Studies on mosquitoes (Diptera: Culicidae) and anthropic environment. 1 - Parity of blood seeking Anopheles (Kertezia) in South-Eastern Brazil

Relata os resultados obtidos em coletas regulares de Anopheles cruzii e An. bellator, mediante o emprego de isca humana e por ocasiäo do crepúsculo vespertino. Objetiva-se, precipuamente, conhecer a paridade de populaçöes dessas espécies, quando em plena tentativa hematófaga, tanto no ambiente intra como peridomiciliar. As coletas foram levadas a efeito no ecossistema primitivo da Mata Atlântica meridional do Brasil, em regiäo do Vale do Ribeira, sudeste do Estado de Säo Paulo, Brasil, durante o período de agosto de 1991 a julho de 1992. Precedeu-se ao exame de amostra, correspondente a 34,4 por cento do total de mosquitos coletados, mediante o emprego da técnica de Polovodova para a observaçäo de dilataçöes ovariolares. Ao lado disso, o desenvolvimento dos ovários foi classificado de acordo com as fases de Christophers e Mer. Os dados obtidos permitiram constatar a dominância de An. cruzii. No entanto, pôde-se detectar maior atividade endofágica por parte de An. bellator, a qual se mostrou três vezes maior do que a apresentada por aquele outro anofelíneo. No que concerne à paridade, houve franco predomínio da nuliparidade (74,6 por cento), com o restante constituído, praticamente, por uniparidade (25,4 por cento). A presença de maior número de dilataçöes ovariolares limitou-se a poucos casos de biparidade em An. cruzii. A paridade apresentada por este revelou-se maior no peridomicílio, reforçando a feiçäo exófila de seu comportamento, ao passo que para An. bellator mostrou-se uniforme em relaçäo dos dois locais de coleta. Observou-se que 17,0 por cento de fêmeas nulíparas revelaram ovários cujo desenvolvimento tinha atingido as fases III a V de Christophers e Mer, o que evidenciou o exercício de hematofagia previamente à primeira oviposiçäo. Dessa maneira, pode-se considerar como sendo de 38,1 por cento para An. cruzii e de 8,7 por cento para An. bellator. Diante disso, conclui-se pela provável existência de discordância gonotrófica, o que permite levantar a hipótese de, entre outros fatores, haver viabilidade de desenvolvimento plasmodiano no organismo desses vetores.

Insects feeding on desert bighorn sheep, domestic rabbits, and Japanese quail in the Santa Rosa mountains of southern California.

Desert bighorn sheep (Ovis canadensis cremnobates), a domestic rabbit (Oryctolagus cuniculus), and Japanese quail (Coturnix japonica) were used as bait animals to collect blood-feeding flies in an area of active blue-tongue and epizootic hemorrhagic disease virus transmission. Precipitin tests were used to confirm the blood source where feasible. Eight species of Culicoides, members of the Leptoconops kerteszi group, Simulium spp., Anopheles franciscanus, and Stomoxys calcitrans were collected from the bighorn sheep. Feeding on the bighorn sheep by Culicoides brookmani (n = 25), C. variipennis (n = 6), C. cacticola (n = 1), and Simulium spp. (n = 3) was confirmed by precipitin testing. Primary species attacking the rabbit were C. brookmani, C. variipennis, and the L. kerteszi group. The quail were attacked primarily by members of the C. copiosus group and the L. kerteszi group.

Sensitivity of a ribosomal RNA gene probe for identification of life stages of Anopheles arabiensis and An. gambiae (Diptera: Culicidae) using three storage methods.

Individual larvae, pupae, female adults, and adult body parts of Anopheles arabiensis Patton and An. gambiae Giles were stored for 1 mo either in isopropanol at room temperature, over a desiccant at room temperature, or at -70 degrees C. DNA was extracted, digested with EcoR1 restriction enzyme, subjected to electrophoresis in agarose gel, transferred to filters, then hybridized to a 32P-labeled rDNA probe. There was no difference among storage treatments in the proportion of correctly identified samples. First instars were not identifiable. Pupae and female adults were more likely to be identified than earlier life history stages. Nonetheless, the probe identified greater than 75% of second instars, 94% of third instars, and 74% of fourth instars. There were no differences between the species in the proportion of identifiable samples for any life history stage.

