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The Indian isolate of Bombyx mori bidensovirus (BmBDV) is a bipartite virus that comprises of a segmented, non-homologous, two linear single-strands of DNA molecules (VD1 and VD2). It is one of the causative agents of the fatal silkworm disease ‘Flacherie’ that causes severe crop loss for the sericulture farmers. Genome analyses of the Indian isolate of BmBDV revealed that it consists of 6 putative ORFs similar to the Japanese and Chinese isolates. VD1 consists of 4 ORFs while VD2 has 2 ORFs that code for 4 non- structural (NS) and 2 structural (VP) proteins, in total. In this study, we investigated, in detail, the impact of BmBDV pathogenesis on growth and development of the silkworm Bombyx mori, at different developmental stages. Mortality rate and weight uptake analyses were also performed on newly ecdysed 4th instar larvae. BmBDV infection was not found to be developmental stage specific and it occurred at all stages. Onset of mortality took place 8 days post infection (dpi) and 100% mortality occurred at 11 dpi. The infected larvae showed a significant difference in weight uptake wherein from 7 dpi the larvae stopped gaining weight and from 8th dpi started demonstrating the typical symptoms of flacherie. Further, the expression pattern of the 6 viral ORFs were also investigated in the newly ecdysed 4th instar BmBDV infected silkworms. Among all the six ORFs, VD2 ORF 1 and 2 revealed the highest transcript numbers, which was followed by VD1 ORF 4 that encodes for the viral DNA polymerase enzyme. This was the first ever attempt to understand the pathogenesis and the expression pattern of all the six ORF transcripts of the Indian isolate of BmBDV.

There is increasing evidence that honeybees (Apis mellifera L.) can adapt naturally to survive Varroa destructor, the primary cause of colony mortality world-wide. Most of the adaptive traits of naturally varroa-surviving honeybees concern varroa reproduction. Here we investigate whether factors in the honeybee metagenome also contribute to this survival. The quantitative and qualitative composition of the bacterial and viral metagenome fluctuated greatly during the active season, but with little overall difference between varroa-surviving and varroa-susceptible colonies. The main exceptions were Bartonella apis and sacbrood virus, particularly during early spring and autumn. Bombella apis was also strongly associated with early and late season, though equally for all colonies. All three affect colony protein management and metabolism. Lake Sinai virus was more abundant in varroa-surviving colonies during the summer. Lake Sinai virus and deformed wing virus also showed a tendency towards seasonal genetic change, but without any distinction between varroa-surviving and varroa-susceptible colonies. Whether the changes in these taxa contribute to survival or reflect demographic differences between the colonies (or both) remains unclear.

The temperate oak tasar silkworm, Antheraea proylei, is frequently infested with Antheraea proylei nucleopolyhedrovirus (AnprNPV) causing tiger band disease. This disease is one of the key factors that obstructs production and productivity of oak tasar sericulture. The current study aimed to investigate the pathogenicity of AnprNPV, its mode of transmission, and detection of AnprNPV in different tissues. Transmission electron micrographs of AnprNPV showed single rod-shaped bodies and occlusion derived virus (ODV) enclosed within multiple envelopes. The infecting AnprNPV displayed tissue tropism with higher copy numbers detected in the insect fat body and ovary. The virus was observed to multiply in all developmental stages of the silkworm such as egg, larva, pupa, and moth, confirming its ability to spread throughout the silkworm lifecycle. Baculovirus isolated from infected A. proylei showed cross-infectivity in other Saturniidae wild silkworm species such as Antheraea pernyi, A. frithi, and Samia ricini, widening their probable host range for infection. Baculoviruses generally display a horizontal mode of transmission, mainly through ingestion of occlusion bodies (OBs); however, the present study revealed a trans-ovum vertical mode of transmission in addition to a horizontal mode. The observations made in this study aid a detailed understanding of the tiger band disease and its causative pathogen AnprNPV, which will support future studies and disease management in oak tasar sericulture.

