rRNA Fragments Research Articles

Systematic Assessment of Small RNA Profiling in Human Extracellular Vesicles.

Extracellular vesicles (EVs) are produced and released by most cells and are now recognized to play a role in intercellular communication through the delivery of molecular cargo, including proteins, lipids, and RNA. Small RNA sequencing (small RNA-seq) has been widely used to characterize the small RNA content in EVs. However, there is a lack of a systematic assessment of the quality, technical biases, RNA composition, and RNA biotypes enrichment for small RNA profiling of EVs across cell types, biofluids, and conditions. We collected and reanalyzed small RNA-seq datasets for 2756 samples from 83 studies involving 55 with EVs only and 28 with both EVs and matched donor cells. We assessed their quality by the total number of reads after adapter trimming, the overall alignment rate to the host and non-host genomes, and the proportional abundance of total small RNA and specific biotypes, such as miRNA, tRNA, rRNA, and Y RNA. We found that EV extraction methods varied in their reproducibility in isolating small RNAs, with effects on small RNA composition. Comparing proportional abundances of RNA biotypes between EVs and matched donor cells, we discovered that rRNA and tRNA fragments were relatively enriched, but miRNAs and snoRNA were depleted in EVs. Except for the export of eight miRNAs being context-independent, the selective release of most miRNAs into EVs was study-specific. This work guides quality control and the selection of EV isolation methods and enhances the interpretation of small RNA contents and preferential loading in EVs.

Molecular Detection of Anaplasma phagocytophilum in Ixodid Ticks Infesting White Fulani Cattle in Zaria and its Environs, Kaduna State, Nigeria.

Anaplasmosis, a disease caused by various species of Anaplasma, poses important economic constraints to animal breeders. In Zaria, molecular detection of tick-borne pathogens infecting cattle is very sketchy. Being hematophagous, ticks are capable of transmitting disease agents such as viruses, bacteria and protozoa. This study was carried out to detect for the presence of Anaplasma pathogen in the various tick species infesting white Fulani breeds of cattle owing to their high population in Zaria and environs using a Polymerase Chain Reaction (PCR). Three hundred and eighty-four (n=384) white Fulani breed of cattle of varying age and sex were sampled from 32 herds in 4 villages (Bomo, Tofu, Ungwan Dabosa and Majeru). The DNA was extracted using Qiagen commercial kit following manufacturer’s instructions. Genomic DNA of the species of ticks was amplified in a semi-nested PCR targeting the 16S rRNA fragment of the Anaplasma spp at expected amplicon size of 711bp. The PCR products were purified using Gel Extraction Kit (Bioland, Scientific LLC) following the manufacturer’s protocol. All sequences were subjected to a Basic Local Alignment Search Tool (BLAST) to determined their identities and assess their homologues and similarities to those in the GenBank. A neighbor-joining tree was generated using the Molecular Engineering Genetic Analysis (MEGA 7.0) to derive the close relationship between the isolates. The result of this study implies that Anaplasma spp (MN044909) is present in all the five species of ticks infesting cattle in Zaria, Kaduna State, Nigeria and pose a high risk on humans and animals health.
 Anaplasmosis, a disease caused by various species of Anaplasma, poses important economic constraints to animal breeders. In Zaria, molecular detection of tick-borne pathogens infecting cattle is very sketchy. Being hematophagous, ticks are capable of transmitting disease agents such as viruses, bacteria and protozoa. This study was carried out to detect for the presence of Anaplasma pathogen in the various tick species infesting white Fulani breeds of cattle owing to their high population in Zaria and environs using a Polymerase Chain Reaction (PCR). Three hundred and eighty-four (n=384) white Fulani breed of cattle of varying age and sex were sampled from 32 herds in 4 villages (Bomo, Tofu, Ungwan Dabosa and Majeru). The DNA was extracted using Qiagen commercial kit following manufacturer’s instructions. Genomic DNA of the species of ticks was amplified in a semi-nested PCR targeting the 16S rRNA fragment of the Anaplasma spp at expected amplicon size of 711bp. The PCR products were purified using Gel Extraction Kit (Bioland, Scientific LLC) following the manufacturer’s protocol. All sequences were subjected to a Basic Local Alignment Search Tool (BLAST) to determined their identities and assess their homologues and similarities to those in the GenBank. A neighbor-joining tree was generated using the Molecular Engineering Genetic Analysis (MEGA 7.0) to derive the close relationship between the isolates. The result of this study implies that Anaplasma spp (MN044909) is present in all the five species of ticks infesting cattle in Zaria, Kaduna State, Nigeria and pose a high risk on humans and animals health.

