16S-23S rRNA Research Articles

Seventy-eight entire mitochondrial genomes and nuclear rRNA genes provide insight into the phylogeny of the hard ticks, particularly the Haemaphysalis species, Africaniella transversale and Robertsicus elaphensis

Hoogstraal and Kim (1985) proposed from morphology, three groups of Haemaphysalis subgenera: (i) the “structurally advanced”; (ii) the “structurally intermediate”; and (iii) the “structurally primitive” subgenera. Nuclear gene phylogenies, however, did not indicate monophyly of these morphological groups but alas, only two mitochondrial (mt) genomes from the “structurally intermediate” subgenera had been sequenced. The phylogeny of Haemaphysalis has not yet been resolved. We aimed to resolve the phylogeny of the genus Haemaphysalis, with respect to the subgenus Alloceraea. We presented 15 newly sequenced and annotated mt genomes from 15 species of ticks, five species of which have not been sequenced before, and four new 18S rRNA and 28S rRNA nuclear gene sequences. Our datasets were constructed from 10 mt protein-coding genes, cox1, and the 18S and 28S nuclear rRNA genes. We found a 132-bp insertion between tRNA-Glu (E) gene and the nad1 gene in the mt genome of Haemaphysalis (Alloceraea) inermis that resembles insertions in H. (Alloceraea) kitaokai and Rhipicephalus (Boophilus) geigyi. Our mt phylogenies had the three species of Amblyomma (Aponomma) we sequenced embedded in the main clade of Amblyomma: Am. (Aponomma) fimbriatum, Am. (Aponomma) gervaisi and Am. (Aponomma) latum. This is further support for the hypothesis that the evolution of eyes appears to have occurred in the most-recent-common-ancestor of Amblyocephalus (i.e. Amblyomminae plus Rhipicephalinae) and that eyes were subsequently lost in the most-recent-common-ancestor of the subgenus Am. (Aponomma). Either Africaniella transversale or Robertsicus elaphensis, or perhaps Af. transversale plus Ro. elaphensis, appear to be the sister-group to the rest of the metastriate Ixodida. Our cox1 phylogenies did not indicate monophyly of the “structurally primitive”, “structurally intermediate” nor the “structurally advanced” groups of Haemaphysalis subgenera. Indeed, the subgenus Alloceraea may be the only monophyletic subgenus of the genus Haemaphysalis sequenced thus far. All of our mt genome and cox1 phylogenies had the subgenus Alloceraea in a clade that was separate from the rest of the Haemaphysalis ticks. If Alloceraea is indeed the sister to the rest of the Haemaphysalis subgenera this would resonate with the argument of Hoogstraal and Kim (1985), that Alloceraea was a subgenus of “primitive” Haemaphysalis. Alectorobius capensis from Japan had a higher genetic-identity to A. sawaii, which was also from Japan, than to the A. capensis from South Africa. This indicates that A. capensis from Japan may be a cryptic species with respect to the A. capensis from South Africa.

Neocylindrospermum variakineticum gen. & sp. nov. (Nostocales, Cyanobacteria), a novel genus separated from Cylindrospermum using a polyphasic method

ABSTRACT The large genus Cylindrospermum is polyphyletic as currently circumscribed, and its cryptic diversity requires detailed taxonomic evaluation. Two novel cyanobacterial strains were isolated by scraping the surface of wet wood above the water level, in the stream below Mae Phun Waterfall in the northern part of Thailand, and investigated with a polyphasic study combining morphological and molecular analyses. The morphological features of these two novel strains fitted the original description of the genus Cylindrospermum but they differed from known species in cell size, exospore features and akinete formation. Phylogenetic analyses based on 16S rRNA, 16S–23S rRNA ITS region, rpoC1 and nifH genes indicated that the novel strains formed a unique clade with strongly supported values from BI and ML methods. This unique clade was distantly separated from the Cylindrospermum sensu stricto clade and other Cylindrospermum-like clades. Molecular analyses showed that the two novel strains had <96.3% 16S rRNA gene sequence similarity and <83.2% 16S–23S rRNA ITS region sequence similarity to known cyanobacterial genera and other related taxa. Furthermore, ITS secondary structures between the 16S–23S rRNA gene in both strains also exhibited singular folding patterns of D1–D1', Box-B and V3 helix, thereby distinguishing them from the closely related taxa. The establishment of Neocylindrospermum gen. nov. with the type species as N. variakineticum sp. nov. is proposed according to the International Code of Nomenclature for Algae, Fungi, and Plants.

