Proof of concept for a novel insecticide bioassay based on sugar feeding by adult Aedes aegypti (Stegomyia aegypti).

Aedes aegypti L. (Stegomyia aegypti) (Diptera: Culicidae) is the principal vector of dengue and yellow fever viruses in tropical and subtropical regions of the world. Disease management is largely based on mosquito control achieved by insecticides applied to interior resting surfaces and through space sprays. Population monitoring to detect insecticide resistance is a significant component of integrated disease management programmes. We developed a bioassay method for assessing insecticide susceptibility based on the feeding activity of mosquitoes on plant sugars. Our prototype sugar-insecticide feeding bioassay system was composed of inexpensive, disposable components, contained minimal volumes of insecticide, and was compact and highly transportable. Individual mosquitoes were assayed in a plastic cup that contained a sucrose-permethrin solution. Trypan blue dye was added to create a visual marker in the mosquito's abdomen for ingested sucrose-permethrin solution. Blue faecal spots provided further evidence of solution ingestion. With the sugar-insecticide feeding bioassay, the permethrin susceptibility of Ae. aegypti females from two field-collected strains was characterized by probit analysis of dosage-response data. The field strains were also tested by forced contact of females with permethrin residues on filter paper. Dosage-response patterns were similar, indicating that the sugar-insecticide feeding bioassay had appropriately characterized the permethrin susceptibility of the two strains.

Host preference of the arbovirus vector Culex erraticus (Diptera: Culicidae) at Sonso Lake, Cauca Valley Department, Colombia.

Culex erraticus (Dyar & Knab) is a competent vector of Eastern equine encephalitis virus and subtype IC Venezuelan equine encephalitis virus, and both St. Louis encephalitis virus and West Nile virus have been isolated from field-collected specimens. Previous bloodmeal analysis studies have shown this species to be a generalist, feeding on a variety of mammals, birds, reptiles, and amphibians. This behavior can bridge arboviral transmission across different vertebrate groups. Our study examined the host preference of Cx. erraticus at Sonso Lake in Colombia. From July to August 2008, blood-engorged mosquitoes were collected from resting boxes, while vertebrate abundance was determined to calculate host preference. Based on mitochondrial DNA analysis of bloodmeals, birds were the predominant hosts (57.6%), followed by mammals (30.8%), and reptiles (6.7%); 9.5% of the bloodmeals were mixed. The most commonly fed upon species were: limpkin, black-crowned night-heron, striated heron, human, and capybara. Forage ratios showed the least bittern, limpkin, Cocoi heron, striated heron, capybara, and black-crowned night heron were preferred hosts across all vertebrates. Of the available avifauna, the least bittern, limpkin, striated heron, Cocoi heron, and black-crowned night heron were preferred, whereas the bare faced ibis, great egret, snowy egret, and cattle egret were under-used. This study shows that while Cx. erraticus is an opportunistic feeder, using diverse vertebrate hosts in the environment, certain avian species are targeted preferentially for bloodmeals.

High Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) prevalence in Triatoma sanguisuga (Hemiptera: Redviidae) in southeastern Louisiana.

From May through November 2007, intensive weekly surveys at the site of a previously reported autochthonous human case of Chagas parasite infection resulted in the collection of 298 Triatoma sanguisuga (Leconte) specimens, of which 60.4% (180) were polymerase chain reaction positive for Trypanosoma cruzi Chagas. All were adults, in a ratio of approximately 1:1 female to male, indicating that the domicile was not colonized, but was a destination for these host-seeking adults. We report on seasonal activity pattern, T. cruzi prevalence in T. sanguisuga, and attempts at insect exclusion and control at the case residence.

Phylogeny and molecular taxonomy of the Rhodniini derived from mitochondrial and nuclear DNA sequences.

Eleven species of Rhodnius and one of Psammolestes were compared by DNA sequence analysis of fragments of the mitochondrial large subunit ribosomal RNA (mtlsurRNA), the mitochondrial cytochrome b (mtCytb), and the D2 variable region of the 28S nuclear RNA (D2), totaling 1,429 base pairs. The inferred phylogeny, using Triatoma infestans as an outgroup, revealed two main clades within the Rhodniini--one, including the prolixus group of species (Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, and Rhodnius nasutus) together with Rhodnius domesticus and Rhodnius neivai, and the other comprising two groups formed by Rhodnius pictipes plus Rhodnius brethesi, and Rhodnius ecuadoriensis plus Rhodnius pallescens. Psammolestes tertius appeared most closely related to the prolixus group. The analysis strongly supports the validity of R. robustus as a species distinct from others of the prolixus group, but suggests higher genetic structuring of R. robustus populations compared to the other species. Although R. robustus has been found naturally infected by Trypanosoma cruzi, the fact that it is apparently entirely sylvatic and unable to establish in homes suggests that it is of no great importance as a Chagas disease vector in humans.

