Evaluation of large volume yeast interfering RNA lure-and-kill ovitraps for attraction and control of Aedes mosquitoes.

Aedes mosquitoes (Diptera: Culicidae), principle vectors of several arboviruses, typically lay eggs in man-made water-filled containers located near human dwellings. Given the widespread emergence of insecticide resistance, stable and biofriendly alternatives for mosquito larviciding are needed. Laboratory studies have demonstrated that inactivated yeast interfering RNA tablets targeting key larval developmental genes can be used to facilitate effective larvicidal activity while also promoting selective gravid female oviposition behaviour. Here we examined the efficacy of transferring this technology toward development of lure-and-kill ovitraps targeting Aedes aegypti (L.) and Aedes albopictus (Skuse) female mosquitoes. Insectary, simulated field and semi-field experiments demonstrated that two mosquito-specific yeast interfering RNA pesticides induce high levels of mortality among larvae of both species in treated large volume containers. Small-scale field trials conducted in Trinidad, West Indies demonstrated that large volume ovitrap containers baited with inactivated yeast tablets lure significantly more gravid females than traps containing only water and were highly attractive to both A. aegypti and A. albopictus females. These studies indicate that development of biorational yeast interfering RNA-baited ovitraps may represent a new tool for control of Aedes mosquitoes, including deployment in existing lure-and-kill ovitrap technologies or traditional container larviciding programs.

Divergent host preferences of above- and below-ground Culex pipiens mosquitoes and their hybrid offspring.

Culex pipiens form pipiens and Cx. pipiens form molestus (Diptera: Culicidae) belong to a cosmopolitan taxonomic group known as the Pipiens Assemblage. Hybridization between these forms is thought to contribute to human transmission of West Nile virus (WNV) in North America. Complementary choice and no-choice landing assays were developed to examine host acceptance by North American Cx. pipiens in the laboratory. Populations collected from above- and below-ground sites in suburban Chicago were identified as forms pipiens and molestus using a polymerase chain reaction-based assay. Avian and human host acceptance was then quantified for the two populations, as well as for their hybrid and backcross offspring. No-choice tests were used to demonstrate that both the pipiens and molestus forms were capable of feeding on human and avian hosts. Choice tests were used to demonstrate that form pipiens females were strongly avian-seeking; an individual's probability of accepting the chick host was 85%. Form molestus females were more likely to accept the human host (87%). Rates of host acceptance by F1 and backcross progeny were intermediate to those of their parents. The results suggest that host preferences in Cx. pipiens are genetically determined, and that ongoing hybridization between above- and below-ground populations is an important contributor to epizootic transmission of WNV in North America.

Insecticide resistance in two Aedes aegypti (Diptera: Culicidae) strains from Costa Rica.

Dengue (family Flaviridae, genus Flavivirus, DENV) and dengue hemorrhagic fever (DHF) are presently important public health problems in Costa Rica. The primary strategy for disease control is based on reducing population densities of the main mosquito vector Aedes aegypti (L.) (Diptera: Culicidae). This is heavily dependent on use of chemical insecticides, thus the development of resistance is a frequent threat to control program effectiveness. The objective of this study was to determine the levels of insecticide resistance and the metabolic resistance mechanisms involved in two Ae. aegypti strains collected from two provinces (Puntarenas and Limon) in Costa Rica. Bioassays with larvae were performed according to World Health Organization guidelines and resistance in adults was measured through standard bottle assays. The activities of beta-esterases, cytochrome P450 monooxygenases, and glutathione S-transferases (GST), were assayed through synergists and biochemical tests, wherein the threshold criteria for each enzyme was established using the susceptible Rockefeller strain. The results showed higher resistance levels to the organophosphate (OP) temephos and the pyrethroid deltamethrin in larvae. The efficacy of commercial formulations of temephos in controlling Ae. aegypti populations was 100% mortality up to 11 and 12 d posttreatment with daily water replacements in test containers. Temephos and deltamethrin resistance in larvae were associated with high esterase activity, but not to cytochrome P450 monooxygenase or GST activities. Adult mosquitoes were resistant to deltamethrin, and susceptible to bendiocarb, chlorpyrifos, and cypermethrin. Because temephos and deltamethrin resistance are emerging at the studied sites, alternative insecticides should be considered. The insecticides chlorpyrifos and cypermethrin could be good candidates to use as alternatives for Ae. aegypti control.

