Reduced survival of a native parasite in the invasive round goby : evidence for the dilution hypothesis ?

With economic globalization, a growing number of exotic species are integrating into food webs outside their historical range, giving rise to the development of novel associations between exotic hosts and local parasites. Depending on the parasite’s ability to survive and undergo transmission, invasive exotic hosts can act as sinks or reservoirs for native parasites, thus either decreasing or increasing their overall abundance in indigenous hosts. Here we evaluate the relative host competence of the invasive round goby (Neogobius melanostomus) for a native acanthocephalan species, Neoechinorhynchus tenellus, in the Great Lakes – St. Lawrence River basin. The second most abundant helminth acquired by the round goby, N. tenellus was found to die prematurely in this novel paratenic (transport) host. On average, nearly half of the cysts found in gobies sampled at 14 localities contained dead and degenerated cystacanths. Parasite remnants in hepatic tissues were surrounded by mast cells indicative of an innate inflammatory host reaction. Conversely, cystacanths of N. tenellus were intact in johnny darters ( Etheostoma nigrum) and logperch (Percina caprodes ), two co-occurring native paratenic hosts. We conclude that the round goby is currently a poor host for N. tenellus relative to indigenous counterparts. As such, this abundant exotic fish could act as a sink and impair the transmission of N. tenellus, possibly resulting in parasite dilution in native fish competitors. The significantly higher intensity and prevalence of infection in johnny darters at a goby-free locality supports this hypothesis. However, this new host-parasite relationship might evolve with time toward an attenuation of the goby immune defense reaction. Indeed, we found a negative correlation between the frequency of cystacanth degradation and time since gobies established in a given locality, with the lowest degeneration rate in the St. Clair River area where the round goby was first recorded in the Great Lakes – St. Lawrence basin. The dilution effect, if it exists, could then be temporary.


