16S-23S rRNA Research Articles

Metabolomic and taxonomic characterization of Haloleptolyngbya lusitanica sp. nov. (Cyanobacteria, Synechococcales)

ABSTRACT The morphological plasticity of cyanobacteria and their widespread ecological dominance in a wide range of habitats highlights the need for in-depth taxonomic studies. This work focused on the taxonomical revision of Leptolyngbya (Cyanophyceae) strains deposited in the ESSACC culture collection and their metabolomic characterization. Although the studied ESSACC strains were morphologically identified as Leptolyngbya sp., the 16S rRNA gene and 16S–23S rRNA ITS analysis revealed that two strains (LMECYA 079 and LMECYA 173) belong to Haloleptolyngbya and represent a new taxonomical unit, genetically unique, ecologically plastic and adapted to both freshwater and thermal habitats, here described as Haloleptolyngbya lusitanica sp. nov. To perform a suspect screening of cyanometabolites in these strains, we used a non-targeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomic approach. Several metabolites were identified in Haloleptolyngbya lusitanica: micropeptin MM978, spumigin 640, oscillatoxin A and anabaenopeptin D. Strains were maintained and grown under the same conditions, revealing the common production of oscillatoxin A by both H. lusitanica strains. Other identified metabolites, however, were strain-specific, such as anabaenoptin D, which was only detected in LMECYA 173. The different cyanometabolite profiles reinforce the notion that cyanobacteria have the ability to adapt to different habitats, which is maintained under long-term culturing conditions.

Refinement of the "Candidatus Accumulibacter" genus based on metagenomic analysis of biological nutrient removal (BNR) pilot-scale plants operated with reduced aeration.

Members of the "Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 "Ca. Accumulibacter" species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most "Ca. Accumulibacter" MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the "Ca. Accumulibacter" diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 "Ca. Accumulibacter" species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized "Ca. Accumulibacter" species for which we propose the new species designation "Ca. Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, "Ca. A. necessarius" and "Ca. A. propinquus" accounted for more than 40% of the "Ca. Accumulibacter" community, whereas the newly proposed "Ca. A. jenkinsii" accounted for about 5% of the "Ca. Accumulibacter" community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the "Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the "Ca. Accumulibacter" species enriched under these conditions, including one novel species for which we propose "Ca. Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for five additional "Ca. Accumulibacter" species further refine the phylogeny of the "Ca. Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

Application of Advanced Molecular Methods to Study Early-Onset Neonatal Sepsis.

Early-onset sepsis (EOS) is a global health issue, considered one of the primary causes of neonatal mortality. Diagnosis of EOS is challenging because its clinical signs are nonspecific, and blood culture, which is the current gold-standard diagnostic tool, has low sensitivity. Commonly used biomarkers for sepsis diagnosis, including C-reactive protein, procalcitonin, and interleukin-6, lack specificity for infection. Due to the disadvantages of blood culture and other common biomarkers, ongoing efforts are directed towards identifying innovative molecular approaches to diagnose neonates at risk of sepsis. This review aims to gather knowledge and recent research on these emerging molecular methods. PCR-based techniques and unrestricted techniques based on 16S rRNA sequencing and 16S-23S rRNA gene interspace region sequencing offer several advantages. Despite their potential, these approaches are not able to replace blood cultures due to several limitations; however, they may prove valuable as complementary tests in neonatal sepsis diagnosis. Several microRNAs have been evaluated and have been proposed as diagnostic biomarkers in EOS. T2 magnetic resonance and bioinformatic analysis have proposed potential biomarkers of neonatal sepsis, though further studies are essential to validate these findings.

Borrelia nietonii sp. nov.: Relapsing Fever Spirochetes Transmitted By the Tick Ornithodoros hermsi Designated Previously as Borrelia hermsii Genomic Group II.

Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined. Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined. Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86-95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3' end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined. Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.

Microscopic and molecular investigation of vector borne haemoprotozoan diseases in dromedary camel of North Gujarat.