Polymerase chain reaction assay to identify all immature stages of two species of the Anopheles quadrimaculatus sibling species complex (Diptera: Culicidae).

A polymerase chain reaction assay that detects differences in the 2nd internal transcribed spacer of ribosomal DNA was tested as a means to identify all immature stages of 2 species in the Anopheles quadrimaculatus sibling species complex. The technique was successful in amplifying DNA from eggs, 1st-4th instars, and pupae of An. quadrilmaculatus Say and An. inundatus Reinert.

Mitochondrial DNA sequence variation among triatomine vectors of Chagas' disease.

Kissing bugs or triatomines (Reduviidae: Triatominae) are vectors of the Chagas' disease agent Trypanosoma cruzi. There is a current need for more sensitive tools for use in discrimination of different bug populations and species, thus allowing a better understanding of these insects as it relates to disease transmission and control. In a preliminary analysis of the mitochondrial large subunit ribosomal RNA (mtlsurRNA) and cytochrome B (mtCytB) genes, we used DNA sequencing to study species identification and phylogeny. In both examined gene regions, about 46% of nucleotide positions exhibited polymorphism. The examined region of mtCytB appears to have evolved more rapidly than the examined region of mtlsurRNA. Phylogenetic analysis of both gene fragments in the examined species produced similar results that were generally consistent with the accepted taxonomy of the subfamily. The two major tribes, Rhodniini and Triatomini, were supported, along with additional clades that corresponded to accepted species complexes within the Rhodnius and Triatoma genera. The one chief exception was that Psammolestes coreodes sorted into the Rhodnius prolixus-robustus-neglectus clade, with bootsrap values of 99% and 81%, respectively, for the mtlsurRNA and mtCytB fragments. All of the individual species examined could be distinguished at both genetic loci.

Role of habitat components on the dynamics of Aedes albopictus (Diptera: Culicidae) from New Orleans.

Monthly sampling of tire pile populations of Aedes albopictus (Skuse) in Orleans Parish, New Orleans, LA, was done in 1995 to determine prevalence of ascogregarine parasites and changes in wing length. Prevalence of Ascogregarina taiwanensis (Lien & Levine) infection was 100% in midsummer and decreased in the fall and spring (60-70%). Wing lengths were longest in the spring and fall and shortest in midsummer. We evaluated the effect of A. taiwanensis infections under high and deficient levels of leaf litter nutrients on mortality, development time, wing length, and reproductive potential of a New Orleans strain of Ae. albopictus. Parasitism and deficient nutrients caused a 35% increase in the rate of larval mortality and significantly extended the development time of females. Parasitized adults were 5% smaller and produced 23% fewer eggs than unparasitized siblings. In addition, abnormal Malpighian tubule morphology and melanization of ascogregarines were seen in adults from nutrient-deficient microcosms. We conclude that ascogregarine infections affect the dynamics of Ae. albopictus by increasing the mortality of immature stages when nutrients supplies are scarce, and by decreasing the reproductive capacity of females under high nutrient conditions.

Effect of nutrient levels and Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) infections on the vector competence of Aedes albopictus (Diptera: Culicidae) for Dirofilaria immitis (Filarioidea: Onchocercidae).

The effect of habitat nutrients and Ascogregarina taiwanensis (Lein & Levine) infection on the vector competence of a New Orleans strain of Aedes albopictus (Skuse) for Dirofilaria immitis (Leidy) were evaluated. Larvae were infected with A. taiwanensis oocysts and reared under high and deficient nutrient conditions using leaf litter as a food source. Ascogregarine-infected and uninfected females were fed on D. immitis-infected blood and examined after 15 d for filariae. Susceptibility to infection with filaria was 60-70% for all females. In groups reared under high nutrients, the infective rate (3rd-stage larvae present) of coinfected females (18%) was significantly greater than females infected only with heartworm (6%). Host mortality following blood meals was significantly less in coinfected (22%) than in heartworm-infected females (37%). Under deficient nutrient conditions, there was no significant difference between the infective rate (8%) or post-blood meal mortality (5%) of coinfected females compared with heartworm-infected females. Prevalence of melanization reactions in coinfected females was significantly higher (31%) than in females infected only with heartworm (6%) at both nutrient levels. It is concluded that high nutrient levels and ascogregarine infection increase the vector competence of Ae. albopictus for D. immitis by enhancing the immune response so that fewer filariae develop, causing lower host mortality. Under low nutrient conditions, the smaller host contains less food reserves for filariae development, and in coinfected females melanization reactions and damage to the Malpighian tubules may reduce vector survival.