Introduction
Host-parasite relationships are evolving ecological interactions between antagonist partners.On their part, hosts tend to evolve immune, physiological and behavioral defenses to eliminate their parasites or minimize their most deleterious effects (Anderson and May 1982;Ebert and Hamilton 1996).In response, parasites develop counter-strategies to evade their host's defense, using tactics such as encapsulation, antigen mimicry or the inhibition of specific immune functions (Sitjà-Bobadilla 2008).Over the long term, coevolution of hosts and parasites can sometimes result in less hostile relationships whereby a cost-benefit tradeoff is reached (Lenskit and May 1994;Alizon et al. 2009).In such cases, pathogens may reduce their virulence and/or hosts may increase their tolerance for unavoidable parasites.
In a globalized world, species invading new habitats outside their native range are likely to shake these long-lasting relationships and provide opportunities for the development of novel hostparasite associations (Lafferty et al. 2005;Nelson et al. 2015b).Indeed, introduced species carry some of their parasites into the invaded habitats, bringing them into contact with a large pool of potential new hosts (Lymbery et al. 2014).Most of these exotic parasites do not find the suitable hosts that would ensure their survival (Dobson and May 1986); a few, however, not only survive but become invasive, sometimes causing significant damage to their new hosts (Taraschewski 2006;Choudhury and Cole 2011;Lymbery et al. 2014).Once established, exotic species can find themselves exposed to local parasites.Although parasite communities in exotic species tend to remain less diverse than conspecific host populations in native habitats (Torchin and Mitchell 2004) and than local competitors in the invaded habitats (Gendron et al. 2012), native parasite acquisition does occur over time and has been reported in a number of recent studies (Telfer et al. 2005;Gendron et al. 2012;Paterson et al. 2012;Sheath et al. 2015).
Until now, efforts to understand the influence of exotic species on native host-parasite dynamics have identified two main divergent outcomes.If an introduced species becomes a competent host to a given native parasite -allowing it to survive and be transmitted further -it may act as a reservoir of infection and eventually lead to an overall increase in the abundance of this parasite among indigenous hosts, termed parasite spillback (Kelly et al. 2009a;Poulin et al. 2011).Alternatively, if a native parasite is acquired by an unsuitable introduced host which acts as a sink (or dead-end) for that parasite, a dilution effect may ensue, whereby indigenous hosts experience a decrease in infection by that parasite (Telfer et al. 2005;Kopp and Jokela 2007;Paterson et al. 2011;Lettoof et al. 2013).
The main objective of this study was to assess the competence of an invasive fish, the round goby (Neogobius melanostomus Pallas, 1814), to host a native parasite, Neoechinorhynchus tenellus (Van Cleave, 1913).The round goby is an introduced Eurasian fish now widespread and abundant in the Great Lakes -St.Lawrence River basin (Brodeur et al. 2011;Kornis et al. 2012) where it is parasitized by a small number of local parasite species (none being exotic), most frequently by the generalist digenetic trematode Diplostomum spp.(Muzzal et al. 1995;Kvach and Stepien 2008;Gendron et al. 2012).Of the twenty-some native helminths acquired so far by gobies in the St. Lawrence River, the acanthocephalan N. tenellus is the second most prevalent and abundant (Gendron et al. 2012).This species is transmitted to fish by the ingestion of infected ostracods (Walkey 1967;Uglem and Larson 1969), an important component of the diet of juvenile round gobies (Gendron et al. 2012).They mature in the digestive tract of their definitive host, usually a piscivorous fish (Amin and Muzzall 2009), releasing embryonated eggs that are passed in the feces and subsequently eaten by ostracods (Walkey 1967;Uglem and Larson 1969;Kennedy 2006).In round gobies however, and in some indigenous benthic percids, N. tenellus rarely establishes in the digestive tract; rather it penetrates the gut and encysts in the viscera as a cystacanth.These fish are utilized as paratenic or transport hosts and are optional components of the parasite's life cycle; they must be consumed by a suitable definitive fish host for cysacanths to develop into adults and reproduce (Kennedy 2006).Considering that the round goby has become a dominant species in the Great Lakes and the St. Lawrence River (Kipp et al. 2012) and is now an important part of the diet of a number of piscivorous fish (Reyjol et al. 2010), it has the potential to influence the population dynamics and transmission of N. tenellus among native fish (causing dilution or spillback), depending on its competence as a host relative to native hosts.To further explore this hypothesis, we compared the prevalence and intensity of infection as well as the survival of N. tenellus in the exotic round goby in the St. Lawrence River and lower Great Lakes with that of two native paratenic hosts, the johnny darter (Etheostoma nigrum Rafinesque, 1820) and the logperch (Percina caprodes Rafinesque, 1818), which presumably have a long-standing association with N. tenellus.

Study area and fish sampling
Fish were sampled in the Great Lakes -St.Lawrence basin during the summer months, between 2006 and 2014, using a beach seine (22.6 m × 1.15 m; 3 mm mesh) pulled by hand or partially deployed from a boat.Samples of round gobies were obtained from 17 sites ranging from Lake St. Clair to Lake St. Pierre (Figure 1, supplementary material Table S1).At 8 of these localities, samples of johnny darter (Etheostoma nigrum) or logperch (Percina caprodes), two native benthic percid fish, were collected as well.In addition, a sample of johnny darter was obtained from a reference area not yet colonized by the round goby.Upon capture, fish were euthanized in a 400 mg/L Eugenol (clove oil) solution (American Veterinary Medical Association 2013), individually bagged and frozen for subsequent parasitological examination.A small number of fish were brought live to our laboratory where they were maintained in indoor tanks for a short period of time until being processed.These were used as a source of voucher parasite specimens and for histological examination of infected tissues.S1.

