Salsuginus seculus (Monogenea: Ancyrocephalidae) and Gyrodactylus gambusiae (Monogenea: Gyrodactylidae) newly recorded infesting Gambusia affinis (Teleostei: Poeciliidae) from New Zealand

The North American western mosquitofish, Gambusia affinis (Baird and Girard, 1853), has been introduced into a number of countries around the world, primarily for the control of mosquito populations. Gambusia affinis is known to harbour several monogenean parasites, which have the potential to be co-introduced to new regions. However, despite the broad non-native distribution of this fish species, their monogenean parasites have been reported from a relatively small number of locations. Parasitological investigations have identified very few freshwater monogenean parasites from freshwater fish in New Zealand. Two monogenean parasites were recorded infesting G. affinis in New Zealand; Salsuginus seculus (Mizelle and Arcadi, 1945) (a new genus to New Zealand) on the gills and Gyrodactlyus gambusiae Rogers and Welborn, 1965, on the gills and surface of the fish. Despite the broad distribution of G. affinis , S. seculus and G. gambusiae have been reported in few countries due, perhaps, to lack of research effort on monogeneans, their small size, enemy-release, or a combination of these factors. Based on the diversity of monogeneans in other countries that are found associated with fish with non-native populations in New Zealand, it is likely that many unrecognised non-native monogeneans are established in New Zealand’s freshwater ecosystems alongside their fish hosts. Further, the number of introduced monogeneans is likely to be even higher than is currently appreciated when considering non-native fish in the aquarium trade and other introduced freshwater fauna, such as terrapins.


Introduction
The western mosquitofish, Gambusia affinis, has spread globally from its native distribution in south-eastern USA and northern Mexico (Ling 2004;Pyke 2008).In attempts to control mosquitoes it has been introduced into other regions of the USA and numerous other countries (Seale 1917;Krumholz 1948;Ling 2004;Jourdan et al. 2021).For example, following initial unsuccessful introductions, G. affinis sourced from non-indigenous populations in Hawaii was established in New Zealand at the Auckland Botanical Gardens in 1930 (now known as the Auckland Domain; McDowall (1990)).Despite initial confidence in the ability of G. affinis to control mosquitoes (Washino 1969), subsequent research has contested the degree of its efficacy (Kramer et al. 1987), and the species is now widely regarded as a harmful invader in many of the ecosystems to which it has been introduced (Rupp 1996;Pyke 2008;Tsang and Dudgeon 2021).
The global spread of non-indigenous fishes provides the opportunity for their parasites to be dispersed with them and thus, expand their distributions.This is illustrated by the establishment of various parasite species with their non-native fish hosts in Hawaii (Font 2003).Similarly, the introduction of grass carp, Ctenopharyngodon idella (Cuvier & Valenciennes, 1844), into New Zealand required treatment to remove parasites following their post-border detection (Edwards and Hine 1974).
Monogenea is a diverse class of Platyhelminthes most commonly found infesting the surfaces of fish, including the gills and fins, and sometimes, but not always, causing disease (Whittington and Chisholm 2008).Most monogeneans are oixenous; that is, they have an obligate relationship with a single host species (Whittington and Chisholm 2008).One monogenean species, Salsuginus seculus, infesting the gills of G. affinis is a member of the family Ancyrocephalidae.Since the initial description of S. seculus in 1945, there have been numerous new distribution records of this species.It has been recorded from G. affinis in various states in the USA, including Texas (Nowlin et al. 1967;Meade and Bedinger 1972), California (Mizelle andArcadi 1945), Oklahoma, Kansas, Arkansas (McAllister et al. 2015), Louisiana (Duobinis-Gray and Corkum 1985), and Hawaii (Font 1997), with the latter being the origin of the New Zealand G. affinis populations.Outside of the USA, S. seculus has been reported from the Bahamas (Hanek and Fernando 1972), though this has been contested due to the absence of G. affinis there, with the related G. manni Hubbs, 1927 and G. puncticulata Poey, 1854 occurring there instead (Nitta and Nagasawa 2014).It has also been reported in Japan by Nitta and Nagasawa (2014), who noted that in spite of parasitological investigation of G. affinis in New Zealand, S. seculus was yet to be located in that country.A second species known to infest G. affinis is a member of the Gyrodactylidae family, Gyrodactylus gambusiae.First observed in 1965 in Florida, little literature exists for this species (Rogers and Welborn 1965;Rubio-Godoy et al. 2010).Distribution records are limited for both species despite the global range of G. affinis.Here, new distributional records of S. seculus and G. gambusiae, both infesting G. affinis in New Zealand, are reported.

