Gill Transcriptome Research Articles

Gill Transcriptome Analysis Revealed the Difference in Gene Expression Between Freshwater and Seawater Acclimated Guppy (Poecilia reticulata).

Guppy (Poecilia reticulata) can adapt to a wide range of salinity changes. To investigate the gene expression changes in the guppy exposed to seawater, we characterized its gill transcriptome using RNA sequencing. Experimental fish were exposed to salinity increase from 0 to 30‰ within 4days, while control fish were cultured in freshwater (0‰ salinity). Seven days after salinity exposure, the gills were sampled and the mortality within 2weeks was recorded. No significant difference in the cumulative mortality at the second week was found between the two groups. Transcriptomic analysis identified 3477 differentially expressed genes (DEGs), including 1067 upregulated and 2410 downregulated genes. These DEGs were enriched in several biological processes, including ion transport, ion homeostasis, ATP biosynthetic process, metabolic process, and immune system process. Oxidative phosphorylation was the most activated pathway. DEGs involved in the pathway "endoplasmic reticulum (ER)-mediated phagocytosis," "starch and sucrose metabolism," and "steroid biosynthesis" were mainly downregulated; chemokines and interleukins involved in "cytokine-cytokine receptor interaction" were differentially expressed. The present results suggested that oxidative phosphorylation had essential roles in osmoregulation in the gills of seawater acclimated guppy, during which the decline in the expression of genes encoding V-ATPases and calreticulin had a negative effect on the phagocytosis and immune response. Besides, several metabolic processes including "starch and sucrose metabolism" and "steroid biosynthesis" were affected. This study elucidates transcriptomic changes in osmotic regulation, metabolism, and immunity in seawater acclimated guppy.

Molecular and cellular characterization of European sea bass CD3ε+ T lymphocytes and their modulation by microalgal feed supplementation.

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and ε chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3ε expression levels increased in head kidney leukocytes, confirming that CD3ε T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3ε+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3ε+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3ε+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3ε+ T cells are involved in nutritional immune responses.

Single-molecule long-read (SMRT) transcriptome sequencing of Mercenaria mercenaria reveals a powerful anti-apoptotic system critical for air exposure endurance

Mercenaria mercenaria is an economically important clam species and exhibits an outstanding resistance to multiple environmental stressors. However, our understanding of their stress adaptability is limited due to a lack of genomic information, such as transcriptome resources. In this study, single-molecule long-read (SMRT) mRNA sequencing was performed to obtain the full-length gill transcriptome reference sequences of M. mercenaria under air exposure stress. In all, 14.5 G subreads were obtained and assembled into 64,603 unigenes, among which 50,613 were successfully annotated. Additionally, 56,295 SSRs, 1457 transcription factors, and 5924 lncRNAs were identified in M. mercenaria transcriptome. Furthermore, numerous apoptosis-related transcripts were identified according to Swiss-Prot annotation and their numbers were counted. We also found that most apoptosis-related transcripts exhibited typical domains of a certain protein family through conserved domain prediction. Additionally, eight typical sequences related to apoptosis pathway were detected by RT-PCR, with the aim to show the sequential variation of gene expression levels under air exposure. These results implied that the complicated apoptosis system, especially the powerful anti-apoptotic system was critical for M. mercenaria to endure air exposure.

Gill transcriptome analysis reveals the molecular response to the acute low-salinity stress in Cyclina sinensis

