What is known about the biological activity of fish cytokines is reviewed. Most of the functional studies performed to date have been in teleost fish, and have focused on the induced effects of cytokine recombinant proteins, or have used loss- and gain-of-function experiments in zebrafish. Such studies begin to tell us about the role of these molecules in the regulation of fish immune responses and whether they are similar or divergent to the well-characterised functions of mammalian cytokines. This knowledge will aid our ability to determine and modulate the pathways leading to protective immunity, to improve fish health in aquaculture.

The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

The earliest jawed vertebrates (Gnathostomes) would likely have had interferon (IFN) genes, since they are present in extant cartilaginous fish (sharks and rays) and bony fish (lobe-finned and ray-finned fish, the latter consisting of the chondrostei, holostei, and teleostei), as well as in tetrapods. They are thought to have evolved from a class II helical cytokine ancestor, along with the interleukin (IL)-10 cytokine family. The two rounds of whole genome duplication (WGD) that occurred between invertebrates and vertebrates (1) may have given rise to additional loci, initially containing an IL-10 ancestor and IFN ancestor, which have duplicated further to give rise to the two loci containing the IL-10 family genes, and potentially the IFN type I and IFN type III loci (2). The timing of the divergence of the IFN type II gene from the IL-10 family genes is not clear but was also an early event in vertebrate evolution. Further WGD events at the base of the teleost fish, and in particular teleost lineages (cyprinids, salmonids), have duplicated the loci further, giving rise to additional IFN genes, with tandem gene duplication within a locus a common occurrence. Finally, retrotransposition events have occurred in different vertebrate lineages giving rise to further IFN loci, with large expansions of genes at these loci in some cases. This review will initially explore the likely IFN system present in the earliest Gnathostomes by comparison of the known cartilaginous fish genes with those present in mammals and will then explore the changes that have occurred in gene number/diversification, gene organization, and the encoded proteins during vertebrate evolution.

The immune response is an energy demanding process, which has effects in many physiological pathways in the body including protein and lipid metabolism. During an inflammatory response the liver is required to produce high levels of acute phase response proteins that attempt to neutralise an invading pathogen. Although this has been extensively studied in both mammals and fish, little is known about how high and low energy reserves modulate the response to an infection in fish which are ectothermic vertebrates. Food withdrawal in fish causes a decrease in metabolic rate so as to preserve protein and lipid energy reserves, which occurs naturally during the life cycle of many salmonids. Here we investigated how the feeding or fasting of Atlantic salmon affected the transcriptional response in the liver to an acute bacterial infection. Total liver RNA was extracted from four different groups of salmon. Two groups were fed or starved for 28 days. One of each of the fed or starved groups was then exposed to an acute bacterial infection. Twenty four hours later (day 29) the livers were isolated from all fish for RNA extraction. The transcriptional changes were examined by micro array analysis using a 17 K Atlantic salmon cDNA microarray. The expression profiling results showed major changes in gene transcription in each of the groups. Enrichment for particular biological pathways was examined by analysis of gene ontology. Those fish that were starved decreased immune gene transcription and reduced production of plasma protein genes, and upon infection there was a further decrease in genes encoding plasma proteins but a large increase in acute phase response proteins. The latter was greater in magnitude than in the fish that had been fed prior to infection. The expression of several genes that were found altered during microarray analysis was confirmed by real time PCR. We demonstrate that both starvation and infection have profound effects on transcription in the liver of salmon. There was a significant effect on the transcriptional response to infection depending on the prior feeding regime of the fish. It is likely that the energy demands on protein synthesis for acute phase response proteins are relatively high in the starved fish which have reduced energy reserves. This has implications for dietary control of fish if an immune response is anticipated.