Population genetic evidence for species A, B, C and D of the Anopheles dirus complex in Thailand and enzyme electromorphs for their identification.

Mixtures of chromosomal forms A, B, C and D in natural populations of Anopheles dirus Peyton & Harrison sensu lato in Thailand show significant positive values of Wright's fixation index for six enzyme-electromorph loci. The mean value of FIS over all loci was found to be +0.28 (SD 0.02), with a range of +0.57 (Odh) to +0.10 (Idh-2). Partitioning electromorph data for the chromosomal forms reduces the mean FIS to 0.03 (SD 0.01), which suggests that positive assortative mating is a characteristic of each form. This supports the hypothesis that the chromosomal/electrophoretic forms A, B, C and D represent four distinct biological species within the An. dirus complex. An example is given of the use of enzyme electromorphs as a means of vector identification during a malaria entomological field study involving a mixture of An. dirus species A and D. Electromorph identifications of 323 sp. A and 161 sp. D were more than 98% correct when cross-referenced to specific DNA probes.

DNA probes to identify members of the Anopheles farauti complex.

DNA probes have been constructed to distinguish between the members of the Anopheles farauti complex of mosquitoes known as species numbers 1, 2 and 3. Partial genomic libraries of the three known species were exposed to labelled total genomic DNA from each species. Colonies showing differential hybridization were selected for further testing. These probes were found which allow identification of the three known species: probe pAf1 (160 bp fragment) hybridizes to DNA from An. farauti nos. 1 and 2; probe pAf2 (95 bp fragment) hybridizes to DNA from An. farauti no. 2 only; and probe pAf3 (1.3 kb fragment) hybridizes strongly to DNA from An. farauti no. 3, less to no. 1 and faintly to no. 2. Increasing the stringency of hybridization reduced the cross-hybridization of probes pAf1 and pAf3. Only radioactively labelled probes were tested. Males and females and individuals from diverse habitats and localities showed the same species/probe hybridization characteristics. This technique allows faster identification of the sibling species than previous methods, and has the added advantage that it allows air-dried and alcohol stored specimens to be identified.

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex.

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.

A quantitative survey of anophelines in six villages of Kheda District, Gujarat.

Sampling of anophelines from six villages of Kheda district, Gujarat was done using four different methods viz. indoor resting, outdoor resting, bovine bait trap and immature collections. A total of 113,487 specimens representing 17 species were collected. An. subpictus (66.51 per cent) was most abundant species followed by An. culicifacies (14.12 per cent) and An. tessellatus (5.24 per cent). Bovine bait collections were found most productive yielding maximum species and highest number of anophelines per unit of collection efforts. Indoor resting collections yielded poorest diversity and greater number of specimens per unit of collection effort than outdoor resting collections. Maximum diversity was observed in outdoor collections. Most of the species exhibited unimodal prevalence.

Anopheline mosquitoes and malaria parasites in Taiwan.

Extensive taxonomic studies of the genus Anopheles were carried out in Taiwan during the first half of the 20th century. As a result, 17 species of Anopheles were identified and reported as occurring in Taiwan. However, the occurrence of two species, An. kochi and An. fluviatilis, in Taiwan is doubtful. Vector competence of the anophelines of Taiwan was also studied both in the laboratory and in the field during the same period of time, and experimental infections of 8 species of Anopheles were carried out by Anazawa in the 1920s. The results showed that all species tested were susceptible to three human plasmodia except for An. sinensis which was refractory to Plasmodium falciparum. The results of dissection of nearly 10,000 anopheline mosquitoes collected from the field by various workers suggested that An. minimus is the chief vector in Taiwan. Crithidial flagellates resembling malaria sporozoites were found in the salivary glands of 4 common species of Anopheles collected from cow-stables. These flagellates may have been misidentified as malaria sporozoites in the earlier investigations. During the first part of the operations for malaria control and eradication, anopheline mosquitoes were collected by day from 1,118 houses scattered over the island. Of the 25,656 specimens of Anopheles collected, nearly 80% were An. minimus, mostly from bedrooms. Since the highly anthropophilic and endophilic An. minimus was determined to be the chief vector of malaria in Taiwan, DDT was applied to the wall surfaces in houses for malaria control and eradication. Before spraying DDT, the rate of P. malariae infections was very high in areas where malaria is endemic. However, these infections were more quickly suppressed by DDT spraying than two other parasite species. Other details of studies on the ecology and behavior of anophelines are also presented.

