First record of Japanese Mystery Snail Cipangopaludina japonica ( von Martens , 1861 ) in Texas

Two Cipangopaludina snails were discovered in Harris County, Texas, USA, during routine fieldwork in October 2015. Dissection yielded one male and one female containing 52 offspring in her brood pouch. Phylogenetic analysis of the cytochrome c oxidase subunit I (COI) gene confirmed both individuals to be Cipangopaludina japonica (von Martens, 1861). This is the first distribution record of C. japonica in Texas. Non-native invasive snails, such as C. japonica, compete with native species and may serve as reservoirs for parasites, prompting the need for increased diligence in monitoring public waterways.

The Japanese Mystery Snail, Cipangopaludina japonica (von Martens, 1861) (Gastropoda: Viviparidae), is a freshwater snail native to eastern Asia (Clench and Fuller 1965;Jokinen 1992) that was first recorded in the United States in the late nineteenth century in California (Wood 1892;Hannibal 1911).The United States Geological Survey's Nonindigenous Aquatic Species Data base (USGS 2016; Appendix, Table A1) only contains records of C. japonica populations for 15 states, but several other state natural resource agencies (DNRs) report populations of C. japonica (Figure 1) that do not appear in the national database (Appendix, Table A2).Cipangopaludina japonica is closely related to, and often confused with or misidentified as, the more widespread and invasive Chinese Mystery Snail, C. chinensis (Gray, 1834), another species that the Asian food markets also brought into the United States in the early 1890s (Jokinen 1982).Cipangopaludina chinensis occurs extensively across the United States with the highest densities found in the Great Lakes and the northeastern United States (Figure 2; Appendix, Table A1).
Accidental or deliberate introductions (Kipp et al. 2016) of these mystery snails into new habitats pose ecological concerns as both species have the ability to reach high population densities (Chaine et al. 2012), compete with native species (Solomon et al. 2010) and alter aquatic ecosystems (Johnson et al. 2009).Like many other freshwater snails, these viviparids also act as intermediate hosts for parasites that pose serious health risks, including the nematode Angiostrongylus cantonensis (Lin and Chen 1980) and Echinostoma cinetorchis, a species of human intestinal fluke (Park et al. 1997;Chung and Jung 1999).The combined threats mystery snails pose to human and environmental health coupled with their invasive history (Wolfert and Hiltunen 1968;Soes et al. 2011) often land both C. chinensis and C. japonica on invasive species watch lists (Kipp et al. 2016).
Both species are also frequently placed in the genus Bellamya versus Cipangopaludina based on morphological features (Smith 2000; Bury et al. 2007), but debate exists on whether this represents a true synonymy (Van Bocxlaer and Strong 2016).The Integrated Taxonomic Information System does not recognize the nomenclature of Bellamya (Havel et al. 2014), but several recent studies chose to use this name for the genus over Cipangopaludina (Smith 2000;Solomon et al. 2010;Soes et al. 2011).Consequently, the taxonomy of both C. chinensis and C. japonica remains unclear as many scientific names are improperly synonymized or used inconsistently (Global Invasive Species Database 2016).
Based on an extensive comparative study of internal morphology, Van Bocxlaer and Strong (2016) argued for the validity of the genus Cipangopaludina to better reflect evolutionary relationships and further called for rigorous molecular studies to resolve the relationships of C. chinensis, C. malleata and C. japonica.Given the similarity of C. japonica to C. chinensis, along with the comparative limited information on C. japonica, we provide background for both species.
Based on morphometric analysis, Cipangopaludina japonica lacks external morphological characters that provide consistent reliable field identifications of sex (Van Bocxlaer and Strong 2016).Field identification of C. japonica and C. chinensis may also prove difficult, as Smith (2000) suggested that C. japonica differs the most from C. chinensis with respect to the embryonic shell.Shells with more acute spires and finer carinae similar life histories and occupy equivalent ecological niches (Jokinen 1982).Both taxa commonly occur in lentic ponds and lakes that have sandy to muddy substrates.Both species feed non-specifically on benthic organic matter (Jokinen 1982).In a study that altered water levels in rice paddy soils, Kurihara and Kadowaki (1988) found that submerged soils provided a suitable habitat in which C. japonica consumed primarily detritus and successfully reproduced.With a generalist diet, long life spans and high fecundity, populations of mystery snails may grow quickly (Stephen et al. 2013).
Based on estimates, female mystery snails may live for approximately five years, with a shorter life expectancy for males of three to four years (Jokinen 1982;Van Bocxlaer and Strong 2016).After their first year, females become reproductively active and release live offspring.Reproduction occurs continuously throughout spring to early summer when females carry embryos that they suggest C. japonica over C. chinensis (Clench and Fuller 1965;Jokinen 1982;Park et al. 1997;Smith 2000).However, those traits only have utility within a small size range (35-45 mm) in both species (Smith 2000).Smith ( 2000 identification and initially froze them at -20°C before dissection to determine sex and whether the female carried any offspring.We preserved foot tissue and offspring found in the brood pouch in 70% ETOH.The shells and offspring are stored as voucher material at the North Carolina Museum of Natural Sciences in Raleigh, North Carolina (NCSM 101740 for the male and NCSM 101741 for the female and brood).
To confirm our morphological identification of the two snails, we sequenced the mitochondrial COI gene to ascertain species identity (Hebert et al. 2003).We ex tracted total genomic DNA from a small piece (~1 mm 2 ) of unpigmented foot tissue using a NucleoSpin® extraction kit (Macherey-Nagel, Pennsylvania, USA).Following the conditions used in Hayes et al. (2009), we amplified a 709 bp fragment of the COI gene by polymerase chain reaction (PCR) using the standard invertebrate primer pair LCO1490 (5ʹ-GGTCAACAAATCATAAAGATATTGG) and HCO2198 (5ʹ-TAAACTTCAGGGTGACCAAAAAATCA) (Folmer et al. 1994).The Institute for Cellular and Molecular Biology at the University of Texas at Austin performed Sanger sequencing in both directions using an ABI BigDye Terminator v.3.1 Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems, Inc.) on an ABI 3730 XL capillary sequencer following the manufacturer's instructions.Using MUSCLE ver. 3.6 (Edgar 2004), we aligned our sequences with Genbank sequences from 47 other Cipangopaludina spp.(Appendix, Table A3) and three Viviparus spp. as outgroups (n = 51 total sequences).We used jModelTest ver.2.1.7(Darriba et al. 2012) to determine the best fit model based on Akaike Information Criterion (AIC) and estimated phylogenetic relationships under maximum likelihood using the TrN+G model in PhyML ver.3.0 (Guindon et al. 2010).Node support was estimated with 1000 bootstrap replicates (Felsenstein 1985).We edited and visualized the consensus tree in FigTree 1. 3.1 (Rambaut 2009).