Distribution pattern of the Microhyla heymonsi group (Anura, Microhylidae) with descriptions of two new species from Vietnam

We provide the first distribution pattern of the Microhyla heymonsi group based on available molecular and morphological data collected from East and Southeast Asia. Our analyses show a high level of genetic diversity in the M. heymonsi group with nine distinct lineages from China, Myanmar, Vietnam, Laos, Malaysia, Thailand, Cambodia, as well as Singapore, and Indonesia. The study also reveals the discovery of two new species in Vietnam, Microhyla hmongorum sp. nov. from Lai Chau Province and Microhyla xodangorum sp. nov. from Kon Tum Province. When comparing the 12S–16S rRNA gene, the genetic divergence between Microhyla xodangorum and other congeners of the Microhyla heymonsi group ranges from 7.5-- to 8.9% (M. cf. heymonsi) and approximately 8.4% between the new species and M. heymonsi s. str. from Taiwan, China. The genetic divergence between Microhyla hmongorum and its congeners ranges from 4.5–5.6% (M. cf. heymonsi) to 8.7% (Microhyla xodangorum). These new findings bring the total number of known species in the genus Microhyla to 48 and the recorded species of Microhyla from Vietnam to 14.

Zarconia navalis gen. nov., sp. nov., Romeriopsis navalis gen. nov., sp. nov. and Romeriopsis marina sp. nov., isolated from inter- and subtidal environments from northern Portugal.

The morphology, 16S rRNA gene phylogeny and 16S-23S rRNA gene ITS secondary structures of three strains of marine Cyanobacteria, isolated from inter- and subtidal environments from north Portugal were studied, resulting in the description of Zarconia navalis gen. nov., sp. nov. (Oscillatoriales incertae sedis), Romeriopsis navalis gen. nov., sp. nov. (Leptolyngbyaceae) and Romeriopsis marina sp. nov., named under the International Code of Nomenclature for algae, fungi, and plants. No diacritical morphological characters were found for the new genera and species. The 16S rRNA gene maximum-likelihood and Bayesian phylogenies supported that the genus Zarconia is a member of the Oscillatoriales, morphologically similar to the genera Microcoleus and Phormidium, but distant from them. The genus Romeriopsis is positioned within the Leptolyngbyaceae (Synechococcales) and is closely related to Alkalinema. The secondary structures of the D1-D1', Box B, V2 and V3 helices corroborate the phylogenetic results. Furthermore, our study supports previous observations of polyphyletic Oscillatoriales families and reinforces the need for their taxonomic revision.

Phylogenetic placement, morphology and gill-associated bacteria of a new genus and species of deep-sea mussel (Mytilidae: Bathymodiolinae) from the South China Sea

