A chromosome-level genome assembly of the Echiura Urechis unicinctus.

Echiura is a distinctive family of unsegmented sausage-shaped marine worms whose phylogenetic relationship still needs strong evidence from the phylogenomic analysis. In this family, Urechis unicinctus is known for its high nutritional and medicinal value and adaptation to harsh intertidal conditions. Herein, we combined PacBio long-read, short-read Illumina and Hi-C sequencing, generating a high-quality chromosome-level genome assembly of U. unicinctus. The assembled genome spans ~1,138.6 Mb with a scaffold N50 of 68.3 Mb, of which 1,113.8 Mb (97.82%) were anchored into 17 pseudo-chromosomes. The BUSCO analysis demonstrated the completeness of the genome assembly and gene model prediction are 93.5% and 91.5%, respectively. A total of 482.1 Mb repetitive sequences, 21,524 protein-coding genes, 1,535 miRNAs, 3,431 tRNAs, 124 rRNAs, and 348 snRNAs were annotated. This study significantly improves the quality of U. unicinctus genome assembly, sets the footsteps for molecular breeding and further study in genome evolution, genetic and molecular biology of U. unicinctus.

Identification and expression analysis of Oxfibrillin gene involved in the regeneration process of Ophryotrocha xiamen (Annelida, Dorcilleidae).

Regeneration of lost body parts is a widespread phenomenon across annelids. However, the molecular inducers of the cell sources for this reparative morphogenesis have not been identified. We have identified a regeneration-related gene Oxfibrillin from the transcriptome analysis of a polychaeta, Ophryotrocha xiamen, which is found to be a well-suited model to study the mechanisms of regeneration. Fibrillins are large glycoproteins that assemble to form the microfibrils and regulate growth factors or other transfer processes. Here, we obtained the 31,274 bp genomic DNA sequences of Oxfibrillin. The coding sequence length was 5784 bp encoding 1927 amino acids with a VWD domain, EGF/cb-EGF domains, a TR domain, and a transmembrane domain. Oxfibrillin was positioned within the subgroup of invertebrates and showed low scores for homology to mammalian fibrillin. In gene expression analysis, Oxfibrillin genes were constantly upregulated during the early regeneration process and then remained stable until the formation of the complete tail which indicated that it might be a vital factor to affect posterior regeneration process. Therefore, the Oxfibrillin of O. xiamen might play important roles in the regeneration process.

Morphological and molecular features of early regeneration in the marine annelid Ophryotrocha xiamen.

Regeneration capability varies in the phylum Annelida making them an excellent group to investigate the differences between closely related organisms. Several studies have described the process of regeneration, while the underlying molecular mechanism remains unclear, especially during the early stage (wound healing and blastema formation). In this study, the newly identified Ophryotrocha xiamen was used to explore the early regeneration. The detailed morphological and molecular analyses positioned O. xiamen within 'labronica' clade. We analyzed the morphological changes during regeneration process (0-3 days post amputation) and molecular changes during the early regeneration stage (1 day post amputation). Wound healing was achieved within one day and a blastema formed one day later. A total of 243 DEGs were mainly involved in metabolism and signal transduction. Currently known regeneration-related genes were identified in O. xiamen which could help with exploring the functions of genes involved in regeneration processes. According to their conserved motif, we identified 8 different Hox gene fragments and Hox5 and Lox2 were found to be absent in early regeneration and during regular growth. Our data can promote further use of O. xiamen which can be used as an experimental model for resolving crucial problems of developmental biology in marine invertebrates.

Potential involvement of the microbiota-gut-brain axis in the neurotoxicity of triphenyl phosphate (TPhP) in the marine medaka (Oryzias melastigma) larvae.

