Identification of six sibling species of the Anopheles maculipennis complex (Diptera: Culicidae) by a polymerase chain reaction assay

Until the eradication of malaria from Europe, members of the Anopheles maculipennis complex had been the major vectors for plasmodial parasites. With the possible reintroduction of Plasmodium species due to climate change and increased travel to and from countries where malaria is endemic, accurate identification of mosquito species will be essential for preventive studies. For this purpose, a diagnostic PCR system to differentiate between six of the seven A. maculipennis sibling species occurring in Europe was developed. The second internal transcribed spacer (ITS2) of the ribosomal DNA was amplified and sequenced for all six species. Based on differences in the nucleotide sequences, species-specific primers were constructed for PCR amplification of mosquito DNA that in combination with a universal primer generate amplification products of different length, each unique for one species. Received: 11 January 1999 / Accepted: 29 April 1999     

The ITS2 ribosomal DNA of Anopheles beklemishevi and further remarks on the phylogenetic relationships within the Anopheles maculipennis group of species (Diptera: Culicidae)

Anopheles beklemishevi specimens from Russia were analysed by their ITS2 ribosomal DNA sequence to amend and to specify the phylogenetic tree of the Anopheles maculipennis species complex. Surprisingly, with 638 base pairs, the ITS2 regions of all the 34 An beklemishevi specimens examined were considerably longer than those of all their sibling species. Sequence alignment with GenBank derived sequences of the other siblings was only possible in the beginning (for approx. 335 bp) and at the end (for approx. 150 bp) of the PCR-amplified DNA fragment, whereas in the middle, the An beklemishevi DNA sequence found no counterpart in sequences of the other siblings. Closer analysis of this intermediate part suggests a duplicated insertion of about 140 bp that has undergone subsequent mutational changes. Due to this large putative insertion, computerized phylogenetic analysis by the Bayesian inference method locates An beklemishevi in a closer relationship to the nearctic than to the palaearctic sibling species. However, when only ITS2 regions are compared, that have corresponding sequences in the other siblings, An beklemishevi forms a lineage with the palaearctic species although it is still most remotely related. It is hypothesized that during the evolution An beklemishevi separated first from the common ancestor of the palaearctic species, which had presumably made its way from the Nearctic to the Palaearctic.     

Seasonal abundance of Anopheles farauti (Diptera: Culicidae) sibling species in far north Queensland, Australia

In the Cairns area of far north Queensland, Australia, the seasonal abundance of Anopheles farauti Laveran sibling species was studied at 6 locations, representing 3 habitat types, between August 1995 and September 1997. A total of 45,401 An. farauti s.l. was collected using CO2 + octenol baited CDC light traps, and consisted of 29,565 An. farauti No. 2, 14,214 An. farauti No. 3, and 1,622 An. farauti s.s. The relative abundance of all 3 species differed significantly by season and location. An. farauti No.2 was the dominant species except in Cairns, where An. farauti s.s. was most abundant, and at Ninds Creek, where An. farauti No. 3 predominated. The dominant species at each location was present year round, although peaks in seasonal abundance were observed. An. farauti s.s. populations were highest during the wet season (January-April). In lowland freshwater swamp habitats and 1 brackish location, An. farauti No. 2 was more abundant during the wet season. However, at the highland freshwater swamp habitat, populations of An. farauti No. 2 were highest during the late dry season and early wet season (October-December). There was a significant positive correlation of both temperature and rainfall with An. farauti s.s. and An. farauti No. 2 trap collections. There was a negative correlation between An. farauti No. 3 and temperature, indicating that this species may be more abundant during cool weather. Although there were significant relationships among weather variables and An. farauti s.l. collections, correlation values were generally low, indicating that other factors may contribute to variability among An. farauti sibling species trap collections.     

Anopheles (Anopheles) forattinii: a new species in Series Arribalzagia (Diptera: Culicidae).

Anopheles (Anopheles) forattinii new species, a member of the Series Arribalzagia, is described with illustrations of the larval and pupal stages, and male and female genitalia. It is contrasted with 2 similar species, An. (Anopheles) costai Fonseca & Ramos and An. (Anopheles) mediopunctatus (Lutz). This species, and An. costai, occur over much of South America where both have been misidentified as An. mediopunctatus, a species presently only known from southeastern Brazil.     

