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We evaluated the potential for mosquitoes collected in the Amazon Basin, near Iquitos, Peru, to become infected with and transmit Murutucu (MURV) and Itaqui viruses (ITQV) (Order Bunyavirales, Family: Peribunyaviridae, Genus: Orthobunyavirus). Viremia levels in Syrian hamsters peaked 2 d after infection with either virus, and both viruses were highly lethal in hamsters with virtually all hamsters dying prior to 3-d postinfection. For almost all of the mosquito species tested some individuals were susceptible to infection and some developed a disseminated infection after oral exposure to either MURV or ITQV. However, only the Culex species (Culex (Culex) coronator Dyar and Knab [Diptera, Culicidae], Culex (Melanoconian) gnomatos Sallum, Huchings, and Ferreira [Diptera, Culicidae], Culex (Mel.) pedroi Sirivanakarn and Belkin [Diptera, Culicidae], and Culex (Mel.) vomerifer Komp [Diptera, Culicidae]) successfully transmitted virus by bite. However, even among these species, only about 37% of the individuals with a disseminated infection successfully transmitted these viruses, indicating a significant salivary gland barrier. Although little is known about the medical or veterinary importance of many members of the genus Orthobunyavirus, we have demonstrated that Culex spp. (Diptera, Culicidae) could be potential vectors.

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Mosquitoes were collected in the Amazon Basin, near Iquitos, Peru, and used in experimental studies to evaluate their susceptibility to strains of eastern equine encephalitis virus (EEEV) that were isolated from mosquitoes captured within 20 km of Iquitos. When fed on hamsters or chickens with a viremia of 4105 plaque-forming units (PFU) of EEEV/ml, Culex pedroi Sirivanakarn and Belkin, Aedesfulvus (Wiedemann), Psorophora albigenu (Peryassu), and Psorophoraferox (Von Humboldt) were susceptible to infection, whereas none of the Aedes serratus (Theobald), Culex vomerifer Komp, Culex gnomatos Sallum, Huchings, and Ferreira, Culex portesi Senevet and Abonnenc, or Culex coronator Dyar and Knab became infected, even though they fed on the same viremic blood sources. When these mosquito species fed on animals with viremias of approximately 10(8) PFU/ml, Cx. pedroi, Ae.II (Brazil-Peru) and a lineage III (Argentina-Panama) isolate of EEEV. This study, combined with the repeated isolation of strains of EEEV from Cx. pedroi captured in the Amazon Basin region of Peru, suggests that Cx. pedroi may be the primary enzootic vector of EEEV in this region.

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As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

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Previous evidence for the presence of chicken anemia virus (CAV) in the gonads of immune specific-pathogen-free chickens raised the question whether this occurs also in commercial breeders. The presence of CAV was investigated by nested PCR in the gonads and spleens of hens from two 55- and 59-week-old, CAV-vaccinated (flocks 2 and 3), and two 48- and 31-week-old non-vaccinated broiler breeder flocks (flocks 1 and 4). In addition, lymphoid tissues of 20-day-old embryos from these hens were also investigated for the presence of CAV. CAV was detected in the gonads and of 5/6 and 11/22 of the vaccinated hens and in some hens also in the spleen alone. Embryos from 7/8 and 5/18 of these hens were positive. In the non-vaccinated flocks, CAV was detected in the gonads of 11/34 and 10/10 hens in flocks 1 and 4, respectively. In addition, 11 birds in flock 1 had positive spleens. CAV DNA was detected in 3/11 and 2/10 of their embryos. CAV-positive gonads and embryos were detected in samples from hens with moderate as well as high VN antibody titers. Vaccinated chickens positive for CAV in the gonads and in their embryos had VN titers ranging from >1:512 to <1:2048. In non-vaccinated chickens, the VN titers of CAV positive chickens ranged from 1:128 to 1:4096. These results demonstrate that CAV genome can remain present in the gonads of hens in commercial broiler breeder flocks even in the presence of high neutralizing antibody titers that have been associated with protection against CAV vertical transmission. It also suggests that transmission to the progeny may occur irrespectively of the level of the humoral immune response in the hens.