Made available in DSpace on 2022-04-28T19:41:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The Aedes aegypti mosquito is the primary vector of Dengue, Chikungunya and Zika causing major problems for public health, which requires new strategies for its control, like the use of entomopathogenic microorganisms. In this study, bacteria from various Amazonian environments were isolated and tested for their pathogenicity to A. aegypti larvae. Following thermal shock to select sporulated Bacillus spp., 77 bacterial strains were isolated. Molecular identification per 16S RNA sequences revealed that the assembled strains contained several species of the genus Bacillus and one species each of Brevibacillus, Klebsiella, Serratia, Achromobacter and Brevundimonas. Among the isolated Bacillus sp. strains, 19 showed larvicidal activity against A. aegypti. Two strains of Brevibacillus halotolerans also displayed larvicidal activity. For the first time, larvicidal activity against A. aegypti was identified for a strain of Brevibacillus halotolerans. Supernatant and pellet fractions of bacterial cultures were tested separately for larvicidal activities. Eight strains contained isolated fractions resulting in at least 50% mortality when tested at a concentration of 5 mg/mL. Further studies are needed to characterize the active larvicidal metabolites produced by these microorganisms and define their mechanisms of action. Programa Multi-Institucional de Pós-Graduação em Biotecnologia—PPGBIOTEC Universidade Federal do Amazonas—UFAM Programa de Pós-Graduação em Biotecnologia e Recursos Naturais da Amazônia—PPGMBT Universidade do Estado do Amazonas—UEA Programa de Pós-Graduação em Biodiversidade e Biotecnologia—PPGBIONORTE Universidade Federal do Amazonas—UFAM Programa de Pós-Graduação em Ciências Biológicas (Entomologia)—PPGENT Instituto Nacional de Pesquisas da Amazônia—INPA Laboratório de Controle Biológico e Biotecnologia da Malária e Dengue—LCBBMD Instituto Nacional de Pesquisas da Amazônia—INPA Faculdade de Ciências Agrárias—FCA Universidade Federal do Amazonas—UFAM School of Agricultural Sciences Department of Bioprocesses and Biotechnology Central Multi User Laboratory São Paulo State University—UNESP Department of Cell and Molecular Biology Microbiology Uppsala University MTEKPrime School of Agricultural Sciences Department of Bioprocesses and Biotechnology Central Multi User Laboratory São Paulo State University—UNESP FAPESP: 2013/11343-6

The Aedes aegypti mosquito is the primary vector of Dengue, Chikungunya and Zika causing major problems for public health, which requires new strategies for its control, like the use of entomopathogenic microorganisms. In this study, bacteria from various Amazonian environments were isolated and tested for their pathogenicity to A. aegypti larvae. Following thermal shock to select sporulated Bacillus spp., 77 bacterial strains were isolated. Molecular identification per 16S RNA sequences revealed that the assembled strains contained several species of the genus Bacillus and one species each of Brevibacillus, Klebsiella, Serratia, Achromobacter and Brevundimonas. Among the isolated Bacillus sp. strains, 19 showed larvicidal activity against A. aegypti. Two strains of Brevibacillus halotolerans also displayed larvicidal activity. For the first time, larvicidal activity against A. aegypti was identified for a strain of Brevibacillus halotolerans. Supernatant and pellet fractions of bacterial cultures were tested separately for larvicidal activities. Eight strains contained isolated fractions resulting in at least 50% mortality when tested at a concentration of 5 mg/mL. Further studies are needed to characterize the active larvicidal metabolites produced by these microorganisms and define their mechanisms of action.

Malaria remains a major public health problem in South America, mostly in the Amazon region. Among newly proposed ways of controlling malaria transmission to humans, paratransgenesis is a promising alternative. Paratransgenesis aims to inhibit the development of parasites within the vector through the action of genetically modified bacteria. The first step towards successful paratransgenesis in the Amazon is the identification of Anopheles darlingi symbiotic bacteria, which are transmitted vertically among mosquitoes, and are not pathogenic to humans.Culturable bacteria associated with An. darlingi and their breeding sites were isolated by conventional microbiological techniques. Isolated strains were transformed with a GFP expressing plasmid, pSPT-1-GFP, and reintroduced in mosquitoes by feeding. Their survival and persistence in the next generation was assessed by the isolation of fluorescent bacteria from eggs, larvae, pupae and adult homogenates.A total of 179 bacterial strains were isolated from samples from two locations, Coari and Manaus. The predominant genera identified in this study were Acinetobacter, Enterobacter, Klebsiella, Serratia, Bacillus, Elizabethkingia, Stenotrophomonas and Pantoea. Two isolated strains, Serratia-Adu40 and Pantoea-Ovo3, were successfully transformed with the pSPT-1-GFP plasmid and expressed GFP. The fluorescent bacteria fed to adult females were transferred to their eggs, which persisted in larvae and throughout metamorphosis, and were detected in adult mosquitoes of the next generation.Serratia-Adu40 and Pantoea-Ovo3 are promising candidates for paratransgenesis in An. darlingi. Further research is needed to determine if these bacteria are vertically transferred in nature.