Parasitological examination
Prior to dissection, standard length was measured to the nearest mm and each fish was weighed to the nearest 0.01 g.The abdominal organs were removed and examined using a stereomicroscope.Squash preparations of liver, kidney, mesenteries and intestinal wall were made to reveal encysted acanthocephalans (cystacanths).The content of the gastro-intestinal tract also was searched for adult worms.Acanthocephalans found were excysted and classified as intact or partially degenerated and counted.Cleaned specimens were fixed in 70% ethanol before being stained in acetocarmine, cleared in clove oil, and mounted on slides.Specimens were identified to species on the basis of morphological criteria following keys in Arai (1989) and taking into account recent redescriptions and revisions within the genus Neoechinorhynchus (Amin 2002;Amin and Muzzall 2009).Morphological measurements were made using the software Leica IM1000 (V4.0) connected to a DC500 digital camera mounted on a Leica DMR microscope.Voucher specimens of N. tenellus (ROMIZ F320; ROMIZ F320) have been deposited in the Royal Ontario Museum (Toronto, Canada).

Histology of parasitized tissues
Round gobies collected in 2009 at Île de la Couvée (ILO), a locality of elevated prevalence of infection by cystacanths, were transported alive to the laboratory and processed for histological examination.Livers of infected fish were rapidly removed and small blocks containing visible cysts were fixed in 10% neutral buffered formalin prior to being sent to a professional histology laboratory (The Centre for Bone and Periodontal Research, McGill University, Montreal, Canada).There, tissues were dehydrated with a graded series of ethanol, embedded in paraffin and sectioned at 4 µm.Liver sections from 15 gobies underwent hematoxylin-eosin (H&E), periodic acid Schiff (PAS), Perls' Prussian blue or Toluidine blue staining and were evaluated qualitatively using the image analysing system previously described.Aspects considered include parasite integrity, cyst structure and signs of immune reaction (degranulation).

Parasite parameters and statistical analyses
Parasitological descriptors are defined in Bush et al. (1997).Prevalence is the percentage of infected fish in a given sample, mean intensity is the mean number of parasites per infected host in a sample whereas mean abundance is the mean number of parasites per host be it infected or not.Fulton's condition factor (K) was calculated as W/L 3 where W is the weight of the fish in gm and L is the standard length in mm (Ricker 1975).Data were analysed using SAS release 9.4 (SAS Institute Inc., Cary, NC, USA).The mean abundance of N. tenellus was compared among localities and between host species through oneway analyses of variances (ANOVA) performed on rank-transformed data using a normal score transformation instead of standard ranks (Conover 1999).When more than two samples were compared, the ANOVA was followed by Tukey-Kramer multiple comparisons of means.Correlation analyses were performed to measure the association between parasite intensity and host-related variables (size, length, condition).When the assumption of linearity could be met (using raw or an arithmetic transformation of data), Pearson's coefficient was calculated.Otherwise, the association was measured using Spearman's rank correlation coefficient.The relationship between infection parameters and time since establishment of the round goby was examined using localities where the documented year of detection was established.