Triphenyl phosphate (TPhP), a prevalent pollutant in the aquatic environment, has been reported to induce neurotoxicity (e.g., a suppression in locomotor activity) in fish larvae, posing a great threat to fish populations. However, the underlying mechanism was not fully revealed. In this study, the Oryzias melastigma larvae (21 dph) were exposed to waterborne TPhP (20 and 100 µg/L) for 7 days and a decreased locomotor activity was found. After exposure, the brain transcriptome and communities of gut microbiota were investigated to explore the potential mechanism underlying the suppressed locomotor activity by TPhP. The results showed that 1160 genes in the brain were dysregulated by TPhP, of which 24 genes were identified as being highly associated with the neural function and development (including nerve regeneration, neuronal growth and differentiation, brain ion homeostasis, production of neurotransmitters and etc), suggesting a general impairment in the central nervous system. Meanwhile, TPhP caused disorders in the gut microbiota. The relative abundance of Gammaproteobacteria and Alphaproteobacteria, which can influence the brain functions of host via the microbiota-gut-brain axis, were significantly altered by TPhP. Furthermore, the Redundancy analysis (RDA) revealed positive correlations between the intestinal genera Ruegeria, Roseivivax and Nautella and the dysregulated brain genes by TPhP. These results suggest that TPhP might impair the central nervous system of the O. melastigma larvae not only directly but also through the microbiota-gut-axis (indirectly), contributing to the suppressed locomotor activity. These findings enrich our mechanistic understanding of the toxicity of TPhP in fish larvae and shed preliminary light on the involvement of microbiota-gut-brain axis in the neurotoxicity of environmental pollutants.

First report of powdery mildew on Astragalus sinicus (Chinese milk vetch) caused by Erysiphe trifoliorum in China.

Astragalus sinicus L., (Chinese milk vetch) is a traditional leguminous green manure that plays a significant role in maintaining paddy soil fertility to enhance yield and the quality of rice in China. It is also found in gardens, roadsides, farms, fields, riverbanks, open wastelands, and is often used as livestock feed. From February 2019 to 2021, severe powdery mildew infections were observed on hundreds of A. sinicus grown in gardens and at roadsides of Fuzhou city, China. The disease incidence was up to 100% on leaves and stems of A. sinicus. White superficial fungal colonies (circular to irregular patches) were present on both sides of the leaves. Hyphae were flexuous to straight, branched, 4 to 8 µm in width, and septate. Hyphal appressoria were lobulate and solitary or in opposite pairs. Conidiophores were erect and straight, hyaline, and 60 to 120 × 8 to 10 µm (n=30). Foot cell was cylindrical, straight to slightly curved, 22 to 38 × 8 to 10 µm, followed by two to three shorter cells. Conidia were cylindrical-oval to doliiform, 30 to 48 × 13.5 to 24 µm with a length/width ratio of 1.6 to 2.4 (n = 30), formed singly, and without fibrosin bodies. Conidial germ tubes were produced subterminal position. No chasmothecia were found in the collected samples. The morphological characteristics of asexual structures were consistent with the descriptions of E. trifoliorum (Wallr.) U. Braun in Braun and Cook (2012). To verify the identification of the pathogen, the ITS and the part of large subunit (LSU) rDNA gene of the isolates were amplified using ITS1/ITS4 and LSU1/ LSU2 primers (Scholin et al. 1994 and White et al. 1990, respectively) and sequences were deposited in GenBank (ITS: MZ021332, MZ021333; LSU: MZ021334, MZ021335). In BLASTn searches, the ITS and LSU sequences were 99 to 100% identical with those of E. trifoliorum parasitic on Lathyrus magellanicus (LC010015), Medicago littoralis (LC270860), Melilotus officinalis (LC009924) and Trifolium spp., (MN216308, KY660821), as well as E. baeumleri (Bradshaw et al. 2021) on Vicia nigricans (LC010014). Pathogenicity test was performed by gently pressing a diseased leaf onto 10 young leaves of three healthy potted plants, while three non-inoculated plants were used as controls. All plants were maintained in a greenhouse at 20 to 25°C, without humidity control, and natural light. Symptoms developed 7 days after inoculation, whereas the control leaves remained symptomless. The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Powdery mildew on A. sinicus has been reported as E. pisi and E. polygoni from Korea and China (Shin, 2000; Tai 1979), respectively. Amano (1986) listed E. pisi and Microsphaera astragali (now E. astragali) on A. sinicus from China and Japan. To our knowledge, this is the first report of powdery mildew caused by E. trifoliorum on A. sinicus in China and in general. E. astragali is the most common and widespread powdery mildew species on Astragalus spp. (Braun and Cook 2012) and would be expected on A. sinicus, but this species is genetically clearly different from E. trifoliorum (Bradshaw et al. 2021). The E. trifoliorum complex (clade) is composed of several morphologically well-distinguishable species, besides E. trifoliorum also including E. baeumleri (on Vicia spp.), E. hyperici (on Hypericum spp.), and E. euonymi (on Euonymus spp.), but based on a combination of sequence plus host identity, the collection on A. sinicus can be assigned to E. trifoliorum (Bradshaw et al. 2021). The information in this study extended the host range of E. trifoliorum as well as future studies on A. sinicus in relation to powdery mildew outbreaks in China. References: Amano (Hirata), K. 1986. Host Range and Geographical Distribution of the Powdery Mildew Fungi. Japan Scientific Societies Press, Tokyo, 741 pp. Bradshaw, M., et al. 2021. Mycologia. (In press) Braun, U., Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, the Netherlands. Scholin, C. A., et al. 1994. J. Phycol. 30:999. Shin, H.D. 2000. Erysiphaceae of Korea. National Institute of Agricultural Science and Technology, Suwon, Korea, 320 pp. Tai, F.L. 1979. Sylloge Fungorum Sinicorum. Sci. Press, Acad. Sin., Peking, 1527 pp. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Sigesbeckia orientalis in China.

