The dynamic gut microbiota of zoophilic members of the Anopheles gambiae complex (Diptera: Culicidae)

The gut microbiota of mosquitoes plays a critical role in the life history of the animal. There is a growing body of research characterising the gut microbiota of a range of mosquito species, but there is still a paucity of information on some members of the Anopheles gambiae complex. In this study, the gut microbiota of four laboratory strains were characterised. SENN (Anopheles arabiensis—insecticide susceptible major vector), SENN DDT (Anopheles arabiensis—insecticide resistant major vector), MAFUS (Anopheles merus—minor vector) and SANGWE (Anopheles quadriannulatus—non-vector) were used in this study. The microbiota of fourth instar larvae, 3-day old, 15-day old non-blood fed and 15-day old blood fed females were characterised by MALDI-TOF mass spectroscopy and 16 s rRNA gene sequencing by next generation sequencing. The four strains differed in species richness but not diversity. The major vectors differ in β-diversity from that of the minor and non-vectors. There was no difference in α- or β-diversity in 15 non-blood fed females and 15-day old females that had 3 blood meals before day 15. These differences may be related to a mixture of the effect of insecticide resistance phenotype as well as a potential relationship to vector competence to a limited extent. Bacterial diversity is affected by species and age. There is also a potential relationship between the differences in gut microbiota and capacity to transmit parasites. This genetic background of the mosquitoes, however, play a major role, and must be considered in this relationship.The National Research Foundation of South Africa Competitive Support for Unrated Researchers and the National Health Laboratory Services Research Trust Development Grant.https://www.nature.com/srepVeterinary Tropical Disease

Exploration and comparison of bacterial communities present in bovine faeces, milk and blood using 16S rRNA metagenomic sequencing

Cattle by-products like faeces, milk and blood have many uses among rural communities; aiding to facilitate everyday household activities and occasional rituals. Ecologically, the body sites from which they are derived consist of distinct microbial communities forming a complex ecosystem of niches. We aimed to explore and compare the faecal, milk and blood microbiota of cows through 16S rRNA sequencing. All downstream analyses were performed using applications in R Studio (v3.6.1). Alpha-diversity metrics showed significant differences between faeces and blood; faeces and milk; but non-significant between blood and milk using Kruskal-Wallis test, P < 0,05. The beta-diversity metrics on Principal Coordinate Analysis and Non-Metric Dimensional Scaling significantly clustered samples by type (PERMANOVA test, P < 0,05). The overall analysis revealed a total of 30 phyla, 74 classes, 156 orders, 243 families and 408 genera. Firmicutes, Bacteroidota and Proteobacteria were the most abundant phyla overall. A total of 58 genus-level taxa occurred concurrently between the body sites. The important taxa could be categorized into four potentially pathogenic clusters i.e. arthropod-borne; food-borne and zoonotic; mastitogenic; and metritic and abortigenic. A number of taxa were significantly differentially abundant (DA) between sites based on the Wald test implemented in DESeq2 package. Majority of the DA taxa (i.e. Romboutsia, Paeniclostridium, Monoglobus, Akkermansia, Turicibacter, Bacteroides, Candidatus_Saccharimonas, UCG-005 and Prevotellaceae_UCG-004) were significantly enriched in faeces in comparison to milk and blood, except for Anaplasma which was greatly enriched in blood and was in turn the largest microbial genus in the entire analysis. This study provides insights into the microbial community composition of the sampled body sites and its extent of overlapping. It further highlights the potential risk of disease occurrence and transmission between the animals and the community of Waaihoek in KwaZulu-Natal, Republic of South Africa pertaining to their unsanitary practices associated with the use of cattle by-products.DATA AVAILABILITY STATEMENT : All FastQ sequence files generated from this work are available from the National Center for Biotechnology Information’s Short Reads Archive, under BioProject number PRJNA777568, Accession numbers SRI168760 -SRI168784. All relevant data are within the manuscript and its Supporting Information files.SUPPLEMENTARY MATERIAL : S1 Fig. A: Alpha diversity box-plots showing Chao1 richness estimates per sample group. *Significant at P < 0,05. B: Alpha diversity box-plots showing Shannon diversity estimates per sample group. *Significant at P < 0,05. C: Alpha diversity box-plots showing Simpson’s diversity estimates per sample group. *Significant at P < 0,05. https://doi.org/10.1371/journal.pone.0273799.s001S2 Fig. UpSetR intersection plot showing number of unique and shared taxa at family level between faeces, milk and blood groups. https://doi.org/10.1371/journal.pone.0273799.s002S1 Table. Read counts tracked through the DADA2 pipeline including ASV counts, richness and genus level-resolved ASVs per sample. https://doi.org/10.1371/journal.pone.0273799.s003S2 Table. Alpha diversity values calculated using Shannon, Simpson and Chao1 indices. https://doi.org/10.1371/journal.pone.0273799.s004S3 Table. Total number of taxa detected per taxonomic rank across bovine faeces, milk and blood. https://doi.org/10.1371/journal.pone.0273799.s005S4 Table. Top 15 abundant taxa with their respective overall rankings and distribution across the three sample groups. https://doi.org/10.1371/journal.pone.0273799.s006S5 Table. Prevalence of potentially pathogenic genera of veterinary significance per sample group. https://doi.org/10.1371/journal.pone.0273799.s007S6 Table. Bacterial taxa shared between bovine faeces, milk and blood and their overall raw and relative abundances. https://doi.org/10.1371/journal.pone.0273799.s008S7 Table. Genus-level taxa exclusively detected and shared between faeces, milk and blood samples. https://doi.org/10.1371/journal.pone.0273799.s009S8 Table. A-C Differentially abundant taxa between blood and faeces; blood and milk and; faeces and milk (Padj < 0,01). https://doi.org/10.1371/journal.pone.0273799.s010S1 Raw images. Gel electrophoresis image of Anaplasma PCR targeting the 16S rRNA gene from blood samples. Image taken under UV transillumination using Enduro™ GOS gel documentation system. Lane 1 = 1 kb DNA ladder; 2–10 = Anaplasma positive samples; 11 = nuclease free H20 (-ve); 12 = A. marginale (+ve). https://doi.org/10.1371/journal.pone.0273799.s011The National Research Foundation – Thuthuka PhD Track Funding Instrument in collaboration with the Tshwane University of Technology.http://www.plosone.orgdm2022Veterinary Tropical Disease

