Karyotype of Brazilian Anopheles albitarsis sensu lato (Diptera:Culicidae)

Anopheles (Nyssorhynchus) albitarsis sensu lato is an important malaria vector in Brazil, especially in the Brazilian Amazon region. Chromosome preparations of fourth-instar larvae of A. albitarsis from Iranduba and Coari (AM) and Ilha Comprida (SP) were analyzed for karyotype determination and to improve cytogenetic identification of this species. Anopheles albitarsis possesses 2n = 6 chromosomes, with two pairs (submetacentric and metacentric) of autosomes and one pair of sex chromosomes, with X-Y dimorphism. The sex pair is homomorphic and acrocentric in females and heteromorphic in males, with a punctiform Y chromosome. Somatic pairing was detected in the prometaphase and metaphase chromosomes of the three A. albitarsis populations. Apparently, sex chromosome evolution in the Culicidae does not function as does evolution in the Culicidae, since it occurs in the subfamily Anophelinae, which possesses heteromorphic sex chromosomes and is regarded as primitive, based on several criteria. These karyotype data on the albitarsis complex reinforce the hypothesis that sex chromosome evolution in the subfamily Anophelinae is conserved, and the variation revealed in the mean size of chromosomes in three populations indicates that selective pressure in these populations is occurring only at a genetic level.     

Parity and age composition for Anopheles darlingi root (Diptera: Culicidae) and Anopheles albitarsis Lynch-Arribálzaga (Diptera: Culicidae) of the northern Amazon Basin, Brazil.

Parity and age composition for Anopheles darlingi and Anopheles albitarsis in the northern Amazon Basin, Brazil, were investigated. Anopheline ovaries and ovarioles were examined in order to determine whether hourly and seasonal parity status for the vectors An. albitarsis and An. darlingi would vary in two different landscapes (forest and savanna/forest) where malaria is endemic in the northern Amazon Basin. A total of 1,199 anophelines (535 An. darlingi and 664 An. albitarsis) was dissected for parity status, ovariole dilatations, and follicular stages. The total number of nulliparous and parous females for both species varied by time of collection, locality, and season. During the rainy season for the first two h of collection, more nulliparous An. albitarsis and An. darlingi females were collected in the first hour (18:00-19:00), but during the second hour (19:00-20:00) more parous females of both species were captured. During the dry season in Copaíbas, more parous females of An. albitarsis were observed in the first hour while more nulliparous females were observed in the second hour. Nulliparous and parous females of both species for both hours were not significantly different at Road 19 in the dry season. This location was characterized by a forest malaria pattern of transmission with higher numbers of parous females and population stability in the dry season. In Copaíbas, the density and parity of An. darlingi increased during the rainy season, and it could be classified as an alluvial malaria pattern of transmission. For Copaíbas, control measures would be more successful if adopted at the transition from dry to rainy season. Further investigation on longitudinal spatio-temporal change in longevity and survival rates would help us to clarify differences in vector competence for An. darlingi and An. albitarsis and add to the understanding of differences regarding prevailing landscapes in malaria epidemiology in the northern Amazon Basin.     

Intragenomic rDNA ITS2 variation in the neotropical Anopheles (Nyssorhynchus) albitarsis complex (Diptera: Culicidae)

We cloned and sequenced the rDNA internal transcribed spacer 2 (ITS2) of 4 species belonging to the neotropical Anopheles (Nyssorhynchus) albitarsis complex, that is, A. albitarsis; A. albitarsis B; Anopheles marajoara, a proven malaria vector; and Anopheles deaneorum, a suspected vector. Even though the ITS2 sequences of these species were very similar (< or =1.17% divergence), we found differences suitable for species identification and intragenomic variation of possible consequence in phylogenetic reconstruction. Variation came from 2 microsatellite regions and a number of indels and base substitutions. The existence of partially correlated subsets of clones in A. albitarsis is hypothesized either to be separate rDNA loci or to be semi-independently evolving portions of a single rDNA locus. No differences were found between males and females, suggesting that similar rDNA arrays exist on both the X and Y chromosomes. In addition, highly variant clones, possibly pseudogenes, were found in A. marajoara from Venezuela.     

