In a malaria endemic area in Orissa state in eastern India baseline (November 1989 to October 1990) malaria incidence ranged from 215 to 328 cases/1,000 population/yr in different groups of villages. In November 1990, nylon bednets treated with deltamethrin at 25 mg/m² were given out in two villages (population 1062), untreated bednets were given out in five villages (population 1,226) and in one village (population 786) nets were not given. Nets were retreated in October 1991 and June 1992 in treated-net villages. The trial continued until October 1992. The treated nets caused significant reduction in indoor resting density, biting (landing), light trap catches, human engorgement rate, and parous rate of malaria vector Anopheles culicifacies Giles as compared with untreated nets or no nets. Untreated nets also caused reductions in biting and indoor density. Treated nets retained insecticidal action well over 6 mo. In the final year, malaria incidence was reduced 8.9% in the no-net village, 34.9% in the villages with untreated nets, and 59.1% in villages with treated nets. The relative risk of malaria and parasite rates declined significantly in villages with treated nets. Pediatric splenomegaly rate did not change in the no-net village, increased significantly in villages with untreated nets, but decreased significantly in those with treated nets. Treated nets also reduced pediatric anemia rates, but Hb concentration increased in all villages. Considering the benefits of treated bednets and development of resistance among vectors to DDT and malathion, bednets treated with deltamethrin could be an effective alternative strategy to control malaria in forested areas in India.

Four strains of Aedes aegypti (L.), one from Cuba and three from Venezuela, were bioassayed for susceptibility to eight insecticides, including the organophosphates, temephos, malathion, fenthion, pirimiphos methyl, and chlorpyrifos, and the pyrethroids, deltamethrin, lambda cyhalothrin and cypermethrin. S, S, S,-tributyl phosphorotrithioate and piperonyl butoxide were used as synergists to assess the involvement of esterases and monooxygenases in organophosphate resistance. Venezuelan strains had low levels of resistance to fenthion and malathion, and moderate to high resistance to temephos, pyrimphos methyl, and chlorpiriphos. All strains were susceptible to the pyrethroids, except the Cuban strain, which had moderate levels of resistance to cypermethrin. Organophosphate resistance in Ae. aegypti is a serious threat to control operations. Integrated strategies for Ae. aegypti control to prevent or delay pyrethroid resistance in Venezuela and Cuba are discussed.

The survival of immature fleas at 25 and 28°C and 40, 55, 75, and 92% RH was studied to test the hypothesis that the difference in microclimatic preferences determines habitat distribution of Xenopsylla conformis Wagner, 1903 and Xenopsylla ramesis Rothschild, 1904. Survival of X. conformis eggs did not depend on either temperature or humidity or both, whereas eggs of X. ramesis survived significantly less at 40% RH than at higher humidities. No larva of either species survived at 40% RH at either temperature. Larval survival of both species at both temperature regimes was significantly lower at 55% humidity than at higher humidities. Maximal survival time of larvae that died before pupation depended on both temperature and humidity in both species. Change of humidity during early stages of the life cycle (from egg to larva) increased the maximal survival time in X. conformis larvae but decreased that in X. ramesis larvae. Pupal survival was higher at higher humidities independent of temperature. Survival of X. conformis pupae was lower than that of X. ramesis pupae when the relative humidity was low. Humidity change on later stages (from larva to cocoon) decreased X. conformis pupal survival and had no effect on X. ramesis pupal survival. The sex ratio of emerged adults was not affected by either temperature or humidity in both species. Changes in humidity between egg and larval environments significantly decreased the percentage of females in X. conformis emergence at 28°C.

Research on domestic and sylvatic triatomines within the community of Carrillo Puerto and neighboring areas of Nayarit, Mexico, documented that Triatoma longipennis (Usinger) and Triatoma picturata (Usinger) were infected with Trypanosoma cruzi (L.) in both habitats. T. picturata was the predominant species in both habitats. Mouse baited-traps increased the effectiveness of collecting sylvatic triatomines, which were difficult to sample by inspecting habitats such as burrows, caves, and cliffs. The colonization of sylvatic and peridomestic habitats by Triatoma, the occurrence of high rates of infection with T. cruzi and the possibility that bugs move between habitats may require modification of current control strategies in Mexico.