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This report describes Trocara virus, a newly recognized member of the genus Alphavirus, that has been isolated from Aedes serratus mosquitoes collected at two widely separated sites in the Amazon Basin. Biological, antigenic and genetic characteristics of the new virus are given. Results of these studies indicate that Trocara virus is the first member of a newly discovered antigenic complex within the family Togaviridae genus Alphavirus. The public health and veterinary importance of Trocara virus is still unknown.

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Mosquitoes collected in the Amazon Basin, near Iquitos, Peru, were evaluated for their susceptibility to epizootic (IAB and IC) and enzootic (ID and IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. After feeding on hamsters with a viremia of approximately 10(8) plaque-forming units of virus per milliliter, Culex (Melanoconion) gnomatus Sallum, Huchings, & Ferreira, Culex (Melanoconion) vomerifer Komp, and Aedes fulvus (Wiedemann) were highly susceptible to infection with all four subtypes of VEE virus (infection rates > or = 87%). Likewise, Psorophora albigenu (Peryassu) and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker) were moderately susceptible to all four strains of VEE virus (infection rates > or = 50%). Although Psorophora cingulata (Fabricius) and Coquillettidia venezuelensis (Theobald) were susceptible to infection with each of the VEE strains, these two species were not efficient transmitters of any of the VEE strains, even after intrathoracic inoculation, indicating the presence of a salivary gland barrier in these species. In contrast to the other species tested, both Culex (Melanoconion) pedroi Sirivanakarn & Belkin and Culex (Culex) coronator Dyar & Knab were nearly refractory to each of the strains of VEE virus tested. Although many of the mosquito species found in this region were competent laboratory vectors of VEE virus, additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of VEE virus are needed to incriminate the principal vector species.

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A checklist of the mosquito fauna encountered during arboviral studies in Iquitos, Peru, is presented. A total of 16 genera, 30 subgenera, and 96 species were identified, including 24 species reported from Peru for the 1st time. Notations on the taxonomy and biology for 28 species are also provided.

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Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was applied to samples from widespread populations of the poorly characterized Anopheles (Nyssorhynchus) albitarsis Lynch-Arribálzaga species complex, and 4 genetically differentiated species were distinguished. A screen of 65 random decamer oligonucleotide primers identified 12 primers, which produced 19 reproducible species-specific genetic markers and 4 markers common to 2 or more species. These markers were correlated in nearly all individuals of each species throughout the ranges sampled, including populations as far apart as 2,500 km. Each individual analyzed was from a different isofemale progeny brood, with associated morphological specimens. These specimens will facilitate studies to relate these species to previously reported chromosomal and enzymatic variation as well as to their feeding behavior and potential as malaria vectors. We hypothesize that 3 of the species have recognized valid names: An. (Nys.) albitarsis Lynch-Arribálzaga, An. (Nys.) marajoara Galvão and Damasceno, and An. (Nys.) deaneorum Rosa-Freitas, whereas the 4th is undescribed.

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Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations of Anopheles albitarsis in Brazil. Two sympatric species, An. deaneorum and An. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (for Had-1, Pgi-1, Pep-1, Mpi-1, and Idh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species of An. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes. An. marajoara form CM had a higher variability (mean heterozygosity, H = 0.22, and percentage of polymorphic loci, P = 66.7) than did form IG and form MA (H = 0.08 in both, and P = 25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates of F statistics, and genetic similarities, we propose that An. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1) An. deaneorum (currently one taxon) and (2) the An. marajoara species complex, which further consists of at least three cryptic forms, marajoara form MA, marajoara form IG, and marajoara form CM.

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Five patients with asexual and sexual parasites of Plasmodium vivax were treated orally with 600 mg chloroquine diphosphate (hour 0) followed with 300 mg at 8, 24 and 48 h later. Primaquine phosphate, 15 mg, was administered concurrently at h 0 and at 24 h intervals for 14 days. Anopheles darlingi were fed before the first dose (h -0.5) and 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 60 and 72 h later. Mosquitoes were examined for oocysts on day 8 and for sporozoites on day 15 after infection. Four of the five patients studied were still infective to mosquitoes from 1-5 h after the first dose of chloroquine plus primaquine. One of these and one other patient, who vomited 15 min after the first dose, became infective again at hours 10 and 12, respectively. Once produced, oocysts in mosquitoes fed on patients before, during and after chloroquine plus primaquine treatment appeared normal and produced sporozoite infected salivary glands. In view of these data, it is concluded that primaquine demonstrated rapid gametocytocidal activity and should be administered concurrently with chloroquine to reduce vivax malaria transmission.