Vector borne haemoprotozoan diseases comprise diverse group of single celled organism transmitted by haematophagus invertebrates. The current study was aimed at identification of major haemoprotozoan (Babesia, Theileria and Trypanosoma) in dromedary camel of North Gujarat region using microscopy and Polymerase Chain Reaction (PCR). A total of 234 blood samples were screened by the microscopic and molecular detection assays. Molecular prevalence studies of Theileria, Trypanosoma spp and Babesia was undertaken using 18s ribosomal DNA, RoTat 1.2 and SS rRNA gene respectively. The data relating to microscopic and molecular prevalence along with associated risk factors were analysed by statistical methods. The overall prevalence of hamoprotozoan disease based on microscopic and molecular investigation was 23.50%. The sensitivity and specificity (95% Confidence Interval) of PCR assay was 100% in comparison to microscopy (45.45% and 100%). The kappa coefficient between PCR and microscopy indicated good level of agreement with a value of 0.704 and SE of 0.159. Despite holding much significance to the animal sector, little work has been undertaken in regional part of India regarding camel parasites. The present paper offers the first preliminary research data investigating haemoprotozoan disease using parasitological and molecular methods in camels in the region.

Prevalence estimation of Tropheryma whipplei in duodenal biopsy tissues of Koreans

Whipple’s disease caused by Tropheryma whipplei is difficult to diagnose because of a broad spectrum of manifestations and non-specific clinical signs. In the current global era, the incidence of duodenal infection/inflammation caused by T. whipplei in Korea may has been underestimated. Here we estimated the prevalence of T. whipplei in duodenal biopsy tissues of Koreans using real-time PCRs (RT-PCRs). A total of 252 duodenal biopsy tissues were collected from Korean patients who underwent esophagogastroduodenoscopy and duodenal biopsy. DNA extracted from the duodenal biopsy tissues was analyzed using three RT-PCRs targeting T. whipplei-specific regions of the 16S–23S rRNA intergenic spacer, hsp65, and Dig15 in parallel. In the samples positive in RT-PCRs, direct sequencing was performed for each RT-PCR target. The prevalence of T. whipplei was estimated based on the RT-PCR and sequencing results. Among the analyzed samples, T. whipplei was not detected. The prevalence of T. whipplei in duodenal biopsy tissues of Koreans was estimated to be less than 0.4%. This is the first study to attempt to detect T. whipplei in duodenal biopsy tissues of Koreans and estimate its prevalence. Our findings infer that while T. whipplei carriers exist in Korea, the incidence of duodenal infection/inflammation caused by T. whipplei is extremely rare.

An integrative description of Mesobiotus mandalori sp. nov. (Eutardigrada, Macrobiotoidea) from Poland

In this paper we provide integrative description (morphological and genetic) of the new eutardigrade species Mesobiotus mandalori sp. nov. from central Poland, found during research on the vertical distribution of Tardigrada in the Czerniejewskie Forests. Mesobiotus mandalori sp. nov. belongs to the harmsworthi group, but it differs from the members of this group in some morphometric characters of the bucco-pharyngeal apparatus, claws and/or eggs. Morphologically, Meb. mandalori sp. nov. is most similar to Meb. pseudocoronatus, but it differs from it by a larger bare egg diameter and smaller number of egg processes on egg circumference. Morphometrical data were supported by results of comparative genetic analysis of four molecular markers: 18S rRNA, 28S rRNA, ITS2 and COI. According to COI the new species is most similar to Meb. skorackii with the genetic distance of 21.1%.https://zoobank.org/urn:lsid:zoobank.org:act:0222E328-CF9E-4D04-8F8C-69C80515FEE2

