The snail Physella acuta is globally invasive (Vinarski 2017) and considered to be an exceptionally efficient invader (Bousset et al. 2014). It is important to recognize that this trait is not universal to all individuals of this snail species. Population studies using cox1 as a marker showed that the global distribution of P. acuta only involves a distinct genetic lineage of this species, termed population A (Ebbs et al. 2018). Another lineage, P. acuta subpopulation B is present only In North America, the native range of this snail species, where it co-occurs with population A (David et al. 2022; Ebbs et al. 2018; Nolan et al. 2014; Vinarski 2017).
Several phenomena can confer unique biological properties to populations within a species that lead to improved fitness under diverse environmental conditions. Natural selection may yield novel genetic variants that are more broadly capable of maintaining homeostasis due to increased plastic response capabilities (Crowl 1990) resulting from altered (epigenetic) regulation of gene expression, changes in metabolic efficiency, possibly resulting from variations in mitogenomes and coordination of nuclear/mitochondrial gene expression (Chapelle and Silvestre 2022; Pozzi and Dowling 2022; Vogt 2021), or the emergence of new gene alleles, including advantageous immune traits. Indications of the latter are provided by the presence in P. acuta of several expanded immune gene families (e.g., fibrinogen-related domain-containing immune factors; FREDs), proposed to confer the capability to tailor immune responses to particular pathogens encountered (Schultz et al. 2018, 2020).
Comparisons of the two different populations (A versus B) within the species with distinct (global versus native range) distribution will enable interpretation of biological features that underlie the invasive potential of P. acuta, and invasiveness in general. The side-by-side presence of both populations in New Mexico (Fig. 1, Fig. 2, also see Ebbs et al. 2018; Nolan et al. 2014) suggests the absence of conditions that constrain expansion of population B beyond the native range.
To prevent confounding effects from working with a field-collected mixture of morphologically indistinguishable snails from both P. acuta populations, this study established laboratory-maintained populations of genetically characterized P. acuta snails to explore the potential correlation between the worldwide distribution patterns of A and B snails and variations in population fitness, as delineated by life history traits (Stearns 1976, 1992). The cox1 marker divided field-collected physid snails that were morphologically indistinguishable into two clades (Fig. 2) that separated the cox1 sequences from the original A- and B-type P. acuta (Nolan et al. 2014).
The selfing reproduction mode of P. acuta as a simultaneous hermaphrodite yielded an initial egg mass that provided DNA for mito-haplotype characterization without harm to the parent snail. A subsequent egg mass from the same individual yielded offspring to initiate a laboratory-maintained population of that particular mito-haplotype (Fig. 1). Analyses of RNA-seq data confirmed that this approach generated two laboratory-maintained populations of P. acuta, genetically characterized to represent both populations A and B. The rDNA cassette (18S-ITS1-5.8S-ITS2-28S) sequence assemblies showed high similarity in nuclear genome-derived genes as a proxy for the nuclear genomes of both populations A and B (Fig. 3). The complete mitogenome sequences from twenty-four snails from the third generation of inbreeding of the laboratory populations confirmed that snails within each different population shared identical mitogenomes, with a ~ 9% sequence difference distinguishing populations A and B from each other. Each of the newly characterized mitogenomes is most similar (although not identical) to the mitogenomic sequences originally described by Nolan et al. (2014). These results underscore the considerable mitogenome sequence variability in P. acuta, also observed elsewhere (David et al. 2022). The combined mitogenomes and rDNA sequences confirmed the morphology-based species identification of the collected physid snails as P. acuta.
The availability of laboratory-maintained populations of genetically defined P. acuta enabled the study of possible association of population-specific geographical distribution patterns (and invasive potentials) with differential population-level fitness. Accordingly, these two P. acuta populations were compared under controlled laboratory conditions for life history parameters: growth pattern, age and size at maturity, and the number of offspring (egg masses) that define fitness (Stearns 1976, 1992). Each parameter differed by small values that were not statistically significant (Fig. 4a, b, c). Snails from population A modestly outperformed population B snails in average growth rate and production of egg masses per snail. Additionally, population A snails showed a younger and less variable age at maturity than population B snails, for both single and paired snails. As noted previously from various freshwater snails that are simultaneous hermaphrodites, a lag time in reproduction was observed from single- versus paired-snails in each P. acuta population (Fig. 4c). Physella acuta prefers reproduction by outcrossing (mating with another partner), but in the absence of a mating partner, it will ultimately engage in selfing (Noël et al. 2016; Tsitrone et al. 2003). This lag time was shorter by ~ 1 week (although not significant) in population A snails compared to population B snails.
Whereas the modest differences in life history features may combine to give A snails a fitness advantage over B snails, the differences were not statistically significant. Accordingly, these laboratory-based studies did not support a hypothesized link of differential fitness with the difference in global distribution patterns or invasive potential for population A versus B P.acuta snails.
Several recent studies convincingly argue that relative to laboratory-based studies (while valuable for controlled experimentation), exposure to variable environments (field-like conditions) reveals more comprehensive, novel aspects of organismal biology (Boughton et al. 2011; Flies and Wild Comparative Immunology Consortium 2020; Martin et al. 2021; Pedersen and Babayan 2011). For example, mice and oysters (Crassostrea hongkongensis) that are exposed to natural field-like environments show greater ranges of biological (immune) responses evoked by a probable plethora of pathogens and variable environmental stressors. So-called dirty mice, living under variable natural conditions, respond to immune challenges with an expanded diversity of immune cells (Kuypers et al. 2021). Oysters in locations with different levels of ocean acidification show distinct immune responses to Vibrio parahaemolyticus infection (Dang et al. 2023).