Mussels in the subfamily Bathymodiolinae are common inhabitants of deep-sea chemosynthetic habitats, but in many places their diversity remains unknown. Here we describe Nypamodiolus samadiae n. gen. et n. sp. (Mytilidae: Bathymodiolinae) based on samples collected from the Haima cold seep in the South China Sea. Phylogenetic analyses based on fragments of three mitochondrial (cox1, 16S rRNA and nad4) and three nuclear (18S rRNA, 28S rRNA and histone H3) genes show that this new species belongs to a new genus, Nypamodiolus, which includes three named species (Myrina longissima Thiele and Jaeckel, 1931, Myrina simpsoni Marshall, 1900, Idasola japonica Habe, 1976) and a few undescribed species. Nypamodiolus n. gen. is characterized by small-sized, modioliform to fan-shaped shells with the umbones in a non-terminal position, and by robust anterior and posterior byssal retractor muscles arranged in roughly one plane. Most closely related to Nypamodiolus samadiae n. sp. is an undescribed species from the northern Papua New Guinea, to which it has a Kimura-2-parameter genetic distance of 11.8% for cox1. Sequencing the V3–V4 region of the microbial 16S rRNA gene reveals two dominant gill-associated bacteria in the new species, including one sulfur-oxidizing Gammaproteobacteria (relative abundance: 43.8–58.8%) and one unclassified Spirochaetes (relative abundance: 23.9–28.1%). Morphologically, N. samadiae n. sp. is similar to N. simpsoni, while can be distinguished by a larger maximum shell size (45.0 mm vs. 40.0 mm), larger length/height ratio (2.6–2.9 vs. 2.1–2.4), broader posterior end, slightly curved dorsal margin, and more anterior umbones.

Phylogenetic characterization and analysis of Sarcocystis buffalonis

Despite its frequent presence in buffaloes, Sarcocystis buffalonis remains as one of the most under studied parasite. In the present study, isolates of S. buffalonis from,Mathura, Uttar Pradesh India were characterized for 18S rRNA (MF595842-MF595844), cox 1 (MG792800-MG792802), 28S rRNA (MH793418-MH793420) and ITS 1 (MH793421-MH793423) genes. Analysis revealed multiple haplotypes for each individual gene viz., 18S rRNA (three haplotypes), cox 1 (two haplotypes), 28S rRNA (two haplotypes) and ITS 1 (single haplotype). The studied Indian sequences showed variable homologies for individual gene loci viz., 18S rRNA (99.3–99.9%); cox 1 (99.8–100.0%); 28S rRNA (99.9–100.0%) and ITS 1 (100.0%) The phylogenetic association between S. buffalonis and closely related Sarcocystis spp. infecting buffaloes and cattle was delineated. All these gene loci placed S. buffalonis close to S. hirsuta. The study has generated A vital phylogenetic data about this erstwhile neglected parasite.

Closing the gap: a new phylogeny and classification of the chemosymbiotic bivalve family Lucinidae with molecular evidence for 73% of living genera

AbstractNew molecular phylogenies of the chemosymbiotic bivalve family Lucinidae, using 18S rRNA, 28S rRNA and cytochrome b genes, include species from genera not previously analysed. Notable additions from Myrteinae are sequences from Rostrilucina, Solelucina and Taylorina species, species of Ustalucina, Gonimyrtea from Leucosphaerinae and additional species of Ctena, Codakia, Lucinoma and Divalucina from Codakiinae. New sequences of Lucininae include the type species of Parvilucina (P. tenuisculpta), Liralucina, Falsolucinoma, Easmithia, Jallenia, Radiolucina and Cardiolucina as well as samples of Loripes orbiculatus from multiple localities. Five major clades, defined as subfamilies, are recognized: Pegophyseminae, Myrteinae, Leucosphaerinae, Codakiinae and Lucininae. Two branches, Fimbriinae and Monitilorinae, are represented by single species. Pegophyseminae are an extremely long-branched group with a sister-group relationship to Leucosphaerinae, while Codakiinae are a sister clade to the Lucininae. In various gene trees, the position of Myrteinae is unstable in relation to Pegophyseminae + Leucosphaerinae, Monitilorinae and Fimbriinae. The Myrteinae are not well resolved, with an ambiguous correlation of molecular and morphological characters. Codakiinae now include Divalucina cumingi, shown to be related to Lucinoma rather than Divaricella and Divalinga of the Lucininae. Leucosphaerinae are a well-supported clade but morphologically disparate, with the positions of Gonimyrtea and Callucina unresolved. Several molecularly distinct subclades are recognized within the Lucininae, especially the Lucinisca, Loripes and Parvilucina groups. Parvilucina species are paraphyletic with P. tenuisculpta, the type species, distinct from the western Atlantic species. Codakia, Ctena and Pegophysema have pan-tropical distributions with former connections disrupted by vicariant events of the closure of the eastern Tethyan and Central American Seaways. Species of Radiolucina, Pleurolucina and Lucinisca are present on either side of the Isthmus of Panama. A new classification of the 96 living lucinid genera is presented, providing a framework for future studies of systematics, ecology, biogeography and bacterial symbioses.

