What Does the Haired Keel on the Shell Whorls of Potamopyrgus antipodarum (Gastropoda, Tateidae) Mean?

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In several European ecosystems, the New Zealand mud snail Potamopyrgus antipodarum (Gray, 1853) (Gastropoda, Tateidae) has been included among the worst invasive species (NENTWIG et al. 2018). GEIST et al. (2022) reported that this invader is currently listed in the aquatic systems of at least 39 countries from five non-native continents. Furthermore, it has been present in Europe for 160 years (BOYCOTT 1936). A wide range of P. antipodarum densities have also been reported in non-native areas; in extreme cases, these densities reach as many as several hundred thousand snails per square metre (LEVRI et al. 2007;GEIST et al. 2022). Such highly abundant populations of P. antipodarum may impact the structure and functioning of the invaded ecosystem (GEIST et al. 2022). The success of P. antipodarum in colonising new areas is due to its tolerance to unfavourable physiochemical conditions (e.g. dissolved oxygen, pH and water temperature), as well as a lack of native enemies and the peculiar features of the shell morphology (ALONSO & CASTRO-DÍEZ 2008, 2012. For example, a solid operculum and a hard shell allow the snails to survive in the digestive systems of fish (ALONSO & CASTRO-DÍEZ 2012). However, the European populations of P. antipodarum show significant fluctuations in their occurrence; in particular, high densities and subsequent extinctions can be observed. It has mainly been postulated that this is the result of low genetic diversity among the individuals (STANICKA et al. 2020).
Little is known about the role of a haired keel on the shell whorls appearing in some populations of P. anipodarum. This morphological variation in P. antipodarum may be stress-induced by changes in environmental conditions, such as variations in the water depth, flow or the appearance of predators and parasites (VERHAEGEN et al. 2018). Based on three years of field research, we would like to consider the presence of a haired keel on the shell whorls of P. antipodarum as an adaptation to stressful environmental conditions.

Materials and Methods
The research areas were three Polish water bodies -Lake Czaplino (53°32N59ON, 16°14N59OE), Lake I³awskie (53°35N37ON, 19°36N54OE) and Lake Sosno (53°20N15ON, 19°20N55OE) -where we have been controlling the presence of P. antipodarum for many years (Fig. 1). The general characteristics of the sampling sites are summarised in Table 1. The water parameters were measured with a core sampler and a MultiLine P4 (WTW) Universal Pocket Sized Meter (Lake Czaplino: conductivity -327 ìS/cm, pH -7.0; Lake I³awskie: conductivity -368 ìS/cm, pH -8.5; Lake Sosno: conductivity -369 ìS/cm, pH -8.3). The collecting of snails and the aforementioned water measurements were carried out once a year -in September 2018, 2019 and 2020 -from a sandy bottom of the littoral zone. Using a core sampler with a diameter of 40 mm (POZNAÑSKA-KAKAREKO et al. 2017), 12 samples were taken at random during each sampling session. The contents of the core samplers were always placed in three containers (Fig. 2). In the laboratory, the snails were isolated and counted on white trays. Then, their densities per m 2 (D) were converted according to the following formula: D = ((B 1 x 10 000 / S) + (B 2 x 10 000 / S) + (B 3 x 10 000 / S)) / 3; B -number of snails in the box; S -the sum of the surface of the four-core sampler, i.e. 50.24 cm 2 .
The preliminary identification of the collected snails as P. antipodarum species was verified based on morphological data (PIECHOCKI & A. STANICKA et al.  . To obtain DNA sequences of the 16S rRNA gene, PCR reactions were run using the S1-Universal (5'-CGGCCGCCTGTTTATCAAAAACAT-3') and S2-Potamo (5'-GTGGTCGAACAGACCAACCC-3') set of primers (STÄDLER et al. 2005). A PCR cocktail and profiles/conditions were used according to (STANICKA et al. 2020). To check the DNA quality, a 3 ìl sample of the PCR product sample was run on a 1.5% agarose gel for 30 min at 100 V. The PCR products were cleaned up using a Clean-up Kit (A&A Biotechnology, Poland). A sequencing reaction was performed in 10 ìl of the reaction mixture, according to the cocktail and profile conditions described in ZAJ¥C et al. (2020). The sequencing products were cleaned up using ExTerminator (A&A Biotechnology, Poland) and were sequenced in one direction. The sequencing reactions were performed by Genomed (Warsaw, Poland). The sequences were deposited in GenBank with the following accession numbers: MK578225, MK578226 and MK578227. Moreover, we observed the shell surfaces of the snails under a stereomicroscope (Motic K-700) to divide the collected individuals into carinatus and regular morphotypes (with and without a haired keel on the shell whorls, respectively), according to BUTKUS et al. (2012). Selected individuals of the carinatus morphotype were photographed using a digital camera, and we then used the images to measure their haired keel with the ImageJ program.