Https://www.micropress.org/microaccess/micropaleontology/issue-404/article-2385

Ostracod crustaceans are among the most abundant microfossils in the geological record, thanks to their strongly calcified shell. Most of the fossils belong to the suborder Cytherocopina, which also has a rich diversity of living species. Unfortunately, many of the living representatives are known only by their shell morphology. Soft body always provides valuable insights into the phylogeny, and sometimes conflicts with currently accepted systematics of this group. We provide a complete description of Coquimba ishizakii Yajima 1978, collected from Jeju Island, South Korea. This is the first report on soft body morphology for the entire genus Coquimba Ohmert 1968, and the first record of this genus from Korea. Its soft body morphology is similar to other representatives of the family Hemicytheridae, where Coquimba currently belongs. After reviewing all records of C. ishizakii, we propose that several of them belong to different species, such as all those from Taiwan (modern and fossil). We also provide 18S rRNA and 28S rRNA sequences for this species, the latter being reported for the first time for the entire family Hemicytheridae.

Recent insights into the morphology, molecular characterization and tissue localization of the caprine Sarcocystis species infecting domestic goats (Capra hiricus): Sarcocystis moulei, Sarcocystis capracanis, and Sarcocystis hircicanis

Ninety-seven (64.67%) out of 150 domestic goats (Capra hiricus) carcasses were found to be infected by Sarcocystis moulei, Sarcocystis capracanis, and Sarcocystis hircicanis sarcocysts. Sarcocystis moulei macrosarcocysts were detected in the cardiac, esophageal, skeletal, lingual, and diaphragmatic muscles of seven goats (4.67%) out of the 150 examined animals, whereas the microscopic Sarcocystis species were found in (90/150 = 60%). Two morphotypes of S. moulei were observed. Morphotype (I) macrosarcocysts were large-sized oval, ovoid, spherical, and measured 2–7 mm in length x 2–6 mm in width. Sarcocystis moulei morphotype (II) macrosarcocysts were spindle-shaped, spheroid, sometimes elongated, and measured 1.8–6 x 0.5–2 mm. By TEM, all S. moulei morphotypes were ultrastructurally the same and had a sarcocyst wall that was characterized by highly branched or cauliflower-like villar protrusions (VP) with dumbbell-like structures. The VP interior was packed with well-developed microtubules in longitudinal and cross arrangements. Sarcocystis moulei cyst wall was 3–6 μm thick. Sarcocystis capracanis microsarcocysts detected herein had a cyst wall that ranged from 4–8 μm in thickness. The VP was upright finger-like or cylindrical. The PVM had electron-dense corrugations in the region of the VP. Few amounts of microfilaments were detected inside the cores of VP. Sarcocystis hircicanis had a thinner cyst wall (~1–3 μm) with hairy long VP that ranged from 1 to 7.5 μm in length. Microtubules were missing inside the cores of the VP. The three caprine Sarcocystis species were molecularly characterized on the level of the 18S rRNA, 28S rRNA, and Cox1 genes.

Margelopsid species search taxonomic home within Corymorphidae and Boreohydridae

Planktonic lifestyle of polyps in representatives of Margelopsidae are very different from all other species in the hydrozoan clade Aplanulata. Their evolutionary origin and phylogenetic position have been the subject of significant speculation. A recent molecular study based only on COI data placed Margelopsidae as a sister group to all Aplanulata, an unexpected result because margelopsid morphology suggests affiliation with Tubulariidae or Corymorphidae. Here we used multigene analyses, including nuclear (18S rRNA and 28S rRNA) and mitochondrial (16S rRNA and COI) markers of the hydroid stage of the margelopsid species Margelopsis haeckelii and the medusa stage of Margelopsis hartlaubii to resolve their phylogenetic position with respect to other hydrozoans. Our data provide strong evidence that M. haeckelii, the type species of Margelopsis, is a member of the family Corymorphidae. In contrast, M. hartlaubii is sister to Plotocnide borealis, a member of Boreohydridae. These results call into question the validity of the genus Margelopsis and the family Margelopsidae. The systematic position of M. haeckelii is discussed in light of the phylogeny of Corymorphidae.

