Co-introduction of metazoan parasites with an invasive host, Micropterus salmoides (Lacépède, 1802) in non-native regions in South Africa
Since the early 18th century the introduction of non-native fish species occurred into South African freshwater systems. Drivers for these introductions included stocking for sports angling, aquaculture, bio-control and the pet trade. Little attention has been given to the co-introduction of symbionts, especially of introduced alien species, that succeeded in overcoming barriers set by introduction into new environments. A typical example in freshwater systems would be fish and their parasites. The movement and introduction of fish hosts typically result in co-introduction of these accompanying parasites and four possible mechanisms: enemy release, dilution, spillback and spill-over, where the latter results in co-invasion of the introduced parasites (Sheath et al., 2015). An example of such a species is the North American native, largemouth bass Micropterus salmoides (Lacépède, 1802) that was introduced into South Africa in 1928 for sport angling and aquaculture. Limited research has been done on the parasites of this fish in South Africa, only including the investigation of mass mortalities of largemouth bass fingerlings (see Du Plessis, 1948) and inclusion in checklists compiled on the helminths of Africa or parasites of freshwater fishes in southern Africa (van As and Basson, 1984; Khalil and Polling, 1999). Other information available include the studies of Barson et al. (2008), Tavakol et al. (2015) and unpublished data of three co-introduced Monogenea from the ureters and gills (see Matla, 2012). The present study investigated the parasite diversity of largemouth bass in South Africa, focusing on populations from the North West, KwaZulu-Natal, Eastern Cape and Western Cape provinces. The populations from the Eastern Cape and Western Cape are believed to be of the first largemouth bass introduced into the country, where it was distributed to other impoundments and freshwater systems throughout the country. This is supported by literature (see Harrison, 1936; McCafferty et al., 2012) and the study of Hargrove et al. (2017). Parasitic communities, especially of the Monogenea are of interest, as these hosts are parasitised by specialist Ancyrocphalidae, that have been co-introduced. To shed light on the diversity of introduced parasites and the possibility or probability of these specialists to spill-over or spillback is investigated, as previous literature did not note or determine if these mechanisms are at play, or failed to identify these parasites up to generic level. An attempt was made to identify these introduced parasites using morphological as well as molecular approaches. To fill the gaps in our knowledge of these specialist monogenean parasites of M. salmoides in the freshwater systems of South Africa, the following hypotheses were proposed: 1) that parasite enemy release did not occur upon the introduction of M. salmoides into South African freshwater systems; 2) that it will be possible to distinguish between parasitic genera and species using three nuclear markers; 3) that there will be a negative correlation between the health of M. salmoides and the intensity of infection with these specialist gill parasites and 4) that no parasite spill-over has occurred to native freshwater fish species. To achieve these hypotheses the main aims of the study was to 1) perform a full macro- and microscopic parasite screening of M. salmoides to determine the parasite diversity of populations from eight localities throughout South Africa (Mooi River system, North West Province (NW); Eerste River catchment (VD) and an closed natural lake, Groenvlei Lake (GV), in the Western Cape; the uMngeni catchment in KwaZulu-Natal (KZN) and two impoundments in the Kariega River system in the Eastern Cape (EC); 2) identify the monogenean parasitic species using both morphological and molecular approaches; 3) to implement a macroscopic necropsy-based fish health assessment to determine health status of the host species from impoundments with the highest infection levels and 4) to investigate the parasite community of native fishes in an impoundment with a specialist gill parasite known to be less host-specific than the other to their centrarchid host, to determine if parasite spill-over has occurred. Micropterus salmoides were collected with the aid of angling and electrofishing techniques from seven impoundments, during October 2015, February 2016, April 2016, October 2016 and April 2017. All other fishes were sampled with the use of gill-, fyke and seine nets, in January and April 2017, from the Boskop Dam, North West. Fish were kept in aerated containers until a dissection and necropsy-based health assessment was performed (recommended by Adams et al., 1993; Fouché, 2016) at the field site, and a macro- and microscopic parasite screening was done. The parasite screening of M. salmoides and morphological characterisation confirmed the presence of eight parasite species from all the populations investigated and consisted of a single protozoan (Trichodina sp.) from the gills, two nematodes (Contracaecum sp. and Spinitectus sp.) from the body cavity and stomach and five ancyrocephalid monogenean parasite species from the gills (Clavunculus bursatus, Onchocleidus dispar, Onchocleidus furcatus, Onchocleidus principalis and Syncleithrium fusiformis). Overall, EC had the highest species richness with five parasite species present, followed by KZN, GV, VD and the lowest in NW. Monogenean species richness was the highest in KZN and the EC, and lower for GV, VD and the lowest in NW with only one species present. The invasive status of the Trichodina sp. and two nematodes are uncertain, but all five monogenean parasites were co-introduced with the invasive M. salmoides and provides new locality records for these species. The lower species richness of the monogenean parasites in South Africa also supports the enemy release hypothesis. Molecular characterisation of the five co-introduced monogenean gill parasites were successful and provides the first molecular data available for these monogenean parasites found on largemouth bass in South Africa. The newly obtained data can potentially serve as a good platform for taxonomic revision of these ancyrocephalids and provide support for future studies in revisions of the phylogeny of the Ancyrocephalidae. The macroscopic and necropsy-based fish health assessment was used and showed that M. salmoides from all localities were in good health condition, with the exception in GV where 46% of the fish had discoloured livers. This may not be linked to parasitic infection, but rather water quality or presence of pollutants in the system. Although the intensity of infection (IF) was the highest in EC, there was a very weak correlation (and not of statistical significance) between white blood cell counts and IF. The absence of a correlation between host health and parasitic infection suggest that the loss in parasite diversity may not be related to the fitness of the fish in the novel environment, but rather the co-evolution of the host and its parasites, this also supports the enemy release hypothesis. All the parasites recorded from the five different native fish species collected from Boskop Dam in the present study represents infection with parasite species known from the specific hosts. The absence of infection with any of the ancyrocephalids from M. salmoides confirms that no spill-over occurred. The possibility that no spill-over has occurred within the past 60 years that this host is present in the Mooi River system, suggest that it is unlikely that any of the Ancyrocephalidae will switch hosts. The possibility of host-switching or spill-over should, however, not be disregarded as little is known about the evolutionary relationship of these parasites with its centrarchid hosts. From the results presented in this study, supplementary knowledge on the invasion status and potential, as well as additional morphological and molecular data is available for these parasite species. The potential for these co-introduced monogeneans to become co-invasive should not be underestimated or assumed from this single study. Future studies should monitor and investigate these parasite species to detect events of spill-over or spillback. Health impacts of these parasites should also be monitored, their presence in South African freshwater systems are still less than a century, and the evolutionary relationship with their hosts are still uncertain.