RNAi knock-downs support roles for the mucin-like (AeIMUC1) gene and short-chain dehydrogenase/reductase (SDR) gene in Aedes aegypti susceptibility to Plasmodium gallinaceum.

The mosquito midgut represents the first barrier encountered by the Plasmodium parasite (Haemosporida: Plasmodiidae) when it is ingested in blood from an infected vertebrate. Previous studies identified the Aedes aegypti (L.) (Diptera: Culicidae) mucin-like (AeIMUC1) and short-chain dehydrogenase/reductase (SDR) genes as midgut-expressed candidate genes influencing susceptibility to infection by Plasmodium gallinaceum (Brumpt). We used RNA inference (RNAi) by double-stranded RNA (dsRNA) injections to examine ookinete survival to the oocyst stage following individual gene knock-downs. Double-stranded RNA gene knock-downs were performed 3 days prior to P. gallinaceum infection and oocyst development was evaluated at 7 days post-infection. Mean numbers of parasites developing to the oocyst stage were significantly reduced by 52.3% in dsAeIMUC1-injected females and by 36.5% in dsSDR-injected females compared with females injected with a dsß-gal control. The prevalence of infection was significantly reduced in dsAeIMUC1- and dsSDR-injected females compared with females injected with dsß-gal; these reductions resulted in a two- and three-fold increase in the number of uninfected individuals, respectively. Overall, these results suggest that both AeIMUC1 and SDR play a role in Ae. aegypti vector competence to P. gallinaceum.

Coadaptation of isoacceptor tRNA genes and codon usage bias for translation efficiency in Aedes aegypti and Anopheles gambiae.

The transfer RNAs (tRNAs) are essential components of translational machinery. We determined that tRNA isoacceptors (tRNAs with different anticodons but incorporating the same amino acid in protein synthesis) show differential copy number abundance, genomic distribution patterns and sequence evolution between Aedes aegypti and Anopheles gambiae mosquitoes. The tRNA-Ala genes are present in unusually high copy number in the Ae. aegypti genome but not in An. gambiae. Many of the tRNA-Ala genes of Ae. aegypti are flanked by a highly conserved sequence that is not observed in An. gambiae. The relative abundance of tRNA isoacceptor genes is correlated with preferred (or optimal) and nonpreferred (or rare) codons for ∼2-4% of the predicted protein coding genes in both species. The majority (∼74-85%) of these genes are related to pathways involved with translation, energy metabolism and carbohydrate metabolism. Our results suggest that these genes and the related pathways may be under translational selection in these mosquitoes.

Comparative analysis of nuclear tRNA genes of Nasonia vitripennis and other arthropods, and relationships to codon usage bias.

Using bioinformatics methods, we identified a total of 221 and 199 tRNA genes in the nuclear genomes of Nasonia vitripennis and honey bee (Apis mellifera), respectively. We performed comparative analyses of Nasonia tRNA genes with honey bee and other selected insects to understand genomic distribution, sequence evolution and relationship of tRNA copy number with codon usage patterns. Many tRNA genes are located physically close to each other in the form of small clusters in the Nasonia genome. However, the number of clusters and the tRNA genes that form such clusters vary from species to species. In particular, the Ala-, Pro-, Tyr- and His-tRNA genes tend to accumulate in clusters in Nasonia but not in honey bee, whereas the bee contains a long cluster of 15 tRNA genes (of which 13 are Gln-tRNAs) that is absent in Nasonia. Though tRNA genes are highly conserved, contrasting patterns of nucleotide diversity are observed among the arm and loop regions of tRNAs between Nasonia and honey bee. Also, the sequence convergence between the reconstructed ancestral tRNAs and the present day tRNAs suggests a common ancestral origin of Nasonia and honey bee tRNAs. Furthermore, we also present evidence that the copy number of isoacceptor tRNAs (those having a different anticodon but charge the same amino acid) is correlated with codon usage patterns of highly expressed genes in Nasonia.

Research ethics. Managing risks of arthropod vector research.

Field research with vectors is an essential aspect of vector biology research and vector-borne disease prevention and control. This type of research, which brings experimental vector manipulations into endemic areas, can present risks to human populations. This paper seeks to stimulate a full discussion within the medical entomology community of the risks associated with vector field research. Such discussions will promote development of a consensus, among investigators, sponsoring agencies and the communities within which the work is done, so that appropriate steps can be taken to minimize and manage the risks, and adequate oversight can be maintained.