An integrative study of morphological and molecular characterization of Diplectanum aequans (Diplectanidae: Monogenea) infecting European sea bass Dicentrarchus labrax (Linnaeus, 1758) from Turkish coasts

The diplectanid monogenean Diplectanum aequans (D. aequans) (Wegener, 1857) is one of the most important ectoparasites in the wild and cultured European sea bass Dicentrarchus labrax (Dic. labrax) (Linnaeus, 1758) worldwide. Presently, there is no information on the molecular data of D. aequans from Turkish coasts. In the present study, diplectanid monogeneans were collected from wild and cultured Dic. labrax in the Black Sea (FAO area 37.4.2) and Aegean Sea (FAO area 37.1.3) coasts of Türkiye, morphologically identified, and genetically characterized by sequencing of 28S rRNA and 18S rRNA fragments. The collected diplectanids were unambiguously identified as D. aequans based on detailed morphological features and sequence characterization of partial 28S rRNA and 18S rRNA genes. The overall prevalence and mean intensity of D. aequans were 100% and 15.1, respectively. This study reports first information of molecular (28S and 18S rRNA) evidence of D. aequans from Dic. labrax in the Turkish coasts. The new 18S and 28S rRNA sequences for D. aequans isolated from the Black Sea and Aegean Sea are genetically characterized. The 28S and 18S rRNA sequences of D. aequans can be used to resolve the phylogenetic positions of species found in the family Diplectanidae from the Black and Mediterranean Sea.

A New Species of Terrestrial-Breeding Frog, Genus Pristimantis (Anura: Strabomantidae), from the Peruvian Yungas of Central Peru

We describe a new species of terrestrial-breeding frog of the genus Pristimantis from the Peruvian Yungas ecoregion of central Peru, Junin Department. The description is based on the observation of morphological features, color patterns of fourteen specimens, bioacoustic traits of the advertisement calls, and a phylogenetic analysis using a sequence fragment of 16S rRNA. The new species is mainly characterized by having a rounded or truncate snout in dorsal view, presence of dorsolateral folds, tympanic membrane and annulus distinct, absence of flash marks on the hidden surfaces of flanks and hindlimbs, and a small size (maximum SVL 15.6 mm in males and 19.3 mm in females). We provide diagnostic traits to differentiate the new species from phylogenetically close relatives, as well as morphologically similar and sympatric species of Pristimantis.

Out of sight, but not out of mind: a name for the Stefania (Anura: Hemiphractidae) from the summit of Murisipán-tepui (Bolívar State, Venezuela)

Previous molecular analyses of the frog genus Stefania have shown that species boundaries in that group are often difficult to delineate when solely based on morphology. As a consequence, “taxonomically cryptic” species are not uncommon in the genus. Several highland Stefania species remain to be described, some potentially critically endangered due to their highly restricted geographic ranges. One case is the microendemic Stefania population from the summit of Murisipán-tepui, a poorly explored table-top mountain in the Los Testigos Massif, a small tepui mountain range located north to the much larger Chimantá Massif in southern Venezuela. That population, mistaken as S. satelles for two decades, was later reported as Stefania sp. 2 and belongs to the S. ginesi clade. The new species is phylogenetically distinct but phenotypically similar to S. satelles, a taxon restricted to its type-locality, i.e. the summit of Aprada-tepui in Venezuela. The new species is described based on morphology and cranial osteology. Molecular divergences with S. satelles are high (> 8%) in the barcoding fragment of 16S rRNA. Amended definitions for the two other described species in the S. ginesi clade (S. ginesi and S. satelles) are also provided. The new species should be listed as critically endangered according to IUCN criteria.