Description of Pseudocalidococcus azoricus gen. sp. nov. (Thermosynechococcaceae, Cyanobacteria), a Rare but Widely Distributed Coccoid Cyanobacteria

Coccoid cyanobacteria represent an important part of cyanobacterial freshwater diversity, with many studied strains in public databases identified as Synechococcus. This is a diverse genus, both morphologically and ecologically, with a global distribution. However, many of the so-called Synechococcus-like cyanobacteria strains could represent several independent genera that require further studies. In this work, four strains of a Synechococcus-like cyanobacteria isolated from freshwater lakes and terrestrial atmophytic habitats on São Miguel and Flores Islands (Azores archipelago) were studied genetically using the 16S rRNA and 16S–23S rRNA ITS, morphologically with light and transmission electron microscopy, and ecologically. A draft genome was produced from the reference strain by Illumina sequencing, which allowed a more complete phylogenetic study and a deeper taxonomic analysis, revealing a divergent phylogenetic evolution and low ANI and AAI values (69.4% and 66.3%, respectively) to Thermosynechococcus, the closest phylogenetic genus. Although morphologically similar to Synechococcus, the 16S rRNA and genome phylogenetic analysis placed the studied strains in a clade sister to Thermosynechococcus, inside the Thermosynechococcaceae. Thus, Pseudocalidococcus azoricus gen. sp. nov. is described as a new coccoid freshwater genus and species from the Azores archipelago. A detailed comparison with similar morphological taxa is provided, supporting the separation of the new genus. The 16S rRNA with a high genetic similarity to other strains from several continents identified as Synechococcus sp. suggests that the new genus probably has a worldwide distribution. Future studies should be performed to clarify the taxonomic identity of those strains.

Phylogeography of an insect inhabiting ‘Sky Islands’: the relationships among genetic structures and geographical characteristics, geohistorical characteristics, and cyclical climate changes

Abstract When gene flow has been restricted between populations, the genetic structure of such species often reflects geohistory and climate changes. Populations of species inhabiting high-altitude regions, known as ‘Sky Islands’, are isolated and exhibit restricted gene flow, so they often have habitat-specific genetic structures that correspond to their surrounding geographical structures. Here we focus on a limnephilid caddisfly, Rivulophilus sakaii, which inhabits the alpine zone of Japan. Phylogenetic analyses were conducted based on the mtDNA COI and 16S rRNA regions, and the nDNA 18S rRNA, 28S rRNA, CAD, EF1-α, and POL-II regions; the results indicated three phylogeographically differentiated intraspecific lineages. Haplotype network and demographic analyses based on the mtDNA COI region suggested the size of the respective isolated populations has stabilized. This suggests that mountain formation in the Japanese Archipelago due to volcanic activity has resulted in barriers to migration and dispersal between high-altitude aquatic insect populations. This was inferred to be an effect of Quaternary climate changes that caused vertical distributional shifts following mountain formation, resulting in repeated connection and fragmentation of the populations. This is important supporting information with regard to discussing the effects and functions of geohistory and climatic changes on the phylogenetic evolution of organisms presently inhabiting interglacial ‘Sky Islands’.

Discovery of Cyanobacteria Associated with Cycads and Description of Three Novel Species in Desmonostoc (Nostocaceae)

Heterocyte-forming cyanobacteria form symbiotic relationships with several lineages of plants. Here, twenty (20) strains of endosymbiotic cyanobacteria (cyanobionts) with Nostoc-like morphologies were isolated from the highly specialized coralloid roots of five host species in Cycadales—Cycas debaoensis, C. fairylakea, C. elongata, Ceratozamia robusta, and Macrozamia moorei. Molecular phylogeny based on the 16S rRNA gene placed these strains into seven different taxa within the Nostocaceae, specifically under the genera Desmonostoc and Dendronalium. The percent dissimilarity and unique patterns in the secondary structures of the D1-D1′, Box-B, V2, and V3 helices, which were based on the 16S–23S rRNA internal transcribed spacer (ITS) regions, supported three distinct species in Desmonostoc. These three morphologically distinct novel species are described in this report: Desmonostoc debaoense sp. nov., Desmonostoc meilinense sp. nov., and Desmonostoc xianhuense sp. nov. Other investigated strains were phylogenetically identified as members of the recently discovered genus Dendronalium and represent the first report of association of that genus with cycads. Our findings suggest that the order Cycadales hosts diverse species of cyanobionts in their coralloid roots and that many potential unreported or novel taxa are present in cycads occurring in their natural habitat and await discovery.

