First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus,important Palaearctic vectors of bluetongue virus

Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.     

Isolation and Characterization of Arsenic-Resistant Bacteria from Contaminated Water-Bodies in West Bengal,India

Bengal Basin is known for severe arsenic contamination. In the present study, we have isolated six bacteria from the arsenic contaminated surface water of Bengal Basin. 16S rDNA sequence analysis identified them as Microbacterium oleivorans, Acinetobacter soli, Acinetobacter venetianus, Acinetobacter junii, Acinetobacter baumannii, Acinetobacter calcoaceticus. All the isolates possess arsenic accumulation potential and high molecular weight plasmid (>10 kb). PCR amplification indicated the presence of arsenic-resistance genes (arsB and aoxB) either in the genome or plasmid or in both in the isolated bacteria (except in Acinetobacter venetianus). Exposure to arsenic affected bacterial growth and induced alteration in cytoplasmic membrane integrity.     

Comparative analysis of midgut bacterial communities in three aedine mosquito species from dengue‐endemic and non‐endemic areas of Rajasthan,India

Dengue viruses are transmitted to humans through the bites of infected female aedine mosquitoes. Differences in the composition and structure of bacterial communities in the midguts of mosquitoes may affect the vector's ability to transmit the disease. To investigate and analyse the role of midgut bacterial communities in viral transmission, midgut bacteria from three species, namely Stegomyia aegypti (= Aedes aegypti), Fredwardsius vittatus (= Aedes vittatus) and Stegomyia albopicta (= Aedes albopictus) (all: Diptera: Culicidae), from dengue‐endemic and non‐endemic areas of Rajasthan, India were compared. Construction and analyses of six 16S rRNA gene libraries indicated that Serratia spp.‐related phylotypes dominated all clone libraries of the three mosquito species from areas in which dengue is not endemic. In dengue‐endemic areas, phylotypes related to Aeromonas, Enhydrobacter spp. and uncultivated bacterium dominated the clone libraries of S. aegypti, F. vittatus and S. albopicta, respectively. Diversity indices analysis and real‐time TaqMan polymerase chain reaction assays showed bacterial diversity and abundance in the midguts of S. aegypti to be higher than in the other two species. Significant differences observed among midgut bacterial communities of the three mosquito species from areas in which dengue is and is not endemic, respectively, may be related to the vectorial capacity of mosquitoes to carry dengue viruses and, hence, to the prevalence of disease in some areas.     

Culicoides aspirated from cattle in Costa Rica, Honduras, Panama and Puerto Rico, and their role as potential vectors of bluetongue viruses

Abstract. In 1991, as part of an epidemiological study of bluetongue viruses (BTV) in the Central American and Caribbean region, eight farms located in Costa Rica, Honduras, Panama and Puerto Rico were sampled for Culicoides spp. attacking cattle. Using cattle bait, 3884 biting midges were collected with an electric aspirator during both crepuscular periods. The predominant species captured was Culicoides insignis Lutz (95%), followed by C.furens (Poey) (3.4%), C.filarifer Hoffman/ C.ocumarensis Ortiz (0.9%), C.lahillei (Iches) (0.7%), C.arubae Fox and Hoffman (<0.1%) and C.gorgasi Wirth and Blanton (<0.1%). Blood-engorged specimens from some of these species were collected and comprised: 18% of all C.insignis , 36% of C.furens , 37% of filarifer/ocumarensis and 25% of C.lahillei. No engorged C.arubae or C.gorgasi were caught. These results confirm earlier findings pointing to C.insignis, C.furens and C.filarifer/ ocumarensis as potential vectors of BTV in the region.     

Gut microbiomes of free‐ranging and captive Namibian cheetahs: Diversity,putative functions and occurrence of potential pathogens

