Can Horton hear the whos? The importance of scale in mosquito-borne disease.

The epidemiology of vector-borne pathogens is determined by mechanisms and interactions at different scales of biological organization, from individual-level cellular processes to community interactions between species and with the environment. Most research, however, focuses on one scale or level with little integration between scales or levels within scales. Understanding the interactions between levels and how they influence our perception of vector-borne pathogens is critical. Here two examples of biological scales (pathogen transmission and mosquito mortality) are presented to illustrate some of the issues of scale and to explore how processes on different levels may interact to influence mosquito-borne pathogen transmission cycles. Individual variation in survival, vector competence, and other traits affect population abundance, transmission potential, and community structure. Community structure affects interactions between individuals such as competition and predation, and thus influences the individual-level dynamics and transmission potential. Modeling is a valuable tool to assess interactions between scales and how processes at different levels can affect transmission dynamics. We expand an existing model to illustrate the types of studies needed, showing that individual-level variation in viral dose acquired or needed for infection can influence the number of infectious vectors. It is critical that interactions within and among biological scales and levels of biological organization are understood for greater understanding of pathogen transmission with the ultimate goal of improving control of vector-borne pathogens.

Widespread evidence for interspecific mating between Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in nature.

Aedes aegypti and Aedes albopictus, two important vectors of the dengue and chikungunya viruses to humans, often come in contact in their invasive ranges. In these circumstances, a number of factors are thought to influence their population dynamics, including resource competition among the larval stages, prevailing environmental conditions and reproductive interference in the form of satyrization. As the distribution and abundance of Ae. aegypti and Ae. albopictus have profound epidemiological implications, understanding the competitive interactions that influence these patterns in nature is important. While evidence for resource competition and environmental factors had been gathered from the field, the evidence for reproductive interference, though strongly inferred through laboratory trials, remained sparse (one small-scale field trial). In this paper we demonstrate that low rates (1.12-3.73%) of interspecific mating occur in nature among populations of these species that have co-existed sympatrically from 3 to 150yrs. Finally this report contributes a new species-specific primer set for identifying the paternity of sperm extracted from field collected specimens.

Aedes aegypti glutamine synthetase: expression and gene structure.

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.

Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene.

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.

Aedes aegypti peroxidase gene characterization and developmental expression.

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).

Cloning and characterization of a chitin synthase cDNA from the mosquito Aedes aegypti.

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 Apyrase gene of the vector mosquito, Aedes aegypti, is expressed specifically in the adult female salivary glands.

The yellow fever mosquito, Aedes aegypti, expresses a gene, Apyrase (Apy), that encodes an ATP-diphosphohydrolase. The product of this gene is a secreted enzyme that facilitates hematophagy by preventing platelet aggregation in the host. Apy gene expression is limited to the cells of the distal-lateral and medial lobes of the adult female salivary glands. Apyrase protein levels, detectable by antibodies, peak in the salivary glands about 4 days after adult emergence and remain high after a blood meal. Primary sequence analysis of a genomic clone encoding apyrase reveals a unique TAAATA sequence and seven introns, as well as other conserved features of eukaryotic genes. The temporal, sex- and tissue-specific expression of the Apy gene is consistent with its role as encoding a platelet anti-aggregation factor that functions to facilitate hematophagy and decrease probing time.

The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5'-nucleotidase family.

The saliva of hematophagous insects contains a variety of pharmacologically active substances that counteract the normal hemostatic response to injury in vertebrate hosts. The yellow-fever mosquito, Aedes aegypti, secretes an apyrase that inhibits ADP-dependent platelet aggregation. Apyrase was purified as an active enzyme from adult female salivary glands and subjected to tryptic digestion, and the resulting peptides were sequenced. The amino acid sequences obtained match the conceptual translation product of a cDNA clone isolated from an adult female salivary gland library. Sequence comparisons indicate similarities with a ubiquitous family of 5'-nucleotidases. The mosquito protein differs from other members of the family by lacking a carboxyl-terminal hydrophobic domain. The apparent conversion of a gene encoding an enzyme involved in a common metabolic event at the cellular level to a gene involved in the antihemostatic response of mosquitoes illustrates one way this particular insect has adapted to the challenges of bloodfeeding.

Glucosamine:fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypti.

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.

A novel member of the RING-finger gene family associated with reproductive tissues of the mosquito, Aaedes aegypti.

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.

Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I.

Flavodoxin and ferredoxin I have both been implicated as components of the electron transport chain to nitrogenase in the aerobic bacterium Azotobacter vinelandii. Recently, the genes encoding flavodoxin (nifF) and ferredoxin I (fdxA) were cloned and sequenced and mutants were constructed which are unable to synthesize either flavodoxin (DJ130) or ferredoxin I (LM100). Both single mutants grow at wild-type rates under N2-fixing conditions. Here we report the construction of a double mutant (DJ138) which does not synthesize either flavodoxin or ferredoxin I. When plated on ammonium-containing medium, this mutant had a very small colony size when compared with the wild type, and in liquid culture with ammonium, this double mutant grew three times slower than the wild type or single mutant strains. This demonstrated that there is an important metabolic function unrelated to nitrogen fixation that is normally carried out by either flavodoxin or ferredoxin. If either one of these proteins is missing, the other can substitute for it. The double mutant phenotype can now be used to screen site-directed mutant versions of ferredoxin I for functionality in vivo even though the specific function of ferredoxin I is still unknown. The double mutant grew at the same slow rate under N2-fixing conditions. Thus, A. vinelandii continues to fix N2 even when both flavodoxin and ferredoxin I are missing, which suggests that a third as yet unidentified protein also serves as an electron donor to nitrogenase.

Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti.

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.

Aedes aegypti dopa decarboxylase: gene structure and regulation.

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.