The non-indigenous parasitic copepod Neoergasilus japonicus ( Harada ) ( Cyclopoida ) from central Mexico : the earliest invasion in continental America

The Eastern Asian ectoparasitic copepod Neoergasilus japonicus (Harada, 1930) is a widely spread species with a high prevalence on many freshwater teleosts. It was recorded for the first time in Europe 45 years ago and continued its invasion to new geographic regions. Its presence in North America was first detected in 1993, followed by only two other continental records. In this work we present an illustrated record of N. japonicus from plankton samples collected during 1990 in a microreservoir of central Mexico. We confirm that this parasitic copepod was already present in continental America at least 3 years before previously known. This is the third published record of this Asian copepod in the Neotropical region. Its occurrence in Mexico is attributed to different events of introduction by human agency. This report supports the notion that N. japonicus is more widespread in Middle America than previously thought and it is intended to aid in the historical monitoring of the invasion of this species.


Introduction
Ergasilids are ectoparasitic copepods infecting different freshwater, estuarine, and coastal teleost families in both natural environments and in culture conditions (Nagasawa and Inoue 2012).Among the 27 genera currently contained in the cyclopoid family Ergasilidae, the genus Neoergasilus is easily distinguished for a thumblike spine on the outer margin of the second exopodal segment of the first legs (Boxshall and Halsey 2004).Neoergasilus japonicus (Harada, 1930) is a widespread species that has been recorded from numerous freshwater fish species mainly in eastern Asia, Europe, and North America (Hudson and Bowen 2002).The spread of this Asian species has been monitored since it was first recorded in Taiwan in the 1920s and was successively reported from other parts of the continent, including China and Russia.It first reached Europe in the 1960s and the latest continental records include Germany, Italy, and Turkey (see Table 1).This copepod was eventually introduced to the Americas, and first reported from Cuba in 1985 by Prieto et al. (1985).The available data also suggest that it was first recorded in North America eight years later (1993), when it was found in Alabama, in the United States (Hayden and Rogers 1998).Shortly after this first record, it was observed in the Great Lakes area (Hudson and Bowen 2002;Kipp et al. 2013).
This species was reported from south-east Mexico as a parasite of three endangered teleost species; it is the southernmost record of N. japonicus in the Americas (Suárez-Morales et al. 2010).During a survey of the aquatic biota of the state of Aguascalientes in central Mexico, several specimens of N. japonicus were recorded from plankton and littoral samples obtained in a microreservoir during 1990, that is, 3 years before the presumed first record of this parasite in North America (Hayden and Rogers 1998).We provide original illustrations of the specimens examined.The relevance of this record is discussed in terms of the distribution of this parasite in Mexico and the Americas.Hudson and Bowen (2002) and Knopf and Hölker (2005)

Material and methods
A total of 5 adult females of N. japonicus were recovered from the examination of more than 800 plankton samples collected from more than 450 localities representing several freshwater environments (i.e.lakes, permanent and ephemeral ponds, large and small reservoirs) in the state of Aguascalientes, central Mexico.The only site where this species was collected was Los Arquitos reservoir (21°55′28″N; 102°23′25″W), visited September 5, 1990.The specimens were fixed and preserved in a 4% formalin solution.Some specimens were dissected under the stereomicroscope in order to perform the taxonomical examination.Selected specimens were deposited in the collection of Zooplankton held at El Colegio de la Frontera Sur (ECOSUR), Unidad Chetumal (ECO-CHZ), Quintana Roo, Mexico.
The Los Arquitos reservoir is located at 1929 m above sea level in the municipality of Jesús María in the state of Aguascalientes, central Mexico.This is a small old reservoir, built in 1900-1901, supplied by the Rio Moricinique, with a total capacity is of 0.54 million m 3 .

Morphology
Total body length average of adult specimens =0.70 mm (range of adult specimens: 0.70-0.73mm, n=2) measured from anterior end of cephalothorax to posterior margin of caudal rami, excluding caudal setae.Length of juvenile specimens: 0.63-0.68mm, n=4.The general morphology of these specimens, including the mouthparts and swimming legs, as described by Hayden and Rogers (1998) and Kim and Choi (2003) (see Figures 1, 2).

Remarks
When compared with values of other adult populations of N. japonicus, our specimens' size average is within the overall range known for this species: 0.70-0.75mm (Kim and Choi 2003;Suárez-Morales et al. 2010).The central Mexico individuals have a size range closest to that of specimens from southeast Mexico (0.67-0.71 mm), but also to those from the Great Lakes area (0.71 mm) (Hudson and Bowen 2002).The Mexican populations are among the smallest known (Suárez-Morales et al. 2010).The specimens from Alabama (0.74-0.79 mm) (Hayden and Rogers 1998), Korea (0.75 mm) (Kim and Choi 2003), and Turkey (0.76 mm) (Soylu and Soylu 2012) are the largest recorded for this species.