Intraspecific hybridization of Anopheles sinensis (Diptera: Culicidae) strains from Thailand and Korea

Anopheles (Anopheles) sinensis [Wiedemann (1828)] is a member of the hyrcanus species group, and it has been incriminated as the natural or experimental malaria vectors in the Republic of Korea, Japan, China, and Indonesia. In Thailand, however, An. sinensis seems to be of little medical importance. Hybridization tests among the three iso-female lines (isolines) of An. sinensis [i.e., Form A (X, Y1) and Form B (X, Y2) (Thailand strain), and Form B (X, Y2) (Korean strain)] were established based on two distinct types of metaphase chromosomes and geographical differences The chromosomal form of the Korean strain was first identified from this study. Results of reciprocal and back crosses indicated that both karyotypic forms of theAn. sinensis Thailand and Korean strains were genetically compatible, and provided viable progenies and completely synaptic polytene chromosomes. The sequences of the rDNA internal-transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit II (COII) among the An. sinensis strains were nearly identical to each other, and the intraspecific sequence variability was very low (0.0-0.6%). Sequence comparisons among the cryptic inter-species (i.e., An. sinensis, An. lesteri, and An. yatsushiroensis), however, revealed extensive divergence, and the intraspecific variability ranged from 12.2 to 34.6%. Therefore, it is concluded from these results and previous vector ability studies that the An. sinensis Forms A and B exhibit cytological polymorphic races that have different vector abilities in their transmission of malaria, depending on their geographical locations.     

Cytogenetic evidence for a complex of species within the taxon Anopheles maculatus (Diptera: Culicidae)

Two largely independent studies of chromosomes from natural populations of Anopheles maculatus provide evidence for several genetic species within the taxon. (1) Polytene chromosome variation shows four different rearrangements of arm 2 and three rearrangements of the X chromosome. There is strong evidence for three species. Two allopatric populations represent either dramatic geographic variation for two independent inversion systems within one of the genetic species, or represent two additional species. Their species status remains unresolved by this work. (2) Heterochromatic variation occurs in both X and Y chromosomes as revealed by Giemsa-banding of mitotic chromosomes from larval brains. The distribution and association of these various sex chromosomes give further evidence of a species complex. A preliminary correlation of these two kinds of chromosomal variation is given.     

Geographic distribution,evolution, and disease importance of species within the Neotropical Anopheles albitarsis Group (Diptera,Culicidae)

The Anopheles albitarsis group of mosquitoes comprises eight recognized species and one mitochondrial lineage. Our knowledge of malaria vectorial importance and the distribution and evolution of these taxa is incomplete. We constructed ecological niche models (ENMs) for these taxa and used hypothesized phylogenetic relationships and ENMs to investigate environmental and ecological divergence associated with speciation events. Two major clades were identified, one north (Clade 1) and one south (Clade 2) of the Amazon River that likely is or was a barrier to mosquito movement. Clade 1 species occur more often in higher average temperature locations than Clade 2 species, and taxon splits within Clade 1 corresponded with a greater divergence of variables related to precipitation than was the case within Clade 2. Comparison of the ecological profiles of sympatric species and sister species support the idea that phylogenetic proximity is related to ecological similarity. Anopheles albitarsis I, An. janconnae, and An. marajoara ENMs had the highest percentage of their predicted suitable habitat overlapping distribution models of Plasmodium falciparum and P. vivax, and warrant additional studies of the transmission potential of these species. Phylogenetic proximity may be related to malaria vectorial importance within the Albitarsis Group.     

A new species of Anopheles (Anopheles) from Namibia (Diptera: Culicidae)

Abstract. The adult, pupa, larva and egg of Anopheles {Anopheles) namibien-sis sp.n. from the Kavango district, Namibia (South West Africa), are described and comparisons made with Anopheles (Anopheles) ziemanni Griinberg. A photomap of the polytene chromosomes from the salivary glands of the fourth stage larvae is presented.     

Multiple blood meals in Anopheles darlingi (Diptera: Culicidae)

Anopheles darlingi is an important vector of human malaria in the Amazon. Adult females of this mosquito species require a blood meal to develop eggs, preferring humans to other blood sources. Although gonotrophic concordance has been described as the norm for An. darlingi, here we report An. darlingi female mosquitoes taking two or more blood meals within their first gonotrophic cycle. Only half of field‐captured adult females fed one blood meal developed follicles to Christophers' stage V. This outcome is dependent on larval nutrition, as 88% of laboratory‐raised well‐nourished females completed the first gonotrophic cycle with only one blood meal, while less nourished females needed additional blood meals. Half of the field‐captured blood‐seeking An. darlingi females had follicles in intermediate (IIIa and IIIb) and final (V) stages of the gonotrophic cycle, supporting the conclusion that An. darlingi blood feed more than once during a gonotrophic cycle. Additionally, we observed females attempting to blood feed a second time during the same day. Additional studies of An. darlingi biting behavior are necessary to accurately estimate Plasmodium sp. entomologic inoculation rates throughout the An. darlingi vast geographical distribution.     