Salinity is an important environmental factor that has a profound impact on the growth, reproduction, survival, and development of mollusk. To investigate the molecular changes in response to acute low-salinity stress, the clams were transferred from 25‰ to 8‰. According to the exposure times of 0, 12, 24, 48, 72, 96, and 120 h, the treatment consisted group T0 (the control), T1, T2, T3, T4, T5, and T6, respectively. Gills were collected, and then transcriptome analysis was conducted. Compared to the control, 3008 DEGs were obtained, including 1127 up-regulated and 1881 down-regulated genes, respectively. Up-regulations of 924, 176, 11, 6, 3, and 7 DEGs, and down-regulations of 1216, 416, 157, 59, 23, and 10 DEGs were observed in T6, T5, T4, T3, T2, and T1 groups, respectively. The related molecular biological processes, and potential functions were explored from enrichment analyses, including energy metabolism, material metabolism, and immune responses. There were 13, 10, 16, 30, 62, and 97 KEGG pathways enriched in T1, T2, T3, T4, T5, and T6 groups, respectively. 13 genes down-regulated, and only one gene up-regulated were found in more than four groups. In addition, the verification of the expression changes of the candidate genes (PCK1, IAP, BIRC2, and LOC102459116) provided insights into responses to salinity change in the gills of the clams. This will be of great value in understanding the molecular basis of low-salinity adaptation in the clam.

Effects of heat stress on histopathology, antioxidant enzymes, and transcriptomic profiles in gills of pikeperch Sander lucioperca

Pikeperch Sander lucioperca is a cool-water fish considered to be a potential aquaculture species. At present, extreme temperature conditions in summer affect the growth and survival of pikeperch cultured in ponds, indicating that the effects of heat stress on pikeperch merits research attention. In this study, pikeperch were heat stressed at 29 °C for up to 48 h. Fish were sampled at different time periods, and changes in gills histopathology and oxidative stress status were determined. The gill transcriptome under heat stress and significantly differentially expressed genes were also analyzed. qPCR was used to detect the expression of antioxidant-related genes and Hsp member family genes in gills of fish subjected to different durations of heat stress. The degree of gill damage increased with prolongation of the heat stress. The activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) and the malondialdehyde (MDA) content in the gills changed significantly in response to continued heat stress. The expression of 252 genes changed significantly under heat stress: 191 genes were up-regulated and 61 genes were down-regulated. KEGG pathway enrichment analysis showed that multiple pathways were involved in the heat-stress response of the pikeperch but mostly involved lipid metabolism (six pathways), followed by amino acid metabolism (four pathways), carbohydrate metabolism (three pathways), and folding, sorting and degradation. The expression of antioxidant-related genes, including CuZn SOD, Mn SOD and CAT, and the heat shock protein (Hsp) member family genes increased during the early period of heat stress, but decreased in the later period. The overall results indicate that the gills of pikeperch are sensitive to heat stress and pikeperch actively responds to heat stress through regulating the antioxidant system, and the expression of Hsp genes and genes involved in energy metabolism. This study improves our understanding of the effects of heat stress on pikeperch, and reveals the mechanism of the heat-stress response in this species.

Effects of salinity on growth, hematological parameters, gill microstructure and transcriptome of fat greenling Hexagrammos otakii

The fat greenling, Hexagrammos otakii, is an emerging aquaculture species and is being evaluated for production in recirculating aquaculture systems (RAS). To better understand the tolerance of H. otakii to varying environmental salinity in actual aquaculture operations, the effects of salinity on survival, growth, hematological parameters, gill microstructure and gill transcriptome were investigated. Juvenile H. otakii (22.0± 1.8 g; 11.2 ± 0.7 cm) were reared at different salinities, 6.7 ± 0.1 PSU (7 PSU), 12.3 ± 0.1 PSU (12 PSU), 18.2 ± 0.1 PSU (18 PSU), 23.9 ± 0.6 PSU (24 PSU), and 28.9 ± 0.8 PSU (29 PSU) in triplicated RASs for five weeks, and the impacts of salinity on these endpoints were evaluated. Fish in the 7 PSU group exhibited significantly lower survival, final weight, feed conversion ratio, and specific growth rate than fish in other treatments. While no statistically significant differences were found among mean white blood cell counts, red blood cell counts and hemoglobin concentrations, mean corpuscular volume was significantly lower in the 7 PSU group than in other groups. At the histological level, there were smaller numbers and smaller volume of mitochondria-rich cells in the 7 PSU group, and the numbers of pavement cells and mucous cells were significantly higher than in other groups. Transcriptome sequencing for a sample of individuals from the 7 PSU, 18 PSU and 29 PSU groups indicated that gene expression in the gills of H. otakii differed significantly among groups. Gene ontology (GO) enrichment showed differential expression between the 7 PSU and 29 PSU groups for seven genes with activities as neurotransmitter transmembrane transporters and neurotransmitter: sodium symporters. Comparing to the 18 PSU group, 16 genes with activities in gas or oxygen transport were down-regulated in the 7 PSU group. No such differences in expression of gas transport genes was found between the 18 PSU and 29 PSU groups. Overall, our exploratory transcriptome analysis suggested that fish in the low-salinity 7 PSU group exhibited high gene expression in the osmoregulation and gas transport pathways. Our results at the histological and gene expression levels suggest why rearing juvenile H. otakii in salinities of 12–29 PSU is optimal for commercial aquaculture production.