Country-wide prevalence of Ascogregarina culicis (apicomplexa: lecudinidae), a protozoan parasite of Aedes aegypti in Trinidad, West Indies.

The prevalence of the gregarine parasite Ascogregarina culicis was determined for Aedes aegypti populations in Trinidad, West Indies. Over 5 months, a total of 5,651 Ae. aegypti larvae were collected and identified from 419 sites throughout Trinidad as part of a national dengue surveillance program. Ascogregarina culicis infections were detected in 13.4% of 1,986 Ae. aegypti larvae dissected. In 7 of 8 counties or major subdivisions where this parasite was detected in Trinidad, the prevalence per locality ranged from 9.6 to 17.1% with an associated parasite density of 19.1-51.9 trophozoites/larva. Confirming that Ae. aegypti populations in Trinidad are highly susceptible to infection with A. culicis parasites, experimental studies showed that a strain of A. culicis from Florida readily infected 3 strains of Ae. aegypti from Trinidad with essentially no associated mortality. This report extends the known geographic range of A. culicis in Ae. aegypti populations and represents one of the first country-wide surveys for this gregarine parasite.

Dirofilaria (Filarioidea: Onchocercidae) infection in Aedes albopictus (Diptera: Culicidae) collected in Louisiana.

First-stage Dirofilaria larvae were found infecting the Malpighian tubules in 3 of 163 Aedes albopictus collected from New Orleans, LA, during June-July 1994. Further studies will be necessary to provide definitive identification of the parasite species, and to better describe the vectorial status of Ae. albopictus.

Evolution of the rDNA spacer, ITS 2, in the ticks Ixodes scapularis and I. pacificus (Acari: Ixodidae).

Evolution of the rDNA spacer, ITS 2, is examined by comparing 17 DNA sequences of the ticks, Ixodes scapularis and I. pacificus. The distribution of fixed interspecific differences and the relative frequency of base changes vs. insertions/deletions (indels) matches the distribution and relative frequency for intraspecifically variable sites. This suggests that most intraspecific variation is not effectively selected against. The base composition of the ITS 2 transcript is G- and U-biased. But, 5-base regions enriched (> 80 per cent) for A or U occur more frequently than expected while G- and C-enriched regions occur less frequently than expected. Enriched sequences may be prone to replication slippage, accounting for the A/T bias in insertions. Slippage-mediated gains and losses of A/T-rich tandem repeats apparently account for most indels. Minimum-energy conformations of the two species' folded transcripts share major structural features. Structural inertia arises from intramolecular base pairing within stems that allows most mutations to be absorbed as new bulges off stems. Yet, there is evidence of selection to maintain the conformation. First, intraspecifically variable sites are concentrated at the ends of stems in loops and intersections, structures that do not contribute to intramolecular base pairing. Moreover, some indels that have become fixed in one species compensate for the presence of conformation-destabilizing indels. However, high rates of sequence evolution within stems and absence of compensatory base evolution contraindicates selective constraint. Degenerate dispersed and tandem copies of two subrepeats, each approximately 20 bases long, may account for much of the ITS 2 sequence. These are approximately inverses of each other and are, consequently, capable of significant intramolecular hydrogen bonding to produce folded transcripts of low energy. Evolution of the ITS 2 sequence may largely entail replication slippage-mediated gains and losses of these repeats or their composite subrepeats.

Variation in ribosomal DNA internal transcribed spacers 1 among eastern populations of Ixodes scapularis (Acari: Ixodidae).

The base sequence of the internal transcribed spacer 1 (ITS 1) of ribosomal DNA of the tick Ixodes scapularis Say (= I. dammini Spielman, Clifford, Piesman & Corwin) was determined to assess genetic divergence between populations along the eastern (Atlantic) seaboard of the United States. Twenty sequences were obtained from localities down the eastern margin of the species's range: 10 from the southeast (Georgia and Florida), seven from the middle east (North Carolina, Maryland), and three from the northeast (Massachusetts, New Jersey, New York). Both the neighbor-joining and parsimony methods cluster most of the southeastern sequences together and most of the middle eastern sequences together but fail to cluster those from the northeast. In addition, an F ratio test revealed significant between-region sequence variation. Thus, there appears to be genetic structuring on at least a macrogeographic scale. Only 23% (SEM = 6.4%) of the sequence variation occurs between regions, with the vast majority of variation, 77% (SEM = 6.4%), being within region. These data, plus other published data, indicate that I. scapularis constitutes a single species. However, the pattern of variation is consistent with restricted gene flow between regions or, alternatively, with recent introgression between northern and southern types in the middle-eastern part of the species's range.

