Insight into Anopheles (Nyssorhynchus) (Diptera: Culicidae) species from Brazil.

Anopheles (Nyssorhynchus) benarrochi s.l., Anopheles (Nyssorhynchus) oswaldoi s.l., and Anopheles (Nyssorhynchus) konderi s.l. collected in Acrelandia, state of Acre, Brazil, were identified based on morphological characters of the male genitalia, fourth-instar larvae, and pupae. Morphological variation was observed in the male genitalia of these species in comparison with specimens from other localities in Brazil. DNA sequence from the nuclear ribosomal second internal transcribed spacer of individuals identified as An. benarrochi s.l. by using male genitalia characteristics showed that the various morphological forms are conspecific but are distinct from An. benarrochi B from Colombia. Anopheles konderi s.l. and An. oswaldoi s.l. both misidentified as An. oswaldoi s.s. (Peryassti) throughout Brazil, may actually comprise at least two undescribed species. Diagnostic morphological characteristics of the male genitalia are provided to distinguish Anopheles benarrochi s.l., Anopheles oswaldoi s.l., and Anopheles konderi s.l. from morphologically similar species. Incrimination of An. oswaldoi s.s. in malaria transmission in Brazil needs further investigation because other undescribed species from Acre may have been confounded with this taxon.

Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA.

The accurate specific identification of ticks is essential for the study, control and prevention of tick-borne diseases. Herein, we determined ribosomal nucleotide sequences of the second internal transcribed spacer (ITS2) of 15 Neotropical hard tick species of the genus Amblyomma Koch found in Brazil. Most of the studied ticks accidentally parasite humans and potentially act as vectors of zoonoses. Lengths of the ITS2 sequences ranged from 956 to 1,207 bp, whereas GC content varied from 62.4 to 66.9%. A matrix of ITS2 divergence was calculated with the ITS2 sequence data obtained showing divergence levels varying from 1.5 to 28.8%. The analysis indicated that this molecular marker can be useful for Amblyomma-specific identification. Phylogenetic inferences based on the ITS2 sequences were used to assess some issues in subgenus taxonomy.

Amazonian malaria vector anopheline relationships interpreted from ITS2 rDNA sequences.

Species identification of anopheline mosquitoes (Diptera: Culicidae) can be problematic because many of them belong to complexes of morphologically similar species, often with contrasted ecology, behaviour and vectorial importance. The application of DNA-based diagnostics has proved to be useful for distinguishing between such species. We determined ribosomal DNA sequences of the second internal transcribed spacer (ITS2) from samples of 16 species of Anopheles captured in the Amazon Basin, Brazil. Length of the ITS2 varied from 323 to 410 base pairs, with GC content ranging from 50.7% to 66.5% and sequence identity from 25% to 99% between species. Maximum-likelihood paup analysis separated two distinct groups of species conforming with the recognized subgenera Anopheles (represented by eiseni, mattogrossensis, mediopunctatus and peryassui) and Nyssorhynchus (represented by 12 spp.). For the latter group, the neighbour-joining tree generated from rDNA sequence ITS2 relationships is compatible with the morphological taxonomic key established for these Amazonian species: albitarsis, aquasalis, benarrochi, braziliensis, darlingi, deaneorum, dunhami, evansae, nuneztovari, oswaldoi, rangeli and triannulatus. These ITS2 sequence data proved to be a useful tool for species identification and, potentially, to solve taxonomic problems.

Gene expression in Plasmodium: from gametocytes to sporozoites.

Completion of the complex developmental program of Plasmodium in the mosquito is essential for parasite transmission, yet this part of its life cycle is still poorly understood. In recent years, considerable progress has been made in the identification and characterization of genes expressed during parasite development in the mosquito. This line of investigation was greatly facilitated by the availability of the genome sequence of several Plasmodium, and by the application of approaches such as proteomics, microarrays, gene disruption by homologous recombination (gene knockout) and by use of subtraction libraries. Here, we review what is presently known about genes expressed in gametocytes and during the Plasmodium life cycle in the mosquito.

Analysis of salivary gland proteins of the mosquito Anopheles darlingi (Diptera: Culicidae).

The salivary proteins of Anopheles darlingi Root, the principal vector of malaria in the Amazon Region, Brazil, were analyzed. Comparison of the protein profiles between adult males and females revealed that most of the polypeptides are present in both sexes, but female-specific polypeptides also were observed. SDS-PAGE analysis of sugar-fed female mosquitoes with ages varying from 1 to 10 d after adult emergence indicated that the proteins start to be accumulated in the first day of life and are present throughout the period analyzed. Analysis of blood-fed mosquitoes showed no differences in salivary proteins when compared with sugar fed ones, suggesting that there is no specific protein induced by blood. The protein profiles of the salivary glands dissected from wild-caught female mosquitoes from different geographical regions of Brazil were compared and some differences were observed.

