Introduction
Early development is a critical stage since the highest mortality of fish occurs in this period (Garrido et al., 2015). The high vulnerability of fish to disease and stressors during larval stages compared to post-larval stages raises a question about the immune capacity of fish larvae before and after the larval - juvenile transition. Overall, the immune repertoire of teleosts and presumably the immune response is more complex than in other vertebrates because of the acquisition and retention of additional gene copies during evolution of immune-related genes as exemplified by the complement genes (Najafpour et al., 2020). Studies of immune-related molecules during larval development tend to take a candidate gene approach, but little information is available about the acquisition of full immune capacities in fish. For example, in the Atlantic cod until 8–10 weeks post-hatching IgM was absent (Schrøder et al., 1998), which suggests the immune repertoire and immune response may be different in early developmental stages. Wang et al. (2008) studied the differential expression of several complement genes, C3, C1r/s, C4, C6, Bf, MBL and MASP in zebrafish (Danio rerio) and proposed the alternative pathway is active and responded to LPS in larval stages. Gilthead sea bream (Sparus aurata) is an important commercial marine species, and disease management and production of high-quality larvae of this species remains a challenge for the aquaculture industry (Muniesa et al., 2020). In the present study, the goal was to use high-throughput sequencing (RNA-seq) of sea bream larvae during flexion and at mid-metamorphosis to prospect the potential immune capacity of fish larvae during very early life stages.
Material and methods
Total RNAs (tRNA) of the whole larvae in flexion and mid-metamorphosis stages were extracted using an E.Z.N.A. Total RNA Kit I (VWR, USA) according to the manufacturer’s instructions. Initially pools composed of 3 sea bream larvae at flexion and 1-2 larvae at mid-metamorphosis were homogenized in lysis buffer using mechanical disruption with two iron beads (5 mm) and a Tissue lyser II (Qiagen, Germany) for 3 cycles (30 Hz) of 30 seconds at room temperature. Paired-end RNA-seq sequencing libraries (2 × 150bp read length) were produced using an Illumina HiSeq xten platform (Novogene, Shanghai, China). After quality control filtering, the reads were mapped to the seabream reference genome (https://www.ncbi.nlm.nih.gov, RefSeq assembly accession: GCF_900880675.1) using the bioconductor package Rsubread (Liao et al., 2013). Analysis of differentially expressed (DE) genes was performed using normalized read counts and by computing moderated t-statistics of differential expression by empirical Bayes moderation of the standard errors in the limma package (Ritchie et al., 2015).
Results
The expression profile of the immune genes involved in both innate and adaptive immunity was analyzed in sea bream at flexion and mid-metamorphosis. Transcriptome comparisons of the two stages of sea bream larvae identified 150 immune-associated gene transcripts that were significantly changed between flexion and mid-metamorphosis. Overall, most of the immune-associated genes involved in innate and adaptive immunity were down-regulated in younger larvae at the flexion stage (≅ 24 dph) compared to the bigger larvae at mid-metamorphosis (≅ 50 dph). Several genes associated with the adaptive immune response such as C1q (e.g., c1qa, c1qb, c1qc) and MHC (e.g., several mr1) were down-regulated in the flexion stage. Gene ontology of the DE genes in 24 dph sea bream larvae (compared to 51 dph) showed enrichment of “Abnormality of complement system” (HP:0005339) and “Complement deficiency” (HP:0004431) terms based on the human phenotype ontology (HPO). Many immune-associated genes were expressed in both flexion and mid-metamorphosis larval stages while some immune gene transcripts such as MHC (e.g., major histocompatibility complex class I-related gene protein-like, XM_030411748), C1q (e.g., complement C1q-like protein 4, XM_030431421) and lectin (e.g., galactose-specific lectin nattectin-like, XM_030422915) were absent at the flexion stage (≅ 24 dph) compared to mid-metamorphosis (≅ 50 dph).
