The genome of the miiuy croaker reveals well-developed innate immune and sensory systems.

The miiuy croaker, Miichthys miiuy, is a representative Sciaenidae known for its exceptionally large otoliths. This species mainly inhabits turbid aquatic environments with mud to sandy mud bottoms. However, the characteristics of the immune system of this organism and its specific aquatic environment adaptations are poorly understood. Thus, we present a high-quality draft genome of miiuy croaker. The expansions of several gene families which are critical for the fish innate immune system were identified. Compared with the genomes of other fishes, some changes have occurred in the miiuy croaker sensory system including modification of vision and expansion of taste and olfaction receptors. These changes allow miiuy croaker to adapt to the environment during the long-term natural selection. The genome of miiuy croaker may elucidate its relatively well-developed immune defense and provide an adaptation model of the species thriving in turbid deep aquatic environments.

Characterization and evolutionary analysis of duplicated C7 in miiuy croaker.

The complement system, as one of the most sophisticated innate immune system, plays an important role in defense against invading microorganisms. The complement component C7 participates in the cytolytic phase of complement activation through a series of polymerization reactions with other terminal complement components. In this study, we derived two C7 genes from the whole genome of miiuy croaker which were the consequence of the fish-specific genome duplication. Our data showed that miiuy croaker C7-1 and C7-2 genes shared same structure domains. The analysis of gene synteny showed that high degree conserved of synteny was retained between miiuy croaker and other teleosts, and miiuy croaker had a relatively closer relationship with fugu. The expression of C7-1 and C7-2 in miiuy croaker healthy tissues revealed that they were ubiquitously expressed in all ten tested tissues. Besides, the immune response of C7-1 and C7-2 were different in spleen with Vibrio anguillarum, Staphylococcus aureus, poly I:C and LPS at 24 h post-injection, respectively. Furthermore, the expression patterns of C7-1 and C7-2 were different in liver, spleen and kidney after infected with V. anguillarum at different time-point. Evolutionary analysis showed that all the ancestral lineages underwent positive selection except for the ancestral lineages of fish C7-2, indicated that the ancestral lineages of fish C7-1 genes undertook more pressures than C7-2 in defense against the invading microorganisms. Meanwhile, a series of maximum likelihood methods were used to explore the evolutionary patterns on extant vertebrates' C7 genes. Three and one positive selection sites were found in extant mammalian C7 genes and fish C7-2 genes, but no positive selection site was found in extant fishes C7-1 genes. The result showed that extant fish C7-2 genes undertook more pressures compared with C7-1. In conclusion, fish C7-1 and C7-2 gene underwent different evolutionary patterns.

Characterization of the duplicate L-SIGN and DC-SIGN genes in miiuy croaker and evolutionary analysis of L-SIGN in fishes.

Dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN/CD209) and liver/lymph node-specific ICAM-grabbing non-integrin (L-SIGN/CD299) which are homologues of DC-SIGN are important members in C-type lectin receptors family as key molecules to recognize and eliminate pathogens in the innate immune system. DC-SIGN and L-SIGN have become hot topics in recent studies which both served as cell adhesion and phagocytic pathogen recognition receptors in mammals. However, there have been almost no studies of DC-SIGN and L-SIGN structure and characters in fish, only DC-SIGN in the zebrafish had been studied. In our study, we identified and characterized the full-length miiuy croaker (Miichthys miiuy) DC-SIGN (mmDC-SIGN) and L-SIGN (mmL-SIGN) genes. The sequence analysis results showed that mmDC-SIGN and mmL-SIGN have the same domains with other vertebrates except primates, and share some conserved motifs in CRD among all the vertebrates which play a crucial role in interacting with Ca(2+) and for recognizing mannose-containing motifs. Gene synteny of DC-SIGN and L-SIGN were analyzed for the first time and gene synteny of L-SIGN was conserved among the five fishes. Interestingly, one gene next to L-SIGN from gene synteny had high similarity with L-SIGN gene that was described as L-SIGN-like in fish species. While only one L-SIGN gene existed in other vertebrates, two L-SIGN in fish may be in consequence of the fish-specific genome duplication to adapt the specific environment. The evolutionary analysis showed that the ancestral lineages of L-SIGN gene in fishes experienced purifying selection and the current lineages of L-SIGN gene in fishes underwent positive selection, indicating that the ancestral lineages and current lineages of L-SIGN gene in fishes underwent different evolutionary patterns. Both mmDC-SIGN and mmL-SIGN were expressed in all tested tissues and ubiquitously up-regulated in infected liver, spleen and kidney at different sampling time points, indicating that the mmDC-SIGN and mmL-SIGN participated in the immune response to defense against bacteria infection.

Genomic characterization and expression pattern of Bf/C2 and C4 in miiuy croaker and molecular evolution analysis on mammals and fishes.

The complement system plays an important role in both innate and adaptive host defense against the invading microorganisms in vertebrates. It can be activated by three pathways: the classical, alternative and lectin pathways. Bf/C2 and C4, as members of complement, play a pivotal role in the activation of the complement system. In our study, we identified Bf/C2 and C4 genes and genomic structure in miiuy croaker, and expression patterns of Bf/C2 and C4 genes was analyzed. In healthy miiuy croaker tissues, Bf/C2 and C4 genes were found to be ubiquitously expressed in all ten tested tissues. Analysis of expression of Bf/C2 and C4 genes after bacterial infection showed a significant up-regulated in liver. The evolutionary analysis showed that the ancestral lineages of Bf/C2 and C4 genes in mammals and fishes experienced positive selection indicated that the ancestors of mammals and fishes had further evolved to adapt to their environment, respectively. A series of maximum likelihood (ML) methods were used to study the evolution on vertebrates' Bf/C2 and C4 genes. One and five positive selection sites were found in mammals of Bf/C2 and C4 genes, but no positive selection site was found in fishes of Bf/C2 and C4 genes, indicating that Bf/C2 and C4 genes in mammals and fishes underwent different evolutionary patterns.

The evolutionary analysis on complement genes reveals that fishes C3 and C9 experience different evolutionary patterns.

Complement is a humoral factor of innate immunity and plays an essential role in altering the host of the presence of potential pathogens and clearing of invading microorganisms. The third complement component (C3) not only is regarded as the crossing of the three pathways of complement activation, but also serves one of the bridges linking innate and acquired immunity. The nine complement component (C9) can combine with C5b, C6, C7 and C8 to form MAC which bounds to the surface of microorganisms to kill them. The evidence of evolution on C3 genes which have multiple functions and plays central role in innate immunity was documented in our previous study. Now we were interested in the evolution of C9 genes which were the terminal complement components. For these reasons, we want to explore the evolutionary patterns of C9 and whether C3 and C9 experience different evolutionary patterns. In our study, we used the sliding window method to separately calculate the values of ω among fishes and mammals of C3 and C9 codons. In order to detect the positive selection sites, we used the maximum likelihood (ML) method to study the evolutionary pattern on C3 and C9 genes. Positive selection sites were detected in mammalian C9 genes and no positive selection sites were detected in fishes C9 genes. However, no positive selection sites were detected in mammalian C3 genes and positive selection sites were detected in fishes C3 genes. The result indicated that C3 and C9 had different evolutionary patterns on mammals and fishes. In conclusion, different living environments lead to different evolutionary patterns on C3 and C9 in mammals and fishes. Besides, different complement components may have different evolutionary patterns on mammals and fishes.