Mosquitoes that do not oviposit their entire batch of eggs in one location, but choose instead to oviposit a few eggs in several different sites exhibit skip oviposition. Although skip oviposition may ensure the greater distribution of progeny from an individual female and reduce sibling competition, it also uses greater maternal energy reserves, may increase the risk of adult female mortality and does not reduce other competition. To test the hypothesis that female Aedes aegypti (L.) do not oviposit all of their eggs at one time and place, we examined females collected with an aspirator resting inside houses in Thailand. Females (n = 384) were dissected and the stage and number of developing oocytes were enumerated. If skip oviposition is a common behavior among female Ae. aegypti, one would expect to find significantly fewer mature eggs (Christophers’ stage V) in gravid females than developing oocytes in half-gravid females (Christophers’ stage IIIa–IVb). No significant differences were found between ovaries containing immature or mature ooctyes, even when the data were stratified by female body size and when immature ooctyes that potentially could undergo resorption (Christophers’ stages IIIa–IIIb) were excluded from the analysis. We found no evidence that skip oviposition over several days is a common behavior among domestic populations of Ae. aegypti at the end of the rainy season in this region of Thailand.

We investigated how temperature and precipitation regime encountered over the life cycle of Aedes albopictus (Skuse) affects populations. Caged populations of A. albopictus were maintained at 22, 26, and 30°C. Cages were equipped with containers that served as sites for oviposition and larval development. All cages were assigned to one of three simulated precipitation regimes: (1) low fluctuation regime - water within the containers was allowed to evaporate to 90% of its maximum before being refilled, (2) high fluctuation regime - water was allowed to evaporate to 25% of its maximum before being refilled, and (3) drying regime - water was allowed to evaporate to complete container dryness before being refilled. Greater temperature and the absence of drying resulted in greater production of adults. Greater temperature in combination with drying were detrimental to adult production. These precipitation effects on adult production were absent at 22°C. Greater temperatures and drying treatments yielded higher and lower eclosion rates, respectively and, both yielded greater mortality. Development time and size of adults decreased with increased temperatures, and drying produced larger adults. Greater temperatures resulted in greater egg mortality. These results suggest that populations occurring in warmer regions are likely to produce more adults as long as containers do not dry completely. Populations in cooler regions are likely to produce fewer adults with the variability of precipitation contributing less to variation in adult production. Predicted climate change in North America is likely to extend the northern distribution of A. albopictus and to limit further its establishment in arid regions.

In southern France, failure to control Culex pipiens L. with Bacillus sphaericus Neide toxin (Bs) was first detected in 1994, at the extreme east of the Languedoc-Roussillon coast. This failure was due to a single recessive mutant, sp-1^R. Two complementary strategies were used to test whether sp-1^R had invaded the Bs-controlled area by 1998. First, a strain (BP) was selected from resistant larvae sampled in the western part of the Bs-controlled area. In BP strain, resistance involved a single recessive gene, sp-2^R, distinct from sp-1^R, that conferred a similarly high resistance in the homozygous state (≈6,000-fold). Combining one copy of sp-1^R and one of sp-2^R conferred a > 100-fold resistance. Second, Bs-resistance was monitored among the offspring of field females crossed to sp-1^RR homozygous males. Females were sampled in 20 localities of southern France and three localities of the Llobregat delta (Barcelona, Spain) where C. pipiens control is also intensive. The 537 females in the study produced enough larvae to infer their genotype: 462 progenies were susceptible and the survival rate of 51 others was explained by the presence of sp-1^R and/or sp-2^R. The remaining 24 cases indicated that other factors could confer resistance when combined with sp-1^R. The current data showed that, even when recessive, resistant mutants can rapidly increase in frequency, providing some interactions that protect them from disappearance. We discuss the consequences of this finding on the current strategies aimed to avoid or delay resistance in the pests controlled with B. sphaericus or B. thuringiensis Berliner toxins.

This study describes the identification of Borrelia parkeri spirochetes in Colorado. Two isolates of B. parkeri (6230 and 6232) were recovered from Ornithodoros parkeri Cooley ticks collected at an inactive prairie dog town in Moffat County. Both isolates were partially characterized by sequencing and subsequent parsimony and neighbor-joining analyses of appropriate regions of the 16S ribosomal RNA, flagellin and P66 genes. Analyses of the 16S gene sequences from the Colorado isolates indicated that they were more closely related to B. parkeri and B. turicatae than to B. hermsii or the other species of Borrelia investigated in this study. Additional analyses of amino acid sequences for flagellin and P66, however, clearly demonstrated that isolates 6230 and 6232 were most closely related to B. parkeri. The possible significance of B. parkeri as an agent of human disease is discussed.