Made available in DSpace on 2022-04-28T19:45:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 As an online encyclopedia, Wikipedia is the world’s largest reference Web site, with 1.7 billion visits per month. Given how easy it is to access and read, students use Wikipedia globally, despite most faculty members’ admonitions. Since 2013, health professional schools worldwide have incorporated Wiki-editing into their formal curricula. These courses impact students by (1) strengthening their ability to evaluate evidence-based content and (2) multiplying their contributions to society through improvements to Wikipedia articles accessed by millions. We showcase several models of incorporating Wikipedia-editing assignments into health professions education worldwide. These successful initiatives can be replicated everywhere. Medical School Sao Paulo State University (UNESP) Children’s Hospital of Eastern Ontario Research Institute School of Medicine & School of Education Tel Aviv University College of Pharmacy University of Manitoba Notre Dame University School of Medicine Uppsala University International League Against Epilepsy Wikipedia Project Medica Superspecialty Hospital South Cliff Dental Group San Francisco School of Medicine University of California Medical School Sao Paulo State University (UNESP)

Objectives Annually, 1.2 million humans are stung by scorpions and severely affected by their venom. Some of the scorpion species of medical importance have a similar morphology to species with low toxicity. To establish diagnostic tools for surveying scorpions, the current study was conducted to generate three mitochondrial markers, Cytochrome Oxidase I (COI gene), 12S rDNA and 16S rDNA for six species of medically important Iranian scorpions: Androctonus crassicauda, Hottentotta saulcyi, Mesobuthus caucasicus, M. eupeus, Odontobuthus doriae, and Scorpio maurus. Results Phylogenetic analyses of the obtained sequences corroborated the morphological identification. For the first time, 12S rDNA sequences are reported from Androctonus crassicauda, Hottentotta saulcyi, Mesobuthus caucasicus and M. eupeus and also the 16S rDNA sequence from Hottentotta saulcyi. We conclude that the mitochondrial markers are useful for species determination among these medically important species of scorpions.

Objective Pebrine, caused by the microsporidium Nosema bombycis, is one of the severe diseases in Thai polyvoltine strains of the silkworm Bombyx mori. Studies showing the presence of Lactobacillus species in the silkworm gut, where the Nosema parasites enter, suggests that these bacteria may have a protective effect. The aim of this study was to investigate the effect of supplementation of Lactobacillus casei on the survival ratio of silkworm larvae challenged with N. bombycis. Results A group of silkworm larvae of the commercial Thai polyvoltine hybrid strain DokBua was supplemented with L. casei on the second day of the 2nd, 3rd, 4th, and 5th instar. When a control group of silkworm larvae were challenged with N. bombycis on the second day of the 4th instar, the survival rate was 68%, but it was 91% for larvae supplemented with L. casei. For those larvae that survived the treatments until pupation, we determined the growth characters larval weight, cocooning ratio, and pupation ratio, and the economic characters cocoon weight and cocoon shell weight. When infected with N. Bombycis, growth characters were significantly higher in larvae also receiving L. casei.