Identification of Anopheles (Nyssorhynchus) albitarsis complex species (Diptera: Culicidae) using rDNA internal transcribed spacer 2-based polymerase chain reaction primes

Anopheles (Nyssorhynchus) marajoara is a proven primary vector of malaria parasites in Northeast Brazil, and An. deaneorum is a suspected vector in Western Brazil. Both are members of the morphologically similar Albitarsis Complex, which also includes An. albitarsis and an undescribed species, An. albitarsis "B". These four species were recognized and can be identified using random amplified polymorphic DNA (RAPD) markers, but various other methodologies also point to multiple species under the name An. albitarsis. We describe here a technique for identification of these species employing polymerase chain reaction (PCR) primers based on ribosomal DNA internal transcribed spacer 2 (rDNA ITS2) sequence. Since this method is based on known sequence it is simpler than the sometimes problematical RAPD-PCR. Primers were tested on samples previously identified using RAPD markers with complete correlation.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

按蚊属一新种(双翅目:蚊科)

记述采自云南思茅市水塘内按蚊1新种,讨论了新种与近缘种的鉴别特征。模式标本保存于云南省寄生虫病防治所。     

Cytogenetic,cross-mating and molecular evidence of four cytological races of Anopheles crawfordi (Diptera: Culicidae) in Thailand and Cambodia

Twenty-nine isolines of Anopheles crawfordi were established from wild-caught females collected from cow-baited traps in Thailand and Cambodia. Three types of X (X₁, X₂, X₃) and four types of Y (Y₁, Y₂, Y₃, and Y₄) chromosomes were identified, according to differing amounts of extra heterochromatin. These sex chromosomes represent four metaphase karyotypes, i.e., Forms A (X₁, X₂, X₃, Y₁), B (X₁, X₂, X₃, Y₂), C (X₂, Y₃) and D (X₂, Y₄). Forms C and D are novel metaphase karyotypes confined to Thailand, whereas forms A and B appear to be common in both Thailand and Cambodia. Cross-mating experiments between the four karyotypic forms indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes. The results suggest that the forms are conspecific and A. crawfordi comprises four cytological races, which is further supported by very low intraspecific variation (mean genetic distance = 0.000–0.018) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA sequences (COI, COII).     

Allozyme variation in Anopheles stephensi Liston from Pakistan (Diptera: Culicidae)

Allozyme variability was analyzed at 16 loci in 11 lines of Anopheles stephensi Liston from Pakistan. Six lines were considered as samples from natural populations. For these lines the mean number of alleles was 1.31-1.63, the degree of polymorphism was 0.188-0.375, the observed heterozygosity was 0.065-0.086, and the genetic distance ranged from 0.001 to 0.016. No population-specific alleles were found. Interbreeding was considerable (mean Fit = 0.183). Differences in allele frequencies were due considerable (mean Fit = 0.183). Differences in allele frequencies were due primarily to local inbreeding (Fis greater than Fst at most loci). The Lahore line, reared for more than 20 generations, had more homozygotes than the other lines. A line refractory to Plasmodium falciparum and a genetic sexing line exhibited decreased allozyme variability. The latter line showed reduced staining intensity at 10 loci. Linkage studies are recommended for the following loci with rare alleles: Acp, Gapdh, Icd-1, Icd-2, Mpi, and Pgd.     

Cladistic analysis of the subgenus Anopheles (Anopheles) Meigen (Diptera: Culicidae) based on morphological characters

In the present study, we used morphological characters to estimate phylogenetic relationships among members of the subgenus Anopheles Meigen. Phylogenetic analyses were carried out for 36 species of Anopheles (Anopheles). An. (Stethomyia) kompi Edwards, An. (Lophopodomyia) gilesi (Peryassú), Bironella hollandi Taylor, An. (Nyssorhynchus) oswaldoi (Peryassú) and An. (Cellia) maculatus Theobald were employed as outgroups. One hundred one characters of the external morphology of the adult male, adult female, fourth-instar larva, and pupa were scored and analyzed under the parsimony criterion in PAUP. Phylogenetic relationships among the series and several species informal groups of Anopheles (Anopheles) were hypothesized. The results suggest that Anopheles (Anopheles) is monophyletic. Additionally, most species groups included in the analysis were demonstrated to be monophyletic.     

Laboratory colonization of Anopheles pseudopunctipennis (Diptera: Culicidae) without forced mating

Anopheles pseudopunctipennis is one of the main malaria vectors in the Andean regions of South America. Few experimental data exist on this species because it is not very available in laboratories due to its eurygamic status that makes colony maintenance difficult. Indeed, individuals do not mate in the confined space of insectary cages. To avoid this problem, forced artificial mating can be used. However, this technique is time consuming, requires a well-trained technician, and is inadequate for easy mass production, which is sometimes necessary for certain experimental works. This study presents a technique based on exposure of adult mosquitoes to a blue stroboscopic light for 20 min during several nights, which encourages them to copulate naturally under laboratory conditions. After some generations, a self-free-mating strain was obtained. The technique is simple, inexpensive and is probably effective whatever the An. pseudopunctipennis strain considered.     

Molecular Variation and Distribution of Anopheles fluviatilis (Diptera: Culicidae) Complex in Iran

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.