Sigesbeckia orientalis L., (St Paul's wort) is an annually grown natural herb of Asteraceae with a long therapeutic history for a wide range of inflammation-related diseases in China (Zhong et al. 2019). In June 2020, typical symptoms of powdery mildew were observed on 30% of wild S. orientalis plants grown along the roadsides and gardens in Minjiang University, Fuzhou, China. Circular to irregular white powdery fungal colonies were observed on both surfaces of the leaves and young stems, causing necrosis and premature senescence. Fungal hyphae were epigenous, flexuous to straight, branched, and septate. Appressoria on the hyphae were nipple-shaped or nearly absent. Conidiophores were straight, 30 to 210× 8 to 12 µm, and produced 3 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindrical, 45 to 75 ×10 to 12 µm, followed by 1 to 2 shorter cells. Conidia were hyaline, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 18 to 23 µm with distinct fibrosin bodies. Germ tubes were produced from a lateral position on the conidia. Chasmothecia were not observed on the infected leaves. Based on anamorph characteristics, fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). For molecular identification, total genomic DNA was extracted (Mukhtar et al. 2018) from fungal colonies on infected leaves of five collections separately. For each DNA sample, the part of LSU and ITS regions were amplified using primers LSU1/LSU2 and ITS1/ITS4 (Scholin et al. 1994; White et al. 1990), respectively. A BLAST search revealed 100 % sequences similarity with P. xanthii sequences reported on Ageratum conyzoides (KY274485), Eclipta prostrata (MT260063), Euphorbia hirta (KY388505), Sonchus asper (MN134013), and Verbena bonariensis (AB462804). Representative sequences (ITS: MZ613309; LSU: MZ614707) of an isolate were deposited in GenBank. The phylogenetic analysis also grouped the obtain sequences into P. xanthii clade. Pathogenicity was confirmed by gently pressing the infected leaves onto young leaves of five healthy one-month-old S. orientalis plants, while three non-inoculated plants were used as controls. All plants were maintained in a greenhouse at 25 ± 2°C. After, seven days, white powdery colonies were observed on inoculated plants, whereas controls remained mildew-free. On inoculated leaves, the fungus was morphologically and molecularly identical to the fungus on the original specimens. P. xanthii has been reported as a significant damaging pathogen on a wide range of plants in China (Farr and Rossman 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. orientalis in China as well as worldwide. S. orientalis is one of the most important commercial Chinese medicinal herbs and the occurrence of powdery mildew is a threat to its production, quality, and marketability. References: Braun, U., and Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 9 October 2021. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Zhong, Z., et al., 2019. Chin. Med. (U. K.) 14, 1-12. 10.1186/s13020-019-0260-y.

Subchronic toxicity of dietary sulfamethazine and nanoplastics in marine medaka (Oryzias melastigma): Insights from the gut microbiota and intestinal oxidative status.