[Plasmodium falciparum travels by train from Ethiopia to Djibouti]. / Plasmodium falciparum voyage en train d'Ethiopie a Djibouti.

To investigate the role of the Djibouti-Ethiopian railway as a potential vehicle for inter-regional spread of malaria vectors and malaria parasites, we performed a double-sided study, both entomological and parasitological, during November 1989, at the frontier post of Guelile where the trains from Ethiopia enter the Republic of Djibouti. No malaria-transmitting mosquitoes were collected either from the daily passenger train or from the weekly vegetables train. One hundred and five passengers entering Djibouti by train from Ethiopia had a thick film examined for malaria parasites. Five smears were positive for Plasmodium falciparum, among them two showed gametocytes. We conclude that the railway may be an effective route for the propagation of the human malaria parasite between Ethiopia and Djibouti. Indeed, passengers infected abroad could import plasmodia into Djibouti and thus become the index cases for local malaria outbreaks, in case the climatic and entomological prerequisites essential for sustaining malaria transmission are present.

[Mosquitoes (Diptera, Culicidae) found in the coastal region from Wejherowo to Darlowo]. / Komary (Diptera, Culicidae) znalezione w pasie przymorskim od Wejherowa do Darlowa.

Species composition Anophelini were investigated in the years 1986/87 and Culicini in 1987/88 at the part of the coastal region. The mosquitoes were caught from April till October, every week, at 47 stations (water reservoirs, free nature and cattle-sheds). Total 16,194 mosquitoes (2452 Anophelini and 13,742 Culicini) were caught. They belong to 26 species of 5 genera: Anopheles (4 spp.), Aedes (15 spp.), Coquillettidia (1 sp.), Culex (1 sp.) and Culiseta (5 spp.). The highest numbers of Anophelini were observed in July (1986) and in September (1987). The aggressiveness toward man showed by Culicini was found to be highest in the second half of May and at the end of July.

Malaria transmission potential of Anopheles mosquitoes in the Mwea-Tebere irrigation scheme, Kenya.

1. Anopheles arabiensis Patton and An. funestus Giles were identified as vectors of Plasmodium falciparum malaria in the Mwea-Tebere irrigation scheme, Kenya. An. arabiensis was the only member of the An. gambiae complex identified from chromosome characteristics. Other Anopheles species found included An. pharoensis Theobald, An. rufipes Gough and An. coustani Laveran. Survival rates per gonotrophic cycle for An. arabiensis averaged 0.37 during the short rains (October-November), 0.49 during the dry season (February) and 0.78 during the long rains (May-June). Vectorial capacities were correspondingly low due to low survival rates and a high degree of zoophily. The average duration of infective life for P. falciparum was 0.2 days for both An. arabiensis and An. funestus. In contrast, entomological inoculation rates were comparatively high: 6-8 infective bites/man/month. An. pharoensis averaged 110 bites/man/night during the short rains; 1/999 (0.1%) was positive by ELISA for P. falciparum circumsporozoite antigen, but the ELISA evidence is not conclusive for vector incrimination. In correspondence with clinical observations, the transmission of P. malariae and P. ovale is unlikely due to the low vector survival rates. The observed anomaly between low vectorial capacities and high entomological inoculation rates demonstrates the importance of accurately estimating vector sporozoite rates to monitor unstable malaria transmission in irrigated areas.

Colonization of rice fields by mosquitoes (Diptera: Culicidae) and larvivorous predators in asynchronous rice cultivation areas in the Philippines.

