Aspects of systematics and host specificity for Gyrodactylus species in aquaculture

Abstract

Of the 430+ extant species of Gyrodactylus, ectoparasitic monogenetic flukes of aquatic vertebrates, Gyrodactylus salaris Malmberg, 1957 is arguably the most well-known. Following the introduction of this species into Norway in the 1970s with consignments of infected Atlantic salmon smolts, Salmo salar L., this species has had a devastating impact on the Norwegian Atlantic salmon population, decimating wild stocks in over 40 rivers. Gyrodactylus salaris is the only OIE (Office International des Epizooties) listed parasitic pathogen of fish and has been reported from 19 countries across Europe, though many of these records require confirmation. The UK, Ireland and some selected watersheds in Finland are currently recognised as G. salaris-free states; however, the threat that this notifiable parasite poses to the salmon industry in the UK and Ireland is of national concern. Current British contingency plans are based on the assumption that if G. salaris were to be introduced, the parasite would follow similar dynamics to those on salmonid stocks from across Scandinavia, i.e. that Atlantic strains of Atlantic salmon would be highly susceptible to infection, with mortalities resulting; that brown trout, Salmo trutta fario L., would be resistant and would lose their infection in a relatively short period of time; and that grayling, Thymallus thymallus (L.), would also be resistant to infection, but would carry parasites, at a low level, for up to 143 days. Two of the objectives of this study were to confirm the current distribution of G. salaris across Europe, and then, to investigate the relative susceptibility of British salmonids to G. salaris, to determine whether they would follow a similar pattern of infection to their Scandinavian counterparts or whether, given their isolation since the last glaciation and potential genetic differences, they would exhibit different responses. It has been almost six years since the distribution of G. salaris across Europe was last evaluated. Some of the European states identified as being G. salaris-positive, however, are ascribed this status based on misidentifications, on partial data resulting from either morphological or molecular tests, or according to records that have not been revisited. Additional Gyrodactylus material from selected salmonids was obtained from several countries to contribute to current understanding regarding the distribution of G. salaris across Europe. From the work conducted in the study, G. salaris is reported from Italy for the first time, alongside three other species, and appears to occur extensively throughout the central region without causing significant mortalities to their rainbow trout, Oncorhynchus mykiss (Walbaum), hosts. The analysis of archive material from G. salaris-positive farms would suggest that G. salaris has been in the country for at least 12 years. Material obtained from rainbow trout from Finland and Germany was confirmed as G. salaris supporting existing data for these countries. No specimens of G. salaris, however, were found in the additional Gyrodactylus material obtained from Portuguese and Spanish rainbow trout, only Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999, a morphologically similar species was found. Gyrodactylus salaris is now reported from 23 out of ~50 recognised states throughout Europe, only 17 of these however, have been confirmed by either morphology or by an appropriate molecular test, and only ten of these records have been confirmed by a combination of both methods. To assess the susceptibility of English and Welsh salmonids to G. salaris, a number of salmonid stocks of wild origin, were flown to the Norwegian Veterinary Institute (NVI) in Oslo, where they were experimentally challenged with G. salaris. Atlantic salmon from the Welsh River Dee, S. trutta fario from the English River Tyne and T. thymallus from the English River Nidd, raised from wild stock in government hatcheries, were flown out and subsequently challenged with G. salaris haplotype A. After acclimation, each fish was infected with ~50–70 G. salaris and marked, so that parasite numbers on individual fish could be followed. The dynamics on individual fish were followed against a control (Lierelva Atlantic salmon). The experiment found that the number of G. salaris on S. salar from the River Dee continued to rise exponentially to a mean intensity (m.i.) of ~3851 G. salaris fish-1 (day 40 post-infection). These salmon were highly susceptible, more so than the Norwegian salmon control (m.i. ~1989 G. salaris fish-1 d40 post-infection) and were unable to regulate parasite numbers. The S. trutta fario and T. thymallus populations, although initially susceptible, were able to control and reduce parasite burdens after 12 (m.i. ~146 G. salaris fish-1) and 19 (m.i. ~253 G. salaris fish-1) days, respectively when peak infections were seen. Although the latter two hosts were able to limit their G. salaris numbers, both hosts carried infections for up to 110 days (i.e. when the experiment was terminated). The ability of S. trutta fario and T. thymallus to carry an infection for long periods increases the window of exposure and the potential transfer of G. salaris to other susceptible hosts. The potential role that brown trout may play in the transmission and spread of G. salaris in the event of an outbreak, needs to be considered carefully, as well as the interpretation of the term “resistant” which is commonly used when referring to brown trout’s susceptibility to G. salaris. The current British surveillance programmes for G. salaris are focused on the screening of Atlantic salmon and on the monitoring of the rainbow trout movements. The findings from this study demonstrate that G. salaris can persist on brown trout for long periods, and suggest that brown trout sites which overlap with