Emphasis Type="Italic"Tetracapsuloides bryosalmo

Authors: Gokhlesh Kumar Subhodeep Sarker Simon MenanteauLedouble Mansour ElMatbouli

Publish Date: 2015/03/20

Volume: 114, Issue: 6, Pages: 2301-2308

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Abstract

Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America The life cycle of the European strain of T bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout Salmo trutta Linnaeus 1758 Little is known about the gene expression in the kidney of brown trout during the developmental stages of T bryosalmonae In the present study quantitative realtime PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T bryosalmonae development PCR primers specific for target genes were designed and optimized and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix Expression of Rab GDP dissociation inhibitor beta integral membrane protein 2B NADH dehydrogenase 1 beta subcomplex subunit 6 and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout while the expression of the ferritin M middle subunit was downregulated significantly These results suggest that host genes involved in cellular signal transduction proteasomal activities including membrane transporters and cellular iron storage are differentially upregulated or downregulated in the kidney of brown trout during parasite development The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoanProliferative kidney disease PKD is an economically important disease affecting various species of salmonids in Europe and North America Hedrick et al 1993 ElMatbouli and Hoffman 1994 The causative agent of PKD is Tetracapsuloides bryosalmonae Anderson et al 1999 Canning et al 1999 Feist et al 2001 which belongs to the phylum Myxozoa and class Malacosporea The T bryosalmonae life cycle alternates between an invertebrate host bryozoan and a vertebrate host salmonids Canning et al 1999 2002 Okamura et al 2011 PKD has been implicated in the decline of wild brown trout Salmo trutta Linnaeus 1758 and other wild populations of salmonids especially in Switzerland Norway and Estonia Wahli et al 2002 Sterud et al 2007 Dash and Vasemägi 2014 Spores of T bryosalmonae develop in the kidney tubules of infected brown trout and are released via urine to infect freshwater bryozoans Morris and Adams 2006 Grabner and ElMatbouli 2008 Low numbers of interstitial presporogonic stages of T bryosalmonae were observed in the kidney samples of brown trout and high numbers of intraluminal sporogonic stages of T bryosalmonae were seen at 8–12 weeks postexposure wpe but it was almost nil at 6 wpe Kumar et al 2013 It has been recently demonstrated that T bryosalmonae is able to persist in chronically infected brown trout and could to infect the bryozoan Fredericella sultana colonies up to 104 wpe AbdElfattah et al 2014Knowledge about gene expression can be used to understand how the parasite affects host renal cell activities and mechanisms during the developmental stages toward explaining pathogenesis of the parasite in targeted tissue Some studies have examined immunerelated gene expression in the kidney of rainbow trout Oncorhynchus mykiss Walbaum 1792 naturally infected with T bryosalmonae Holland et al 2003 Gorgoglione et al 2013 Kumar et al 2015 However very little is known about gene expression in brown trout only few genes involved in cellular stress growth hemoglobin and calcium metabolism have been examined in the kidney of brown trout during T bryosalmonae developmental stages Kumar et al 2014A number of genes of interest were selected based on our previous study where we compared transcriptomes from the kidneys of infected and noninfected brown trout by suppressive subtractive hybridization and discovered transcripts in the kidneys of brown trout Kumar et al 2014 Afterward the relative gene expression of these genes in the kidney of infected brown trout was examined at different time points of T bryosalmonae development New results on the expression of brown trout genes would offer insights on the modulation of renal tissue signal transduction proteasomal activities including membrane transporters as well as the role of proteins involved in cellular iron storage in gene regulatory mechanisms in brown trout during parasite developmentInfected kidney samples were originated from our previous study Kumar et al 2014 Briefly 60 brown trout were infected with the spores of T bryosalmonae released from mature sacs of parasite from laboratoryinfected Fredericella sultana colonies and maintained at 165 ± 1 °C during the whole experimental period Additional 30 brown trout were held as a noninfected fish Posterior kidneys were sampled from both infected n = 10 and noninfected groups n = 5 at different time points such as 6 8 10 and 12 wpe Infection in individual kidney sample was first confirmed by quantitative realtime PCR qPCR of T bryosalmonae according to the method described by Grabner and ElMatbouli 2009 At 6 wpe parasite load was low while at 8–12 wpe parasite load was high in the kidney of infected brown trout Kumar et al 2013 Additionally T bryosalmonae stages were determined in the kidney samples using antiT bryosalmonae monoclonal antibody Aquatic Diagnostics and antibody–antigen reaction was visualized using a Dako EnVision+ SystemHRP AEC kitBased on work previously conducted in our laboratory Kumar et al 2014 where we identified transcripts in the kidney of brown trout which exhibited signal transduction ion transporter and cellular iron activities based on this we selected six genes of interest in the present study Rab GDP dissociation inhibitor beta gammasecretase subunit PEN2 integral membrane protein 2B ITM2B NADH dehydrogenase 1 beta subcomplex subunit 6 NDUFB6 26S protease regulatory subunit S10B PSMC6 and ferritin middle M subunitTetracapsuloides bryosalmonae stages in the kidney of brown trout a Interstitial presporogonic parasite stages arrows b intraluminal sporogonic parasite stages arrows in the renal tubule Parasite stages were visualized by immunohistochemistry using antiT bryosalmonae monoclonal antibody IgG1 isotype P01 and counterstained with hematoxylin

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