Made available in DSpace on 2021-06-25T10:20:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Uppsala Universitet Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Vetenskapsrådet Background: Malaria remains a major public health problem in South America, mostly in the Amazon region. Among newly proposed ways of controlling malaria transmission to humans, paratransgenesis is a promising alternative. Paratransgenesis aims to inhibit the development of parasites within the vector through the action of genetically modified bacteria. The first step towards successful paratransgenesis in the Amazon is the identification of Anopheles darlingi symbiotic bacteria, which are transmitted vertically among mosquitoes, and are not pathogenic to humans. Methods: Culturable bacteria associated with An. darlingi and their breeding sites were isolated by conventional microbiological techniques. Isolated strains were transformed with a GFP expressing plasmid, pSPT-1-GFP, and reintroduced in mosquitoes by feeding. Their survival and persistence in the next generation was assessed by the isolation of fluorescent bacteria from eggs, larvae, pupae and adult homogenates. Results: A total of 179 bacterial strains were isolated from samples from two locations, Coari and Manaus. The predominant genera identified in this study were Acinetobacter, Enterobacter, Klebsiella, Serratia, Bacillus, Elizabethkingia, Stenotrophomonas and Pantoea. Two isolated strains, Serratia-Adu40 and Pantoea-Ovo3, were successfully transformed with the pSPT-1-GFP plasmid and expressed GFP. The fluorescent bacteria fed to adult females were transferred to their eggs, which persisted in larvae and throughout metamorphosis, and were detected in adult mosquitoes of the next generation. Conclusion: Serratia-Adu40 and Pantoea-Ovo3 are promising candidates for paratransgenesis in An. darlingi. Further research is needed to determine if these bacteria are vertically transferred in nature. Programa de Pós-Graduação em Biotecnologia Universidade Federal do Amazonas - PPGBIOTEC / UFAM MTEKPrime Universidade Estadual do Amazonas - MBT UEA Universidade Estadual do Amazonas - BIONORTE UEA Laboratório de Malária E Dengue Instituto Nacional de Pesquisas da Amazônia INPA Department of Ecology Swedish University of Agricultural Sciences (SLU), Box 7044 School of Agricultural Sciences Department of Bioprocesses and Biotechnology Central Multi User Laboratory São Paulo State University (UNESP) Department of Cell and Molecular Biology Microbiology Uppsala University, Box 596 School of Agricultural Sciences Department of Bioprocesses and Biotechnology Central Multi User Laboratory São Paulo State University (UNESP) CAPES: 001 Vetenskapsrådet: 348-2012-622

Pebrine is one of the devastating diseases mostly caused by notorious Nosema - a microsporidian infecting silkworms. Identification of novel genes associated with the pathogen plays a key role in developing a reliable diagnostic tool for the detection of disease. Targeting potential biomarkers will help in developing strategies for fast and efficient control measures, which can prevent the spread of infection. This study was to identify genes present commonly in Nosema such as Nosema bombycis, Nosema mylitta, Nosema assamensis and Nosema ricini infecting Indian silkworms in order to find potential gene markers for early pebrine disease diagnosis. Real time PCR was used to validate the genes active early during the infection cycle, confirming the expression of genes and their order of expression. 16S rRNA and β-tubulin were found to be expressed early in infection followed by PTP1 and PTP2, PTP3, SWP5 and MetAP2 genes. These identified molecular markers can be used in addition to conventional gene primers which are traditionally used for the detection of pebrine.

Objectives: Hard ticks (Acari: Ixodidae) are ectoparasites of human and animals and transmit a wide range of pathogens. The species identification of ticks is normally based on morphological keys. However, considering the importance of proper identification of the species and their bio-ecological characteristics, relying on morphological characteristics alone can be questionable. In this study, ITS2 and COI were selected for phylogenetic evaluation of Iranian species belonging to three genera: Dermacentor, Hyalomma and Rhipicephalus. Results: The ticks were from 10 populations from three different climatic and zoogeographical zones of Iran. The study of important morphological characteristics revealed notable differences in these characteristics between different populations of D. marginatus species.Contrary to the results of ITS2 sequence analysis which giving additional support for the view that D. niveus and D. marginatus should be viewed as one species only, the sequence analysis of COI and phylogeny favored the separation of the two species of D. niveus and D. marginatus. Given the greater importance of COI in identifying and distinguishing species, the Being two species should be considered.

The midgut microbiota of disease vectors plays a critical role in the successful transmission of human pathogens. The environment influences the microbiota composition; however, the relative mosquito-species contribution has not been rigorously disentangled from the environmental contribution to the microbiota structure. Also, the extent to which the microbiota of the adult sugar food source and larval water can predict that of the adult midgut and vice versa is not fully understood. To address these relationships, larvae and adults of Anopheles gambiae and Aedes albopictus were either reared separately or in a co-rearing system, whereby aquatic and adult stages of both species shared the larval water and sugar food source, respectively. Despite being reared under identical conditions, clear intra- and interspecies differences in midgut microbiota-composition were observed across seven cohorts, collected at different time points over a period of eight months. Fitting a linear model separately for each OTU in the mosquito midgut showed that two OTUs significantly differed between the midguts of the two mosquito species. We also show an effect for the sugar food source and larval water on the adult midgut microbiota. Our findings suggest that the mosquito midgut microbiota is highly dynamic and controlled by multiple factors.

