Bacteria of the genus Wolbachia constitute a group of intracellular and maternally inherited micro-organisms that are widespread in arthropods, inducing several reproductive disorders such as cytoplasmic incompatibility in their hosts. Considering relevant biological implications related to the presence of Wolbachia in several insect orders, for example its potential role as mechanism for rapid speciation and as vehicle to drive genetic markers in wild populations of vectors of medical and veterinary interest, we carried out an extensive polymerase chain reaction survey to detect Wolbachia in several species of mosquito belonging to genera involved in the transmission of pathogens. Five species out of 26 tested have shown to be infected; for four of them this is the first evidence of the Wolbachia infection. A phylogenetic analysis was also performed, positioning the five Wolbachia strains in the phyletic subdivision B.

Anopheles gambiae Giles has been implicated as a major vector of malaria in Africa. A number of paracentric chromosomal inversions have been observed as polymorphisms in wild and laboratory populations of this species. These polymorphisms have been used to demonstrate the existence of five reproductive units in West African populations that are currently described as incipient species. They have also been correlated with various behavioral characteristics such as adaptation to aridity and feeding preference and have been associated with insecticide resistance. Two paracentric inversions namely 2La and 2Rb are highly ubiquitous in the wild and laboratory populations sampled. Both inversions are easily conserved during laboratory colonization of wild material and one shows significant positive heterosis with respect to Hardy-Weinberg proportions. Inversion 2La has previously been associated with dieldrin resistance and inversion 2Rb shows an association with DDT resistance based on this study. The stability and maintenance of these inversions as polymorphisms provides an explanation for the transmission and continued presence of DDT and dieldrin resistance in a laboratory strain of An. gambiae in the absence of insecticide selection pressure. This effect may also be operational in wild populations. Stable inversion polymorphism also provides a possible mechanism for the continual inheritance of suitable genetic factors that otherwise compromise the fitness of genetically modified malaria vector mosquitoes.

Suction traps (30 cm diameter) were more effective for non-attractant sampling of flying adult Culex tarsalis Coquillett than were smaller CDC (5.5 cm diameter), Malaise or ramp traps. Comparative catch in suction traps operated in a variety of vegetation types indicated that females congregated along elevated ecotones and were significantly less abundant flying over low vegetation or under and over elevated vegetation. Most females taken at upland orchards or Tamarisk tree lines were unfed (97%, n = 5,278) and similar in reproductive condition to host-seeking females. Blood fed and gravid females and males were only abundant near emergence sites. Pyrocide 7396 (Pyrethrin 5%, PBO 25%) was applied at the label rate of 5 oz/min by truck mounted Pro-Mist ultra low volume (ULV) equipment and particle drift measured by bioassay. ULV particles dispersed well downwind over low vegetation, between citrus orchard rows, and under date orchard canopy, but did not penetrate citrus orchards or vineyards when rows were perpendicular to wind direction. Particles did move up and over vegetation contacting sentinel mosquitoes placed above the canopy. The congregation of adult mosquitoes at vegetative ecotones and within orchard vegetation may afford protection from ground applied ULV particles, negatively impacting control. These data may explain why repeated applications often fail to interrupt encephalitis virus transmission.

Member(s) of the Anopheles barbirostris group Reid, particularly Anopheles barbirostris and Anopheles campestris Reid are the suspected vectors of Plasmodium vivax in Pa Rai (Aranyaprathet, Sa Kaeo province). To determine if An. barbirostris, An. campestris, or both, are present in Pa Rai and to determine their potential to transmit malaria, a field and laboratory study was conducted. Isofemale colonizations of wild caught mosquitoes captured by landing catches were made for species confirmation and to determine the mosquito life cycle. Pupal morphology indicated all mosquitoes were An. campestris. During the late rainy season (October and November), An. campestris populations comprised 78.6% of all females captured by human landing catches and 7.1% of mosquitoes in a cow-baited trap. The biting activity cycle peaked between 2000 and 0100 hours and was highest (17.6 bites per person per hour) at 2300 hours. More An. campestris bit people indoors (nine bites per person per hour) than outdoors (four bites per person per hour). Immature An. campestris were found in ponds, swamps, rice-fields, puddles, marshes, ground pools, and pits with open sunlight to partial shade. The time from egg hatch to adult was 18–47 d and 14–22 d under laboratory (25.0–27.0°C) and ambient (26–32°C) conditions, respectively. The fecundity of An. campestris ranged from 173 to 311 eggs. Based on experimental infections, An. campestris was able to support the sporogonic cycle of P. vivax with 76.2 and 23.8% oocyst and sporozoite formation rate, respectively. An. campestris shows high potential as a malaria vector in Pa Rai.