The Atlantic Salmon Gill Transcriptome Response in a Natural Outbreak of Salmon Gill Pox Virus Infection Reveals New Biomarkers of Gill Pathology and Suppression of Mucosal Defense

The salmon gill poxvirus (SGPV) is a large DNA virus that infects gill epithelial cells in Atlantic salmon and is associated with acute high mortality disease outbreaks in aquaculture. The pathological effects of SGPV infection include gill epithelial apoptosis in the acute phase of the disease and hyperplasia of gill epithelial cells in surviving fish, causing damage to the gill respiratory surface. In this study, we sampled gills from Atlantic salmon presmolts during a natural outbreak of SGPV disease (SGPVD). Samples covered the early phase of infection, the acute mortality phase, the resolving phase of the disease and control fish from the same group and facility. Mortality, the presence and level of SGPV and gill epithelial apoptosis were clearly associated. The gene expression pattern in the acute phase of SGPVD was in tune with the pathological findings and revealed novel transcript-based disease biomarkers, including pro-apoptotic and proliferative genes, along with changes in expression of ion channels and mucins. The innate antiviral response was strongly upregulated in infected gills and chemokine expression was altered. The regenerating phase did not reveal adaptive immune activity within the study period, but several immune effector genes involved in mucosal protection were downregulated into the late phase, indicating that SGPV infection could compromise mucosal defense. These data provide novel insight into the infection mechanisms and host interaction of SGPV.

Crowding reshapes the mucosal but not the systemic response repertoires of Atlantic salmon to peracetic acid

Knowledge of the impact of aquaculture chemotherapeutants on fish physiology is scarce. This is particularly relevant for peracetic acid (PAA), a widely used oxidative disinfectant in aquaculture. The chemical behaviour in water is well studied but knowledge about the physiological consequences for fish is limited. The present study investigated the transcriptomics, morphology, and physiology of Atlantic salmon (Salmo salar) responses to PAA and explored how crowding prior to exposure influenced these responses. Post-smolts were subjected to crowding by reducing the water volume thereby increasing the density for 1 h before they were exposed to 4.8 ppm PAA for 30 min. The exposed fish were allowed to recover for 2 weeks (w), with samplings carried out at 4 h and 2 w post-exposure (p.e.). There were four treatment groups in total: no crowding/control; no crowding/PAA; crowding/control; and crowding/PAA. The physiological changes were documented at the mucosal (i.e., skin and gills) and systemic (i.e., plasma) levels. The overall external welfare score was in good status in all experimental groups. The treatments did not dramatically affect the number of mucous cells in both the skin and the gills. Branchial histomorphology was in a fairly good condition, despite the increased occurrence of epithelial lifting in the crowded groups at 2 w p.e. The gill transcriptome was affected by crowding, PAA, and their combinations more than the skin, as manifested by the number of differentially expressed genes (DEG) in the former. In general, individual stimuli and their combinations elicited strong transcriptional responses in the gills at 4 h p.e. and a marked recovery was observed 2 w thereafter. Crowding altered the dynamics of transcriptional response to PAA especially at 4 h p.e. and the two mucosal tissues demonstrated a contrasting profile – a higher number of DEGs in the gills without crowding history, while higher skin DEGs were observed in the group subjected to crowding prior to exposure. Plasma metabolomics identified 639 compounds, and the metabolomic changes were affected mainly by crowding and sampling time, and not by PAA exposure. The results revealed the ability of salmon to mobilise physiological countermeasures to PAA exposure that were differentially influenced by crowding, and that such an effect was remarkably exhibited at the mucosa rather than in the circulating metabolome.