Mindful of limitations inherent to laboratory-based studies, laboratory-reared snails were also exposed to field conditions for a comparative study of the fitness of rewilded P. acuta populations A and B, relative to laboratory-maintained control snails. The rewilding trials were conducted for one- or two-weeks, appropriate time intervals to elicit environmental responses in P. acuta (e.g., Camargo and Alonso 2017; De Castro-Català et al. 2013; Prieto-Amador et al. 2021; Spyra et al. 2019). The considerably different abiotic conditions between the field environment and maintenance in the laboratory were still within the ranges for water chemistry that are naturally preferred by P. acuta (Spyra, 2010). The field environment showed considerable variability in temperature and pH. In contrast, laboratory conditions exposed snails to more pronounced variations in water hardness and elevated ammonia levels. The rewilded environment also contains more diverse pathogens (microorganisms and metazoan parasites; Johnson and Paull, 2011), as exemplified by the natural presence in the field of digenean trematodes, specialized parasites of P. acuta (Kraus et al. 2014). The screening of sentinel and experimental snails, however, did not detect any snails harboring trematodes such that the direct impact of trematode infections on intrinsic snail fitness (De Jong-Brink 1995; Hall et al. 2007) was excluded from this study.
The rewilding approach not only tested the impact of different environmental conditions on the fitness of P. acuta but also tracked the fitness of cohorts of snails over time. The comparisons during one- and two-week intervals of groups of rewilded snails and laboratory-maintained controls yielded population-level data, informing a more nuanced view of the relative fitness of populations A and B of P. acuta that was not provided by the initial laboratory-based study of fitness in individual snails.
Rewilding led to a significant, up to 2.5-fold increase in population-level fecundity (total egg mass production by cohorts of 20 snails, not replacing snails that died during the experiment) as compared to laboratory controls. For this fitness parameter, performance under laboratory conditions during 1 week of A and B P. acuta was similar with a small (not significant) advantage for population A (Fig. 5a). With rewilding, certainly combined with an increased 2-week experimental time interval, population A snails displayed significantly higher fitness than population B P. acuta. First, the survival rate of both populations was usually similar in these same trials, except for dramatic crashes with ≥ 55% mortality in snails of population B kept under rewilding conditions that did not similarly impact population A P. acuta (1 of 4 1-week, and 2 of 3 2-week trails, respectively). Secondly, whereas the realized fecundity of both populations was usually similar in both laboratory controls and rewilding trials, population A significantly outperformed population B in 3 out of 4 instances (once under laboratory conditions, twice for rewilding). The single occurrence of greater realized fecundity of laboratory-maintained population B P. acuta was marginally significant.
This study of life history-determining fitness parameters indicated that the population-level fitness, as it emerges under several environmental conditions, is frequently similar for snails of population A and population B, albeit with indications of a slight (not significant) advantage for population A P. acuta. Although such modest differences may become biologically meaningful, similar fitness levels may explain why both populations of P. acuta persist side-by-side in the environmental conditions that prevail in the native range of the species P. acuta. The use of more variable, unpredictable stressors through the application of the rewilding approach also yielded an expanded perspective for further interpretation of the apparent potential for invasiveness of the globally distributed P. acuta with mito-haplotype A relative to population B, restricted to the native range. The fitness traits shared by both populations A and B are equally adequate to maintain homeostasis by effectively responding to stressors that are part of the natural conditions in the native range. The experimentally observed instances of population crashes of snails from population B, but not from population A (in adjacent cages in the field) suggest that rare or extreme environmental stressors can defeat the response capabilities of population B snails. For instance, elevated temperatures may exert deleterious effects on animal fitness (Bozinovic et al. 2011; Godwin et al. 2020; Sepulveda and Moeller 2020). However, no correlation was revealed between any particular environmental parameter and the observed fitness of P. acuta, as shown in the graph (Online Resource 3). Physella acuta from population A showed a higher degree of response plasticity, enabling the maintenance of fitness even under more severe stress conditions, akin to those also encountered in invaded ranges.
Definition of the traits that convey fitness advantage(s) may provide an improved understanding of genetic profiles associated with the invasiveness potential of (populations within) particular species. The mitochondrial haplotypes that distinguish populations A and B of P. acuta, showing approximately a 9% difference in overall sequence composition, see tables (Online Resource 2, Online Resource 4), could potentially influence metabolic rates and the coordination of nuclear-mitochondrial gene expression, resulting in divergent individual behaviors and fitness. Similar phenomena have been documented in other organisms, ranging from protozoans to metazoans (Brand et al. 2023, Papier et al. 2022). The fact that populations A and B showed similar fitness levels in several experimental trials suggests that other features may also have a role, such as population-specific gene alleles and differences in the regulation of nuclear gene expression. The observed differential population-level fitness motivates in-depth exploration of the underlying biological processes governing these differences between the two P. acuta populations, with ongoing analysis of available RNA-seq data to characterize the biological factors that shape the fitness of distinct populations within this snail species.