Rewilding shows differential fitness of Physella acuta snail populations with different invasive potential

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Within this species, however, cox1 haplotype markers distinguished a globally invasive population (A) from a non-invasive population (B) restricted to North America, the native range of P. acuta . This study investigated whether invasiveness is associated with differential population fitness. Field-collected P. acuta were genetically characterized to establish laboratory populations representing mito-haplotypes A and B. While the nuclear rDNA cassette (7,023 nt) differed only by 0.03% between populations A and B, the mitogenome haplotypes differed in size (14,383 vs 14,333 bp) and sequence content (~ 9%). Under controlled laboratory conditions, growth rate, age at maturity, size at maturity, and reproductive output did not show fitness differences between populations A and B (3 trials). Population fitness was also studied using a rewilding approach. Survival and fecundity of A and B snails were evaluated during one- or two-week intervals among cohorts of 20 laboratory-bred P. acuta adult snails in flow-through cages in the laboratory or exposed to natural field conditions. Only modest differences in fitness parameters were indicated under laboratory conditions, providing no clear association of population fitness with global distribution patterns. Under field conditions, however, population level fecundity differed with population A having a 3-fold greater fitness than population B in 5 of 7 trials (survival in 3 trials; realized fecundity in 2 trials). Whereas laboratory-based studies indicated only minor differences, the rewilding approach showed significant differential fitness between P. acuta populations A and B that differ in invasiveness. Physella acuta realized fecundity fitness invasiveness mitogenome hermaphrodite Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The current distribution of the invasive freshwater snail Physella acuta extends far beyond the native range of North America (Vinarski 2017 ). Due to natural dispersal (van Leeuwen et al. 2013) and human activities, including the aquarium trade (Duggan 2010 ), P. acuta is established across all continents except Antarctica. As a simultaneous hermaphrodite, capable of reproduction by selfing (Bousset et al. 2004 ), P. acuta can efficiently colonize new habitats, persisting because of considerable tolerance to a range of environmental conditions (Min et al. 2022 ; Spyra 2010 ), including anthropogenic pollution (Alonso-Trujillo et al. 2020 ; Spyra et al. 2019). Bousset et al. ( 2014 ) proposed that P. acuta ‘can be considered an excellent biological model for analyzing bioinvasions at a vast geographic scale.’ Invasive potential, however, is not a common feature of P. acuta as a species. Previous research has unveiled two divergent mito-haplotypes (A and B) among North American P. acuta snails (Nolan et al. 2014 ). Haplotype markers identify globally invasive P. acuta as members of population A, distinct from snails of a non-invasive population B that is restricted to North America, the native range of this species (David et al. 2022; Ebbs et al. 2018 ; Vinarski 2017 ). Considering that populations A and B occur side-by-side in the same North American habitats (Ebbs et al. 2018 ; Nolan et al. 2014 ), both are assumed to have equal opportunity for dispersal. Remarkably, only a subpopulation within the species P. acuta is globally invasive. This difference in invasive potential likely reflects inherent differences between P. acuta populations A and B regarding the ability to respond to environmental changes, such as those that may be encountered when entering novel habitats. The environment contains diverse and variable abiotic and biotic stimuli that challenge the homeostasis of any organism (Hambrook and Hanington 2021 ; Pandey et al. 2021 ; Pinaud et al. 2021; Romero et al. 2009 ). Alterations in a current habitat or environmental differences in a newly entered habitat may amount to environmental stress, with selective pressures possibly impacting reproductive output and survival, causing sharp reductions that lead to variations in the biographical ranges of particular organisms (Cunanan et al. 2018; Coutellec and Caquet 2017 ; Selye 1950 ). The evolution of biological adaptations (behavioral and genetic traits) enables organisms to better cope with environmental stressors and successfully colonize and persist in a specific environment. For instance, physid snail species display varying degrees of predator avoidance behavior, consisting of vigorous shell shaking and detachment from the substratum when encountering leeches (Frieswijk 1957 ), and P. acuta is thought to utilize expanded gene families to optimize immune responses against various pathogens (Schultz et al. 2020 ). In turn, these adaptations shape survival, growth patterns, and reproduction, key life history traits that define the biological fitness of (populations of) an organism (Stearns 1976 , 1992 ). Unique ecological challenges and selective pressures may lead to distinct adaptations within a species. For example, laboratory-maintained Lymnaea stagnalis snails comprised two populations with different lectin-based immune recognition capabilities (van der Knaap et al. 1983 ). Furthermore, the natural distribution of the marine prosobranch snail Chlorostoma funebralis is determined by the expression levels of population-specific stress response proteins contributing to thermal tolerance (Gleason and Burton 2015 , 2016 ). These phenomena could give rise to populations with varied fitness that all flourish within their native range, while specific populations have the capability for expanding into habitats