Response of the PI3K‐AKT signalling pathway to low salinity and the effect of its inhibition mediated by wortmannin on ion channels in turbot Scophthalmus maximus

The PI3K-AKT signalling pathway is a crucial part of the signal transduction network and is involved in many important physiological processes and the stress response to environmental alteration. In this study, gill and kidney transcriptome data demonstrated that the PI3K-AKT signalling pathway plays an important role in the stress response of the turbot Scophthalmus maximus when exposed to low salinity. Low-salinity stress led to down-regulation of gene expression, protein content and phosphorylation of AKT1 at Thr308 of the PI3K-AKT signalling pathway. However, the abundance and phosphorylation of AKT1 protein at Ser473 and Thr308 were detected only in the gill and not in the kidney. Inhibition of the PI3K-AKT signalling pathway mediated by wortmannin showed that wortmannin remarkably inhibited PI3K gene expression and protein synthesis as well as phosphorylation of AKT1 at Thr308 with or without low-salinity stress. In the context of pathway inhibition, suppressed expression levels of ion channel genes demonstrated that the PI3K-AKT signalling pathway acts on osmoregulation by positively regulating ion transport.

Gill transcriptomes reveal expression changes of genes related with immune and ion transport under salinity stress in silvery pomfret (Pampus argenteus).

Salinity is a major ecological factor in the marine environment, and extremely important for the survival, development, and growth of fish. In this study, gill transcriptomes were examined by high-throughput sequencing at three different salinities (12ppt as low salinity, 22ppt as control salinity, and 32ppt as high salinity) in an importantly economical fish silvery pomfret. A total of 187 genes were differentially expressed, including 111 up-regulated and 76 down-regulated transcripts in low-salinity treatment group and 107 genes differentially expressed, including 74 up-regulated and 33 down-regulated transcripts in high-salinity treatment group compared with thecontrol group, respectively. Some pathways including NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, Toll-like receptor pathway, cardiac muscle contraction, and vascular smooth muscle contraction were significantly enriched. qPCR analysis further confirmed that mRNA expression levels of immune (HSP90A, IL-1β, TNFα, TLR2, IP-10, MIG, CCL19, and IL-11) and ion transport-related genes (WNK2, NPY2R, CFTR, and SLC4A2) significantly changed under salinity stress. Low salinity stress caused more intensive expression changes of immune-related genes than high salinity. These results imply that salinity stress may affect immune function in addition to regulating osmotic pressure in silvery pomfret.

Transcriptome changes for Nile tilapia (Oreochromis niloticus) in response to alkalinity stress

