mosquitoes that are absent in naive mosquitoes. The peptides are potently active against Gram-positive bacteria and against one of the Gram-negative bacteria that were tested. The amino acid sequences clearly show that the three peptides are novel isoforms of the insect defensin family of antibacterial peptides. They differ from each other by one or two amino acid residues. We present here the complete amino acid sequences of the three isoforms and the activity spectrum of the predominant Aedes defensin.

Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F2 populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs[2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filarial nematode Brugia malayi and the yellow fever virus.

Aedes albopictus and Aedes aegypti are members of the mosquito family Culicidae and share a haploid chromosome complement of three. Although a genetic linkage map based on restriction fragment length polymorphism (RFLP), markers exists for Ae. aegypti, the extent of synteny and linkage order conservation between the two species was unknown. A comparative linkage map for Ae. albopictus was constructed based mainly on cDNA clones from Ae. aegypti. Nearly all Ae. aegypti probes hybridized to Ae. albopictus DNA at high stringency. For eighteen RFLP markers tested, the linkage group and linear order appears to be identical for the two species. 78% of the loci tested exhibited significant deviations from the expected segregation ratio in at least one of the test crosses. An excess of heterozygote genotypes was recovered with most loci. This probably reflects the effects of lethal loci on survival of F2 progeny homozygous for the parental genotypes. These results demonstrate that comparative linkage maps based on common DNA markers provide a basis for rapidly developing linkage maps for various mosquito species, and the opportunity to examine the significance and function of orthologous quantitative trait loci associated with mosquito vector competence for disease transmission.

Phenol oxidase (PO) and dopachrome conversion enzyme (DCE) were partially purified from Aedes aegypti larvae by ammonium sulfate fractionation. PO from A. aegypti functions in the hydroxylation of monophenols (e.g., tyrosine and tyramine) to their related o-diphenols, and the oxidation of o-diphenols (e.g., L-dopa, dopamine, N-acetyldopamine) to their respective o-quinones. Partially purified DCE showed high specificity toward dopachrome generated from dopa with the L-configuration. The combined effects of PO and DCE significantly accelerated melanization pathways when L-dopa was used as substrate. Significant DCE activity also was detected in hemolymph samples from adult, female A. aegypti, and undoubtedly plays a role in melanotic encapsulation reactions.

We report on the initial characterization of an 84-kDa polypeptide that is differentially expressed in Aedes aegypti during melanotic encapsulation immune reactions against filarial worms. [35S]Methionine-labeled hemolymph from mosquitoes inoculated with saline, parasites that are melanized, or parasites that are not melanized was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results show that the level of the 84-kDa polypeptide increases considerably in those mosquitoes undergoing encapsulation reactions against parasites but remains down-regulated in those mosquitoes exposed to parasites that are not melanized or are undergoing wound healing responses (saline-inoculated). Experiments involving glycosidase treatment of hemolymph samples indicate that this polypeptide is not heavily glycosylated. Amino acid microsequencing was performed and two internal sequence fragments (15 continuous amino acids and 12 noncontinuous amino acids) were obtained. Analysis of these sequences to known sequences in a protein database did not yield any conclusive information as to the identify of the 84-kDa polypeptide. Therefore, degenerate oligonucleotide primers were designed, based on the sequence of the 15-amino-acid fragment, and used with the polymerase chain reaction (PCR) to amplify from A. aegypti genomic DNA the region between the primers. The PCR product was cloned and sequenced to verify that the nucleic acid sequence matched the known protein sequence. Screening of an A. aegypti cDNA library with this small PCR-generated clone resulted in the selection of an approximately 540-bp clone. Northern analysis with this larger cDNA clone indicates that it hybridizes to an approximately 2.0-kb message that is differentially expressed in mosquitoes undergoing melanotic encapsulation reactions against filarial worms. Furthermore, sequencing of this approximately 540-bp clone showed that it contained the 15-amino-acid sequence that had been used to design the degenerate PCR primers, indicating that an appropriate clone was selected. However, sequence analysis of this clone at the protein and nucleic acid level did not provide any conclusive answers to the identity or function of the 84-kDa polypeptide.

Several studies have demonstrated a genetic basis for variation in susceptibility of Aedes aegypti to Plasmodium gallinaceum. Although 25 yr ago it was reported that P. gallinaceum susceptibility in Ae. aegypti is determined primarily by a single autosomal dominant gene, evidence for additional genetic factors has emerged. Two sublines, 1 refractory and 1 of intermediate susceptibility to P. gallinaceum, have been selected from the Moyo-In-Dry strain (MOYO) of Ae. aegypti. Prior to selection, the MOYO population was 20.3% refractory. Genetic crosses of the highly susceptible Rockefeller strain (ROCK) and the 2 selected sublines of the MOYO strain provide evidence for a complex mode of inheritance of Plasmodium susceptibility in Ae. aegypti.