Hosts
Unknown, but the reservoir is inhabited by two introduced fish species, the black tilapia Oreochromis mossambicus (Peters, 1852) and carp Cyprinus carpio Linnaeus, 1758.

Discussion
Most of the research about this species deals with parasitic infection and its effects on natural and cultured fish populations (Nagasawa and Inoue 2012; Nagasawa and Obe 2013), but this ergasilid is also important in other aspects.The gravid females of N. japonicus, unlike most other ergasilids, are frequently found as part of the plankton community (Pónyi and Molnar 1969;Hayden and Rogers 1998;Alfonso and Belmonte 2010).In addition, pre-adult individuals are freeliving, thus increasing the relevance of this species in the water column biota (Urawa et al. 1991).Tuuha et al. (1992) suggested that direct parasitological surveys may not reveal the presence of this parasite as they are usually loosely attached on the fins of their hosts.This small, inconspicuous species can easily be overlooked by casual examination of parasitized fish (Hudson and Bowen 2002) and there is a limited availability of taxonomic expertise and identification keys.These factors suggest that this parasite is probably more widespread than currently recognized.Parasitological surveys of freshwater fish should be complemented with examination of plankton samples, particularly in detecting the occurrence of this small, widespread parasitic copepod.
Neoergasilus japonicus has been recorded from a wide variety of freshwater fish families worldwide, including cyprinids, percids, centrarchids, ictalurids, and cichlids (Pónyi and Molnar 1969;Lescher-Moutoué 1979;Mugridge et al. 1982;Tuuha et al. 1992;Hayden and Rogers 1998;Hudson and Bowen 2002;Knopf and Hoelker 2005;Suárez-Morales et al. 2010).Neoergasilus japonicus can easily change hosts in freshwater systems (El-Rashidy and Boxshall 2009).It is also able to parasitize a wide variety of fish even in limited geographic areas (Kim and Choi 2003) and they can use both native and alien fish species as hosts (Suárez-Morales et al. 2010; Nagasawa and Uyeno 2012).These adaptive features have probably accelerated their spread worldwide.
It is a well-known fact that aquaculture is one of the most relevant means for species introductions (Taylor et al. 2001).Living non-indigenous fish, mainly cyprinids, were probably the vector by which N. japonicus reached Mexico, but this parasite is currently infecting native, endangered teleost species as well (Suárez-Morales et al. 2010).The occurrence of this species in southeast Mexico (Suárez-Morales et al. 2010) suggests an anthropogenic introduction as this is an area with intense aquacultural activity (Zambrano and Macías-García 1999).The present results suggest that N. japonicus was first introduced in central Mexico and this was probably also a result of human agency related to aquaculture.In the area of Aguascalientes, central Mexico, many reservoirs and microreservoirs have long been used for fish culture and at least 13 introduced teleost species have been recorded in the state (Martínez and Rojas 1996).
The expansion of the parasite distributional range is a by-product of the rapidly increasing rates of fish introductions related to aquacultural activities worldwide (Taylor et al. 2001;Reid and Hudson 2008;Gozlan et al. 2010).Neoergasilus japonicus has been known to disperse over long distances rather quickly, spreading across Europe in 20 years and then moving towards the Americas over a span of less than 10 years (Hanek 1968;Hudson and Bowen 2002; Table 1).Our finding of this species in central Mexico from samples collected in 1990 reveals that it was already present in the continent at least 3 years before Hayden and Rogers' (1998) record, at that time recognized as the first known record in continental America.There is an older American record, from Cuba (Prieto et al. 1985), where it was recorded from different teleost species.The species remained unrecorded from adjacent Caribbean islands and from the American mainland for nearly 5 years before it was detected (this survey) on the continent.During the 1960s and 1980s, Cuba received a strong aquacultural influence from the Soviet Union, including the introduction of different fish species (Coto 2006).In Russia N. japonicus was recorded in the 1950s (Dogiel and Akhmerov 1952;Gussev and Smirnova 1964) so it is possible that the introduction of N. japonicus in Cuba resulted from this process.Because of the isolation of Cuba, it is unlikely that this invasion was a stepping stone for further introductions in the Americas.The Mexican records are likely the result of independent colonization events and are more likely to represent intermediate steps of the spreading of this copepod in continental America, where it was probably introduced by aquaculture-related activities.
Currently, the southernmost record (16°N) of this species in the Americas is from the state of Chiapas, in southern Mexico.According to Suárez-Morales et al. (2010) it is expected that N. japonicus will eventually advance to other areas of the Neotropical region.It is probable that it is already present but undetected in these areas.Fish and plankton samples from the Neotropical region including Central and South America should be examined with more detail in order to determine the true distribution of this copepod.This illustrated report is intended to aid in the historical monitoring of the spreading process of this parasitic species.

Figure 3 .
Figure 3. Chronological sequence and geographic distribution of records of N. japonicus in the Americas.