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.     

The Anopheles maculipennis complex (Diptera: Culicidae) in Iran: molecular characterization and recognition of a new species

Mosquitoes of the Anopheles maculipennis complex were collected in nine provinces of Iran (Esfahan, Fars, Gilan, Golestan, Kohkiluyeh va Boyerahmad, Mazandaran, Tehran, Azarbaijan-e Gharbi and Zanjan) between June 1983 and September 2002. The nuclear rDNA ITS2 sequences of 86 specimens were compared with those of seven species of the complex available in GenBank. Three genetically distinct species of the complex were distinguished: A. maculipennis Meigen, A. sacharovi Favre and a previously unrecognized species. The last species is most similar to, but clearly distinct from, A. martinius Shingarev and A. sacharovi. The taxonomy of A. martinius and A. sacharovi is critically reviewed, and justification is provided for formally recognizing the third species as Anopheles persiensis sp.n. The new species is the first culicid to be characterized and named principally on the basis of DNA evidence. Anopheles persiensis was collected only in the northern Caspian Sea littoral provinces of Gilan and Mazandaran, and it seems likely that this species could be responsible for malaria transmission in this region that was previously attributed to A. maculipennis. A species-specific RFLP-PCR assay based on ITS2 sequences was developed to facilitate further studies of the three species in Iran.     

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.     

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.     

Cytogenetic analysis of the species composition and inversion structure of populations of malarial mosquitoes in the Astrakhan region

A cytogenetic analysis of Anopheles mosquitoes in the Astrakhan region was carried out. Three species of Anopheles were identified. An. messeae lives everywhere and prevails in all of the areas of research, An. hyrcanus is found in the southwest of the region, and An. maculipennis in the northern part of the region. The populations of An. messeae show a high level of inversion polymorphism for the sex chromosome and the third autosome. A clear clinal trend of an increase in chromosomal rearrangements XL1, 3R1, and 3L1 and a decrease in the frequency of evolutionary source alternatives was revealed in laraval hemipopulations of the species from south to north.     

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 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.     

Genetic distance of the malarial mosquitoes, Anopheles beklemishevi and Anopheles messeae (Diptera, Culiccidae), and their intraspecific polymorphism

Allele frequencies at enzyme loci have been studied in Finnish populations of two species of the Anopheles maculipennis complex: A. beklemishevi and A. messeae. A. beklemishevi is spread over central and northern Finland, whereas A. messeae is found in southwestern and central Finland. The allele frequencies of these two species exhibit both similarities and differences. The results indicate that the two species do not interbreed in the nature. The allele frequencies at two loci--Hydroxybutyrate dehydrogenase (Hbdh) and Superoxide dismutase-2 (Su-2)--are almost totally different and adult individuals of the two species can be reliably diagnosed by these allelic differences. The genetic distance, D, between A. beklemishevi and A. messeae is 0.35. This value is compared with corresponding distances between other dipterans studied.     

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.     

Localization of repetitive DNA sequences in the pericentromeric heterochromatin of malarial mosquitoes of the "Anopheles maculipennis" complex

Distribution of eight fragments of conserved repetitive DNA from pericentromeric heterochromatin of chromosome 2 of Anopheles atroparvus has been investigated by in situ hybridization on polytene chromosomes of An. atroparvus and An. messeae. We have shown that heterochromatic regions of all chromosomes both in An. atroparvus and An. messeae vary in combinations of, at least, conserved repeats. Some repeats have been found only in pericentromeric heterochromatic regions of chromosomes 2 (clones Atr2R-46a, Atr2R-73, Atr2R-85a in An. atroparvus and Atr2R-25 in An. messeae). Others have been found in two (clones Atr2R-25a and Atr2R-90 in An. atroparvus, Atr2R-25a in An. messeae) and more (clones Atr2R-118, Atr2R-136 in An. atroparvus, Atr2R-73 in An. messeae) pericentromeric heterochromatic regions of chromosomes. DNA comparison of pericentromeric heterochromatic regions of chromosomes in species of the "Anopheles maculipennis" complex is species- and chromosome-specific, due, in particular, to different maintenance of conserved repeates.     

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real‐time PCR assays

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking. Between 2010 and 2013, a total of 1445 mosquitoes of the An. maculipennis complex were collected at 72 different sites in Germany. The samples comprise 722 single individuals as well as 723 individuals in 90 pools of up to 25 mosquitoes. All samples were analysed with newly developed species‐specific qPCR assays for the identification of the four German species using nucleotide differences within the internal transcribed spacer 2 (ITS2) ribosomal DNA. All gathered data were used for species distribution modelling. The overall prevalence of An. messeae s.l. was highest with 98.89% of all pools; An. daciae with 6.93% of all individuals and An. messeae s.s. with 69.53%. The prevalence of the other two species was relatively low: An. maculipennis s.s. with 13.30% of all individuals (6.67% of all pools) and An. atroparvus with 1.80% of all individuals (1.11% of all pools).     