Although the significance of the gut microbiome for host health is well acknowledged, the impact of host traits and environmental factors on the interindividual variation of gut microbiomes of wildlife species is not well understood. Such information is essential; however, as changes in the composition of these microbial communities beyond the natural range might cause dysbiosis leading to increased susceptibility to infections. We examined the potential influence of sex, age, genetic relatedness, spatial tactics and the environment on the natural range of the gut microbiome diversity in free‐ranging Namibian cheetahs (Acinonyx jubatus). We further explored the impact of an altered diet and frequent contact with roaming dogs and cats on the occurrence of potential bacterial pathogens by comparing free‐ranging and captive individuals living under the same climatic conditions. Abundance patterns of particular bacterial genera differed between the sexes, and bacterial diversity and richness were higher in older (>3.5 years) than in younger individuals. In contrast, male spatial tactics, which probably influence host exposure to environmental bacteria, had no discernible effect on the gut microbiome. The profound resemblance of the gut microbiome of kin in contrast to nonkin suggests a predominant role of genetics in shaping bacterial community characteristics and functional similarities. We also detected various Operational Taxonomic Units (OTUs) assigned to potential pathogenic bacteria known to cause diseases in humans and wildlife species, such as Helicobacter spp., and Clostridium perfringens. Captive individuals did not differ in their microbial alpha diversity but exhibited higher abundances of OTUs related to potential pathogenic bacteria and shifts in disease‐associated functional pathways. Our study emphasizes the need to integrate ecological, genetic and pathogenic aspects to improve our comprehension of the main drivers of natural variation and shifts in gut microbial communities possibly affecting host health. This knowledge is essential for in situ and ex situ conservation management.     

Isolation and characterization of sulphate-reducing bacteria <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> from Vajreshwari thermal springs in Maharashtra,India

Sulphate-reducing bacteria (SRB) in the thermal springs of Vajreshwari were investigated with combined microbiological and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 25 to 55 °C and could use pyruvate, lactate and ethanol as electron donors. Desulfoviridin was detected in all the isolates. The partial 16S rRNA and dissimilatory sulphite reductase (DSR) gene sequences of five representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris.     

First molecular identification of the vertebrate hosts of Culicoides imicola in Europe and a review of its blood‐feeding patterns worldwide: implications for the transmission of bluetongue disease and African horse sickness

Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens that affect wildlife, livestock and, occasionally, humans. Culicoides imicola (Kieffer, 1913) is considered to be the main vector of the pathogens that cause bluetongue disease (BT) and African horse sickness (AHS) in southern Europe. The study of blood‐feeding patterns in Culicoides is an essential step towards understanding the epidemiology of these pathogens. Molecular tools that increase the accuracy and sensitivity of traditional methods have been developed to identify the hosts of potential insect vectors. However, to the present group's knowledge, molecular studies that identify the hosts of C. imicola in Europe are lacking. The present study genetically characterizes the barcoding region of C. imicola trapped on farms in southern Spain and identifies its vertebrate hosts in the area. The report also reviews available information on the blood‐feeding patterns of C. imicola worldwide. Culicoides imicola from Spain feed on blood of six mammals that include species known to be hosts of the BT and AHS viruses. This study provides evidence of the importance of livestock as sources of bloodmeals for C. imicola and the relevance of this species in the transmission of BT and AHS viruses in Europe.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Molecular phylogeny of the higher taxa of Odonata (Insecta) inferred from COI, 16S rRNA, 28S rRNA,and EF1‐α sequences

In this study, we sequenced both two mitochondrial genes (COI and 16S rRNA) and nuclear genes (28S rRNA and elongation factor‐1α) from 71 species of Odonata that represent 7 superfamilies in 3 suborders. Phylogenetic testing for each two concatenated gene sequences based on function (ribosomal vs protein‐coding genes) and origin (mitochondrial vs nuclear genes) proved limited resolution. Thus, four concatenated sequences were utilized to test the previous phylogenetic hypotheses of higher taxa of Odonata via Bayesian inference (BI) and maximum likelihood (ML) algorithms, along with the data partition by the BI method. As a result, three slightly different topologies were obtained, but the BI tree without partition was slightly better supported by the topological test. This topology supported the suborders Anisoptera and Zygoptera each being a monophyly, and the close relationship of Anisozygoptera to Anisoptera. All the families represented by multiple taxa in both Anisoptera and Zygoptera were consistently revealed to each be a monophyly with the highest nodal support. Unlike consistent and robust familial relationships in Zygoptera those of Anisoptera were partially unresolved, presenting the following relationships: ((((Libellulidae + Corduliidae) + Macromiidae) + Gomphidae + Aeshnidae) + Anisozygoptera) + (((Coenagrionidae + Platycnemdidae) + Calopterygidae) + Lestidae). The subfamily Sympetrinae, represented by three genera in the anisopteran family Libellulidae, was not monophyletic, dividing Crocothemis and Deielia in one group together with other subfamilies and Sympetrum in another independent group.     

