Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins

Neospora caninum is a parasite with veterinary relevance, inducing severe disease in dogs and reproductive disorders in ruminants, especially cattle, leading to major losses. The close phylogenetic relationship to Toxoplasma gondii and the lack of pathogenicity in humans drives an interest of the scientific community toward using N. caninum as a model to study the pathogenicity of T. gondii .

Characterisation of the Complete Mitochondrial Genome of Critically Endangered Mustela lutreola (Carnivora: Mustelidae) and Its Phylogenetic and Conservation Implications

In this paper, a complete mitochondrial genome of the critically endangered European mink Mustela lutreola L., 1761 is reported. The mitogenome was 16,504 bp in length and encoded the typical 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes, and harboured a putative control region. The A+T content of the entire genome was 60.06% (A > T > C > G), and the AT-skew and GC-skew were 0.093 and −0.308, respectively. The encoding-strand identity of genes and their order were consistent with a collinear gene order characteristic for vertebrate mitogenomes. The start codons of all protein-coding genes were the typical ATN. In eight cases, they were ended by complete stop codons, while five had incomplete termination codons (TA or T). All tRNAs had a typical cloverleaf secondary structure, except tRNASer(AGC) and tRNALys, which lacked the DHU stem and had reduced DHU loop, respectively. Both rRNAs were capable of folding into complex secondary structures, containing unmatched base pairs. Eighty-one single nucleotide variants (substitutions and indels) were identified. Comparative interspecies analyses confirmed the close phylogenetic relationship of the European mink to the so-called ferret group, clustering the European polecat, the steppe polecat and the black-footed ferret. The obtained results are expected to provide useful molecular data, informing and supporting effective conservation measures to save M. lutreola.

Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus

The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV’s cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.

Ubiquitous but Ignored? A Case of Water Beetle in Southeastern Europe

Although freshwater habitats, especially springs, are widely recognized as top-priority habitats for monitoring and conservation procedures, their fauna, especially water beetles, are still poorly studied in the southeastern part of Europe. Thus, the dominant water beetle in these habitats, E. bosnica (Zaitzev, 1908) (Insecta: Coleoptera: Elmidae), has been completely ignored and misidentified. This study represents the first review of its taxonomy and its population and ecological traits. Both published and unpublished data are presented and discussed, as well as the results of field sampling in 46 springs and other waterbodies conducted in this region from 2004 to 2019. The identification characters of the male genitalia and the first DNA barcode of the species are presented. The results confirm the close phylogenetic relationship of E. bosnica with E. aenea (Müller, 1806) and E. rioloides (Kuwert, 1890). The species proved to be a useful environmental descriptor and can easily be used as a biological indicator due to its easy identification. The species shows remarkable sensitivity to environmental conditions and inhabits sites that are potentially under increased anthropogenic pressure and could disappear at an alarming rate. Thus, karstic habitats should be included in future conservation and monitoring procedures in this part of Europe.

Three new species of Byrsopteryx Flint microcaddisflies from Peru (Insecta: Trichoptera) including DNA-based larval associations

In this paper, we have described and illustrated three new species of Byrsopteryx from Peru: Byrsopteryx inti, sp. nov. Byrsopteryx mamaocllo sp. nov., and Byrsopteryx mancocapac sp. nov. Larvae of the latter two were also associated to male specimens based on comparison of a fragment of COI gene and pharate male identification. Byrsopteryx inti sp. nov. and Byrsopteryx mamaocllo sp. nov. share a unique feature: a semi-dome process formed by a thickened area on male forewings. The three species can be easily identified by wing coloration and male genitalia. Furthermore, Byrsopteryx inti sp. nov. can be recognized by its sternum VIII with a median digitate process on posterior margin, slightly capitate; and by long dorsolateral processes from segment VIII, which cross each other apically in dorsal view. Byrsopteryx mamaocllo sp. nov. can be distinguished by sternum VIII bearing a pair of short, posterior, spinelike processes, which are curved inwards and bordered by a rounded, membranous structure, and by a pair of short, heavily sclerotized, dorsolateral processes. Byrsopteryx mancocapac sp. nov. can be distinguished by strong spine-like processes arising dorsally from subgenital plate and by sternum VIII with posterior margin divided into two plate-like lobes. Larvae of B. mamaocllo sp. nov. and B. mancocapac sp. nov. are similar to other Byrsopteryx larvae known. They can be distinguished from each other by the shape of the operculum formed by terga VIII and IX, and number of setae on the second abdominal pleurite. Maximum likelihood analyses of 20 COI sequences, including nine Byrsopteryx species, placed B. inti sp. nov. and B. mamaocllo sp. nov. as sister species and related to a clade including B. gomezi, B. tapanti, and B. esparta, while B. mancocapac sp. nov. was found as sister to B. abrelata. Despite the close phylogenetic relationship found between B. inti sp. nov. and B. mamaocllo sp. nov., they are separated by 14.9% minimum K2P divergence of COI. The highest intraspecific distance observed was 1.4% for B. mancocapac sp. nov. individuals. Although the Peruvian caddisfly fauna has around 320 known species and almost a third of them are microcaddisflies, in this paper we present the first descriptions of Byrsopteryx species for the country.

Genomic comparison and phenotypic profiling of small colony variants of Burkholderia pseudomallei

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific.