with different environmental conditions. This study aims to analyze life history traits of two P. acuta populations (A and B) to investigate a potential association of differential population-level fitness within this snail species with the evident invasive potential of P. acuta. A well-defined genetic background was established to allow meaningful interpretation of population-level fitness in morphologically indistinguishable P. acuta snails that co-occur in the field throughout the native range. Field-collected P. acuta snails were genotyped as representative of populations A or B. Progeny produced by individual snails through selfing was used to establish laboratory populations representing A and B mito-haplotypes to evaluate population-level fitness in subsequent laboratory experiments. Studies in the laboratory provide useful views of stress responses (Feder and Hofmann 1999 ), however, an artificial, constant experimental environment fails to capture natural situations involving an unpredictable environment with various stressors that challenge organismal fitness (Eads et al. 2008 ). Integrating both laboratory and field-based investigations through exposure to a (semi)natural environment may reveal novel biological features from study organisms (see Beura et al. 2016; Leung et al. 2018; Lin et al. 2020; Martin et al. 2021 ; Pedersen and Babayan 2011 ; Seppälä et al. 2021 ). For example, neonatal immune function differs dramatically between laboratory-maintained mice and so-called “ dirty mice ” from the wild or obtained from pet stores (Hamilton et al. 2020 ). Accordingly, differential fitness within P. acuta was also studied using a rewilding approach. Specifically, laboratory-bred P. acuta from genetically defined populations A and B were exposed temporarily to natural conditions in cages at a field site, to record life history parameters for comparison relative to a P. acuta (control) group maintained under laboratory conditions. This approach provided increased sensitivity for investigating differential population-level fitness associated with the success and invasiveness of P. acuta in nature. Materials and Methods Collection, haplotype characterization, and maintenance of snail populations Physella acuta snails were field collected at Shady Lakes Commercial Fishing Facility, Albuquerque, New Mexico (35.216 N 106.6 W) in June 2020. The snails were kept individually in 150 ml tanks with artificial spring water at room temperature and fed lettuce, supplemented weekly with shrimp-based feed pellets (Nolan et al. 2014 ). Initially, snails were identified as P. acuta based on morphological features that include a gray body, thread-like tentacles, digitations along the mantle edge, and a sinistral shell exhibiting a "fawn" coloration with five distinct whorls and an oval-shaped aperture, (Paraense and Pointier 2003 ). For sequence-based identification and characterization of mito-haplotype of individual snails, genomic DNA was extracted from recently produced snail egg masses using a CTAB-based method (Winnepenninckx et al. 1993 ). Amplicons from the mitochondrial cox1 gene sequence, resulting from PCR (TaqGold) using primers LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG − 3’ and HC02198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3’ (Folmer et al. 1994 ) were sequenced (BigDye 3.1, ABI). Resulting sequences were aligned (Clustal X, Thompson et al. 2003 ) for phylogenetic analysis (RaxML, Kozlov et al. 2019 ) to determine the mito-haplotype of snails as A or B (Ebbs et al. 2018 ). Following mito-haplotype characterization, single snails were allowed to reproduce via selfing ( P. acuta is a simultaneous hermaphrodite), using single egg masses to initiate uniparental populations representing mito-haplotypes A and B (Fig. 1 ). All snails were kept in 20 L tanks as described above. The mito-haplotype and rDNA cassette of 24 snails (representing each population, 13 A and 11 B) were characterized by RNA-seq (see below). All experiments employed reproductively mature (indicated by egg-laying) snails with a shell length ≥ 5 mm, experimentally determined as described below. Laboratory fitness Snails from each population were evaluated for fitness under laboratory conditions employing three of the seven traits of life history theory listed by Stearns ( 1976 , 1992 ): growth pattern, age (size) at maturity, and fecundity (number of egg masses produced). Growth rates of each P. acuta population were determined starting with ten newly hatched snails (1 mm shell length) kept in 20 L tanks. The shell length (apex to outside edge of aperture) of each snail was recorded using a ruler (to the closest half mm), twice a week over a 76-day time interval. Three separate trials were performed. Data points were averaged, and curve fitting was used to plot and compare growth rates. Physella acuta is a simultaneous hermaphrodite that preferentially reproduces by outcrossing in the presence of other snails, or by selfing in isolation. The latter may be associated with a delay in reproduction, a so-called lag time, (Tsitrone et al. 2003 ). Newly hatched snails (shell length ~ 1 mm) were kept separately (n = 5) or as pairs (2 x 5) of the same population (A or B) in separate containers (150 ml). The number of days was recorded until an egg mass was first produced, and the shell length of single or paired snails in that container was measured (to the closest half mm) to determine the minimum size at which snails were reproductively mature. Three trials were performed. Fecundity was measured as the average number of total egg masses produced over a 108-day interval by individual snails from P. acuta populations A and B. Ten newly hatched snails (1 mm) from each population were separated into