PCR-based gene synthesis with overlapping unisense-oligomers asymmetric extension supported by a simulator for oligonucleotide extension achieved 1kbp dsDNA.

We formulated a method to synthesize 1kbp DNA fragments using 'oligomer unidirectional joining method' via asymmetric extension supported by a simulator for oligonucleotide extension (AESOE). In this study, trials were conducted on 41 sets of different genomic pieces of ten flaviviral genomes, and 31 bacterial 16s rRNA fragments with sizes ranging from 500 bases to 1.0kbp. Synthetic gene production was found to be successful in all those sets. The synthesis method has three steps: the first step is a seven-linked AESOE, the second step is the linking of the 400-base fragments from the first step, and the third step is the final amplification. Our present approach is highly reproducible and may no longer require optimization of oligomer design.

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa.

Lung infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AECs) deliver microRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AECs transfer multiple distinct short RNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%), and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S ribosomal RNA (rRNA) external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AECs in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD downregulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with ciprofloxacin-resistant P. aeruginosa infections.NEW & NOTEWORTHY Human RNA fragments transported in extracellular vesicles interfere with Pseudomonas aeruginosa drug efflux pumps. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

Multiplexed Nanopore-Based Nucleic Acid Sensing and Bacterial Identification Using DNA Dumbbell Nanoswitches.

Multiplexed nucleic acid sensing methods with high specificity are vital for clinical diagnostics and infectious disease control, especially in the postpandemic era. Nanopore sensing techniques have developed in the past two decades, offering versatile tools for biosensing while enabling highly sensitive analyte measurements at the single-molecule level. Here, we establish a nanopore sensor based on DNA dumbbell nanoswitches for multiplexed nucleic acid detection and bacterial identification. The DNA nanotechnology-based sensor switches from an "open" into a "closed" state when a target strand hybridizes to two sequence-specific sensing overhangs. The loop in the DNA pulls two groups of dumbbells together. The change in topology results in an easily recognized peak in the current trace. Simultaneous detection of four different sequences was achieved by assembling four DNA dumbbell nanoswitches on one carrier. The high specificity of the dumbbell nanoswitch was verified by distinguishing single base variants in DNA and RNA targets using four barcoded carriers in multiplexed measurements. By combining multiple dumbbell nanoswitches with barcoded DNA carriers, we identified different bacterial species even with high sequence similarity by detecting strain specific 16S ribosomal RNA (rRNA) fragments.

Taxonomy of Antarctic Buccinoidea (Gastropoda: Neogastropoda) revisited based on molecular data

The superfamily Buccinoidea is the most speciose group of Neogastropoda within the Antarctic Convergence, with ~70 species classified in 21 genera, but is still poorly represented in molecular phylogenies. The first molecular data on the group presented in the recent phylogeny of the Buccinoidea (Kantor et al. 2022) lacked many important lineages, thereby limiting inference of the relationships of Antarctic Buccinoidea. We revisited relationships of the Antarctic Buccinoidea, involving recently collected molecular-grade samples from the bathyal and abyssal depths of the Scotia Sea, the Weddel Sea and adjacent regions. Our data set includes 25 species (including six genera studied on a molecular basis for the first time), sequenced for five phylogenetic markers: the barcode fragment of cox1 gene, fragments of ribosomal 12S, 16S rRNA and 28S and nuclear H3 genes. Based on phylogenetic reconstructions, we synonymise the genus Lussitromina with Falsitromina and reassign the latter from Cominellidae to Prosiphonidae. We confirm the placement of four further genera, Drepanodontus, Germonea, Parabuccinum and Spikebuccinum in Prosiphonidae. We detect a previously unrecognised deep lineage of the family Prosiphonidae and describe this as the new genus Scotiabuccinum. The genus Parabuccinum, previously recorded in the Magellanic province and off the eastern coast of Argentina was reported for the first time within the Antarctic Convergence. We discover four previously unrecognised species of Antarctic Prosiphonidae and these are the first Buccinoidea from Antarctic waters described based on molecular data. According to our data, all but two species of Antarctic Buccinoidea belong to the family Prosiphonidae. Seven of the nineteen Recent Antarctic Prosiphonidae genera (36%) cross the boundaries of the Convergence and eight Antarctic genera are monotypic (42%). Currently no Buccinoidea species are known to occur both within and outside the Antarctic Convergence. ZooBank: urn:lsid:zoobank.org:pub:DEEA1599-C951-414E-9098-563EAD10BD57

