Oligodeoxynucleotide primers, based on amino acid sequences conserved in known acetylcholinesterases (AChEs), were used in reverse-transcription polymerase chain reaction (RT-PCR) with mRNA from Boophilus microplus (Canestrini) as the template. Primer walking and rapid amplification of cDNA ends (RACE) techniques were used to complete the cDNA sequence identified by RT-PCR. The complete B. microplus cDNA sequence contained an open reading frame encoding a 620 amino acid protein with a 20 amino acid signal peptide at the N-terminus targeting the protein for the secretion pathway. BLAST searches of GenBank using the presumptively encoded protein revealed highest sequence similarity to AChEs. The presumptively encoded protein was of similar size and structural properties to other identified AChEs, including the presence of the catalytic triad (Ser, Glu, His) and appropriate placement of internal cysteines to yield three internal disulfide bonds corresponding to those of known AChEs. Putative conserved domains identified the sequence as a member of the carboxylesterase family, pfam00135.8, of which AChE is a member. This cDNA therefore presumptively encodes a third transcribed AChE (AChE3) cDNA of B. microplus. Comparison of the three AChE cDNA sequences expressed in B. microplus demonstrated no discernible nucleotide sequence homology and relatively low amino acid sequence homology, strongly suggesting that they are not alleles of one another. The potential presence of multiple expressed AChEs in B. microplus suggests alternative mechanisms for development of resistance to pesticides that target AChE. The homology-based identification of a third expressed AChE in B. microplus is a surprising result and strongly implies the need for confirmation of gene identity for presumptive AChEs.

The dispersal pattern of the widow spider Latrodectus hasseltii (Araneae: Theridiadae), in Osaka Prefecture, Japan, was analyzed from accumulated survey data of spider distribution. All confirmed infested points from 1996 to 2002 were plotted on a map, and infested points in each survey year were connected with the nearest previously confirmed points. Expansion patterns of the confirmed habitats were analyzed by year and dispersion distances were classified by using spider diagram, one of method of geographical information system spatial analysis. L. hasseltii moves by walking step by step on the ground, mainly in coastal areas during early stages of infestation, and also can be conveyed over distances by various vehicles, mainly in inland areas. These analyses show L. hasseltii does not move by ballooning.

Bacterial endosymbionts with significant homology to Francisella tularensis (γ-proteobacteria) have been described from at least five species of ticks in three different genera, including two North American Dermacentor species [D. andersoni Stiles and D. variabilis (Say)]. The evolutionary relationships among the Francisella-like endosymbionts (FLE) from different hosts and between FLE and the arthropod-borne pathogen F. tularensis are not known. A 1,169-base fragment of the 16s rDNA and a 713-base fragment of the F. tularensis 17-kDa lipoprotein gene homolog of the FLE of six North American Dermacentor tick species [D. anderson, D. variabilis, D. albipictus (Packard), D. occidentalis Marx, D. hunteri Bishopp, and D. (Anocentor) nitens Neumann] and of Amblyomma maculatum Koch and Ornithodoros porcinus (Murry 1877, sensu Walton 1979) as outgroups, were subjected to phylogenetic analysis. These gene phylogenies were compared with a phylogeny of the same tick species constructed from a 435-base fragment of the tick mitochondrial 16s rDNA. Although the phylogenies of the FLE and their tick hosts are parallel at the genus level, the Dermacentor FLE are unresolved at the species level. The FLE and the Dermacentor ticks show little sign of co-speciation, possibly indicating that the association between these endosymbiont and the Dermacentor ticks is of a relatively recent origin. Several ticks were co-infected, either with two FLE with divergent 17-kDa lipoprotein gene sequences or with FLE and an unidentified species of spotted fever group rickettsia (α-proteobacteria). Infection with FLE does not seem to have precluded infection with either a second closely related γ-proteobacterial symbiont or with a second less closely related α-proteobacterial symbiont.

Anopheles sundaicus s.l. is a principal malaria vector taxon on islands and along the coastal areas of Southeast Asia. It has a wide geographical distribution and exhibits a high level of ecological and behavioral variability. Study of this taxon is crucial for understanding its biology and implementing effective vector control measures. We compared populations of An. sundaicus from Vietnam, Thailand, and Malaysian Borneo by using two mitochondrial DNA markers: cytochrome oxidase I and cytochrome b. Genetic divergence, geographic separation, and cladistic analysis of relationships revealed the presence of two cryptic species: Anopheles sundaicus s.s. on Malaysian Borneo and An. sundaicus species A in coastal areas of Thailand and Vietnam. A polymerase chain reaction (PCR) assay was developed to easily identify these two species throughout their geographic distributions. The assay was based on sequence characterized amplified region derived from random amplified polymorphic DNA. This PCR identification method needs to be validated and adapted for the recognition of other possible species in the Sundaicus Complex.

Eight Triatoma dimidiata populations from different geographic regions were compared along with related species using traditional morphometry of head characters. A method for removing allometric change was used for the intraspecific comparisons, and scaling for the isometric change of size was used for the interspecific comparisons. The intraspecific comparison showed significant head shape differences between T. dimidiata populations correlating with geography, showing a separation between the northern, intermediate, and southern collections (more evident in females), and supporting the idea that this species includes several evolutionarily divergent populations. The positioning of one sylvatic group from Guatemala did not correlate with geography, because it was more closely related to a distinct population of Colombia. This sylvatic group was found in caves, while the Colombian specimens, although collected in houses, may have migrated from nearby caves. Evolutionary and/or ecological influences could be responsible for the head shape similarities between these two groups: a common ancestral origin of both populations or a morphological convergence caused by similar environmental pressures. The interspecific comparisons included four other regional species of the same genus, three of them belonging to the phyllosoma complex (T. pallidipennis, T. mexicana, and T. ryckmani, the latter provisionally) and the fourth one classified in the protracta complex (T. nitida). Both complexes were readily separated by their head dimensions, even after size adjustment, and our data support inclusion of T. dimidiata within the phyllosoma complex.

