Observations on sporozoite detection in naturally infected sibling species of the <Emphasis Type="Italic">Anopheles culicifacies</Emphasis> complex and variant of <Emphasis Type="Italic">Anopheles stephensi</Emphasis> in India

Sporozoites were detected in naturally infected sibling species of the primary rural vector Anopheles culicifacies complex in two primary health centres (PHCs) and a variant of the urban vector Anopheles stephensi in Mangalore city, Karnataka, south India while carrying out malaria outbreak investigations from 1998–2006. Sibling species of An. culicifacies were identified based on the banding patterns on ovarian polytene chromosomes, and variants of An. stephensi were identified based on the number of ridges on the egg floats. Sporozoites were detected in the salivary glands by the dissection method. Of the total 334 salivary glands of An. culicifacies dissected, 17 (5.08%) were found to be positive for sporozoites. Of the 17 positive samples, 11 were suitable for sibling species analysis; 10 were species A (an efficient vector) and 1 was species B (a poor vector). Out of 46 An. stephensi dissected, one was sporozoite positive and belonged to the type form (an efficient vector). In malaria epidemiology this observation is useful for planning an effective vector control programme, because each sibling species/variant differs in host specificity, susceptibility to malarial parasites, breeding habitats and response to insecticides.     

Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species

Abstract A polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north‐eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR‐RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980 ), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR‐RFLP assay can detect species not included in the initial set‐up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non‐vector anophelines in South‐east Asia.     

Isolation and characterization of microsatellite markers from malaria vector,Anopheles culicifacies

Anopheles culicifacies, an important vector in the Indian subcontinent is a complex of five sibling species of which four are vectors. We describe the isolation of 31 microsatellite markers from the recently recognized isomorphic species A of which 13 were characterized in sympatric populations of Anopheles culicifacies isomorphic species A and B. The allele frequencies ranges from two to 12 in species A and two to seven in species B. Species A being a vector, and that these markers can be used in closely related species, makes the isolation of these markers important to study population structure of all sibling species in this complex.     

Anopheles culicifacies Y-chromosome dimorphism indicates sibling species (B and E) with different malaria vector potential in Sri Lanka

In Sri Lanka, malaria is transmitted mainly by Anopheles culicifacies Giles sensu lato (Diptera: Culicidae). In India, this nominal taxon comprises sibling species A, B, C, D and E, distinguished by their chromosome morphology. Species B (identified by polytene chromosome sequence Xab, 2g1 + h1) is not such an efficient vector of malaria as other members of the An. culicifacies complex in India. All specimens of An. culicifacies s.l. examined from Sri Lanka possess Xab, 2g1 + h1 polytenes, previously interpreted as species B, despite their important vector status. Recently, species E was described from Rameshwaram Island (Tamil Nadu, India) between Sri Lanka and the Indian mainland, where both species B and E are sympatric. Species B and E share polytene sequence Xab, 2g1 + h1 but differ by the mitotic Y-chromosome being acrocentric in species B, submetacentric in species E, the latter implicated as vector of vivax malaria. From May 1999 to January 2000, we surveyed Y-chromosomes of male progeny from An. culicifacies Xab, 2g1 + h1 females collected from cattle bait in diverse malarious districts of Sri Lanka: Badulla, Monaragala, Puttalam and Trincomalee. Karyotypes of readable quality were obtained from 42/83 families examined, with overall proportions 24% acrocentric and 76% submetacentric Y-chromosome carriers, both types being sympatric in at least 3/4 localities sampled. By analogy with the situation on Rameshwaram Island, we interpret these observations to demonstrate widespread presence of two members of the An. culicifacies complex in Sri Lanka, their karyotypes being compatible with species B and E, the latter predominant and having greater vector potential.     

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species

Background: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. Objectives: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. Methodology: In achieving these objectives, twenty two ITS2 sequences (~370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. Results and discussions: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Conclusions: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.     