Virus infection by the Bombyx mori nucleopolyhedrovirus (BmNPV) is the most severe disease in Thai sericultural practice of polyvoltine silkworms. Here, we characterized a newly isolated BmNPV isolated from the Mahasarakham province in Thailand (BmNPV-MSU). The purity and morphology of BmNPV-MSU were examined using light microscopy and scanning electron microscopy. The polyhedral inclusion bodies (PIBs) of BmNPV-MSU appeared in tetragonal, hexagonal, octagonal, and globular forms. The virions were both single and multiple embedded as observed by transmission electron microscopy. We also determined the virulence of BmNPV-MSU for six different Thai polyvoltine strains by LC50 and time to death after infection. The LC50 values of Nang Lai, NK04, and Sam Rong strains were 5.05–1.52 × 107 PIBs per ml and mortality peaked 7- to 8-d after inoculation. For Nang Noi, SP2, and RE05 strains the LC50 values were 7.91–1.82 × 106 PIBs/ml and mortality peaked 4–5 d after inoculation, thus having lower chance of survival to infection by BmNPV-MSU.

Fleas are important vectors of diseases such as murine typhus, tularaemia, hymenolepiasis and plague. The presence of active foci and history of human- and flea-transmitted plague in northwest Iran prompted the present group to collect and identify fleas from human and livestock dwellings across West Azerbaijan Province. Adult fleas were collected and identified using routine taxonomic keys. Species designation was confirmed by sequencing the cytochrome oxidase subunit I (COI). Of the collected specimens (n = 989), 104 were collected off-host (30 from human dwellings and 74 in light traps) and the rest were found on hosts (107 on animals and 778 by human bait). Of these fleas, 394 (40%) were male and 595 (60%) were female. The collected specimens belonged to the species Ctenocephalides canis, Ctenocephalides felis, Ctenocephalides orientis and Pulex irritans (all: Siphonaptera: Pulicidae). The amplified COI fragment, in addition to confirming the morphological identification of species, showed good efficacy in separating the different species in the phylogenetic analysis. In addition to the identification of fleas from human and livestock dwellings using morphological and molecular characteristics, the current paper represents the first report of the presence of C. orientis in northwest Iran. This finding suggests that changing climate conditions may have expanded the distribution of this species.

"Pe ' brine" is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the beta-tubulin gene in the pe ' brine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 x 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV)

A Gram-stain-negative, rod-shaped strain, Braz8T, isolated from larvae of Anopheles darlingi was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Braz8T was related most closely to species of the genus Thorsellia , with 95.6, 96.5 and 96.6 % similarity to the type strains of Thorsellia anophelis , Thorsellia kandunguensis and Thorsellia kenyensis , respectively, and formed a separate branch in the phylogenetic tree next to the monophyletic cluster of the genus Thorsellia . Chemotaxonomic data supported the allocation of the strain to the family Thorselliaceae . The major fatty acids were C18 : 1 ω7c, C16 : 0 and C14 : 0. The quinone system was composed of ubiquinones Q-8 and Q-7 (1 : 0.3), the predominant polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and the polyamine pattern showed the major compound putrescine. However, qualitative and quantitative differences in the major polyamine, polar lipid profile and fatty acid patterns distinguished strain Braz8T from species of the genus Thorsellia . Phylogenetic analysis based on 16S rRNA gene sequences, average nucleotide identity, DNA–DNA hybridization, multilocus sequence analysis as well as physiological and biochemical tests distinguished strain Braz8T both genotypically and phenotypically from the three Thorsellia species but also showed its placement in the family Thorselliaceae . Thus, strain Braz8T is considered to represent a novel species of a new genus most closely related to the genus Thorsellia, for which the name Coetzeea brasiliensis gen. nov., sp. nov. is proposed. The type strain of Coetzeea brasiliensis is Braz8T (=LMG 29552T=CIP 111088T).

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.

