Studies Of Melanopsin In The Mudskipper Boleophthalmus Pectinirostris

The burrow-dwelling species of the mudskipper Boleophthalmus pectinirostris is widely distributed in the marine intertidal regions.In our previous study,during the spawning season,the female mudskipper stock exhibits a semilunar spawning pattern,variations of plasma steroid hormones,melatonin,melatonin recepter genes of mtnr1a1.4 in the diencephalon and mtnr1a1.7 in the ovary,were synchronized obviously with semilunar periodicity.In addition,the peaks of semilunar rhythm coincided with the timing of spawning in this species.To improve our understanding of linkage between melanopsin and melatonin,and semilunar spawning physiology in this species,in this study,using molecular biological method,we cloned all melanopsin subtypes(opn4m1,opn4m3,opn4x1,opn4x2)from B.pectinirostris,and analyzed their tissue distribution,diurnal,semilunar and seasonal variations.In addition,we studied the external structure and cytoarchitecture of B.pectinirostris brain under optical microscope.The main results and conclusions are as follows:1.The brain of B.pectinirostris can be mainly divided into three parts:the forebrain.midbrain and hindbrain.The forebrain is composed of the olfactory bulbs,telencephalon and diencephalon.Telencephalon is well developed and composed of two hemispheres.The midbrain contains the optic tectum and other components.The hindbrain consists of cerebellum and myelencephalon.2.The full sequence of opn4m1 was 2299 bp,which encoded a protein of 496 amino acids,flanked by 187 bp of 3' UTR and 621 bp of 5' UTR.The cloned complete sequence of opn4m3 was 3381 bp,encoding a protein of 546 amino acids.5'and 3' UTR regions were 126 and 1614 bp.The full length of the opn4xl cDNA was 1588 bp,including encoding 419 amino acids,176 bp in the 5' UTR and 152 bp in the 3' UTR.The 2668 bp opn4x2 cDNA codes for a 516-aa potein,there were 900 bp and 217 bp in the 5' and 3' UTR regions,respectively.Phylogenetic analysis of known melanopsin genes in vertebrates further verified that the four melanopsin subtypes cloned from B.pectinirostris can be divided into two branches,the mammalian-like(opn4m)and the Xenopus-like(opn4x)melanopsins,designated as opn4ml?opn4m3?opn4xl?opn4x2,respectively.3.Quantitative real-time PCR was used to analyze the expression of the four melanopsin subtypes in different tissues of B.pectinirostris.The results showed that all four melanopsin subtypes had different abundances in different tissues of female,and expressed significantly higher in the eyes than other tissues(brain,skin,gill,kidney,muscle,spleen,ovary),while the four melanopsin subtypes were not observed in the liver and intestine.4.Diurnal expression of melanopsin mRNAs showed similar patterns in both brain and eyes,low in the morning(04:30-10:30).The same subtype showed various diurnal expressions in different brain regions,especially the opn4m3 mRNAs displaying the opposite trends in the telencephalon and diencephalon.5.During peak spawning period,the transcript levels of opn4m3 and opn4xl in the telencephalon displayed obviously semilunar rhythms in the female mudskippers,exhibiting two cycles within a lunar month,each one with a peak.Moreover,the peaks coincided with the timing of spawning,the plasma levels of melatonin and steroid hormones in this species.The transcript levels opn4m3 and opn4xl did not show any semilunar expression patterns in the diencephalon and eyes.6.Using real time PCR,we found that levels of opn4m3 and opn4x2 did not significantly change in the telencephalon and diencephalon during the peak spawning(May and July).The opn4ml mRNA was significantly up-regulated in the telencephalon during May,but not in the diencephalon.In the telencephalon,the expression of opn4xl was the lowest before the spawning season(March),while during the peak spawning season(from April to June)opn4xl was significantly up-regulated and reached the highest level.However,in the diencephalon,the transcript level of opn4xl increased significantly in April,was down-regulated from May to June,and was significantly up-regulated in July.Taken together,the results from the present study suggested that melanopsin subtypes in telencephalon may be the regulation factor of semi-lunar spawning activity in the mudskipper B.pectinirostris;light information is transferred to the HPG axis through the telencephalon or acts directly on melatonin to modulate spawning of female mudskippers.