Oviposition site selection has been recognized as critical both for the survival and population dynamics of mosquitoes. Volatile substances released from larval habitats have been implicated as potential olfactory cues mediating oviposition. In our continuing studies of cues involved in oviposition site selection, we collected material from the larval habitats of Anopheles albimanus Wiedemann and Anopheles vestitipennis Dyar & Knab, i.e., cyanobacterial mats and Typha domingensis Pers. litter, respectively. The volatile compounds were extracted by freeze-drying the material and trapping the volatilized material on a −55°C titanium condenser. For oviposition trials conducted with wild-caught females, the tested volatile materials were pipetted onto filters floating on the surface of distilled water in Teflon beakers that were placed within oviposition cages. For both species, volatile materials in low concentrations increased oviposition, assessed as egg density, whereas there was a shift to reduced oviposition at higher concentrations. Volatile effect was strongly habitat/species-specific as shown by reciprocal treatment tests.

A comparative study of human-landing catches (HLCs) and odor-baited entry traps (OBETs) for sampling malaria vectors was conducted in two different bioclimatic areas of Senegal, the Sahelian and Sudano-Guinean phytogeographic zones, from September to December 2002. Mosquitoes were collected by the two methods both indoors and outdoors. The reliability of OBET samples was tested by comparing the two methods. Overall, HLC was more effective indoors and for surveying the anopheline fauna. Both methods were effective in sampling the four known malaria vectors in Senegal [Anopheles gambiae s.s., An. arabiensis Patton, An. funestus Giles, and An. nili (Theobald)], and mosquito age structures and infectivity rates did not differ between methods.

Family-based phenotypic selection was used to breed two genetic strains of Aedes aegypti L. that differ in susceptibility to infection with dengue serotype 2 virus (DEN-2) strain JAM1409. A Dengue 2 Susceptible on 3 chromosomes (D2S3) strain was bred from Ae. aegypti aegypti and Ae. aegypti formosus P₁ parents to have a high midgut infection rate (MIR) and a high disseminated infection rate (DIR). A Dengue 2 Midgut Escape Barrier (D2MEB) strain was bred from D2S3 and Houston P₁ parents to have a high MIR and a low DIR. After selection in the F₂ generation, single strand conformation polymorphism genotypes were determined at cDNA marker loci throughout the genome to test for Mendelian ratios and thereby identify regions containing deleterious or lethal alleles. Both strains were orally challenged with two other DEN-2 genotypes, two DEN-1 genotypes, one DEN-3 genotype, and two DEN-4 genotypes. There were significant differences in MIR and DIR for the different virus strains in both the D2S3 and the D2MEB mosquito lines.

The endosymbiont Wolbachia, extensively occurring in arthropods, usually causes reproductive distortions of the host, such as mosquitoes. In past years, detection of Wolbachia in host tissues has highly relied on transmission electron microscopy (TEM) that is tedious and usually unable to gain satisfactory results without experienced techniques and expensive instruments. Polymerase chain reaction (PCR) recently has become popular in Wolbachia identification. However, necessity of DNA extraction from host individuals or dissected tissues has limited its application in extensiveness and versatility. At present, in situ hybridization has increased its role in examination of various microbes. This report provides a technique for rapid detection and localization of Wolbachia in tissues dissected from mosquitoes and possibly other infected organisms. To detect Wolbachia and to localize them in host tissues more precisely, in situ hybridization by using digoxigenin (DIG)-labeled probes was invented and applied to Wolbachia detection in this study. The results showed that Wolbachia preferentially aggregate in ovarioles, which is consistent with previous observations by TEM. The endobacteria also were detected in salivary glands, mostly in lateral lobes. Ultrastructurally, Wolbachia has been shown to occur in the cytoplasma of salivary gland cells.

As a part of an inventory of bats in abandoned mines at the municipality of Guanaceví, Durango, Mexico, a sample of long-eared bats (genus Corynorhinus) was collected and ectoparasites were taken. Twenty-three specimens of Corynorhinus mexicanus Allen, 1916, and 18 of Corynorhinus townsendii (Cooper, 1937) were collected in four sampling periods coincident with the seasons. In total, 98 ectoparasites of 10 species and seven families were examined. Five species are recorded for the first time on C. mexicanus and four on C. townsendii. Macronyssus cyclaspis and Trichobius corynorhini had the highest frequency of infestation in both bats. Differences in number of arthropods per bat among seasons were nonsignificant for both species.