individual 150 ml containers. Twice a week, egg masses produced by each snail were enumerated and removed. Three trials were performed. Physella acuta fitness in rewilding experiments Cohorts of snails from each population were evaluated simultaneously for fitness under field and laboratory conditions, employing two of the seven traits of life history theory as listed by Stearns ( 1976 , 1992 ): survival (number of live snails after experimental exposures), and fecundity, considering both population-level- and realized fecundity (see definitions below). Populations of A and B P. acuta snails were rewilded by placing genetically- characterized, laboratory-bred P. acuta snails temporarily under natural conditions. This rewilding approach examines how organisms function under actual natural environmental conditions, supplementing observations made under controlled laboratory conditions (Leung et al. 2018; Lin et al. 2020; Pedersen and Babayan 2011 ). The relative fitness of populations A and B in the field and the laboratory was investigated using groups of adult siblings of each type of snail (n = 20). Snails were housed in flow-through cages (20 L) with two 12 x 15 cm screens with 1 mm mesh size. Snails were exposed to both experimental conditions (laboratory and field) for either one- or two-week intervals. Two field sites were used: one experiment was done at Shady Lakes (the original snail-collection site), and the others at the Rio Grande Nature State Park (35.128 N 106.685 W). Physella acuta occurs naturally at both locations. In the laboratory, the flow-through cages holding P. acuta snails were kept in a 150 L artificial pond containing snail-conditioned water. Every second day, all snails were fed lettuce and fitness parameters were recorded. Survival was determined by the number of individual P. acuta snails that were actively moving or firmly adhering to substrates in the cages. Inactive snails were examined to confirm death, and empty shells were removed from the cages. Fecundity was tracked by enumerating and removing all egg masses in each cage. Population fecundity was determined as total egg masses produced by the snail population in a cage, starting at n = 20, and irrespective of the demise of individual snails, during the complete one- or two-week exposure interval. Data recorded after the first week of two-week exposure intervals and results from the one-week exposures were combined for analysis. Realized fecundity was calculated every second day from the number of egg masses divided by the number of living snails in a cage. Four one-week trials and three two-week trials were performed. Water chemistry for rewilding experiments To ascertain variations between the environmental conditions experienced by field and laboratory populations of P. acuta snails, several environmental parameters were recorded. These included water temperature, nitrite and nitrate levels, general hardness (calcium and magnesium content), German carbonate hardness, and pH, which were monitored every second day throughout all trials using the freshwater master test kit from API. DNA and RNA Extraction from Physella acuta Following each rewilding experiment, RNA was extracted from each surviving P. acuta snail from populations A and B, and from both field and laboratory exposures. Individual whole snails were placed in 1.5 ml tubes, disrupted with a pestle, and mixed with 1 ml Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). To accommodate the screening of all field-exposed snails for naturally incurred parasites (see below), the Trizol protocol was modified (according to the manufacturer’s instructions) to also extract DNA in addition to RNA. The quality of the extracted nucleic acids was evaluated spectrophotometrically (NanoDrop 6000, Thermo Scientific). Evaluation of rewilded snails for trematode parasites Field exposure may lead to trematode parasite infection in P. acuta snails. The exclusion of infected snails from this study is important because these parasites impact snail host neurophysiology and general fitness (e.g. De Jong-Brink 1995 ). Half of the rewilded snails served as sentinel snails to determine the risk of parasite infections for field-exposed P. acuta . After a rewilding trial, these snails were maintained in 20 L tanks in the laboratory and checked after 3- and 6 weeks for the shedding of cercaria as an indication of a digenean trematode infection. DNA was extracted from rewilded snails (see above) for PCR screening against potential trematode parasite infections. Genbank entries of 28S -derived sequences from digenean trematodes that employ P. acuta as a host were aligned (Clustal X) and conserved regions were used to design primers (5’ to 3’) DT_FP_28S: CACTTATCAAGTGTTGTGC and DT_RP_28S: CTACACCACAGACTATTGG. The PCR reactions used TaqGold (as above) with 4 µl of the DNA extracted from rewilded P. acuta as the template in a 50µl reaction, Tm 55°C for a total of 40 cycles with a final extension at 72°C for 5 min. Presence of amplicons was checked by agarose gel electrophoresis. Finally, 18S , 28S , 16S , and cox1 sequences of digenean trematodes known to infect P. acuta were retrieved from GenBank, see table (Online Resource 1) and blasted against raw RNA-seq reads collected from rewilded snails, available from this study. RNA-seq Individual snail RNA samples of 24 snails (7 field-exposed, 6 laboratory-kept snails from population A; 5 field-exposed, 6 laboratory-kept snails from population B) from a one-week rewilding experiment performed at the Shady Lakes field site were used for commercial library preparation and Illumina NovaSeq RNA-Seq sequencing (Novogene). Raw RNA-Seq paired-end (PE150) reads were trimmed with Trimmomatic (Bolger et al. 2014 ), and adapters were removed