We identified the two snails as a species of nonnative Cipangopaludina based on shell shape and size.All native viviparids in the U.S. are smaller and possess different shell forms than C. japonica and C. chinensis (Burch 1982).Based on the acute spires and more ovate apertures (Lu et al. 2014;Hirano et al. 2015), we assigned both specimens putatively to C. japonica.Dissection revealed one male (Figure 3) and one female (Figure 4) with 52 offspring in her brood pouch.Offspring showed slightly depressed embryonic whorls but not enough to be indicative of C. chinensis.The female shell (62.8 mm length and 43.9 mm width) was larger than the male (53.1 mm length and 38.0 mm width) and both were larger and had thinner shells than most native viviparids (Burch 1982).
For genetic analysis, we edited and assembled our COI sequences in Geneious 7.1 (Biomatters Ltd.).Sequences from the male and female were 658 bp in length and 100% identical (GenBank accession KX259343).Phylogenetic analysis yielded a single tree (log likelihood = -2106.8749,model = TrN + G) clustering both snails in a clade with other C. japonica specimens (Figure 5), confirming our field identification.The C. japonica clade was well supported (> 50%) and clearly separate from specimens of C. chinensis.
Our collection of two individual Cipangopaludina japonica represents the first record of this species in Texas (USGS 2016).To our knowledge, this work presents the first published sequence of the COI gene for C. japonica obtained from a United States population.The USGS Nonindigenous Aquatic Species Database lists collections in 15 states but our state-by-state examination of DNR websites also discovered a number of states that reported introductions of C. japonica not present in the national database.Lack of centralized reporting raises questions as to the closest source populations for introductions.Cipangopaludina chinensis introductions appear better documented, including a widespread distribution within the Saint John River in New Brunswick, Canada (McAlpine et al. 2016).
The closest known collection of C. japonica to our field site occurs approximately 518 km away in Lake Ardmore, Oklahoma (USGS 2016).Overall, our discovery of large, reproducing adults in a degraded urban pond raises serious ecological concerns about the potential of C. japonica to survive overland exposure (Havel et al. 2014), and even perhaps overwinter.Wide environmental tolerances would enable the species to establish new populations more easily, particularly in areas already invaded by other, non-native invasive mollusks such as P. maculata (Karatayev et al. 2009).Non-native invasive snails, such as C. japonica, compete with native species (Johnson et al. 2009) and may serve as reservoirs for parasites (Lin and Chen 1980;Chung and Jung 1999), prompting the need for increased diligence in monitoring public waterways.
Although we identified our specimens as C. japonica, the majority of published research includes individuals morphologically identified as C. chinensis.We therefore used these studies for comparison purposes when addressing issues of spread and ecological impact.In terms of their potential to spread, both C. japonica and C. chinensis can likely survive long periods of overland exposure.Both C. japonica and C. chinensis possess an operculum that partially functions in preventing water loss.In one experiment, large C. chinensis juveniles (greater than 25 mm) survived up to four weeks of air exposure while smaller juveniles (less than 10 mm) showed greater sensitivity to air exposure and low humidity (Havel 2011).In another experiment, adult C. chinensis survived an average of 44.6 days (± 4.9 as 1 SD) when left vulnerable to air exposure (Havel et al. 2014).Due to anatomical similarities between the two species, C. japonica can also likely survive long periods of overland exposure.Quantifying the ability of non-native freshwater snails to survive air exposure and overland transport provides direct insight into their ability to colonize new bodies of water and impact native species.
In terms of ecological impacts, initial evidence from one study suggests that C. chinensis negatively affects native species.In a mesocosm experiment, C. chinensis decreased sediment periphyton, significantly increased the ratio of N:P in the water column, and negatively influenced the abundance of the gastropod Lymnaea stagnalis (Johnson et al. 2009).Given their morphological similarities and shared ecological roles, we might expect similar survival abilities and ecological impacts for C. japonica.Kurihara and Kadowaki (1988) reported that C. japonica grazed heavily on sedimentassociated detritus, likely altering nutrient cycles.Furthermore, populations of C. chinensis occur at high densities and establish sustaining populations rapidly, which may also occur for C. japonica.One of the few population estimates for C. chinensis calculated 52,280 snails per 0.1 km 2 and up to 253,570 snails in a Nebraska reservoir (Chaine et al. 2012).In Long Island, NY, a population of C. chinensis increased from an estimated 150 individuals to an estimated 950 individuals within a two-year time period (McCann 2014).These high population levels could also be reached by C. japonica where anecdotal evidence exists in which fishermen have reported catching up to two tons of C. japonica from a single seine haul (Wolfert and Hiltunen 1968).
Survival probabilities of C. chinensis depend on size, with snails over 35 mm in length having almost a 100% probability of survival based on mark-recapture studies (McCann 2014).Higher survival rates for larger individuals likely apply to C. japonica and fecundity in this species increases predictably with female body size (Van Bocxlaer and Strong 2016).Distribution of mystery snails across northern and southern latitudes in the United States suggests the ability of this group to tolerate a wide range of temperatures (Karatayev et al. 2009;Solomon et al. 2010).Further investigation of the community pond area and connected waterways in Texas could provide insight into the distribution of C. japonica as well as the source of introduction (Karatayev et al. 2009).
Besides the predictable negative effects on native species and ecosystems (Solomon et al. 2010;Soes et al. 2011), non-native viviparids pose genuine disease risks.If consumed, Cipanopaludina spp.may transfer human intestinal fluke, Echinostoma cinetorchis (Jokinen 1982;Chung and Jung 1999) or serve as a vector for angiostrongyliasis (Lin and Chen 1980).Overall, the discovery of C. japonica in Texas highlights the ability of mystery snails to survive in urban waterways and indicates the likelihood that populations may spread successfully across the southern United States.Furthermore, our study affirms the need for genetic identification of species introductions to reduce taxonomic confusion and supports the need for improved monitoring programs coupled with a comprehensive centralized database to better track non-native invasive aquatic species.