This report presents the outcome of the joint work of PhD students and senior researchers working with DNA-based biodiversity assessment approaches with the goal to facilitate others the access to definitions and explanations about novel DNA-based methods. The work was performed during a PhD course (SLU PNS0169) at the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. The course was co-organized by the EU COST research network DNAqua-Net and the SLU Research Schools Focus on Soils and Water (FoSW) and Ecology - basics and applications. DNAqua-Net (COST Action CA15219, 2016-2020) is a network connecting researchers, water managers, politicians and other stakeholders with the aim to develop new genetic tools for bioassessment of aquatic ecosystems in Europe and beyond. The PhD course offered a comprehensive overview of the paradigm shift from traditional morphology-based species identification to novel identification approaches based on molecular markers. We covered the use of molecular tools in both basic research and applied use with a focus on aquatic ecosystem assessment, from species collection to the use of diversity in environmental legislation. The focus of the course was on DNA (meta)barcoding and aquatic organisms. The knowledge gained was shared with the general public by creating Wikipedia pages and through this collaborative Open Access publication, co-authored by all course participants.

The microbiota in mosquito breeding waters can affect ovipositing mosquitoes, have effects on larval development, and can modify adult mosquito-gut bacterial composition. This, in turn, can affect transmission of human pathogens such as malaria parasites. Here, we explore the microbiota of four breeding sites for Anopheles darlingi, the most important malaria vector in Latin America. The sites are located in Manaus in the Amazon basin in Brazil, an area of active malaria transmission. Using 16S rRNA gene sequencing by MiSeq, we found that all sites were dominated by Proteobacteria and Firmicutes and that 94% of the total number of reads belonged to 36 operational taxonomic units (OTUs) identified in all sites. Of these, the most common OTUs belonged to Escherichia/Shigella, Staphylococcus, and Pseudomonas. Of the remaining 6% of the reads, the OTUs found to differentiate between the four sites belonged to the orders Burkholderiales, Actinomycetales, and Clostridiales. We conclude that An. darlingi can develop in breeding waters with different surface-water bacteria, but that the common microbiota found in all breeding sites might indicate or contribute to a suitable habitat for this important malaria vector. Title in dissertation list of articles: Characterization of bacterial communities in different breeding waters of Anopheles darlingi from the Amazon basin malaria-endemic area.

Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these two Heterobasidion species was investigated. Five different culture media with different culture age were investigated to study the effect of substrate composition and culture age for metabolite production. Bacterial cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was analysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the antifungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps.

Several mosquito species are vectors of disease; however, to understand their role in disease transmission, accurate species identification is of particular importance. Morphological identification is the main method used, but molecular techniques have emerged as a tool for the identification of closely related species. In this study, mosquitoes from the West Azerbaijan Province in northwestern Iran were characterized on the basis of their rDNA-ITS2 sequences. Nine populations of 6 species of mosquitoes belonging to the genera Anopheles, Culex, Culiseta, and Ochlerotatus were studied. To the best of our knowledge, ITS2 sequences of Culiseta longiareolata and Culex hortensis have been reported for the first time. In addition, ITS2 sequences of Culex theileri and Ochlerotatus caspius have been reported for the first time in Iran. Phylogenetic analysis based on ITS2 showed that subfamilies Anophelinae and Culicinae of the family Culicidae could be differentiated successfully and subgenera Anopheles and Cellia of the genus Anopheles were separated. The analysis showed that the genera Culex, Culiseta, and Ochlerotatus have diverged separately.

International audience; In many species, animals live in highly structured groups. In these groups, individual differences in the number and identity of social contacts (alters) of each ego define the social network structure of the animal group. The composition of groups can be disturbed by grouping animals according to age or production stage, which can in turn induce stress. We investigated whether the preference of two animals to stay together depends on the sociality of each individual (defined by the time it spends close to other animals) or on the composition of the group. We observed 158 dairy cows distributed in 6 pens during 17 weeks. The precise positions of the cows were monitored with positional loggers 24/7 in two groups with fixed independent populations and during the formation of new groups with varying population. In fixed groups, the sociality of a cow was maintained over the entire observation period, but this trait depended on the area contacts occurred (inside vs. outside the resting area). When introducing foreign individuals into social groups, the sociality of individual cows was maintained independently of the group; this sociality was therefore not necessarily influenced by the time spent in the group but by the social characteristics of individual cows. During the formation of new groups, newly introduced cows dynamically interacted with resident ones, forming a few strong short-lasting contacts between newcomers and resident cows. However, a few long-lasting interactions occurred between resident and newcomers and the whole network is weakened. Our study suggests that each cow has its own sociality independent of the group and tend to establish relations with specific partners when the population is fixed. In addition, when introducing cows into a group, the social network of the group is weakened, with absence of strong links between newcomers and resident cows and a decrease in the links between resident cows for at least 2 weeks.

