Intrapopulation polymorphism in Anopheles messeae (An. maculipennis complex) inferred by molecular analysis

We evaluated the internal transcribed spacer two (ITS2) sequence to detect intraspecific polymorphism in the Palearctic Anopheles maculipennis complex, analyzing 52 populations from 12 countries and representing six species. For An. messene, two fragments of the cytochrome oxidase I (COI) gene were also evaluated. The results were compared with GenBank sequences and data from the literature. ITS2 analysis revealed evident intraspecific polymorphism for An. messeae and a slightly less evident polymorphism for An. melanoon, whereas for each of the other species, 100% identity was found among populations. ITS2 analysis of An. messeae identified five haplotypes that were consistent with the geographical origin of the populations. ITS2 seems to be a reliable marker of intraspecific polymorphism for this complex, whereas the COI gene is apparently uninformative.     

Ribosomal DNA ITS2 sequences differentiate six species in the Anopheles crucians complex (Diptera: Culicidae)

Anopheles crucians Wiedemann (sensu lato) was investigated for the presence of cryptic species using rDNA ITS2 sequences. This complex of species presently contains the named species An. crucians, An. bradleyi King, and An. georgianus King. Adult female mosquitoes were collected at 28 sites in Alabama, Florida, Georgia, North Carolina, Mississippi, and Louisiana, resulting in 245 progeny broods. Species were identified using preliminary morphological characters, and the internal transcribed spacer two (ITS2) was amplified from all broods. The result was five distinct sizes of amplification product, and based on morphological characters, one of the size classes was suspected to consist of two species. All six putative species were then sequenced: five directly, and the sixth, because of extreme intragenomic (each individual with many variants) size variability, cloned. The ITS2 sequences were markedly distinct for all six species. Species designations and ITS2 sequence lengths (base pairs in parentheses) were A (461), B (1,000+), C (204), D (293), E (195), and An. bradleyi (208). Species B showed both large intraspecific and intragenomic sequence variability and is distinguished by having the longest ITS2 found so far in an Anopheles. Based on these data, we found that all species could be identified with polymerase chain reaction (PCR) using a mixture of four primers in a single reaction. Members of this complex were often found in sympatry, with the adults of five species collected at a single site in central Florida.     

Molecular identification of the malaria vectors Anopheles anthropophagus and Anopheles sinensis (Diptera: Culicidae) in central China using polymerase chain reaction and appraisal of their position within the Hyrcanus group

In central China, Anopheles anthropophagus is considered the primary malaria vector and Anopheles sinensis is a secondary vector. Identification of these two cryptic species would facilitate studies on malaria transmission and the application of control measures. At present, the only reliable morphological markers occur in the egg stage, making this approach impractical for any large scale field studies. In this study, we report on the development of a polymerase chain reaction (PCR)-restriction fragment length polymorphism procedure involving the ribosomal DNA ITS2 region for discrimination of these species. The PCR-amplified product size of the ITS2 was 574 bp for An. anthropophagus and 594 bp for An. sinensis. Diagnostic restriction fragment length polymorphisms appeared with the restriction enzymes RsaI or HinfI. This diagnostic PCR was tested on mosquitoes collected from different locations throughout China. Specimens identified morphologically as An. anthropophagus in the adult and egg stage from one location in Quangdong Province were found to be An. sinensis, while specimens from Liaoning Province, which were variable in their egg morphology, were found to be An. anthropophagus. The presence of An. anthropophagus in Liaoning Province extends the range of this species north to 42 degrees N. The ITS2 spacer sequence was used in a maximum parsimony phylogenetic reconstruction of six members of the Hyrcanus group, two members of the Lesteri subgroup, and one member of the Nigerrimus subgroup, with the resulting molecular groupings at odds with the current morphological groupings.     

Integration of Anopheles beklemishevi (Diptera: Culicidae) in a PCR assay diagnostic for palaearctic Anopheles maculipennis sibling species

A few years ago a PCR-based assay for a quick and reliable identification of six palaearctic sibling species of the Anopheles maculipennis complex was presented making use of differences in the nucleotide sequence of the ITS2 ribosomal mosquito DNA. An. beklemishevi, which is distributed in Scandinavia and Russia only, has now been integrated into this test after analysis of its ITS2 region which turned out to be much longer than those of the other sibling species. Three oligonucleotides putatively specific for An. beklemishevi were constructed and tested in combination with a universal genus-specific primer for the amplification of an An. beklemishevi-specific ITS2 DNA-fragment. Two of the three oligos generated accurate and specific PCR products, even when used in a multiplex PCR together with the specific primers for the other six sibling species. Cross-hybridization of the primers to heterologous culicid DNA was never observed. The amplicons that identify An. beklemishevi consist of 554 and 735 bp, respectively, and are easily distinguished from those specific for the other sibling species after gel electrophoresis.     

