Genome Survey Sequencing of an Iconic ‘Trophy’ Sportfish, the Roosterfish Nematistius pectoralis: Genome Size, Repetitive Elements, Nuclear RNA Gene Operon, and Microsatellite Discovery

The ‘Pez Gallo’ or the Roosterfish, Nematistius pectoralis, is an ecologically relevant species in the shallow water soft-bottom environments and a target of a most lucrative recreational sport fishery in the Central Eastern Pacific Ocean. According to the International Union for Conservation of Nature, N. pectoralis is assessed globally as Data Deficient. Using low-coverage short Illumina 300 bp pair-end reads sequencing, this study reports, for the first time, the genome size, single/low-copy genome content, and nuclear repetitive elements, including the 45S rRNA DNA operon and microsatellites, in N. pectoralis. The haploid genome size estimated using a k-mer approach was 816.04 Mbp, which is within the range previously reported for other representatives of the Carangiformes order. Single/low-copy genome content (63%) was relatively high. A large portion of repetitive sequences could not be assigned to the known repeat element families. Considering only annotated repetitive elements, the most common were classified as Satellite DNA which were considerably more abundant than Class I-Long Interspersed Nuclear Elements and Class I-LTR Retroviral elements. The nuclear ribosomal operon in N. pectoralis consists of, in the following order: a 5′ ETS (length = 948 bp), ssrDNA (1835 bp), ITS1 (724 bp), a 5.8S rDNA (158 bp), ITS2 (508 bp), lsrDNA (3924 bp), and a 3′ ETS (32 bp). A total of 44 SSRs were identified. These newly developed genomic resources are most relevant for improving the understanding of biology, developing conservation plans, and managing the fishery of the iconic N. pectoralis.

Molecular cytogenetic study of Eranthis (Ranunculaceae)

45S and 5S ribosomal DNA were originally localized on chromosomes of five species of winter aconits,namely, Eranthis cilicica, E. hyemalis (section Eranthis), E. pinnatifida, E. stellata и E. tanhoensis (section Shibateranthis).Fluorescence in situ hybridization was performed with oligonucleotide DNA probes Oligo-pTa71-2 and Oligo-5S rDNAof wheat that are complementary to 45S and 5S ribosomal DNA. In addition, oligonucleotide DNA probe (Oligo-5.8SrDNA-Ran, 50 b) for localization of 45S rDNA was designed and tested. This probe is based on the 5.8S rDNA sequencesof some species of fam. Ranunculaceae taken from GenBank. A specific hybridization of the Oligo-5S rDNA and Oligo5.8S rDNA-Ran probes with the chromosomes of Eranthis was shown. The use of the Oligo-pTa71-2 probe did not localizeclusters of 45S rDNA on chromosomes of studied species.

First Report of Southern Blight Caused by Sclerotium delphinii on Euonymus fortunei in China