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World ecosystems differ significantly and a multidisciplinary malaria control approach must be adjusted to meet these requirements. These include a comprehensive understanding of the malaria vectors, their behavior, seasonal distribution and abundance, susceptibility to insecticides (physiological and behavioral), methods to reduce the numbers of human gametocyte carriers through effective health care systems and antimalarial drug treatment, urban malaria transmission versus rural or forest malaria transmission, and the impact of vaccine development. Many malaria vectors are members of species complexes and individual relationships to malaria transmission, seasonal distribution, biting behavior, etc. is poorly understood. Additionally, malaria patients are not examined for circulating gametocytes and both falciparum and vivax malaria patients may be highly infective to mosquitoes after treatment with currently used antimalarial drugs. Studies on the physiological and behavioral effects of DDT and other insecticides are inconclusive and need to be evaluated.

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A study of peridomestic man-biting culicines in the Amazon Basin was conducted from January through December, 1987. Fifteen species of mosquitoes from six genera were collected by volunteers in all-night human-bait indoor and outdoor collections at five houses in and near the town of Costa Marques, Rondônia, Brazil. Culex quinquefasciatus and members of the Mansonia titillans/indubitans Group comprised 61 and 33%, respectively, of all culicines collected from human-bait outside houses and 62 and 35%, respectively, of those collected from volunteers inside houses in the town. In rural areas, Cx. quinquefasciatus was less abundant and only comprised 2 and 5% of the culicines, respectively, collected inside and outside houses. Mansonia titillans/indubitans Group comprised 75% and 86% of the culicines collected inside and outside houses, respectively, from rural residences. Culex quinquefasciatus and members of the Mn. titillans/indubitans Group were more endophilic than other culicines collected. Nocturnal and seasonal biting rhythms for the more common culicines are described.

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World ecosystems differ significantly and a multidisciplinary malaria control approach must be adjusted to meet these requirements. These include a comprehensive understanding of the malaria vectors, their behavior, seasonal distribution and abundance, susceptibility to insecticides (physiological and behavioral), methods to reduce the numbers of human gametocyte carriers through effective health care systems and antimalarial drug treatment, urban malaria transmission versus rural or forest malaria transmission, and the impact of vaccine development. Many malaria vectors are members of species complexes and individual relationship to malaria transmission, seasonal distribution, bitting behavior, etc. is poorly understood. Additionaly, malaria patients are not examined for circulating gametocytes and both falciparum and vivax malaria patients may be highly infective to mosquitoes after treatment with currently used antimalarial drugs. Studies on the physiological and behavioral effects of DDT and other insecticides are inconclusive and need to be evalusted

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Seven anopheline species from Costa Marques, Rondonia, Brazil were compared with Anopheles darlingi for susceptibility to infection by Plasmodium vivax. Laboratory-reared F1 progeny of field-collected An. darlingi and the test anopheline species were fed at the same time on the same patients, all of whom had gametocytes in peripheral blood before treatment. Mosquitoes were dissected on day 8 after infection for oocysts and on days 14-16 after infection for sporozoites. The mean numbers of P. vivax oocysts and the percent of salivary gland infections for An. darlingi and An. deaneorum were similar and far exceeded those found in the other anopheline species tested. Anopheles albitarsis and An. mediopunctatus were less susceptible to infection by oocyst measurements than An. darlingi. However, for oocyst-infected An. albitarsis and An. mediopunctatus, the percent of mosquitoes with salivary gland infections and the numbers of sporozoites in the salivary glands were similar to An. darlingi. Anopheles triannulatus and An. oswaldoi were both susceptible to P. vivax infection, but the sporozoite infection rates and the numbers of sporozoites observed in the salivary glands were very low. Anopheles braziliensis and An. benarrochi both developed oocysts, but were never observed to have sporozoites in the salivary glands. These studies implicate some anopheline species as potential malaria vectors, but also show that species previously incriminated by ELISA techniques are not vectors of malaria parasites in Costa Marques, Rondonia, Brazil.