Discussion and conclusion
The significant change in many immune-related gene transcripts between larvae at flexion and mid-metamorphosis suggests that the immune capacity of sea bream larvae changes during development. The clear down-regulation of most of the immune-associated gene transcripts in larvae at flexion is indicative of a very low immune capacity in very early stages of sea bream. This is coherent with the observed increase in a suite of immune-associated genes (C3, C1r/s, C4, Bf, MBL and MASP) in zebrafish larvae after hatching (Wang et al., 2008). The lack of immune competence in early life stages leading to severe mortality was also proposed for the Indian major carp, Labeo rohita, which had low mean antibody levels up to 3-weeks post-hatch (Swain et al., 2006). Most innate immune genes showed an increase in transcription around hatching and first feeding in Atlantic cod, Gadus morhua (Seppola et al., 2009). Taking into consideration the results about complement in our transcriptome study of sea bream larvae and the study of the zebrafish complement response where the competency of the alternative pathway was suggested (Wang et al., 2008), it seems likely that activation of complement by the classical and lectin pathways does not occur in early development. The absence of gene transcripts related to the adaptive response at flexion in sea bream and their upregulation at mid-metamorphosis (≅ 50 dph) suggests the adaptive response emerges later in the development of sea bream larvae. Overall, the transcriptome data suggests that although many gene transcripts related to the immune system are present in the flexion stage, levels are very low and the larvae are unlikely to be immunocompetent. The levels of immune-related gene expression increased during development and achieved significantly higher expression at mid-metamorphosis. The results of this study about the immune status of sea bream larvae as revealed by their transcriptome will contribute to improve the management and efficacy of immunostimulants and vaccines.
Acknowledgements
Supported by the European Union Horizon2020 Programme (PerformFISH, grant nº 727610) and the Portuguese Foundation for Science and Technology (FCT) project UID/Multi/04326/2020 and CRESC Algarve 2020 and COMPETE 2020 through project EMBRC.PT ALG-01-0145-FEDER-022121. PISP has a contract with the University of Algarve, funded by FCT, with reference L57/2016/CP6321/CT 0015.
References
Garrido, S., Ben-Hamadou, R., Santos, A.M.P., Ferreira, S., Teodósio, M.A., Cotano, U., Irigoien, X., Peck, M.A., Saiz, E., Ré, P., 2015. Born small, die young: Intrinsic, size-selective mortality in marine larval fish. Sci. Rep. 5, 17065. https://doi.org/10.1038/srep17065
Liao, Y., Smyth, G., Shi, W., 2013. The Subread aligner: fast, accurate and scalable read mapping by seed-and-voteI. Nucleic Acids Res. 41, e108.
Muniesa, A., Basurco, B., Aguilera, C., Furones, D., Reverté, C., Sanjuan‐Vilaplana, A., Jansen, M.D., Brun, E., Tavornpanich, S., 2020. Mapping the knowledge of the main diseases affecting sea bass and sea bream in Mediterranean. Transbound. Emerg. Dis. 67, 1089–1100. https://doi.org/10.1111/tbed.13482
Najafpour, B., Cardoso, J.C.R., Canário, A.V.M., Power, D.M., 2020. Specific Evolution and Gene Family Expansion of Complement 3 and Regulatory Factor H in Fish. Front. Immunol. 11, 2945. https://doi.org/10.3389/FIMMU.2020.568631
Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., Smyth, G.K., 2015. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47. https://doi.org/10.1093/nar/gkv007
Schrøder, M.B., Villena, A.J., Jørgensen, T.O., 1998. Ontogeny of lymphoid organs and immunoglobulin producing cells in Atlantic cod (Gadus morhua L.). Dev. Comp. Immunol. 22, 507–517. https://doi.org/10.1016/S0145-305X(98)00030-5
Seppola, M., Johnsen, H., Mennen, S., Myrnes, B., Tveiten, H., 2009. Maternal transfer and transcriptional onset of immune genes during ontogenesis in Atlantic cod. Dev. Comp. Immunol. 33, 1205–1211. https://doi.org/10.1016/J.DCI.2009.06.013
Swain, P., Dash, S., Bal, J., Routray, P., Sahoo, P.K., Sahoo, S.K., Saurabh, S., Gupta, S.D., Meher, P.K., 2006. Passive transfer of maternal antibodies and their existence in eggs, larvae and fry of Indian major carp, Labeo rohita (Ham.). Fish Shellfish Immunol. 20, 519–527. https://doi.org/10.1016/J.FSI.2005.06.011
Wang, Z., Zhang, S., Wang, G., 2008. Response of complement expression to challenge with lipopolysaccharide in embryos/larvae of zebrafish Danio rerio: Acquisition of immunocompetent complement. Fish Shellfish Immunol. 25, 264–270. https://doi.org/10.1016/J.FSI.2008.05.010