Antibiotics and nanoplastics are two prevalent pollutants in oceans, posing a great threat to marine ecosystems. As antibiotics and nanoplastics are highly bioconcentrated in lower trophic levels, evaluating their impacts on marine organisms via dietary exposure route is of great importance. In this study, the individual and joint effects of dietborne sulfamethazine (SMZ) and nanoplastic fragments (polystyrene, PS) in marine medaka (Oryzias melastigma) were investigated. After 30 days of dietary exposure, 4.62 mg/g SMZ decreased the Chao1 index (60.86% for females and 26.85% for males) and the Shannon index (68.95% for females and 65.05% for males) and significantly altered the structure of gut microbial communities in both sexes. The female fish exposed to 4.62 mg/g SMZ exhibited higher intestinal sod (43.5%), cat (38.5%) and gpx (39.6%) transcripts, indicating oxidative stress in the gut. PS alone at 3.45 mg/g slightly altered the composition of the gut microbiota. Interestingly, the mixture of SMZ and PS caused more modest effects on the gut microbiota and intestinal antioxidant physiology than the SMZ alone, suggesting that the presence of PS might alleviate the intestinal toxicity of SMZ in a scenario of dietary co-exposure. This study helps better understand the risk of antibiotics and nanoplastics to marine ecosystems.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Bidens pilosa in China.

Bidens pilosa L., (spanish needle), is a wild, flowering plant of Asteraceae, that is grown in gardens, fields, roadsides, and riverbanks in Fuzhou, China. It is also used in traditional folk medicine for a broad range of ailments in China. In March 2019 and 2020, hundreds of B. pilosa growing along the roadsides, and gardens in the districts of Minhou and Jinshan were observed to be severely affected by a powdery mildew with approximately 80% disease incidence. Symptoms appeared as circular to irregular small white, powdery patches, typically on the adaxial sides of leaves and progressed to coalescent colonies on the leaves. As the disease developed, the infected leaves became wilted and senesced. Mycelia on leaves were superficial and solitary appressoria were slightly to distinctly nipple-shaped. Conidiophores were erect, 120 to 230 × 10 to 12 µm, and produced two to five conidia in chains with a sinuate outline. Foot-cells were erect, cylindrical, and 60 to 110 µm long. Conidia were hyaline, ellipsoid to barrel-shaped, 26 to 40 × 18 to 24 µm, and devoid of distinct fibrosin bodies. Germ tubes were long and produced at the perihilar position of the conidia. No chasmothecia were observed. Morphological characteristics overlapped with Golovinomyces ambrosiae, G. cichoracearum, and G. spadiceus (Braun and Cook 2012) on hosts within the Asteraceae tribe Heliantheae (Takamatsu et al. 2013). For molecular identification, ITS and IGS regions as well as partial LSU of two representative collections (MJU-IM019- MJU-IM020), were amplified using ITS1/ITS4, IGS-12a/ NS1R and LSU1/LSU2 primers (Carbone & Kohn, 1999; Scholin et al. 1994; White et al. 1990), respectively. The resulting sequences were deposited in GenBank (ITS: MW965777, MW965778; LSU: MW965787, MW965788; IGS: MW981256, MW981257). A BLAST search revealed 99 to 100 % sequence similarity to G. ambrosiae sequences (KX987303, AB769421, AB077689, AB769426, AB077643, and AB769425). Phylogenetic analysis of ITS, LSU and IGS also grouped obtained sequences within the G. ambrosiae complex (Qiu et al. 2020). Pathogenicity was confirmed through inoculation by gently pressing infected leaves onto leaves of five healthy, potted, young B. pilosa plants, while five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25 ± 2°C. Inoculated plants developed symptoms after 7 to 10 days, whereas the control plants remained symptomless. The morphology of the resulting fungus on inoculated plants was identical to that originally observed on diseased plants. Podosphaera spp., have been reported on B. pilosa (Farr & Rossman 2021) from North America, Africa, and Asia. To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on B. pilosa in China and Asia. Wild populations of B. pilosa may be the primary source of powdery mildew inoculum for commercial Asteraceae members and may warrant consideration in the control of this disease. References: Braun, U., and Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, The Netherlands. Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 18 April 2021. Qiu, P. L., et al. 2020. BMC Microbiol. 20:1. Scholin, C. A., et al. 1994. J. Phycol. 30:999. Takamatsu, S., et al. 2013. Mycologia 105:1135. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Cuphea hyssopifolia (Lythraceae) in Mainland China.