Morphological and molecular description of new species of squat lobster (Crustacea: Decapoda: Galatheidae) from the Solomon and Fiji Islands (South‐West Pacific)

The family Galatheidae is among the most diverse families of anomuran decapod crustaceans, and the South‐West Pacific is a biodiversity hot spot for these squat lobsters. Attempts to clarify the taxonomic and evolutionary relationships of the Galatheidae on the basis of morphological and molecular data have revealed the existence of several cryptic species, differentiated only by subtle morphological characters. Despite these efforts, however, relationships among genera are poorly understood, and the family is in need of a detailed systematic review. In this study, we assess material collected in different surveys conducted in the Solomon Islands, as well as comparative material from the Fiji Islands, by examining both the morphology of the specimens and two mitochondrial markers (cytochrome oxidase subunit I, COI, and 16S rRNA). These two sources of data revealed the existence of eight new species of squat lobster, four of which were ascribed to the genus Munida, two to the genus Paramunida, one to the genus Plesionida, and the last species was ascribed to the genus Agononida. These eight species are described along with phylogenetic relationships at the genus level. Our findings support the taxonomic status of the new species, yet the phylogenetic relationships are not yet fully resolved. Further molecular analysis of a larger data set of species, and more conserved genes, will help clarify the systematics of this group. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 465–493.     

内蒙古原始森林桦树皮内生和表生乳酸菌分离鉴定及其特性

[背景]随着乳酸菌产业的快速发展,不同环境乳酸菌群落结构的分析和菌种资源的开发利用已经成为该领域的研究热点.从不同环境、不同来源分离、保藏乳酸菌对于优良菌种的筛选和开发利用具有重要意义.[目的]阐明内蒙古原始森林桦树皮中乳酸菌的组成,并获得丰富的野生型乳酸菌资源,为优良菌株的筛选和工业化应用提供核心菌株,从原始森林桦树...     

Phosphate solubilisation and plant growth promoting potential by stress tolerant Bacillus sp. isolated from rhizosphere of apple orchards in trans Himalayan region of Himachal Pradesh

Phosphate‐solubilising ability and co‐production of plant growth promoting traits of stress tolerant Bacillus subtilis CB₈A isolated from apple rhizosphere was tested under in vitro conditions against a wide range of temperature (30–45°C), pH (7–9) and salt (0–5%) stresses. Under the extremes of temperature (45°C), pH‐9 and salt concentration (5%), production of soluble phosphate, indole acetic acid, siderophore and antifungal activity against Dematophora necatrix were reduced by 71.09%, 75.29%, 90.3% and 88.47%, respectively. Per cent decrease in P‐solubilisation at extreme temperature (45°C) and normal pH (7) without salt concentration was 36.23%; at extreme pH (9) and normal temperature (37°C) without salt concentration was 23.45% and at extreme salt concentration (5%), optimum temperature (37°C) and pH (7) was 36.7%. P‐solubilisation by CB₈A was inversely correlated with pH (r = ?0.78) and positively correlated with siderophore production (r = 0.81), indole acetic acid (r = 0.58) and antifungal activity (r = 0.63). Gluconic acid (1.43%) and citric acid (0.67%) were detected as major organic acids. P‐solubilisation and nitrogen fixing abilities of B. subtilis CB₈A were confirmed by amplification of gdh and nifH genes. The ability of CB8A showing plant growth promoting rhizobacteria (PGPR) traits at a wide range of temperature, pH and varying salt concentration can be exploited for developing multifunctional biofertiliser in apple orchards.     

Diversity of spore‐forming bacteria in cattle manure,slaughterhouse waste and samples from biogas plants

Aims: As biowaste intended for biogas production can contain pathogenic micro‐organisms, the recommended treatment is pasteurization at 70°C for 60 min. This reduces pathogens such as Salmonella spp., whereas spore‐forming bacteria (Bacillus spp. and Clostridium spp.) survive. Most spore‐forming bacteria are harmless, but some can cause diseases such as blackleg, botulism and anthrax. In this study, the effect of the biogas process on Bacillus spp. and Clostridium spp. was investigated. Methods and Results: We analysed 97 faecal samples, 20 slaughterhouse waste samples and 60 samples collected at different stages in the biogas process. Bacillus spp. and Clostridium spp. were quantified and subcultured. The isolates were identified by biochemical methods and by 16S rRNA gene sequencing. Phylogenetic trees were constructed from the sequences obtained from isolates from the samples. Clostridium botulinum/Clostridium spp. and Clostridium sordellii were found both before and after pasteurization, but not after digestion (AD). Some of the isolated strains probably represented new members of the genera Clostridium and Bacillus. Conclusion: After digestion, the numbers of clostridia decreased, but none of the pathogenic bacteria did, whereas Bacillus spp. remained constant during the process. Significance and Impact of the Study: Biogas is gaining in importance as an energy source and because the residues are used as fertilizers, we needed to study the prevalence of pathogenic bacteria in such material.     