Effect of COVID-19 pandemic on incidence of mycobacterial diseases among suspected tuberculosis pulmonary patients in Tehran, Iran.

Recent pandemic of coronavirus SARS-CoV-2 (COVID-19) caused limitations in the country's strategies to fight against mycobacterial infections. The aim of this study was to compare the suspected tuberculosis (TB) pulmonary patients before and during the COVID-19 pandemic (January 2018-December 2021) who were referred to the National Reference TB Laboratory (NRL TB), Tehran, Iran. The mycobacterial isolated strains were identified and compared with previous data. A total of 16,899 clinical samples collected from 7041 suspected pulmonary TB patients were received from 2018 to 2021. Primary isolation of Mycobacterium isolates was done on Löwenstein-Jensen medium. Then, the DNA was extracted from acid-fast bacillus culture-positive samples and identification was performed by IS6110, Hsp65, and 16S-23S rRNA genes using polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and nested PCR methods. A total of 11679 specimens (69.1%) from 4866 suspected TB patients were collected in 2018-2019 and 5220 specimens (30.8%; from 2175 patients) in 2020-2021. Out of 11679 specimens, 2046 samples that belong to 852 patients were infected with Mycobacterium tuberculosis, and the remaining were non-TB Mycobacterium (NTM) species (n = 244) isolated from 102 patients. The cultures for 12894 specimens were either negative (76.3%) or contaminated (845/16899; 5%). A comparison of the total number of patients who were referred for diagnosis and treatment (954/666 patients, P > 0.05) showed a 30.1% reduction during the COVID-19 pandemic. Although, with these low number of patients, the significant increases of NTM species (P < 0.05) among suspected TB pulmonary patients were observed. Besides, new species of NTM, for example, Mycobacterium peregrinum and Mycobacterium montefiorense, were detected. For the past 20 years, these two species were not reported from pulmonary patients in Iran. During the pandemic of COVID-19, the TB diagnosis network became irregular, as a consequence, many patients could not reach the treatment center, and this could increase the circulation of mycobacterial diseases (TB and NTM). The study shows the emergence of new opportunistic NTM species also.

Genome analysis and phenotypic characterization of Halomonas hibernica isolated from a traditional food process with novel quorum quenching and catalase activities

Traditional food processes can utilize bacteria to promote positive organoleptic qualities and increase shelf life. Wiltshire curing has a vital bacterial component that has not been fully investigated from a microbial perspective. During the investigation of a Wiltshire brine, a culturable novel bacterium of the genus Halomonas was identified by 16S rRNA gene (MN822133) sequencing and analysis. The isolate was confirmed as representing a novel species (Halomonas hibernica B1.N12) using a housekeeping (HK) gene phylogenetic tree reconstruction with the selected genes 16S rRNA, 23S rRNA, atpA, gyrB, rpoD and secA. The genome of the new isolate was sequenced and annotated and comparative genome analysis was conducted. Functional analysis revealed that the isolate has a unique phenotypic signature including high salt tolerance, a wide temperature growth range and substrate metabolism. Phenotypic and biochemical profiling demonstrated that H. hibernica B1.N12 possesses strong catalase activity which is an important feature for an industrial food processing bacterium, as it can promote an increased product shelf life and improve organoleptic qualities. Moreover, H. hibernica exhibits biocontrol properties based on its quorum quenching capabilities. Our work on this novel isolate advances knowledge on potential mechanistic interplays operating in complex microbial communities that mediate traditional food processes.