Two quantitative trait loci (QTL) affecting susceptibility of the mosquito Aedes aegypti to the filarial worm parasite Brugia malayi were identified using restriction fragment length polymorphism (RFLP) markers. The first locus, fsb[1,LF178], resides within a 10 cM interval on chromosome 1 and exhibits a recessive effect with respect to susceptibility. The second locus, fsb[2,LF98], resides within a 9 cM interval on chromosome 2 and exhibits an additive effect on susceptibility. Significant epistasis was detected between these loci, although the effect of fsb[2,LF98] was dependent on the genetic background of the mosquito strains. Suggestions for a standard QTL nomenclature are included.

We report construction of a genetic linkage map for the mosquito, Aedes aegypti, based on restriction fragment length polymorphisms (RFLPs). The map consists of 50 DNA markers that identify 53 loci covering 134 map units across three linkage groups. Determination of linkage associations between RFLP markers and several mutant marker loci allowed for partial integration of the RFLP markers with an existing classical genetic linkage map for A. aegypti. The RFLP markers include 42 random cDNA clones, three random genomic DNA clones, and five cDNA clones of known genes. We discuss the influence of autosomal sex determination, characteristic of culicine mosquitoes, in relation to its observed influence on segregation ratios. This has important ramifications for future efforts to identify quantitative trait loci associated with the ability of these mosquitoes to transmit various pathogens and parasites to man and other animals.

Comparisons were made of in vivo labeled polypeptides from Aedes aegypti strains refractory to either Brugia malayi or Dirofilaria immitis. There does not seem to be a generalized "anti-parasite" polypeptide response that mosquitoes refractory to filarial worm infection produce following bloodfeeding. Instead, it seems that any response produced by these mosquitoes is localized to the tissue in which the filarial parasite develops.

Translation products were used to monitor changes in gene expression between different strains and developmental stages of Aedes aegypti. Total RNA was isolated from fourth stadium larvae, male and female pupae, and male and female adults collected at fixed time intervals following pupation and ecdysis. Differences in RNA populations were assessed by in vitro translation followed by one- and two-dimensional polyacrylamide gel electrophoresis and fluorography. Variations in gene expression between sexes and during development were examined in Liverpool (LVP) and Rockefeller (RKF) Ae. aegypti strains. Sex-related differences consisted primarily of differing lengths of expression for certain polypeptides, although two-dimensional electrophoresis revealed changes in intensity of a 44-kD polypeptide with a pI of 7.9 between males and females. Fourth stadium larvae, pupae, and adults expressed different translation products, which probably correlated with developmental differences. Strain-related differences were observed between LVP and RKF.

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity in hemocytes and cell-free plasma perfused from 7-, 14-, 21-, and 28-day-old Aedes aegypti mosquitoes were compared. A progressive decrease of enzyme activity was detected as mosquito age increased, and this decrease was significant in both hemocytes and cell-free plasma when mosquitoes were 28 days old as compared with that found in 7-day-old mosquitoes. There was no significant difference in total hemolymph protein as mosquito age increased. Although this decreased MPO and DPO activity might be partially responsible for the reduced immune response against filarial worms previously reported for older mosquitoes, other factors undoubtedly play a significant role.

Brugia malayi and Wuchereria bancrofti are mosquito-borne parasitic nematodes responsible for lymphatic filariasis in approximately 90 million people. The genetic control of the susceptibility of Aedes aegypti mosquitoes to B. malayi was well defined 30 years ago, but no data have since been provided regarding the gene products responsible for susceptibility or refractoriness or both. We addressed this problem by assessing polypeptide synthesis in thoracic tissue, the developmental site of this parasite, in susceptible and refractory strains of A. aegypti. Polyacrylamide gel electrophoresis of radiolabeled polypeptides synthesized in vivo were compared between (i) established susceptible and refractory strains and (ii) a refractory strain newly isolated from the established susceptible strain. Six polypeptide differences recognized by SDS/PAGE and two-dimensional gel electrophoresis were seen only in the refractory strains after they took a blood meal. A seventh polypeptide was present in those refractory mosquitoes that had ingested sucrose but increased in intensity after blood-feeding. The presence of parasites in the blood meal was not necessary to stimulate the synthesis of these polypeptides. These refractory strain-associated molecules may mediate genetically determined variation in susceptibility.