We analyzed 64 quantitative and qualitative morphological characters in different populations of Lutzomyia (Nyssomyia) umbratilis Ward & Fraiha from Venezuela (Gran Sabana, Bolívar/BO) and Brazil (Serra do Navio, Amapá/AP, Manaus, Amazonas/AM, Monte Dourado, Pará/PA, and Peixoto de Azevedo, Mato Grosso/MT). Based on an analysis of qualitative characters, no differences were observed that could distinguish between the populations. Parametric tests were used to verify differences between sample means and variance homogeneity. The analysis-of-variance (ANOVA) test showed the existence of differences between character means in the five populations. In the graphic representation of the Student-Newman-Keuls test (Student–Newman–Keuls), we observed that 77% of the analyzed characters displayed disjunctions between the Venezuelan and Brazilian populations. Considering only the Brazilian population averages, we concluded that they are statistically different. When the Student–Newman–Keuls test was applied, no pattern was found that could separate them. However, in part, the phenetic analysis showed some evidences of heterogeneity in MT population.

A new Lutzomyia species in the subgenus Trichophoromyia, L. pabloi, is described and illustrated. A description of the previously unknown female of L. howardi Young is also presented. These specimens were captured in the Amazon region of Colombia.

Persistence and diuretic shedding of hepatitis B virus (HBV) by mosquitoes (Diptera: Culicidae) was studied by using infectious blood feedings, intrathoracic inoculations, and detection of virus by polymerase chain reaction (PCR) and Southern hybridization. Results showed that both Anopheles stephensi Liston and Ochlerotatus triseriatus (Say) shed HBV during diuresis for up to 72 h after feeding on an HBV-positive serum drawn from a human donor. HBV did not persist in the bodies of either An. stephensi or Oc. triseriatus past 72 h by infectious feeding or intrathoracic inoculation of HBV suspension. Viral dissemination did not occur by infectious feeding in An. stephensi or Oc. triseriatus, or by intrathoracic inoculation in An. stephensi, Oc. triseriatus, or Culex quinquefasciatus Say. These results suggest that HBV could be transmitted to humans by a stercorarian route, especially if mosquitoes that fed on an HBV-positive human are interrupted during feeding and move to another person to resume feeding.

Genetic variability and divergence were estimated for populations of Anopheles rangeli Gabaldón, Cova-Garcia & Lopes, Anopheles nuneztovari Gabaldón cytotype A, and Anopheles dunhami Causey from the Brazilian Amazon using isozyme electrophoresis. These species are included in the Oswaldoi subgroup, subgenus Nyssorhynchus (Diptera: Culicidae). Thirteen enzymes yielded a total of 22 loci, of which 14 were monomorphic in the three species. Three diagnostic loci (Gpi-1, Hk-1, and Me) and a strong differentiation in the Mdh locus were found between An. rangeli and An. nuneztovari. Five diagnostic loci (Mdh, Gpi-1, Hk-1, Gpd, and Me) separated An. rangeli from An. dunhami, whereas one diagnostic locus (Gpd) separated An. nuneztovari from An. dunhami. Moderate differentiation was observed in the Est-5 and Pgm loci between An. rangeli and An. nuneztovari, and between An. nuneztovari and An. dunhami. Anopheles dunhami had the highest values for three indices of genetic variability, whereas An. rangeli showed the lowest values for mean number of alleles per locus and mean heterozygosity. Nei’s genetic distance was highest between An. rangeli and An. dunhami (0.280) and lowest between An. nuneztovari and An. dunhami (0.072). Between An. rangeli and An. nuneztovari the genetic distance was 0.237. Anopheles dunhami and An. nuneztovari are sister species very closely related and may have a recent evolutionary origin. Anopheles rangeli probably diverged before the other two species separated. This is the first record of An. dunhami in Coari (Amazonas, Brazil) and only the third collection site of this species since its description in 1945.