Ribosomal DNA sequence markers differentiate two species of the Anopheles maculatus (Diptera: Culicidae) complex in the Philippines

The Anopheles maculatus Theobald complex includes important vectors of malaria. Based on chromosomal and morphological evidence, two species in this complex occur in the Philippines. Because separation of these species, An. dispar Rattanarithikul & Harbach and An. greeni Rattanarithikul & Harbach, is problematic due to the difficulty or unreliability of the identification methods currently available, we sought a molecular technique for identifying these two species. We sequenced two regions of nuclear ribosomal DNA; the second internal transcribed spacer (ITS2) and the third domain (D3) of the 28S gene, from An. maculatus sensu lato (s.l.) collected throughout the Philippines. Two sequence groups were identified that corresponded morphologically to An. dispar and An. greeni. Four percent of the 318-320 bp ITS2 and 2.5% of the 367 bp D3 differed between the two species. No evidence of intraspecific variation in sequences was found. From the sequence data, we developed a more reliable and easier method for identifying An. dispar and An. greeni, based on a HaeII restriction fragment-length polymorphism in a polymerase chain reaction amplified fragment of ITS2. This method will facilitate future vector studies, which will be necessary, as previous data collected on An. maculatus s.l. in the Philippines is unreliable given the multispecies nature of this taxon.     

Identification of Anopheles daciae in Germany through ITS2 sequencing

Until the middle of the twentieth century, malaria was frequently endemic in parts of Germany; Anopheles maculipennis complex species were considered the primary vectors. Three species of this complex have been identified in Germany: A. maculipennis s.s., Anopheles messeae and Anopheles atroparvus; the last predominantly from the coastal regions of Northern Germany. Anopheles daciae is a recently described member of the A . maculipennis complex and resembles the well-characterised species A. messeae, although the two species can be distinguished through their egg morphology and sequencing of the internal transcribed spacer 2 (ITS2) region of their nuclear rDNA. In this study, we harvested larval and adult mosquito samples from five breeding sites and ten CO₂ trap collection sites in the Upper Rhine Valley of Southwestern Germany to analyse the complement of anopheline species present. Mosquito ITS2 DNA was extracted and polymerase chain reaction (PCR)-amplified using established protocols. Genomic analysis was performed by a species-diagnostic restriction fragment length polymorphism assay as well as by sequencing of PCR products; the data obtained were aligned against nucleic acid sequences from English mosquitoes retrieved from GenBank. Additionally, the larval breeding sites of A. messeae were characterised through water quality measurement. Forty-seven samples were successfully processed, of which 6 were identified as A. daciae and 41 as A. messeae. All samples of A. daciae, which has not previously been found in Central Europe, originated from one CO₂ trap collection site in Dettenheim, close to Karlsruhe, Southwestern Germany. The identification of this malarial vector in a novel area may have implications for the re-emergence of disease subsequent to climatic changes.     

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 Galv?o & 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.     

Molecular characterization of hard and soft ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, four hard tick species and one soft tick species, namely, Dermacentor marginatus, Haemaphysalis punctata, Haemaphysalis parva, Ixodes ricinus, and Dermanyssus gallinae, from south-western Romania were characterized genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), using a hard tick, Haemaphysalis longicornis, from China for comparative purposes. The ITS rDNA was amplified by polymerase chain reaction (PCR) and sequenced from individual ticks. The lengths of the ITS-1 sequences were 238–1819 bp, and the lengths of ITS-2 were 137–1695 bp, respectively, for all ticks sequenced. While sequence variation within a hard tick species was 0–1.5%, nucleotide differences between hard tick species ranged 2–25.2%, indicating that ITS rDNA sequences provide genetic markers for the differentiation of hard ticks from Romania. Hence, a PCR-linked restriction fragment length polymorphism approach was developed for their unequivocal differentiation based on ITS-1 rDNA. This is the first characterization of ticks from Romania using a genetic approach, which provides the foundation for further studies on ticks in Romania and has implications for studying the population genetic structure of the Romanian ticks and for identification and differentiation of closely related ticks. An erratum to this article can be found at     

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.     