The isolation of novel terrestrial Streptomyces strains with antimicrobial and cytotoxic properties

Streptomyces are very important Gram-positive filamentous bacteria because they can produce many beneficial secondary metabolites with various biological activities. The study aims to isolate and identify novel Streptomyces strains from terrestrial habitats in the United Arab Emirates (UAE) with antimicrobial and cytotoxic properties. The antimicrobial efficacy was assessed using the disk and well susceptibility methods. Crude ethyl acetate extracts of active-producing strains were used to determine the antimicrobial activity by minimum inhibitory concentration (MIC) against E. coli, S. aureus, C. albicans, S. cerevisiae, B. subtilis, and E. coli-ESBL (extended-spectrum Beta-Lactamase), and cytotoxic properties against HeLa cells by MTT assay and DAPI staining. The novelty of the strains was determined by sequencing the 16S rRNA fragment. Results showed that antimicrobial activity was observed for CSK1, CSK3, CSW2, CSU1, CSU2, and CSG1 strains, with zones of inhibition ranging between 16 and 35 mm. Minimum inhibitory concentrations of ethyl acetate extracts ranged from 0.21 to 12.17 µg/mL, with the highest inhibitory effect against S. aureus ranging between 0.21 and 0.29 µg/mL. Some strains (CSK3, CSW2, CSU1 and CSG1) also displayed cytotoxic activity against the HeLa cancer cell line with IC50 values ranging between 3.46 and 9.74 µg/µL, and apparent DNA fragmentation and chromatin condensation. This study indicates that Streptomyces sp. strains isolated from different soil habitats in the UAE can produce antimicrobial compounds that can treat microbial infections. In addition, some strains’ cytotoxicity suggests that they are producing bioactive compounds that can lead to drug discovery.

A new species of the genus Catillopecten (Bivalvia: Pectinoidea: Propeamussiidae): morphology, mitochondrial genome, and phylogenetic relationship

Catillopecten is a small genus of deep-sea glass scallops, but its diversity is poorly known in many parts of the world ocean. We described C. margaritatus n. sp. (Pectinoidea: Propeamussiidae), and performed morphological analyses and DNA sequencing, and estimated the divergence time of scallops based on samples collected from Haima cold seep in the South China Sea. Morphologically, the new species can be distinguished from congeneric species by its large shell size, relatively small auricle length, absence of monocrystal aerials, presence of longitudinal radial ridges on the left valve, and the alternated rounded striae and distal and proximal growth lines of prisms on the right valve. Anatomically, this new species can be distinguished from C. vulcani by its anteriorly located auriculate gills, compared to the centrally located lamellar gills of the latter, and the different locations of the pericardium. Sequence comparison and phylogenetic analysis based on the 18S rRNA fragments supported the placement of the new species in Catillopecten. We also report the mitogenome of C. margaritatus n. sp. as the only reported mitogenome of the family Propeamussiidae, which differs from those of other scallops substantially in gene order arrangement. Divergence time estimation revealed that Propeamussiidae and Pectinidae diverged in the early Carboniferous, while Catillopecten and Parvamussium diverged during the late Cretaceous to early Eocene. Finally, we presented a key to the species of Catillopecten.

Glyphosate treatment mediates the accumulation of small discrete 5'- and 3'-terminal fragments of 18S rRNA in plant cells.