Although malaria was officially declared eradicated from Europe in 1975, its former vectors, mainly members of the Anopheles maculipennis (Meigen) complex, are still distributed throughout the continent. The present situation of Anophelism without malaria indicates that current socio-economic and environmental conditions maintain the basic case reproduction number, Ro, below 1. Recently, it has been speculated that predicted climate changes may increase anopheline abundance and biting rates (as well as reduce the Plasmodium parasite extrinsic incubation period), allowing the reemergence of malaria transmission in Europe. As a preliminary step toward predicting future scenarios, we have constructed models to test whether the current distribution of the five former European malaria vectors [An. atroparvus (Van Thiel), An. labranchiae (Falleroni), An. messeae (Swellengrebel & De Buck), An. sacharovi (Favr) and An. superpictus (Grassi)] can be explained by environmental parameters, including climate. Multivariate logistic regression models using climate surfaces derived from interpolation of meteorological station data (resolution 0.5 × 0.5°) and remotely sensed land cover (resolution 1 × 1 km) were fitted to 1,833 reported observations of the presence and absence of each species across Europe. These relatively crude statistical models predicted presence and absence with a sensitivity of 74–85.7% and specificity of 73.4–98.1% (with climate a significantly better predictor than land cover type). A geographically independent validation of the models gave a sensitivity of 72.9–88.5% and a specificity of 72.7–99.6%. This allowed us to generate risk maps for each species across Europe. Assuming that high risk equates with the potential for high abundance, these models should permit the development of risk maps for European mosquitoes under future climate scenarios. These techniques would be equally useful for estimating the risk of reemergence in other nonendemic areas such as the United States and Australia, as well as changes to risk within endemic areas.

American canine hepatozoonosis (ACH) caused by Hepatozoon americanum Vincent-Johnson, Macintire, Lindsay, Lenz, Baneth, and Shkap is an emerging, often fatal, tick-borne protozoal disease of dogs in the United States of America. Dogs acquire infection by ingesting ticks that contain oocysts. To understand the invertebrate (definitive) host range of H. americanum, experiments were carried out using four ixodids, Rhipicephalus sanguineus (Latreille), Dermacentor variabilis Say, Amblyomma americanum (L.), and Amblyomma maculatum Koch. Laboratory-reared nymphal ticks were fed on dogs that were either naturally or experimentally infected with H. americanum; when these ticks molted to the adult stage they were either fed to susceptible dogs or were dissected and examined for the presence of oocysts. Mature H. americanum oocysts were found in >90% of A. maculatum (both males and females), whereas oocysts were not found in any of the other three species. These results confirm that A. maculatum is an excellent host and vector for H. americanum and also suggest that this apicomplexan may have a narrow invertebrate host range, at least among ixodid ticks that are likely candidate vectors in the United States.

Ochlerotatus j. japonicus, a recent introduction to the United States, was studied to determine its capability to serve as a vector of La Crosse (LAC) virus. A field-collected population of Ochlerotatus triseriatus, the primary vector of LAC virus, was similarly tested for comparison. After Oc. j. japonicus ingested virus from hamsters with viremias of 10^3.6–5.4 plaque-forming units (PFU)/ml of blood, its estimated transmission rates were 35–88%. These rates were slightly lower than, though similar to, those for Oc. triseriatus, 75–100%. Viral titers in Oc. j. japonicus peaked at ≈10^5.5 PFU/mosquito about 7 d after ingesting a blood meal in which the concentration of LAC virus was 10^5.4 PFU/ml of blood; virus had disseminated from the midgut in 100% (8/8) of these specimens. These data, combined with the close association between the habitats of Oc. j. japonicus and Oc. triseriatus and the reported expansion of the range of this newly discovered species in the eastern United States, indicate that Oc. j. japonicus could function as an additional vector of LAC virus.

