Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture1,2. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates3,4, but we show that Atlantic cod has lost the genes for MHCII, CD4 and Ii that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions5. We find a highly expanded number of MHCI genes and a unique composition of its Toll-like receptor (TLR) families. This suggests how the Atlantic cod immune system has evolved compensatory mechanisms within both adaptive and innate immunity in the absence of MHCII. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.
The aim of the present study was to develop a cell culture system for studying the proliferation and differentiation of preadipocytes isolated from Atlantic salmon adipose tissue. The expression of proliferating cell nuclear antigen (PCNA) was used as a marker for cell proliferation. The cells started to proliferate within 48 h after seeding and continued to proliferate throughout the culture period of 2 wk. Undifferentiated preadipocytes showed a fibroblast-like morphology with a homogeneous cytoplasm devoid of lipid droplets. At confluence, an exogenous lipid mixture was added to the cell cultures. The preadipocytes became larger and rounder during the subsequent days, and the cytoplasm gradually filled with lipid-rich droplets. These droplets were revealed by oil red O staining. Immunocytochemical staining showed that differentiated adipocytes expressed detectable levels of the three regulatory proteins associated with adipocyte differentiation: peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer binding protein alpha (C/EBPalpha), and leptin. The cells also showed activity of glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8), a biochemical marker of adipocyte differentiation. The morphological and biochemical data presented here show that fish preadipocytes have properties that are similar to those of preadipocytes in mammals. We conclude therefore that salmon adipose tissue contains a sizable population of preadipocytes. Exogenous lipids promote the activation of adipose-related genes and induce the differentiation of fish preadipocytes in vitro.
Optimization of reference genes for real-time polymerase chain reaction (PCR) studies in fish is strongly needed. We systematically tested beta-actin (ACTB), 18S rRNA (18S), beta(2)-microglobulin (B2M), elongation factor 1-alpha (EF1A), RNA polymerase I and II (RPL1/2), and glycerol 6-phosphate dehydrogenase (G6PDH) for stability in salmon immune relevant tissues and kidney cells (ASK) infected with infectious salmon anemia virus (ISAV), plus in tissues from fish fed thia fatty acids. Transcription of all genes was unchanged in infected and thia fatty acid-treated tissues versus normal tissues. Between tissues, 18S and EF1A were most stable, RPL1 and RPL2 were intermediate, and G6PDH and ACTB and B2M were the least stable. However, only 18S had constant expression in infected cells; the rest significantly down-regulated. Implications of this finding were demonstrated when normalizing major histocompatibility complex (MHC) class I expression in ASK. Software predictions supported a proper normalization is obtained combining 18S, EF1A, and RPL1 in vivo, but for in vitro viral infection assays we recommend using 18S.
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