Colonization of rice fields by mosquitoes and larvivorous predators was studied in asynchronous rice cultivation areas in the Philippines. Dipper samples were taken from rice fields at six phases of maturity (fallow, ploughed, nursery, newly transplanted, after tillering, mature). All phases were present concurrently at each of two study sites. Abundance levels of the vishnui subgroup of Culex and of the genus Anopheles were high in ploughed fields, nurseries, and newly transplanted fields; this was primarily because of the concentration of Culex vishnui. Theobald and Anopheles vagus Doenitz in those fields with short, sparse vegetation. Dytiscidae, Anisoptera, and Zygoptera were more abundant in fallow or mature fields. The abundance of aquatic predators decreased at the onset of ploughing and then recovered slowly as rice plants grew. The abundance of surface predators showed a similar pattern, but less conspicuously. The abundance of nonpredators (other than mosquitoes) was relatively stable with regard to rice field phases. Alleviation of mortality by predation was considered to be a secondary cause of increased mosquito abundance in the fields under early phases of rice cultivation.

Differentiation of Anopheles gambiae and An. arabiensis (Diptera: Culicidae) by ELISA using immunoaffinity-purified antibodies to vitellogenin.

Yolk proteins (vitellogenin and vitellin) proved to be excellent marker molecules for separating Anopheles gambiae Giles and An. arabiensis Patton, two morphologically indistinguishable members of the An. gambiae species complex. A rabbit polyclonal antibody directed against An. gambiae yolk proteins was made species-specific by removing immunoglobulins that crossreacted with An. arabiensis by immunoaffinity chromatography. The resultant antibody was 400 times more sensitive to An. gambiae and was employed as the secondary antibody in a modified double antibody "sandwich" ELISA, which also used monoclonal antibodies to anopheline vitellogenin as the primary or coating antibody. This ELISA easily differentiated soluble yolk protein samples from An. gambiae and An. arabiensis. A field study with 628 females of An. gambiae complex collected in western Kenya demonstrated that the ELISA results were 98.4% in agreement with the standard cytotaxonomic method.

Algunas observaciones sobre la contaminación de las aguas y la presencia de Anopheles albimanus Wiedemann, 1821 y Culex nigripalpus Teobald, 1901 / Some observation on water contamination and presence of Anopheles albimanus Wiedemann, 1821 and Culex nigripalpus Theobald, 1901

El estudio se efectuó en una presa de la provincia La Habana desde marzo de 1986 a febrero de 1987. Se realizaron colectas de larvas relacionándolas con factores químicos y bacteriológicos del agua. Se colectaron 9 especies de culicidos. Culex nigripalpus y Anopheles albimanus resultaron las más abundantes. Los niveles de contaminación variaron y fueron siempre de origen fecal. Culex nigripalpus presentó asociación con la demanda biológica de oxígeno, la demanda química, de oxígeno y el número más probable de coliformes totales y Anopheles albimanus con coliformes totales, fecales y el oxígeno disuelto del agua. Estas asociaciones variaron con la distancia entre los puntos de vertimiento y muestreo

A simple and rapid technique for identification of large numbers of individual mosquitoes using DNA hybridization.

A general method for obtaining species-specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.

Field studies on inversion polymorphism in Anopheles superpictus from southern Italy.

Polytene chromosome studies were carried out on various population samples of Anopheles superpictus from different localities in Southern Italy. More than 7,000 female specimens, mostly obtained from daytime collections of indoor resting mosquitoes, were successfully scored for nurse cell polytene chromosomes. Night biting samples were also examined in some localities. Only one chromosomal polymorphism, due to a paracentric inversion involving the central third of the 2L arm, was recorded in all samples. In all localities, the inverted 2La arrangement showed remarkably stable frequency although the populations examined were isolated from each other and at least some of them have presumably been subject to bottle neck in recent years because of insecticide treatments or ecological changes affecting the availability of breeding places. Departures from the Hardy-Weinberg expectations, indicating an excess of heterokaryotypes, were noted and critically analysed by comparing samples obtained simultaneously in the same locality from different cow sheds, from different sections of the same cow shed and from night and day catches in the same cow shed. The phenomenon was not found uniformly distributed among indoor resting samples: significant departures from the Hardy-Weinberg expectations were observed in some cow sheds but not in others situated nearby or even adjacent to them. These results did not support the hypothesis that the excess of heterokaryotypes is due to their greater longevity or to differential mortality in the preimaginal stages. It is suggested that different karyotypes may react differently to microclimatic specific conditions, since the Hardy-Weinberg disequilibrium was mostly observed in samples from resting sites that were more lit and subject to wider climatic variation.