Despite the detection of West Nile (WN) virus in overwintering Culex pipiens L. in New York in February 2000, the mechanism by which this virus persists throughout the winter to initiate infections in vertebrate hosts and vectors the following spring remains unknown. After a blood meal, parous mosquitoes generally do not survive until spring and gonotrophic dissociation occurs in only a small percentage of the population. To investigate vertical transmission as a means of viral survival during interepizootics, we intrathoracically inoculated Cx. pipiens and Aedes albopictus (Skuse) with WN virus and subsequently tested their F₁ progeny for the presence of virus. Among the Cx. pipiens, we recovered virus from two of 1,417 adult progeny that had been reared at 18°C for a minimal filial infection rate (MFIR) of ≈1.4/1,000 and four of 1,873 adult progeny reared at 26°C (MFIR = 2.1/1,000). The mean titer of the positive pools was 10^5.6 plaque-forming units (PFU)/ml (=10^5.9 PFU/mosquito for positive mosquitoes) of virus. Overall, the MFIR was ≈1.8/1,000 for Cx. pipiens. Although reports indicate that Ae. albopictus vertically transmit various viruses in the Japanese encephalitis virus complex, we did not detect WN virus in any of > 13,000 F1 progeny of WN virus-inoculated specimens. Female Cx. pipiens that are vertically infected during the late summer season and then survive the winter could serve as a source of WN virus to initiate an infection cycle the following spring.

Modifications of exposure time, substrate, and formulation were made to the Food and Agriculture Organization Larval Packet Test (LPT) to determine a combination suitable for measuring the susceptibility of Boophilus microplus (Canestrini) to amitraz. Exposure time influenced the slope of the dose–response when paper was used as a substrate for amitraz. However, time did not influence the dose–response slope when nylon fabric was used as an amitraz substrate. Formulated amitraz produced results with less deviation from the log-probit model than technical amitraz. The combination of formulated amitraz and nylon fabric as a substrate for amitraz produced results that best fit the log-probit model. The modified FAO procedure (formulated amitraz/nylon substrate combination) was used to assay a Brazilian strain of B. microplus and a Panamanian strain of Rhipicephalus sanguineus (Latreille). Resistance ratios (95% CI) of 26.3 (25.7–26.9) and 7.3 (5.5–9.9) were calculated for the B. microplus and R. sanguineus strains, respectively. A discriminating dose of 0.03% amitraz was determined for B. microplus. This technique will help to locate amitraz resistant tick populations, provide data for improved control practices, and aid in the discovery of resistance mechanisms through synergist studies and verification of molecular techniques.

Oestrus ovis (Linné 1761) larvae are obligatory parasites of the nasal and sinus cavities of sheep and goats. Infestation is prevalent in hot and dry regions, such as Mediterranean countries. The current work was developed to establish the chronobiology of O. ovis in Sardinia, to determine the most suitable time for chemoprophylaxis. A survey was carried out during 1998, and sheep heads were collected monthly from local flocks. A total of 443 heads was examined, and the prevalence of oestrosis was 73.8%. We collected 2,691 larvae (mean = 6.07 ± 9.52), and the intensity was greatest in November. The humoral immune response against the nasal bot fly was analyzed by means of an indirect-ELISA using second-instar O. ovis excretory and secretory antigens. A seasonal variation in the antibody levels was observed, increasing from April and peaked in June and in September. A significant correlation was observed between first instar intensity and the mean relative humidity (r² = 0.120; P < 0.05), and between second-instar intensity and the mean temperature (r² = 0.241; P < 0.05). Three periods in the chronobiology of O. ovis were defined: diapause (October–February), the active phase of the endogenous cycle (March–September) and the exit phase (May–September). Our results showed that treatment in October–November was suitable, because first instars were in diapause, preventing the development of first into second instars, and second into third instars.

Land crab burrows are larval mosquito habitats of major significance in the Pacific region. They are constituted by a sinuous tunnel leading to a chamber in contact with the water table, where mosquito larvae proliferate. Controlling larvae in these sites is difficult, because the configuration of burrows prevents the use of standard techniques. An experiment was carried out in French Polynesia to control Aedes polynesiensis Marks and Culex spp. breeding in burrows of the land crab Cardisoma carnifex (Herbst). The technique was based on the crab’s behavior, which involves the crab carrying food into its burrow. It was shown that appetizing baits impregnated with an insecticide were carried by crabs into the flooded chamber of their burrows. A field treatment of burrows was carried out by sowing insecticide impregnated baits on the ground. The treatment coverage was almost perfect and the easy implementation of the technique enabled large areas to be treated in a short time. The bait was developed by compacting various flours, which easily incorporate a large variety of insecticide formulations. Although the baits can be easily stocked, a reliable insecticide is still to be found. The results indicate that our technique could be a method of choice for treating crab burrows.