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity was assessed in hemocytes, cell-free plasma and complete hemolymph collected from Aedes aegypti Liverpool strain, intrathoracically inoculated with saline alone, immune activated by the inoculation of Dirofilaria immitis microfilariae (mff), and uninoculated. Enzyme activities between groups were compared using a radiometric hydroxylation assay (MPO) and a high pressure liquid chromatography with electrochemical detection assay (DPO). There were no significant differences in enzyme activity in hemocytes, cell-free plasma, and complete hemolymph between uninoculated and saline-inoculated controls. Both MPO and DPO activity of mosquito hemocytes and complete hemolymph from immune-activated mosquitoes were significantly increased at 12 and 24 h postinoculation as compared with the enzyme activity from saline-inoculated mosquitoes, but no significant increase in enzyme activity was detected in cell-free plasma from immune-activated mosquitoes. Increases of MPO and DPO activity in hemocytes and hemolymph following immune activation were proportional, thereby suggesting that a single enzyme might react with both monophenols and o-diphenols within the hemolymph of A. aegypti. Results also suggest that augmented phenol oxidase activity associated with melanotic encapsulation reactions is associated primarily with hemocytes.

Hemocytes from adult, female Aedes aegypti, intrathoracically inoculated with microfilariae (mf) of the nematode Dirofilaria immitis, were compared to saline-inoculated and uninoculated controls using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 125I-labeling, and wheat germ agglutinin (WGA) binding techniques. Activation of wound healing and/or melanotic encapsulation responses by the inoculation of saline or mf into the host hemocoel induced alterations in the hemocyte activity of these mosquitoes. Protein assays of whole hemocyte lysates revealed that hemocytes from saline- and mf-inoculated mosquitoes had higher protein concentrations than uninoculated controls. Many polypeptides were seen within all three hemocytes preparations when stained with silver nitrate, but there was an overall increase in protein synthesis in hemocytes from inoculated mosquitoes. In addition, a 200-kDa polypeptide was uniquely expressed in hemocytes from inoculated mosquitoes. There were several prominent surface proteins labeled with 125I, and several of these increased dramatically in intensity during wound healing and/or a melanotic encapsulation response. Similar results were seen in two-dimensional separations. A set of basic polypeptides comigrated with an acidic polypeptide resulting in a surface protein of approximately 80-90 kDa that increased in inoculated mosquitoes. Hemocytes from inoculated mosquitoes exhibited a group of three acidic polypeptides, whereas hemocytes from uninoculated mosquitoes exhibited only one of these protein fragments. Three surface polypeptides bound 125I-labeled WGA, and binding of WGA to hemocyte surface polypeptides was successfully inhibited by the incubation of cells with the lectin and its competing sugar.

[35S]Methionine-labeled hemolymph polypeptides from adult, female Aedes aegypti Liverpool strain mosquitoes inoculated with the microfilariae of the filarial nematode Dirofilaria immitis were compared with those from saline-inoculated and uninoculated controls by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. SDS-PAGE analysis of cell-free hemolymph collected via perfusion at 6, 12, 24, 48, 72, and 96 hr postinoculation (PI) detected the enhanced expression of an 84-kDa polypeptide. This polypeptide, expressed constitutively in the hemolymph of all three groups of mosquitoes, increased considerably in inoculated mosquitoes as time progressed as compared with uninoculated controls. Moreover, the 84-kDa polypeptide was expressed at higher levels in D. immitis-inoculated mosquitoes than in saline-inoculated controls. This stimulation of de novo biosynthesis of the 84-kDa polypeptide in inoculated mosquitoes may play a role in the immune response of mosquitoes. Since it is likely that the wound healing response in insects involves many of the same chemical processes as occur in melanotic encapsulation reactions of mosquitoes against filarial worms, the preferential expression of the 84-kDa polypeptide in saline-inoculated mosquitoes seen in this study may reflect a wound healing response. The greater increase in synthesis of this protein in D. immitis-inoculated mosquitoes may reflect production of melanotic material required for parasite destruction as well as for wound healing.