Water-storage containers are common in households where access to water is scarce and often act as breeding sites for vector mosquitoes. Bacteria in these containers may be important for attracting or repelling ovipositing mosquitoes. We hypothesized that bacterial community composition in water-storage containers would represent either inhibitory or suitable environmental conditions for mosquito larvae. To investigate this, we characterized the bacterial community composition in water-storage containers and correlated these communities to Aedes and Anopheles larval densities. Water samples were collected over two years from 13 containers in an Indian village and analyzed by high throughput 16S rRNA gene amplicon sequencing. Comparisons of bacterial community composition between water with and without mosquito larvae showed that Xanthomonadaceae, Comamonadaceae and Burkholderiaceae were more common (P 0.05) in absence of larvae, while Lachnospiraceae, Synechococcaceae, Alcaligenaceae and Cryomorphaceae were more common (P 0.05) in presence of larvae. Indicator analysis identified operational taxonomic units designated as CL500-29 marine group (Acidimicrobiaceae) and FukuN101 (Microbacteriaceae) for absence and presence of larvae, respectively. These results contribute to the understanding of which bacteria, directly or indirectly, can be linked to absence or presence of mosquitoes around households and set the basis for potential measures to be taken against these vector mosquitoes.

The diet of entomophagous coccinellids is mainly based on aphids and other food sources such as pollen, nectar, or fungal spores. Knowledge of their foraging behavior on plants infected by powdery mildew and their survival on fungal spores is currently limited. In this study, we investigated the olfactory response of Coccinella septempunctata to odor emission of barley plants infected by powdery mildew and their survival on fungal spores in the presence or absence of aphids. Odors released by powdery-mildew infected plants were more attractive for ladybirds compared to those of uninfected controls. After 3 days, the survival rate of ladybirds feeding only on powdery-mildew spores was less than 50%, while for ladybirds feeding exclusively on Rhopalosiphum padi aphids, the survival rate was close to 90%. After 15 days, the highest survival rate (almost 80%) was observed for ladybirds feeding on plants with both aphids and powdery mildew. Molecular analyses confirmed the presence of fungal spores in ladybird guts when feeding either on powdery mildew or on a mixed diet. Our results provide new insights into foraging behavior of entomophagous coccinellids revealing the potential of powdery mildew to be utilized as important non-essential food in a mixed diet, but also its lethal effect if consumed alone.

The pine weevil is one of the most important pest insects of conifer reforestation areas in Europe. Female pine weevils cover their eggs with chewed bark and feces (frass) resulting in avoidance be ...

Antheraea pernyi Iflavirus (ApIV) is a virus that is the likely causative agent of A. pernyi Vomit Disease (AVD). The virus is maintained in A. pernyi populations as an unapparent infection by as yet unknown transmission routes. The mechanisms by which an infection results in AVD are also not yet known. Both these factors were investigated in this study. A. pernyi larvae were fed or injected with ApIV, but only injection led to active replication and spread of ApIV to the head, epidermis, hemocytes, gut, fatbody, ovary and testis. Twenty percent of ApIV-injected pupae produced ApIV-infected offspring as adults, proving that ApIV can be transmitted vertically. As a confirmation of vertical transmission occurring also in nature, 53 A. pernyi couples were collected from areas where ApIV has been detected. Eleven of these couples produced ApIV-infected offspring, again pointing to a vertical transmission of ApIV.