Materials and methods
From April 2022 to March 2023, G. affinis were collected from Knighton and Oranga Lake, two interconnected ponds on the University of Waikato campus, Hamilton (37°47′08.7″S;175°18′53.3″Eand 37°47′12.4″S;175°18′57.2″E,respectively).Fish sampling was carried out by placing minnow traps around the edges of the ponds and retrieving them approximately 24 hours later.Upon collection, G. affinis were transferred to the laboratory and examined immediately.For each location, the two lakes and the stream connecting them, 50 fish specimens were examined for parasites.Hand netting was also utilised to capture more fish if the traps had failed to capture sufficient fish to meet an ideal minimum of 50 individuals to examine.Fish were humanely euthanised using an anaesthetic overdose of benzocaine and promptly examined for parasites to ensure that fresh material was being observed.Following examination of the outer surface of each fish, the gills were extracted using forceps and examined using an Olympus SZ40 dissecting microscope.When a parasite was observed, it was removed from the body or gills using fine needles.The live parasite, immersed in water, had a coverslip placed on it and was observed for photography and morphometric identification using a DMRE Leica microscope.Photographs and measurements were taken of monogeneans infesting G. affinis on 29 and 31 August, 28 September, 5 October 2022, and 7 January, 25 and 30 March 2023.By examining freshly euthanised fish, parasites were located on account of their movement.Further, examining live parasites helped to mitigate contraction of their bodies as may happen when specimens are fixed in alcohol (Nitta and Nagasawa 2014).For S. seculus, measurements were taken of the length and width of the body, the haptor size, and structures of the haptor, including the hamuli (total length, deep root length, superficial root length, blade length), as well as the penis and accessory piece.Additionally, the number of marginal hooks was recorded.These measurements were recorded according to Murith and Beverly-Burton (1985), who set out a process for measuring sclerotised structures of Salsuginus species.These measurements were compared with the species' original description (Mizelle and Arcadi 1945) and a subsequent distributional record in Japan (Nitta and Nagasawa 2014) to confirm the species' identity.Measurements for G. gambusiae, and confirmation of the species, were made following the species' description in Rogers and Welborn (1965).Measurements for G. gambusiae included the length and width of the body as well as the haptor size, hamuli lengths (root lengths were not taken as this species does not have structures like those of S. seculus), bar widths and lengths, and number of marginal hooks.Measurements were taken for those structures that were clearly visible without chemical staining of the specimen, and which are the most relevant for the identification of monogeneans.

Discussion
The morphologies of the two parasite species reported here were overall consistent with the descriptions reported in the literature for S. seculus (Mizelle and Arcadi 1945;Nitta and Nagasawa 2014) and G. gambusiae (Rogers and Welborn 1965).Further, being found to infest G. affinis is also a key diagnostic feature that confirms the identity of these species; as monogeneans are highly host specific (Poulin 1992;Whittington et al. 2000), identification of the host species aids their identification greatly.Of particular note, G. gambusiae specimens were found not only on the fins, but also on the gills of G. affinis.Rogers and Welborn (1965) reported this species to infest the fins and body of the fish only.However, examination of the gills was not mentioned in their methodology.Since their study, no literature has been published to note the greater range of attachment on G. affinis and as such these observations are the first to note the occurrence of this monogenean on the gills.That so few G. gambusiae were observed is also noteworthy.Why G. gambusiae has low prevalence in the current study may be due to seasonal changes in abundance, with sampling not coinciding with peak infestation.Perhaps more likely, low infestation rates may be due to this species occurring at low frequencies on G. affinis in general.Studies on the populations of other gyrodactylid species have found greater numbers and higher infestation prevalence than seen here (e.g., Scott and Anderson 1984;Mo 1992).The literature on G. gambusiae, in particular, is not extensive, not extending greatly beyond its initial description, with no studies existing on their prevalence, intensity, or population dynamics.This may indicate that their prevalence and abundance are low globally, and as such, they are rarely sighted.
These new distributional records indicate that S. seculus and G. gambusiae have been previously overlooked in New Zealand.It is likely that these parasites accompanied G. affinis when it established in the Auckland Botanic Gardens in 1930, from mainland USA via Hawaii.As such, they have likely existed there for some 90 years.Nitta and Nagasawa (2014) noted that in spite of intense parasitological investigation of G. affinis in New Zealand by Zhang (2012), S. seculus had not been found there.Indeed, the investigation conducted by Zhang (2012) found no parasites on or in G. affinis at all.However, only a small subset of those collected were dissected and their gills inspected by this author, with the rest digested with an HCl and pepsin solution to search for internal parasites.Additionally, the bulk of sampling in that study was undertaken over the winter months, when parasite numbers might have been reduced.This makes it likely that these monogeneans would have been missed by that author.