Quantitative trait loci determining autogeny and body size in the Asian tiger mosquito (Aedes albopictus).

The majority of mosquito species require a blood meal to stimulate vitellogenesis and subsequent oviposition (anautogeny), but some autogenous individuals complete their first ovarian cycle without a blood meal. Autogeny may be facultative or obligatory. In this study, we selected for an autogenous strain in the Asian tiger mosquito Aedes albopictus and examined an F(1) intercross population for quantitative trait loci (QTL) determining the autogeny trait as well as wing length as a proxy for body size. Using composite interval mapping, we identified four QTL for each trait and observed considerable overlap in genome positions between each QTL for autogeny (follicle size) and wing length. Most QTL were minor in magnitude, individually explaining <10% of the phenotypic variation. Alleles from the autogenous parent generally showed a dominance or overdominance effect on both phenotypes. Strong genetic and phenotypic correlations indicate that autogeny and wing length are determined by up to four clusters of tightly linked genes or the potential pleiotropic effects of single genes. Although females from the autogenous strain produced approximately fivefold more eggs following a blood meal than through autogeny, we suggest that the maintenance of alleles for autogeny in natural populations is likely due to balancing selection. Autogeny should be favored under conditions of limited host availability for blood feeding or increased defensive behavior by the host and adequate larval nutrition. Correlation between autogeny and body size may reflect an increased ability for larger females to accumulate sufficient nutrient reserves to support oogenesis without the requirement for a blood meal.

Identification of a polymorphic mucin-like gene expressed in the midgut of the mosquito, Aedes aegypti, using an integrated bulked segregant and differential display analysis.

The identification of putative differentially expressed genes within genome regions containing QTL determining susceptibility of the mosquito, Aedes aegypti, to the malarial parasite, Plasmodium gallinaceum, was investigated using an integrated, targeted approach based on bulked segregant and differential display analysis. A mosquito F2 population was obtained from pairwise matings between the parasite-susceptible RED strain and the resistant MOYO-R substrain. DNA from female carcasses was used to genotype individuals at RFLP markers of known chromosomal position around the major QTL (pgs 1). Midguts, dissected 48 hr after an infected blood meal, were used to prepare two RNA bulks, each representing one of the parental genotypes at the QTL interval. The RNA bulks were compared by differential display PCR. A mucin-like protein gene (AeIMUC1) was isolated and characterized. The gene maps within the pgs 1 QTL interval and is expressed in the adult female midgut. AeIMUC1 RNA abundance decreased with time after blood meal ingestion. No differential expression was observed between the two mosquito strains but three different alleles with inter- and intrastrain allelic polymorphisms including indels and SNPs were characterized. The AeIMUC1 gene chromosome location and allelic polymorphisms raise the possibility that the protein might be involved in parasite-mosquito interactions.

Evidence for genetic hitchhiking effect associated with insecticide resistance in Aedes aegypti.

Information on genetic variation within and between populations is critical for understanding the evolutionary history of mosquito populations and disease epidemiology. Previous studies with Drosophila suggest that genetic variation of selectively neutral loci in a large fraction of genome may be constrained by fixation of advantageous mutations associated with hitchhiking effect. This study examined restriction fragment length polymorphisms of four natural Aedes aegypti mosquito populations from Trinidad and Tobago, at 16 loci. These populations have been subjected to organophosphate (OP) insecticide treatments for more than two decades, while dichlor-diphenyltrichlor (DDT) was the insecticide of choice prior to this period. We predicted that genes closely linked to the OP target loci would exhibit reduced genetic variation as a result of the hitchhiking effect associated with intensive OP insecticide selection. We also predicted that genetic variability of the genes conferring resistance to DDT and loci near the target site would be similar to other unlinked loci. As predicted, reduced genetic variation was found for loci in the general chromosomal region of a putative OP target site, and these loci generally exhibited larger F(ST) values than other random loci. In contrast, the gene conferring resistance to DDT and its linked loci show polymorphisms and genetic differentiation similar to other random loci. The reduced genetic variability and apparent gene deletion in some regions of chromosome 1 likely reflect the hitchhiking effect associated with OP insecticide selection.

Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti.

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.

Mapping a quantitative trait locus involved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae.

A Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium, whereas wild-type mosquitoes are usually susceptible. This encapsulation trait can also be observed by studying the response of refractory and susceptible strains to intrathoracically injected CM-Sephadex beads. We report the results of broad-scale quantitative trait locus (QTL) mapping of the encapsulation trait using the bead model system. Interval mapping using the method of maximum likelihood identified one major QTL, Pen1. The 13.7-cM interval containing Pen1 was defined by marker AGH157 at 8E and AGH46 at 7A on 2R. Pen1 was associated with a maximum LOD score of 9.0 and accounted for 44% of the phenotypic variance in the distribution of phenotypes in the backcross. To test if this QTL is important for encapsulation of Plasmodium berghei, F2 progeny were infected with P. berghei and evaluated for degree of parasite encapsulation. For each of the two markers that define the interval containing Pen1, a significant difference of encapsulation was seen in progeny with at least one refractory allele in contrast with homozygous susceptible progeny. These results suggest that Pen1 is important for melanotic encapsulation of Plasmodium as well as beads.

Integration of the Aedes aegypti mosquito genetic linkage and physical maps.

Two approaches were used to correlate the Aedes aegypti genetic linkage map to the physical map. STS markers were developed for previously mapped RFLP-based genetic markers so that large genomic clones from cosmid libraries could be found and placed to the metaphase chromosome physical maps using standard FISH methods. Eight cosmids were identified that contained eight RFLP marker sequences, and these cosmids were located on the metaphase chromosomes. Twenty-one cDNAs were mapped directly to metaphase chromosomes using a FISH amplification procedure. The chromosome numbering schemes of the genetic linkage and physical maps corresponded directly and the orientations of the genetic linkage maps for chromosomes 2 and 3 were inverted relative to the physical maps. While the chromosome 2 linkage map represented essentially 100% of chromosome 2, approximately 65% of the chromosome 1 linkage map mapped to only 36% of the short p-arm and 83% of the chromosome 3 physical map contained the complete genetic linkage map. Since the genetic linkage map is a RFLP cDNA-based map, these data also provide a minimal estimate for the size of the euchromatic regions. The implications of these findings on positional cloning in A. aegypti are discussed.

Nucleotide sequence of a middle repetitive DNA from honey bees.

The nucleotide sequence of a 264-bp clone, pAfr3.4, which represents a related, tandemly arrayed, middle repetitive DNA family within various subspecies of the honey bee (Apis mellifera L.), has been determined.

Vitelline envelope genes of the yellow fever mosquito, Aedes aegypti.

Vitelline envelope genes from the mosquito Aedes aegypti were analyzed with respect to their DNA sequences, genomic representation, temporal and spatial expression profiles and response to 20-hydroxyecdysone. Genomic clones of three vitelline envelope genes, 15a-1, 15a-2 and 15a-3 were isolated. Southern analysis indicates that all three genes are represented by a single copy in the genome. The deduced amino acid sequences of all three vitelline envelope genes contain a conserved region of 46 residues that overlaps with a region that is conserved in four Drosophila melanogaster vitelline envelope genes. DNA was sequenced flanking the 15a-1, 15a-2 and 15a-3 coding regions. A 360 bp sequence 5' of the 15a-2 coding region was identified with 72% identity to a sequence upstream of the Ae. aegypti VgA1 vitellogenin gene. The temporal patterns of 15a-1, 15a-2 and 15a-3 expression, as determined by Northern analysis, were similar. The spatial patterns of expression, as determined by whole-mount in situ hybridization, differed between the three genes. 15a-1 and 15a-3 were only expressed in the middle and posterior regions of the follicle, while 15a-2 was also expressed at the anterior region. Vitelline envelope gene expression was higher in ovaries that were dissected at 0, 2 and 10 h following a blood meal and then incubated in vitro for 10 h in medium containing 10(-5) M 20-hydroxyecdysone, compared to ovaries that were incubated without hormone.

Evidence for two distinct members of the amylase gene family in the yellow fever mosquito, Aedes aegypti.