Under many kinds of stress, eukaryotic cells rapidly decrease the overall translation level of the majority of mRNAs. However, some molecular mechanisms of protein synthesis inhibition like phosphorylation of eukaryotic elongation factor 2 (eEF2), which are known to be functional in animals and yeast, are not implemented in plants. We suggest that there is an alternative mechanism for the inhibition of protein synthesis in plant cells and possibly, in other eukaryotes, which is based on the discrete fragmentation of 18S rRNA molecules within small ribosomal subunits. We identified four stress-induced small RNAs, which are 5'- and 3'-terminal fragments of 18S rRNA. In the present work, we studied the induction of 18S rRNA discrete fragmentation and phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) in germinated wheat embryos in the presence of glyphosate, which imitates the condition of amino acid starvation. Using northern and western blotting, we have shown that stress-induced 18S rRNA fragments started to accumulate in wheat embryos at glyphosate concentrations that did not evoke eIF2α phosphorylation. It was also found that cleavage of 18S rRNA near the 5'-terminus began much earlier than eIF2α phosphorylation, which became noticeable only at higher concentration (500 μM) of glyphosate. This result suggests that discrete fragmentation of 18S rRNA may constitute a regulatory mechanism of mRNA translation in response to stress and may occur in plant cells in parallel with and independently of eIF2α phosphorylation. The identified small 5'- and 3'-terminal fragments of 18S rRNA that accumulate during various stresses may serve as stress resistance markers in the breeding of economically important plant crops.

Identification of a new Sarcocystis sp. in marsh deer (Blastocerus dichotomus) from wetlands of Argentina.

The marsh deer (Blastocerus dichotomus) is the largest South American native deer species and is listed as "Vulnerable" by IUCN due to the population reduction. As part of a conservation and disease surveillance program, muscle samples from 14 marsh deer found dead in 2016 and 2017 in northeast Argentina were obtained at necropsy. Samples from each animal were processed as pooled muscles (heart, diaphragm, tongue and hindlimb) by homogenization and direct microscopical observation to detect intracellular Sarcocystis spp. cysts. Sarcocysts were observed in six samples, and several cysts recovered from two samples were processed by transmission electron microscopy. The cysts were thin-walled and showed a cyst-wall ultrastructure with ribbon-like protrusions similar to other species using cervids as intermediate host and canids as definitive hosts. Genomic DNA from individual sarcocysts from three marsh deer were successfully amplified by PCR of 18S rRNA and COI gene fragments and further sequenced. Sequence comparison revealed a 99.3-100% identity among them and only 93.7-96.6% and 88.8-89.7% identity at 18S rRNA and COI markers, respectively, with other Sarcocystis spp. Despite morphological similarities, the high sequence divergence at 18S rRNA and COI fragments allowed the assumption that Sarcocystis sp. from marsh deer is a different species from others using cervids as intermediate hosts. Therefore, we propose the name Sarcocystis blastoceris n. sp. for the species infecting marsh deer.

Detection of parasite-derived tRNA and rRNA fragments in the peripheral blood of mice experimentally infected with Leishmania donovani and Leishmania amazonensis using next-generation sequencing analysis.

All prokaryotes and eukaryotes, including parasites, release extracellular vesicles or exosomes that contain selected proteins, lipids, nucleic acids, glycoconjugates, and metabolites. Leishmania exosomes are highly enriched in small RNAs derived from the rRNAs and tRNAs of the protozoan parasite species. Here, using plasma exosomes isolated by a kit and next-generation sequencing, we report the detection of fragments of parasite-derived rRNAs and tRNAs in the peripheral plasma samples of mice experimentally infected with Leishmania donovani and Leishmania amazonensis, the causative agents of Old World visceral leishmaniasis and New World disseminated cutaneous leishmaniasis, respectively. Detected RNA molecules of 28S rRNA, 5.8S rRNA, tRNA-Glu, and tRNA-Thr were common to both plasma samples of mice inoculated with L. donovani and L. amazonensis, whereas tRNA-Ile and tRNA-Trp were only detected in L. amazonensis-infected mice. The detected rRNAs and tRNA isotypes were matched with the exosomal components reported in a previous key study. Our preliminary results suggested that parasite-derived small RNAs were circulating in the blood of mice infected with Leishmania species, providing a better understanding of the roles of exosomal components in leishmaniasis and also new insights into exosome-based biomarkers for Leishmania infection.