Occurrence of an unusual polymerase chain reaction product during identification of Anopheles gambiae sibling species (Diptera: Culicidae)

 Using the polymerase chain reaction (PCR) for species differentiation within the Anopheles gambiae complex, we examined several hundred Cameroonian mosquito specimens. Applying an approved routine protocol for DNA extraction and PCR conditions, apart from the indubitable identification of most of the specimens, we came across ten PCR products that did not correspond in length to any of the hitherto known amplification products of the sibling species. Sequencing experiments showed the ten products to be of identical length (117 bp) and nucleotide sequence. The total sequence of the novel product is included in the PCR product specific for A. melas, which is known to occur in the same collection area as the ten unidentifiable mosquito specimens. On alignment of the novel PCR product sequence and the A. melas one starting at the 3"-end primer annealing site, the last 20 nucleotides of the novel product, reflecting the sequence of the 2^nd PCR primer, showed only 60% homology with the then-corresponding A. melas DNA site. Explanations for the occurrence of the unusual PCR products are given and discussed. Received: 10 July 1995 / Accepted: 27 October 1995     

Molecular and cytogenetic evidence of three sibling species of the Anopheles barbirostris Form A (Diptera:Culicidae) in Thailand

Nine isoline colonies of Anopheles barbirostris Form A, derived from individual isofemale lines from Chiang Mai, Phetchaburi, and Kanchanaburi, were established in our insectary at Chiang Mai University. All isolines shared the same mitotic karyotype (X₁, X₂, Y₁). Molecular analysis of deoxyribonucleic acid (DNA) sequences and polymerase chain reaction (PCR) products of ITS2, COI, and COII regions revealed three distinct groups: A1 (Chiang Mai), A2 (Phetchaburi), and A3 (Kanchanaburi). Crossing experiments among the three groups exhibited strong reproductive isolation, producing low and/or non-hatched eggs, and inviable and/or abnormal development of the reproductive system of F₁-progenies. Asynaptic regions along the five polytene chromosome arms of F₁-hybrid larvae clearly supported the existence of three sibling species within A. barbirostris Form A, provisionally named species A1, A2, and A3.     

PCR identification and distribution of Anopheles daciae (Diptera,Culicidae) in Germany

Based primarily on nucleotide polymorphisms in the internal transcribed spacer 2 (ITS2) of the ribosomal DNA, Anopheles daciae was recently described as an additional member of the Maculipennis Group of species, separate from Anopheles messeae with which it had previously been confused due to morphological and genetic similarity. Species differentiation between A. messeae and A. daciae was possible only by ITS2 polymerase chain reaction (PCR) amplification followed by DNA sequencing or RFLP analysis. In addition to its siblings, Anopheles maculipennis, Anopheles atroparvus and A. messeae, A. daciae has been shown to occur in Germany, although with limited distribution. We here describe additional collection sites for this species in Germany, showing concentrations in East Germany and the northern Upper Rhine Valley in Southwest Germany. A species-specific multiplex PCR assay is presented that is able to differentiate the four Maculipennis Group sibling species occurring in Germany plus Anopheles sacharovi, Anopheles melanoon and Anopheles labranchiae. The correct identification and detailed knowledge of the biology of A. daciae are of relevance since it might be a vector of disease agents, as suggested by the vector potential of its siblings and the recent finding of an A. daciae female infected with Dirofilaria repens in southern Germany.     

Reproduction-longevity trade-off in Anopheles gambiae (Diptera: Culicidae)

Reduced survival and future reproduction due to of current reproduction is a trade-off known as the cost of reproduction. Surprisingly, only a few studies have assessed the cost of reproduction in arthropod disease vectors, despite its effect on longevity, and thus on vectorial capacity. We evaluated the cost of reproduction on survival of Anopheles gambiae Giles by comparing mosquitoes that were denied exposure to the other sex, hereafter named virgins, and those that were allowed exposure to the other sex and mating, hereafter named mated. Merely 6 d of exposure to females with mating activity reduced male survival from a median of 17 d in virgins to 15 d in mated, indicating that male mating cost is substantial. The increase in mortality of mated males began several days after the exposure to females ended, indicating that mating is not associated with immediate mortality risk. Notably, body size was negatively correlated with male mortality in mated males, but not in virgins. The rate of insemination declined after 4 d of exposure to females, indicating that male mating capacity is limited and further supporting the hypothesis that mating is costly for males. Consistent with previous studies, female survival on sugar alone (median=16 d) was shorter than on blood and sugar (median=19 d), regardless if she was mated or virgin. Overall, survival of mated females was lower than that of virgins on a diet of blood and sugar, but no difference was found on a diet of sugar only. However, the cost of reproduction in females remains ambiguous because the difference in survival between virgin and mated females was driven by the difference between virgin (median=19 d) and uninseminated females exposed to males (median=17 d), rather than between virgin and inseminated females (median=19 d). Accordingly, sperm and seminal fluid, egg development, and oviposition have negligible cost in terms of female survival. Only exposure to males without insemination decreased female survival. Nonetheless, if exposure to males under natural conditions is also associated with reduced survival, it might explain why females remain monogamous.     