using fastp (Chen et al. 2018 ) employing Galaxy (Pond et al. 2009). The resulting RNA-seq data were used to set up a local blastable database (Sequenceserver, Priyam et al. 2019). The transcriptomic data were examined for parasite-derived sequences to screen for parasite-infected snails (described above). RNA-seq data was used to assemble the mitogenomes for each individual snail (see Bunkóczi et al. 2011; Forni et al. 2019 ; Smith 2013 ) employing MitoBIM (Hahn et al. 2014) with the P. acuta A and B mitogenomes (GenBank NC_023253, JQ390526; Nolan et al. 2014 ) as references. Additionally, BBDUK (Guzman and D’Orso 2017 ) was used to collect RNAseq raw reads similar to these reference mitogenomes for assembly using MEGAHIT (Li et al. 2015 ). Any discrepancies between the resulting assemblies were reconciled using ras paired-end sequence data. Automated mitogenome annotation (MITOS; Bernt et al. 2013; Donath et al. 2019) was checked using criteria established by Fourdrilis et al. ( 2018 ) through Snapgene (Dotmatics). The nuclear rDNA cassette of each of the 24 snails was reconstructed manually from RNA-seq data by iterative blasting, initiated with P. acuta ITS sequences (GenBank KF316326-9). Multiple sequence alignments (Clustal X) were checked by eye to compare the mitogenomes and rDNA cassettes of each snail population among individuals and between populations. Statistical analyses Growth patterns, reproductive output, and size at maturity (single snails) of populations A and B within the laboratory were compared using a one-tailed t-test. The age at maturity of A and B snails (both kept single and in pairs) was compared using a Kruskal-Wallis test. Differences between abiotic conditions in the field and laboratory for P. acuta were determined using one-tailed t-tests for water temperature, nitrite and nitrate levels, degree of general hardness, degree of carbonate hardness, and pH. Various statistical tests were applied to assess the life history parameters studied in the rewilding experiments between A and B snail populations under laboratory and field conditions. One-tailed t-tests were utilized for realized fecundity, chi-squared tests for population-level fecundity, and a rank test (via the R package 'survival'; accessed through https://astatsa.com/LogRankTest/ , Vasavada 2016 ) for comparing survival in rewilding experiments. All abiotic environmental parameters were plotted as bell curves to identify extreme conditions experienced by snails in the laboratory and during rewilding over one- and two-week trials. Extreme high and low values were defined by values differing by greater than one standard deviation from the mean. The range of each abiotic parameter recorded during a specific trial was plotted onto the bell curve to check for potential associations between environmental variation and recorded fitness (survival and realized fecundity). Results Identification of Physella acuta from the field and laboratory populations The initial morphology-based identification of field-collected snails as Physidae was further developed by identification as Physella acuta by phylogenetic analysis of cox1 sequences amplified by PCR from genomic DNA extracted from embryos in single egg masses produced by individual snails via selfing. Of 23 P. acuta collected in the same location, ten snails exhibited mito-haplotype A and 13 mito-haplotype B (Fig. 2 ). Two separate laboratory-maintained populations representing each mito-haplotype (A or B) were initiated with progeny produced by selfing from one individual of these field-collected P. acuta snails (Fig. 1 , Fig. 2 ). RNA-seq data collected from 24 third-generation snails of the inbred laboratory populations were used to assemble the rDNA cassette for individual snails (Fig. 3 a). The sequences (7,023 bp); 13 from population A and 11 from B were identical within the populations but differed by two single nucleotide polymorphisms (0.03%) between populations A and B. BLAST searches showed that the rDNA sequences had the highest similarity to previous GenBank entries for P. acuta . Assembly and analysis of full-length mitogenomes showed identical sequences within the populations, while showing a 9.3% in sequence content between populations A and B (Fig. 3 b). Specific genes within these mito-haplotypes displayed different levels of sequence identity, ranging from 95% for cox1 to 72% for nad4L , see table (Online Resource 2). Furthermore, the mitogenome of population A was most similar to the previously characterized mitogenome of type A P. acuta . Likewise, the mitogenome of population B was most similar to the previously described mitogenome of P. acuta type B (NC_023253, JQ3905261; Nolan et al. 2014 ). Fitness in the laboratory Physella acuta snail populations A and B showed only minor differences in fitness parameters when maintained under controlled and consistent environmental conditions in the laboratory. The average growth rate of individual snails (both by size measurements and fitted curve comparisons) and the average total number of egg masses per snail over time were not significantly different (Fig. 4 a, b). The age of snails at maturity (first production of egg masses), either kept as individuals or in pairs, showed some variation but was not significantly different between populations A and B (Fig. 4 c). Within each population, however, there was a lag time in reproduction between single and paired snails. On average, single snails from population A first produced an egg mass 8 days later than paired snails; this difference was 14 days for population B. In both instances, the time differences due to the observed lag time were not statistically significant (ANOVA, A snails p = 0.880; B snails p = 0.585). Regarding size, individually kept