Search

Additional file 2: Fig S1. Genome-wide Hi-C interaction map. Fig S2. Divergence distributions of four TE sequences predicted by the de novo method (a) and comparison of the distribution of several features in the final gene set for seven fish species (b). TE, transport elements; DNA, DNA transposons; LTR, long terminal repeats; LINE, long interspersed nuclear elements; SINE, short interspersed nuclear element. The seven fish species: Misgurnus anguillicaudatus, Carassius auratus, Cyprinus carpio, Danio rerio, Sinocyclocheilus graham, S. rhinocerous, and Triplophysa tibetana. Fig S3. The construction of maximum likelihood tree of fos gene and generation of the fos (ID: Mis0086400.1) knockout loach (fos−/−) by CRISPR/Cas9 technology. (a) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of fos gene in fish species. Different branch colors represent different species. Red words indicate the knockout of fos gene in loach genome. (b) Schematic position of the CRISPR/Cas9 target site for fos gene knockout. (c) The transcription level of fos in livers of wild-type loach (WT) and fos−/− loach. (d) A statistics of survival rate of fertilized eggs of WT loach and heterozygous F1 generation (F1 generation self-crossed). *** extremely significant difference (p < 0.001). hpf, hours post fertilization; fos, Proto-oncogene c-Fos. The gene in red color is a positively selected gene in the loach genome. Fig S4. Identification and expression analysis of air-breathing- and digestion/absorption- related genes in loach Misgurnus anguillicaudatus. (a) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of hb gene family. Different color backgrounds represent different genes. Different branch colors and inner circle colors represent different species. hbb, hemoglobin subunit beta; hba, hemoglobin subunit alpha. (b) Expression levels of hbb and hba in posterior intestines of the loach under air exposure. (c) qPCR validation of RNA-seq data of loach posterior intestines under air exposure. (d) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of air-breathing-related genes. Different branch colors represent different species. (e) Maximum likelihood tree of digestion/absorption-related genes (TBLASTN (with e-value of 1×10−5)). Atp1a, sodium/potassium-transporting ATPase subunit; ryr, ryanodine receptor. Different color backgrounds represent different genes. Different branch colors represent different species. Npr2, atrial natriuretic peptide receptor 2; f9, coagulation factor IX; hspb1, heat shock protein beta-1; hyou1, hypoxia up-regulated protein 1; ptafr, platelet-activating factor receptor; scx, basic helix-loop-helix transcription factor scleraxis; galnt8, polypeptide N-acetylgalactosaminyltransferase 8; krt13, keratin, type I cytoskeletal 13 Cytokeratin-13; gp2, pancreatic secretory granule membrane major glycoprotein; smco3, single-pass membrane and coiled-coil domain-containing protein 3; ccl5, C-C motif chemokine 5; ifi44, interferon-induced protein 44; cldn5, claudin-5 Transmembrane protein deleted in VCFS; hspb1, heat shock protein beta-1; vegfr1 (flt1), vascular endothelial growth factor receptor 1; ctgf, connective tissue growth factor CCN family member 2. Fig S5. Identification of FMO and UGT gene families. (a) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of FMO gene family. Different colors represent different FMO gene families and different branch colors represent different species. (b) Maximum likelihood tree (TBLASTN (with e-value of 1×10−5)) of UGT gene family. Different colors represent different UGT gene families and different inner circle colors represent different species. Fig S6. qPCR validation of RNA-seq data from the drug stress trial. Fig S7. The expression levels of ugt genes in liver tissues of the loach under five drug stress. Mis115330.1 and Mis0135560.1 (ugt2a2), UDP-glucuronosyltransferase 2A2; Mis0072610.1 (ugt2a1), UDP-glucuronosyltransferase 2A1. Fig S8. Tissue expression analysis and the knockout of fmo5 (ID: Mis0185930.1) gene (fmo5−/−) in loach Misgurnus anguillicaudatus genome. (a) Tissue expression analysis of fmo5 (ID: Mis0185930.1) of the loach. (b) Schematic position of the CRISPR/Cas9 target site for fmo5 gene knockout. (c) The mRNA and protein expression levels of fmo5 in livers of wild-type (WT) and fmo5−/− loach. Different letters above error bars indicate significant difference among different tissues (p < 0.05); *** extremely significant difference (p < 0.001). Fig S9. Survival rates of wild-type (WT) and fmo5deletion (fmo5−/−) loach under five drug stress.