The involvement of insects in Reticuloendotheliosis virus (REV) transmission was examined by testing insects trapped at commercial farms and by controlled feeding experiments using mosquitoes, Culex pipiens L. and house flies, Musca domestica L. We established sensitive methods of REV detection, including reverse transcription-polymerase chain reaction (PCR) for REV-LTR and REV-gag genes, REV antigenemia measurements by enzyme-linked immunosorbent assay, and virus isolation in tissue cultures. A variety of blood-sucking species of insects were trapped at farms with infected poultry and tested, but none were positive. To rule out the possibility of PCR inhibition by insect RNA, spiking experiments were conducted and no interference was observed. Because Cx. pipiens mosquitoes were trapped frequently at farms, we performed feeding experiments with mosquito females fed on a REV-containing tissue culture medium and chicken blood mixture. Virus was detected in the mosquitoes up to 5 h postfeeding, compared with 96 h in the feeding mixture, indicating that Cx. pipiens can only harbor REV for a short period. House flies were suspected to be involved in the virus transmission because they frequently were trapped on positive farms. In contrast to mosquitoes, REV was harbored within the house fly digestive tract for up to 72 h and could infect chickens, as demonstrated by seroconversion and by detection of viral gag-sequence in the cloaca. The current study is supportive for the role of house flies as a mechanical vector of REV among poultry.

We show that the spatial-temporal variability of human West Nile (WN) cases and the transmission of West Nile virus (WNV) to sentinel chickens are associated with the spatial-temporal variability of drought and wetting in southern Florida. Land surface wetness conditions at 52 sites in 31 counties in southern Florida for 2001–2003 were simulated and compared with the occurrence of human WN cases and the transmission of WNV to sentinel chickens within these counties. Both WNV transmission to sentinel chickens and the occurrence of human WN cases were associated with drought 2–6 mo prior and land surface wetting 0.5–1.5 mo prior. These dynamics are similar to the amplification and transmission patterns found in southern Florida for the closely related St. Louis encephalitis virus. Drought brings avian hosts and vector mosquitoes into close contact and facilitates the epizootic cycling and amplification of the arboviruses within these populations. Southern Florida has not recorded a severe, widespread drought since the introduction of WNV into the state in 2001. Our results indicate that widespread drought in the spring followed by wetting during summer greatly increase the probability of a WNV epidemic in southern Florida.

Protozoan parasites in the genus Leishmania are ingested by sand flies with blood and multiply in the gut until they are transmitted to a vertebrate host when the sand fly blood feeds again. Infections of the enzootic vector Phlebotomus papatasi Scopoli result in distended midguts with no spontaneous gut contractions. Using a P. papatasi hindgut contraction bioassay, a paralytic factor sensitive to trypsin, chymotrypsin, proteinase-K, and heating at 56°C was detected in crude lysates of Leishmania major promastigotes. Application of parasite lysate to isolated hindguts resulted in reversible, dose-dependent inhibition of spontaneous contractions. Mean volume of isolated midguts and hindguts increased by 50–60% after application of L. major lysate. L. major paralytic factor was purified 10⁴-fold over the total protein preparation and yielded a hydrophobic 12-kDa peptide. Myoinhibitory activity eluted as a single peak in reverse phase-high-pressure liquid chromatography. Tandem mass spectrometry resulted in 15 amino acid sequences, three of them sharing 45–73% homology with short hypothetical gene products of undefined function from Pseudomonas, Halobacterium, and Drosophila. This unique protozoan peptide mimics the function of endogenous insect neuropeptides that control visceral muscle contractions. By this novel mechanism, parasites persist in the expanded, relaxed midgut after blood meal and peritrophic matrix digestion. This allows time for development and migration of infective forms, facilitating sand fly vector competence and parasite transmission.