Transcriptome and comparative genomics analyses reveal new functional insights on key determinants of pathogenesis and interbacterial competition in Pectobacterium and Dickeya spp.

Soft-rot Enterobacteriaceae (SRE), typified by Pectobacterium and Dickeya genera, are phytopathogenic bacteria inflicting soft-rot disease in crops worldwide. By combining genomic information from 100 SRE with whole-transcriptome data sets, we identified novel genomic and transcriptional associations among key pathogenicity themes in this group. Comparative genomics revealed solid linkage between the type I secretion system (T1SS) and the carotovoricin bacteriophage (Ctv) conserved in 96.7% of Pectobacterium genomes. Moreover, their coactivation during infection indicates a novel functional association involving T1SS and Ctv. Another bacteriophage-borne genomic region, mostly confined to less than 10% of Pectobacterium strains, was found, presumably comprising a novel lineage-specific prophage in the genus. We also detected the transcriptional coregulation of a previously predicted toxin/immunity pair (WHH and SMI1_KNR4 families), along with the type VI secretion system (T6SS), which includes hcp and/or vgrG genes, suggesting a role in disease development as T6SS-dependent effectors. Further, we showed that another predicted T6SS-dependent endonuclease (AHH family) exhibited toxicity in ectopic expression assays, indicating antibacterial activity. Additionally, we report the striking conservation of the group 4 capsule (GFC) cluster in 100 SRE strains which consistently features adjacently conserved serotype-specific gene arrays comprising a previously unknown organization in GFC clusters. Also, extensive sequence variations found in gfcA orthologs suggest a serotype-specific role in the GfcABCD machinery.Supplemental file 1: Overall synteny among strains (Fig. S1); gene conservation (Fig. S2); representativeness of serotype-specific block sizes within GFC region in SRE genomes (Fig. S3); sequence alignment (Fig. S4).Supplemental file 2: Differential expression summaries of Pcb1692 and D. dadantii during in planta infection (Table S1).Supplemental file 3: Gene neighborhood screenings and differential expression summary of T6SS elements in Pcb1692 (Table S2).Supplemental file 4: Identification, gene neighborhood screening, and differential expression of carotovoricin in SRE (Table S3).Supplemental file 5: Conservation and differential expression of PcbPr1 bacteriophage in Pcb1692 (Table S4).Supplemental file 6: Differential expression of R. solanacearum Rasop1 and -2 prophages during infection (Table S5).Supplemental file 7: Conservation, annotation, and gene-neighborhood screenings of GFC regions in SRE (Table S6).Supplemental file 8 : Species abbreviations and genome build accessions (Table S7).The National Research Foundation (NRF), South Africa, through Competitive Funding for Rated Researchers (CFRR 98993), NRF Bioin-formatics and Functional Genomics (BFG 93685), and the NRF Research Technology and Transfer Fund (RTF 98654). D.Y.S. received an NRF BFG postdoctoral fellowship. D.B.-R. received a University of Pretoria postdoctoral fellowship. N.M. was funded by NRF Freestanding, PSA, and University of Pretoria Postgraduate bursaries. C.K.T. was funded by a University of Pretoria bursary.http://aem.asm.orghj2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Genome-wide identification of potato long intergenic noncoding RNAs responsive to <i>Pectobacterium carotovorum</i> subspecies <i>brasiliense</i> infection