Atlantic salmon or rainbow trout sites are also included within surveillance programmes and that the role that brown trout could play in disseminating infections needs to be factored into contingency/management plans. Throughout the course of the study, a number of parasite samples were sent to the Aquatic Parasitology Laboratory at Stirling for evaluation. Some of these samples represented Gyrodactylus material that were associated with fish mortalities, but the species of Gyrodactylus responsible appeared to be new to science. A further aspect of this study was, therefore, to investigate these Gyrodactylus related mortalities in aquaculture stock and to describe the species found in each case, which may represent emerging pathogens. The two new species, Gyrodactylus orecchiae Paladini, Cable, Fioravanti, Faria, Di Cave et Shinn, 2009 and Gyrodactylus longipes Paladini, Hansen, Fioravanti et Shinn, 2011 on farmed gilthead seabream, Sparus aurata L., were collected from several Mediterranean farms. The finding of G. orecchiae in Albania and Croatia was associated with 2–10% mortality of juvenile stock and represents the first species of Gyrodactylus to be formally described from S. aurata. Subsequently, G. longipes was found in Bosnia-Herzegovina and Italy, and at the Italian farm site, it occurred as a mixed infection with G. orecchiae, but these infections did not appear to result in any loss of stock. Unconfirmed farm reports from this latter site, however, suggest that a 5–10% mortality of juvenile S. aurata was also caused by an infection of Gyrodactylus, which is suspected to be G. longipes. Additional samples of Gyrodactylus from a gilthead seabream farm located in the north of France have been morphologically identified as G. longipes, extending the geographical distribution of this potentially pathogenic species to three countries and three different coasts. In addition to these samples, some specimens of Gyrodactylus from a Mexican population of rainbow trout were sent for evaluation. These latter specimens were later determined to be a new morphological isolate/strain of Gyrodactylus salmonis (Yin et Sproston, 1948), a notable pathogen of salmonids throughout North America. The current material was of particular interest as it extends the current geographic range of this parasite from Canada and the USA to the south-eastern region of Mexico. This new Mexican isolate was genetically identical with G. salmonis from Canada and USA, although small morphological differences were evident in the marginal hook sickle shape, which allows to discriminate between the two strains. The results from this study are important as they reflect a similar situation in Europe with G. salaris and Gyrodactylus thymalli Žitňan, 1960, two morphological different but genetically similar species. Discriminating G. salaris from other species of Gyrodactylus infecting salmonids is difficult and, according to OIE, the identification should be based on a combination of data resulting from morphological and molecular approaches. The impact of Gyrodactylus salaris in Norway currently costs £38 million p.a., including loss of revenue from tourism and angling restrictions, and also the cost of on-going surveillance programmes and river treatments. The infection in certain rivers is removed through the addition of either 100 ppb biocide rotenone, which kills all the fish that are host to the parasite, or by a 10–14 day-treatment with 100 µg L-1 aluminium sulfate, which removes the parasite but does not kill its salmon host. If G. salaris were to enter the UK, it is unlikely that either of these compounds would be used because of the human health concerns (i.e. potential links to Parkinson’s and Alzheimer’s diseases) linked to their use. There are, however, very few compounds that could be used as alternatives for the control of wild infections, and there is little research investigating possible replacements. To begin exploring alternatives, a minor component of the study was to explore the effectiveness of two compounds: bronopol (2-bromo-2-nitropropane-1,3-diol) - a broad spectrum disinfectant - and tannic acid - a natural polyphenol that is released from the breakdown of plant material. The evaluation of bronopol was conducted against two strains of G. salaris from Atlantic salmon and on a single population of Gyrodactylus arcuatus Bychowsky, 1933 from three-spined sticklebacks, Gasterosteus aculeatus aculeatus L., as a continuous exposure and for 1 hour only. The results showed that there was a significant increase in the mortality rate of G. salaris as the dose of bronopol increased, but as time progressed, the influence of dose on mortality decreased. Bronopol had a statistically significant (p<0.001) greater effect on G. salaris than it did on G. arcuatus. The analysis suggested that the 1 hour-LC50 for G. salaris was ~384 ppm bronopol, while that needed to kill 50% of G. arcuatus within a 1 hour window of exposure was ~810 ppm bronopol. The trial with tannic acid represented a preliminary assessment and its effects as a continuous exposure and as a 10 minute treatment on G. salaris only were determined. The effect of tannic acid caused the tegument of G. salaris to lift away and the 1 hour-LC50 for tannic acid was <100 ppm although lower doses administered over long periods of time (i.e. 10–14 days as is currently used for aluminium sulfate) may have greater impacts on the survival of the parasite population. While these results demonstrate that bronopol could be used to control infections of G. salaris in confined aquaria, this does not mean that this advocates its use in river systems, as there are a plethora of logistic, economic and environmental considerations to take into account. The study does, however, take important steps towards investigating alternative control agents for use in the event of an outbreak, and both these products are worthy of further evaluation.