Haematozoan parasites of the lizard Ameiva ameiva (Teiidae) from Amazonian Brazil: a preliminary note

Three different haematozoan parasites are described in the blood of the teiid lizard Ameiva ameiva Linn. from North Brazil: one in the monocytes and the other two in erythrocytes. The leucocytic parasite is probably a species of Lainsonia Landau, 1973 (Lankesterellidae) as suggested by the presence of sporogonic stages in the internal organs, morphology of the blood forms (sporozoites), and their survival and accumulation in macrophages of the liver. One of the erythrocytic parasites produces encapsulated, stain-resistant forms in the peripheral blood, very similar to gametocytes of Hemolivia Petit et al., 1990. The other is morphologically very different and characteristically adheres to the host-cell nucleus. None of the parasites underwent development in the mosquitoes Culex quinquefasciatus and Aedes aegypti and their behaviour in other haematophagous hosts is under investigation. Mixed infections of the parasites commonly occur and this often creates difficulties in relating the tissue stages in the internal organs to the forms seen in the blood. Concomitant infections with a Plasmodium tropiduri-like malaria parasite were seen and were sometimes extremely heavy.

Health matters: What you need to know - malaria

Malaria is a serious, sometimes fatal, disease spread by mosquitoes. It is common in many tropical countries and is caused by a parasite called Plasmodium. This two page leaflet covers the types of parasite that carry malaria, where it is most commonly found and the preventative measures you can take to avoid malaria.

Aedes aegypti resistance to temephos during 2001 in several municipalities in the states of Rio de Janeiro, Sergipe, and Alagoas, Brazil

For more than 30 years temephos, an organophosphate insecticide, has been the sole larvicide used in Brazil in the control of Aedes aegypti. Organophosphates were also used for adult control, being replaced by pyrethroids since l999. In this same year, the Brazilian Health Foundation started the coordination of the Ae. aegypti Insecticide Resistance Monitoring Program. In the context of this program, our group was responsible for the detection of temephos resistance in a total of 12 municipalities in the states of Rio de Janeiro (RJ), Alagoas (AL), and Sergipe (SE) during 2001. In each municipality, a pool of mosquitoes collected from different districts was used, with the exception of Rio de Janeiro city, where eight districts have been separately evaluated. Exposure of larvae to the diagnostic dose of temephos revealed resistance in all localities examined, with mortality levels ranging from 4% (Pilares district, Rio de Janeiro, RJ) to 61.9% (Campos dos Goytacazes, RJ). Quantification of mortality showed resistance ratios from 6.1 (Aracaju, SE) to 16.8 (São Gonçalo, RJ and Penha district, Rio de Janeiro, RJ). The national dengue control program is presently using these data to subside insecticide resistance management.

Association of human leukocyte antigen DQ1 and dengue fever in a white Southern Brazilian population

Dengue is an infectious disease of viral etiology transmitted by the mosquitoes Aedes aegypti, A. albopictus, and A. scutellaris. It can develop either as a benign form or as a severe hemorrhagic form. Previous work showed an association of the hemorrhagic form with human leukocyte antigens (HLA), suggesting a role of genetic factors in disease susceptibility. Nevertheless, data on HLA association with the classical form of the disease is scarce in literature. Sixty-four patients and 667 normal individuals, living in the state of Paraná, Southern Brazil, were used as test and control group, respectively. The patients developed the disease during a virus 1 dengue outbreak either in Maringá city in 1995 (47) or in Paranavaí city in 1999 (17). The diagnostic was confirmed through serology and/or viral culture. HLA class I and II typing was performed by the classical microlynfocitotoxicity test using monoclonal antisera and fluorobeads. Qui-square statistical analysis confirmed a positive association with HLA-DQ1 (76.6% vs 57.7%; p = 0.005243; pc = 0.026215). HLA-DR1 also presented an increased frequency in the test group, not statistically significant after p correction though (32.8% vs 15.9%; p = 0.005729; pc = 0.080206). In conclusion, genetic factors may play a role on the susceptibility to the classical dengue, virus 1, in the Brazilian population. Further independent studies should be performed in the Brazilian population to confirm these preliminary data.

