Molecular characterization of the Anopheles maculipennis complex during surveillance for the 2004 Olympic Games in Athens

Specimens belonging to the Anopheles maculipennis complex were collected as larvae or resting adults from May 2003 to November 2004 in the area of the Athens 2004 Olympic Rowing Centre in Schinias, Attiki, Greece, and identified by morphological and molecular analyses. Of the 201 specimens collected, 199 were found to be Anopheles sacharovi Favre and two were An. maculipennis Meigen s.s. on the basis of similarity to published sequence data for the rDNA internal transcribed spacer (ITS2) region and the mitochondrial cytochrome c oxidase I gene (COI). Sequence data from a number of specimens were obtained for both genes and compared with corresponding GenBank data derived from diverse geographical areas. A high degree of homology in ITS2 sequences was found in both species, ranging from 99.5% to 100% in An. sacharovi and 99.4% to 100% in An. Maculipennis, with no intraspecific variation in either of the two species in our study. The degree of homology in the COI sequences was 94.8-99.8% in An. sacharovi and 95.0-99.8% in An. maculipennis. The 522-bp fragment produced a rather high degree of intrapopulation polymorphism for An. sacharovi, generating nine different haplotypes, five of which were singletons. Intraspecific variation for these sequences ranged from 0.2% to 1.4%, but was much lower (0.77%) for the two An. maculipennis sequences. These findings represent the first characterization of the An. maculipennis complex in the area of Schinias.     

Morphological and molecular characterization of Anopheles (Anopheles) sacharovi Favre, a primary vector of malaria in the Middle East

Abstract. Combined morphological and molecular studies were conducted on Anopheles ( Anopheles ) sacharovi Favre, an important malaria vector of the Palaearctic Maculipennis Complex. Specimens collected in Greece and Iran were identified on the basis of morphology and confirmed by correlation of their nuclear internal transcribed spacer 2 (ITS2) rDNA sequences with those available in GenBank. The progeny of females collected in Greece were used for detailed morphological study. The morphology of the adults, pupa, fourth-instar larva and egg are described and distinguished from those of An. maculipennis , the nominotypical member of the Maculipennis Complex. In addition to sequence data for the nuclear ITS2 region, partial sequence data are provided for the mitochondrial cytochrome oxidase I gene. The taxonomy, systematics, bionomics and distribution of the species are reviewed. This work provides the first fully integrated assessment of An. sacharovi as a basis for comparative studies of the Maculipennis Complex.     

Mosquitoes of the Anopheles maculipennis group (Diptera: Culicidae) in Romania, with the discovery and formal recognition of a new species based on molecular and morphological evidence

Mosquitoes of the Anopheles maculipennis group were collected in five districts of Romania (Constant,a, Giurgiu, Ilfov, Mehedint,i and Suceava) between March 2000 and June 2003. Two hundred and ninety-seven specimens were identified by molecular methods. Nuclear rDNA ITS2 sequences of 178 specimens were compared with GenBank sequences for nine known Palaearctic species of the group, and 119 specimens were identified using an ITS2 PCR-RFLP assay developed during the study. Five genetically distinct species of the group were identified: A. atroparvus van Thiel, A. maculipennis Meigen, A. melanoon Hackett and A. messeae Falleroni and a previously unrecognized species. The new species, herein formally described and named A. daciae sp. n., was collected in the Black Sea coastal region and plains adjacent to the Danube River in southern Romania. Anopheles daciae is most similar to and sympatric with A. messeae. It is contrasted with A. messeae and characterized on the basis of unique nuclear ITS2 and mitochondrial COI DNA sequences and morphological characters of the eggs. The larval, pupal and adults stages of the two species were also compared, but no reliable characters were found to distinguish them. It seems likely that A. daciae is more widespread in eastern Europe and the Balkan States, and could be responsible for malaria transmission in these regions that is currently attributed to A. messeae. Anopheles melanoon is reported from Romania for the first time.     