Genetic aspects of sexual behavior in malaria mosquitoes on the basis of specific acoustic signals at mating

Acoustic characteristics were studied in two species of the "Anopheles maculipennis" species complex, A. messeae and A. atroparvus. The species were found to clearly differ in sound frequencies, which was assumed to play a key role in species identification during mating in regions of their sympatric distribution. The sound spectrum in A. messeae was far more diverse than in A. atroparvus, which was associated with intraspecific inversion polymorphism of the former. Mosquitoes with the inversion combinations that were most common in populations of the central region of the A. messeae species area specifically differed in acoustic signal spectrum from each other. Hence, sound communication within the species was considered to be the main mechanism that is responsible for sexual partner selection and determines the chromosome associations observed earlier in individual karyotypes. Since males carrying different inversion combinations significantly differed in acoustic characteristics, females were assumed to play a main role in selecting the sexual partner.     

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.     

Estimated resistance of the malaria mosquito Anopheles messeae s.l. to the insecticide malathion

Resistance to agricultural pesticides is an important and insufficiently studied concern for pest and disease vector research. We determined the malathion resistance of species in the Anopheles maculipennis mosquito group in a habitat near Novosibirsk, Russia. Most of the 851 individuals we measured were members of the Anopheles messeae s.l. complex (An. messeae and An. daciae species). The LC₅₀ value for malathion was 0.052 mg/L for the mixed specimens, and we failed to find any differences between species. The LC₅₀ value was within the range of values for malathion resistance of Anopheles stephensi and Culex quinquefasciatus. As the main resistance mechanism to organophosphate and carbamate insecticides is a single mononucleotide substitution in the ace‐1 gene, we searched for this mutation in An. messeae s.l. and An. beklemishevi by restriction analysis. This mutation was not found in 347 of the specimens. We sequenced the ace‐1 gene fragment for 24 specimens from four species of the Anopheles maculipennis group, including An. messeae, An. daciae, An. atroparvus, and An. beklemishevi. These specimens harbored a nucleotide substitution in the triplet where a mutation can lead to insecticide resistance, but this substitution would make it difficult for the resistance to develop. Since the studied specimens belong to branches of the Palearctic portion of the Anopheles maculipennis group, we suspect that all other Palearctic species of this group would have difficulties harboring the ace‐1 mutation that would lead to organophosphate and carbamate resistance.     

The utility of internally transcribed spacer 2 DNA sequences of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma

The usefulness of the internally transcribed spacer 2 (ITS2) of the nuclear ribosomal gene complex is tested for providing taxonomic characters to identify Trichogramma species. The ITS2 sequences of a group of sibling species of the T. deion/T. pretiosum complexes were determined. A simple and precise identification key to the species of these assemblages was constructed using as taxonomic characters the size of the ITS2 and the difference in restriction length polymorphism of species with similarly sized ITS2. Individual wasps can be identified by amplification of their ITS2 with general primers, determining the size of the PCR product using standard agarose electrophoresis, followed in some species by a DNA-digestion with a restriction enzyme. Because this system works well for a number of closely related species we are hopeful that similar PCR-based identification can be extended to all species of the genus once their ITS2 sequences have been determined. The advantage of this identification system over the morphology-based system is that non-specialists are able to quickly and cheaply identify individual specimens. In addition, species specific primers were tested for the two most common species of these groups (i.e. T. pretiosum and T. deion). These primers can be used either as a direct identification tool or as a method to confirm the identification using the general key. The phylogeny of this group of wasps was also analyzed based on the ITS2 sequence.     

Systemic reorganization of the architectonics of polytene chromosomes in the onto- and phylogenesis of malaria mosquitoes

Cardinal differences in the spatial organization of polytene chromosomes in the nuclei of salivary gland cells (malphigian tubules) and nurse ovarian cells in malarial mosquitoes of Anopheles maculipennis complex are found. The chromosomes have typical chromocentric organization in salivary gland cells and malphigian tubules. In contrast to these cells, the trophocytes have no single chromocentre and some of their chromosomes are rigidly attached to the nucleus envelope with the centromeric parts or with the particular loci. The loci-specificity of the X-chromosome intercalary parts' attachment to the nucleus envelope is found in Anopheles messeae.     

Molecular comparison of topotypic specimens confirms Anopheles (Nyssorhynchus) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.     

Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.     

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.