P. acuta snails first reached maturity at 6.5 mm for population A and 7 mm for population B (Fig. 4 d). When kept as pairs, P. acuta of both populations first produced an egg mass at 5 mm in size. These differences in size, nor the other fitness parameters tested under this section, were not statistically significant at 𝛼= 0.05. Rewilding experiments Relative to the laboratory maintenance, the abiotic environmental conditions at the field site were significantly different (p-values ≤ 0.05). Water temperature, pH and water hardness (general hardness, German carbonate hardness) had higher values in the field (Fig. 5 a-d). Ammonia levels were higher in the laboratory (Fig. 5 e), whereas nitrates and nitrites were not detected (Fig. 5 f, g). Temperature and pH were more variable in the field, and water hardness and ammonia levels were more so in the laboratory. Parasite infections were not observed in sentinel snails during rewilding experiments, nor were infections detected from experimental snails, either by PCR or analyses of RNA-seq data. The population-level fecundity of rewilded snails in populations A and B was three times higher than laboratory-maintained snails of the same populations at one- and two-week intervals. When comparing A and B laboratory-maintained snails, the total and average numbers of egg masses in 7 trials over one week did not differ significantly (Fig. 6 a, Table 1 ). In one of these trials, however, population B produced 112 egg masses, significantly more than the 77 from population A (p = 0.011). Under rewilding conditions, the average number of egg masses per one-week trial did not differ (p = 0.123). However, population A snails produced more egg masses in total over all seven trials than snails from population B (p < 0.001), with population A significantly outperforming B in 3 of 7 trials (p ≤ 0.05). More pronounced differences were recorded from the three trials of two-week time intervals. Under laboratory conditions, population A produced more egg masses than population B, both for the combined total egg masses (p < 0.001) and the average number over three trials (p = 0.003), with population A significantly outperforming B in 2 of 3 trials (p ≤ 0.05). These trends were more prominent under field conditions, with both greater total (p < 0.001) and average (p < 0.001) numbers of egg masses recorded from rewilded snails of population A versus B. Population A generated significantly more total egg masses than B in 2 of 3 trials, p ≤ 0.05 (Fig. 6 b, Table 1 ). Table 1 Cumulative population-level fecundity (total number of egg masses) of 20 Physella acuta snails from population A and B, laboratory maintained or rewilded, over 7 one- and 3 two-week trials ONE WEEK LABORATORY A LABORATORY B REWILDED A REWILDED B TRIAL 1 57 50 101 61 TRIAL 2 69 68 110 139 TRIAL 3 77 112 157 117 TRIAL 4 44 37 162 167 TRIAL 5 85 56 156 164 TRIAL 6 41 26 203 124 TRIAL 7 31 9 202 135 TWO WEEKS TRIAL 5 160 109 329 324 TRIAL 6 69 43 333 176 TRIAL 7 94 53 361 218 Comparison of survival in the laboratory during one- and two-week intervals (Fig. 7 ) revealed minimal snail mortality with modestly higher survival rates for A snails compared to B snails (trials 1, 2, 3, and 5), except for trial 4 (two dead A snails versus no mortality of B snails), and equal numbers of population A and B survived in trials 6 and 7. These laboratory trials showed no significant differences in survival rates. In contrast, rewilded snails displayed significant differences in survival, with A snails outperforming B snails in trials 1, 6, and 7. Trials 2, 3, and 4 each showed a single mortality for population A, with all B snails surviving (p > 0.05, no significant differences). Notably, no rewilded snails from A and B populations died in trial 5. During either one- or two-week intervals, the realized fecundity of both populations was similar in 5 of 7 trials under laboratory conditions. Significant differences were evident in trial 3, with B snails outperforming A snails (p = 0.048, marginally significant), and in trial 4, with greater realized fecundity for A snails compared to B snails (p < 0.001). Rewilding in the field showed that the realized fecundity of A snails was similar to B snails, with minor differences (p < 0.05), except in trials 3 and 4, where A snails had significantly higher realized fecundity compared to B snails (p values < 𝛼). Analysis of environmental variation did not indicate any association between differential fitness and the abiotic parameters tested, for example see graph (Online Resource 4), otherwise not shown. Discussion 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. 4 a, 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. 4 c). 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. 5 a). 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. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethics declarations Conflicts of interest The authors declare that they have no conflicts of interest. Permits The Energy, Minerals and Natural Resources Department, State Park Division, New Mexico provided permits for field research. Funding Kevin A. McQuirk acknowledges graduate research scholarships from the Biology Department and the Graduate and Professional Student Association of the University of New Mexico. Maria G. Castillo was supported by NIH 5SC2AI133645. Author Contributions Kevin A McQuirk, Maria G Castillo and Coen M Adema contributed to the study conception and design. Material preparation, data collection and analysis were performed by Kevin A McQuirk, Julie DeCore, Maria G Castillo, and Coen M Adema. The first draft of the manuscript was written by Kevin A McQuirk and Coen M Adema and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgments . We thank the superintendents of the Rio Grande Nature Park in Albuquerque, New Mexico for access to the Discovery Pond. Field research permits were provided by the Energy, Minerals and Natural Resources Department, State Park Division, New Mexico. Maria G. Castillo was supported by NIH 5SC2AI133645. Kevin A. McQuirk acknowledges graduate research scholarships from the Biology Department and the Graduate and Professional Student Association of the University of New Mexico. 