Relative Abundance and Plasmodium Infection Rates of Malaria Vectors in and around Jabalpur,a Malaria Endemic Region in Madhya Pradesh State,Central India

BackgroundThis study was undertaken in two Primary Health Centers (PHCs) of malaria endemic district Jabalpur in Madhya Pradesh (Central India).MethodsIn this study we had investigated the relative frequencies of the different anopheline species collected within the study areas by using indoor resting catches, CDC light trap and human landing methods. Sibling species of malaria vectors were identified by cytogenetic and molecular techniques. The role of each vector and its sibling species in the transmission of the different Plasmodium species was ascertained by using sporozoite ELISA.ResultsA total of 52,857 specimens comprising of 17 anopheline species were collected by three different methods (39,964 by indoor resting collections, 1059 by human landing and 11,834 by CDC light trap). Anopheles culicifacies was most predominant species in all collections (55, 71 and 32% in indoor resting, human landing and light trap collections respectively) followed by An. subpictus and An. annularis. All five sibling species of An. culicifacies viz. species A, B, C, D and E were found while only species T and S of An. fluviatilis were collected. The overall sporozoite rate in An. culicifacies and An. fluviatilis were 0.42% (0.25% for P. falciparum and 0.17% for P. vivax) and 0.90% (0.45% for P. falciparum and 0.45% for P. vivax) respectively. An. culicifacies and An. fluviatilis were found harbouring both P. vivax variants VK-210 and VK-247, and P. falciparum. An. culicifacies sibling species C and D were incriminated as vectors during most part of the year while sibling species T of An. fluviatilis was identified as potential vector in monsoon and post monsoon season.ConclusionsAn. culicifacies species C (59%) was the most abundant species followed by An. culicifacies D (24%), B (8.7%), E (6.7%) and A (1.5%). Among An. fluviatilis sibling species, species T was common (99%) and only few specimens of S were found. Our study provides crucial information on the prevalence of An. culicifacies and An. fluviatilis sibling species and their potential in malaria transmission which will assist in developing strategic control measures against these vectors.     

On the conspecificity ofAnopheles fluviatilis species S withAnopheles minimus species C

Anopheles fluviatilis andAn. minimus complexes, each comprising of at least three sibling species, are closely related and important malaria vectors in Oriental Region. RecentlyAn. fluviatilis species S, which is a highly efficient malaria vector in India, has been made conspecific withAn. minimus species C (senior synonym) on the basis of homology in 335 base pair nucleotide sequence of D3 domain of 28S ribosomal DNA(rDNA). We examined the conspecificity of these two nominal species by obtaining and analysing the DNA sequences of nuclear ribosomal loci internal transcribed spacer 2 (ITS2) and D2-D3 domain of 28S rDNA (28S-D2/D3) from those ofAn. fluviatilis S andAn. minimus C. We found that the sequences ofAn. fluviatilis S are appreciably different from those ofAn. minimus C with pair-wise distance (Kimura-2-parametre model) of 3.6 and 0.7% for loci ITS2 and 28S-D2/D3, respectively. Pair-wise distance and phylogenetic analyses using ITS2 sequences of members of Minimus and Fluviatilis Complexes revealedthat An. fluviatilis S is distantly related toAn. minimus C as compared to any other members of the Fluviatilis Complex. These findings suggest that the two nominal species,An. fluviatilis S andAn. minimus C, do not merit synonymy. The study also confirms that the reported speciesAn. fluviatilis X is synonym with species S.     

Transcriptional analysis of an immune-responsive serine protease from Indian malarial vector, <Emphasis Type="Italic">Anopheles culicifacies</Emphasis>

Background  

The main vector for transmission of malaria in India is the Anopheles culicifacies mosquito species, a naturally selected subgroup of which is completely refractory (R) to transmission of the malaria parasite, Plasmodium vivax;     

Disappearance of malaria vector Anopheles sundaicus from Chilika Lake area of Orissa State in India