First report of Sarcocystis spp. (Apicomplexa, Sarcocystidae) in Lagostomus maximus (Desmarest, 1917) (Rodentia, Chinchillidae) in Argentina

Sarcocystis is a genus of intracellular parasitic protozoa that infects various species of mammals, birds, and reptiles worldwide. At least 46 Sarcocystis species naturally infect rodents as intermediate hosts producing tissue cysts. This study aimed to provide the first report and molecular characterisation of Sarcocystis spp. in muscles from plains viscacha (Lagostomus maximus) in Argentina. Muscle samples of 53 plains viscachas from three provinces of Argentina were processed by homogenisation and optical microscopy to detect tissue cysts. Positive samples were analysed by PCR-sequencing, using the following markers: 18S rRNA, ITS1, and coxI. The 18S rRNA and coxI consensus sequences were aligned with other sequences from Sarcocystis spp., and phylogenetic trees were constructed. Of all animals processed, 13.2% (7/53) harboured Sarcocystis sp. cysts. 18S rRNA consensus sequences were obtained from four muscle samples and one individual cyst, and they showed 99.88-100% similarity, except for the cyst sequence, which showed 97.11% homology. Similarities of only 96-97% were recorded in the 18S rRNA fragment with other Sarcocystis spp. whose sequences are available in the GenBank. The five coxI fragment sequences obtained were 100% identical and showed an identity of 99.41-99.48% with S. canis. For ITS1 only short and low-quality sequences were obtained. In the phylogenetic trees, all the sequences from plains viscachas were positioned together in a branch separated from other Sarcocystis spp. These results could be related to new Sarcocystis spp. producing sarcocysts in plains viscachas. Besides, comprehensive cyst morphological analysis using TEM from the new Sarcocystis species will allow a description of the cyst wall ultrastructure. In this sense, further studies are needed to deepen these findings and elucidate other potential intermediate and possible definitive hosts.

Fatal Sarcocystis calchasi hepatitis in a captive Indian ringneck parakeet (Psittacula krameri manillensis)

An adult Indian ringneck parakeet (Psittacula krameri manillensis) from an outdoor aviary in Sacramento, California was found dead on the nest box. Postmortem examination showed firm, enlarged, yellow‑tinged liver and splenomegaly. Multifocal to coalescing, acute necrosis with macrophages, lymphocytes, plasma cells, and periportal ductular reaction were seen on liver histology with extra- and intracellular schizonts and merozoites. A few schizonts and lymphohistiocytic inflammation were seen in the spleen. Toxoplasma gondii, Sarcocystis neurona, S. falcatula and Neospora caninum were ruled out by immunohistochemistry. PCR of the liver for Sarcocystis spp. Based on the positive amplification/PCR of ITS1 segment and sequencing of 28S rRNA fragment, S. calchasi was confirmed. The splanchnic presentation of S. calchasi in this parakeet resembles the acute infection described experimentally in domestic pigeons (Columba livia f. domestica) and cockatiels (Nymphicus hollandicus). Since large populations of red-tailed hawks (Buteo jamaicensis) and Cooper's hawk (Accipiter cooperi), the likely definitive hosts of S. calchasi in North America, inhabit the Sacramento area, their presence near outdoor aviaries may account for the source of S. calchasi infective sporocysts.

Analysis of genetic diversity and population structure of Babesia gibsoni.

Babesia gibsoni is a tick-borne apicomplexan protozoan causing canine babesiosis. This parasite has diploid sexual reproduction in ticks, during which genetic exchanges can occur leading to increased genetic diversity, which is an important factor in adapting to environmental changes. Exploring the genetic variation of B. gibsoni population can provide a foundation for understanding the patterns of disease transmission and developing babesiosis control strategies. Partial 18S rRNA fragment sequences were obtained from 11 B. gibsoni isolates collected from different regions in China and 117 publicly available sequences were from 12 geographical areas including China. The genetic variation, demographic expansion and population structure were examined. A total of 34 haplotypes were identified among B. gibsoni populations. Analysis of molecular variance, pairwise Fst and structure analysis showed that high genetic variation within populations, low genetic differentiation and obvious mixture haplotype were apparent in a single continent, but higher genetic differentiation was detected across different continents. Neutrality tests implied that B. gibsoni populations had experienced population extension. These findings will contribute to understand the genetics and evolution of B. gibsoni and will be useful for formulating effective management strategies to prevent and control this parasite.