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Patients with asexual and sexual parasites of Plasmodium vivax were treated orally with 600 mg of chloroquine diphosphate at hour zero, followed by 300 mg at 8, 24 and 48 hr. Anopheles darlingi were fed before the first dose (-0.5 hr) and at 0.5, 1, 2, 4, 6, 8, 9, 10, 11, 12, 20, 24, 36, 48, 60, and 72 hr later. Mosquitoes were examined for oocysts on day 8 and sporozoites on day 15 after infection. The frequency of infected mosquitoes and the mean number of oocysts were lower in mosquitoes that fed on patients 2-4 hr after the initial dose of chloroquine than in mosquitoes fed before treatment and at 0.5 and 1 hr. This sporontocidal effect was temporary, since the frequency of infected mosquitoes and the mean number of oocysts increased in mosquitoes fed 4-8 hr after the first dose. Nearly all mosquitoes fed on patients after the third dose of chloroquine, at 24 hr, were negative for P. vivax oocysts. Oocysts in mosquitoes fed on patients before, during, and after chloroquine treatment appeared normal and produced sporozoites. We conclude that chloroquine affects either the gametocytes, fertilization, zygotes, and/or ookinetes of P. vivax, but not subsequent stages of development.

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Five anopheline species, Anopheles deaneorum, An. albitarsis, An. triannulatus, An. oswaldoi, and An. mediopunctatus were compared to An. darlingi for susceptibility to infection by P. falciparum in Costa Marques, Rondonia, Brazil. Laboratory reared F1 An. darlingi and anopheline test species were allowed to feed at the same time on falciparum malaria patients who had gametocytes in their blood, and who had not yet been treated with quinine. Mosquitoes were dissected and examined for oocysts on day 9, and for sporozoites on days 16-20 after feeding. Anopheles mediopunctatus had higher mean numbers of oocysts and oocyst positive rates than An. darlingi. The oocyst positive rate and the mean number of oocysts in An. deaneorum and An. darlingi were similar. Anopheles triannulatus and An. oswaldoi had fewer oocysts than An. darlingi. The salivary gland sporozoite infection rate was similar for An. mediopunctatus and An. deaneorum and much lower for An. triannulatus and An. oswaldoi when compared to An. darlingi. Anopheles albitarsis developed oocysts, but sporozoites did not invade the salivary glands. In relative levels of susceptibility to P. falciparum, An. darlingi was equal to An. mediopunctatus which was greater than An. deaneorum, which was greater than An. triannulatus, which was greater than An. oswaldoi.

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Mosquito collections were made in and near Costa Marques, Rondonia, Brazil, to determine anopheline anthropophilic/zoophilic behavior. Collections from a non-illuminated, bovine-baited trap and indoor and outdoor human-bait collections were compared. Anopheles darlingi and Anopheles deaneorum were more anthropophilic than the other anophelines collected. The remainder of the Anopheles species were collected much more frequently in bovine-baited traps than in human-bait collections. Anopheles darlingi and An. deaneorum were more frequently collected inside houses than the other anopheline species. But, when collections were made in a house with numerous openings in the walls, there were few differences in the percentages of each species biting man indoors versus outdoors. Anopheles darlingi was the predominant mosquito collected, both inside and outside houses, and had the strongest anthropophilic feeding behavior of the anophelines present.

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Thirteen species of anopheline mosquitoes were collected in all-night human-bait indoor and outdoor collections at 5 houses from July 1986 through December 1987 in and near the town of Costa Marques, Rondonia, Brazil. Anopheles darlingi and An. deaneorum accounted for more than 92 and 6%, respectively, of all anophelines collected from human-bait outside houses and 90 and 9%, respectively, inside houses in the town. The diversity of anophelines was greater in human-bait collections in rural areas. However, An. darlingi still accounted for more than 90% of all the anophelines collected from human-bait inside and outside rural houses. Data on nocturnal and seasonal biting rhythms for the more common anophelines are given.

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Anopheles deaneorum, an important malaria vector and a member of the Anopheles albitarsis complex, was colonized by an artificial mating technique. Morphological and behavioral differences between An. deaneorum and An. albitarsis from Costa Marques, Rondonia, Brazil, are discussed. The essential methods and colonization techniques are described. Immature development and mortality rates were reduced when dried grass was added to larval rearing pans. Males frequently mate more than once using the force mating technique. However, insemination and larval eclosion rates decline as males are successively mated with unmated females.

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