Nile tilapia is an important economic fish in the world because of its fast growth, high meat yield and strong adaptability. It is more adaptable to high alkalinity than common freshwater fish and provides valuable material for developing alkaline-tolerant strains and understanding the adaptation mechanism of fish to extreme environmental stress. In this study, we employed high throughput RNA sequencing to reveal the tissues (gill, kidney and liver) transcriptome differences of O. niloticus at different carbonate alkalinities (FW, AW40 and AW60). A total of 1,369,381,790 raw reads were obtained, including 496,441,232 reads in FW group, 437,907,696 reads in AW40 and 435,032,862 reads in AW60. In addition, 484,555,626 reads in gill, 451,618,224 reads in kidney and 433,207,940 reads in liver. A large number of stress-regulated changes were detected comprehensively. We focused on 3 significantly change pathways (steroid biosynthesis, drug metabolism and protein digestion/absorption) and 17 DEGs (HMG-CoA reductase, UDP-glucuronosyltransferase, and carbonic anhydrase etc.) which were shared among compared groups (AW40 vs FW, AW60 vs FW, AW40 vs 60 AW60) in gill, kidney and liver, respectively. These pathways/genes are sensitive to alkalinity stress and crucial to the alkalinity adaptation of tilapia. Overall, we found a large number of candidate genes, which encode important regulators of stress tolerance and ultimately contribute to future alkaline-tolerant fish breeding. Among these genes, lipid metabolism (involving signal transduction), detoxification and immune related genes are more prominent to the response and adaptability of fish to alkalinity stress.

Applying a gene-suite approach to examine the physiological status of wild-caught walleye (Sander vitreus).

Molecular techniques have been increasingly used in a conservation physiology framework to provide valuable information regarding the mechanisms underlying responses of wild organisms to environmental and anthropogenic stressors. In the present study, we developed a reference gill transcriptome for walleye (Sander vitreus), allowing us to pair a gene-suite approach (i.e. multiple genes across multiple cellular processes) with multivariate statistics to examine the physiological status of wild-caught walleye. For molecular analyses of wild fish, the gill is a useful target for conservation studies, not only because of its importance as an indicator of the physiological status of fish but also because it can be biopsied non-lethally. Walleye were non-lethally sampled following short- (~1.5months) and long-term (~3.5months) confinement in the Delta Marsh, which is located south of Lake Manitoba in Manitoba, Canada. Large-bodied walleye are confined in the Delta Marsh from late April to early August by exclusion screens used to protect the marsh from invasive common carp (Cyprinus carpio), exposing fish to potentially stressful water quality conditions. Principal components analysis revealed patterns of transcript abundance consistent with exposure of fish to increasingly high temperature and low oxygen conditions with longer holding in the marsh. For example, longer-term confinement in the marsh was associated with increases in the mRNA levels of heat shock proteins and a shift in the mRNA abundance of aerobic to anaerobic metabolic genes. Overall, the results of the present study suggest that walleye confined in the Delta Marsh may be exhibiting sub-lethal responses to high temperature and low oxygen conditions. These results provide valuable information for managers invested in mediating impacts to a local species of conservation concern. More broadly, we highlight the usefulness of pairing transcriptomic techniques with multivariate statistics to address potential confounding factors that can affect measured physiological responses of wild-caught fish.

Diving into divergence: Differentiation in swimming performances, physiology and gene expression between locally-adapted sympatric cichlid fishes.

Sympatric speciation occurs without geographical barriers and is thought to often be driven by ecological specialization of individuals that eventually diverge genetically and phenotypically. Distinct morphologies between sympatric populations occupying different niches have been interpreted as such differentiating adaptive phenotypes, yet differences in performance and thus likely adaptiveness between them were rarely tested. Here, we investigated if divergent body shapes of two sympatric crater lake cichlid species from Nicaragua, one being a shore-associated (benthic) species while the other prefers the open water zones (limnetic), affect cruising (Ucrit ) and sprinting (Usprint ) swimming abilities - performances particularly relevant to their respective lifestyles. Furthermore, we investigated species differences in oxygen consumption (MO2 ) across different swimming speeds and compare gene expression in gills and white muscle at rest and during exercise. We found a superior cruising ability in the limnetic Amphilophus zaliosus compared to the benthic Amphilophus astorquii, while sprinting was not different, suggesting that their distinct morphologies affect swimming performance. Increased cruising swimming ability in A.zaliosus was linked to a higher oxygen demand during activity (but not rest), indicating different metabolic rates during exercise - a hypothesis supported by coinciding gene expression patterns of gill transcriptomes. We identified differentially expressed genes linked to swimming physiology, regulation of swimming behaviour and oxygen intake. A combination of physiological and morphological differences may thus underlie adaptations to these species' distinct niches. This complex ecological specialization probably resulted in morphological and physiological trade-offs that contributed to the rapid establishment and maintenance of divergence with gene flow.