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle that is endemic throughout large areas of the United States. Cattle that survive acute infection become life-long persistently infected carriers. In the intermountain west the Rocky Mountain wood tick, Dermacentor andersoni Stiles, is the most common vector of A. marginale. Male D. andersoni acquire A. marginale when feeding on persistently infected cattle and biologically transmit it when they transfer from infected to susceptible hosts. Host-seeking adult D. andersoni were collected from four widely separated natural populations and tested for susceptibility to midgut colonization with A. marginale. Male ticks were fed on calves persistently infected with a strain of A. marginale naturally transmitted by D. andersoni. Gut infection rates ranged from 12.5% of ticks collected from a mountain site near Hamilton, MT, to 62.5% of ticks from a rangeland site near Riley, OR. Sites near Miles City, MT, and Kamloops, British Columbia, Canada, had intermediate levels of susceptibility. The infection rates differed significantly among populations, and the same populations sampled in two consecutive years were not significantly different from one year to the next. Although there was variation among the populations in the size of ticks, size was unrelated to acquisition of gut infection. Quantitative polymerase chain reaction (PCR) demonstrated that there was no significant difference between populations in the mean number of genome copies in the guts of infected ticks. A. marginale from infected ticks was genotyped to confirm that they were all infected with the laboratory strain, and a sample of 682 field-collected D. andersoni was surveyed for A. marginale by nested PCR; none were found to be naturally infected. Infection of the gut is an essential constituent of vector competence for A. marginale; in this study, we have demonstrated significant variation among populations in this key component of vector competence.

The flea and rodent samples studied in this project were collected from field study sites in New Mexico from winter 1998 to spring 2001. During this period, 155 small rodents (14 different species) were live-trapped and combed for the presence of fleas. A total of 253 fleas were collected, comprising 21 species. Two of the 253 fleas collected were polymerase chain reaction (PCR) positive for the Rickettsia 17-kDa protein gene. These two fleas were both Anomiopsyllus nudata Baker, each collected from an individual Neotoma albigula Hartley, on two occasions. Individual fleas positive for the Rickettsia 17-kDa protein gene were then tested with primers targeting the rickettsial genes for citrate synthase (gltA) and two major outer membrane proteins (ompA and ompB). The nucleotide sequences of the PCR products of these two fleas were identical to each other and were 100% (394/394), 100% (1150/1150), 99.8% (469/470), and 99.3% (818/824) similar to the corresponding sequences of the 17-kDa, gltA, ompA, and ompB genes of Rickettsia felis, respectively. Flea homogenates of individual PCR-positive fleas were inoculated into shell vials seeded with Vero cells, and the Giménez stain technique was used to demonstrate the presence of Rickettsia-like organisms in detached cells found in aspirated medium 19 d after inoculation. These cells were harvested and tested by PCR, targeting portions of the 17-kDa and gltA genes, resulting in products 100% identical to R. felis. This work comprises the first report of R. felis detection in a flea species (A. nudata) endemic to the New World.

Birds belonging to 59 species (n = 1,206) were live captured in Piemonte, northwestern Italy, in 2001. Ixodes ricinus (L.) larvae were collected from 59 birds belonging to nine species, and nymphs were recovered on 79 birds belonging to 10 species. Eurasian blackbirds, Turdus merula L., had significantly higher levels of infestation by ticks than other passerine species. Larval I. ricinus of blackbirds peaked in summer, when prevalence was 39% (95% confidence interval 24.2–55.5) and mean number of ticks per host was 3.3 (1.6–7.2), whereas nymphs peaked in spring, when prevalence was 72.2% (54.8–85.8) and mean number of ticks per host was 6.9 (4.4–10.7). Immature I. ricinus were coincidentally aggregated on blackbirds, with 15 blackbirds feeding 67.4% of nymphs and 40.3% of larvae, and coinfestation by both stages was relatively high in summer: Kappa = 0.64 (0.40–0.88). Borrelia burgdorferi sensu lato was identified by polymerase chain reaction (PCR) in 58.3% (35.9–78.5) of larvae with engorgement ratio ≥3 that were collected from blackbirds. Larvae that were collected from other passerine species gave negative PCR results. Sixteen of 21 PCR-positive samples belonged to B. garinii (76.2%), and five (23.8%) were Borrelia valaisiana. Results of this study suggest that blackbirds play an important role as hosts for immature I. ricinus and as reservoir of Borrelia garinii in northwestern Italy.

Medical importance of the Gulf Coast tick, Amblyomma maculatum Koch, has been highlighted with the recent discovery of a new rickettsial pathogen associated with this tick. Accordingly, distribution and seasonal collection records of A. maculatum were assessed in Mississippi by using three sources of data: sampling of experimental field plots, national tick collection records, and Mississippi Department of Health human tick biting records. A. maculatum was collected in 17/82 (21%) Mississippi counties, mostly in central and southern regions. Estimates of seasonal activity were made using collection data for 2,217 adult, 11 nymphal, and 426 larval specimens. Adult A. maculatum were collected during March through November, with a peak during late July through early August. Nymphs were collected during February through August, and larvae were collected once during each of June, September, and October. Small sample sizes of the immature stages precluded determination of their peak activity.