Euonymus fortunei is an evergreen shrub-vine in the family Celastraceae, widely used as a groundcover or a vine to climb walls, or traditional herbal medicine in China. In August 2019, typical southern blight symptoms that included basal stem rot and the presence of sclerotia in rotted tissue were observed on E. fortunei in Kunshan city, Jiangsu province, China. Disease incidence was estimated at approximately 15 to 20%; meanwhile, approximately 30 to 40% of diseased plants died. The infected plants showed brown to dark stem necrosis near the base, leaf yellowing and wilting. White mycelia and white to dark reddish-brown sclerotia were observed at the base of the stem and rotten tissue. To isolate the causal organism, infected stem tissue and sclerotia collected from diseased plants in a median strip in Kunshan (31°23'40"N, 120°54'57"E) were disinfected with 70% ethanol for 2 to 3 sec, followed by 2 min in 5% NaClO, rinsed three times with sterile water, then plated on potato dextrose agar (PDA) medium, and incubated at 25°C. Isolated colonies were subcultured by needle tip transfer 3 days later. Isolates had white mycelia on PDA, with a radial growth rate of 15.2 to 18.7 mm/day. White and orange sclerotia were developed after 5 to 8 days and eventually turned dark reddish-brown. The sclerotia were globoid or irregular with surface markings (1.4 to 4.3 mm diam.; mean = 2.59 mm; n = 50) on PDA, and the average number of sclerotia produced per Petri dish ranged from 35 to 85 (mean = 52; n = 10). Microscopic observations found septal hyphae and clamp connections. These morphological features were identical to the description of Sclerotium delphinii (syn. Sclerotium rolfsii var. delphinii) (Mukherjee et al. 2015; Punja and Damiani 1996; Stevens 1931). A representative isolate YKY2020.01 was stored in the Key laboratory of National Forestry and Grassland Administration on Ecological Landscaping of challenging Urban Sites in Shanghai. For molecular identification, DNA of the isolate YKY2020.01 was extracted using the Fungal DNA Kit (OMEGA bio-tek, China). The internal transcribed spacer region (ITS fragment including ITS1, 5.8S rDNA, and ITS2 region) was amplified with primers ITS1/ITS4 (White et al. 1990), and then sequenced by Sangon Biotech (Shanghai, China). BLAST analysis in NCBI found the ITS sequence of YKY2020.01 (MW916955) was 99.84% similar to S. delphinii strain CBS272.30 (MH855140). Phylogenetic analysis using maximum likelihood (ML) method placed isolate YKY2020.01 in the same clade as S. delphinii. To evaluate pathogenicity, hyphal blocks (0.7 cm diam.) were placed at the base of the stem of healthy E. fortunei (n = 5 plants). Five healthy plants were inoculated by uncolonized agar blocks as controls. All plants were kept in a greenhouse with a temperature range from 21 to 25.6°C (mean = 24.9°C) and relative humidity of 50%. Inoculated plants were symptomatic after 3 days and wilted after 12 days. Symptoms in inoculated plants were similar to those observed under natural conditions, whereas the control group remained asymptomatic. The fungal pathogen was reisolated from symptomatic tissue and confirmed as S. delphinii. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on E. fortunei in China and worldwide. This finding provides concise and practical information on the newly emerged disease of E. fortunei, which is beneficial for future disease management. References: Mukherjee, A. K., et al. 2015. J. Plant Pathol. 97:303. Punja, Z. K. and Damiani, A. 1996. Mycologia 88:694. Stevens, F. L. 1931. Mycologia 23:204. White, T. J., et al. 1990. Page 315 in PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Funding: This work was supported by the Key Project of Science and Technology Commission of Shanghai Municipality (19DZ1204102).

Genomic Insights into the Biology of Digenetic Fluke, Explanatum explanatum Infecting Domestic Water Buffalo, Bubalus bubalis from India: New Targets for Drug Designing

Explanatum explanatum is a digenetic trematode mostly found parasitizing bile duct and the gall bladder of domestic buffaloes in India .The parasite fabricates productivity loss and occasionally turns out as severe clinical disease leading to granulomatous nodules in the bile duct mucosa, glandular hyperplasia and thickening of the blood vessels. Ribosomes are macromolecular machinery for cell protein synthesis in all organisms. The present study deals with the identification of different DNA motif in 5.8S rDNA to block the activity of ribosome translocation, thereby hindering the process of protein synthesis within the parasite and thus serving as a novel tool for controlling its growth. Moreover, this segment of gene remains evolutionary conserved thus, enhancing the rate of success in designing potent drug molecules for these identified motifs in the genome of E. explanatum. This is first report of the identification of position of DNA motifs in 5.8S rDNA of E. explanatum from India and an attempt to provide new insights for further designing of new potent drugs for its efficient treatment as the parasite has developed tolerance power for already available drugs and the significant loss caused by it to the livestock and economy. Our study will form the foundation for future in-depth analysis of the parasite biology and development, immune evasion strategies, virulence and long-term survival within the definitive host. Our findings aim to provide a better understanding of the parasite genome, the search for new drug design research and thus, can prove as a vital tool for improving animal health that would ultimately succor to meet the ever increasing demand for food.

Identification of Fungi Involved in Onychomycosis in Patients of a Spanish Rural Area

Onychomycosis is one of the most frequent reasons for visiting podiatrist clinics. Complementary tests and the accurate identification of the infectious agents are key issues for a successful treatment of onychomycosis. This is particularly important when lifestyle, age and immunodepressed patients increase the prevalence of non-dermatophyte fungal infection. In this paper, we describe issues related to onychomycosis prevalence in a population of patients, mostly with rural lifestyles, visiting a podiatry clinic in a rural area of Spain. A total of 51 cases were studied with an average age of 65.96 ± 21.28 years (the youngest being 16 years and the oldest being 95 years). Fungal agents were isolated using conventional sampling and microbiological culture techniques. The results obtained with these techniques were compared with the results obtained with a direct methodology using molecular biology, by PCR and nucleotide sequencing of the ITS-5.8S rDNA fragment. The classical culture methodology confirmed the infection in 76.5% of the samples (n = 39), while the PCR confirmed the infection in 84.3% (n = 51) of the nails, although the difference between these results did not show statistical significance (p = 0.388). We found a high variability in agents, with more yeasts than dermatophytes as etiological agents of onychomycosis. However, only among individuals older than 65 years, was the difference between yeasts (82%) and dermatophytes (18%) was statistically significant (p = 0.004). Among the agents of non-dermatophyte onychomycosis, we found predominantly fungi (yeasts) of the Candida genus, interestingly with no isolates of Candida albicans, and moulds of the Aspergillus genus.