Comparative transcriptome analysis of Triplophysa yarkandensis in response to salinity and alkalinity stress

Triplophysa yarkandensis, a fish belonging to the family Nemacheilidae, is distributed in the Tarim River, China, immediately north of the Qinghai-Tibet Plateau. Due to increasing salinity and alkalinity in the Tarim River, the habitats of T. yarkandensis have been seriously altered. To identify the genes and pathways that are important for responding to salinity and alkalinity stress, the gill transcriptomes of fish living under different salinity and alkalinity conditions were obtained using RNA sequencing. A total of 1,123,448,964 clean reads were obtained and assembled into 177,271 unigenes, with an average length of 1703 bp. Around 13,526 unigenes showed differential expression when comparing different salinity concentrations with the controls, 6967 of which were upregulated and 6559 were downregulated. When comparing different alkalinity concentrations with the controls, there were 17,475 unigenes that showed differential expression, of which 10,457 were upregulated and 7018 were downregulated. Only 146 unigenes were both differentially expressed in salinity and alkalinity groups compared to the control. The results of KEGG enrichment showed that there were five upregulated and 12 downregulated pathways in fish subject to salinity treatment. For fish exposed to alkalinity treatment, 15 pathways were upregulated and 13 downregulated. There were four upregulated and four downregulated pathways that were shared by fish subject to salinity and alkalinity treatments. To our knowledge, this is the first study on the T. yarkandensis transcriptome; the information presented here will provide further understanding of the fish's response to salinity and alkalinity stress, as well as further insight into the T. yarkandensis genome.

Tissue specific alpha-2-Macroglobulin (A2M) splice isoform diversity in Hilsa shad, Tenualosa ilisha (Hamilton, 1822).

The present study, for the first time, reported twelve A2M isoforms in Tenualosa ilisha, through SMRT sequencing. Hilsa shad, T. ilisha, an anadromous fish, faces environmental stresses and is thus prone to diseases. Here, expression profiles of different A2M isoforms in four tissues were studied in T. ilisha, for the tissue specific diversity of A2M. Large scale high quality full length transcripts (>0.99% accuracy) were obtained from liver, ovary, testes and gill transcriptomes, through Iso-sequencing on PacBio RSII. A total of 12 isoforms, with complete putatative proteins, were detected in three tissues (7 isoforms in liver, 4 in ovary and 1 in testes). Complete structure of A2M mRNA was predicted from these isoforms, containing 4680 bp sequence, 35 exons and 1508 amino acids. With Homo sapiens A2M as reference, six functional domains (A2M_N,A2M_N2, A2M, Thiol-ester_cl, Complement and Receptor domain), along with a bait region, were predicted in A2M consensus protein. A total of 35 splice sites were identified in T. ilisha A2M consensus transcript, with highest frequency (55.7%) of GT-AG splice sites, as compared to that of Homo sapiens. Liver showed longest isoform (X1) consisting of all domains, while smallest (X10) was found in ovary with one Receptor domain. Present study predicted five putative markers (I-212, I-269, A-472, S-567 and Y-906) for EUS disease resistance in A2M protein, which were present in MG2 domains (A2M_N and A2M_N2), by comparing with that of resistant and susceptible/unknown response species. These markers classified fishes into two groups, resistant and susceptible response. Potential markers, predicted in T. ilisha, placed it to be EUS susceptible category. Putative markers reported in A2M protein may serve as molecular markers in diagnosis of EUS disease resistance/susceptibility in fishes and may have a potential for inclusion in the marker panel for pilot studies. Further, challenging studies are required to confirm the role of particular A2M isoforms and markers identified in immune protection against EUS disease.