BACKGROUND: Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression in both mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of long intergenic nocoding RNAs (lincRNAs) in plant defence responses are yet to be fully explored. RESULTS: In this study, we used strand-specific RNA sequencing to identify 1113 lincRNAs in potato (Solanum tuberosum) from stem tissues. The lincRNAs are expressed from all 12 potato chromosomes and generally smaller in size compared to protein-coding genes. Like in other plants, most potato lincRNAs possess single exons. A time-course RNA-seq analysis between a tolerant and a susceptible potato cultivar showed that 559 lincRNAs are responsive to Pectobacterium carotovorum subsp. brasiliense challenge compared to mock-inoculated controls. Moreover, coexpression analysis revealed that 17 of these lincRNAs are highly associated with 12 potato defence-related genes. CONCLUSIONS: Together, these results suggest that lincRNAs have potential functional roles in potato defence responses. Furthermore, this work provides the first library of potato lincRNAs and a set of novel lincRNAs implicated in potato defences against P. carotovorum subsp. brasiliense, a member of the soft rot Enterobacteriaceae phytopathogens. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2967-9) contains supplementary material, which is available to authorized users

Discovery and profiling of small RNAs responsive to stress conditions in the plant pathogen <i>Pectobacterium atrosepticum</i>

BACKGROUND: Small RNAs (sRNAs) have emerged as important regulatory molecules and have been studied in several bacteria. However, to date, there have been no whole-transcriptome studies on sRNAs in any of the Soft Rot Enterobacteriaceae (SRE) group of pathogens. Although the main ecological niches for these pathogens are plants, a significant part of their life cycle is undertaken outside their host within adverse soil environment. However, the mechanisms of SRE adaptation to this harsh nutrient-deficient environment are poorly understood. RESULTS: In the study reported herein, by using strand-specific RNA-seq analysis and in silico sRNA predictions, we describe the sRNA pool of Pectobacterium atrosepticum and reveal numerous sRNA candidates, including those that are induced during starvation-activated stress responses. Consequently, strand-specific RNA-seq enabled detection of 137 sRNAs and sRNA candidates under starvation conditions; 25 of these sRNAs were predicted for this bacterium in silico. Functional annotations were computationally assigned to 68 sRNAs. The expression of sRNAs in P. atrosepticum was compared under growth-promoting and starvation conditions: 68 sRNAs were differentially expressed with 47 sRNAs up-regulated under nutrient-deficient conditions. Conservation analysis using BLAST showed that most of the identified sRNAs are conserved within the SRE. Subsequently, we identified 9 novel sRNAs within the P. atrosepticum genome. CONCLUSIONS: Since many of the identified sRNAs are starvation-induced, the results of our study suggests that sRNAs play key roles in bacterial adaptive response. Finally, this work provides a basis for future experimental characterization and validation of sRNAs in plant pathogens. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2376-0) contains supplementary material, which is available to authorized users

Transcriptome profiling of potato plant stems challenged with Pectobacterium carotovorum subsp. brasiliense and elucidation of the role of small RNAs in Pectobacterium survival mechanisms

