Water temperature is one of the most important factors in fish physiology; thus, it is important to identify genes that respond to changes in water temperature. In this study, we identified a warm- temperature acclimation-associated 65-kDa protein (Wap65) in the Kumgang fat minnow Rhynchocypris kumgangensis, a small, cold-freshwater fish species endemic to Korea. Kumgang fat minnow Wap65-1 (kmWap65-1) was cloned using polymerase chain reaction (PCR)-based strategies, and was found to be highly homologous with teleost Wap65-1 and mammalian hemopexin, a heme-binding protein that transfers plasma heme into hepatocytes. kmWap65-1 mRNA was expressed mainly in the liver and its expression levels were significantly increased by both short- and long-term exposure to high temperature, which was evaluated by real-time quantitative PCR. Furthermore, the expression levels of kmWap65-1 were highly elevated by exposure to bacterial lipopolysaccharide. These results indicate that kmWap65-1 expression is associated with environmental stresses such as increases in water temperature and bacterial infection. J. Exp. Zool. 325A:65-74, 2016. © 2015 Wiley Periodicals, Inc.

Warm temperature acclimation-associated 65-kDa protein (Wap65) is known to respond to elevated water temperatures and the corresponding gene from several fish species has been cloned. Expression of Wap65 gene is induced by various physiological stresses, such as increase in water temperature, immune response and heavy metal exposure. Two isolated Wap65 genes, Wap65-1 and Wap65-2, display distinct tissue distribution and physiological functions despite high sequence homology. In a previous study, we identified the Wap65-1 gene (kmWap65-1) from Kumgang fat minnow, Rhynchocypris kumgangensis, a small freshwater fish endemic to Korea. The kmWap65-1 gene showed sequence homology with teleost Wap65-1 and mammalian hemopexin, and was highly expressed in response to increased water temperature and bacterial lipopolysaccharide (LPS) exposure. Here, we isolated kmWap65-2 from liver tissue of Kumgang fat minnow and compared the expression profiles of both kmWap65 genes following exposure to various physiological stresses, including thermal changes, bacterial challenge, and environmental toxins. Notably, while kmWap65-1 expression was significantly increased in response to high water temperature, LPS, cadmium, and iron, kmWap65-2 displayed no alterations in expression at high water temperature. However, kmWap65-2 expression was upregulated slightly in response to LPS and highly in presence of copper, bisphenol A, and estradiol. Based on the collective findings, we propose that kmWap65-1 and kmWap65-2 are multifunctional proteins with distinct functions that could serve as useful biomarkers for assessing physiological stress and associated responses in Kumgang fat minnow.

Watermelon fruit rind color (RC) and bloom formation (BF) affect product value and consumer preference. However, information on the candidate gene(s) for additional loci involved in dark green (DG) RC and the genetic control of BF and its major chemical components is lacking. Therefore, this study aimed to identify loci controlling RC and BF using QTL-seq of the F2 population derived by crossing ‘FD061129’ with light-green rind and bloom and ‘SIT55616RN’ with DG rind and bloomless. Phenotypic evaluation of the F1 and 219 F2 plants indicated the genetic control of two complementary dominant loci, G1 and G2, for DG and a dominant locus, Bf, for BF. QTL-seq identified a genomic region on Chr.6 for G1, Chr.8 for G2, and Chr.1 for Bf. G1 and G2 helped determine RC with possible environmental effects. Chlorophyll a-b binding protein gene-based CAPS (RC-m5) at G1 matched the highest with the RC phenotype. In the 1.4 cM Bf map interval, two additional gene-based CAPS markers were designed, and the CAPS for a nonsynonymous SNP in Cla97C01G020050, encoding a CSC1-like protein, cosegregated with the BF trait in 219 F2 plants. Bloom powder showed a high Ca2+ concentration (16,358 mg·kg−1), indicating that the CSC1-like protein gene is possibly responsible for BF. Our findings provide valuable information for marker-assisted selection for RC and BF and insights into the functional characterization of genes governing these watermelon-fruit-related traits.

Heat shock proteins (Hsps) are molecular chaperones that respond to various external and internal cellular stresses. Hsp90 is an abundant cellular heat shock protein. It has two isoforms, Hsp90α and Hsp90β. In fish, expression of the isoforms is augmented by various stress signals, including thermal change, salinity, pH, ammonia, infection, and environmental pollutants. In this study, we isolated both isoforms of Hsp90, designated rkHsp90α and rkHsp90β, from the liver of Kumgang fat minnow, Rhynchocypris kumgangensis, a small freshwater fish that is endemic to Korea. Sequences of the isolated isoforms showed homology with the corresponding isoforms of teleost Hsp90. Both isoforms of rkHsp90 were highly expressed in the liver compared to other tissues, including brain, gastrointestinal tract, gills, and muscle. Water temperature elevation induced increased hepatic and muscular expression of rkHsp90α, but not rkHsp90β. Both isoforms did not respond to lipopolysaccharide challenges. Exposure to environmental pollutants promoted expression of rkHsp90β, but not rkHsp90α. The collective findings support the proposal that rkHsp90α and rkHsp90β act as molecular chaperones that respond to distinct cellular stresses. Both could serve as useful biomarkers for assessing cellular stress in Kumgang fat minnow.