Effect of the Aedes fluviatilis saliva on the development of Plasmodium gallinaceum infection in Gallus (gallus) domesticus

Effect of Aedes fluviatilis saliva on the development of Plasmodium gallinaceum experimental infection in Gallus (gallus) domesticus was studied in distinct aspects. Chickens subcutaneously infected with sporozoites in the presence of the mosquito salivary gland homogenates (SGH) showed higher levels of parasitaemia when compared to those ones that received only the sporozoites. However, the parasitaemia levels were lower among chickens previously immunized by SGH or non-infected mosquito bites compared to the controls, which did not receive saliva. High levels of anti-saliva antibodies were observed in those immunized chickens. Moreover, 53 and 102 kDa saliva proteins were recognized by sera from immunized chickens. After the sporozoite challenge, the chickens also showed significant levels of anti-sporozoite antibodies. However, the ability to generate anti-sporozoites antibodies was not correlated to the saliva immunization. Our results suggest that mosquito saliva components enhance P. gallinaceum parasite development in naive chickens. However, the prior exposure of chickens to salivary components controls the parasitemia levels in infected individuals.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.

Evaluation of methoprene effect on Aedes aegypti (Diptera: Culicidae) development in laboratory conditions

Several Brazilian Aedes aegypti populations are resistant to the larvicidae temephos. Methoprene, that inhibits adult emergence, is one of the alternatives envisaged by the Brazilian Dengue Control Program (PNCD). However, at Brazil vector infestation rates are measured through larvae indexes and it has been claimed that methoprene use in the field could face operational problems. In order to define a standardized protocol, methoprene effect was evaluated in laboratory conditions after continuous exposure of larvae (Rockefeller strain) to a methoprene formulation available to the PNCD. Methoprene-derived mortality occurs mainly at the pupa stage and pupa development is inversely proportional to methoprene concentration. Number and viability of eggs laid by treated and control females are equivalent. A methoprene dose-dependent delay in the development was noted; however, b correlations were found for total mortality or adult emergence inhibition if data obtained when all control mosquitoes have emerged are compared to data obtained when methoprene-treated groups finish development. The cumulative record of total methoprene-induced mortality at the time control adults emerge is proposed for routine evaluation of field populations. Mortality of all specimens, but not of larva, could account for adult emergence inhibition, confirming the inadequacy of larvae indexes to evaluate methoprene effect.

Laboratory estimation of the effects of increasing temperatures on the duration of gonotrophic cycle of Anopheles albimanus (Diptera: Culicidae)

The increase of malaria transmission in the Pacific Coast of Colombia during the occurrence of El Niño warm event has been found not to be linked to increases in the density of the vector Anopheles albimanus, but to other temperature-sensitive variables such as longevity, duration of the gonotrophic cycle or the sporogonic period of Plasmodium. The present study estimated the effects of temperature on duration of the gonotrophic cycle and on maturation of the ovaries of An. albimanus. Blood fed adult mosquitoes were exposed to temperatures of 24, 27, and 30°C, held individually in oviposition cages and assessed at 12 h intervals. At 24, 27, and 30°C the mean development time of the oocytes was 91.2 h (95% C.I.: 86.5-96), 66.2 h (61.5-70.8), and 73.1 h (64-82.3), respectively. The mean duration of the gonotrophic cycle for these three temperatures was 88.4 h (81.88-94.9), 75 h (71.4-78.7), and 69.1 h (64.6-73.6) respectively. These findings indicate that both parameters in An. albimanus are reduced when temperatures rose from 24 to 30°C, in a nonlinear manner. According to these results the increase in malaria transmission during El Niño in Colombia could be associated with a shortening of the gonotrophic cycle in malaria vectors, which could enhance the frequency of man-vector contact, affecting the incidence of the disease.

Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.