The specific status of the head and body lice of humans has been debated for more than 200 yr. To clarify the specific status of head and body lice, we sequenced 524 base pairs (bp) of the cytochrome oxidase I (COI) gene of 28 head and 28 body lice from nine countries. Ten haplotypes that differed by 1–5 bp at 11 nucleotide positions were identified. A phylogeny of these sequences indicates that these head and body lice are not from reciprocally monophyletic lineages. Indeed, head and body lice share three of the 10 haplotypes we found. F_ST values and exact tests of haplotype frequencies showed significant differences between head and body lice. However, the same tests also showed significant differences among lice from different countries. Indeed, more of the variation in haplotype frequencies was explained by differences among lice from different countries than by differences between head and body lice. Our results indicate the following: (1) head and body lice do not represent reciprocally monophyletic lineages and are conspecific; (2) gene flow among populations of lice from different countries is limited; and (3) frequencies of COI haplotypes can be used to study maternal gene flow among populations of head and body lice and thus transmission of lice among their human hosts.

The experimental vector competence of five laboratory-reared ixodid tick species representing three genera [Amblyomma americanum (L.), Boophilus microplus (Canestrini), D. andersoni Stiles, D. occidentalis Marx, and D. variabilis (Say)] for Babesia equi (Laveran 1901) was evaluated by delayed transfer of male ticks from infected to susceptible equids or by infesting the latter animals with adult ticks previously fed as nymphs on infected equids. After feeding for 5, 6, or 13 d on acquisition hosts, ticks were forcibly removed and held off the host at 26°C, ≈93% RH, and a photoperiod of 14:10 (L:D) h for 6, 12, or 27 d. Intrastadial transmission to susceptible ponies by D. variabilis males, and transstadial transmission to susceptible burros by B. microplus adults, was demonstrated by blood smear and indirect immunofluorescence serology. The data indicated that male D. variabilis and adult B. microplus, tick species that occur on equids in North America and, in the case of the latter tick, also extensively in tropical and subtropical regions of the world, may be competent natural vectors of B. equi

Strategies for controlling cat fleas, Ctenocephalides felis felis (Bouché), have undergone dramatic changes in the past 5 yr. With the advent of on-animal treatments with residual activity the potential for the development of insecticide resistance increases. A larval bioassay was developed to determine the baseline susceptibility of field-collected strains of cat fleas to imidacloprid. All four laboratory strains tested showed a similar level of susceptibility to imidacloprid. Advantages of this bioassay are that smaller numbers of fleas are required because flea eggs are collected for the test. Insect growth regulators and other novel insecticides can also be evaluated. Using a discriminating dose, the detection of reduced susceptibility in field strains can be determined with as few as 40 eggs.

The in vitro maintenance technique described in this article has been used successfully to rear Cimex lectularius (L.) by feeding for >2 yr all nymphal stages and adults through parafilm “M” sealing film on different types of blood. Using this feeding technique, the subsequent egg production of female bedbugs was remarkably high. The blood was maintained at 37°C to enhance the attachment of the bugs. The effect of anticoagulation methods for the blood meal was investigated, and heparinized blood was found the most suitable for feeding bugs. All stages of the bugs fed weekly on blood in the artificial feeding system remained attached for up to 0.5–1.0 h, until completion of their blood meals, and all reached engorged weights. More than 90% of the bugs fed artificially on whole blood, and they molted or laid eggs successfully.