Polyphasic Characterization and Taxonomic Evaluation of a Bloom-Forming Strain Morphologically Resembling Radiocystis fernandoi (Chroococcales, Cyanobacteria) from Lake Erhai, China

Microcystis-dominated blooms cause environmental and ecological impacts worldwide. However, it is sometimes challenging to correctly identify Microcystis and its related genera. Radiocystis fernandoi Komárek et Komáková-Legnerová 1993, characterized by large cells and correspondingly large colonies with gas vesicles, has been frequently found in cyanobacterial blooms in Brazil. However, its molecular and phylogenetic identity is unknown because no valuable DNA sequences are available in GenBank. In this study, a strain with R. fernandoi morphology was successfully isolated from a cyanobacterial bloom sample in Lake Erhai, a subtropical plateau lake in China. Molecular characterization and phylogenetic analyses based on 16S rRNA and cpcBA genes revealed that the strain is different from the type species of the genus Radiocystis but formed a well-supported clade with the Microcystis species. The comparative results of the ITS region between 16S–23S rRNA genes also showed that the strain had very similar secondary structures to the species of Microcystis, supporting it as a new member of the genus Microcystis. Therefore, we propose the transfer of Radiocystis fernandoi into Microcystis fernandoi comb. nov. The correct identification and further taxonomic procedure for the bloom-forming cyanobacterial genera, such as Microcystis, Radiocystis, and Sphaerocavum, are also discussed in this study.

Molecular Detection of Tick-Borne Bacteria from Amblyomma (Acari: Ixodidae) Ticks Collected from Reptiles in South Africa.

Reptiles are hosts for various tick species and tick-associated organisms, many of which are zoonotic. However, little is known about the presence and diversity of tick-borne bacteria infecting reptiles and their ticks in South Africa. Amblyomma ticks (n = 253) collected from reptiles were screened for the presence of Coxiella, Anaplasma, Rickettsia, and Borrelia species by amplification, sequencing and phylogenetic analysis of the 16S rRNA, 23S rRNA, gltA, OmpA, and Flagellin genes, respectively. This study recorded the presence of reptile associated Borrelia species and Coxiella-like endosymbiont in South Africa for the first time. Furthermore, a spotted fever group Rickettsia species was observed in 7 Amblyomma marmoreum and 14 Amblyomma sylvaticum from tortoises of genera Kinixys and Chersina. Francisella-like endosymbiont was observed from 2 Amblyomma latum collected from the Mozambique spitting cobra, Naja mossambica. Coxiella burnetii and Anaplasma spp., were not detected from the current samples. Although the direct evidence that reptiles can act as reservoir hosts remains to be determined, observations from this study provide indications that reptilian ticks may play a role in the transmission of pathogenic bacteria to homothermic animals. Furthermore, the absence of Anaplasma spp., and C. burnetii does not mean that these pathogens should be completely neglected.

The nuclear-encoded plastid ribosomal protein L18s are essential for plant development.

Plastid ribosomal proteins (PRPs) are necessary components for plastid ribosome biogenesis, playing essential roles in plastid development. The ribosomal protein L18 involved in the assemble of 5S rRNA and 23S rRNA, is vital for E. coli viability, but the functions of its homologs in plant plastid remain elusive. Here, we characterized the functions of the plant plastid ribosomal protein L18s (PRPL18s) in Arabidopsis and rice. AtPRPL18 was ubiquitously expressed in most of the plant tissues, but with higher expression levels in seedling shoots, leaves, and flowers. AtPRPL18 was localized in chloroplast. Genetic and cytological analyses revealed that a loss of function of AtPRPL18 resulted in embryo development arrest at globular stage. However, overexpression of AtPRPL18 did not show any visible phenotypical changes in Arabidopsis. The rice OsPRPL18 was localized in chloroplast. In contrast to AtPRPL18, knockout of OsPRPL18 did not affect embryo development, but led to an albino lethal phenotype at the seedling stage. Cytological analyses showed that chloroplast development was impaired in the osprpl18-1 mutant. Moreover, a loss-function of OsPRPL18 led to defects in plastid ribosome biogenesis and a serious reduction in the efficiency of plastid intron splicing. In all, these results suggested that PRPL18s play critical roles in plastid ribosome biogenesis, plastid intron splicing, and chloroplast development, and are essential for plant survival.