Results
Round gobies were infected by N. tenellus at 12 out of 14 localities sampled in the St. Lawrence River and at 2 out of 4 localities in the Great Lakes (Table S2, Figure 2).Overall, the parasite was present in 27% of the gobies examined at a mean intensity of 3.2.However, infection levels varied considerably from one locality to another (Figure 2), reaching highs at Beauharnois (BEA; prevalence: 85%; mean intensity: 3.7) and Île de la Couvée (ILO; prevalence: 93%; mean intensity: 6.6) where as many as 36 acanthocephalans were found in a single fish host.The native johnny darter was also a frequent host for N. tenellus (Table S2, Figure 3).The prevalence and intensity of infection in darters fluctuated among sites around an overall mean prevalence of 30% and a mean intensity of 2.7.At Île aux Ours (IAO), a site where the round goby has not yet established, the mean abundance of acanthocephalans in the johnny darter was significantly higher (P<0.0001)than at all other localities.Each fish examined from IAO was parasitized by N. tenellus with a mean intensity of 4.33 (Table S2).In comparison, N. tenellus was less prevalent and abundant in the tissues of logperch (Table S2, Figure 3).Only 3% of all logperch examined in this study were parasitized by this acanthocephalan species.As in the round goby, the prevalence and mean intensity were more elevated at Île de la Couvée (ILO), with values of 18% and 1.6 respectively.
Only one adult N. tenellus was found in the intestine of the round goby.In both the invasive host and the native fish hosts studied, the acanthocephalans remained at an immature stage (cystacanth).They were found encapsulated in the body cavity in association with the intestinal wall or mesenteries or, more frequently, within the liver (Figure 4).Except for one specimen, all acanthocephalans excysted from the logperch and the johnny darters showed no signs of degradation.In contrast, cystacanths regularly were found partly degenerated in the round goby (Figure 4).Cysts of degraded acanthocephalans were surrounded by mast cells and free granules (degranulation) were visible in the infected region (Figure 5).In several instances, the proboscis hard parts were the only remaining identifiable structures in the cysts (Figure 5d).The frequency of degraded acanthocephalans in round gobies fluctuated between 0% and 66% (47% on average) depending on locality (Figure 2) and was negatively correlated (P=0.005;r=-0.84) with the estimated time since gobies became established in a given locality (Figure 6).Indeed, the lowest levels of parasite degradation occurred at two localities in the St. Clair River area, where the round goby was first reported in the Great Lakes at the beginning of the 1990s.
Morphometric measurements of the three fish hosts are presented in Table S2.We found no significant relationship between the level of infection by N. tenellus and the size, weight or condition in the round goby and the two native species except at Île aux Ours (IAO) where the condition index of johnny darters significantly decreased with the intensity of infection (rho=-0.57,P=0.0068).

