Preliminary Study On Skin Pattern Formation And Gene Expression Of Two Connexins Cx39.4 And Cx41.8 In Mandarin Fish (Siniperca Chuatsi)

Patterns are the important external morphological characteristics in fish and play an important role in survival and reproduction.Mandarin fish(Siniperca chuatsi)is a unique precious freshwater fish in China,its special pattern are composed of one brown strip on the head dorsal,two brown stripes on the head lateral across the eyes to the bottom of the dorsal fin,two wide brown vertical bands on both sides of the trunk,and some brown spots on the back of the trunk.In this paper,pigment development and pattern formation were observed microscopically in mandarin fish at early stages.The distribution and arrangement of skin pigment cells in different pattern regions were observed by light and electron microscopes.Genes expression of two gap junction proteins Cx39.4 and Cx41.8 were detected at different skin pattern types by real time PCR technology,which could provide some cellular and molecular information for the formation of skin patterns in mandarin fish.The main results were as follows:(1)In order to understand morphological changes in the early development in mandarin fish,CCD-Adapter anatomical microscope was used to observe the pigment cells formation,distribution and main patterns(stripe,band and spot)formation in the early stage(embryo stage-40 days old).The results showed that melanophores were first observed in the yolk sac and oil balls at embryonic stage,xanthophores appear on the head at the pre-hatching stage.After hatching,melanophores developed most prominently and erythrophores appeared behind the eye and in the front of trunk.In the later laval stages,xanthophores increased throughout the whole body and melanophores continued to develop and pattern formation began.Early color pattern formation process of mandarin fish included: Overhead strip: the black spots at the head dorsal increased at 6 days old,these spot extended forward and backward at 18 days old,the black strip at the head dorsal was formed at 23 days old.Eye strip: the melanin above the gill cover increased at 10 days old,the hind strip part was formed from the upper edge of gill cover to the upper back at 12 days old,the front strip part formed from the upper snout to the front of the eye at 17 days old.Trunk band: some melanin appeared at the upper dorsal at 5 days old,the dorsal melanin and the abdominal melanin were connected at 14 days old.Trunk spots: one small black spot appeared at the bottom of the tail at 8 days old,three irregular black patches appeared in the front of the tail at 15 days old,five round black spots formed in the back of the trunk at 25 days old.The results showed that melanophores,xanthophores and erythrophores appeared in skin of mandarin fish at early stage,the whole body was dominated by black color,the main strips,band and spots gradually formed in different modes at the later larval stages.(2)In order to understand the different types and arrangement of pigment cells in different skin pattern areas of mandarin fish,the skin from pattern area,non-pattern area and their junction area were observed by light and electron microscopes.The results showed that the skin contained melanophores,xanthophores,erythrophores and iridophores,which were mainly distributed in the epidermis and dermis.The pattern area(eye stripe,trunk band,trunk spots)contained melanophores in both the epidermis and dermis.The non-pattern area only contained a small amount of melanophores in the dermis.In the trunk pattern area(band,strips),the pigment cells of dermis was obviously distributed.From the outside to the inside,it was xanthophores,erythrophores,melanophores and iridophores,the reflective platelets in iridophores were long and neatly arranged horizontally.In the non-patterned areas of the trunk were xanthophores,erythrophores and iridophores from the outside to the inside,the reflective small plates in iridophores were short and irregularly arranged.The dermis of the eye stripe contained these four pigment cells,and the number of pigment cells in this region was small and irregularly arranged,and the melanophores were large.At the junction area,the number of melanophores in the dermis gradually decreased toward the non-patterned area,while the number of iridophores gradually increased.The result showed,the pigment cells types,distribution and arrangement were distinctly different among pattern areas,non-pattern area and junction area.(3)Connexin(Cx)is one of important factors in connection and information exchange between pigment cells,and it also involved in the formation of fish skin patterns.The skin tissues of two different pattern types of mandarin fish(Common type: the pattern contains several distinct strips,band and spots;Irregular type: patterns are short,broken,irregular stripes)were sectioned for observation and gene expression of Cx39.4 and Cx41.8 were detected by RT-PCR technology.The results showed that the melanophores were loosely and irregularly distributed in trunk skin of the irregular type.In common types,the gene expression levels of Cx39.4 and Cx41.8 in the trunk area(bands,spots and non-pattern area)were higher than those in the head area(eye strip).The gene expression level of Cx39.4 at the irregular type was not significantly different to the trunk areas(bands,spots and non-pattern area)of common type,but the gene expression level of Cx41.8 was significant lower than the common type(trunk and head).It was speculated that the gene expression level of the Cx41.8 was related to the arrangement and distribution of melanophores,which consequently affected the formation of skin pattern.