Additional file 1: Table S1. Statistics of assembly and annotation of loach Misgurnus anguillicaudatus genome. (1) Statistics of Illumina HiSeq sequencing data of the loach genome. (2) 17-kmer analysis for estimation of the loach genome size. (3) Hi-C library sequencing data of the loach. (4) Statistics of repeated sequence classification of the loach genome. (5) Statistics of gene annotation of the loach genome. (6) Statistics of gene functional annotation of the loach genome. Table S2. Detailed gene information related to mud-dwelling behavior and intestinal evolution (air-breathing and digestion/absorption) of loach Misgurnus anguillicaudatus. (1) GO enrichment analysis of the expanded myosin complex genes in the loach genome. (2) KEGG enrichment analysis of the positively selected gene involved in osteoclast differentiation in the loach genome. (3) GO enrichment analysis of the expanded gene families involved in oxygen transport in the loach genome. Red and black gene IDs present hbb and hba genes, respectively. (4) KEGG enrichment analysis of the positively selected genes involved in VEGF signaling pathway in the loach genome. (5) Expression analysis of some DEGs in posterior intestine transcriptomes between Leptobotia elongate (LE, without air-breathing) and the loach (MA, with intestinal air-breathing) (referred from our previous study). (6) Summary of detected microRNAs and target genes involved in vascular biology of loach posterior intestines (referred from our previous study). (7) The DEGs involved in intestinal air-breathing and nutrient uptake of the loach (referred from our previous studies) (8) Expression analysis of five key DEGs in posterior intestine transcriptomes of the loach between the control (C_chang) and air exposure (T_chang) group. Un means Mis0158000.1, which is a new gene and its KEGG annotation is interleukin 1 beta (Il1b). (9) KEGG enrichment analysis of the contracted gene families involved in digestion/absorption of the loach. (10) KEGG enrichment analysis of the expanded genes involved in digestion/absorption of the loach. hbb, hemoglobin subunit beta; hba, hemoglobin subunit alpha; VEGF, vascular endothelial growth factor; DEGs, differentially expressed genes; Il1b, Interleukin-1 beta; cldn5, Claudin-5 Transmembrane protein deleted in VCFS; hspb1, heat shock protein beta-1; vegfr1 (flt1), vascular endothelial growth factor receptor1; ctgf, Connective tissue growth factor CCN family member 2; hif1a, hypoxia-inducible factor 1-alpha. Table S3. Genes involved in detoxification function of loach Misgurnus anguillicaudatus. (1) The expanded genes involved in the detoxification function of the loach genome. (2) KEGG enrichment analysis of DEGs involved in the xenobiotics biodegradation and metabolism between the control and five drug stress groups. Red and black gene IDs present fmo and ugt genes, respectively. DEGs, differentially expressed genes. Table S4. Gene annotations for the zebrafish gene clusters in this study. Fos, proto-oncogene c-Fos; hba, hemoglobin subunit alpha; hbb, hemoglobin subunit beta; ryr, ryanodine receptor; atp1a, sodium/potassium-transporting ATPase subunit alpha; cldn5, claudin-5; vegfr1, vascular endothelial growth factor receptor 1; hspb1, heat shock protein beta-1; ctgf, connective tissue growth factor CCN family member 2; fmo, dimethylaniline monooxygenase [N-oxide-forming]; ugt, UDP-glucuronosyltransferase. Table S5. Primers used