Discussion
Neoechinorhynchus tenellus is one of the few native parasites acquired by the exotic round goby since its introduction in the Great Lakes -St.Lawrence River basin in the 1990s (Gendron et al. 2012).Prevalence of infection by this parasite can reach as much as 93% locally and mean intensity as many as 7 worms per fish.However, results presented herein indicate that N. tenellus experiences high mortality in its novel host.Indeed, we found that, on average, half of the acanthocephalans encysted in the internal organs of gobies from the St. Lawrence River, were dead and degraded.In the Black Sea, the Sea of Azov and the other parts of its Eurasian range, the round goby is commonly parasitized by several marine, brackish and/or freshwater acanthocephalans, including species of the genus Acanthocephalus, Acanthocephaloides, Pomphorhynchus, Telosenti (Kvach (2002(Kvach ( , 2005(Kvach ( , 2006) ) and occasionally Neoechinorhynchus (N.rutili Miller, 1880) (Özer 2007).These helminths, which often dominate the parasite communities of gobies locally, either encyst in the viscera and remain as cystacanths and/or develop into gravid adults in the intestine then using gobies as definitive hosts (e.g.N. rutili; A. propinquus Dujardin, 1845).To our knowledge, there is no published mention of degradation or any other signs of premature death of cystacanths infecting gobies in their native habitats or other parts of their extended distribution in Europe (Kvach and Winkler 2011;Emde et al. 2012).
Parasite death in host tissue may go unnoticed, overlooked or be considered irrelevant to reporting unless it is the focus of research.In this study, cystacanth degradation was clearly a phenomenon unique to gobies whereas virtually all acanthocephalans recovered from two native paratenic fish hosts -the johnny darter and the logperchwere intact.Moreover, histological assessment revealed mobilization of mast cells and their degranulation around cystacanth remnants in gobies, suggestive of a vigorous host reaction to infection.Mast cells are inflammatory cell types involved in the innate immune response of fish to parasitic helminths, including acanthocephalans (Dezfuli et al. 2008).
Similar results were reported by Kelehear and Jones (2010) who studied the establishment of native larval nematodes (Spirurida) in the invasive cane toad (Rhinella marina=Bufo marinus Linnaeus, 1758) in Australia.These authors found evidence of a marked immune response in the host tissues, including cellular infiltration and fibrotic reaction around the parasites leading to calcification and parasite destruction.In contrast, nematode infection rarely provoked such exacerbated reaction in native frog hosts, presumably reflecting the long evolutionary association between them.In another study where metamorphs of cane toads were experimentally exposed to native lungworms (Rhabdias spp.), infective larvae were able to penetrate the skin and migrate through the body of the exotic toads but none reached the target tissue -the lungs -where they mature and reproduce (Nelson et al. 2015b).Most immature nematodes establishing in non-target tissues did not survive and many were found disintegrating and invaded by immune cells.
Using a combination of field survey and experimental infection, Paterson et al. (2013) also observed low establishment and survival of two generalist native trematodes in exotic salmonids, Salmo trutta (Linnaeus, 1758) and Oncorhynchus mykiss (Walbaum, 1792), in New Zealand.The authors concluded that the introduced fish were poor hosts for both native helminths and that they could contribute to reducing trematode transmission to the main native hosts over the long term.Our results also suggest that the exotic round goby is a poorly competent paratenic host for N. tenellus in the St. Lawrence River, relative to indigenous fish hosts fulfilling the same ecological function for the parasite.With the increasing density of round gobies in the St. Lawrence River (up to 6 gobies/m 2 : Kipp et al. (2012)) and their juveniles feeding heavily on ostracods (Gendron et al. 2012) -the intermediate hosts of N. tenellus -we suspect that this exotic fish could act as a sink or dead-end for a large number of these acanthocephalans.Assuming that a significant part of the available larval pool of N. tenellus ends up in the goby, premature death of cystacanths before gobies are consumed by suitable definitive hosts should result in a net loss of infective stages from the system, ultimately causing a decrease in the overall parasite population size.
This phenomenon, termed dilution effect, has been described in a number of host-parasite systems after changes in species composition and/or richness following events such as bioinvasion (Keesing et al. 2006;Johnson et al. 2008;Kelly et al. 2009b;Johnson and Thieltges 2010;Lettoof et al. 2013).For instance, cane toad invasion in Australia was associated with a general reduction in parasite burden in co-occurring native frogs (Lettoof et al. 2013).In New Zealand streams, Kelly et al. (2009b) found that the introduced brown trout diluted infection by helminths in native fish in direct relation to its abundance.Density-dependent dilution effects were reported in a few other epidemiological studies that were also replicated experimentally (Nelson et al. 2015a).Experiments with mussels raised in laboratory mesocosms revealed that non-indigenous hosts acted as decoys for the infective stages of native parasites, thus reducing the risk of indigenous hosts becoming infected, with greater reductions of infection levels at higher invader densities (Thieltges et al. 2008).
The round goby now occurs in a variety of habitats throughout the St. Lawrence River and the Great Lakes.