Rubidium marking of Anopheles stephensi (Diptera: Culicidae)

Immature Anopheles stephensi Liston were reared in untreated water and water containing eight 2-fold dilutions of rubidium (Rb) from 1,000 to 7.8 ppm to determine the concentration that allowed reliable detection and produced the least toxic effects as measured by adult emergence, weight, and survival. The amount of Rb detected in mosquitoes increased positively with increasing concentrations in the rearing water. Concentrations > or = 31.2 ppm Rb in the rearing water provided high and consistent detection levels of > or = 3,500 ppm Rb/mg of adult mosquito. There were no adverse effects of Rb on the weights of mosquitoes. However, increased Rb concentrations in the rearing water were associated with decreased emergence and survival. After 8 d, percentage emergence from Rb concentrations of 0-31.2 ppm was > or = 50%. At day 21, Rb concentrations of 0-31.2 ppm showed < or = 29% reduction of female survival compared with controls. The recommended concentration for reliable Rb detection with minimal toxic effects in An. stephensi was 32 ppm.     

Bloodmeal hosts of Anopheles species (Diptera: Culicidae) in a malaria-endemic area of the Brazilian Amazon

Hosts of blood-fed anophelines (Diptera: Culicidae) were determined in three riverine villages, 1.5-7.0 km apart, along the Matapí River, Amapá state, Brazil, by enzyme-linked immunosorbent assay midgut analysis for IgG of common vertebrates. Anopheles marajoara Galv?o & Damsceno and Anopheles darlingi Root had higher human blood indices (HBI) than Anopheles nuneztovari Gabaldón, Anopheles triannulatus (Neiva and Pinto), and Anopheles intermedius (Chagas), which were relatively zoophilic. HBIs of An. darlingi varied significantly among villages, attributable to a low proportion of human-fed mosquitoes in Santo Ant?nio. Significantly higher incidence of An. marajoara and An. nuneztovari fed on pig blood at two villages, associated with a low number of pigs in Santo Ant?nio. The incidences of bovine blood varied significantly among villages for all three of the most common anopheline species. The incidence of mixed meals ranged from 7.1 to 27.6% among common species, and, for An. marajoara, varied significantly among villages. This study demonstrates differences in host selection patterns among villages only a few kilometers apart, which may be influenced by host availability and have important epidemiological consequences.     

Dichotomous electrophoretic taxonomic key for identification of sibling species A, B, and C of the Anopheles quadrimaculatus complex (Diptera: Culicidae)

Samples of 17 populations of Anopheles quadrimaculatus Say from Florida, Alabama, Arkansas, Louisiana, Mississippi, Tennessee, New York, and New Jersey were analyzed for genetic variability at 33 enzyme loci. Statistical analysis of electromorph frequency distributions indicated that sympatric sibling (morphologically indistinguishable) species occurred in about 59% of the populations tested. The association of polytene chromosome and electrophoretic patterns of individual field-collected females confirmed species-specific diagnostic allozymes, which were useful in identifying sibling species A, B, and C and in estimating the proportions of each species at the 17 collection sites. A dichotomous electrophoretic key is presented for the identification of sibling species of the An. quadrimaculatus complex. The electrophoretic method is better than the ovarian polytene chromosome method, because mosquitoes of both sexes and females irrespective of their gonotrophic condition can be identified.     

Molecular evidence for similarity between Anopheles hyrcanus (Diptera: Culicidae) and Anopheles pseudopictus (Diptera: Culicidae), sympatric potential vectors of malaria in France

Malaria was a former public health problem in the Camargue, southeastern France, where members of the Hyrcanus group were recently described as the main malaria potential vectors. However, the systematic status in this group, which includes at least two sympatric sibling species, Anopheles hyrcanus (Pallas) and Anopheles pseudopictus Grassi as well as a morphologically intermediate form in the Camargue, is unclear. Indeed, both species have been alternatively considered as separated or synonymous species. We examined sequence variation of the internal transcribed spacer (ITS) 2 and domain-3 (D3) of 28S ribosomal DNA and the cytochrome oxidase subunit I and II (COI and COII) genes of mitochondrial DNA of the Hyrcanus group mosquitoes from the Camargue and Turkey to infer the taxonomic status of the members of this group. DNA sequence analysis of ITS2 and D3 showed no difference between either species or geographical origin (mean pairwise genetic distances d = 0.000-0.003). The COI and COII sequences between French specimens also were nearly identical (d = 0.001-0.002), whereas French and Turkish Anopheles were genetically distinct (d = 0.009-0.014). The distinction between populations of the two areas, supported, respectively, by four and five fixed mutations, attested the differentiation by the distance. Finally, the high degree of genetic similarity, despite morphological differences between An. hyrcanus, An. pseudopictus, and an intermediate form, suggests that these three taxa may belong to a single species in the Camargue.     