Taxonomy, genetic diversity, and phylogeny of the Antarctic mud dragon, Polacanthoderes (Kinorhyncha: Echinorhagata: Echinoderidae)

A new species Polacanthoderes shiraseae sp. nov. from three Antarctic regions (off Cape Darnley, off Totten Glacier, and in Lützow-Holm Bay) is described. In addition, type species of Polacanthoderes, Polacanthoderes martinezi, is redescribed. The new species is distinguished from P. martinezi by the presence of conspicuously thick lateroventral acicular spines on segments 8 and 9 and the presence of sublateral small acicular spines on segment 7. Both P. martinezi and P. shiraseae sp. nov. occasionally show intraspecific morphological variations in the position of some small acicular spines. The K2P genetic distances based on the mitochondrial cytochrome c oxidase subunit 1 gene sequences in P. shiraseae sp. nov. are 0–1.5%, equivalent to those in other echinoderid species. Phylogenetic analyses of Polacanthoderes based on the nuclear 18S rRNA and 28S rRNA gene sequences support the inclusion of the genus in Echinorhagata/Echinoderidae and suggest that the genus represents the most basal group of this order/family.

Bartonella spp. detection in laelapid (Mesostigmata: Laelapidae) mites collected from small rodents in Lithuania.

The genus Bartonella contains facultative Gram-negative intracellular bacteria from the family Bartonellaceae that can cause diseases in humans and animals. Various Bartonella species have been detected in rodents' ectoparasites, such as fleas, ticks, mites, and lice. However, the role of laelapid mites (Mesostigmata: Laelapidae) as carriers of Bartonella spp. needs to be confirmed. We aimed to investigate the presence of Bartonella spp. in laelapid mites collected from small rodents in Lithuania using real-time PCR targeting the transfer-messenger RNA/tmRNA (ssrA) gene and to characterize Bartonella strains using nested PCR and sequence analysis of the 16S-23S rRNA intergenic transcribed spacer region (ITS). A total of 271 laelapid mites of five species (Laelaps agilis, Haemogamasus nidi, Eulaelaps stabularis, Myonyssus gigas, and Hyperlaelaps microti) were collected from five rodent species (Apodemus flavicollis, Apodemus agrarius, Clethrionomys glareolus, Micromys minutus, and Microtus oeconomus) during 2015-2016. Bartonella DNA was detected in three mite species L. agilis, M. gigas, and Hg. nidi with an overall prevalence of 11.4%. Sequence analysis of the 16S-23S rRNA ITS region revealed the presence of Bartonella taylorii in L. agilis, Hg. nidi, and M. gigas, and Bartonella grahamii in L. agilis. Our results suggest that laelapid mites are involved in the maintenance of rodent-associated Bartonella spp. in nature. To the best of the authors' knowledge, this is the first study to demonstrate the presence of Bartonella spp. DNA in laelapid mites from small rodents.