The phylogenetic relationships among 18 species of Triatominae were inferred based on mitochondrial DNA (mtDNA) sequences. The species of Triatoma included 11 belonging to the infestans complex [T. infestans (Klug), T. guasayana Wygodzinsky & Abalos, T. sordida (Stål), T. platensis Neiva, T. brasiliensis Neiva, T. rubrovaria (Blanchard), T. vitticeps (Stål), T. delpontei Romaña & Abalos, T. maculata (Erichson), T. patagonica Del Ponte, and T. matogrossensis Leite & Barbosa] and four others of the same genus but of different complexes [T. circummaculata (Stål), T. protracta (Uhler), T. dimidiata (Latreille), and T. mazzottii Usinger]. As possible outgroups we used Mepraia spinolai Mazza, Panstrongylus megistus (Burmeister), and Rhodnius prolixus Stål. We analyzed mtDNA fragments of the 12S and 16S ribosomal RNA genes from each of the 18 species, as well as of the cytochrome oxidase I (COI) gene from nine. The 12S, 16S, and COI gene sequences were analyzed individually and combined. All of the phylogenetic analyses unambiguously supported two clusters: one including T. infestans, T. platensis, and T. delpontei, and the other T. sordida and T. matogrossensis. Inclusion of T. circummaculata into the infestans complex was confirmed, although this is in disagreement with the morphological classification. On the other hand, our analyses showed that T. dimidiata is closely related to a phyllosoma complex species, T. mazzottii. This is consistent with the tentative classification previously made based on morphological characters. The issue of the monophyly of the genus Triatoma remains unresolved.

The developmental timing of Ixodes pacificus Cooley & Kohls, the primary vector of the Lyme disease spirochete and the agent of human granulocytic ehrlichiosis in the far-western United States, was determined under field and laboratory conditions. During their seasonal peaks of abundance, each of the three parasitic stages of I. pacificus, both fed and unfed, was placed inside silk-screen packets. These packets were apportioned between four topographic exposures of two hilltop sites in northwestern California. The sites differed in vegetational composition and elevation: the first (elevation 390 m) was dominated by woodland-grass, the second (elevation 914 m) by chaparral. The timing of oviposition, larval eclosion, molting, and mortality were recorded in the field every 2–3 wk for 2.5 yr. Microenvironmental temperatures were measured on all four exposures at both sites. Accelerated developmental rates of all three stages were correlated with warmer soil temperatures and the time of placement in the field. In the laboratory, replete female I. pacificus maintained under uniform environmental conditions sustained constant preovipositional and prehatch periods independent of date-of-feeding. In the field, all unfed stages survived through one active feeding season with most larvae and nymphs remaining in behavioral diapause between late summer and early spring. No life stage survived through two active feeding periods which suggests that cohorts do not overlap. We concluded that I. pacificus takes a minimum of 3 yr to complete its life cycle in northwestern California.

Ixodes pacificus, life history, microhabitat, development, Lyme disease

Transtadial persistence and stercorarial shedding of hepatitis B virus (HBV) in common bed bugs, Cimex lectularius L., was studied by using experimental infectious blood feedings, infectious intrathoracic inoculations, and virus detection by polymerase chain reaction and Southern hybridization. Results showed that HBV persisted after an infectious blood meal in bed bug bodies for up to 35 d after the infectious blood meal. It was passed transtadially through one molt regardless of instar, was shed in fecal droplets for up to 35 d after the infectious blood meal, but was not passed transovarially. In bugs inoculated intrathoracically, HBV was detected for 21 d postinoculation. Previous studies detected the hepatitis B surface antigen found on both infectious and noninfectious particles in bed bugs. In this study, the presence of nucleic acids amplified from a conserved core region of the viral genome in bodies and feces of C. lectularius suggests that the HBV virus may be mechanically transmitted in feces or when bugs are crushed, during feeding.

A Hermes-based transposable element transformation system incorporating an enhanced green fluorescent protein (EGFP) marker was used to produce two transgenic lines of Culex quinquefasciatus (Say). The transformation frequency was ≈12% and transformation of Culex was shown to be dependent on the presence of Hermes transposase. Injected Culex embryos were treated with four different heat shock regimes, two of which produced transformed individuals. These individuals were mated with wild-type mosquitoes and produced offspring which expressed the dominant EGFP gene in Mendelian ratios predicted for the stable integration of a gene at a single locus. The two transformed lines displayed distinct patterns of phenotypic expression, the expression of which has remained stable after fifteen generations. In these transgenic lines both the Hermes element and flanking plasmid DNA integrated into the Culex genome, as has been previously seen in Hermes-mediated transgenic strains of Aedes aegypti (L.). The high frequency of Culex transformation together with the dependence on the presence of Hermes transposase suggests that, as for Ae. aegypti, this mode of transposition into the germ-line genome occurs by an alternate mechanism to the cut and paste type of transposition seen for this element in other insect species and in the somatic nuclei of mosquitoes. This is the first report of the genetic transformation of a species in the genus Culex and demonstrates that this medically important mosquito species can now, along with several other Culicine and Anopheline mosquito species, be genetically manipulated.