On ranges of the malaria mosquitoes (Diptera: Culicidae: Anopheles) of the Maculipennis complex on the territory of Russia

Maps and distribution data are provided for the seven mosquito species of the genus Anopheles, the maculipennis group: Anopheles atroparvus, A. beklemishevi, A. maculipennis, A. messeae, A. malanoon, A. sacharovi and A. subalpinus.     

Residual mosquitoes in the northern Adriatic seacoast 50 years after the disappearance of malaria

The Northern Adriatic Sea littoral was heavily malarious; intensive land drainages, agricultural development and socioeconomic improvement were the key factors which led to malaria eradication, sped up by indoor insecticide spraying, achieved soon after World War II. Regular observations on anophelism were carried out by the Istituto Interprovinciale per la Lotta Antimalarica nelle Venezie from middle 20's until early 60's. The main vector was Anopheles sacharovi, a species which typically bred in coastal brackish swamps; other species were An. atroparvus (which was a probable secondary vector) and the usually strictly zoophilic An. maculipennis, An. melanoon, An. messeae and An. subalpinus. From 1995 to 1997 surveys were carried out in order to review the genus Anopheles in the coastal area of Friuli-Venezia Giulia and Veneto regions. A total of 11,346 females were collected from animal shelters (cow-shed, pigsties, horse stables) of 52 sites along 180 km of coast crossing 5 provinces (from North: Gorizia, Udine, Venezia, Padova and Rovigo). All specimens belonging to the An. maculipennis complex were scored for the presence of the differential characters of An. sacharovi, the only species of the complex morphologically characterized at the adult stage. The examination of morphological characters of single egg batches obtained from field collected females was the main diagnostic tool for the other species. Species identification was subject to confirmation by larval chaetotaxy analysis (number of branches of antepalmate hairs of IV and V abdominal segments) in representative samples of laboratory-reared mature larvae, while biochemical analysis (enzyme electrophoresis) on some samples of identified females was performed in the laboratory of Prof. L. Bullini and Dr. R. Cianchi of the University of Rome "La Sapienza" and partly in our laboratory. No An. sacharovi female was recorded. The examination of 6,361 single ovipositions led to the identification of three species of the An. maculipennis complex: An. atroparvus, An. maculipennis and An. messeae; An. claviger s.str. was also recorded. Larval chaetotaxy examination carried out on 1,608 larvae and the biochemical identification of 467 females confirmed the previous diagnosis based on egg characters. The relative frequency of the three species varied depending on the site: An. maculipennis was the most abundant species north of Venice; south of Venice, and particularly in the Po river delta, the most abundant species were An. atroparvus and, in some sites, An. messeae. In view of the high density recorded for An. atroparvus in some sites (corresponding to various thousands females in a single animal shelter), the vectorial capacity values may be significant and should be assessed.     

Systemic reorganization of the architectonics of polytene chromosomes in the onto- and phylogenesis of malarial mosquitoes. II. Species specificity in the pattern of chromosome relations with the nuclear envelope of nutrient ovarian cells

Essential differences in the architecture of the chromosomes between the 7 species of Anopheles maculipennis complex are found. The system of chromosomes' attachment to the nuclear envelope is invariant within particular species, each of the species studied, together with homosequential A. maculipennis and A. subalpinus differing one from another. The spatial organization of nutrse ovarian cell chromosomes in experimental hybrids (A. maculipennis X A. subalpinus and A. sacharovi X A. matrinius) shows species-specificity pattern of parental species. Thus, the spatial organization of interphase nucleus is the invariant species sign, and from the author's point of view, this phenomenon is due to penetration of new type mutations--systemic mutations (according to Richard Goldschmidt), directly connected with speciation.     