Molecular confirmation of the specific status of Anopheles halophylus (Diptera: Culicidae) and evidence of a new cryptic species within An. triannulatus in central Brazil

Anopheles halophylus Silva-do-Nascimento & Louren?o-de-Oliveira was recently described using morphological and biological variants in specimens previously identified as Anopheles triannulatus (Neiva & Pinto). Because these two species occur in sympatry in central Brazil, we used allozymes to determine the extent of gene flow to confirm that they are different species. Of 11 allozyme loci analyzed, one (Mpi) was found to be diagnostic for An. halophylus and An. triannulatus, confirming their specific status. This locus revealed a second sibling species within An. triannulatus sensu lato. An. halophylus and the new undescribed species were confirmed using random amplified polymorphic DNA markers that showed moderate genetic divergence among these three sympatric and closely related taxa (D = 0.145-0.428). Moreover, this marker indicates that An. halophylus and the new species are more closely related to each other than either is to An. triannulatus.     

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.     

Characterization of <Emphasis Type="Italic">Oesophagostomum</Emphasis> spp. from pigs in China by PCR-based approaches using genetic markers in the internal transcribed spacers of ribosomal DNA

In the present study, samples of Oesophagostomum spp. collected from pigs from different geographical localities in mainland China were characterized genetically by polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) and restriction fragment length polymorphism (PCR-RFLP) techniques using genetic markers in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The second internal transcribed spacer (ITS-2) was amplified from 51 individual nodule worms by PCR, and the amplicons were analyzed by SSCP. With the exception of slight microheterogeneity, SSCP analyses displayed two distinct banding profiles that allowed the identification of all Oesophagostomum spp. samples examined into two groups, the first one represented O. dentatum, and the second one may represent O. quadrispinulatum. Then, the entire ITS was amplified from individual samples, and the amplicons were digested with restriction endonuclease Pst I. The results of RFLP analyses were consistent with that of SSCP. Sequence analysis of ITS rDNA supported the identification and differentiation of Chinese Oesophagostomum spp. samples into two species, namely, O. dentatum and O. quadrispinulatum. These PCR-based approaches provide useful complementary tools to traditional methods for the accurate identification of Oesophagostomum spp. (irrespective of developmental stage) and have implications for studying the ecology and population genetic structures of these parasites and for the prevention and control of the diseases they cause.     

Differentiation of phylogenetically related slowly growing mycobacteria by their gyrB sequences

The conventional methods for identifying mycobacterial species are based on their phenotypic characterization. Since some problematic species are slow growers, their taxonomy takes several weeks or months to identify. The ribosomal DNA (rDNA) sequence-based identification strategy has been adopted to solve this problem. More recently, the gyrB sequences have been shown to be useful phylogenetic markers for the identification of species. We determined the gyrB sequences of 43 slowly growing strains belonging to 15 species in the genus Mycobacterium. The frequencies of base substitutions in the gyrB sequences were comparable to those in the 16S-23S rDNA internal transcribed spacer (ITS) sequences. The ITS sequences of four species belonging to the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, and M. microti) were 100% identical, while four synonymous substitutions were found in the gyrB sequences of these strains. Based on the differences found in the gyrB sequences, we developed PCR and PCR-restriction fragment length polymorphism methods to discriminate these species.     

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.     

Rediscovery of Anopheles (Cellia) clowi (Diptera: Culicidae), a rarely recorded member of the Anopheles punctulatus group

Anopheline specimens collected in Papua New Guinea were morphologically identified as the rarely recorded Anopheles clowi Rozeboom & Knight. Amplification of the rDNA ITS2 region of this material revealed a fragment of 750 bp confirming its placement in the Anopheles punctulatus group. This group contains 12 species and includes the major malaria vectors in the islands of the southwest Pacific. Digestion of the ITS2 with the restriction enzyme MspI produced restriction fragment-length polymorphism with bands at 380, 300, and 150 bp, a pattern shared by no other members of this group. Phylogenetic analysis involving the sequencing of a 2 kb region of the rDNA 18S gene indicated that An. clowi was monophyletic and basal to the rest of the group and showed considerable independent evolution from the other members. This is the first record of An. clowi in Papua New Guinea and only the third collection of this species since its discovery in 1945.     

Sequence analysis of the rDNA internal transcribed spacer 2 and polymerase chain reaction identification of Anopheles fluminensis (Diptera: Culicidae: Anopheles) in Bolivia

Anopheles fluminensis Root is a member of the Arribalzagia Series in the subgenus Anopheles. We report the first record of this species in the department of Cochabamba, Bolivia. This species was sampled from two locations in the foothills of the eastern Andes Mountains within the Chapare Valley. Larvae were collected in fast-flowing, shaded streams at the edges of rocky pools. We provide the first sequence data for the rDNA of An. fluminensis, a partial sequence of the 5.8S and the internal transcribed spacer 2 (ITS2). The ITS2 of An. fluminensis, sequenced from two individuals at one site, was at least 596 bp, had 56.5% GC, and included three large repeats (approximately equal to 125 bp each). We describe a polymerase chain reaction protocol and species-specific primers for identifying this species in the Chapare Valley, Bolivia.     