Integrative Studies on a New Ciliate Campanella sinica n. sp. (Protista, Ciliophora, Peritrichia) Based on the Morphological and Molecular Data, With Notes on the Phylogeny and Systematics of the Family Epistylididae

During an investigation on freshwater peritrichs, a new colonial sessilid ciliate, Campanella sinica n. sp., was isolated from aquatic plants in an artificial freshwater pond in Qingdao, China. Specimen observations of this species were performed both in vivo and using silver staining. C. sinica n. sp. is characterized by the appearance of the mature colony, which is up to 2 cm high and contains more than 1,000 zooids, the asymmetric horn-shaped zooids, strongly everted and multi-layered peristomial lip, the slightly convex peristomial disc, and the well-developed haplokinety and polykinety, which make more than four circuits of the peristome before descending into the infundibulum. The small subunit ribosomal DNA (SSU rDNA), 5.8s rDNA and its flank internal transcribed spacers (ITS1-5.8s rDNA-ITS2), and large subunit ribosomal DNA (LSU rDNA) are sequenced and used for phylogenetic analyses which reveal that the family Epistylididae Kahl, 1933 is non-monophyletic whereas the genus Campanella is monophyletic and nests within the basal clade of the sessilids. The integrative results support the assertion that the genus Campanella represents a separate lineage from other epistylidids, suggesting a further revision of the family Epistylididae is needed. We revise Campanella including the transfer into this genus of a taxon formerly assigned to Epistylis, which we raise to species rank, i.e., Campanella ovata (Nenninger, 1948) n. grad. & n. comb. (original combination Epistylis purneri f. ovataNenninger, 1948). In addition, we provide a key to the identification of the species of Campanella.

Comparative analysis of the characteristics of Haplorchis taichui mitochondrial genome with Metagonimus yokogawai and its ribosomal transcription unit with H. pumilio (family Heterophyidae)

Minute intestinal flukes, Haplorchis taichui and H. pumilio, belong to the family Heterophyidae (Trematoda: Platyhelminthes), which have been studied very limited, especially the molecular markers of the mitochondrial genomes (mtDNA) and the ribosome transcription units (rTU or rDNA). We have obtained the complete mitochondrial genome of H. taichui and the coding part of ribosome transcription unit of H. taichui and H. pumilio of Vietnam. Nucleotide and amino acid data were compared between H. taichui and Metagonimus yokogawai for genomic/gene composition, codon usage (skew/skewness), and tandem repeat units (TRU). The complete mtDNA of H. taichui (strain Htai-QT3-VN) with the length of 15,120 bp and M. yokogawai (15,258 bp; Korea; KC330755) contains 36 genes, including 12 protein-coding genes (cox1, cox2, cox3, nad1, nad2, nad3, nad4L, nad4, nad5, nad6, atp6 and cob), 2 ribosomal RNA genes (rRNA); 22 transfer RNA (tRNA or trn) and a noncoding region (NCR) between trnE and trnG, divided into 2 sub-regions containing 5 TRUs (182–183 bp/TRU). H. taichui (Vietnam and Laos) uses A = 19.56%, T = 39.71%, G = 28.34%, C = 12.39% (A + T is 59.27% ​​and G + C is 40.73%) for mtDNA construction, whose skew/skewness value at A+T is negative (–0,340) and G+C is positive (0.392); for 12 protein-coding genes (PCGs) is similar; but for the mito-ribosomal genes (MRGs, of 16S/rrnL and 12S/rrnS) it is less for A+T (57.22%) and more for G+C (42.78%). M. yokogawai had lower A+T (mtDNA/55.68%; PCGs/55.96%; MRGs/54.15%) and higher G+C usage rate than H. taichui. H. taichui of Vietnam and Laos has 10,164 bp encoding for 3,376 amino acids to construct 12 PCGs with the mostly used codons as Phenylalanine (Phe-TTT) and Leucine (Leu-TTG), and the leastly used codonsas Glutamine (Gln-CAA), Arginine (Arg-CGC). Additional condon, Thr-ACA/ACC can be added as the least used in M. yokogawai. The rTU (from 5 '18S to 3' 28S) of H. taichui (7,268 bp) and H. pumilio (7,416 bp) were identified with 5 genomic regions including 18S rDNA, ITS1, 5.8S rDNA, ITS2 and 28S rDNA. The 18S and 5.8S genes of both species were of the same length (1,992 bp for 18S, 160 bp for 5.8S), but different for 28S genes (3,875 bp for H. taichui and 3,870 bp for H. pumilio). ITS1 in H. taichui (797 bp) and ITS2 in H. pumilio (280 bp) do not contain TRUs, whilst ITS1 in H. pumilio (1,106 bp) contains 5 TRUs(136 bp for 3 TRU and 116 bp for 2 TRUs); and ITS2 in H. taichui (444 bp) contain 3 TRUs (83–85 bp/each).