Cryptic species Anopheles daciae (Diptera: Culicidae) found in the Czech Republic and Slovakia

Parasitology Research - We report the distribution of mosquitoes of the maculipennis complex in two distinct areas of the Czech Republic (Bohemia and South Moravia) and in one locality of...     

Diving ability of Anopheles gambiae (Diptera: Culicidae) larvae

Anopheles gambiae Giles larvae usually live near the surface of shallow and temporary aquatic habitats. How deep the larvae can dive and how long they can submerge may be related to feeding efficiency and predator avoidance. This study examined diving behavior of An. gambiae larvae in the laboratory. We recorded diving depths and larval mortality of second and fourth instars in clean water and muddy water by using deep water (32-cm) and shallow water (20-cm) columns. In deep water columns with clean water, we found that 2% of second instars and 6% of fourth instars died from diving, whereas 3% of second instars and 11% of fourth instars died in muddy water. The fourth instars dived deeper in muddy water than in clean water. The mortality rates of the fourth instars subjected to diving stimulations were significantly higher than those in the shallow water columns. Therefore, larval diving behavior may offer the benefits of predator avoidance and food acquisition but also incur energetic costs and increased mortality.     

Potential distribution of two species in the medically important Anopheles minimus complex (Diptera: Culicidae)

Anopheles minimus Theobald (=An. minimus A) and possibly Anopheles harrisoni Harbach & Manguin (=An. minimus C) are important malaria vector species in the Minimus Complex in Southeast Asia. The distributions of these species are poorly known, although detailed information could benefit malaria vector incrimination and control. We used published collection records of these species and environmental geospatial data to construct consensus ecological niche models (ENM) of each species' potential geographic distribution. The status of the Indian taxon An. fluviatilis S as a species distinct from An. harrisoni has been debated in the literature, so we tested for differentiation in ecological niche characteristics. The predicted potential distribution of An. minimus is more southerly than that of An. harrisoni: Southeast Asia is predicted to be more suitable for An. minimus, and China and India are predicted more suitable for An. harrisoni, so An. harrisoni seems to dominate under cooler conditions. The distribution of An. minimus is more continuous than that of An. harrisoni: disjunction in the potential distribution of the latter is suggested between India and Southeast Asia Anopheles fluviatilis S occurrences are predicted within the An. harrisoni ecological potential, so we do not document ecological differentiation that might reject conspecificity. Overall, model predictions offer a synthetic view of the distribution of this species complex across the landscapes of southern and eastern Asia.     

Insecticide resistance in Anopheles funestus (Diptera: Culicidae) from Mozambique

Malaria control in southern Mozambique is currently by indoor residual carbamate insecticide treatment, with pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles funestus s.s. to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 19 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. funestus from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A high level of pyrethroid resistance was detected in An. funestus populations in southern Mozambique. An. funestus outside Maputo province were still susceptible to pyrethroids. An. funestus from six localities also were resistant to carbamate insecticides propoxur and bendiocarb. Both pyrethroid and carbamate resistance occurred in five of these six localities. Mosquitoes from five of the localities with elevated p450 estimates, compared with the insecticide-susceptible Durban strain, were pyrethroid-resistant. The only exception to this trend was Mozal, which had elevated p450 estimates but full pyrethroid susceptibility by bioassay. The lack of cross-resistance between pyrethroids and DDT in Mozambican An. funestus suggests that a kdr-type target site resistance mechanism has not been selected. Low levels of insecticide-insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in all populations tested. The high level of metabolically based pyrethroid resistance has implications for current malaria control programs in Mozambique.     

Reassociation kinetics of Anopheles gambiae (Diptera: Culicidae) DNA.

The renaturation kinetics of nuclear DNA from the G3 colony of Anopheles gambiae Giles was studied to estimate the genome size and to determine the proportion of repeated sequences. An. gambiae has a haploid DNA content of 0.27 picograms or 2.6 x 10(8) basepairs. Analysis of reassociation kinetics indicated that the genome is composed of 61% single-copy and 33% repetitive sequences, with 6% foldback sequences.