We investigated the effect of different densities (0, 20, or 40) of developing larval Aedes triseriatus (Say) on bacterial abundance, bacterial productivity, and leaf fungal biomass in a microcosm experiment. Larvae in the low-density treatment developed normally, but larvae at the high density were significantly slower to develop. Both bacterial abundance (direct microscopic counts) and bacterial productivity (³H-leucine incorporation rates) on leaf material were significantly lower in the presence of larvae. Bacterial abundance in the water column did not change significantly with treatment, but bacterial productivity varied with time and declined significantly at both larval densities. Bacteria on the walls and bottom of the containers also were less abundant and significantly less productive in the presence of larvae. Aside from presence/absence effects, there was no clear evidence that larval impacts were density-dependent. Leaf-associated fungal biomass, as measured by ergosterol levels, varied with time but was not significantly affected by any treatment, suggesting most fungal tissue was incorporated in the leaf matrix and unavailable to larvae. Based upon estimated biomass accrual and respiration of larvae, it appears that bacterial biomass and production were insufficient to account for carbon demands of growing larvae. Because fungal biomass and leaf mass likely contributed little to gross larval demands, other carbon sources (e.g., protozoa and extracellular microbial components) were probably used by larvae. Although apparently insufficient for all larval carbon demands, bacterial and leaf fungal biomass may be adequate for other larval nutritional needs (i.e., nitrogen and essential lipids).

Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 μl of a 10⁴ pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23–30°C. Following 7, 10, 14, and 28 d application, 10 mosquitoes each were removed from the lure, pooled, and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7, 10, 14, 21, and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after application to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation.

The larvae of Plodia interpunctella (Hübner), commonly known as the Indian meal moth, often cause enormous losses in stored food supplies. We present three clinical case reports of accidental infestation by P. interpunctella larvae in two domestic cats and one parakeet. A larva gained entry into the avian host and subsequently migrated to the brain. It was alive, covered with “silk-like” fibers and confirmed to be a fourth instar. Plodia interpunctella larvae were excised with forceps from the subcutaneous tissues of the ear and neck of two cats in a different household. Previous reports of infestation by P. interpunctella larvae in vertebrates are unknown.

The blood feeding of mosquitoes and black flies from Hereford cattle and ponies treated with commercial formulations of permethrin was evaluated using an animal enclosure trap sample system that allowed comparison of insect blood-feeding levels between treated and nontreated animals. Blood feeding of both Aedes dorsalis Meigen and A. melanimon Dyar from heifers treated with pour-on concentrate and whole body spray treatments was reduced significantly by 79–88% at 4 d posttreatment, with apparent but not significant reductions of 61–68% at 11 d posttreatment. Simulium bivittatum Malloch and S. griseum Coquillett blood feeding was reduced significantly by 96% to >99% at 4 d posttreatment, but apparent reductions of 30–87% at 11 d posttreatment were not significant. Blood feeding of S. bivittatum from ponies treated with a permethrin fly wipe was reduced significantly by 98 and 87% at 1 and 7 d posttreatment, respectively. No evidence of treatment-induced mortality was observed for recently blood-fed female mosquitoes or black flies captured from treated animals and held for 24 h. The potential benefit of using permethrin to protect livestock from insect-transmitted pathogens was estimated with a model based on level of host attack, pathogen infection rate in the vector, and suppression of blood feeding. Suppression of blood feeding by 90% is predicted to prevent the exposure of a host to a pathogen for up to10 d at 1,000 insect feedings per d when the vector population infection rate is one insect per 1,000. If insect feedings are lower (100/d) and the insect infection rate remains at one per 1,000, protection is predicted for 100 d. In contrast, a 90% suppression of blood feeding is predicted to provide protection for less than 1 d at 1,000 feeding per day and a vector infection rate of one insect per 100.

Respiratory gas exchange was measured in various developmental stages of the flea Xenopsylla conformis mycerini (Rothschild, 1904) originating from the central Negev in Israel. Gas exchange in fleas was measured using a flow-through respirometry system that monitored CO₂ emission. Lowest metabolic rates were found in the cocooned stage, which included prepupae, early stage pupae, late stage pupae, and preemerged adults. Newly emerged adults and feeding larvae had metabolic rates 2.5–3.0 times greater than those for the cocooned stages. Highest rates of gas exchange were found in feeding fleas. The low metabolic rates of the cocooned stages is thought to contribute to their ability to survive for longer periods than free-living larvae and adults.