Mute Swans (Cygnus olor) were observed using tailwind as a support for high-speed water transportation on three different occasions in three different locations in Sweden. With the wings arched over the back, they traveled similar to 100 m in an inlet of the Baltic Sea in Stockholm, several hundred meters in Lake Hjalstaviken in Enkoping, and similar to 350 m in Lake Krankesjon in Lund. The speed of the movement was estimated to be much higher than normally seen for swimming swans. The first observation included two Mute Swans traveling one after another in the same direction, the second observation was of one single individual traveling towards a group of conspecifics, and the third observation was of a single individual traveling by itself. This behavior may serve as a means of medium-distance water transportation in this heavy bird species.

In vector mosquitoes, the presence of midgut bacteria may affect the ability to transmit pathogens. We have used a laboratory colony of Aedes aegypti as a model for bacterial interspecies competition and show that after a blood meal, the number of species (culturable on Luria-Bertani agar) that coexist in the midgut is low and that about 40% of the females do not harbor any cultivable bacteria. We isolated species belonging to the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas, and we also determined their growth rates, antibiotic resistance, and ex vivo inhibition of each other. To investigate the possible existence of coadaptation between midgut bacteria and their host, we fed Ae. aegypti cohorts with gut bacteria from human, a frog, and two mosquito species and followed the bacterial population growth over time. The dynamics of the different species suggests coadaptation between host and bacteria, and interestingly, we found that Pantoea stewartii isolated from Ae. aegypti survive better in Ae. aegypti as compared to P. stewartii isolated from the malaria mosquito Anopheles gambiae.

The West Nile virus (WNV) transmission cycle includes a wide range of migratory wetland birds as reservoirs, mosquitoes as biological vectors, and equines and humans as dead-end hosts. Despite the presence of potential vector species, there is no information about the existence of WNV in mosquito vectors in Iran. The Iranian West Azerbaijan Province is located in the northwestern part of Iran and has borders with Turkey, Iraq, Armenia, and the Republic of Azerbaijan. The current study was conducted to identify the wetland mosquitoes of the West Azerbaijan Province and their infection with WNV. In this study, 2143 specimens were collected, comprising 1541 adults and 602 larvae. Six species belonging to four genera were collected and identified: Anopheles maculipennis sensu lato (s.l.), Culex (Cx.) hortensis, Cx. pipiens s.l., Cx. theileri, Culiseta longiareolata, and Aedes (Ae.) (Ochlerotatus) caspius. In total, 45 pools of mosquitoes were examined. Two of the adult pools collected from the same location showed the presence of WNV in Ae. (Och.) caspius, from Sangar, Makoo County, as confirmed by PCR and sequencing. Due to the discovery of WNV in the mosquito population of the region, and the presence of wetlands and significant populations of migratory birds, the health sector should carefully monitor the factors involved in the cycle of this disease.

Lepidopteran larvae are regularly infected by baculoviruses during feeding on infected plants. The differences in sensitivity to these infections can be substantial, even among closely related species. For example, the noctuids Cotton bollworm ( Helicoverpa zea ) and Tobacco budworm ( Heliothis virescens ), have a 1000-fold difference in sensitivity to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection. Recent data were interpreted to indicate that the lepidopteran immunoglobulin protein, Hemolin , is synthesized upon viral injection and therefore to participate in anti-viral responses. To investigate whether Hemolin synthesis is affected by a natural viral infection, specific transcription in fat bodies and hemocytes of H. zea and H. virescens larvae was monitored following per os infection with the baculovirus HzSNPV ( H. zea single nucleopolyhedrovirus). Both moths showed the same expression pattern as seen in uninfected animals and coincided with ecdysone responses, previously known to induce Hemolin expression. In contrast, injection of lyophilized Micrococcus luteus resulted in increased Hemolin expression supporting a role for Hemolin as an immuno-responsive protein in these species. The combined data are consistent with the suggestion that while Hemolin seems to participate in the response to virus infection in the superfamily Bombycoidea, this is not true in the Noctuoidea.

The microbial flora associated with Anopheles darlingi Root (Diptera: Culicidae), a major Neotropical malaria vector, was investigated for the development of a paratransgenesis-based approach to control malaria transmission in Brazil. Female mosquitoes were collected using human land catches and captured insects provided a bloodmeal. The controlled blood feeding resulted in increased detection of mosquito bacterial population because it was possible to retrieve bacterial DNA from all blood-fed mosquitoes. The 16S sequences of bacteria recovered, include some closely related to those found in other vector mosquitoes, including Aeromonas, Pantoea and Pseudomonas species.