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Trends Genet 9:407. https://doi.org/10.1016/0168-9525(93)90102-n Supplementary Files ESM1.pdf ESM2.pdf ESM3.pdf ESM4.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3994352","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285068279,"identity":"ba1bb6c8-6013-45a0-8cbd-60ab25c9d9a6","order_by":0,"name":"Kevin Arthur McQuirk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBACCTBpwGDAD+Ezk6BFsoE0LUBNBgeI1SI57fDD2zwF94yNb+Qe3cBQYZ3YQEiLtHSasTWPQbGZ2Y28tBsMZ9IJa5GTzmGT5jFIsDG7kWN2g7HtMAlajGeAtPwjQos0VIuZgQRISwMRWiRnpxlbzjFIMJY488bsRsKxdGOCWiRuJz+88eZPgmF/O9CWDzXWsgS1gLXBWQnEKEfVMgpGwSgYBaMAGwAADe44FfI7qC4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0009-4128-3127","institution":"The University of New Mexico - Albuquerque: The University of New Mexico","correspondingAuthor":true,"prefix":"","firstName":"Kevin","middleName":"Arthur","lastName":"McQuirk","suffix":""},{"id":285068280,"identity":"cf111410-521d-4f7c-a883-c4b2d82c55b4","order_by":1,"name":"Juliana DeCore","email":"","orcid":"","institution":"University of New Mexico - Albuquerque: The University of New Mexico","correspondingAuthor":false,"prefix":"","firstName":"Juliana","middleName":"","lastName":"DeCore","suffix":""},{"id":285068281,"identity":"6f265095-7705-4acd-a2df-a5b1d1dcf6e0","order_by":2,"name":"Maria Castillo","email":"","orcid":"","institution":"New Mexico State University","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Castillo","suffix":""},{"id":285068282,"identity":"a12d1629-1e61-40d2-b7a4-f295aaf10b18","order_by":3,"name":"Coen Adema","email":"","orcid":"https://orcid.org/0000-0002-6250-5499","institution":"University of New Mexico - Albuquerque: The University of New Mexico","correspondingAuthor":false,"prefix":"","firstName":"Coen","middleName":"","lastName":"Adema","suffix":""}],"badges":[],"createdAt":"2024-02-27 16:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3994352/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3994352/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53912560,"identity":"715d3fd1-6d05-4f38-8265-5ad4fb52f2ff","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21208,"visible":true,"origin":"","legend":"\u003cp\u003eFlow diagram depicting establishment of mito-haplotype-specific populations of \u003cem\u003eP. acuta\u003c/em\u003e in the laboratory. Egg masses produced by selfing by individually housed snails were used to characterize the parental mito-haplotype by \u003cem\u003ecox1\u003c/em\u003esequencing. Selfed offspring from individual snails were then used to establish uniparental snail lines with either A or B mito-haplotypes in the laboratory\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/1a0cfa7a7dbe4ba185ee924c.png"},{"id":53912568,"identity":"ed91d83b-7710-445f-86fb-b68774fa3a0a","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76557,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree analysis of \u003cem\u003ecox1\u003c/em\u003e sequences shows that 23 field-collected physid\u003cem\u003e \u003c/em\u003esnails distribute across two clades representing two mito-haplotypes A and B, previously reported for \u003cem\u003eP. acuta \u003c/em\u003e(NC_023253 and JQ390526). The simplified maximum likelihood tree shows bootstrap values \u0026gt;65 for 1,000 replicates. The outgroups include \u003cem\u003ecox1\u003c/em\u003e sequences from \u003cem\u003ePhysella anatine \u003c/em\u003e(AY651177)\u003cem\u003e, \u003c/em\u003eLymnaeidae: \u003cem\u003ePseudosuccinea columella \u003c/em\u003e(ON953198) and \u003cem\u003eLymnaea stagnalis \u003c/em\u003e(HQ926919); Planorbidae \u003cem\u003eBulinus truncatus \u003c/em\u003e(OP233112), \u003cem\u003eAncylus fluviatilis \u003c/em\u003e(MT883692), and \u003cem\u003eHebetancylus excentricus \u003c/em\u003e(GU391113), and the basal gastropod \u003cem\u003ePyramidella dolabrata \u003c/em\u003e(AY345054). Stars mark the individual snails used to establish laboratory populations with mito-haplotypes A or B\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/a1ceb16c849bceaee8f4d78f.png"},{"id":53912561,"identity":"2b4d9e0a-d85d-493d-955b-51f6762ce6a3","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":167087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e rDNA cassette of \u003cem\u003ePhysella acuta \u003c/em\u003esnail populations A and B (5’ →3’). The assembled sequences include part of the External Transcribed Spacer (ETS), 18S, Internal Transcribed Spacer 1 (ITS1), 5.8S, ITS2, and 28S. The sequences from populations A and B differ by two SNPs, one each located in ITS1 and ITS2 (dotted lines). \u003cstrong\u003eb\u003c/strong\u003e The mitochondrial genomes from \u003cem\u003ePhysella acuta\u003c/em\u003e A and B populations, with arrows depicting interval and strand orientation of protein-encoding genes (black), ribosomal RNA genes (gray), and tRNA genes (white). Note size difference between the mitogenomes of \u003cem\u003eP. acuta \u003c/em\u003eA and B populations\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/6dcb5935b9de14861ac4ec50.png"},{"id":53912562,"identity":"adc77378-1faa-4517-a2f6-12fcc9146530","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54193,"visible":true,"origin":"","legend":"\u003cp\u003eFitness of \u003cem\u003ePhysella acuta\u003c/em\u003e