Genomic DNA fragments encoding a salivary gland-specific alpha-amylase gene, Amylase I (Amy I), and an additional amylase, Amylase II (AmyII) of the yellow fever mosquito, Aedes aegypti, were isolated and characterized. Two independently isolated DNA fragments, G34-F and G34-14A, encode polymorphic alleles of Amy I. A 3.2 kilobase (kb) EcoR I fragment of G34-F, F2, has been sequenced in its entirety and contains 832 base pairs (bp) of the 5'-end, non-coding and putative promoter regions that are adjacent to 2.4 kb of the Amy I coding region. One intron, 59 bp in length, is found towards the 3'-end of the clone. A third genomic clone, 3A, corresponding to Amy II, was sequenced and shown not to contain the primary DNA sequence that encodes the 260 amino acid region that uniquely characterizes the amino terminal end of the Amy I product. Amy I was assigned by restriction fragment length polymorphism (RFLP) mapping to chromosome 2 (23.0 cM) and Amy II to chromosome 1 (44.0 cM). Amy I and Amy II are highly polymorphic and there may be multiple linked copies at each locus. Comparisons between Amy I and Amy II are presented for the putative promoter and conceptual translation products. The identification of two distinct amylase genes and their separate linkage assignments provides evidence for a multigene family of alpha-amylases in Ae. aegypti.

The suitability of restriction fragment length polymorphism markers for evaluating genetic diversity among and synteny between mosquito species.

Restriction fragment length polymorphism (RFLP) markers derived from the yellow fever mosquito, Aedes aegypti, were used in hybridizations to genomic DNA of the following mosquito species: Ae. albopictus, Ae. togoi, Armigeres subalbatus, Culex pipiens, and Anopheles gambiae. Interspecific hybridization with Ae. aegypti probes varied from 50% (An. gambiae) to 100% (Ae. albopictus) under high stringency conditions. We demonstrated the usefulness of using RFLP profiles to examine genetic diversity between mosquito populations; Ae. aegypti RFLP markers were used to examine genetic relatedness between 10 laboratory strains of Ae. aegypti as well as between nine populations representing four Cx. pipiens subspecies. These results indicate that many Ae. aegypti RFLP markers should have direct applicability in gaining a better understanding of genome structure in other mosquito species, including RFLP linkage mapping and determinations of genetic relatedness among field populations.

Reinterpretation of the genetics of susceptibility of Aedes aegypti to Plasmodium gallinaceum.

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.

Complete mitochondrial DNA sequence and amino acid analysis of the cytochrome C oxidase subunit I (COI) from Aedes aegypti.

The complete sequence of the yellow fever mosquito, Aedes aegypti, mitochondrial cytochrome c oxidase subunit 1 gene has been identified. The nucleotide sequence codes for a 512 amino acid peptide. The AeCOI sequence is A + T rich (68.6%) and the codon usage is highly biased toward a preference for A- or T-ending triplets. The A. aegypti COI peptide shows high homology, up to 93% identity, with several other insect sequences and a phylogenetic analysis indicates that the A. aegypti sequence is closely related to two other mosquito species, Anopheles gambiae and A. quadrimaculatus. Comparisons of the nucleotide sequence for four A. aegypti laboratory strains revealed single nucleotide polymorphisms, with 25 nucleotide sites showing SNPs between strains. All SNPs occurred as synonymous transitions such that the peptide sequence is conserved among A. aegypti strains. RT-PCR analysis showed that COI is expressed at similar levels in all developmental stages and tissues.

Impact of road networks on the distribution of dengue fever cases in Trinidad, West Indies.

This study examined the impact of road networks on the distribution of dengue fever cases in Trinidad, West Indies. All confirmed cases of dengue hemorrhagic fever (DHF) observed during 1998 were georeferenced and spatially located on a road map of Trinidad using Geographic Information Systems software. A new digital geographic layer representing these cases was created and the distances from these cases to the nearest classified road category (5 classifications based on a functional utility system) were examined. The distance from each spatially located DHF case to the nearest road in each of the 5 road subsets was determined and then subjected to an ANOVA and t-test to determine levels of association between minor road networks (especially 3rd and 4th class roads) and DHF cases and found DHF cases were located away from forests, especially 5th class roads). The frequency of DHF cases to different road classes was: 0% (1st class roads), 7% (2nd class roads), 32% (3rd class roads), 57% (4th class roads) and 4% (5th class road). The data clearly demonstrated that both class 3 and class 4 roads account for 89% of nearby dengue cases. These results represent the first evidence of dengue cases being found restricted between forested areas and major highways and would be useful when planning and implementing control strategies for dengue and Aedes aegypti mosquitoes.