Previous studies indicated that two distinct chromosomal forms of Anopheles nuneztovari Gabaldón, cytotypes B and C, occurred on the west and east of the Latin American Andes Mountains, respectively. To determine the taxonomic status of Anopheles (Nyssorhynchus) nuneztovari in Colombia, link-reared specimens were collected from four sites: in the departments of Chocó (La Pacurita) and Valle (Sitronella) in the west, and Norte de Santander (Guaramito and Tibú) in the east. Nuclear ITS2 sequences were generated for 46 individuals. Only two specimens (4.4%) showed divergent haplotypes, varying from the consensus by a single-base polymorphism (0.18%). These results suggest that populations of An. nuneztovari corresponding to cytotypes (B and C) are conspecific.

Two genera of parasitic cheyletid mites, Smileycheles Fain, 1979 (monotypic) and Thryonomycheyla Fain, 1972 (two species), are synonymized with the genus Sciurocheyla Volgin, 1969. An emended diagnosis of the genus Sciurocheyla is provided. Sciurocheyla squamosa (Domrow & Baker, 1963), type species of the genus, and Sciurocheyla camerounensis (Fain, 1979), new combination, are redescribed. Females, males, teleo- and protonymphs of Sciurocheyla funisciurus, new species, collected from Funisciurus pyrrohopus F. Cuvier in tropical Africa are described and illustrated.

For the first time, the reduviid bug Triatoma ryckmani Zeledón and Ponce (Hemiptera; Reduviidae) was recorded to inhabit the epiphyte Tillandsia xerographica Rohweder (Bromeliaceae) in the semiarid region of Guatemala. These bromeliads grow mainly in drought-resistant trees with rough bark such as Pereskia lychnidiflora (Cactaceae). In our study site, we investigated 30 T. xerographica, and 53 specimens of T. ryckmani were found. Most T. ryckmani (68.5%) were unfed. Ants (Formicidae) were the predominant (92.2%) insect taxon in T. xerographica. Other insects such as Blattidae (3.0%), Reduviidae (T. ryckmani: 2.5%), Blaberidae (2.2%), Gryllidae (0.1%), and Acrididae (0.1%) were recorded in the bromeliads. T. xerographica is illegally commercialized without previous inspection. This may cause accidental introduction of T. ryckmani to houses and to other countries.

In the first part of this study, monthly infestation by ticks was evaluated on dogs from December 2000 to November 2002 in the rural area of Taiaçupeba, São Paulo. Adults of Amblyomma aureolatum (Pallas) were found on dogs in all months, with a mean prevalence per month of 46.9 ± 15.7% (range, 25–80%). The mean tick relative abundance per month was 2.4 ± 2.7 ticks (range, 0.5–14 ticks), and the mean tick mean intensity per month was 4.7 ± 4.2 ticks (range, 1.5–23.3 ticks). No A. aureolatum immature ticks were found on dogs. In the second part of this study, we studied the life cycle of A. aureolatum in the laboratory. We tested the suitability of six host species for the immature stages and dogs for the adult stage. Tick developmental periods were observed at different temperatures (23, 25, or 27°C), always with RH >95%, which were satisfactory for all free-living developmental stages of the tick life cycle. Chickens and guinea pigs were the most suitable hosts for larvae and nymphs (recovery rates, 18.4–52.2%). Dogs were highly suitable for adult ticks (all females exposed to them were recovered and laid eggs) but were unsuitable for the immature ticks (recovery rates, 0–10%). Based on published host records for A. aureolatum, our results indicate that dogs and birds are primary hosts for adult and immature stages, respectively, of A. aureolatum in nature. In addition, wild guinea pigs are indicated as another potential primary host for immature ticks.

We observed the survivorship and distribution of larvae and pupae of Anopheles gambiae s.l. Giles immature stages in three habitats (rock pools, swamp, and puddles) in Banambani village, Mali, West Africa, during the mid-rainy season of 2000. Horizontal life tables were constructed for immatures in the laboratory. Times spent in the various immature stages were determined, and laboratory survival was measured. Vertical life tables were obtained from each habitat. We found large day-to-day variation for age class composition within habitats across days. The swamp samples had small but statistically significant different distributions in some instar stages compared with rock pools and puddles as affected by precipitation history. There were obviously unstable age distributions in the swamp and puddles and to some extent in rock pools. There were more individuals in some later age classes than in earlier ones. The daily survival estimates using an exponential decay model were 0.807 in rock pools, 0.899 in the swamp, 0.818 in puddles, and 0.863 in the overall village. Possible reasons for the departure from stable age distribution were cannibalism, predation and other complex interactions, rainfall effects, sampling bias, and differences in physicochemical properties of the water in the habitats.

Differences in midgut microbial communities inhabiting Culicoides spp., insect vectors of virus pathogens, may affect the variation observed in the ability of these biting midges to propagate arthropod-borne viruses. As a first step toward addressing this hypothesis, midgut bacterial communities were compared between Culicoides species expected to be efficient and inefficient vectors of virus pathogens. We used 16S rDNA sequence and restriction fragment information to provisionally identify 36 bacterial genera from guts of wild adult female biting midges, Culicoides sonorensis Wirth and Jones and Culicoides variipennis (Coquillet), from two geographical locations. Bacterial identification was made by sequence analysis of 16S rDNA fragments and by terminal restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S rDNA fragments from adult guts. Of 36 bacterial genera identified, 12 had been previously identified in other insects: Comomonas, Enterobacter, Klebsiella, Acinetobacter, Pseudomonas, Stenotrophomonas, Staphylococcus, Chryseobacterium, Moraxella, Acholeplasma, Flavobacterium, and Rickettsia. Significant differences in bacterial community composition were found between all three groups of wild adult females analyzed: live-trapped C. sonorensis, laboratory-emerged C. sonorensis, and laboratory-emerged C. variipennis.