Investigating seed bank potential of crustose coralline algae using DNA metabarcoding.

To examine the potential for the autogenic ecosystem engineers, crustose coralline algae (CCA), to serve as seed banks or refugia for life stages of other species, it is critical to develop sampling protocols that reflect the diversity of life present. In this pilot study on two shallow water species of CCA collected from Raoul Island (Kermadec Islands; Rangitāhua) New Zealand, we investigated two preservation methods (ethanol vs. silica gel), sampled inner and outer regions of the crusts, and used DNA metabarcoding and seven genes/gene regions (16S rRNA, 18S rRNA, 23S rRNA, cox1, rbcL, and tufA genes and the ITS rRNA region) to develop a protocol for taxa identification. The results revealed immense diversity, with typically more taxa identified within the inner layers than the outer layers. As highlighted in other metabarcoding studies and in earlier work on rhodoliths (nodose coralline algae), reference databases are incomplete, and to some extent, the use of multiple markers mitigates this issue. Specifically, the 23S rRNA and rbcL genes are currently more suitable for identifying algae, while the cox1 gene fares better at capturing the diversity present inclusive of algae. Further investigation of these autogenic ecosystem engineers that likely act as marine seed banks is needed.

Stigonema lichenoides sp. nov. (Nostocales, Сyanobacteria): новый вид, выделенный из лишайника Ephebe lanata

A new cyanobacterium species Stigonema lichenoides Patova, Novakovskaya, Shalygin et Shadrin, sp. nov., a photobiont of the cyanolichen Ephebe lanata, has been described. The samples were collected from rocky substrates in the mountain tundra of the Northern Urals. A detailed morphological description is given and differences between this species and related taxa of the genus Stigonema are discussed. A molecular phylogenetic analysis of the studied samples was performed using 16S–23S rRNA sequences. The correlation of morphological and molecular data, as well as the comparison of the sequences obtained from the samples with data available in the NCBI database, allowed the identification of a new species of the genus Stigonema.

Phytoplankton Ecology in an Oilsands End Pit Lake

Alberta oilsands mining and extraction have produced over 1 trillion litres of tailings wastewater (AER 2021) containing several compounds of concern (Cossey et al. 2021). End-pit lakes are a low-cost, long-term proposed strategy of tailings reclamation that sequester tailings in a mined-out pit under a freshwater cap. Through dilution and biogeochemical processes, the water cap should over time develop into a functional ecosystem integrable with the local watershed (Cossey et al. 2021, Saborimanesh 2021). Established in 2012, Base Mine Lake is currently the only full-scale pilot end pit lake developed by the Alberta oilsands industry and requires further investigation to validate end pit lakes as a tailings reclamation technology (Cossey et al. 2021). The first stage of reclamation requires the development of a phytoplankton community, which serves as the base of the aquatic food web (CEMA 2012). The primary objective of this study was to characterize the phytoplanktxon community over time in BML from 2016 to 2021 to determine how community composition and abundances shift over seasons and years. Characterization used Illumina gene sequencing targeting 16S rRNA, 18S rRNA, and 23S rRNA gene amplicons, giving relative abundance data over time for phytoplankton. Cell count data was used to verify gene sequencing results. The phytoplankton community composition and diversity in Base Mine Lake was compared to those of a freshwater reservoir and a tailings pond. Analysis of gene sequencing data revealed that major genera of phytoplankton included Cryptomonas (Cryptophyceae), Choricystis (Trebouxiophyceae), Euglena (Euglenales), and Synechococcus (Synechococcales), all of which appear to exhibit seasonal blooms during 2016-2021 (Fig. 1). Sequencing analysis also indicated that Base Mine Lake and its freshwater input source Beaver Creek Reservoir shared many of the same genera but different strains/species of those genera. This suggested that the distinct conditions in each aquatic site may have selected for distinct strains. Diversity analyses of gene sequencing data revealed that phytoplankton diversity in Base Mine Lake was intermediate between that of its freshwater input reservoir and a tailings pond. There currently exists no low-cost, large-scale treatment method that fully reclaims tailings water (Cossey et al. 2021, Saborimanesh 2021). End-pit lakes retain tailings until recalcitrant compounds are degraded to near-environmental levels (Saborimanesh 2021, CEMA 2012), but further research is required before end-pit lakes can be approved as a viable reclamation technology (Cossey et al. 2021). Although research is available now on native microbial communities in tailings waters, knowledge on their contributions to an aquatic microbial food web is limited (Saborimanesh 2021). This proposed research is the first of its kind to examine the contribution of phytoplankton to end pit lake food web ecology. This will advance knowledge of end-pit lakes as a reclamation strategy in effort to reduce the environmental footprint of tailings water.