Results
All P. antipodarum 16S rRNA sequences belonged to the same haplotype and showed a 100% similarity to P. antipodarum (MG979469, SHARBROUGH et al. 2018), confirming the morphological identification.
The carinatus morphological form (Fig. 3) was detected only in the specimens from Lake I³awskie, and it was the only morphotype of P. antipodarum in this location. Their haired keel had an average length of 121 (SE 2) µm. At Lake I³awskie, where the carinatus morphotype was noted, individuals of P. antipodarum were recorded only in the first year of the study. By contrast, only their empty shells were detected in the following two years. In the other two research areas, where no haired keels on the shell whorls of P. antipodarum were recorded, the individuals of P. antipodarum were recorded during all three years (Table 2).

Discussion
There are known cases where trematode larvae in the snail body can modify the shell morphology of their hosts (in terms of the shape, size or ornamentation) (LAGRUE et al. 2007). ¯BIKOWSKA & BIKOWSKI (2005) emphasised that the morphology of snail shells can be affected by parasites only in cases where they play the role of the first intermediate host. Nevertheless, it is well known that outside their native range, infections of P. antipodarum with sporocysts, rediae or cercariae are extremely rare (EVANS et al. 1981;GÉRARD & LE LANNIC 2003;BIKOWSKI &¯BIKOWSKA 2009;STANICKA et al. 2020).
Shell polymorphisms may have a genetic basis (VERHAEGEN et al. 2018), and regular and carinatus morphotypes might result from two independent invasion events, as was suggested by BUTKUS et al. (2012). However, both morphotypes collected in the present study belonged to the one haplotype (STAN-ICKA et al. 2020). This suggests that the presence of the two morphotypes might not be solely attributed to a genetic basis, but could also have been promoted by a plastic response to the environment. Accordingly, variations in the shell morphology could reflect the adaptive responses to abiotic and biotic factors, and may be the result of phenotypic plasticity in conjunction with evolutionary changes (HAASE 2003;KRIST-NER & DYBDAHL 2013).
It is well known that the ability of organisms to form inducible defences in response to environmental changes is based on their phenotypic plasticity (SPITZE & SADLER 1996). We did not find a population exhibiting both morphotypes in the investigated research areas. It is most likely that the analysed physicochemical parameters of the habitats did not have a decisive influence on the morphotype formation, because these parameters seemed similar in the habitats with and without carinatus morphotypes (especially the conductivity, which is of particular importance for P. antipodarum (LARSON et al. 2020)). However, the impact of unfavourable environmental factors (other than the water quality, e.g. the type of substrate, water flow and composition of co-inhabitants) on creating the carinatus morphological form of these snails seems to be the most probable conclusion, because in the next two years of research at Lake I³awskie only the empty shells of P. antipodarum were recorded. This proves the constant presence of a stress factor for P. antipodarum in this environment. It is also unlikely that the destruction of the population would be caused by any short-term event, such as a spillover of pollutants or pesticides, because our qualitative observations indicated the presence of other species of snails. HOLOMUZKI & BIGGS (2006) suggested two reasons for the emergence of carinatus forms: (i) a higher probability of snail dislocation caused by flow; and (ii) for protection against being eaten by fish. Laboratory experiments have indicated that the spines collected seston, making the individuals of the carinatus morphotype more prone to flow-induced dislodgment than individuals of the regular morphotype (HOLOMUZKI & BIGGS 2006). This seems very beneficial for these snails, because P. antipodarum naturally avoids strong currents by wandering into safer habitats (HOLOMUZKI & BIGGS 2006). It is also well known that water movement may affect animal populations (HUBENDICK 1958). In addition to the movement of water, such as the action of waves or currents, issues related to changes in the water level should be mentioned (HUBENDICK 1958), which were most often observed at Lake I³awskie during seasonal observations among the examined lakes (personal observations). At the sampling site, individual representatives of other species of prosobranch snails with small shell sizes were found. Nevertheless, these snails belonging to the genus Valvata and Bithynia inhabited the neighbouring habitat that was heavily overgrown with elodeids, which may indicate their attempt to avoid unfavourable water movements. On the other hand, I³awskie Lake is abundantly inhabited by numerous species of fish, with the most abundant being bream, tench and pike (as reported by a local fish farm). Therefore, it can be expected that predation could have caused the appearance of carinatus forms here. Generally, it is challenging to link this with the destruction of the snail population, because if one individual of P. antipodarum remains in the set-  ting that is enough for the population to recover (SCHREIBER et al. 1998). However, such a change probably requires a huge investment of energy, and in the event of an additional negative factor, such as the aforementioned water level fluctuations, we can assume that such a population would collapse. In conclusion, the presence of a haired keel on the shell whorls may be considered as a type of snail defensive reaction; however, long-term field and experimental research are necessary to identify the exact cause of its occurrence. Learning the exact purpose of the appearance of the carinatus morphotype may prove useful in developing tools to combat invasions of P. antipodarum.