The possible presence of sand flies (Diptera: Psychodidae) among colonies of Gunnison’s prairie dogs, Cynomys gunnisoni Baird, was investigated on or near ranches with histories of vesicular stomatitis (VS) in domestic livestock in the mid-Rio Grande River Valley, NM, and west-slope region of Colorado. Sampling was conducted at six locations, three in each region, using three methods: standard down-draft suction/light traps, up-draft suction/light traps (both supplemented with CO₂), and burrow-emergence traps. Two male and four female sand flies, Lutzomyia apache, Young and Perkins were recovered from prairie dog colony and sand-sagebrush association habitats on a cattle ranch in Socorro County, NM. Sand flies were captured only in up-draft suction/light traps. This record is of interest for the following reasons: (1) sand flies have not been reported from the Rio Grande River Valley of New Mexico, (2) sand flies are biological vectors of VS viruses in other areas, (3) the mid-Rio Grande River Valley has been a focus of VS virus activity in domestic livestock during recent outbreaks, and (4) the source of vesicular stomatitis viruses that infect livestock in the western United States is unknown, but may involve introductions of virus from Mexico or an endemic cycle maintained by vector insects.

A total of 3,382 cat fleas, Ctenocephalides felis (Bouché), was taken from 164 of the 200 stray cats examined. It was observed that cat fleas preferred specific areas on the cat. A significantly higher mean number of fleas was found on the area of head plus neck than on the ventral part of the body. More specifically, the mean number of fleas was highest on both of the neck and dorsal areas. However, in terms of the density of fleas, the neck had more fleas than the dorsal area did. The fewest fleas were found infesting the legs and tail. Distribution of fleas on the cat may well be explained by the various grooming patterns of the cat, and the knowledge of flea distribution may be valuable for application of on-animal flea control procedures.

Newly designed primers amplified a 365-bp fragment including the 3′ end of mitochondrial cytochrome b (cyt b). Field collected specimens of the sand fly, Lutzomyia longipalpis (Lutz and Neiva), from two Brazil locations provided test DNA. The primers, based on consensus sequences from a taxonomic spectrum of arthropods, have also amplified DNA successfully from Lutzomyia shannoni Dyar, Lutzomyia cruzi (Mangabeira), and the mosquito Aedes albopictus (Skuse). Single strand conformation polymorphism (SSCP) was used to recognize the presence of L. longipalpis haplotypes; these were verified subsequently by sequencing the polymerase chain reaction products. The sensitivity of SSCP was demonstrated by (1) the detection of single nucleotide differences in SSCP variants and (2) no sequence variation in specimens with the same SSCP mobility. The application of new primers and the SSCP technique provided a valuable addition to available population genetic tools: they increased the efficiency of detection of variability in the cyt b gene and decreased the time required for screening large numbers of specimens to detect nucleotide variation.

The black soldier fly, Hermetia illucens (L.), is a nonpest tropical and warm-temperate region insect that is useful for managing large concentrations of animal manure and other biosolids. Manure management relying on wild fly oviposition has been successful in several studies. However, confidence in this robust natural system was low and biological studies were hampered by the lack of a dependable source of eggs and larvae. Larvae had been reared easily by earlier investigators, but achieving mating had been problematic. We achieved mating reliably in a 2 by 2 by 4-m screen cage in a 7 by 9 by 5-m greenhouse where sunlight and adequate space for aerial mating were available. Mating occurred during the shortest days of winter if the sun was not obscured by clouds. Adults were provided with water, but no food was required. Techniques for egg collection and larval rearing are given. Larvae were fed a moist mixture of wheat bran, corn meal, and alfalfa meal. This culture has been maintained for 3 yr. Maintainance of a black soldier fly laboratory colony will allow for development of manure management systems in fully enclosed animal housing and in colder regions.

Many studies investigating the ecology and evolution of mosquitoes rely on morphometric measurements related to body size to estimate fecundity. Determining the most reliable estimators of fecundity is therefore an important methodological consideration. We compared the relative accuracy of pupal mass and wing length in predicting the fecundity (number of mature stage IV follicles of the first gonotrophic cycle) for (1) Aedes albopictus (Skuse) reared in the laboratory, (2) Aedes geniculatus (Oliver) reared in the laboratory, and (3) A. geniculatus that completed their larval development in water-filled tree holes (their natural habitat) in the field. In all cases, pupal weight and wing length provided highly significant and equally accurate indicators of fecundity. Furthermore, the measurement error (1 − repeatability) for both traits was ≤1%. Our results indicate that either pupal mass or wing length can be used with confidence to predict fecundity in these two aedine mosquitoes.