Genomic diversity and taxonomic marker for Arcobacter species.

Arcobacter was recognized as an emerging enteropathogen and controversies regarding its classification persisted. This study aimed to reevaluate the taxonomy of Arcobacter utilizing the 16S rRNA gene, 23S rRNA gene, single-copy orthologous genes, as well as genomic indices such as Average Nucleotide Identity (ANI) and in silico DNA-DNA hybridization (isDDH). The taxonomy of this genus was reevaluated in this study using multiple indices with a dataset of 371 genomes comprising 34 known species and 14 potentially new species. Good discrimination could be achieved only in some species but not for the species with higher sequence similarity using the comparisons of the 16S rRNA gene and 23S rRNA gene sequences. A high-accuracy phylogenomic approach for Arcobacter was established using 84 single-copy orthologous genes obtained through various bioinformatics methods. One marker gene (gene711), which was found to possess the same distinguishing ability as ANI, isDDH, and single-copy orthologous methods, was identified as a reliable locus for inferring the phylogeny of the genus. The effective species classification was achieved by employing gene711 with a sequence similarity exceeding 96%, even for species like A. cloacae, A. lanthieri, and A. skirrowii, which exhibited ambiguous classification using ANI and isDDH. Additionally, excellent subspecies categorizing among A. cryaerophilus could be distinguished using gene711. In conclusion, this framework strategy had the potential advantage of developing rapid species identification, particularly for highly variable species, providing a novel insight into the behavior and characteristics of Arcobacter.

Biodiversity of symbiotic microalgae associated with meiofaunal marine acoels in Southern Japan.

Acoels in the family Convolutidae are commonly found with microalgal symbionts. Convolutids can host green algal Tetraselmis and dinoflagellates within the family Symbiodiniaceae and the genus Amphidinium. The diversity of these microalgae has not been well surveyed. In this study, we used PCR and culture techniques to demonstrate the biodiversity of Tetraselmis and dinoflagellates in symbiosis with meiofaunal acoels. Here, 66 acoels were collected from seven localities around Okinawa, Ishigaki, and Kochi, Japan. While convolutids were heavily represented in this sampling, some acoels formed a clade outside Convolutidae and are potentially a new family of acoels harboring symbiotic microalgae. From the acoels collected, a total of 32 Tetraselmis and 26 Symbiodiniaceae cultures were established. Molecular phylogenies were constructed from cultured material (and from total host DNA) using the 18S rRNA gene (Tetraselmis) and 28S rRNA gene (dinoflagellates). The majority of Tetraselmis sequences grouped within the T. astigmatica clade but strains closely related to T. convolutae, T. marina, and T. gracilis were also observed. This is the first report of Tetraselmis species, other than T. convolutae, naturally associating with acoels. For dinoflagellates, members of Cladocopium and Miliolidium were observed, but most Symbiodiniaceae sequences formed clusters within Symbiodinium, grouping with S. natans, or sister to S. tridacnidorum. Several new Symbiodinium sequences from this study may represent novel species. This is the first molecular record of Miliolidium and Symbiodinium from acoels. Microalgal strains from this study will provide a necessary framework for future taxonomic studies and research on symbiotic relationships between acoels and microalgae.