Field evaluation of effectiveness of the BG-Sentinel, a new trap for capturing adult Aedes aegypti (Diptera: Culicidae)

In recent years, the development of new tools to gather field information about vector ecological parameters has increased. This report evaluated the BG-Sentinel Trap (BGS-Trap), a promising new attempt to improve collection of the dengue vector, Aedes aegypti. The efficacy of the BGS-Trap was compared with the CDC backpack aspirator, one of the commonest used methods for capturing adult mosquitoes. BGS-Traps captured significantly more Ae. aegypti males (chi2 = 21.774, df = 1, P < 0.05) and females (chi2 = 56.007, df = 1, P < 0.05) than CDC aspirator during all days of field collection. However, CDC aspirator was significantly more efficient to capture Culex quinquefasciatus males (chi2 = 5.681, df = 1, P < 0.05) and females (chi2 = 6.553, df = 1, P < 0.05). BGS-Traps captured host-seeking females (varying between 68.75 to 89.8%) in detriment of females in other behavioral and physiological stages. BGS-Traps proved to be efficient and can be used for monitoring adult mosquito populations.

Susceptibility of Anopheles aquasalis and An. darlingi to Plasmodium vivax VK210 and VK247

The susceptibility of Anopheles aquasalis (F3 generation) and An. darlingi (F1 generation) to Plasmodium vivax circumsporozoite protein phenotypes from a limited number of blood samples of malaria patients in Belém, state of Pará, Brazil, was examined. A polymerase chain reaction was used to determine the P. vivax phenotypes in blood samples and the blood-fed infected mosquitoes were dissected and tested by ELISA. In all patient infections, more infected An. aquasalis and An. darlingi were positive for VK210 compared with VK247.

Winter survival of immature instars of Mansonia indubitans Dyar & Shannon and Mansonia titillans Walker (Diptera: Culicidae), in Buenos Aires, Argentina

We conducted a whole year research on the ecology of Mansonia indubitans and Ma. titillans in Macáes Pond, Costanera Sur Reserve, Buenos Aires, Argentina. The usage of different floating plants by immature instars and their overwintering was analized. The percentage of usage of the available floating macrophytes (Pistia, Limnobium, and Salvinia) by the larvae and pupae was studied. Also, we defined positivity (P+) as the percentage of plants with immature instars for each plant genus on a monthly basis. Ma. immature instars were captured throughout the year and Pistia was the resource most commonly exploited by the mosquitoes. The percentage of fourth-instar larvae and pupae on Pistia roots with respect to total immature instars captured was assessed on a monthly and seasonal basis. The proportion of fourth-instar larvae and pupae from both species of Mansonia on water lettuce roots, showed significant differences between months and seasons. Our results suggest that the populations of Ma. indubitans and Ma. titillans in Macáes Pond, survive during winter mainly as fourth-instar larvae.

Incrimination of Anopheles (Nyssorhynchus) rangeli and An. (Nys.) oswaldoi as natural vectors of Plasmodium vivax in Southern Colombia

Malaria transmission in the Southern Colombian state of Putumayo continues despite the absence of traditional vector species, except for the presence of Anopheles darlingi near the southeastern border with the state of Amazonas. In order to facilitate malaria vector incrimination in Putumayo, 2445 morphologically identified Anopheles females were tested for natural infection of Plasmodium vivax by ELISA. Specimens tested included An. apicimacula (n = 2), An. benarrochi B (n = 1617), An. darlingi (n = 29), An. mattogrossensis (n = 7), An. neomaculipalpus (n = 7), An. oswaldoi (n = 362), An. peryassui (n = 1), An. punctimacula (n = 1), An. rangeli (n = 413), and An. triannulatus (n = 6). Despite being overwhelmingly the most anthropophilic species in the region and comprising 66.1% of the mosquitoes tested, An. benarrochi B was not shown to be a vector. Thirty-five An. rangeli and one An. oswaldoi were naturally infected with P. vivax VK210. Sequence data were generated for the nuclear second internal transcriber space region of 31 of these 36 vivax positive mosquitoes (86.1%) to confirm their morphological identification. An. oswaldoi is known to be a species complex in Latin America, but its internal taxonomy remains unresolved. Herein we show that the An. oswaldoi found in the state of Putumayo is genetically similar to specimens from the state of Amapá in Brazil and from the Ocama region in the state of Amazonas in Venezuela, and that this form harbors natural infections of P. vivax. That An. rangeli and this member of the An. oswaldoi complex are incriminated as malaria vectors in Putumayo, is a novel finding of significance for malaria control in Southern Colombia, and possibly in other areas of Latin America.

Germline transformation of Aedes fluviatilis (Diptera:Culicidae) with the piggyBac transposable element

The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.