Malaria has declined around Chilika Lake (85°20′ E, 19°40′ N) in Orissa State, India, from hyperendemicity in the 1930s to hypoendemicity during recent decades. Six decades ago, 21 spp. of Anopheles mosquitoes (Diptera: Culicidae) were recorded from this area, including the well known Indian malaria vectors An. culicifacies Giles, An. fluviatilis James, An. maculatus Theobald, An. stephensi Liston and An. sundaicus (Rodenwaldt), the last formerly regarded as the main vector locally. Surveys of Chilika area during 1995–96 found 8 spp. of culicine plus 14 spp. of anopheline mosquitoes, the latter comprising An. subpictus Grassi sensu lato, An. hyrcanus (Pallas) s.l., An. vagus Dönitz, An. annularis van der Wulp s.l., An. culicifacies Giles s.l., An. aconitus Dönitz, An. varuna Iyengar, An. barbirostris van der Wulp s.l., An. philippinensis Ludlow, An. ramsayi Covell, An. jeyporiensis James, An. pallidus Theobald, An. tessellatus Theobald and An. karwari James in decreasing order of abundance. Among indoor‐resting female mosquitoes, the anthropophilic index was 4–7% and some species (An. culicifacies, An. subpictus, An. vagus) tended to enter houses for resting after blood‐feeding outside. Females of potentially infective age (three‐parous) were obtained for An. culicifacies (11%) and An. annularis (< 2%), the more abundant established vector in this coastal area, but not for small samples of An. subpictus and An. vagus. Anophelines reported previously but not found in our survey were An. fluviatilis, An. jamesii Theobald, A. maculatus, An. splendidus Koidzumi, An. stephensi, An. theobaldi Giles and the former main vector An. sundaicus.     

<Emphasis Type="Italic">Eriocaulon epedunculatum</Emphasis>, a new species of <Emphasis Type="Italic">Eriocaulaceae</Emphasis> from the Western Ghats,India

Summary  A new species of Eriocaulon, E. epedunculatum (Eriocaulaceae) is described and illustrated from the Western Ghats, India.     

Anopheline species and their Plasmodium infection status in Aligarh,India

Malaria is a global issue and India contributes substantially to global malaria incidence. Information related to malaria vectors is very limited in Aligarh. The environmental and climatological situations permit the continual breeding of vectors in permanent breeding sites. This study was designed with the aim to screen all the anophelines species and possible malaria vectors in three different localities of Aligarh. Anopheles mosquitoes were collected from three different localities (Fort, Jalali and Tappal) during peak malaria transmission season (July to November) by using mouth aspirator and CDC light traps. Enzyme-linked immunosorbent assay (ELISA) was done to detect Plasmodium falciparum, Plasmodium vivax-210 and P. vivax-247 circumsporozoite proteins (CSP) from the collected female species. A total of 794 female anopheline mosquitoes belonging to 7 species were collected by different methods. Circumsporozoite protein–enzyme-linked immunosorbent assay was performed with 780 anopheline mosquitoes out of which 13 mosquitoes were positive in CSP–ELISA. Thus, the overall infection rate was 1.66% (13/780). Four (0.51%) mosquitoes belonging to three species were positive for P. falciparum, 7 (0.89%) mosquitoes belonging to three species were positive for VK 210 and 2 (0.25%) mosquitoes belonging to Anopheles culicifacies and Anopheles stephensi species were positive for VK 247. No mixed infection was found in this study. According to species, the highest infection rate was observed in An. culicifacies (7/288, 2.43%) followed by An. stephensi (2.40%) and Anopheles annularis (1.98%). An. culicifacies and An. stephensi were previously incriminated as malaria vectors in Aligarh. There was, however, no previous report in favor of infections in An. annularis in Aligarh. The on-going Malaria Control Program in India needs up to date information on malaria vectors. A major challenge is the lack of knowledge about vectors and their role in malaria transmission. Findings of this study suggested that in the absence of major malaria vectors there is a possibility that other Anopheles species may have been playing a role in malaria transmission in Aligarh.     

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).     

Pseudobithynia guldeni sp. n., a new gastropod species from the Mediterranean region of Turkey (Gastropoda: Truncatelloidea)

A new Bithyniidae species Pseudobithynia guldeni sp.n. is described from the Mediter-ranean region of Turkey based on shell and male genitalia morphology. An identification key for the species of Pseudobithynia of Turkey is given.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:FA659D77-5B02-4C2F-9A6D-D61E676778E1     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

<Emphasis Type="Italic">Colletotrichum echinochloae</Emphasis>, a new species on Japanese barnyard millet (<Emphasis Type="Italic">Echinochloa utilis</Emphasis>)

Five isolates of a species of Colletotrichum were collected from Japanese barnyard millet (Echinochloa utilis) in Japan. Although the fungus had once been identi-fied as C. graminicola sensu lato, it was clearly different from C. graminicola isolated from maize (Zea mays) in its falcate and short conidia, 18.0–22.2 μm in length, cultural characteristics, and specific pathogenicity to E. utilis. Moreover, molecular phylogenetic analyses using sequences of rDNA-ITS, HMG, and SOD2 indicated a monophyly of the isolates. A new species, Colletotrichum echinochloae, is then proposed based on the morphological, pathological, and molecular characteristics.     