Diversity and molecular identification of endosymbionts of the white-flies Bemisia tabaci and Trialeurodes vaporariorum

The infection of insects with symbiotic bacteria has significant implications for the evolution and ecology of the hosts. Maternally inherited symbionts associated with B. tabaci and T. vaporariorum whiteflies play a vital role in their fitness and survival. Whitefly symbionts have been identified in many countries, but no study has been undertaken in Iraq and the UK. For the first time in both countries, the molecular identification and diversity of the symbionts of both whiteflies have been investigated in the present study. Fourteen populations of B. tabaci from Iraq and twenty populations of T. vaporariorum from the UK were used to detect and identify seven common endosymbiont bacteria associated with whitefly using the 16S rRNA and 23S rRNA nuclear markers. All females and males of B. tabaci harboured one primary symbiont, Portiera aleyrodidarum, and almost all of both sexes of all B. tabaci species have the two secondary symbionts Hamiltonella sp. and Rickettsia sp. The primary symbiont P. aleyrodidarum was also detected in both sexes of T. vaporariorum, whereas only one secondary symbiont, Arsenophonus sp., was detected in almost all females but not in the males. Additionally, an investigation into genetic diversity using three genes of the Arsenophonus sp. populations showed no variation among different populations. The results supported the notion that Arsenophonus sp. might play an important role in the survival of T. vaporariorum females and maybe a killer of male whiteflies. Also, secondary symbionts Hamiltonella sp. and Rickettsia sp. with B. tabaci could support their host’s fitness and survival. These findings reveal the endosymbionts associated with B. tabaci and T. vaporariorum in Iraq and the UK, respectively. Further investigation is needed to understand the roles of these symbionts in both countries

Genotypes and virulence factors in Staphylococcus aureus isolated from dairy cows with subclinical mastitis in Serbia

This study aimed to determine the prevalence of Staphylococcus aureus subclinical mastitis and to genotype the S. aureus isolates using the 16S–23S rRNA intergenic spacer (RS-PCR) method. In addition, the genes responsible for adherence, biofilm formation, host evasion, tissue necrosis, methicillin resistance, and enterotoxin production of S. aureus were investigated. The overall prevalence of S. aureus subclinical mastitis in lactating cows was 5.4% (95% confidence interval, CI=4.7–6.1%). An increased risk of S. aureus intramammary infection was observed on small family farms (odds ratio, OR=4.2, 95% CI=2.6–6.6, P < 0.001) and medium-sized farms (OR=3.5, 95% CI=2.2–5.7, P < 0.001). The RS-PCR analysis revealed 44 genotypes and genotype variants, of which 15 new genotypes and five new variants were detected within small and medium-sized farms. S. aureus isolates of new genotypes and genotype variants carried the clfA gene responsible for adherence at a lower frequency (64.8%) and enterotoxin-producing genes sea (20.4%), seb (14.8%) and sec (14.8%) at a higher frequency than the other known genotypes (P < 0.001), and were confirmed to carry the sej and sep genes. The spa gene was detected in all S. aureus isolates, whereas none harbored bap, ser, or tsst-1 genes. Methicillin-resistant strains of S. aureus (MRSA) were also detected, with a higher prevalence (19.2%) on large farms with more than 50 cows (P < 0.001). Using molecular techniques as diagnostic tools provides a better understanding of intramammary staphylococcal infections’ occurrence, spread, and eradication.