Localities not yet colonized by the species are few, which complicated the comparison of N. tenellus infection in indigenous hosts between habitats with and without gobies.Only one of our sampling sites turned out to be goby-free.Interestingly, the prevalence and mean intensity of N. tenellus in johnny darter at that locality was more than double those at all other sites where darters co-occurred with gobies.This observation suggests that the introduction of an invasive incompetent host has resulted in native paratenic hosts becoming less parasitized.However, more extensive surveys of the abundance of N. tenellus in indigenous fish including yet-to-be invaded areas in tributaries of the St. Lawrence River, should be conducted before concluding that the round goby has induced a dilution effect.
Further research should also assess the infection levels in the obligatory hosts of N. tenellus including its definitive hosts.Given that definitive hosts of N. tenellus are piscivorous fish such as the northern pike and the walleye (Amin and Muzzall 2009), a significant portion of their infections probably is acquired through the consumption of infected fish and less through direct ingestion of the invertebrate intermediate hosts (ostracods), making paratenic hosts key components in the life cycle of this parasite.For a reduction in N. tenellus infection to occur in definitive hosts, the increase in the abundance of gobies should be paralleled with a decrease in the abundance of native paratenic hosts.In the Great Lakes, darters and other benthic fish were found to decline significantly following gobies invasion presumably due to competition for food and space (Lauer et al. 2004) and the same appears to be occurring now in the St. Lawrence River.In our seines, gobies typically outnumbered johnny darters and logperch (following a ratio of 10 to 1) in areas where these two indigenous percids used to be abundant (data not shown).Therefore, it appears that competent indigenous hosts (johnny darters), which play an important role in the transmission of N. tenellus to its definitive host, are being replaced with a poor exotic host (round gobies).The net influence of gobies on the infection dynamics of definitive hosts (dilution or amplification) will very much depends on the relative rates of predation of gobies vs other paratenic hosts along with the specific survival rates of cystacanths in gobies, which is difficult to predict from existing information and will need to be addressed in further studies.
Parasite dilution may represent one of the few benefits conveyed by exotic species to invaded communities (Kopp and Jokela 2007;Kelly et al. 2009b).Here, a reduction in the intensity of infection by N. tenellus driven by the round goby could hypothetically improve the health of native fish competitors.Generally speaking, acanthocephalans are not responsible for major vertebrate host die-offs, although they can on occasion reach densities at which they cause epizootics (Kennedy 2006).Nonetheless, they regularly induce local damage to their fish hosts manifested as inflammation and accumulation of fibrous tissues at the site of infection, as well as gut perforation and damage to internal organs as the parasite migrates toward the sites of encystment in paratenic hosts (Dezfuli et al. 2008).Intestinal occlusions may also occur in highly infected definitive hosts and result in significant weight loss.A few studies have linked infection by acanthocephalan to a reduction in host body condition (Kennedy 2006;Thilakaratne et al. 2007), an effect seen here in johnny darters at the gobyfree locality.At that site, N. tenellus dominated the parasite fauna of johnny darters, accounting for 50% of the total number of helminths infecting their tissues, whereas the acanthocephalan represented less than 10% of johnny darters' parasite load at sites where they coexisted with round gobies (A.D. Gendron and D. Marcogliese, unpublished data).
This novel host-parasite relationship likely will evolve over the years, most probably toward a reduction in the defense mounted by the exotic host against the native parasite.Our results suggest that this is already occurring in the Great Lakes, where the degradation rate of cystacanths in gobies is significantly reduced compared to what is observed in more recently established populations in the St. Lawrence River.Although this needs to be confirmed by more extensive surveys, a rapid co-adaptation would not be surprising considering the circumpolar distribution of Neoechinorhynchus and the fact that other species of this genus were found to infect the round goby in its European native range (Özer 2007).As it progressively develops into a more competent paratenic host, the influence of the round goby in the transmission of N. tenellus in the Great Lakes -St.Lawrence basin might eventually switch from a putative dilution effect to a spillback effect.Round gobies could then facilitate the dissemination of that native parasite and increase the intensity and prevalence of infection in its main predators -saugers, walleyes and northern pikes (Reyjol et al. 2010) -which are known definitive hosts of N. tenellus (Amin and Muzzall 2009).

Figure 2 .
Figure 2. Infection of round gobies (Neogobius melanostomus) by Neoechinorhynchus tenellus in the Great Lakes -St.Lawrence basin.Locations shown are those where at least one goby was infected by the parasite.A. Bars reflect the intensity of infection expressed as the mean number of acanthocephalans -intact and degraded -per infected fish ± SEM.B. Prevalence is the percent fish parasitized by N. tenellus.

Figure 3 .
Figure 3.Comparison in the level of infection by Neoechinorhynchus tenellus between the round goby (Neogobius melanostomus) and the two native fish hosts, the johnny darter (Etheostoma nigrum) and the logperch (Percina caprodes).A. Prevalence: each bar displays the percentage of fish parasitized by N. tenellus.B. Intensity of infection is expressed as the average number of N. tenellus among infected fish.Error bars shown are SEM.The round goby was found at all locations but one (IAO).

Figure 4 .
Figure 4. Death and degeneration of cystacanths in tissues of the exotic round goby (Neogobius melanostomus) (A) in comparison to the native johnny darter (Etheostoma nigrum) (B) and logperch (Percina caprodes) (C).Stacked bars cumulate the mean number of whole (intact) and degraded cystacanths for each fish host (all locations pooled).Error bars refers to SEM.