This study was conducted as part of a field ecology study of arboviral activity in the Amazon Basin, Peru, to determine the taxonomy, frequency, seasonal, and vertical distributions of potential mosquito vectors. In addition, the relative efficiency of human-landing collections and dry ice-baited Centers for Disease Control (CDC)-type light traps was determined for collecting mosquitoes. A total of 70 species of mosquitoes from 14 genera were collected from June 1996 through December 1997 at a forested site near Puerto Almendras, ≈20 km west-southwest of Iquitos, Peru. Three species [Psorophora (Janthinosoma) albigenu (Peryassu), Ochlerotatus (Ochlerotatus) fulvus (Wiedemann), and Ochlerotatus (Ochlerotatus) serratus (Theobald)] accounted for 70% of all mosquitoes captured in human-landing collections. Overall, biting activity occurred throughout the 24-h cycle but was higher during the daytime, primarily because of large populations of two day-biting species, Ps. albigenu and Oc. serratus. Oc. fulvus was active throughout the 24-h cycle but was more frequently collected during the evening. Oc. fulvus, Ps. albigenu, Culex (Melanoconion) pedroi Sirivanakarn & Belkin, and a mixture of Culex (Melanoconion) vomerifer Komp, and Culex (Melanoconion) gnomatos Sallum, Huchings & Ferreira, accounted for 73% of the mosquitoes captured during darkness by human collectors. In general, Ochlerotatus spp. and Psorophora spp. were more commonly captured in human-landing collections, whereas most Culex spp. were more frequently collected in the dry ice-baited CDC-type light traps. In general, mosquito populations were lowest from June through August when river levels were at their lowest. Two large population peaks occurred in November–December and in February–March as a result of “flood water” mosquito populations (e.g., Ps. albigenu). These data provide a better understanding of the taxonomy, population density, and seasonal distribution of potential mosquito vectors within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies.

We studied the relationship between climate and Ixodes ricinus L. tick behavior by following every day the proportion of ticks questing in a tick population placed in polyamide mesh-delimited arenas in the field. Simultaneously, the phenology of the questing density of nymphs and adults was studied by sampling ticks in a close location. At any time during the year, the proportion of questing adults was significantly higher (mean 24%) than the proportion of questing nymphs (mean 12%). The proportion of questing nymphs and adults decreased stepwise with time. The proportion of questing adults partially recovered after each decrease. In contrast, the proportion of questing nymphs was strongly reduced during a single short period in June and did not recover even partially. Decrease in the proportion of questing ticks was strongly related either to a peak in saturation deficit or to a drop in maximal relative humidity. No increase in the proportion of questing nymphs was observed in the arenas during autumn, although an autumn peak of nymphs was observed at the sampling location close to the arenas. This suggests that the autumn peak of nymphs observed in nature was due to newly emerged spring-fed larvae and not to reactivated spring active nymphs.

The presence of cryptic species within Anopheles minimus s.l. Theobald, one of the most widespread malaria vectors in Southeast Asia, was suggested on the basis of behavioral heterogeneities observed within this taxon. Subsequently, two species, A and C, were recognized. However, the existence of these cryptic species did not explain all observed behavioral heterogeneities within this complex. Besides, data on the behavior of vectors are essential to understand the dynamics of disease transmission and thus evaluate the appropriateness of vector control measures. Different collection methods were used to collect Anopheles species from several localities in Southeast Asia to assess the inter- and intraspecific behavioral divergences of An. minimus A and C. Collection results were subjected to a correspondence analysis. The members of the An. minimus complex were identified by use of the octanol dehydrogenase allozyme profiles or the polymerase chain reaction-restriction fragment length polymorphism assay. Large intraspecific behavioral differences were observed among populations of An. minimus A. These populations belong to the same species on the basis of the applied genetic markers. In northern Vietnam, species A tended to be more zoophilic, whereas in the study sites of south central Vietnam, Cambodia, and Laos it showed marked antropophilic behavior when cattle were scarce. In the most northern study site, An. minimus A showed noteworthy endophilic behavior. An. minimus C was primarily zoophilic and based on this behavior, its role in malaria transmission is questionable. However, it was only found in one locality, so that intraspecific behavioral variation could not be assessed. An. minimus A is able to change its host preference in function of local situations in host availability. Hence, its role in malaria transmission can differ from region to region. Similarly, the impact of vector control on this species may differ between localities. In conclusion, intraspecific behavioral differences in Anopheles species can occur and these behavioral heterogeneities, albeit important for disease transmission and control, are not a priori indicative for the presence of cryptic species.

Recent advances have demonstrated that, in the absence of postmating barriers to hybridization, reproductive isolation between different forms of Anopheles gambiae sensu stricto is maintained by strong assortative mating. The forms of An. gambiae s.s. and the sister species An. arabiensis commonly form mixed swarms in which they mate. This raises the question as to how individuals recognize mates of their own species or form within swarms. It has been proposed that wingbeat frequency is used as a cue to discriminate potential mates. This has important implications for prospective genetic control programs. We used a photosensor to record the transient waveforms generated by individuals An. arabiensis and from the M and S molecular forms of An. gambiae s.s. as they flew through a beam of light. We found no significant between-species or between-form differences in the fundamental harmonic—equivalent to wingbeat frequency—either in males or females collected from sympatric populations in Mali, West Africa. However, there were significant differences in the amplitude of the first and third harmonics in females and of the first and second harmonics in males. Whereas these results suggest some morphological or behavioral differences between species and forms, the extensive overlap in the distributions of harmonic amplitudes does not point to them as reliable cues for assortative mating. Combining all waveforms parameters into a discriminant analysis did not yield characteristic scores either for males or females. Thus, our results do not support the wingbeat hypothesis of premating isolation in the An. gambiae complex.