Cuphea hyssopifolia (Mexican heather) is a popular evergreen perennial shrub used for ornamental and medicinal purposes. Due to its high ornamental value, it is often used as a ground cover in parks and gardens in China. During February and March 2019 & 2020, powdery mildew was observed on C. hyssopifolia in the districts of Minhou and Jinshan of Fuzhou, China. Disease incidence was 70% but of low severity with only a few older leaves showing yellowing and wilting. Sparse irregular patches of white superficial powdery mildew observed on both sides of mature and young leaves. The powdery mildew fungal appressoria that occurred on epigenous hyphae, were indistinct to nipple-shaped, hyaline, and smooth. Conidiophores were erect, smooth, 80 to 210 × 10 to 12 µm, and produced two to eight crenate-shaped conidia in chains. Foot-cells of conidiophores were straight, cylindric, and 30 to 65 × 10 to12 µm. Conidia were hyaline, smooth, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 16 to 20 µm with distinct fibrosin bodies. Germ tubes were simple to forked and produced from the lateral position of the germinating conidia. No chasmothecia were observed on the surface of infected leaves. Based on the morphology of the imperfect state, the powdery mildew fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). To confirm fungal identification, total DNA was extracted (Mukhtar et al., 2018) directly from epiphytic mycelia on infected leaves collected from both districts. Internal transcribed spacer (ITS) regions and the partial large subunit (LSU) rDNA were amplified using primers ITS1/ITS4 and LSU1/LSU2 (Scholin et al. 1994, White et al. 1990), respectively. The sequences were deposited in GenBank (ITS: MW692364, MW692365; LSU: MW699924, MW699925). The ITS and LSU sequences were 99 to 100 % identical to those of P. xanthii in GenBank, (ITS: MT568609, MT472035, MT250855, and AB462800; LSU: AB936276, JX896687, AB936277, and AB936274). Koch's postulates were completed by gently pressing diseased leaves onto leaves of five healthy potted C. hyssopifolia plants that were held in a greenhouse at 24 to 30°C without humidity control. Five non-inoculated plants served as controls. Inoculated plants developed symptoms after 6 to 10 days, whereas the controls remained symptomless. The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Previously, Podosphaera sp. has been reported on C. rosea in the United Kingdom (Beales & Cook 2008) and P. xanthii on C. hyssopifolia in Taiwan (Yeh et al. 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on C. hyssopifolia in mainland China. Our field observations suggest that the P. xanthii infections would be a potential threat to the health of C. hyssopifolia in China. References: Beales, P. A., and Cook, R. T. A. 2008. Plant Pathol. 57:778. Braun, U., Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Yeh, Y. W., et al. 2021. Trop. Plant Pathol. 46:44.

Transcriptome Profiling Revealed Multiple rquA Genes in the Species of Spirostomum (Protozoa: Ciliophora: Heterotrichea).

Adaptation to life at different oxygen tensions plays a role in protozoan ecology and controls the distribution of different species in anoxic habitats. The ciliate genus Spirostomum inhabiting fresh or low salinity water globally where these species are considered as bioindicators. Under anaerobic or low oxygen conditions, the rhodoquinol-dependent pathway has been reported in the species from the class Heterotrichea. With the help of RNA sequencing (RNAseq) data, Spirostomum spp., are suitable for deep molecular investigations on rquA for rhodoquinone (RQ) biosynthesis. In this study, Spirostomum ambiguum, Spirostomum subtilis, and Spirostomum teres collected from densely vegetated freshwater habitat in Fuzhou, China, explored the evidence of rquA. Based on transcriptome analysis, two to three RquA proteins were identified in S. ambiguum, S. teres, and S. subtilis, respectively. The presence of a key Motif-I of RquA and mitochondrial targeting signals (MTS), also confirmed the identity of these as RquA. Furthermore, Spirostomum RquA proteins could be sorted into two groups based on their conserved amino acid (CAA) residues. Phylogenetic analysis also exhibited RquA division into two subclades contained RquA1 and RquA2/RquA3 and supports two to three paralogs of rquA genes in the genomes Spirostomum spp. Additional transcriptomes and genomes analysis of Blepharisma spp., and Stentor spp., respectively, also revealed at least two paralogs of rquA in members of the class Heterotrichea. The present study provides evidence for the presence of RquA and rhodoquinol dependent fumarate reduction pathway in Spirostomum species potentially use to respire in the oxygen-depleted habitats and two to three diverse rquA genes.

Molecular mechanisms of an antimicrobial peptide piscidin (Lc-pis) in a parasitic protozoan, Cryptocaryon irritans.