Sequence analysis of the second internal transcribed spacer of ribosomal DNA in Anopheles oswaldoi (Diptera: Culicidae).

Sequence divergence in the second internal transcribed spacer (ITS2) of ribosomal DNA was examined for female specimens of Anopheles oswaldoi Peryassu from 7 localities in South America. The lengths of ITS2 for all mosquitoes ranged from 348 to 356 nucleotides. After alignment of these sequences, similarity ranged from 87 to 100%. Divergence was within the range of inter-specific differences for members of anopheline species complexes. Therefore, specimens were placed into 4 groups that may correspond to at least 4 cryptic species. One is probably related to An. oswaldoi sensu stricto and another to Anopheles konderi Galvão & Damasceno. The other 2 groups may correspond to species for which morphological identification remains to be clarified. These data provide evidence that An. oswaldoi comprise a complex of cryptic species and that DNA identification may help to resolve the taxonomic questions related to this group.

Analysis of ITS2 DNA sequences from Brazilian Anopheles darlingi (Diptera: Culicidae).

Specimens of Anopheles darlingi Root, the major vector of malaria in Brazil, were collected from several states in Brazil: Sao Paulo (Dourado), Bahia (Itabela), Rondônia (Porto Velho), Roraima (Boa Vista), and Acre (Plácido de Castro). Sequence divergence in the 2nd internal transcribed spacer (ITS2) was examined. The ITS2 sequences of mosquitoes captured in the Amazon region (Porto Velho, Boa Vista and Plácido de Castro) and in the northeast of Brazil (Itabela) were almost identical; however, a 4-5% sequence divergence was observed in the ITS2 of mosquitoes captured in the southeast (Dourado). Further analysis is needed to determine if these differences indicate that Dourado population may be a separate species.

Comparative susceptibility of two members of the Anopheles oswaldoi complex, An. oswaldoi and An. konderi, to infection by Plasmodium vivax.

We compared the susceptibility of Anopheles oswaldoi and An. konderi to infection by Plasmodium vivax based on the proportion of mosquitoes presenting oocysts and sporozoites. Anophelines were captured in the State of Acre and Rondônia, Brazilian Amazon, and used to obtain F1 progenies. After emergence of adults, male genitalia of mosquitoes of each family were dissected in order to identify them as either An. oswaldoi or An. konderi. F1 progenies of field-captured An. oswaldoi, An. konderi and An. darlingi (used as control) were fed simultaneously on P. vivax-infected blood. Mosquitoes were dissected on day 10-12 after feeding and examined for the presence of oocysts and sporozoites. Both An. oswaldoi and An. konderi developed oocysts in the midguts, however, the percentage of oocyst-positive mosquitoes for An. oswaldoi (13.8%) was higher than for An. konderi (3.3%), and only An. oswaldoi developed salivary infection with sporozoites (6.9% of positivity). Infection rates in An. darlingi ranged from 22.5% to 30.0% for both oocysts and sporozoites. These results indicate that An. oswaldoi can transmit P. vivax and suggest that it is more susceptible than An. konderi. Although An. oswaldoi is an exophilic and zoophilic species, it may be involved in malaria transmission as possibly occurred in the State of Acre.

Correlation between positive serology for Plasmodium vivax-like/Plasmodium simiovale malaria parasites in the human and anopheline populations in the State of Acre, Brazil.

Antibodies against the Plasmodium vivax-like/P. simiovale malaria parasite circumsporozoite repeat peptide (APGANQEGGAA)3 were determined by enzyme-linked immunosorbent assay (ELISA) in 120 sera randomly collected in 1994 from adults in 3 localities of the malaria endemic area in the State of Acre, Brazil; antibody was detected in 18 (15%). A 'sandwich' ELISA using monoclonal antibody (mab) Pam 172, directed against the same peptide, was carried out on 1207 Anopheles oswaldoi, 12 of which (1.0%) were positive, and 168 A. deaneorum, 2 of which (1.2%) were positive. This is the first report of serological detection of the P. vivax-like parasite in anophelines and the first report linking anopheline to human serology for this parasite in the same geographical area. It is an additional indication that A. oswaldoi is a malaria vector in Acre.

Seasonal variation of anti-Plasmodium falciparum antibodies directed against a repetitive peptide of gametocyte antigen pfs2400 in inhabitants in the State of Amapá, Brazil.