populations in the laboratory. \u003cstrong\u003ea\u003c/strong\u003e Average growth of laboratory-maintained \u003cem\u003ePhysella acuta \u003c/em\u003esnails in populations A (white squares) and B (gray hexagons) over 76 days. x-axis: Time in days. y-axis: Shell size in millimeters. Measurements are connected by fitted curves (black for A snails, gray for B snails), with logistical fitted functions and R-squared values provided. \u003cstrong\u003eb\u003c/strong\u003eAverage number of egg masses produced over 108 days by individual snails from population A (white) and population B (gray). y-axis: Number of egg masses produced. \u003cstrong\u003ec\u003c/strong\u003e Time to maturity for single (individually housed) and paired snails of A (white) and B (gray) populations. y-axis: Time (days) elapsed before initial egg mass production. \u003cstrong\u003ed\u003c/strong\u003e Size at maturity (first egg mass production) for individually housed snails of populations A (white) and B (gray). y-axis: Size in millimeters\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/b7d5797dc5c0e0f7be838249.png"},{"id":53912548,"identity":"6b115d6f-72ab-4765-ad64-150bcee1204f","added_by":"auto","created_at":"2024-04-02 06:56:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34503,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of abiotic conditions recorded during experimental exposure of snails to conditions in the laboratory (LAB) and during rewilding (FIELD), \u003cstrong\u003ea-g \u003c/strong\u003eranges for temperature, general hardness, German carbonate hardness, pH, NH4, NO2, and NO3, calculated from data recorded every other day during four 1 week - and three 2 week trials\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/e95b0b383c846c5a3f19beca.png"},{"id":53912569,"identity":"375f0e7c-6c78-422e-975e-f5a0bff5c6e6","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":365140,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative population-level fecundity, determined as total egg mass number from starting populations of 20 snails without replacement of deceased snails for \u003cstrong\u003ea \u003c/strong\u003eseven 1 week trials and \u003cstrong\u003eb \u003c/strong\u003ethree 2 week trials. The data above the stippled line in \u003cstrong\u003ea\u003c/strong\u003e were recorded after the first week of three 2-week trails. Bars show population-level fecundity of laboratory A and B, and rewilded A and B populations. Values above bars indicate the cumulative egg mass production (top) and average population-level fecundity (bottom) from \u003cem\u003eP. acuta \u003c/em\u003esnail populations A and B for each experimental treatment group, with different letters indicating groups that display significant differences\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/56f725d4220e654c6e2a5a39.png"},{"id":53912575,"identity":"cb488cec-5d24-4dcb-8fc0-4aab39fc1c42","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":82461,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival and realized fecundity for laboratory-maintained (top) or rewilded (bottom) \u003cem\u003eP. acuta\u003c/em\u003e snails from populations A and B from 7 trials. Snails were evaluated over one-week or two-week periods. Significant differences are shown with an asterisk and p-value\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/752fdf45ba6cdbbe1bf51d92.png"},{"id":61946638,"identity":"bf0002ac-6cb8-49f1-99b6-360cdbd12134","added_by":"auto","created_at":"2024-08-07 11:39:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1454841,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/76859894-8d62-4e04-87aa-b571391babba.pdf"},{"id":53912544,"identity":"66fbf4f2-4ea1-40a0-a881-a9f372ab4241","added_by":"auto","created_at":"2024-04-02 06:56:09","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":55487,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/45bdf13cb46b06def58f5bc9.pdf"},{"id":53912564,"identity":"d91dcfa7-5334-42fd-b7ba-984909972947","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":101283,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/9b3cdcb636ab5726545262b5.pdf"},{"id":53912566,"identity":"236f4499-d29f-45d9-b46b-ed1340529c53","added_by":"auto","created_at":"2024-04-02 06:56:12","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":77432,"visible":true,"origin":"","legend":"","description":"","filename":"ESM3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/7bab83e18023ff5bd97cfb08.pdf"},{"id":53913078,"identity":"d52aae4b-6210-4a23-8820-99e6205158e3","added_by":"auto","created_at":"2024-04-02 07:04:12","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":33460,"visible":true,"origin":"","legend":"","description":"","filename":"ESM4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3994352/v1/9d13bf5ce9ec9be7bbbac66f.pdf"}],"financialInterests":"","formattedTitle":"Rewilding shows differential fitness of Physella acuta snail populations with different invasive potential","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe current distribution of the invasive freshwater snail \u003cem\u003ePhysella acuta\u003c/em\u003e extends far beyond the native range of North America (Vinarski \u003cspan citationid=\"CR145\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Due to natural dispersal (van Leeuwen et al. 2013) and human activities, including the aquarium trade (Duggan \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), \u003cem\u003eP. acuta\u003c/em\u003e is established across all continents except Antarctica. As a simultaneous hermaphrodite, capable of reproduction by selfing (Bousset et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), P. \u003cem\u003eacuta\u003c/em\u003e can efficiently colonize new habitats, persisting because of considerable tolerance to a range of environmental conditions (Min et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Spyra \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), including anthropogenic pollution (Alonso-Trujillo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Spyra et al. 2019). Bousset et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) proposed that \u003cem\u003eP. acuta\u003c/em\u003e \u0026lsquo;can be considered an excellent biological model for analyzing bioinvasions at a vast geographic scale.