Identification of the midgut microbiota of An. stephensi and An. maculipennis for their application as a paratransgenic tool against malaria

The midgut microbiota associated with Anopheles stephensi and Anopheles maculipennis (Diptera: Culicidae) was investigated for development of a paratransgenesis-based approach to control malaria transmission in Eastern Mediterranean Region (EMR). Here, we present the results of a polymerase chain reaction (PCR) and biochemical-based approaches to identify the female adult and larvae mosquitoe microbiota of these two major malaria vectors, originated from South Eastern and North of Iran. Plating the mosquito midgut contents from lab-reared and field-collected Anopheles spp. was used for microbiota isolation. The gram-negative and gram-positive bacterial colonies were identified by Gram staining and specific mediums. Selected colonies were identified by differential biochemical tests and 16S rRNA gene sequence analysis. A number of 10 An. stephensi and 32 An. maculipennis adult mosquitoes and 15 An. stephensi and 7 An. maculipennis larvae were analyzed and 13 sequences of 16S rRNA gene bacterial species were retrieved, that were categorized in 3 classes and 8 families. The majority of the identified bacteria were belonged to the γ-proteobacteria class, including Pseudomonas sp. and Aeromonas sp. and the others were some closely related to those found in other vector mosquitoes, including Pantoea, Acinetobacter, Brevundimonas, Bacillus, Sphingomonas, Lysinibacillus and Rahnella. The 16S rRNA sequences in the current study aligned with the reference strains available in GenBank were used for construction of the phylogenetic tree that revealed the relatedness among the bacteria identified. The presented data strongly encourage further investigations, to verify the potential role of the detected bacteria for the malaria control in Iran and neighboring countries.     

Morphological and molecular characterization of Anopheles (Anopheles) maculipennis Meigen, type species of the genus and nominotypical member of the Maculipennis Complex

Abstract. Adults and larvae of Anopheles maculipennis Meigen were collected in Greece in May and June 2001. Larvae and the progeny of wild‐caught females were individually reared to obtain samples of all life stages for integrated morphological and molecular study. Specimens were identified on the basis of egg morphology and correlation of their ITS2 rDNA sequences with those in GenBank. The adult, pupal, larval (fourth‐instar) and egg stages are described, and all stages except the egg are illustrated. Partial sequence data are provided for the mitochondrial cytochrome c oxidase I (COI) gene and complete sequences for the nuclear ITS2 region. Additionally, the bionomics and distribution of the species are reviewed, and its taxonomy and systematics are discussed. This study provides the first fully integrated morphological and molecular assessment of An. maculipennis, the nominotypical member of the Maculipennis Complex, and the type species of genus Anopheles, and serves as a foundation for further studies of the complex and subfamily Anophelinae.     

Checklist of Iranian mosquitoes (Diptera: Culicidae).

The mosquito fauna of Iran includes seven genera, 64 species, and three subspecies. The records of 12 other species should be verified. There are 24 species in the most recent checklist of Iranian Anopheles. Two species, An. peditaeniatus and An. fluviatilis species V, have been reported since. An. atroparvus, An. labranchiae, and An. martinius of the Maculipennis Group, and An. cinereus, An. nigerrimus, and An. rhodesiensis rupicola were recorded previously but are not included in the checklist. The checklist of Iranian Culicinae includes ten species of the tribe Aedini, but there are some records of four other species: Aedes aegypti, Ochlerotatus berlandi, Oc. chelli, and Oc. dorsalis. The genus Culex includes 19 species, excluding Cx. impudicus, which has not been collected recently, and some doubtful records of Cx. univittatus, Cx. vishnui, and Cx. vagans. The genus Culiseta includes five species and the genera Coquillettidia and Uranotaenia each include one species in Iran. No information is available for the An. subpictus, Oc. caspius, Oc. detritus, and Oc. pulcritaris species complexes in Iran. The An. claviger and Cx. pipiens complexes and the An. hyrcanus group require review.     

