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.     

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。     

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.     

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 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.     

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.     

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     

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.     

Identification of two cryptic species in the Anopheles (Cellia) annularis complex using ribosomal DNA PCR-RFLP

Anopheles (Cellia) annularis Van der Wulp is a complex of two sibling species provisionally designated as species A and B and can only be differentiated on the basis of the paracentric inversion in the ovarian polytene chromosomes. To analyze the distribution of these two species and to develop a molecular method for the identification of these two cryptic species, we sequenced the ribosomal DNA internal transcribed spacer 2 (ITS2) and domain 3 (D3) of A. annularis specimens collected from Sonapur (Assam), Jabalpur (Madhya Pradesh), Ranchi (Jharkhand), and Ghaziabad (Uttar Pradesh). We did not find any sequence variation among the specimens collected from Assam, Madhya Pradesh, and Jharkhand states, whereas two types of sequences were obtained from the specimens collected from the state of Uttar Pradesh, which correspond to species A and B of the A. annularis complex. Species A was more prevalent among the all four regions studied. The ITS2 sequence of species A showed unique restriction sites for MvaI and Eco24I, while species B displayed HinfI and NruI sites. Similarly, the D3 sequence of species A showed unique restriction site for Alw26I, while species B showed a unique KpnI site. In this study, we report for the first time the development of ribosomal DNA polymerase chain reaction-restriction fragment length polymorphism methods for identifying these two cryptic species of the Annularis complex.     

Revisited ITS2 phylogeny of Anopheles (Anopheles) Hyrcanus group mosquitoes: reexamination of unidentified and misidentified ITS2 sequences

The Anopheles (Anopheles) Hyrcanus group of mosquitoes is very important for human health because some are malarial vector mosquitoes. Despite their pathological importance, some unidentified internal transcribed spacer 2 (ITS2) sequences have been reported from members of this group, and their phylogenetic relationships have been rarely understood. In the present study, 84 ITS2 sequences for the Hyrcanus group members were retrieved from GenBank. The detailed sequence comparison unambiguously revealed that the unknown1 sequences of Li et al. (Zootaxa 939:1–8, 2005), YM-2004 (or sp.2) of Ma and Xu [J Med Entomol 42(4):610–619, 2005], Anopheles sp. of Ree et al. (2005), and Anopheles lesteri reported by Gao et al. [J Med Entomol 41(1):5–11, 2004] are identical to Anopheles belenrae [Rueda (Zootaxa 941:1–26, 2005)] and that YM-2003 (or sp.1) of Ma and Xu [J Med Entomol 42(4):610–619, 2005] is closely related to A. lesteri. In addition, some candidate species that may be synonymized are suggested in addition to the possibility that A. lesteri may be divided into at least three types: A, B, and C. The neighbor joining, maximum parsimony, and maximum likelihood trees show that the 23 examined Hyrcanus group members could be divided into four subgroups. The phylogenetic relationships among them are generally well resolved with high bootstrapping values (60–100%), and they are consistently supported by all three trees without any conflicts. These results may strongly suggest that the morphology-based groupings of the Hyrcanus group members should be seriously reconsidered. Further study must be conducted to address the following issues: the three or more multitypified A. lesteri; the unidentified YM-2003 closely related to A. lesteri; and the synonymies of Anopheles peditaeniatus/Anopheles nigerrimus, Anopheles kunmingensis/Anopheles liangshanensis, Anopheles pullus/Anopheles junlianensis, and Anopheles engarensis/Anopheles kleini. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Ministry of Science and Technology (MOST) of Korea (R01-2004-000-10930-0) awarded to UWH.     

The phylogeny of the tomato leaf mould fungus Cladosporium fulvum syn. Fulvia fulva by analysis of rDNA sequences

The nucleotide sequence of part of the ribosomal DNA from races of the fungal tomato pathogen Cladosporium fulvum and other Cladosporium species have been determined. Comparisons of the internal transcribed spacer regions (ITS1 and ITS2) of several C. fulvum races showed complete sequence homology suggesting a recent evolutionary divergence. Comparisons of these nucleotide sequences in the ITS region with those of other Cladosporium species showed the close relationship within the Cladosporium genus. Using the nucleotide sequence of part of the 18s ribosomal subunit from these isolates and comparing them with sequences of some Ascomycetes, Basidiomycetes and Chytridiomycetes, obtained from GenBank, we infer the phylogeny of the Cladosporium species studied here. Our analysis shows that the Cladosporia form a monophyletic group which falls within the order Ascomycotina.     

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.     

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     

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.     

Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Leptosphaeria maculans from different pathogenicity groups

The regions coding for the 5.8s rRNA and the flanking internal transcribed spacers (ITS1 and ITS2) from nine isolates of the blackleg pathogen Leptosphaeria maculans and one isolate of Sclerotinia sclerotiorum were amplified by the polymerase chain reaction and sequenced. Five of the L. maculans isolates were highly virulent to Brassica plants, two were weakly virulent and two were isolated from the cruciferous weed Thlaspi arvense. The 5.8s DNA sequences of all L. maculans isolates were identical. However, there were major differences in both ITS1 and ITS2 sequences that correlated with the pathogenicity grouping. Phylogenetic analysis of the ITS sequences by both parsimony and maximum-likelihood methods indicated that each pathogenicity group was statistically different from each other with the weaklyvirulent isolates being more closely related to the Thlaspi than to the highly-virulent isolates. The relationships of L. maculans to other fungi, based on a comparison of the 5.8s rDNA sequences, are discussed.     