BACKGROUND: Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture fish species and cause high lethality and economic loss. Current methods of controlling this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. Piscidins with broad spectrum antibacterial, antifungal and antiviral activities were found to have potent activity against C. irritans. Little, however, has been understood about the killing mechanisms of piscidins in parasites. RESULTS: In total, 57.12, 50.44, 55.86 and 47.87 million raw reads were generated from untreated theront and trophont, and piscidin (Lc-pis) treated theront and trophont libraries, respectively. After de novo assembly, 966,609 unigenes were generated with an average length of 420 bp: among these, 618,629 unigenes showed identity with sequences in one or more databases, with some showing to be significantly manipulated by Lc-pis treatment. The species classification showed that more than 25.8% unigenes from trophonts were homologous to the large yellow croaker (Larimichthys crocea) and less than 3.8% unigenes from theronts were matched. The homologous unigenes demonstrated that the tissue from host could exist in trophonts and might be transported to parasite via vesicular transports. Our analysis showed that regulatory transcripts were involved in vesicular trafficking. Among transcripts induced by Lc-pis, most genes up-regulated in treated and untreated theronts were involved in cell migration and apoptosis related pathways. Few transcripts were found to be down-regulated in treated and untreated trophonts related to cell structure and migration after treatment. CONCLUSIONS: This is the first transcriptome analysis of C. irritans exposed to Lc-pis, which enhanced the genomic resources and provided novel insights into molecular mechanisms of ciliates treated by cationic antimicrobial peptide. Our comprehensive transcriptome analysis can facilitate the identification of potential drug targets and vaccines candidates for controlling this devastating fish pathogen.

Excavating differentially expressed antimicrobial peptides from transcriptome of Larimichthys crocea liver in response to Cryptocaryon irritans.

Larimichthys crocea, the special marine economy fish, owns the largest annual yield for a single species in China. One of the most significant factors affecting large yellow croaker culture is the diseases, especially the threat of marine white spot disease which caused by a protozoan Cryptocaryon irritans. Antimicrobial peptides (AMPs) have been demonstrated to be active against bacterium, fungi and parasites, showing their potential usefulness in aquaculture as substitutes for antibiotics. Many researches have been carried out about the AMPs concentrating on the activity resist on C. irritans, and piscidin-like of L. crocea owning widely antibacterial spectrum and strong activity against C. irritans was screened in our team. In the paper, taking advantage of the large yellow croaker hepatic comparison transcriptome in response to C. irritans at 3d post infection, seven kinds of AMPs have been excavated from the differently expressed genes, including LEAP2 like, LEAP-2A, hepcidin, hepcidin-like, piscidin-5-like, piscidin-5-like type 4 and bactericidal permeability increasing protein (BPI). Hepcidin, hepcidin-like, piscidin-5-like, piscidin-5-like type4 and BPI were up-regulated to protect large yellow croaker from being damaged by C. irritans infection; while LEAP2 like and LEAP-2A were down-regulated, they might be as a negative-feedback regulation factor or some other regulatory mechanisms to adjust the immune response in the process of C. irritans infection. The differential expression changes were verified with quantitative real-time PCR (qRT-PCR) to illustrate the reliability of the sequenced data. Hearteningly, piscidin-5-like type 4 was a novel type which was high similar to other piscidin-5-like types. Interestingly, the infection may well cause alternative splicing of LEAP-2A mRNA, which was a surprised phenomenon and finding after C. irritans infection, but more further study was needed to be conducted. Therefore, the data showed that these AMPs were involved in the immune response to the C. irritans infection. In all, these results implied that the immune response of AMPs to C. irritans infection was a complex and sophisticated regulatory process.

A novel stimulator of interferon gene (STING) from Larimichthys crocea and their involvement in immune response to ectoparasite Cryptocaryon irritans infection.