Xanthommatin is the primary ommochrome eye pigment in mosquitoes. The terminal step in xanthommatin biosynthesis, involving oxidation of 3-hydroxykynurenine (3HK), can proceed enzymatically by phenoxazinone synthase or by nonenzymatic auto-oxidation of 3HK. The relative contributions of these pathways, however, are unclear. We isolated a novel Culex pipiens mutant (crimson) that could be used to address this question. Homozygous crimson embryos exhibit no visible eyespot; first-instar larval ocelli are colorless. Eyes gradually turn red through immature development. Teneral crimson adults possess red eyes that darken to wild-type ≈5 d after emergence. Crosses indicate that crimson is sex-linked and fully recessive. Addition of xanthommatin precursors to rearing water did not rescue wild-type phenotype and suggested that the mutation is in the terminal step of ommochrome biosynthesis. Crimson expression was not temperature sensitive. Thin-layer chromatography demonstrated teneral crimson adults lacked xanthommatin. Teneral and aged wild-type adults exhibited low-mobility black ommochrome spots; aged crimson adults exhibited low-mobility brown-red ommochrome spots. Absorbance spectroscopy of eye extracts indicated teneral adult crimson eyes lacked xanthommatin but had abnormally high levels of 3HK, whereas extracts of 10-d-old crimson adults had depleted levels of 3HK and detectable levels of xanthommatin. Light microscopy indicated that eyes of young (3 d old) wild-type adults had a high concentration of pigment granules. Eyes of teneral crimson adults had no pigment granules. Eyes of 20-d-old crimson adults had low levels of pigment granules. We suggest two possible mechanisms for the crimson mutation: (1) transport of 3HK into the pigment cells and/or pigment granules is slow, with normal oxidation of 3HK into xanthommatin, or (2) 3HK is transported normally into pigment cells/granules but is not immediately oxidized to xanthommatin, resulting in 3HK hyper-accumulation and slow nonenzymatic production of xanthommatin after adult emergence.

Anopheles crucians Wiedemann (sensu lato) was investigated for the presence of cryptic species using rDNA ITS2 sequences. This complex of species presently contains the named species An. crucians, An. bradleyi King, and An. georgianus King. Adult female mosquitoes were collected at 28 sites in Alabama, Florida, Georgia, North Carolina, Mississippi, and Louisiana, resulting in 245 progeny broods. Species were identified using preliminary morphological characters, and the internal transcribed spacer two (ITS2) was amplified from all broods. The result was five distinct sizes of amplification product, and based on morphological characters, one of the size classes was suspected to consist of two species. All six putative species were then sequenced: five directly, and the sixth, because of extreme intragenomic (each individual with many variants) size variability, cloned. The ITS2 sequences were markedly distinct for all six species. Species designations and ITS2 sequence lengths (base pairs in parentheses) were A (461), B (1,000 ), C (204), D (293), E (195), and An. bradleyi (208). Species B showed both large intraspecific and intragenomic sequence variability and is distinguished by having the longest ITS2 found so far in an Anopheles. Based on these data, we found that all species could be identified with polymerase chain reaction (PCR) using a mixture of four primers in a single reaction. Members of this complex were often found in sympatry, with the adults of five species collected at a single site in central Florida.

The efficacy of a single treatment with a pour-on application or a subcutaneous injection of the macrocyclic lactone endectocide, doramectin, was evaluated in separate trials on Hereford heifers infested with Boophilus microplus (Canestrini). Significantly fewer ticks per calf were recovered from both groups of treated animals than from the complimentary untreated calves. The mean weights of engorged females and egg masses from both pour-on–treated and injectable-treated calves were also significantly smaller than the complimentary variables for the two groups of untreated calves. Among the treated groups, the mean weight of females from calves treated with the subcutaneous injection was 55% less than females from cattle that received the pour-on treatment and the weights of egg masses were 71% lighter than those from the pour-on–treated group. The estimates of percentage control for the two treatments were 88.6 for the pour-on formulation and a notably higher 99.8 for the injectable formulation. To obtain estimates of the effect of the treatments on the parasitic stages of the tick, cattle were infested with B. microplus larvae at three weekly intervals beginning 18 d pretreatment to ensure that, on the day of treatment, ticks in all three parasitic stages (adult, nymph, larva) would be on the cattle. The effect of the treatments on each parasitic stage was estimated by partitioning detached females into three groups by noting in which of the three 7-d intervals after detachment of engorged females began that detachment occurred. There was no difference for either the pour-on or injectable in the effect of the specific treatment on each parasitic stage. The persistent efficacy of the pour-on treatment against larvae placed on the hosts 1 wk after treatment was zero. The persistent efficacy of the injectable treatment ranged from 100 to 82.1% (mean, 93.7%) against the larvae placed on calves the first 3 wk after treatment and was still 44% against the fourth weekly posttreatment infestation. The injectable doramectin is a potential alternative to the coumaphos product now used as a precautionary treatment at USDA, Veterinary Services, Livestock Import Stations, for cattle exported from Mexico.

Insecticide resistance and cross-resistance was determined for three strains of Culex quinquefasciatus Say in the southeastern United States. HAmCq and MAmCq strains were collected in 2002 from Huntsville and Mobile, AL. The VBFmCq strain was collected in 1998 from Vero Beach, FL. VBFmCq, HAmCq, and MAmCq larvae showed resistance to permethrin with resistance ratios of 13, 100, and 940, respectively, compared with the susceptible S-Lab strain. Levels of resistance in HAmCq and MAmCq larvae were 200- and 830-fold to resmethrin and 4- and 70-fold to malathion, respectively. VBFmCq, HAmCq, and MAmCq strains all demonstrated a great ability to develop tolerance and/or cross-resistance to different insecticides, including deltamethrin (50-, 100-, and 300-fold), chlorpyrifos (150-, 33-, and 720-fold), fipronil (10-, 5-, and 15-fold), and imidacloprid (7.5-, 5- and 10-fold, respectively). Comparison of resistance ratios for pyrethroids, organophosphates, and imidacloprid at LC₅₀ and LC₉₀ and gradual slopes of dose-response curves indicated that VBFmCq, HAmCq, and MAmCq were heterozygous in response to these insecticides. All three strains showed high levels of susceptibility to Bacillus thuringiensis variety israelensis (Bti) and spinosad, although these mosquitoes had been extensively exposed to Bti. Thus, we conclude that Bti and spinosad may be valuable for the management of Cx. quinquefasciatus, especially in situations where local strains are highly resistant to other insecticides.