[Entomological results of the malaria program of the Pasteur Institute in the Malagasy Highland Plateaux in 1987-1988]. / Résultats entomologiques du programme paludisme de l'Institut Pasteur,sur les hauts-plateaux malgaches, en 1987-1988.

An entomological survey carried out between october 1987 and july 1988 in Manarintsoa, a village 30 Km West of Antananarivo, shows that An. gambiae s.l. and An. funestus are both vectors of malaria. The sporozoite rate was estimated at 0.71%, and the annual risk about 2 infectious bites per person. In all, more than 16,000 mosquitoes, belonging to at least 15 species, were caught over a period of 294 nights.

Identification of Plasmodium falciparum-infected mosquitoes using a probe containing repetitive DNA.

A cloned repetitive DNA sequence (rep20) was evaluated as a diagnostic probe specific for Plasmodium falciparum sporozoites using experimentally infected mosquitoes squashed directly on nylon filters. Head/thorax portions of mosquitoes, killed 14-16 days after ingesting P. falciparum-infected blood, gave positive signals when examined for the presence of P. falciparum sporozoite DNA by hybridisation. This correlated with the number of oocysts found in a sample of the same batch of mosquitoes examined by dissection. No positive signals were obtained with 50 Plasmodium berghei-infected mosquitoes probed with the rep20 sequence. The results indicate that a probe containing rep20 may be useful in the rapid and specific incrimination of vectors carrying P. falciparum sporozoites. The value of repetitive DNA in the diagnosis of malaria is discussed.

Comparison of DNA probe and cytogenetic methods for identifying field collected Anopheles gambiae complex mosquitoes.

A recently developed DNA probe method was compared with the standard cytogenetic method for identifying the species of individual mosquitoes in the Anopheles gambiae complex. The complex consists of 6 morphologically indistinguishable sibling species that include the major African malaria vectors. Half-gravid, field collected mosquitoes were split into 2 portions: the abdomen was preserved for ovarian nurse cell cytotaxonomy and the head/thorax portion was desiccated for DNA extraction. Cytogenetic examination of the Kenya specimens showed 88 An. gambiae and 108 An. arabiensis. The Zimbabwe specimens consisted of 6 An. gambiae and 55 An. Quadriannulatus. All samples of the 3 species were polymorphic for the major chromosomal inversions previously recorded in field specimens from eastern and southern Africa, indicating that the collections reflected natural levels of intraspecific variation in the field populations sampled. Approximately 97% of the cytologically identified mosquitoes were also identified to species by the DNA probe method, and in every case the DNA probe and cytogenetic methods of species identification produced concordant results.

Research on parasitic arthropods of the Lake Zarnowieckie environs carried out in 1981-1985. Part II. Mosquitoes (Diptera, Culicidae).

In the years 1981-1985 a total of 19,651 specimens mosquitoes were collected in the area of investigation--Lake Zarnowieckie environs. In that amount 15,072 specimens were identified. There were found 26 species belonging to 4 genera: Anopheles, Aedes, Culex and Culiseta. Some of the species found, such as Aedes cantans, Aedes punctor or Culex pipiens are troublesome pests and particularly aggressive to man, possible vectors of many diseases. In the cowsheds there were found 12 species, the most numerous were the specimens of Anopheles genera (92.1% of the collection). The species captured in the open area by means of the man bait were 17 in number. The highest aggressiveness to man was shown by Ae. punctor, Ae. cinereus, Ae. cantans and Ae. annulipes. In the water bodies among the preimaginal forms caught there identified were 22 species. The majority of them were: Culex pipiens and Aedes punctor. A seasonal activity of the Culicidae preimaginal forms was also observed. The considerable environmental changes caused by the construction of the nuclear power plant, and the work of the pump-storage power plant are, as it seems, the main cause of the changes in the species domination observed in this region. Any finau conclusions, however, can be drawn after the further investigations are completed.