Molecular Epidemiology and Microbiological Characteristics of Cryptococcus gattii VGII isolates from China

Cryptococcus gattii (C. gattii) is a fungal pathogen that once caused an outbreak of cryptococcosis on Vancouver Island, and had spread worldwide, while few data were available in China. In this study, seven clinical isolates of C. gattii VGII were collected from 19 hospitals, Multi-locus Sequence Typing (MLST) analysis and whole-genome sequencing (WGS) was performed, and combined with published data for phylogenetic analysis. In addition, in vitro antifungal susceptibility testing, phenotypic analysis, and in vivo virulence studies were performed, subsequently, histopathological analysis of lung tissue was performed. C.gattii VGII infected patients were mainly immunocompetent male, and most of them had symptoms of central nervous system (CNS) involvement. MLST results showed that isolates from china exhibited high genetic diversity, and sequence type (ST) 7 was the major ST among the isolates. Some clinical isolates showed a close phylogenetic relationship with strains from Australia and South America. All clinical isolates did not show resistance to antifungal drugs. In addition, there was no correlation between virulence factors (temperature, melanin production, and capsule size) and virulence while in vivo experiments showed significant differences in virulence among strains. Lung fungal burden and damage to lung tissue correlated with virulence, and degree of damage to lung tissue in mice may highlight differences in virulence. Our work seeks to provide useful data for molecular epidemiology, antifungal susceptibility, and virulence differences of C. gattii VGII in China.

Lemnaceae and Orontiaceae Are Phylogenetically and Morphologically Distinct from Araceae

Duckweeds comprise a distinctive clade of pleustophytic monocots that traditionally has been classified as the family Lemnaceae. However, molecular evidence has called into question their phylogenetic independence, with some authors asserting instead that duckweeds should be reclassified as subfamily Lemnoideae of an expanded family Araceae. Although a close phylogenetic relationship of duckweeds with traditional Araceae has been supported by multiple studies, the taxonomic disposition of duckweeds must be evaluated more critically to promote nomenclatural stability and utility. Subsuming duckweeds as a morphologically incongruent lineage of Araceae effectively eliminates the family category of Lemnaceae that has been widely used for many years. Instead, we suggest that Araceae subfamily Orontioideae should be restored to family status as Orontiaceae, which thereby would enable the recognition of three morphologically and phylogenetically distinct lineages: Araceae, Lemnaceae, and Orontiaceae.

Comparative Genomics and Pathogenicity Analysis of Two Bacterial Symbionts of Entomopathogenic Nematodes: The Role of The GroEL Protein in Virulence

Bacteria of the genera Xenorhabdus and Photorhabdus are symbionts of entomopathogenic nematodes. Despite their close phylogenetic relationship, they show differences in their pathogenicity and virulence mechanisms in target insects. These differences can be explored by the analysis of the pangenome, as it provides a framework for characterizing and defining the gene repertoire. Here, we report the genome of strain SC 0516. In addition, we performed the first pangenome analysis of 91 strains of Xenorhabdus and Photorhabdus, obtaining a total of 23,603 gene clusters and a core genome of 348 genes. Phylogenetic analysis performed with the core genome showed that our strain belonged to the X. nematophila group. Biological tests showed that whole cells of X. nematophila SC 0516 were more virulent than those of P. luminescens HIM3 when both were injected into Galleria mellonella larvae. In addition, we cloned and expressed the GroEL proteins of both bacteria, as this protein has been previously indicated to show insecticidal activity in the genus Xenorhabdus. Cpn60-Xn was found to be the most toxic at all concentrations tested, with an LC50 value of 102.34 ng/larva. Sequence analysis suggested that the Cpn60-Xn toxin was homologous to Cpn60-Pl; however, Cpn60-Xn contained thirty-five differentially substituted amino acid residues that could be responsible for its insecticidal activity.

Molecular Characterization and Population Genetic Structure of Fagopyrum Species Cultivated in Himalayan Regions

Fagopyrum spp. (buckwheat) is a dicotyledonous pseudocereal crop mainly cultivated in the north-western Himalayan regions for its highly nutritional, antioxidant and therapeutic values. In the present investigation, molecular characterization was performed by using ISSR (inter simple sequence repeat) markers on 42 accessions of four buckwheat species (Fagopyrum esculentum, F. sagittatum, F. tataricum and F. kashmirianum). The 12 pre-screened ISSR primers amplified 102 bands, and amongst them 85 bands exhibited polymorphism with an average polymorphism of 82.73%. The results revealed that Shannon’s information indices (I) and Nei’s genetic diversity (H) were low for F. tataricum (I = 0.1028 ± 0.2307; H = 0.0707 ± 0.1617) and high for F. esculentum (I = 0.1715 ± 0.2622; H = 0.1164 ± 0.1796). It was estimated that within the accessions of Fagopyrum species, the species diversity (HT) and mean diversity (HS) were 0.3200 and 0.1041, respectively. Molecular variance partitioning by AMOVA also indicated a significant genetic differentiation accounting for 73% among and 27% within the accessions of Fagopyrum species. Overall, accessions of F. esculentum had the greatest distance from the other accessions of buckwheat species, which includes F. sagittatum, F. tataricum and F. kashmirianum as revealed by FST distance and Nei’s unbiased genetic distance. The dendograms based on UPGMA and PCoA segregated 42 accessions of four buckwheat species into three major groups. This study clearly reveals a considerable amount of genetic diversity at the intra-specific level in F. esculentum, F. sagittatum and F. kashmirianum accessions. The factors responsible for it are diverse geographical conditions, pollinating behavior and cultivation practices adapted in these regions. The study also indicated a close phylogenetic relationship between F. tataricum and F. kashmirianum.