A cytoskeletal actin gene in the mosquito Anopheles gambiae.

Five actin genes have been identified in the mosquito Anopheles gambiae, and a constitutively expressed actin gene has been chosen for detailed analysis. We have physically mapped and sequenced this gene and six associated cDNAs, including translated coding regions, as well as the 5' and 3' flanking sequences. Analysis of stage-specific RNA shows this gene to be present in all stages of mosquito development and in an established A. gambiae cell line, thus indicating a cytoskeletal actin. In the sequence of the translated coding region and in pattern of expression, this gene is very similar to the cytoskeletal actin genes of Drosophila melanogaster, and in sequence, equally similar to the Artemia cytoskeletal actin gene 403 (99.2% identity among the three amino acid sequences). Sequencing of this A. gambiae actin gene (designated act1D for its location in chromosome division 1D) and selected cDNAs shows that it possesses three alternative leader sequences; thus the gene appears to have three alternative promoters. These promoters should ultimately prove useful in the production of transgenic constructs for constitutive expression.

Investigation of the validity of species status of Ixodes dammini (Acari: Ixodidae) using rDNA.

The two internal transcribed spacers (ITS1 and ITS2) of rDNA of three members of the Ixodes ricinus "complex" (Acari: Ixodidae) were sequenced. Sequence variation was assessed for the North American species I. scapularis, I. dammini, and I. pacificus at three levels: within individual/population, between individuals of different geographic origin within a species, and between species. Both spacers are highly variable, particularly with regard to small deletions and additions which may arise via replication slippage. Homogenization of rDNA multigene arrays for particular sequence variants appears to occur at a relatively rapid rate, since I. pacificus sequences differ from the others at numerous invariant sites, facilitating the use of these sequences to assess sibling species relationships. Based on maximum parsimony and two distance methods (unweighted pair-group with arithmetic means and neighbor-joining), sequence variation in ITS1 and ITS2 suggests that I. scapularis and I. dammini are not distinct species and that even individuals from geographically isolated locations are very similar. Individuals from geographically separated populations of I. pacificus appear to be relatively less closely related to each other but distinct from those of I. scapularis/dammini. In I. scapularis/dammini, diversity within and between individuals from geographic populations contributed equally to total sequence diversity.

The internal transcribed spacers of ribosomal DNA in five members of the Anopheles gambiae species complex.

The primary and secondary structure of the internal transcribed spacers of ribosomal DNA (ITS1 and ITS2) and their utility for phylogenetic analysis of closely related species were examined using the Anopheles gambiae complex as a model. Restriction mapping revealed an unusual architectural feature in the ITS1 of several members of an An. gambiae cryptic species complex. Multiple spacer lengths are prevalent in An. merus and An. melas and are due to variable numbers of a repeated 250 bp sequence. Secondary structure analysis indicated that the repeat forms a helix and loop that may be involved in rDNA processing. Intra- and interspecific polymorphism within the species complex were further examined by DNA sequencing of forty-eight ITS2 clones obtained by polymerase chain reaction from individuals of the five species. Interspecies variation in the approximately 426 bp ITS2 sequence ranged between 0.4% and 1.6%; intraspecies variation ranged from 0.07% in An. arabiensis to 0.43% in An. gambiae. Intraindividual variation ranged from 0% in four individuals to a high of 0.4% in one An. quadriannulatus specimen. None of the variants were shared between species. The low level of variation supports the hypothesis that species of the complex evolved recently.

Sequence and secondary structure comparisons of ITS rDNA in mosquitoes (Diptera: Culicidae).

Sequences of the internal transcribed spacers (ITS1 and ITS2) of the mosquito Aedes aegypti, and the ITS2 of six related species, A. simpsoni, A. albopictus, A. vexans, A. triseriatus, Haemagogus mesodentatus, and Psorophora ferox are reported. Intraspecific variation in A. aegypti ITS1 is 1.07% among four clones from three individuals, and in the ITS2 is 1.17% among 15 clones from four individuals. In A. simpsoni, intraspecific ITS2 variation is 0.46% among 10 clones from a single individual. Alignment of the ITS2 sequence of the seven species reveals several homologous domains. Secondary structure predictions for the ITS2 region indicate that these domains base pair to form a core region central to several stem features. The sequence outside the ITS2 homologous domains tends to be GC-rich and characteristically slippage generated; these areas preserve or add to the stem length of the predicted secondary structures. These ITS2 intraspacer variable regions resemble previously described expansion segments of the 28S gene region. Evolutionary analysis of the ITS2 of these species, using both sequence and secondary structure information, leads to the prediction of divergence in the mosquito tribe Aedini that is not clearly reflected in current taxonomic designations.