Abstract

   Hybrid oysters often show heterosis in growth rate, weight, survival
   and adaptability to extremes of salinity. Oysters have also been used
   as model organisms to study the evolution of host-defense system. To
   gain comprehensive knowledge about various physiological processes in
   hybrid oysters under low salinity stress, we performed transcriptomic
   analysis of gill tissue of Crassostrea sikamea ♀ × Crassostrea
   angulata♂ hybrid using the deep-sequencing platform Illumina HiSeq. We
   exploited the high-throughput technique to delineate differentially
   expressed genes (DEGs) in oysters maintained in hypotonic conditions. A
   total of 199,391 high quality unigenes, with average length of 644 bp,
   were generated. Of these 35 and 31 genes showed up- and
   down-regulation, respectively. Functional categorization and pathway
   analysis of these DEGs revealed enrichment for immune mechanism,
   apoptosis, energy metabolism and osmoregulation under low salinity
   stress. The expression patterns of 41 DEGs in hybrids and their
   parental species were further analyzed by quantitative real-time PCR
   (qRT-PCR). This study will serve as a platform for subsequent gene
   expression analysis regarding environmental stress. Our findings will
   also provide valuable information about gene expression to better
   understand the immune mechanism, apoptosis, energy metabolism and
   osmoregulation in hybrid oysters under low salinity stress.

Introduction

   Hybridization enhances genetic variance, allowing ecesis of unexploited
   niches. Following recombination, ‘transgressive’ quantitative variation
   is effected resulting in more extreme traits than either of the
   parents. Therefore, hybridization not only plays an important role in
   speciation but also forms the backdrop for evolutionary innovations
   [[38]1]. Hybridization breeding, which is defined as the mating of
   animals from different species, strains or inbred lines, facilitates
   genetic rearrangements, which may have strong selective value in
   aquaculture [[39]2, [40]3]. There are several instances where the
   resulting hybrid offsprings show hybrid vigor or heterosis in growth
   rate, weight, survival and adaptability to extremes of temperature,
   salinity, etc. [[41]4–[42]8]. Recently, many attempts have been made at
   hybridization between various Crassostrea species. For instance,
   successful fertilization has been reported for the following crosses
   amongst others- C. hongkongensis × C. ariakensis [[43]9], C. ariakensis
   ×C. angulata [[44]10], C. ariakensis × C. sikamea [[45]11], and C.
   angulata × C. sikamea [[46]12]. Previous studies have mostly focused on
   the survival and growth rate of these hybrids under various
   environmental conditions [[47]9–[48]12].

   Salinity is one of the environmental factors which directly affect the
   survival, growth, and physiological function of oysters
   [[49]13–[50]16]. Oysters are generally euryhaline mollusks, although
   the preferred salinity range varies from species to species [[51]11].
   The Kumamoto oyster (C. sikamea) and the Portuguese oyster (C.
   angulata) have different preferences of optimum salinities, although