The finding of these parasites leaves the question as to how many more non-indigenous monogenean species are yet to be found in New Zealand.To date, around 5000 monogenean species have been described globally; however, the number of species overall may exceed 25000 (Kuchta et al. 2020).As of 2009, only 25 monogenean species had been recognised in New Zealand, including only three from freshwater fishes (Hine et al. 2000); the rest were all from marine fish species (Johns et al. 2009).Ctenopharyngodon idella possessed two of these freshwater monogenean species (Dactylogyrus ctenopharyngodonis Achmerow, 1952 and Gyrodactylus ctenopharyngodontis Ling, 1962), and was the only non-native freshwater fish reported to host monogeneans (Hine et al. 2000).Prior to release in New Zealand, grass carp were kept in tanks and were treated for parasites, which they were reportedly laden with.Once it was deemed that these infestations were under control, they were released into outdoor ponds (Edwards and Hine 1974).However, subsequent research has not confirmed whether these parasites had been definitively eliminated.The third monogenean reported from freshwater fish in New Zealand was an undescribed Gyrodactylus species on the native kōaro, Galaxias brevipinnis Günther, 1866.There have been no apparent additions since this time.These facts demonstrate that Monogenea have been understudied in New Zealand, not just those from non-native fishes, but in general.There are 21 established nonindigenous freshwater fish species in New Zealand, each of which is likely to possess further unreported monogeneans.Parasites for a number of individual fish species are listed in "Parasites of North American Freshwater Fishes" by Hoffman (1999); from these lists, monogenean numbers for some non-native freshwater fish species living in New Zealand waters are as follows: 44 species of monogeneans from Carassius auratus (Linnaeus, 1758), 32 from Cyprinus carpio Linnaeus, 1758, 1 from C. idella (distinct from the 2 reported by Hine et al. (2000) from New Zealand), 4 from G. affinis (including the two reported here), 7 from Ameiurus nebulosus (Lesueur, 1819), 6 from Poecilia reticulata (Peters, 1859), 9 from Onocorhyncus mykiss (Walbaum, 1792), and 6 from Salmo trutta (Linnaeus, 1758).Together, this totals 111 monogenean species found infesting these fish species.This represents a large number of monogenean species with the potential to have entered New Zealand along with their fish hosts, which have not been reported.Even more introduced monogeneans may yet to be reported in New Zealand when organisms in the freshwater aquarium trade are considered, as well as animals such as the red-eared terrapin, Trachemys scripta elegans (Wied-Neuwied, 1839), which are kept as pets but are commonly released into the wild.In Japan, for example, red-eared terrapin are known to carry two introduced monogeneans (Oi et al. 2012).The establishment of non-native parasites in New Zealand freshwaters has been noted as an area that is understudied, and not just for monogeneans (Lane et al. 2022).Not only are there likely to be more monogenean species introduced along with their hosts, previously unrecorded in New Zealand, it is also likely to be the case for other geographic regions.
The number of countries G. affinis has been introduced to far exceeds the number of countries from which these monogenean parasites have been recorded.It is possible that monogeneans, in particular, have gone unnoticed on freshwater fish due in part to a lack of research effort on this taxonomic group.Nevertheless, the enemy-release hypothesis (Keane and Crawley 2002) predicts that host species may be liberated from their parasites, and has been hypothesised as a reason why parasites have not been observed infesting G. affinis in New Zealand (Zhang 2012).Indeed, loss of parasites in this manner may have prevented other species entering New Zealand, explaining the lack of records for those taxa.Enemy-release may be responsible for G. affinis in some countries not being infested with S. seculus and G. gambusiae.In New Zealand, however, this is not the case.The mosquitofish established in New Zealand were first introduced from Texas into Hawaii (Seale 1917), and then subsequently the Hawaiian fish were introduced into New Zealand in 1930 (McDowall 1990).This stepwise introduction would presumably increase the chances of the parasites being lost along the way, yet this has not happened for these species.Gyrodactlyus gambusiae being found in New Zealand indicates that this species should be found infesting G. affinis in Texas and Hawaii, where G. affinis introduced into New Zealand originated from.However, this species has not been reported from either of these areas.When it comes to establishing populations in new areas, monogeneans may have a great advantage over other parasite groups by virtue of their life cycle.Many other parasites require multiple hosts to complete their life-cycle, which would preclude them from establishing populations in new countries, unless it coincided with the introduction of multiple host animals.Monogeneans, on the other hand, can enter countries with their single host, which allows them to complete their life-cycles as normal.As such, both S. seculus and G. gambusiae will also most likely be found in many other countries where G. affinis exists.This fact also illustrates the importance of parasitological surveys, as it is evident that enemy-release may not apply equally to all parasites and their hosts.As monogeneans do not typically cause disease in their hosts (Whittington and Chisholm 2008), it is unlikely that introduced host populations would see decreases in death rates, or increases in birth rates or population growth rates, on the basis of becoming free of these parasites.
Changes in climate and the establishment of non-native parasites are likely to have diverse ecological ramifications that may be revealed with more research conducted in this area.The record of S. seculus and G. gambusiae living on G. affinis in New Zealand, alongside estimates of a great many more unreported monogeneans, highlights a lack of attention to invaders in this taxonomic group.Further study in this area will no doubt highlight a much greater number of freshwater parasite species in New Zealand than are currently recognised in the scientific literature.