Field efficacy of repellent formulations containing picaridin (1-methyl-propyl 2-(2-hydroxyethyl)-1-piperidinecarboxylate) or deet (N,N,-diethyl-3-methylbenzamide) against mosquitoes in Northern Territory, Australia, was evaluated. The following repellent treatments were evaluated: 19.2% picaridin (Autan Repel Army 20), a solution of 20% deet in ethanol, and 35% deet in a gel (Australian Defense Force [ADF]). The predominant mosquito species were Culex annulirostris Skuse (57.8%), Anopheles meraukensis Venhuis (15.4%), and Anopheles bancroftii Giles (13.2%). The protection provided by repellents against Anopheles spp. was relatively poor, with 19.2% picaridin and ADF deet providing >95% protection for only 1 h, whereas 20% deet provided <95% protection at 1 h after repellent application. In contrast, the repellents provided good protection against Cx. annulirostris, with 19.2% picaridin providing >95% protection for 5 h and both deet formulations providing >95% protection for 7 h when collections ceased. This study provides additional field data showing tolerance of Anopheles spp. for repellents. The response of field populations of Cx. annulirostris, an important vector of arboviruses in Australia, to repellents containing deet and picaridin is reported for the first time.

The insect repellents N,N-diethyl-3-methylbenzamide (Deet) and the racemate and 1S,2′S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (AI3-37220) were tested against Anopheles albimanus Wiedemann and Aedes aegypti (L.) in laboratory human-volunteer assays. Estimated skin doses of Deet or racemic AI3–37220 required to reduce biting by 95% in Ae. aegypti were 2.3 and 3.5 × 10^–2 μmol/cm² skin, respectively, whereas estimated doses for 95% bite reduction of An. albimanus in an ≈40-yr-old laboratory colony established from El Salvador were 5 times higher at 12 × 10^–2 μmol Deet/cm² skin and >20 × 10^–2 μmol/cm² skin for AI3-37220. In tests with the 1S,2′S stereoisomer of AI3-37220, a newly established colony of An. albimanus from Belize bit less aggressively than El Salvador An. albimanus. However, the Belize-derived mosquitoes were as resistant as the old El Salvador colony to repellent effects of 1S,2′S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide. Earlier workers surmised that usual skin doses of Deet would offer only limited protection against An. albimanus in the field. Our findings support this speculation, but they also indicate that doses of Deet higher than those needed for protection against Ae. aegypti might offer reasonable protection against An. albimanus. Results indicate that neither racemate nor 1S,2′S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide offer as much protection as Deet against An. Albimanus, despite being highly effective against Ae. aegypti.

In the laboratory, three microbial mosquito larvicidal products consisting of Bacillus thuringiensis ssp. israelensis de Barjac (Bti), Bacillus sphaericus (Neide) (Bsph) (strain 2362), and the University of California Riverside (UCR) recombinant (producing toxins of both Bacillus sphaericus and Bacillus thuringiensis ssp. israelensis) were bioassayed against larvae of Culex quinequefasciatus Say (susceptible and resistant to Bsph 2362), and Aedes aegypti (L.). Bti proved highly effective against Cx. Quinequefasciatus susceptible and resistant strains, with LC₅₀ values of 0.009 and 0.011 ppm and LC₉₀ values of 0.057 and 0.026 ppm for Bsph-susceptible and -resistant strains, respectively. Bti was also highly active against Ae. eagypti with LC₅₀ and LC₉₀ values of 0.014 and 0.055 ppm, respectively. The UCR recombinant was equally active against both Bsph-susceptible and -resistant strains of Cx. Quinquefasciatus; LC₅₀ values were 0.005 and 0.009 and LC₉₀ values were 0.030 and 0.043 ppm, respectively. Bti and the UCR recombinant essentially showed similar activity against Bsph-susceptible and -resistant strains. UCR recombinant showed high toxicity against Ae. eagypti with LC₅₀ and LC₉₀ values of 0.023 and 0.064 ppm, respectively. Bsph was highly active against susceptible strain of Cx. quinequefasciatus with LC₅₀ and LC₉₀ values of 0.006 and 0.024 ppm, respectively. Bsph exhibited little toxicity against Ae. eagypti larvae and also no toxicity to Bsph resistance. In the field, we evaluated four experimental corn grit formulations of Bti (VBC 60021), Bsph (VBC 60022), UCR recombinants VBC 60023 (7.89%), and VBC 60024 (1.87%) in simulated field (microcosms) against Bsph-susceptible Culex mosquitoes. Bti and low-concentrate UCR recombinant showed similar initial activity as well as persistence. Both materials provided high-to-moderate level of control for 2–7 d posttreatment at low treatment rates. At low dosages, residual activity of Bti and UCR recombinant lasted for <7 d. Bsph and high-concentrate UCR recombinant (VBC 60023), however, were more effective against natural populations of Culex and achieved longer control (7–21 d) than the other two materials.

Seventeen analogs of the repellent compounds N,N-diethyl-3-methylbenzamide (DEET) and N,N-diethylphenylacetamide (DEPA) were evaluated in vitro for repellency against laboratory-reared Aedes aegypti (L.) and Anopheles stephensi Liston mosquitoes by using a modified membrane blood feeding test system. The system was a valuable tool for comparing the effective concentrations of the repellent analogs. Additionally, this method used easily controlled test conditions, allowing completion of the many test replications necessary to evaluate all of the repellent analogs over a period of several years. One compound, N,N-diethyl-2-[3-(triflouromethyl)phenyl]acetamide, provided significantly better repellency than DEET against Ae. aegypti and slightly improved efficacy against An. stephensi. Eight of the analogs were as effective as or slightly more repellent than DEET against both species. Seven analogs were less effective than DEET and one compound, N,N-diethyl-3-hydroxybenzamide, was as a poor repellent. Overall, two DEPA analogs and a single DEET analog provided better repellency than DEET against both mosquito species and warrant future laboratory and field evaluation.