Gill transcriptomic analysis in fast- and slow-growing individuals of Mytilus galloprovincialis

The molecular basis underlying the mechanisms at the origin of growth variation in bivalves is still poorly understood, although several genes have been described as upregulated in fast-growing individuals. In the present study, we reared mussel spat of the species Mytilus galloprovincialis under diets below the pseudofaeces threshold (BP) and above the pseudofaeces threshold (AP). After 3 months, F and S mussels from each condition were selected to obtain 4 experimental groups: FBP, SBP, FAP and SAP. We hypothesized that the nurturing conditions during the growing period would modify the molecular basis of their growth rate differences.To decipher the molecular mechanisms underlying the growth variation, the gill transcriptomes for the four mussel groups were analysed. Gene expression analysis revealed i) a low number (12) of genes differentially expressed in association with diet and ii) 117 genes differentially expressed by the fast- and slow-growing mussels. According to Biological Process GO term analysis transcriptomic differences between the F and S mussels were mainly based on the upregulation of: response to the stimulus, growth and cell activity. Regarding the KEGG terms, carbohydrate metabolism and the Krebs cycle were upregulated in F mussels, whereas biosynthetic processes were upregulated in S mussels. In accordance with their larger gill surface area and higher rates of feeding and growth, the F individuals overexpressed genes in their gill tissues, and these were involved in i) growth (insulin-like growth factors and myostatin); ii) maintenance of the structure and functioning of extracellular matrix (collagen, laminin, fibulin and decorin); iii) filtration and ciliary activity (mucin, fibrocystin, dynein and tilB homolog protein genes); iv) aerobic metabolism (citrate synthase and carbonic anhydrase); and v) the immune-system, probably in association with haemocytes. In contrast, S individuals overexpressed a different series of genes pertaining to immune system (leucine-rich repeat protein and galectin), along with genes involved in the response to cellular stress (Heat shock protein (HSP24) and metalloendopeptidase) as well as anaerobic metabolism (C4-dicarboxylate transporter). These results might suggest that S individuals would have a greater prevalence of pathogens/diseases or a higher susceptibility to the pathogens.

Divergent transcriptomic signatures in response to salinity exposure in two populations of an estuarine fish.

In estuary and coastal systems, human demand for freshwater, climate change‐driven precipitation variability, and extreme weather impact salinity levels, reducing connectivity between mesohaline coastal fish populations and potentially contributing to genomic divergence. We examined gill transcriptome responses to salinity in wild‐caught juveniles from two populations of Sacramento splittail (Pogonichthys macrolepidotus), a species of conservation concern that is endemic to the San Francisco Estuary, USA, and the lower reaches of its tributaries. Recent extreme droughts have led to salinities above the tolerance limits for this species, creating a migration barrier between these populations, which potentially contributed to population divergence. We identified transcripts involved in a conserved response to salinity; however, the more salinity‐tolerant San Pablo population had greater transcriptome plasticity (3.6‐fold more transcripts responded than the Central Valley population) and a response consistent with gill remodeling after 168 hr of exposure to elevated salinity. The reorganization of the gill in response to changing osmotic gradients is a process critical for acclimation and would facilitate enhanced salinity tolerance. We detected an upregulation of receptors that control the Wnt (wingless‐type) cell signaling pathway that may be required for an adaptive response to increases in salinity, patterns not observed in the relatively salinity‐sensitive Central Valley population. We detected 62 single nucleotide polymorphisms (SNPs) in coding regions of 26 transcripts that differed between the populations. Eight transcripts that contained SNPs were associated with immune responses, highlighting the importance of diversity in immune gene sequences as a defining characteristic of genomic divergence between these populations. Our data demonstrate that these populations have divergent transcriptomic responses to salinity, which is consistent with observed physiological differences in salinity tolerance.