Tyrosine and catecholamines have been implicated as substrates for the encapsulation reactions involved in the immune response of mosquitoes to microfilariae (mff). Identification and quantitation of tyrosine and catecholamines present in Aedes aegypti hemolymph plasma were accomplished by ion-pair high-pressure liquid chromatography with electrochemical detection at either +650 or +850 mV vs Ag/AgCl. Tyrosine, dopamine, and N-beta-alanyldopamine were detected in the hemolymph plasma of naive A. aegypti. Although no differences in these compounds were observed in hemolymph plasma from A. aegypti inoculated with Dirofilaria immitis mff, the chromatogram showed a single major peak (PI) (65 microM, expressed as dopamine equivalents) that was not present in naive hemolymph plasma. Saline-inoculated controls contained only 5% of the PI in immune reactive hemolymph plasma. A high concentration of PI (127 +/- 39 microM) was also detected after treatment of hemolymph plasma with mild alkaline conditions (pH 9.0), indicating that it is normally present as an electrochemically inert form in naive mosquitoes. High concentrations of PI were also detected in the naive hemolymph plasma from three other mosquito species, but no PI was found in A. trivittatus under any conditions. PI did not cochromatograph with any of the catecholamines commonly thought to be involved in immune responses of dipterans against metazoan parasites, suggesting that it may be a unique substrate for these reactions. The biological relevance of PI was evidenced by its appearance in the hemolymph plasma of two strains of D. immitis-inoculated A. aegypti.

The effect of gamma irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15-20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15-20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External gamma irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in gamma-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff.

Hemocytes were perfused from uninoculated, saline-inoculated, and Dirofilaria immitis microfilariae-inoculated Aedes trivittatus and assessed for their capacity to bind wheat germ agglutinin (WGA). Approximately one-fourth of perfused hemocytes were WGA positive in all groups of mosquitoes at all time periods tested. Results suggest A. trivittatus has a larger inherent population of immune-competent hemocytes as compared with Aedes aegypti and, therefore, is faster and more effective in killing microfilariae by melanotic encapsulation reactions.

The inherent ability of Brugia malayi and Brugia pahangi (Nematoda) to establish successful relationships with the mosquitoes Armigeres subalbatus and Aedes aegypti Liverpool strain was evaluated. Brugia pahangi microfilariae (mff) avoided the immune response and developed normally in A. subalbatus exposed to the parasite by an infective bloodmeal, whereas nearly 85% of B. malayi were destroyed by the immune response. Because A. aegypti supports the development of both filarial worm species but destroys intrathoracically inoculated B. pahangi isolated from jird blood, blood-isolated B. malayi were inoculated into A. aegypti, and the immune response was compared with that observed against B. pahangi. The response against B. malayi was significantly more rapid and effective than the response against B. pahangi. Similar results were obtained when blood-isolated B. pahangi or B. malayi were inoculated into A. subalbatus. Microfilariae of both species were able to avoid immune destruction in A. aegypti if they were allowed to penetrate the Liverpool midgut in vitro prior to inoculation. Most B. pahangi that had first penetrated an Armigeres midgut prior to inoculation into A. subalbatus were able to avoid the immune response, but by day 3 postinoculation, less than 40% of the B. malayi, treated in the same manner, were able to escape the immune response. Genetic susceptibility of mosquitoes to infection by filarial worms and potential mechanisms of immune evasion/suppression are discussed regarding B. malayi and B. pahangi.

Aedes trivittatus were experimentally exposed through a membrane to a concentration of trivittatus (TVT) virus in dog defibrinated blood of 10(3.4)-10(3.5) SMICLD 50/0.025 ml. Transovarial and transstadial transmission was demonstrated. Fifty-five percent of the infected mosquitoes tested transovarially transmitted TVT virus. TVT virus was isolated from 16% of the F1 generation progeny examined, including 19% of the larvae, 23% of adult females, and 10% of adult males.

Monophenol oxidase (MPO) activity in hemocytes collected from Aedes aegypti Liverpool strain and Aedes trivittatus intrathoracically inoculated with saline alone, inoculated with Dirofilaria immitis microfilariae (mff), or from uninoculated mosquitoes was compared using a radiometric tyrosine hydroxylation assay. Hemocyte MPO activity in mff-inoculated (= immune-activated) mosquitoes was significantly increased at 24 hr postinoculation (PI) in A. aegypti and at 6, 12, and 24 hr PI in A. trivittatus as compared with saline-inoculated controls. Baseline and immune-activated levels of hemocyte MPO activity in A. trivittatus were significantly higher compared with those seen in A. aegypti. Baseline hemocyte population levels were similar in both species, but immune activation did not elicit increases in total hemocyte populations in A. trivittatus as has been demonstrated for A. aegypti. Likewise, immune activation by the inoculation of mff did not significantly alter plasma MPO activity in A. trivittatus as compared with uninoculated or saline-inoculated mosquitoes. Plasma MPO activity in A. aegypti, however, appears to constitute a major component of the immune response. The importance of phenol oxidase(s) in the immune response of mosquitoes against mff and the relationship of observed differences in MPO activity to differences in immunological capability between A. aegypti and A. trivittatus are assessed.