Abstract

   White spot syndrome virus (WSSV) is one of the most devastating
   pathogens of cultured shrimp, responsible for massive loss of its
   commercial products worldwide. The oriental river prawn Macrobrachium
   nipponense is an economically important species that is widely farmed
   in China and adult prawns can be infected by WSSV. However, the
   molecular mechanisms of the host pathogen interaction remain unknown.
   There is an urgent need to learn the host pathogen interaction between
   M. nipponense and WSSV which will be able to offer a solution in
   controlling the spread of WSSV. Next Generation Sequencing (NGS) was
   used in this study to determin the transcriptome differences by the
   comparison of control and WSSV-challenged moribund samples, control and
   WSSV-challenged survived samples of hepatopancreas in M. nipponense. A
   total of 64,049 predicted unigenes were obtained and classified into 63
   functional groups. Approximately, 4,311 differential expression genes
   were identified with 3,308 genes were up-regulated when comparing the
   survived samples with the control. In the comparison of moribund
   samples with control, 1,960 differential expression genes were
   identified with 764 genes were up-regulated. In the contrast of two
   comparison libraries, 300 mutual DEGs with 95 up-regulated genes and
   205 down-regulated genes. All the DEGs were performed GO and KEGG
   analysis, overall a total of 85 immune-related genes were obtained and
   these gene were groups into 13 functions and 4 KEGG pathways, such as
   protease inhibitors, heat shock proteins, oxidative stress, pathogen
   recognition immune receptors, PI3K/AKT/mTOR pathway, MAPK signaling
   pathway and Ubiquitin Proteasome Pathway. Ten genes that valuable in
   immune responses against WSSV were selected from those DEGs to furture
   discuss the response of host to WSSV. Results from this study
   contribute to a better understanding of the immune response of M.
   nipponense to WSSV, provide information for identifying novel genes in
   the absence of genome of M. nipponense. Furthermore, large number of
   transcripts obtained from this study could provide a strong basis for
   future genomic research on M. nipponense.

Introduction

   White spot syndrome disease is one of the most destructive viral
   disease in global shrimp aquaculture, causing considerable economic
   losses every year and has been estimated at more US$8 billion since
   2000 [[40]1–[41]9]. The disease is caused by white spot syndrome virus
   (WSSV), a double-stranded DNA virus in the genus Whispovirus, family
   Nimaviridae [[42]10]. Since its first appearance in Taiwan in 1992, the
   virus has quickly spread and affected many aquaculture areas worldwide
   [[43]11, [44]12]. Almost all decapod crustaceans, including shrimps,
   crabs, lobsters and crayfish, are considered susceptible to this virus
   [[45]13, [46]14]. The oriental river prawn Macrobrachium nipponense
   could be effectively infected by WSSV through oral administration and
   intramuscular injection [[47]15, [48]16].

   The Oriental river prawn, Macrobrachium nipponense is one of the most
   important economic species that is farmed widely in China [[49]17,
   [50]18], with annual yields exceeding 265 061 metric tonnes [[51]19].
   Compared with penaeid shrimps, freshwater prawns, especially M.
   nipponense, were generally considered to be less prone to disease in
   culture [[52]16, [53]20]. Macrobrachium nipponense, like other
   crustaceans, lack an acquired immune system and rely totally on the
   innate defense system to resist pathogen invasion [[54]21]. The
   hepatopancreas of crustaceans is the main immune organ, playing an
   important role in health, growth and survival and has been used as a
   monitor organ for the overall assessment the health [[55]22, [56]23].
   Macrobrachium nipponense can be infected by WSSV and the natural WSSV
   prevalence level of M. nipponense was about 8.3% [[57]15, [58]24]. In
   our present study, M. nipponense has a more effective immune reponse to
   WSSV infection than L. vannamei and the hepatopancreas in M. nipponense
   can be affected by WSSV easily [[59]16]. In addition, M. nipponense can
   normally survive with carrying WSSV. The survived M. nipponense with
   WSSV reveals that the host conducted a successful and efficient immune
   responses to against WSSV infection. While, the moribund M. nipponense
   due to WSSV infection reveals the host have made a ultimate immune
   responses to against WSSV. Elucidation of the immune mechanism of WSSV
   infection in M. nipponense will be helpful to other crustaceans
   aquaculture.

   In recent years, attempts have been conducted to investigate the
   effects of WSSV infection on shrimp transcriptome using cDNA
   microarray, the suppression subtractive hybridization (SSH), expressed
   sequence tag analysis (EST) and so on [[60]25–[61]27]. However,
   microarrays and subtractive hybridization methods are impeded by
   background and cross-hybridization problems, and only the relative
   abundance of transcripts is measured [[62]28, [63]29]. The EST
   sequencing technique is laborious, and it has limitations in sampling
   the depth of the transcriptome, which could possibly loss the detection
   of transcripts with low abundance [[64]30]. The next generation
   sequencing (NGS), a far superior technology, was introduced in 2004
   [[65]31, [66]32]. The expressed sequences produced using NGS
   technologies are usually 10- to100-fold greater than the number
   identified by traditional Sanger sequencing technologies in much
   shorter times [[67]33, [68]34]. This platform provides an efficient way
   to rapidly generate large amounts of data with low cost [[69]35,
   [70]36]. As a powerful method, comparative transcriptome analysis has
   been used to discover the molecular basis underlying specific
   biological events, such as immune processes during infection [[71]37,
   [72]38]. However, no information is available on the gene expression
   profiles of M. nipponense with a WSSV infection.

   In the present study, we applied the next generation sequencing and
   bioinformatics techniques for analyzing the transcriptome differences
   from the hepatopancreas of M. nipponense experimentally infection with
   WSSV among the survived, moribund and normal control prawns without
   previously annotated genomes as references. It will demonstrate some