ACKNOWLEDGEMENTS
A grant from the Keck Foundation for molecular biology research at Southwestern University provided the funding for this project.The Howard Hughes Medical Institute (HHMI) Inquiry Based Initiative at

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List 12(5): 1973, 1 October 2016 doi: http://dx.doi.org/10.15560/12.5.1973ISSN 1809-127X © 2016 Check List and Authors Notes oN GeoGraphic DistributioN later release June through October (Jokinen 1982).Mature females release an average of 25 offspring per year and 130 offspring during their lifetime, although as many as 133 offspring have been documented in a single individual of C. chinensis (Stephen et al. 2013) and 178 offspring from one C. japonica specimen (Van Bocxlaer and Strong 2016).Offspring measure approximately 5 mm in size at birth.Juveniles can reach 35 mm in size after their first year and adults can measure up to 65 mm in size

Figure 1 .
Figure 1.Distribution of Cipangopaludina japonica collections reported in the United States (USGS 2016).Records of C. japonica exist for fifteen states on the USGS Nonindigenous Aquatic Species Database (AL, CA, DE, FL, IN, KS, MA, MD, MI, MS, NE, OH, OK, PA, WI) although several other state departments of natural resources (ID, MN, NY, NC, OR, PA, SC, VA, WA, WI) include information about introductions on their websites (hatched states).A black star represents our collection of C. japonica in Harris County, Texas.The closest occurrence of C. japonica to our present collection occurs approximately 518 km away in Lake Ardmore, OK (USGS 2016).

Figure 2 .
Figure 2. Distribution map of Cipangopaludina chinensis collections reported in the United States (USGS 2016).Cipangopaludina chinensis occurs extensively across the United States with the highest densities found in the Great Lakes and the northeastern United States.A black star represents our collection of C. japonica in Harris County, Texas.

Table A1 .
Continued.Perez et al. | First record of Cipangopaludina japonica in Texas

Table A1 .
Continued.Perez et al. | First record of Cipangopaludina japonica in Texas

Table A1 .
Continued.Perez et al. | First record of Cipangopaludina japonica in Texas

Table A1 .
Continued.Perez et al. | First record of Cipangopaludina japonica in Texas