Chlamydiaceae-Like Bacterium in Wild Magellanic Penguins (Spheniscus magellanicus)

The family Chlamydiaceae is comprised of obligate intracellular bacteria, some of which are significant pathogens of humans and domestic animals. Magellanic penguins (Spheniscus magellanicus) are susceptible to Chlamydia psittaci outbreaks in captivity, and serological surveys detected antibodies against C. psittaci (or antigenically similar organisms) in wild populations of this species. To investigate the occurrence of Chlamydiaceae in wild Magellanic penguins, 167 cloacal swabs were collected from apparently healthy individuals at four breeding colonies along the coast of Chubut, Argentina. Real-time PCR revealed the presence of DNA from Chlamydiaceae-like bacteria in 61 cloacal swabs (36.5%). Detection did not correlate to nutritional status or to meaningful hematological abnormalities. Isolation in monkey kidney cells was unsuccessful, but DNA sequences for the 16S–23S rRNA region were obtained from one sample. Phylogenetic analysis showed a close relationship to unidentified Chlamydiaceae-like bacteria found in chinstrap penguins (Pygoscelis antarcticus) in Antarctica and seagulls in France and the Bering Sea, and to Chlamydiifrater spp. isolated from flamingos in France. Further studies are necessary to clarify the taxonomy and investigate the epidemiology, pathogenicity, and zoonotic potential of this group of Chlamydiaceae-like bacteria.

The novel marine cyanobacterium Nunduva sanctimaloensis sp. nov. (Nostocales, Cyanobacteria) from rocky shores and its reproduction through modified monocytes

Rocky intertidal regions are diverse as they provide many unique habitats for the growth of cyanobacteria. From outcrops battered by waves, tidal pools, and periods of desiccations due to tides, these regions offer distinct environments and much of their cyanobacterial diversity remains unexplored. To help elucidate cyanobacteria from this habitat, one nostocalean cyanobacterium from Brittany, France was isolated and cultured. Typical morphological features including filaments with heteropolar trichomes, basal or intercalary heterocytes, false branching, and long hyaline apical hairs suggested the genus Nunduva. The combination of 16S rRNA gene and 16S-23S rRNA internal transcribed spacer (ITS), along with diagnosable morphological characteristics supports a distinct taxon from previously described species. Herein, the novel false branching cyanobacterium Nunduva sanctimaloensis sp. nov. is described along with its reproduction through modified monocytes.

Eimeria spp. (Eimeriidae) in the migratory whooper swan (Cygnus cygnus) Linnaeus, 1758 (Anatidae) from Sanmenxia Swan Lake National Urban Wetland Park in the middle reaches of the Yellow River in China.

This study i dentifies four Eimeria spp. recorded from fecal samples of migratory whooper swans (Cygnus cygnus) in Sanmenxia Swan Lake National Urban Wetland Park in Sanmenxia city in the middle reaches of the Yellow River, China. Eimeria hermani, Eimeria nocens, Eimeria stigmosa, and Eimeria magnalabia were compatible in all characteristic features with their respective original descriptions. In addition to the preliminary morphological identification, this study provides a preliminary genotypic identification of these four Eimeria spp. via sequencing of the 18S rRNA, 28S rRNA, and COI gene loci that are suitable for the genotypic differentiation of these coccidia. This is the first report of molecular data for the four Eimeria spp. in migratory whooper swans. Finally, this study discusses the environmental risks of these coccidia for migratory whooper swans in Sanmenxia Swan Lake National Urban Wetland Park.

Screening of endophytic fungi from Cremastra appendiculata and their potential for plant growth promotion and biological control.