Ultrasensitive Hybridization Chain Reaction-Assisted Multisite Exonuclease III Amplification Strategy Combined with a Direct Quantitative Fluorescence Lateral Flow Technique for Multiple Bacterial 16S rRNA Detection.

Accurate and in-time detection of bacteria conduces to preventing their rapid spread around the environment, while a nucleic acid test (NAT) is a powerful tool for early diagnosis of pathogens. Herein, we propose a hybridization chain reaction (HCR)-mediated multisite exonuclease III (Exo-III) amplification strategy (HCR/Exo-III amplifier) to achieve the one-pot and ultrasensitive isothermal amplification of bacterial 16S rRNA and a portable fluorescence detection device (PFD) to directly read signals in a lateral flow assay (LFA). In detail, the target-initiated HCR products present multiple binding sites for triggering the Exo-III amplifier that produces numerous target amplicons. Following that, the target amplicons travel up on the strip and bridge between the DNA-CdTe/CdS probes and the capture DNA to form a positive fluorescence line. After that, the strip is inserted into the PFD to accomplish the fluorescence signal reading. The constructed HCR/Exo-III amplifier-based PFD-LFA implemented the simultaneous and specific detection of three bacteria with a detection limit of a few tenths of fM for synthetic 16S rRNA fragments and dozens of CFU/mL for Staphylococcus aureus, Listeria monocytogenes, and Salmonella typhimurium in pure cultures. The sensing platform features isothermal amplification, convenient operation, and good economy, displaying great potential for on-site testing toward multiple nucleic acid analytes.

Micropodarke fujianensis n. sp. (Annelida: Hesionidae) from Fujian, China

We describe M. fujianensis n. sp. (Annelida: Hesionidae), collected from the coast of Fujian Province, China, based on morphological and molecular analyses. Micropodarke fujianensis n. sp. is characterized by having two pairs of minute eyes, and neurochaetae of middle segments without basal long teeth. The K2P distances of COI, 16S rRNA, 18S rRNA, 28S rRNA, and histone H3 fragments diverged from the corresponding sequences of the closest related species of Micropodarke in GenBank and BOLD Systems by 22.3%, 10.7%, 0, 1.15%, and 1.1%, respectively. Among the Micropodarke species with such DNA sequences for comparison, M. fujianensis n. sp. is most closely related to M. cf. dubia-a species collected from Daya Bay, China. An updated key to Micropodarke species is provided.

Rhizobium tropici and Riboflavin Amendment Condition Arbuscular Mycorrhiza Colonization in Phaseolus vulgaris L.

Phaseolus vulgaris L. (Fabaceae) forms symbioses with arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing rhizobia (NFB). The tripartite relationship uses molecular singals to establish intracellular symbioses in roots. The goal of this study was to determine if Rhizobium tropici CIAT 899 and exogenous riboflavin (vitamin B2) have an effect on AMF species selection and root colonization of P. vulgaris. Using SSU rRNA fragment amplification of DNA extracted from P. vulgaris roots, we found that the presence of R. tropici altered the relative distribution of AMF species. Dominikia bernensis (Ohel) was the most abundant AMF species in P. vulgaris roots but when R. tropici was co-inoculated, Glomus species dominated. Rhizobacteria such as R. tropici, secrete riboflavin and could affect AMF symbiosis. Addition of 50 μM riboflavin to P. vulgaris, increased plant growth (28%), dry nodule weight (18%), AMF colonization (248%) and mycorrhizal vesicle frequency (56%) in bean roots. 3.12 and 12.5 µM riboflavin favored the presence of Glomus macrocarpum in P. vulgaris roots. This work provides the basis to further study of rhizobial and mycorrhizal co-inoculation of Phaseolus vulgaris bean.