The bacteria Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus produce insecticidal toxins used to control mosquito larvae throughout the world. Unfortunately, there are few alternative insecticides with similar activity and environmental safety, which may limit the long-term success of these insecticides. Bacillus thuringiensis subsp. jegathesan is another bacterium with toxins that are active against mosquitoes and has potential for development as a commercial product. B. t. subsp. jegathesan would be ineffective if cross-resistance was detected or if treated mosquito populations evolved resistance. B. t. subsp. jegathesan was evaluated for its potential for selecting insecticide resistance in Culex quinquefasciatus Say. Susceptibility changes in mosquitoes selected with the wild-type strain were compared with susceptibility changes in mosquitoes selected with Cry11B, a component toxin of B. t. subsp. jegathesan. Resistance was detected in generation 18 in the Cry11B-selected colony, reached a maximum of 38-fold, and was present through generation 40. The B. t. subsp. jegathesan-selected colony evolved 13-fold resistance in generation 22, but resistance declined to 2.3-fold in generation 26 and remained low throughout the study. Cry11B-selected mosquitoes showed no significant resistance to the wild-type bacterium, whereas B. t. subsp. jegathesan-selected mosquitoes expressed significant resistance to Cry11B. Both colonies displayed cross-resistance to component toxins of B. t. subsp. israelensis, but they lacked cross-resistance to that wild-type strain. The patterns of resistance and cross-resistance in this study are consistent with the patterns previously observed in mosquitoes selected with B. t. subsp. israelensis and suggest that B. t. subsp. jegathesan might also be at low risk for resistance.

Culex quinquefasciatus and Mansonia annulifera are abundant in the rural areas of Kuttanadu, Kerala, India. Bloodmeal identification for individuals of these species collected in this region was determined by the agar-gel precipitin test. A total of 2,328 blood smears from Cx. quinquefasciatus (1,148) and Ma. annulifera (1,180) was tested. Results showed that Cu. quinquefasciatus and Ma. annulifera were highly anthropophilic and that human feeding accounted for 74 and 66% of the total bloodmeals tested. Feeding on cattle accounted for only 1.5 and 2.1% of Cx. quinquefasciatus and Ma. annulifera bloodmeals, respectively. This study showed the high anthropophilic feeding rates of Cx. quinquefasciatus and Ma. annulifera collected from an endemic belt of Malayan filariasis, where epidemiological studies revealed the coexistence of Bancroftian and Malayan filariasis.

Genetically altering the disease vector status of insects using recombinant DNA technologies is being considered as an alternative to eradication efforts. Manipulating the endogenous immune response of mosquitoes such as the temporal and special expression of antimicrobial peptides like cecropin may result in a refractory phenotype. Using transgenic technology a unique pattern of expression of cecropin A (cecA) in Anopheles gambiae was created such that cecA was expressed beginning 24 h after a blood meal in the posterior midgut. Two independent lines of transgenic An. gambiae were created using a piggyBac gene vector containing the An. gambiae cecA cDNA under the regulatory control of the Aedes aegypti carboxypeptidase promoter. Infection with Plasmodium berghei resulted in a 60% reduction in the number of oocysts in transgenic mosquitoes compared with nontransgenic mosquitoes. Manipulating the innate immune system of mosquitoes can negatively affect their capacity to serve as hosts for the development of disease-causing microbes.

A 2-yr entomological study was carried out in Kerala, south India, to identify the mosquito vectors of Japanese encephalitis (JE) virus and to determine their seasonal abundance and infection. In total, 150,454 mosquitoes belonging to five genera and 18 species were collected from vegetation surrounding cattle sheds and pigsties in villages at dusk. Culex tritaeniorhynchus Giles (66.7%) was the most abundant species, with increases in numbers associated with rice cultivation. JE virus isolations were made from Cx. tritaeniorhynchus and Mansonia indiana Edwards. Based on high abundance and frequent JE virus infection, Cx. tritaeniorhynchus seems to be the most important vector, whereas Ma. indiana is probably a secondary vector.

After-hatching and hatching year, mourning doves were infected by inoculation with either western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses; some birds in each group also were treated with the immunosuppressant cyclophosphamide before and during infection. Cyclophosphamide treatment significantly increased the WEE viremia but did not alter the antibody response. In contrast, cyclophosphamide-treated and -untreated doves did not develop a detectable SLE viremia but became antibody positive. Antibody peaked at 10 wk after inoculation for both viruses and remained detectable in most birds throughout the 26-wk study. When treated with cyclophosphamide the following spring, birds did not relapse and develop a detectable viremia. Previously infected birds were protected when challenged with conspecific virus (i.e., none produced a detectable viremia), but there was no anamnestic antibody response to reinfection. In agreement with our failure to detect relapses, all birds were negative for viral RNA when sera, spleen, lung, and kidney tissues were tested by reverse transcriptase-polymerase chain reaction after necropsy. Our results indicated that adult mourning doves were an incompetent host for SLE virus and probably do not serve as a suitable overwintering or dispersal host for either WEE and SLE viruses.

The mechanism by which arboviruses bypass the basal lamina of mosquito midgut cells and enter the body cavity has been unclear. Experiments using Venezuelan equine encephalitis viral replicon particles, which express the green fluorescent protein gene in cells, indicate the operation of tissue conduits, possibly involving tracheae and visceral muscles, that facilitate virus movement through the basal lamina. Ultrastructural studies of the midgut reveal evidence for possible complete penetration of the basal lamina by tracheal cells and regions of modified basal lamina associated with visceral muscle. The modified basal lamina closely resembles proventricular matrix material known to allow virus passage.