From October 1997 to September 1998, an entomological survey was carried out in Manhiça, Mozambique, to describe the anopheline population and intensity of malaria transmission. Ten different huts were randomly selected for entomological surveillance throughout the year. CDC light trap collections were conducted during three nights each month. Additional knockdown spraying catches were carried out in the morning, after the last catch. A total of 17,245 Culicinae and 1,251 Anophelinae were collected during the study. There was substantial house to house variation and seasonality in the distribution of Anophelinae population, with a peak in April towards the end of the warm and rainy season. Four species of genus Anopheles (Diptera: Culicidae) were described: Anopheles funestus Giles, Anopheles tenebrosus Dönitz, Anopheles arabiensis Patton, and Anopheles merus Dönitz. An. funestus constitutes 72.3% of the anopheline population. The estimated sporozoite rate was 1.2% and the average entomological inoculation rate for the area was 15 infective bites per person per year.

International travel to tropical countries accounts for an increasing incidence of imported diseases. An unusual case of furuncular myiasis due to Cordylobia anthropophaga (Blanchard) is reported in northern France in a 9-mo-old infant, after a 4-mo stay in Congo. A review has been made of the major cases of imported furuncular myiasis due to Cordylobia, as well as identification of second larval instars and management of the myiasis.

RESUMÉ Les trajets internationaux ont pour résultat une augmentation des pathologies importées des zones tropicales. Les auteurs présentent un cas peu courant de myiase furonculeuse causée par des larves de Cordylobia anthropophaga (Blanchard) sur un jeune enfant de neuf mois qui revenait du congo. Une revue de la littérature concernant les cas d’importation de myiases furonculeuses à Cordylobia est faite. L’identification a été possible à partir de larves au second stade. Le traitement de la myiase est également présenté.

Fourteen fresh animal carcasses were monitored throughout decomposition in a mixed flatwood forest in East Baton Rouge Parish, LA, from 5 October to 7 December 1999 (fall) and from 18 January to 30 March 2000 (winter). Species composition and residency patterns of necrophilous insects were documented for four animal species per seasonal experiment: one Louisiana black bear (threatened species), two white-tailed deer, two alligators, and two swine (experimental reference). Results suggested variation in species composition associated with temperature (fall versus winter conditions) and carcass type. In total, 89 species from 39 families and three classes were manually collected from the seven fall carcasses. Ninety-five species from 38 families and three classes were collected at the seven winter carcasses. Overall arthropod diversity was greatest for fall deer and winter swine carrion. Fall alligator carcasses were associated with fewer taxa than the three mammal species during both seasons. The hairy maggot blow fly, Chrysomya rufifacies (Macquart), was the dominant species of the fall study, impacting developmental rates and overall carrion community structure. The winter study was characterized by prolonged carcass decomposition and reduced insect activity due to fluctuating ambient temperatures.

Studies on viruses of zoonotic importance in certain villages around Pune were undertaken between December 2000 and January 2002. A total of 1,138 adult ticks belonging to six different species were collected off domestic animals and processed for virus isolation. Six virus isolates were obtained. All six isolates were identified as Ganjam virus by Quick Complement Fixation test and reverse transcriptase-polymerase chain reaction using RNA nucleocapsid gene amplification. Five isolates were from the pools of adult Hemaphysalis intermedia ticks, and one isolate was from a pool of adult Rhipecephalus hemaphysaloides. This is the first report of isolation of Ganjam virus from Maharashtra state of India.

A series of in vitro and in vivo bioassays were conducted to assess the ectoparasiticide activity of isopropyl-4-nitro-2,6-bis(trifluoromethyl)-1-benzimidazole-carbamate, an experimental benzimidazole-carbamate class compound. This compound was less potent than permethrin against ectoparasiticide-susceptible larvae of the lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae); larvae of the southern cattle tick, Boophilus microplus (Canestrini); and adult stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae) in vitro, but it was significantly more potent than permethrin against the Santa Luiza strain of B. microplus known to possess high-level resistance to amitraz and pyrethroids. In contrast, the benzimidazole-carbamate was substantially more efficacious than permethrin when applied topically onto rats that were infested with A. americanum nymphs. These results suggest that this experimental compound may be a viable candidate ectoparasiticide that retains significant activity against resistant B. microplus and also suggests that the benzimidazole-carbamate chemistry may be useful for addressing the growing problem of resistance in ectoparasites.