Identification of a piscine reovirus-related pathogen in proliferative darkening syndrome (PDS) infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline.

The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100% in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS.

Critical swimming speed of brown trout (Salmo trutta) infested with freshwater pearl mussel (Margaritifera margaritifera) glochidia and implications for artificial breeding of an endangered mussel species.

Unionid freshwater mussels need to attach to a host fish for completion of their life cycle. It remains unclear whether the relationship between these mussels and their host fishes can be considered parasitic, mutualistic, or commensal. Herein, we studied the effects of Margaritifera margaritifera infestation on Salmo trutta, the most important host of this endangered mussel species in Central Europe. Glochidial load of host fish increased with increasing glochidial concentration, but the highest ratios of encysted glochidia to exposed glochidia were found at low concentration (15,000 glochidia L(-1)) during infestation. Host fish mortality occurred at infestation rates of ~350 glochidia per g fish weight and was highest (60%) at the highest infestation rates (~900 glochidia per g fish weight). On a sublethal level, swimming performance of hosts was inversely related to infestation rates, with infestation of ~900 glochidia per g fish weight reducing critical swimming speed of S. trutta significantly by ~20% compared to infestation with 6 glochidia per g fish weight. The high mortality and the impaired swimming capability of highly infested hosts indicate a parasitic interaction between M. margaritifera and its host. For conservation and reintroduction of M. margaritifera via glochidia-infested S. trutta, we recommend glochidial loads of 5-100 glochidia per g fish weight, while for artificial breeding of juvenile M. margaritifera under laboratory conditions, higher infestation rates of up to 300 glochidia per g fish weight are ideal to balance high yields of mussels and welfare of host fishes.