We assessed the effects of sympatric (occupying the same or overlapping geographic areas) and allopatric (occurring in separate geographic areas) isolates of Anaplasma phagocytophilum on the survival of Ixodes scapularis Say larvae that were derived from ticks collected in Bridgeport, CT. Seven isolates of A. phagocytophilum, originating from different geographic regions of the United States, were tested: four isolates from the northeast (Bridgeport, Dawson, Gaillard, and NY-8), two from the Midwest (Webster and Sp-Is), and one from California (MRK). BALB/c mice were infected with each of the seven isolates via exposure to infected I. scapularis nymphs, whereas uninfected nymphs fed upon control mice. Both infected and control mice were infested with uninfected larvae at 1, 2, 3, 4, 6, and 9 wk after nymphal infestation. The molting success in cohorts of infected and uninfected ticks was calculated as the percentage of larvae successfully molting into nymphal stage, and the prevalence of infection in molted nymphs was determined by polymerase chain reaction. In ticks that became infected with the Bridgeport or Sp-Is isolates, the molting success decreased with an increase in the prevalence of infection. Ticks that fed upon mice infected with six allopatric isolates (Dawson, Gaillard, NY-8, Sp-Is, Webster, and MRK) showed significantly lower levels of survival than those fed upon control mice, regardless of the prevalence of infection, whereas in ticks fed upon mice infected with a sympatric isolate (Bridgeport), the overall molting success was similar to the control. Thus, some but not all of the A. phagocytophilum isolates have adverse effects on ticks. Ticks exposed to harmful isolates may experience higher levels of bacterial metabolism, and/or reduced quality of their blood meal, thereby reducing their survival. Noted differences between isolates may be due to the origin of a particular isolate and/or the degree of coadaptation between the pathogen and its vector on the population level.

Granite rocks is a very abundant material in Mexico because they are used habitually as borders for fields. The current work established the significance of this ecotype as a colonization site for triatomines of the Phyllosoma complex. Seven sites, arbitrary selected, in San Martin de Hidalgo municipality (Mexico) were investigated in April 2002 by using 210 mouse bait-traps left during the night in wall hollows. One hundred and seventy-two triatomines of all life stages were collected from the seven sites. Triatomines adhered to the tape that covered the traps or were found near them, and 36.6% of the traps collected triatomines. The principal species was Triatoma longipennis Usinger (1939) and low numbers of Triatoma picturata Usinger (1939) were found. The nymphal population was very young, probably corresponding to the reproductive period in April (end of the dry season). The infection rate of the triatomines for Trypanosoma cruzi was 49%. Because collecting triatomines in the field is laborious and time-consuming, the mouse bait-trap method found to be practical to assess the population of triatomines within a sylvatic habitat.

Malaria, both Plasmodium falciparum (Welch) and Plasmodium vivax (Grassi & Feletti), has reemerged as a significant public health disease issue in Peru, especially in forested areas in the eastern part of the country. The spread of Anopheles darlingi Root, the principal South American malaria vector, into new areas of Peru is thought to be a factor in this resurgence. However, epidemiological evidence suggests that in malaria endemic areas of eastern Peru where An. darlingi does not occur, other species are involved in malaria transmission. The objective of this study was to analyze Anopheles species collected from 11 provinces within four departments in eastern Peru during 2001 and 2002 for infections with P. falciparum and P. vivax. More than 84,000 Anopheles mosquitoes representing 13 species were tested by enzyme-linked immunosorbent assay for the presence of Plasmodium circumsporozoite (CS) proteins. Of these, only An. darlingi and Anopheles benarrochi Gabaldón, Cova García & López were found positive. In total, 14 (0.98%) of 1,432 pools of An. darlingi were positive for Plasmodium species; specifically 10 (0.70%) pools were positive for P. falciparum, two (0.14%) were positive for P. vivax VK210, and two (0.14%) were positive for P. vivax VK247 proteins. Nine (0.14%) of 6,323 pools of An. benarrochi were positive for Plasmodium; five (0.08%) of 6,323 pools were positive for P. falciparum, two (0.03%) were positive for P. vivax VK247, one (0.02%) was positive for mixed P. vivax VK210/VK247 infections, and one (0.02%) was positive for mixed P. falciparum and P. vivax VK210 CS-proteins. Although infection rates in An. benarrochi were significantly lower (0.14%) than rates found for An. darlingi (0.98%), our data suggest that An. benarrochi may play a role in transmitting and maintaining Plasmodium species in various malaria endemic areas of eastern Peru.

The species composition and population dynamics of adult mosquitoes in a wetland near Iuka, MS, were analyzed over a 6-yr period (1997–2002) and reverse transcription-polymerase chain reaction (PCR) detection rates of arboviruses determined during five of those years. Blood meals of three likely vector species were identified using a PCR-based method that allows identification of the host to species. Culex erraticus (Dyar & Knab) composed 51.9% of the population during the 6-yr period with 295 females collected per trap night. Eastern equine encephalomyelitis (EEE) virus was detected in six genera of mosquitoes [Coquillettidia perturbans (Walker), Culex restuans Theobald, Culex salinarius Coquillett, Culex erraticus (Dyar & Knab), Anopheles crucians Wiedemann, Anopheles quadrimaculatus Say, Aedes vexans (Meigen), Ochlerotatus triseriatus Say, and Psorophora ferox Humboldt) with positive pools occurring in 1998, 1999, and 2002. Culiseta melanura Coquillett occurred at a low level (<1%) and was not infected. Saint Louis encephalitis virus was detected once in a single pool of Cx. erraticus in 1998. Neither West Nile virus nor LaCrosse virus was found. Minimum infection rates per 1000 females tested of competent vectors of EEE virus were variable and ranged from 0.14 for Cx. erraticus to 40.0 for Oc. triseriatus. Thirty-nine species of birds were identified in the focus with blood-engorged mosquitoes found to contain meals (n = 29) from eight avian species. The majority of meals was from the great blue heron, Ardea herodias L. (n = 55%), but when bird abundance data were adjusted for avian mass, the brown-headed cowbird, Molothrus ater (Boddaert); blue jay, Cyanocitta cristata (L.); and northern mockingbird, Mimus polyglottos (L.), were overrepresented as hosts.