in this study. qPCR, quantitative PCR; # indicated T7 promoter sequences. Fos, proto-oncogene c-Fos; fmo5, dimethylaniline monooxygenase [N-oxide-forming] 5 (Mis0185930.1); hbb, hemoglobin subunit beta; hba, hemoglobin subunit alpha; atf6, cyclic AMP-dependent transcription factor; eif2ak3, eukaryotic translation initiation factor 2-alpha kinase 3; chop, DNA damage-inducible transcript 3 protein; muc2, mucin-2; Mis0185950.1, Mis0185940.1, Mis0185920.1, Mis0185970.1, Mis0186000.1, Mis0186010.1 (fmo5), dimethylaniline monooxygenase [N-oxide-forming] 5; Mis0115330.1, Mis0115330.1, Mis0135560.1 (ugt2a2), UDP-glucuronosyltransferase 2A2; Mis0072610.1 (ugt2a1), UDP-glucuronosyltransferase 2A1; npr2, atrial natriuretic peptide receptor 2; f9, coagulation factor IX; hspb1, heat shock protein beta-1; hyou1, hypoxia up-regulated protein 1; ptafr, platelet-activating factor receptor; scx, basic helix-loop-helix transcription factor scleraxis; galnt8, polypeptide N-acetylgalactosaminyltransferase 8; krt13, keratin, type I cytoskeletal 13 Cytokeratin-13; gp2, pancreatic secretory granule membrane major glycoprotein; smco3, single-pass membrane and coiled-coil domain-containing protein 3; ccl5, C-C motif chemokine 5; ifi44, interferon-induced protein 44; atf4, cyclic AMP-dependent transcription factor 4; ugt1a1, UDP-glucuronosyltransferase 1-1. Table S6. A summary of sequencing data used in genome assembly and annotation of Misgurnus anguillicaudatus.

Resumen del trabajo presentado en el XVIII Congreso Nacional de Acuicultura, celebrado en Cádiz (España) del 21 al 24 de noviembre de 2022., [EN]: Gammarids are arising as promising candidates for feed formulation in aquaculture due to their capacity to be fed on a wide range of sidestreams while maintaining adequate levels of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA). The main goal of this work was to elucidate how different conditions (temperature and diet) modulate the expression of LC-PUFA biosynthesis and energy metabolism related genes. Individuals of the marine gammarid Gammarus locusta were fed on different diets (3) and reared at several temperatures (4), their RNA was sequenced, and the raw data were used to build a reference transcriptome for differential gene expression (DGE) analysis. Genes related to LC-PUFA and energy metabolism were downregulated in the gammarids fed low LC-PUFA diets and reared at suboptimal temperatures., [ES]: Los gamáridos son prometedores candidatos para la formulación de pienso en acuicultura debido a su capacidad para alimentarse de una amplia gama de subproductos mientras mantienen niveles adecuados de ácidos grasos poliinsaturados de cadena larga (≥C20) (LC-PUFA). El objetivo principal de este trabajo fue dilucidar cómo diferentes condiciones (temperatura y dieta) afectan a la expresión de genes relacionados con la biosíntesis de LC-PUFA y el metabolismo energético. Ejemplares del gamárido marino Gammarus locusta se alimentaron con diferentes dietas (3), a distintas temperaturas (4), se secuenció su ARN y los datos se usaron para construir un transcriptoma de referencia para el análisis de la expresión génica diferencial (DGE). Los resultados indican que los genes relacionados con LC-PUFA y el metabolismo energético están subexpresados en los gamáridos alimentados con dietas pobres en LC-PUFA y cultivados a temperaturas subóptimas, SIDESTREAM (ERA-NET BlueBio, Grant ID 68), IMPROMEGA RTI2018-095119-B-100.