The electrophoretic polymorphism of loci encoding for 10 enzymes was studied in Culex p. quinquefasciatus Say from six localities of Vietnam. The analysis of 11 “neutral genes” showed that differentiation among samples was low, but significant (Fst = 0.06), and significantly related to geographic distance between sample sites. These results are similar to those observed in other countries (Europe and west Africa). A single type of overproduced esterases (A2-B2) was observed, and its frequency was high (60–100%) in all samples. This situation is in sharp contrast with that observed in other countries of South East Asia (China, South Korea and Japan), where two or more types of overproduced esterases have been reported. A map summarizing the geographic distribution of Asian Cx. p. quinquefasciatus with overproduced esterases is provided.

The forensic entomology case described herein is the first such case documented in Thailand. A mummified corpse of a 32-yr-old man was discovered in a forested habitat, with the larvae of six species of flies (Diptera) found in association with the corpse at the time of its discovery, i.e., those of Hydrotaea (=Ophyra) spinigera Stein (family Muscidae), Piophila casei (L.) (family Piophilidae), Megaselia scalaris (Loew) (family Phoridae), Sargus sp. (family Stratiomyidae), and larvae of two unidentified flesh fly species (family Sarcophagidae). The presence and age of the larval specimens of P. casei, M. scalaris, and H. spinigera gave entomological evidence that the postmortem interval for the corpse was 3–6 mo. This report also documents some of the forensically important fly species that occur in Thailand.

Transmission electron microscopy (TEM) was used to study two types of endocrine cells from the midgut of adult female Lutzomyia longipalpis (Lutz & Neiva). Endocrine cells rarely have been observed in Nematocera, even using TEM, and were present in small numbers dispersed among the monolayer of midgut digestive epithelial cells. Triangular shaped “closed” cells were observed along the basement membrane, bounded on each side by digestive cells; these cells closed distally before reaching the epithelial lumen. These endocrine cells appeared to deliver active granules that were secreted through a cellular membrane into the hemolymph. A second cell type occupied a similar position to the closed cells, but opened into the midgut lumen via microvilli, where the secretory products may be delivered. Each cell type possessed both electron-lucent and electron-dense vesicles with secretory granules which may indicate different stages in maturation and activity. These granular secretory products are probably peptidergic substances, with secretion mediated by diet via basal and baso-lateral receptors that were bound to membranes or microvilli.

The eggs of Aedes lineatopennis (Ludlow) are described by means of a scanning electron microscopy. The Ae. lineatopennis egg has a rather boat-shaped, fragmented micropylar collar, and membrane-like wall enclosing many tubercles of exochorionic sculpture.

The morphology of third-instar Piophila casei (L.) is described by means of scanning electron microscopy. Features of the anterior cephalic region and creeping welts that are used in larval skipping and creeping, respectively, are highlighted. Morphological features classically used for larval identification are also illustrated.

Holstein heifers in a confined feedlot setting on a southern California dairy were either sprayed individually along the ventral midline using 0.2% permethrin (250 ml/animal) (two pens) or were not treated (two pens). Treatments (n = 6 dates) were applied every 2 wk during the peak fall bluetongue virus transmission season (22 August–29 October). Animals seronegative for bluetongue virus antibodies at the initial bleeding on 15–18 September (n = 106 in the treatment pens and n = 117 in the control pens) were bled again for testing 2 mo later (12–13 November). Seroconversion rates were not significantly different: 56% for the treated animals and 48% for the controls (P > 0.2). The area has many essentially contiguous, confinement dairies with wastewater ponds that produce large numbers of Culicoides sonorensis Wirth & Jones, the primary bluetongue virus vector. Further, these dairies presumably provided a large reservoir of virus-infected cattle to infect vectors in the immediate area. Under these severe virus challenge conditions, permethrin applied at 2-wk intervals failed to reduce exposure to bluetongue virus.

The salivary proteins of Anopheles darlingi Root, the principal vector of malaria in the Amazon Region, Brazil, were analyzed. Comparison of the protein profiles between adult males and females revealed that most of the polypeptides are present in both sexes, but female-specific polypeptides also were observed. SDS-PAGE analysis of sugar-fed female mosquitoes with ages varying from 1 to 10 d after adult emergence indicated that the proteins start to be accumulated in the first day of life and are present throughout the period analyzed. Analysis of blood-fed mosquitoes showed no differences in salivary proteins when compared with sugar fed ones, suggesting that there is no specific protein induced by blood. The protein profiles of the salivary glands dissected from wild-caught female mosquitoes from different geographical regions of Brazil were compared and some differences were observed.