An epidemic of dengue during 2001 in Northwestern Peru reemphasized the need for efficient, accurate, and economical vector surveillance. Between November 1998 and January 1999, we carried out extensive entomological surveys in two neighborhoods of ≈600 contiguous houses located in the Amazonian city of Iquitos, providing a unique opportunity to evaluate the Aedes aegypti (L.) rapid assessment survey strategy. Based on Pan American Health Organization recommendations, this strategy is used by the Peruvian Ministry of Health (MOH). In our analysis all household locations, including closed and unoccupied houses, were georeferenced and displayed in a geographic information system, which facilitated simulations of MOH surveys based on hypothetical systematic sampling transects. Larval, pupal, and adult mosquito indices were calculated for each simulation (n = 10) and compared with the indices calculated from the complete data set (n = 4). The range of indices calculated from simulations was moderately high, but included actual indices. For example, simulation ranges for house indices (HI, percentage of infested houses from complete survey) were 38–56% (45%), 36–42% (38%), 21–34% (30%), and 13–33% (27%) in four surveys. HI, Breteau index, pupae per hectare, adult index, and adults per hectare were more robust entomological indicators (coefficient of variation [CV]/mean = 0.1–2.9) than the container index, pupae per person, pupae per house, adults per person, and adults per house (CV/mean >20). Our results demonstrate that the MOH’s Ae. aegypti risk assessment program provides reasonable estimates of indices based on samples from every house. However, it is critical that future studies investigate the association of these indices with rates of virus transmission to determine whether sampling variability will negatively impact the application of indices in a public health context.

We conducted a carrion succession study within a restricted urban backyard in the city of Vienna, Austria (16° 22′E, 48° 12′N) from May to November 2001 to analyze sequence and composition of the local carrion visiting fauna. Two medium sized clothed domestic pig carcasses (Sus scrofa Linnaeus), were used as surrogate human models. In total, 42 arthropod species from the families Calliphoridae, Sarcophagidae, Sepsidae, Piophilidae, Muscidae, Fanniidae, Sphaeroceridae, Phoridae, Drosophilidae, Anthomyiidae, and Lauxaniidae (Diptera), Formicidae, Braconidae, Pteromalidae, and Vespidae (Hymenoptera), Silphidae, Staphylinidae, Histeridae, Cleridae, and Dermestidae (Coleoptera), as well as species from the orders Isopoda and Acari were collected during the decomposition of these carcasses. A significant feature in this study was the high abundance of Calliphora vomitoria (L.) and Chrysomya albiceps (Wiedemann). In the experiment conducted May to June, larvae and adults of C. vomitoria outnumbered all other blow fly species, followed by Protophormia terraenovae (Robineau-Desvoidy), C. vicina Robineau-Desvoidy, and Lucilia sericata (Meigen). C. vomitoria is generally considered to be rural in distribution, where it prefers shaded locations. The presence of this species in rural as well as in urban habitats in Austria precludes this species as biogeographic indicator. In the study beginning in August large numbers of female adults of the nonindigenous blow fly C. albiceps began oviposition at day 3 after placement of the cadaver. The predatory second and third instars of C. albiceps larvae subsequently almost monopolized the cadaver. C. albiceps is generally described as tropical and subtropical species. The observed northward expansion of its range beyond southern Europe obviously decreases the value of C. albiceps in estimating place of death, in that it is no longer exclusive to southern regions. Our results clearly show, that caution must be used when drawing conclusions from succession data generated in different geographic areas. Moreover, this study demonstrates, that arthropod mediated decomposition of a 44 kg exposed pig carcass in a central European urban habitat can be completed within 3 wk.

Efficacy of multiple dip treatments (one to three) at 0.3% active ingredient (AI) coumpahos applied at 7- or 10-d intervals was determined against organophosphate (OP)-resistant Boophilus microplus (Canestrini). None of the treatments totally prevented repletion of all females. In the 7 d after one treatment, the percentage of reduction of female ticks was 24.8%, whereas in the 10 d after one treatment, the reduction of female numbers was 47.1%. Application of two or three dips at either interval provided substantially higher reductions in female numbers than either single treatment (range 80.2–88.8%). Percentage reduction of the index of fecundity of females (designated as percentage of control) in the 7 d after one dip provided 46.8% control, whereas control in the 10 d after one treatment was 65.6%. Again, application of two or three dips at either interval provided substantially higher control (range 92.9 to >99.9%). Although control levels after two or three dips were similar, only application of three dips at either interval provided a high enough level of control (>99.5%) to ensure that the U.S. Boophilus eradication program would not be at risk of inadvertently dispersing viable ticks. Thus, although no treatment regime prevented repletion of all treated females, application of three dips at either interval virtually eliminated production of reproductively active females, thereby eliminating the possibility that ticks would become established. It should be noted that ticks possessing different OP resistance mechanisms than ticks in this study could have higher reproductive capabilities than were observed against these treatment regimes.

House dust mite allergens cause allergy and asthma in sensitized individuals. The allergens they produce are known to resist decay under natural household conditions and are thought to accumulate until removed. We sought to evaluate the effects of high temperature (96°C) generated by a hard surface cleaner on live mite populations of American house dust mites, Dermatophagoides farinae Hughes and their allergens in carpet and mattresses. Statistically significant (P < 0.05) mite mortality (100%) was observed in response to treatment in both textile surfaces. Similar effects were observed on Der f 1 fecal allergen. Allergen reductions in carpet with two or five pass treatment regimes were 61.4 and 100%, respectively. These results demonstrate the potential of employing a hard surface steam cleaner as a novel method to eliminate house dust mite populations and their allergens in a residential setting and appear particularly promising as an environmental control strategy.

Two Amblyomma longirostre adult male ticks were collected from a Brazilian porcupine Coendou prehensilis L. in the state of Rondonia, Western Amazon, Brazil. The two ticks were pooled for DNA extraction and tested for the presence of rickettsial DNA by amplifying portions of the gltA, 17-kDa, ompA, and ompB rickettsial genes by polymerase chain reaction (PCR). Portions of the four genes were amplified from the sample and subsequently sequenced. These results indicated the presence of a Rickettsia strain infecting A. longirostre, which was designated as strain Aranha. Compared with homologous ompA rickettsial sequences, “Rickettsia amblyommii” ompA seemed to be the closest relative to Aranha (similarity values: 99.0–99.3%). Phylogenetic analyses of more conserved genes including 17-kDa and gtlA partial sequences indicated that this Rickettsia sp. is a spotted fever group rickettsia. The partial ompB sequence of strain Aranha was distinct from all homologous sequences available in GenBank. Although our ompA analysis suggested a very close molecular phylogenetic relationship of Aranha with “R. amblyommii,” we cannot at this time determine if Aranha is a new strain of “R. amblyommii” or a new Rickettsia species in South America.