The marine white spot disease caused by protozoan ectoparasite Cryptocaryon irritans is a severe problem to the large yellow croaker farming industry. To understand the molecular immune mechanisms and improve its immune capacity are particular important. STING, one of the important second messengers in innate immune response process, plays pivotal roles in defensing against different pathogenic microorganisms. Many reports have pointed that STING could not only combine the uncovered dsDNA, ssDNA directly in the cytoplasm from the pathogens or biology itself, but it also could recognize cyclic diguanylate monophosphate (c-di-GMP), cyclic diadenylate monophosphate (c-di-AMP). Based on the STING sequence, a variant of the L. crocea STING (termed LcSTING2) was found by accident. RACE was used to clone the full-length cDNA of LcSTING2 which contained a 5'- UTR of 154 bp, a 3'-UTR of 592 bp and an ORF of 1227 bp encoding 408 amino acids. The predicted protein molecular weight (Mw) was 45.83 KDa and the estimated theoretical isoelectric point (pI) was 6.24. The deduced protein of LcSTING2 contains no signal peptide. One transmembrane motif (TM) in the N-terminal region, a TMEM173 domain and five putative motifs (RXR) found in resident endoplasmic reticulum proteins were also conserved. According to the partial genomic sequence, the unknown sequences were amplified, it contained 6 exons and 5 introns, and all the exon-intron boundaries were in accordance with classical GT-AG rule (GT/intron/AG). The similarity shared with fishes was higher than other high vertebrates. qRT-PCR results showed that LcSTING (two variants) distributed in all examined tissues, and it was the most abundant in gill. After challenged by C. irritans, LcSTING mRNA only appeared instantaneous up-regulation during the infective stage of theronts in the head kidney and was also up-regulated during the whole infectious cycle of C. irritans in the liver, which implied LcSTING gene was likely to be involved in the defensing against C. irritans infection, it is the first time to explore the STING taking part in the immune response to ectoparasite rather than bacterium or viruses.

Analysis of liver and gill miRNAs of Larimichthys crocea against Cryptocryon irritans challenge.

The white-spot disease caused by marine ciliate Cryptocryon irritans hindered the sustainable development of large yellow croaker Larimichthys crocea industry. Better understandings about the parasite-host interactions in the molecular level will facilitate the prevention of mass mortality of the L. crocea caused by white-spot disease. MicroRNAs (miRNAs) are a class of small RNA molecules about 20-22 nucleotides which post-transcriptionally regulated many protein-coding genes and involved in many biological processes, especially in host-pathogen responses. In this study, we identified known and novel miRNAs in the gill and liver of L. crocea challenged by C. irritans by high throughput sequencing using Solexa technology. The data were further studied to screen differentially expressed miRNAs, and predict their target genes. The differential expression (p < 0.05) between libraries was observed in 103 miRNAs in liver tissue, among which 65 and 38 were conserved and novel miRNAs, 67 and 36 were up- and down-regulated miRNAs. While in gill tissue, 122 significant differentially expressed miRNAs were identified, among which 83 and 39 were conserved and novel miRNAs, 79 and 43 were up- and down-regulated miRNAs. In addition, these differentially expressed miRNAs target a series of genes which involved in many important biological processes including immune response. Here via deep sequencing, we for the first time characterize L. crocea miRNAs in response to C. irritans challenge, the results should help for better understandings about the immune response of the L. crocea under C. irritans challenge.

Molecular characterization and expression analysis of interferon-gamma in the large yellow croaker Larimichthys crocea.

The large yellow croaker Larimichthys crocea is an important mariculture fish species in China, and the bacterium Vibrio harveyi (V. harveyi) and the ciliate protozoan Cryptocaryon irritans (C. irritans) are the two major pathogens in its aquaculture sector. Interferon-gamma (IFN-γ) plays important roles in regulating both innate and cell mediated immune responses as an inflammatory cytokine. In this study, we obtained the nucleotide sequence of IFN-γ from the large yellow croaker (LcIFN-γ). The phylogenetic relationship tree of 18 available IFN-γ genes was constructed based on their sequences. Expression analyses in 10 various tissues were conducted after the croaker challenged with V. harveyi and C. irritans, respectively. Real time PCR analysis showed that the expression of LcIFN-γ was observed broadly in health individuals. After injected with V. harveyi, the 10 tissues had a higher expression of IFN-γ at the first day (1 d); only spleen, muscle, intestine, heart and skin had higher expressions after infected with C. irritans at 1 d. Major immune tissues (skin, gill, head kidney and spleen) and detoxification tissue (liver) were sampled at 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 5 d and 7 d to understand the expression trends of LcIFN-γ after challenged with C. irritans. The expressions of LcIFN-γ in skin and gill (the primary immune organs) showed a clear correlative relationship with the life cycle of C. irritans.