Pectobacterium carotovorum subsp. brasiliense, a necrotrophic phytopathogen belonging to the soft rot Enterobacteriaceae (SRE) family is responsible for causing tuber soft rot and blackleg diseases of stems in potato plants. In recent years, P. c. brasiliense, has emerged as a soft rot pathogen of significance, potentially threatening potato production globally. To date, P. c. brasiliense is the most aggressive soft rot phytopathogen isolated from potato in South Africa. Currently effective chemical control measures are unavailable once soft rot pathogens have established disease in potato plants and/or harvested tubers. Therefore, this study sought to determine the molecular basis of quantitative resistance in potato stems challenged with P. c. brasiliense. In addition, this thesis explores some of the regulatory mechanisms important in the adaptation of Pectobacterium species to harsh nutrient-deficient environments such as plant xylem vessels. Determining the activated defense responses in potato stems is key in deciphering potential control approaches against pectobacteria as these soft rot pathogens colonize vascular tissues during infection of plants. Currently, no transcriptome-wide studies have been applied in the P. c. brasiliense and potato stem interaction to understand inducible defense responses within potato stems. In chapter 2, by implementing a time-course RNA-seq analysis, our study revealed important signaling pathways suggested to contribute to the potato defense transcriptome against P. c. brasiliense infection. Comparison of transcriptomes between a susceptible potato cultivar (Solanum tuberosum cv Valor) and tolerant cultivar (S. tuberosum cv BP1) following P. c. brasiliense inoculation revealed that the MAPK signaling cascades and ethylene hormonal pathway are central to potato defense responses against this pathogen. Specifically, genes encoding MPK3 protein kinase, and MKS1; ethylene biosynthetic and signaling pathways such as ACC, ERF2 and EIN3 genes were up-regulated in the tolerant cultivar within the time-course. Furthermore, expression of downstream defense-related genes was enhanced in S. tuberosum cv BP1, including transcription factors such WRKY33, MYB83, and several ethylene-responsive binding factors (ERFs); as well as various secondary wall biosynthetic genes for lignification and cellulose biosynthesis, for example, IRX9 and CESA8, respectively. In chapter 3, a bioinformatics analysis using strand-specific RNA sequencing allowed the identification of 1113 potato long intergenic noncoding RNA (lincRNAs) from stem tissues. Long noncoding RNAs (lncRNAs) have been implicated in diverse regulatory roles in eukaryotes. Recently, defense-related lncRNAs have been identified in Arabidopsis and wheat. In this thesis we identified 559 potato lincRNAs that were differentially expressed (DE) in both cultivars compared to mock-inoculated controls, following inoculation by P. c. brasiliense. Furthermore, co-expression analysis associated 17 of these lincRNAs with 12 potato defense-related genes. These results suggest that lincRNAs possibly have functional roles in potato defence responses. Future work will focus on characterization of these lincRNAs in order to understand their specific functional roles, particularly in potato defense mechanisms. In chapter 4, regarding potential regulatory mechanisms employed by Pectobacterium species during survival under nutrient-limiting conditions, we described 137 sRNA transcripts in P. atrosepticum genome. About 62% of the identified sRNAs are conserved within the SRE. Furthermore, 68 sRNAs were differentially expressed when comparing P. atrosepticum cells under growth-promoting and starvation conditions; with 47 sRNAs up-regulated under nutrient-deficient conditions. Thus, since many starvation-induced sRNAs were identified, these findings highlighted that sRNAs play key roles in adaptive responses in the genus Pectobacterium.Thesis (PhD)--University of Pretoria, 2016.Microbiology and Plant PathologyPhDUnrestricte

Chloranthus glaber Makino

原著和名: センリャウ科名: センリョウ科 = Chloranthaceae採集地: 千葉県 千葉市 千葉大学 (下総 千葉市 千葉大学)採集日: 1971/5/27採集者: 萩庭丈壽整理番号: JH008984国立科学博物館整理番号: TNS-VS-95898

Rubus lambertianus Ser.

原著和名: シマバライチゴ科名: バラ科 = Rosaceae採集地: 長崎県 島原市 眉山 (肥前 島原市 眉山)採集日: 1975/1/3採集者: 萩庭丈壽整理番号: JH009571国立科学博物館整理番号: TNS-VS-95957

Additional file 2: Table S6. of Genome-wide identification of potato long intergenic noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection

List of RT-PCR primers used in this study. (XLSX 8 kb