Climate change and the spread of vector‐borne diseases: using approximate Bayesian computation to compare invasion scenarios for the bluetongue virus vector Culicoides imicola in Italy

Bluetongue (BT) is a commonly cited example of a disease with a distribution believed to have recently expanded in response to global warming. The BT virus is transmitted to ruminants by biting midges of the genus Culicoides, and it has been hypothesized that the emergence of BT in Mediterranean Europe during the last two decades is a consequence of the recent colonization of the region by Culicoides imicola and linked to climate change. To better understand the mechanism responsible for the northward spread of BT, we tested the hypothesis of a recent colonization of Italy by C. imicola, by obtaining samples from more than 60 localities across Italy, Corsica, Southern France, and Northern Africa (the hypothesized source point for the recent invasion of C. imicola), and by genotyping them with 10 newly identified microsatellite loci. The patterns of genetic variation within and among the sampled populations were characterized and used in a rigorous approximate Bayesian computation framework to compare three competing historical hypotheses related to the arrival and establishment of C. imicola in Italy. The hypothesis of an ancient presence of the insect vector was strongly favoured by this analysis, with an associated P ≥ 99%, suggesting that causes other than the northward range expansion of C. imicola may have supported the emergence of BT in southern Europe. Overall, this study illustrates the potential of molecular genetic markers for exploring the assumed link between climate change and the spread of diseases.     

Promising Biological Indicator of Heavy Metal Pollution: Bioluminescent Bacterial Strains Isolated and Characterized from Marine Niches of Goa,India

In present study, several marine water samples collected from the North Goa Beaches, India for isolation of luminescent bacterial species. Isolates obtained labelled as DP1-5 and AB1-6. Molecular characterization including identification of a microbial culture using 16S rRNA gene based molecular technique and phylogenetic analysis confirmed that DP3 & AB1 isolates were Vibrio harveyi. All of the isolates demonstrated multiple metal resistances in terms of growth, with altered luminescence with variable metal concentration. Present investigations were an attempt towards exploring and reporting an updated diversity of bioluminescent bacterial species from various sites around the Goa, India which would be explored in future for constructing luminescence based biosensor for efficiently monitoring the level of hazardous metals in the environment.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0531-y) contains supplementary material, which is available to authorized users.     

Assessment of microbial diversity in the rhizosphere of Pinus roxburghii (Sarg.) and bio‐inoculant potential of selected pine bacterial isolates for wheat varieties based on cultureindependent and culture‐dependent techniques

• Evidence is lacking regarding compatibility of pine bacteria as bio‐inoculants for crops. The diversity and abundance of rhizosphere bacteria of Pinus roxburghii has never been investigated with simultaneous application of culture‐dependent and culture‐independent techniques. The present study was aimed to isolate, characterise, check the bio‐inoculant potential of pine bacteria and assess rhizosphere bacterial diversity using culture‐independent advanced approaches.
• Forty bacteria isolated from the rhizoplane of P. roxburghii growing in a cold climate at high altitude in Murree, were morphologically characterised; nine were identified by 16S rRNA sequence analyses and used in experiments. Diversity and abundance of the 16S rRNA gene and nif H gene in the rhizosphere was assessed by cloning, RFLP analysis, 454‐amplicon pyrosequencing and qPCR.
• The bacterial isolates significantly improved dry weight of shoot, root, root area, IAA and GA₃ content, number of grains plant^?1, weight of grains plant^?1 in wheat varieties Chakwal‐50 and Fareed‐06 under axenic and field conditions. The number of 16S rRNA sequences (2979) identified by pyrosequencing shared similarity with 13 phyla of bacteria and archaea.
• The results confirm the existence of diverse bacteria of agricultural and industrial importance in the rhizosphere and compatibility of rhizoplane bacteria as bio‐inoculants for wheat varieties.