Molecular taxonomy of members of the Anopheles hyrcanus group from Thailand and Indonesia

During studies of malaria vectors in Indonesia and Thailand, several specimens identified by field staff as members of the Anopheles barbirostris group (Diptera: Culicidae) were found to belong to the Anopheles hyrcanus group, as shown by marked differences in the size of the nuclear rDNA second internal transcribed spacer (ITS2) between the barbirostris (~1500 bp) and hyrcanus (~600 bp) groups. Identification of the species concerned required a more detailed study of ITS2 sequences and subunit I of the mitochondrial DNA cytochrome oxidase gene (COI). A phylogenetic analysis, based on Bayesian methods, revealed that the hyrcanus group specimens comprised five distinct clades, two of which corresponded with known species, Anopheles peditaeniatus and Anopheles sinensis. The remaining specimens formed three additional clades, for which there are no similar sequences in GenBank and which cannot be linked to previously described species. The misidentification of hyrcanus group species has important implications for malaria vector control; more comprehensive studies employing gene sequences are required to clarify the number of species in the group, their distribution and vector status.     

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.     

Single nucleotide polymorphism analysis of the ITS2 region of two sympatric malaria mosquito species in Sweden: Anopheles daciae and Anopheles messeae

Four species of the Anopheles maculipennis complex have previously been recorded in Sweden. A recent addition to the complex is Anopheles daciae, which is considered to be closely related to, but distinct from Anopheles messeae. The original designation of An. daciae was based on five genetic differences (161, 165, 167, 362 and 382) in the second internal transcribed spacer (ITS) 2 of the ribosomal RNA. Further studies have shown that only two nucleotide differences (362 and 382) robustly separate the species. Thirty-three An. maculipennis complex mosquitoes were collected in the province of Uppland, Sweden. All were An. daciae but showed double peaks for three variable positions (161, 165 and 167). When cloned, the intra-individual nucleotide variation was almost exclusively fixed with either TTC or AAT, originally diagnostic for An. messae and An. daciae, respectively. To further investigate the intra-individual variation, nine An. daciae and 11 An. messeae were collected in southern Sweden and their ITS2 fragments were amplified and sequenced using Illumina MiSeq sequencing (Illumina, Inc., San Diego, CA, USA). For the diagnostic nucleotide 382 no intra-individual variation could be detected. However, although each An. daciae specimen carried several ITS2 sequence variants for the four other nucleotides, there was no intra-individual variation in the An. messeae specimens.     

Redescription and remarks on the species Minniza persica (Pseudoscorpiones: Olpiidae) from Iran

Minniza persica, which has been described briefly by Beier in 1951 on the basis of specimens from Hormozgan and Mazandaran provinces of Iran, was recently collected again from Hormozgan and Fars provinces and is described and illustrated here. The subspecies M. persica deminuta Beier is regarded as synonymous with the nominate subspecies.     

Erysimum damirliense sp. nov. (Brassicaceae) from Iran

Erysimum damirliense, a new species of Brassicaceae from Zanjan and Ardebil provinces (northwest Iran) is described and illustrated. The new species resembles E. uncinatifolium and E. elbrusense, but is easily recognized by its life form, basal leaf margin, indumentum of cauline leaves, number of flowers in the main raceme, fruit width and style length. Phylogenetic analysis of internal transcribed spacer (ITS) DNA sequences confirm that the new species is distinct from morphologically similar species.     

Molecular characterization of Iranian Trichogrammatids (Hymenoptera: Trichogrammatidae) and their Wolbachia endosymbiont

During 2009–2010, a field survey of native Trichogramma species was carried out in six provinces of Iran, including Khorasan Razavi, Tehran, Mazandaran, Guilan, Golestan, and Qom. In this study, a molecular method for identifying Trichogramma and for determining the prevalence of Wolbachia in those species was used. Based on ITS2 (internal transcribed spacer 2) sequence, 14 populations were identified as the species T. embryophagum, T. evanescens, or T. brassicae. Wolbachia infection in these Trichogrammatids was detected using wsp gene sequencing. The highest infection rates in Trichogramma were found in Mazandaran and Golestan provinces. There was no evidence of infection in Trichogramma species in Guilan and Qom provinces. Of the three infected populations, two populations of T. evanescens were infected with only one Wolbachia strain from sib subgroup and one population was superinfected. Here, we report the first data on molecular characterization of Iranian Trichogrammatids and their Wolbachia-endosymbionts.     