Antibodies to the Pfs2400 gametocyte antigen have been shown to inhibit the development of Plasmodium falciparum in anophelines and therefore this antigen is a candidate for a transmission-blocking vaccine. To test seasonal variation of these antibodies under field conditions, sera from 72 individuals were collected twice, first during the long-rains season with low malaria transmission and then, 6 months later, during the short-rains season, when transmission is high. This study was conducted in several localities in the State of Amapá, Brazil. All but three individuals had a positive indirect fluorescent antibody test (IFAT) with asexual forms of P. falciparum. Most of them did not report malaria attacks during the period between the first and second sampling. Their sera were tested by IFAT with P. falciparum gametocytes. The overall positivity of this test did not vary between seasons, and was 47.2 (34/72) and 48.6% (35/72), respectively. The sera were also tested by ELISA with the Pfs2400 repeat peptide. The positivity rate dropped from 29.2 (21/72) to 15.3% (11/72) and the mean absorbancies from 0.623 to 0.354, when we compared the results of the long-rains and short-rains seasons. Fifteen out of the 21 ELISA positive sera turned negative, with no change of geometric mean of titres (GMT) of asexual IFAT, while five negatives became ELISA positive on second sampling, with increase of GMT. Soon after the second sampling a malaria outbreak was reported in one of the localities. These results point toward a relatively short persistence of anti-Pfs2400 repeat peptide antibodies, under natural field conditions. A gametocyte antigen booster before a high transmission period might contribute towards lowering malaria incidence by eliciting a partially effective antibody response.

[Infection of Anopheles (Kerteszia) cruzii by Plasmodium vivax and Plasmodium vivax variant VK247 in the municipalities of São Vicente and Juquitiba, São Paulo]. / Infecção do Anopheles (Kerteszia) cruzii por Plasmodium vivax e Plasmodium vivax variante VK247 nos Municípios de São Vicente e Juquitiba, São Paulo.

Sporadic cases of autochthonous malaria have been recorded in São Paulo State, located in the Southeast region of Brazil. These cases are characterized by their benign course, low parasitemia, and mild symptomatology and have been identified as vivax malaria. Little is known about the symptoms and immune response elicited in humans by the variants Plasmodium vivax VK247 and P. vivax-like human malaria parasites. These variants are transmitted by Anopheles (Kerteszia) cruzii, one of the most common species of mosquitoes in the Southeast of Brazil. The objective of the study described in this paper was to investigate infection in anophelines using ELISA immunoenzymatic assay with specific monoclonal antibodies directed against the repetitive regions of the circumsporozoite protein in classic P. vivax, P. brasilianum/P. malariae, and P. vivax VK247. Between 1991 and 1993, mosquitoes were collected in São Vicente and Juquitiba, municipalites located in a remnant of the Brazilian Atlantic forest in São Paulo State, an ecosystem rich in plants of the Bromeliaceae family. These plants function as nurseries for immature forms of anophelines of the subgenus Kerteszia. Of 1,117 An. (Ker.) cruzii captured in São Vicente, 0.179% were positive for classic P. vivax. In Juquitiba, of 1,161 An. (Ker.) cruzii, 0.086% were positive for P. vivax VK247, confirming the presence of this variant in the region. Although the infection rate is low, the high density of these mosquitoes and their voracity (they exhibit 24-h biting activity) could compensate for the low percentage of infected specimens.

Detection of anti-Plasmodium falciparum antibodies directed against a repetitive peptide of the gametocyte antigen Pfs2400 in malaria patients in Brazil.

Sera collected from 164 individuals who had clinical Plasmodium falciparum malaria and came from several areas of Brazil where malaria is endemic were tested for the presence of anti-gametocyte antibodies. Antibodies directed against P. falciparum gametocytes were detected, by IFAT, in the sera of 67.1% of these patients. The prevalence of these antibodies was significantly higher in patients who had undergone multiple attacks of malaria than in those who were experiencing their first attack at the time of serum collection. Although circulating gametocytes were detected in 22% of the patients at this time, there was no difference in the percentages of IFAT positivity between apparent gametocyte 'carriers' and 'non-carriers'. All sera were also tested by ELISA, using a dimer of the nonamer peptide [PEE(L/V)VEEV(I/V)]2, which represents a tandem consensus repeat of the P. falciparum gametocyte antigen, Pfs2400, a target of transmission-blocking antibodies. ELISA demonstrated that 32.9% of the patients had antibodies that reacted with this peptide. Positive ELISA reactions were significantly more frequent amongst the sera of patients who had had multiple malaria attacks than in those undergoing their first malaria episode; positivity was lower in the gametocyte 'carriers' than in their 'non-carriers'. These results demonstrate that anti-gametocyte antibodies, which have already been shown to have potential transmission-blocking activity, are naturally elicited in Brazilian patients, the highest rates of seropositivity occurring after multiple malaria attacks.