Resumen del trabajo presentado en Aquaculture Europe, celebrado en Rimini (Italia) del 27 al 30 de septiembre de 2022., [Introduction]: The common ragworm Hediste diversicolor is an omnivorous, burrowing polychaete with a broad tolerance to environmental conditions such as temperature, salinity and oxygen levels. This nutritious annelid has received a great deal of attention lately for its potential as an extractive species in IMTA systems to recapture high-value compounds from fed aquaculture particulate side streams (sludge) and its suitability for subsequent utilisation as a novel, omega-3 rich aquafeed ingredient (Wang et al., 2019). It has recently been shown that this species has the capacity for endogenous production of omega-3 long-chain polyunsaturated fatty acids (Kabeya, et al., 2020), however it is yet not understood how environmental cues affects this ability. We conducted two sets of experiments to assess the combined effects of diet, temperature and salinity on growth, survival and total body fatty acid composition in H. diversicolor juveniles. [Materials and Methods]: Polychaetes (H. diversicolor) were collected from Leangen Bay, Trondheim, Norway (63°26′24.5″N, 10°28′27.7″E). To investigate the effects of diet and temperature on growth, survival and fatty acid (FA) composition of H. diversicolor, worms were fed mixes of solid biogas digestate (SBD) and salmon aquaculture sludge (SS) along a 4-step feed gradient ranging from pure SBD to pure SS, and a 5-step temperature gradient ranging from 5.8 to 17.1 °C, for 15 days, using fish feed as a control (Experiment 1). A second experiment was conducted to investigate the effects of salinity and temperature on the same variables. Here, the worms were fed the diet which yielded the highest growth rates in the first experiment (33:66 % SS:SBD) along 5-step salinity and temperature gradients ranging from 5 to 40 ppt and 7.7 to 17.9 °C, respectively, for a duration of 28 days. In both experiments, worms were fed isonitrogenous diets equalling 30 % of the worms' total body nitrogen per day (Wang et al., 2019). Both experiments were conducted in a temperature gradient table (modified after Thomas et al. (1963)) using a 18h:6h light:dark cycle. Worms (n=7-8) were stocked in glass beakers (800 mL) containing an eight cm thick layer of sand and sand- and bagfiltered (1 µm) seawater from the Trondheim fjord collected at 60 m depth. The worms were allowed to evacuate their guts in clean seawater for minimum 4 hours before each sampling and weighing. Water exchange and feeding was conducted every second day. Data analyses were performed using the inbuild statistical package of SigmaPlot v. 14.5., [Materials and Methods]: Polychaetes (H. diversicolor) were collected from Leangen Bay, Trondheim, Norway (63°26′24.5″N, 10°28′27.7″E). To investigate the effects of diet and temperature on growth, survival and fatty acid (FA) composition of H. diversicolor, worms were fed mixes of solid biogas digestate (SBD) and salmon aquaculture sludge (SS) along a 4- step feed gradient ranging from pure SBD to pure SS, and a 5-step temperature gradient ranging from 5.8 to 17.1 °C, for 15 days, using fish feed as a control (Experiment 1). A second experiment was conducted to investigate the effects of salinity and temperature on the same variables. Here, the worms were fed the diet which yielded the highest growth rates in the first experiment (33:66 % SS:SBD) along 5-step salinity and temperature gradients ranging from 5 to 40 ppt and 7.7 to 17.9 °C, respectively, for a duration of 28 days. In both experiments, worms were fed isonitrogenous diets equalling 30 % of the worms' total body nitrogen per day (Wang et al., 2019). Both experiments were conducted in a temperature gradient table (modified after Thomas et al. (1963)) using a 18h:6h light:dark cycle. Worms (n=7-8) were stocked in glass beakers (800 mL) containing an eight cm thick layer of sand and sand- and bagfiltered (1 µm) seawater from the Trondheim fjord collected at 60 m depth. The worms were allowed to evacuate their guts in clean seawater for minimum 4 hours before each sampling and weighing. Water exchange and feeding was conducted every second day. Data analyses were performed using the inbuild statistical package of SigmaPlot v. 14.5., This work was funded by the H2020 BlueBio ERA-NET COFUND (grant no. 817992) / NRC - Norwegian Research Council (#311701); SIDESTREAM. The experiments were carried out within the framework of the national research infrastructure “Norwegian Center for Plankton Technology” (#245937/F50).