The expression of villin 1-like protein is involved in the microvilli formation of lamellar ionocytes in gills of euryhaline milkfish (Chanos chanos)

The gill ionocytes involved in the intracellular homeostasis of the teleosts in different environments. The morphologies and functions of ionocytes in the seawater (SW) fish, the apical membrane of the ionocytes is hole type which forms a pit for ion excretion. In the freshwater (FW) fish, the apical membrane of the ionocytes is flat surface with larger opening, being equipped with numerous microvilli for ion absorption. Milkfish is a euryhaline species, which can survive in environments with hypo- or hyper-salinity and important fish farming in southern Taiwan. Our previous study reported that changes of microvilli in gill ionocytes of the milkfish exposed to the environments with different salinities or low-temperature. Therefore, this study explored the effects of environmental salinity and low-temperature on the expression of villin homologos proteins to illustrate the remodeling mechanism of the apical morphologies in gill ionocytes. Two mRNA sequences, villin 1-like (VILL) and villin 1 were identified from the database of milkfish transcriptome. The higher expression of VILL was found in gills from FW milkfish when compared to the SW individuals. In contrast, the expression of villin 1 was constant in the milkfish gills. The double immunofluorescence staining revealed that the apical region of FW-type ionocytes exhibited the VILL signals in the gill lamellae of FW milkfish but not the SW ionocytes. Observation of scanning electron microscope (SEM) showed that the apical morphology of the FW-type ionocytes with VILL expression was flat-type with microvilli in gill lamellae of the FW milkfish. In contrast, the ionocytes without VILL expression were hole-type and squint-type in the gill filaments of the SW and FW milkfish, respectively. The present study evaluated the effects of low temperature (18°C) on the VILL expression in the apical membrane morphology of ionocytes. Compared with the control (28°C) FW milkfish, the reduce expressions of VILL were found in gills of the FW milkfish exposed to 18°C. In contrast, the expression of villin 1 was constant in the 28°C and 18°C groups. The apical VILL signals in lamellar ionocytes decreased in the 18°C groups. The SEM observation showed that the lamellar ionocytes was squint-type in the low temperature. Meanwhile, the microvilli sparsely occurred. The present study illustrated the VILL is involved in the formation of microvilli in the absorptive ionocytes of milkfish for ionoregulatory function in the different environments.魚類為了適應不同的環境會藉由離子細胞來調節維持體內離子的恆定。在海水環境下的離子調節細胞具有排出離子的功能，在淡水環境下則有吸收離子的功能，其型態也會有所不同;海水型的離子調節細胞的頂膜型態表面狹小內凹成洞;而淡水型離子調節細胞頂膜平坦微凸，表面積大並具有微絨毛結構。虱目魚(Chanos chanos)是台灣南部相當重要的養殖魚種，屬於廣鹽性硬骨魚類，無論在高張的海水環境或低張的淡水環境下皆可以存活，非常適合用來探討養殖魚種對於鹽度與溫度逆境所產生的生理反應。此外，之前的研究也發現，虱目魚鰓上離子調節細胞在低鹽逆境以及低溫淡水逆境下，其形態微絨毛的結構皆會改變，因此，本篇研究試以了解在低鹽以及淡水低溫逆境下，虱目魚鰓上絨毛蛋白的表現，探討其對於離子調節細胞頂膜型態之影響。 從虱目魚次世代定序的轉錄體資料庫中發現，虱目魚具有二型絨毛蛋白基因，分別為 villin1-like (VILL) protein 和 villin 1。本研究首先比較馴養於淡水與海水環境下的虱目魚，淡水虱目魚的鰓上 VILL 基因與蛋白表現量高於海水魚，而 villin 1 的表現量則沒有差異。其次利用雙重免疫螢光染色，發現 VILL 會分佈在淡水虱目魚鰓薄板上離子調節細胞頂膜區域;之後利用掃描式電子顯微鏡觀察虱目魚鰓上離子細胞的型態變化，發現分佈在鰓薄板上的離子細胞，在淡水常溫下其細胞開口大且平坦，並具有微絨毛的結構;相對的，分佈在鰓絲上無 VILL表現的離子細胞在淡水環境下呈縫型，而在海水環境下無 VILL 表現的細胞則呈洞型。此外，本研究將常溫(28°C)淡水馴養虱目魚轉移置低溫(18°C)的環境下一週，在低溫環境下鰓上 VILL 基因與蛋白質表現量皆會下降，在鰓薄板上離子調節細胞頂膜 VILL 的表現有減少的趨勢，此細胞的開口則呈縫型而微絨毛結構也減少。總和以上結果，發現虱目魚為適應環境鹽度或溫度變化時 VILL 的表現參與鰓上離子調節細胞型態包括頂膜微絨毛生成都有變化，說明絨毛蛋白可能參與廣鹽性虱目魚鰓薄板離子調節細胞微絨毛消長的機制。中文摘要 ...........i Abstract ........... ii Introduction ...........1 Aim ...........7 Materials and Methods...........8 Results ........... 14 Discussion ...........18 Conclusion ...........24 Table and figures...........25 References ...........4