CHARACTERIZATION OF FUSARIUM SPECIES ASSOCIATED WITH MANGO MALFORMATION DISEASE IN SOUTHERN SENEGAL

In Senegal mango production has long existed in traditional forms, especially in the south and center of the country. It accounts for 60% of the countrys fruit production, with an estimated annual output of 150,000 tons. Despite a positive balance sheet in recent years, mango orchardsareaffected by numerous phytosanitary constraints like diseases and insect pests. Among the diseases, mango malformation is considered one of the most deadly for the mango tree. Since its discovery in 2009 in the south of Senegal, the disease has continued to spread in this area with incursions towards the northern part of the country. Few studies were dedicated to that pathogen in the Senegalese context. This study was carried out to help bring more light in the identity of the pathogen(s) causing this disease. Therefore 13 isolates of Fusarium isolates obtainedfrom mango malformation tissueswere characterized using morphological criteria and the molecular analysis. The 5.8S rDNA region of the internal transcribed spacer (ITS) was amplifiedusing ITS1 and ITS4 primer pairs. Sequence analysis and other analytical studiesshowed that the malformation of mango tree in southern Senegal is relatedto a diversity of Fusarium species. The morphological and molecular analysisallowed to identify several Fusarium strains with a very high degree of similarity (99.08 to 99.82%) with the species in the NCBI database ranged.

Genetic Diversity of Symbiotic Green Algae of Paramecium bursaria Syngens Originating from Distant Geographical Locations

Paramecium bursaria (Ehrenberg 1831) is a ciliate species living in a symbiotic relationship with green algae. The aim of the study was to identify green algal symbionts of P. bursaria originating from distant geographical locations and to answer the question of whether the occurrence of endosymbiont taxa was correlated with a specific ciliate syngen (sexually separated sibling group). In a comparative analysis, we investigated 43 P. bursaria symbiont strains based on molecular features. Three DNA fragments were sequenced: two from the nuclear genomes—a fragment of the ITS1-5.8S rDNA-ITS2 region and a fragment of the gene encoding large subunit ribosomal RNA (28S rDNA), as well as a fragment of the plastid genome comprising the 3′rpl36-5′infA genes. The analysis of two ribosomal sequences showed the presence of 29 haplotypes (haplotype diversity Hd = 0.98736 for ITS1-5.8S rDNA-ITS2 and Hd = 0.908 for 28S rDNA) in the former two regions, and 36 haplotypes in the 3′rpl36-5′infA gene fragment (Hd = 0.984). The following symbiotic strains were identified: Chlorella vulgaris, Chlorella variabilis, Chlorella sorokiniana and Micractinium conductrix. We rejected the hypotheses concerning (i) the correlation between P. bursaria syngen and symbiotic species, and (ii) the relationship between symbiotic species and geographic distribution.

A new species of Fomitiporia (Hymenochaetales) from Australia

A new species of Fomitiporia, F. tasmanica, is described from Tasmania (Australia) based on morphological examination and phylogenetic analysis of the nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA and nuc 28S rDNA D1-D2 domains. The new species is characterized by perennial, resupinate basidiocarps, very small pores (10–12 per mm), a dimitic hyphal system, presence of hymenial setae and cystidioles, and subglobose basidiospores measuring 5.5–6.6 × 5–6 µm.