Intraspecific variation of second internal transcribed spacer of nuclear ribosomal DNA among populations of Anopheles (Kerteszia) cruzii (Diptera: Culicidae)

Anopheline species of the subgenus Kerteszia, including Anopheles (Kerteszia) cruzii Dyar & Knab (Diptera: Culicidae), are bromeliad-malaria vectors in the Atlantic rain forest of Brazil. Morphological, genetic, and molecular polymorphisms among different populations of An. cruzii have been reported, and it has been suggested that this taxon includes a complex of cryptic species. Specimens of An. cruzii were collected in the states of SHo Paulo and Santa Catarina, from locations where autochthonous malaria cases have been reported during the last decade. The second internal transcribed spacers (ITS2) of nuclear ribosomal DNA of the captured specimens were sequenced and compared with each other. Intraspecific ITS2 sequence polymorphisms were identified, and nucleotide divergence among specimens varied from 0.3 to 0.9%. The number of nucleotides in the ITS2 sequences of these mosquitoes varied from 327 to 334, and the CG contents varied from 61.7 to 62%. The data provide further indication of An. cruzii being a complex of cryptic species.     

Length heterogeneity in ITS 2 and the methylation status of CCGG and GCGC sites in the rRNA genes of the genus Peronosclerospora

Summary The polymerase chain reaction (PCR) was used with primers complementary to conserved flanking sequences to amplify the internal transcribed spacer 2 (ITS 2) of the rDNA repeat units of five Peronoscleropora isolates, one each of P. sorghi, P. maydis, P. sacchari and tow of P. zeae. In contrast to the situation found in mostfungi that have been examined, length heterogeneity was evident in each sample. The rDNA composition of the amplified bands was confirmed by Southern hybridizations using an ITS 2 amplified from P. sorghi and cloned rDNA from Neurospora crassa as probes. Length heterogeneity was also detected in genomic DNA digests using the same probes. In addition to one dominant fragment for each isolate, there were several less frequent fragments of different sizes, and the isolate(s) for each species had a unique banding pattern for ITS 2. The absence of 5-methylcytosine residues in CCGG and GCGC sequences in the ribosomal genes of these four Peronosclerospora species was demonstrated by the production of identical banding patterns with ribosomal DNA probes following digestion of genomic DNA with MspI and HpaII, and by complete digestion with CfoI.     

A novel family of repetitive DNA sequences amplified site-specifically on the W chromosomes in Neognathous birds

A novel family of repetitive DNA sequences was molecularly cloned from ApaI-digested genomic DNA of two Galliformes species, Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris), and characterized by chromosome in-situ hybridization and filter hybridization. Both the repeated sequence elements produced intensely painted signals on the W chromosomes, whereas they weakly hybridized to whole chromosomal regions as interspersed-type repetitive sequences. The repeated elements of the two species had high similarity of nucleotide sequences, and cross-hybridized to chromosomes of two other Galliformes species, chicken (Gallus gallus) and blue-breasted quail (Coturnix chinensis). The nucleotide sequences were conserved in three other orders of Neognathous birds, the Strigiformes, Gruiformes and Falconiformes, but not in Palaeognathous birds, the Struthioniformes and Tinamiformes, indicating that the repeated sequence elements were amplified on the W chromosomes in the lineage of Neognathous birds after the common ancestor diverged into the Palaeognathae and Neognathae. They are components of the W heterochromatin in Neognathous birds, and a good molecular cytogenetic marker for estimating the phylogenetic relationships and for clarifying the origin of the sex chromosome heterochromatin and the process of sex chromosome differentiation in birds.     

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the A and B chromosomes of the Korean field mouse (<Emphasis Type="Italic">Apodemus peninsulae</Emphasis>, Muridae,Rodentia)

Three novel families of repetitive DNA sequences were molecularly cloned from the Korean field mouse (Apodemus peninsulae) and characterized by chromosome in-situ hybridization and filter hybridization. They were all localized to the centromeric regions of all autosomes and categorized into major satellite DNA, type I minor, and type II minor repetitive sequences. The type II minor repetitive sequence also hybridized interspersedly in the non-centromeric regions. The major satellite DNA sequence, which consisted of 30 bp elements, was organized in tandem arrays and constituted the majority of centromeric heterochromatin. Three families of repetitive sequences hybridized with B chromosomes in different patterns, suggesting that the B chromosomes of A. peninsulae were derived from A chromosomes and that the three repetitive sequences were amplified independently on each B chromosome. The minor repetitive sequences are present in the genomes of the other seven Apodemus species. In contrast, the major satellite DNA sequences that had a low sequence homology are present only in a few species. These results suggest that the major satellite DNA was amplified with base substitution in A. peninsulae after the divergence of the genus Apodemus from the common ancestor and that the B chromosomes of A. peninsulae might have a species-specific origin.