嗜人按蚊rDNA—ITS2基因的克隆鉴定及SNP分析

目的克隆和分析嗜人按蚊核糖体DNA（rDNA）第2内转录间隔区（ITS2）序列，研究嗜人按蚊rDNA-ITS2序列的种属特异性及不同个体rDNA-ITS2序列的单核苷酸多态性（Single Nuclear Polymorphism，SNP）。方法用DNA提取试剂盒从嗜人按蚊中提取DNA摸板，利用蚊虫5．8s和28S序列的保守性设计特异引物进行聚合酶链反应以扩增嗜人按蚊rDNA-ITS2基因，扩增产物经回收纯化后连接到TA载体，经amp^＋LB固体平板筛选的阳性克隆。经amp^＋LB液体培养基培养后进行PCR鉴定、EcoRI酶切鉴定和序列分析。结果经PCR反应扩增出全长556bp的嗜人按蚊rDNA-ITS2基因。纯化后重组克隆经PCB鉴定可重现556bp的特异性条带，其酶切产物亦与目的基因PCR产物位置相同。嗜人按蚊6个克隆的同一性为99．6％，其rDNA-ITS2基因单核苷酸存在颠换和插入，在556的范围内有一个A→T，一个G→T的颠换；一个T的插入。结论嗜人按蚊rDNA-ITS2序列在种间高度保守，但不同个体间也存在一定的SNP多态性。     

Molecular epidemiology and population genetics in Leishmania

Polymorphic DNA sequences have been amplified using different PCR-based techniques and used for species identification, strain discrimination and population genetic studies in Leishmania. A PCR fingerprinting method that uses single non-specific primers generates species-specific banding patterns with some intraspecies variation. This approach can be used to identify Leishmania species and also to discriminate strains of different Leishmania species. Cultivation of the parasites is, however, mandatory. PCR-restriction fragment length polymorphism of the internal transcribed spacer (ITS) in the ribosomal operon differentiates all Leishmania species, except members of the L. donovani and L. brasiliensis complexes. ITS-single-strand conformation polymorphism or ITS sequencing can detect strain specific-variation (except in L. infantum); culturing is not required. Species of Leishmania exhibit different degrees of genetic variation (L. tropica > L. aethiopica > L. major > L. donovani). Population analysis using co-dominant DNA markers developed by sequence-confirmed amplified region analysis revealed a primarily clonal structure in a L. donovani population from Sudan and suggested that occasional recombination events may occur in this population.     

鲁道夫对盲囊线虫rDNA ITS遗传标记的研究

本实验通过对鲁道夫对盲囊线虫(Contracaecum rudolphii)rDNA的第一及第二内转录间隔区(ITS-1及ITS-2)进行PCR扩增、PCR-SSCP分析及序列分析,以明确ITS-1及ITS-2是否可作为C.rudolphii分子分类的遗传标记.结果发现C.rudolphii rDNA ITS序列存在种间差异,并且差异显著,但是种内序列一致,没有差异.本实验证明C.rudolphii确为由两个种(C.rudolphii A和C.rudolphii B)组成的复合种,ITS-1及ITS-2可作为两个姊妹种的遗传标志.     

Multilocus DNA sequence comparisons rapidly identify pathogenic molds

The increasing incidence of opportunistic fungal infections necessitates rapid and accurate identification of the associated fungi to facilitate optimal patient treatment. Traditional phenotype-based identification methods utilized in clinical laboratories rely on the production and recognition of reproductive structures, making identification difficult or impossible when these structures are not observed. We hypothesized that DNA sequence analysis of multiple loci is useful for rapidly identifying medically important molds. Our study included the analysis of the D1/D2 hypervariable region of the 28S ribosomal gene and the internal transcribed spacer (ITS) regions 1 and 2 of the rRNA operon. Two hundred one strains, including 143 clinical isolates and 58 reference and type strains, representing 43 recognized species and one possible new species, were examined. We generated a phenotypically validated database of 118 diagnostic alleles. DNA length polymorphisms detected among ITS1 and ITS2 PCR products can differentiate 20 of 33 species of molds tested, and ITS DNA sequence analysis permits identification of all species tested. For 42 of 44 species tested, conspecific strains displayed >99% sequence identity at ITS1 and ITS2; sequevars were detected in two species. For all 44 species, identifications by genotypic and traditional phenotypic methods were 100% concordant. Because dendrograms based on ITS sequence analysis are similar in topology to 28S-based trees, we conclude that ITS sequences provide phylogenetically valid information and can be utilized to identify clinically important molds. Additionally, this phenotypically validated database of ITS sequences will be useful for identifying new species of pathogenic molds.