<Emphasis Type="Italic">Kazachstania wufongensis</Emphasis> sp. nov., an ascosporogenous yeast isolated from soil in Taiwan

A new yeast species, Kazachstania wufongensis, is proposed in this paper based on six strains isolated from soil in Taiwan. The species may produce one to four ellipsoidal ascospores in each ascus, directly transformed from diploid cells. Genus assignment and distinction of the species from other recognized species of Kazachstania is based on morphological and physiological characteristics, and on phylogenetic analysis of nucleotide sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene. Sequence analysis of the D1/D2 domains of the LSU rRNA gene reveals that K. wufongensis is a member of the Kazachstania exigua complex, and its phylogenetically closest relatives are K. exigua, K. barnettii, K. bulderi, and K. turicensis. The species can be further differentiated from the other phylogenetically related species based on internal transcribed spacer sequence and electrophoretic karyotype. Therefore, the new species Kazachstania wufongensis sp. nov. is proposed. The type strain of this new species, which was isolated from forest soil in Wufong, Hsinchu, Taiwan, is FN21S03^T (=CBS 10886^T = BCRC 23138^T).     

Two new species of Apsilochorema Ulmer, 1907 (Trichoptera: Hydrobiosidae) from India

Two new species Apsilochorema (Apsilochorema) shalimarensis sp. n. and Apsilochorema (Apsilochorema) sainii sp. n. are described and illustrated from India. With these new additions, the genus Apsilochorema Ulmer, 1907 is now represented by 14 species from India.http://www.zoobank.org/urn:lsid:zoobank.org:pub:AAA8C469-850E-4778-BCF1-3FF5B429E559     

<Emphasis Type="Italic">Candida patagonica</Emphasis> sp. nov., a new species of yeast from cellar surfaces

A novel anamorphic yeast species belonging to the genus Candida has been isolated from cellar surfaces in North Patagonia. Morphological and physiological observation and phylogenetic analysis were performed. Pseudomycelium was plentifully produced. No sexual reproduction was observed. From sequence analysis of the 26S rDNA D1/D2 region, Candida bituminiphila and Zygoascus hellenicus were the closest species with 40 and 79 bp substitutions, respectively. C. bituminiphila differed physiologically from the novel species in its ability to assimilate sucrose and erythritol, in not fermenting any sugars, in growing without some vitamin compounds, and in growing at 40°C. All these data support the hypothesis that the new yeast, named Candida patagonica, is a novel species related to C. bituminiphila. The type strain is UNCOMA 159.5 (= CECT 12029 = CBS 10443).     

Phylogeny and taxonomy of powdery mildew fungi of Erysiphe sect. Uncinula on Carpinus species

A phylogenetic analysis of the Erysiphe with uncinuloid ascoma appendages (Erysiphe section Uncinula, Erysiphales, Ascomycota) on Carpinus spp. was done using sequences of the rDNA ITS regions and the D1/D2 domains of the 28S rDNA. These results, combined with morphological data, revealed a complex consisting of several distinct taxa. These included the already described Erysiphe carpinicola on C. japonica distinguishable from the Erysiphe sp. on C. betulus and C. tschonoskii as well as the one on C. laxiflora. Thus, it was shown that Oidium carpini, described from Europe on Carpinus betulus, the powdery mildew with uncinula-like ascomata, recently found in Europe on this host, as well as an Erysiphe on C. tschonoskii in Japan, described previously as E. carpinicola, all belong to a single new species, named E. arcuata in this paper. As the powdery mildew on C. laxiflora was also distinct from other known species, it is named E. carpini-laxiflorae in this paper. The already described E. pseudocarpinicola and Erysiphe sp. on Carpinus cordata are two additional taxa, which are morphologically and genetically distinguished from the other species of Erysiphe sect. Uncinula on Carpinus spp.