Transcriptome analysis of gill from Lateolabrax maculatus and aqp3 gene expression

The Lateolabrax maculatus is an important marine fish in China that can adapt to a wide range of water salinities. The gill is the main organ involved in fish osmoregulation and in this study, a cDNA library of the L. maculatus gill was constructed to understand gill function in Lateolabrax maculatus and it adaptation to salinity. A total raw data of 5.3G base pairs was obtained, followed removal of low-quality and linker DNA sequences and it yielded a total of 23,038,590 clean reads. After splicing and clustering, 31,976 unigenes were obtained. The average transcript length was 1327 bp and N50 was 2057 bp. In comparisons with other tissues analysis of the gill transcriptome revealed a high level of relative expression of apolipoprotein E, CD74, keratin, type I cytoskeleton 13, carbonic anhydrase, betaine-homocysteine S-methyltransferase, and aquaporin 3 (AQP3) genes. The L. maculatus aqp3 (Lm-aqp3) gene was specific for the gills Lm-aqp3 and this gene has a typical transmembrane structure of six alpha helices and a water channel domain. Phylogenetic analysis indicated that Lm-aqp3 clusters with the homologue genes from Labrus bergylta and Dicentrarchus labrax. Quantitative RT-PCR confirmed that Lm-aqp3 was highly abundant in the gills and low expressed in other tissues (stomach, liver, brain, spleen and testis). Analysis of Lm-aqp3 in freshwater aquaculture fish revealed that gene expression was significantly statistical higher than in seawater (20 ppt) fish, suggesting that Lm-aqp3 may play an important role in water metabolism and osmotic pressure regulation in L. maculatus.

High throughput screening of small immune peptides and antimicrobial peptides from the Fish-T1K database

Analyses of transcriptomic datasets have the potential to reveal genetic markers underlying ecological adaptations. In the present study, we leverage the expanding dataset generated by the Fish-T1K Project (Transcriptomes of 1000 Fishes) to characterize small peptides that may be implicated in the immune system of fishes. We focused our analyses on sequences smaller than 360 bp obtained from gill transcriptomes of 87 ray-finned fishes (Actinopterygii). Functional annotation of short transcripts revealed that the number of small immune peptides varied significantly among the studied species. High-throughput screening of antimicrobial peptides (AMPs) with homologous searches was used to characterize the composition of innate immune defense factors present in fishes. We analyzed the putative effects of habitat, climatic zone and genetic system on the distribution of small peptides among species. Our results highlight the utility of large transcriptomic datasets such as Fish-T1K to explore patterns of variation at macroevolutionary scales and to discover novel peptides that may be used for further investigation and drug development.

RNA-Seq Analysis for Assessing the Early Response to DSP Toxins in Mytilus galloprovincialis Digestive Gland and Gill.

The harmful effects of diarrhetic shellfish poisoning (DSP) toxins on mammalian cell lines have been widely assessed. Studies in bivalves suggest that mussels display a resistance to the cytogenotoxic effects of DSP toxins. Further, it seems that the bigger the exposure, the more resistant mussels become. To elucidate the early genetic response of mussels against these toxins, the digestive gland and the gill transcriptomes of Mytilus galloprovincialis after Prorocentrum lima exposure (100,000 cells/L, 48 h) were de novo assembled based on the sequencing of 8 cDNA libraries obtained using an Illumina HiSeq 2000 platform. The assembly provided 95,702 contigs. A total of 2286 and 4523 differentially expressed transcripts were obtained in the digestive gland and the gill, respectively, indicating tissue-specific transcriptome responses. These transcripts were annotated and functionally enriched, showing 44 and 60 significant Pfam families in the digestive gland and the gill, respectively. Quantitative PCR (qPCR) was performed to validate the differential expression patterns of several genes related to lipid and carbohydrate metabolism, energy production, genome integrity and defense, suggesting their participation in the protective mechanism. This work provides knowledge of the early response against DSP toxins in the mussel M. galloprovincialis and useful information for further research on the molecular mechanisms of the bivalve resistance to these toxins.