Characterization and comparative analysis of DNA from the pericentric heterochromatin of chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)

The minilibrary containing DNA sequences from the diffuse pericentric heterochromatin from the right arm of Anopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the minilibrary fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the An. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of An. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific minilibrary. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.     

Molecular variation, systematics and distribution of the Anopheles fluviatilis complex in southern Asia

Species of the Anopheles fluviatilis James and Anopheles minimus complexes (Diptera: Culicidae) are difficult to distinguish morphologically. Members of the two complexes have been confused and, consequently, their distributions and roles in malaria transmission are uncertain. We identified numerous mosquitoes from China, Thailand, Vietnam, India, Nepal, Pakistan and Iran by single-strand conformation polymorphism (SSCP) and/or sequencing, and analysed the variation in the 28S D3 region of ribosomal DNA for members of the Minimus Subgroup and also the internal transcribed spacer region 2 (ITS2) for members of the An. fluviatilis complex. The D3 region is highly conserved between taxa and therefore could serve as a standard for molecular diagnosis of the subgroup members. D3 sequence, bionomics and malaria transmission data provide further evidence that An. fluviatilis S in India is conspecific with An. minimus C in South-east Asia. An. fluviatilis T has three ITS2 haplotypes (designated T1, T2 and Y) and its distribution in India, Nepal, Pakistan and Iran is confirmed. An. fluviatilis U is well defined on cytogenetic grounds in Uttar Pradesh, India, but is very close to An. fluviatilis T and the two species may hybridize in some regions. Variant ITS2 sequences suggest the possible existence of two additional taxa within the An. fluviatilis complex, one in Iran and another in India, provisionally designated An. fluviatilis forms V and X, respectively. The distributions of members of the An. fluviatilis and An. minimus complexes in south-central Asia are summarized.     

Genetic differentiation in the African malaria vector, Anopheles gambiae s.s., and the problem of taxonomic status

Of the seven recognized species of the Anopheles gambiae complex, A. gambiae s.s. is the most widespread and most important vector of malaria. It is becoming clear that, in parts of West Africa, this nominal species is not a single panmictic unit. We found that the internal transcribed spacer (ITS) of the X-linked rDNA has two distinct sequences with three fixed nucleotide differences; we detected no heterozygotes at these three sites, even in areas of sympatry of the two ITS types. The intergenic spacer (IGS) of this region also displays two distinct sequences that are in almost complete linkage disequilibrium with the distinct ITS alleles. We have designated these two types as S/type I and M/type II. These rDNA types correspond at least partly to the previously recognized chromosomal forms. Here we expand the geographic range of sampling to 251 individuals from 38 populations. Outside of West Africa, a single rDNA type, S/type I, corresponds to the Savanna chromosomal form. In West Africa, both types are often found in a single local sample. To understand if these findings might be due to unusual behavior of the rDNA region, we sequenced the same region for 46 A. arabiensis, a sympatric sibling species. No such distinct discontinuity was observed for this species. Autosomal inversions in one chromosome arm (2R), an insecticide resistance gene on 2L, and this single X-linked region indicate at least two genetically differentiated subpopulations of A. gambiae. Yet, rather extensive studies of other regions of the genome have failed to reveal genetic discontinuity. Evidently, incomplete genetic isolation exists within this single nominal species.     

Ribosomal DNA difference between species A and D of the Anopheles dims complex of mosquitoes from China

Abstract. Species A and D of the Anopheles dints complex were found in China. Ribosomal DNA second internal transcribed spacers (ITS2) of both species A and D were sequenced and found to be 716 and 710 base-pairs in length, respectively, with 699c GC content. No evidence of intraspecific variation was detected in the ITS2 sequence of species A, whereas the sequence of species D showed variation at one position in the ITS2. A large number of simple repeat motifs were dispersed throughout the ITS2 sequences. The level of interspecific difference was 5.4% of the nucleotide sequences. Some of the interspecific differences were located in regions with subrepeat structure.