Biocontrol fungi are widely used to promote plant growth and pest control. Four fungi were isolated from Cremastra appendiculata tubers and screened for plant growth-promoting and antagonistic effects. Based on the morphological characterization and ITS, 18S rRNA and 28S rRNA gene sequencing analysis, the fungi were identified to be related to Colletotrichum gloeosporioides (DJL-6), Trichoderma tomentosum (DJL-9), Colletotrichum godetiae (DJL-10) and Talaromyces amestolkiae (DJL-15). The growth inhibition tests showed that the four isolates had different inhibitory effects on Colletotrichum fructicola, Alternaria alternata and Alternaria longipes, among which DJL-9 showed the highest inhibitory activity. Their culture filtrates (especially that of DJL-15) can also inhibit pathogens. Four isolates were positive for the production of indole-3-acid (IAA) and β-1,3-glucanase and possessed proteolytic activity but were negative for the production of iron siderophore complexes. The four fungi showed strong nitrogen fixation and potassium dissolution abilities. In addition to DJL-9 being able to solubilize phosphate, DJL-10 was able to produce chitinase and cellulase. Pot experiments indicated that the four fungi increased the germination rate of C. appendiculata and soybean seeds and increased soybean radicle growth and plant biomass. Among them, DJL-6 had a better growth-promoting effect. Therefore, we successfully screened the biocontrol potential of endophytes from C. appendiculata, with a focus on preventing fungal diseases and promoting plant growth, and selected strains that could provide nutrients and hormones for plant growth.

White-tailed eagle (Haliaeetus albicilla) as the definitive host of Sarcocystis lutrae in the Czech Republic.

The white-tailed eagle, Haliaeetus albicilla, has been involved in the life cycle of several Sarcocystis species as the intermediate and definitive host. To date, it has been supposed that the eagle might play the role as the definitive host for S. Lutrae, and, herein, we tried to elucidate it based on morphometric and molecular analyses. One out of two eagles harbored oocysts (17.0-17.4 × 11.3-11.9 μm) and sporocysts (11.3-12.3 × 8.3-9.3 μm) in the intestinal mucosa, whose sequences at 18S rRNA, 28S rRNA, ITS1, and cox1 showed similar identity (97.64-100%) to published sequences of S. lutrae from other hosts. The presence of sporulated oocysts in the lamina propria of villi confirms that S. lutrae truly infects the white-tailed eagle. The white-tailed eagle is confirmed as the definitive host of S. lutrae in the Czech Republic.

Bioactive Earthworm Peptides Produced by Novel Protease-Producing Bacillus velezensis PM 35 and Its Bioactivities on Liver Cancer Cell Death via Apoptosis, Antioxidant Activity, Protection Against Oxidative Stress, and Immune Cell Activation.

Earthworms have long been used as traditional medicine. The purposes of this research were to create bioactive peptides from the unique Amynthas arenulus earthworm (PAAEs) and test their potentials on liver cancer bioprophylactic activity, antioxidant, oxidative stress protection, and immune cell activation. This earthworm had a high protein content ratio, at 55.39%. Besides, PM 35 is one out of 58 bacteria isolated from the earthworm carcasses that exhibited the highest protease and yield protein production which was chosen as the protease-producing bacteria to hydrolyze the protein. The genera were identified by 16S rRNA and 16S–23S rRNA comparison and confirmed as Bacillus velezensis PM 35. The response surface methodology was applied to optimize these hydrolysis parameters, i.e., the enzyme/substrate (E/S) concentration ratio [1%–3% (v/v)] and time (1–3 h) of the hydrolyzing earthworm’s proteins. The optimal hydrolyzing conditions were 3% (v/v) of E/S concentration ratio and 3 h of hydrolysis time, which found protein-hydrolysate yield (24.62%) and degree of hydrolysis (85.45%) as the highest. After being challenged in the gastrointestinal tract-resistant model, these PAAEs (MW <3 and 3–5 kDa) induced liver cancer cell (HepG2) death via apoptotic action modes (cell morphological change and DNA fragmentation). The PAAEs (MW <3 kDa) exhibited significant antioxidant activity via DPPH, ABTS, and FRAP with IC50 values of 0.94, 0.44, and 6.34 mg/ml, respectively. The PAAEs (MW < 3 kDa) were non-cytotoxic and protected the mouse fibroblast cells (L929) against oxidative stress. These PAAEs (MW < 3 kDa, 0.2 mg/ml) stimulated the B lymphocytes (122.3%), and T lymphocytes (126.7%) proliferation. This research suggests that PAAEs can be used in a variety of applications, especially in the food and pharmaceutical industries.