\u0026rsquo;\u003c/p\u003e \u003cp\u003eInvasive potential, however, is not a common feature of \u003cem\u003eP. acuta\u003c/em\u003e as a species. Previous research has unveiled two divergent mito-haplotypes (A and B) among North American \u003cem\u003eP. acuta\u003c/em\u003e snails (Nolan et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Haplotype markers identify globally invasive \u003cem\u003eP. acuta\u003c/em\u003e as members of population A, distinct from snails of a non-invasive population B that is restricted to North America, the native range of this species (David et al. 2022; Ebbs et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vinarski \u003cspan citationid=\"CR145\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering that populations A and B occur side-by-side in the same North American habitats (Ebbs et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nolan et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), both are assumed to have equal opportunity for dispersal. Remarkably, only a subpopulation within the species \u003cem\u003eP. acuta\u003c/em\u003e is globally invasive. This difference in invasive potential likely reflects inherent differences between \u003cem\u003eP. acuta\u003c/em\u003e populations A and B regarding the ability to respond to environmental changes, such as those that may be encountered when entering novel habitats.\u003c/p\u003e \u003cp\u003eThe environment contains diverse and variable abiotic and biotic stimuli that challenge the homeostasis of any organism (Hambrook and Hanington \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pandey et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pinaud et al. 2021; Romero et al. \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Alterations in a current habitat or environmental differences in a newly entered habitat may amount to environmental stress, with selective pressures possibly impacting reproductive output and survival, causing sharp reductions that lead to variations in the biographical ranges of particular organisms (Cunanan et al. 2018; Coutellec and Caquet \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Selye \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e1950\u003c/span\u003e). The evolution of biological adaptations (behavioral and genetic traits) enables organisms to better cope with environmental stressors and successfully colonize and persist in a specific environment. For instance, physid snail species display varying degrees of predator avoidance behavior, consisting of vigorous shell shaking and detachment from the substratum when encountering leeches (Frieswijk \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1957\u003c/span\u003e), and \u003cem\u003eP. acuta\u003c/em\u003e is thought to utilize expanded gene families to optimize immune responses against various pathogens (Schultz et al. \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In turn, these adaptations shape survival, growth patterns, and reproduction, key life history traits that define the biological fitness of (populations of) an organism (Stearns \u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e1976\u003c/span\u003e, \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Unique ecological challenges and selective pressures may lead to distinct adaptations within a species. For example, laboratory-maintained \u003cem\u003eLymnaea stagnalis\u003c/em\u003e snails comprised two populations with different lectin-based immune recognition capabilities (van der Knaap et al. \u003cspan citationid=\"CR139\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Furthermore, the natural distribution of the marine prosobranch snail \u003cem\u003eChlorostoma funebralis\u003c/em\u003e is determined by the expression levels of population-specific stress response proteins contributing to thermal tolerance (Gleason and Burton \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These phenomena could give rise to populations with varied fitness that all flourish within their native range, while specific populations have the capability for expanding into habitats with different environmental conditions.\u003c/p\u003e \u003cp\u003eThis study aims to analyze life history traits of two \u003cem\u003eP. acuta\u003c/em\u003e populations (A and B) to investigate a potential association of differential population-level fitness within this snail species with the evident invasive potential of \u003cem\u003eP. acuta.\u003c/em\u003e A well-defined genetic background was established to allow meaningful interpretation of population-level fitness in morphologically indistinguishable \u003cem\u003eP. acuta\u003c/em\u003e snails that co-occur in the field throughout the native range. Field-collected \u003cem\u003eP. acuta\u003c/em\u003e snails were genotyped as representative of populations A or B. Progeny produced by individual snails through selfing was used to establish laboratory populations representing A and B mito-haplotypes to evaluate population-level fitness in subsequent laboratory experiments. Studies in the laboratory provide useful views of stress responses (Feder and Hofmann \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), however, an artificial, constant experimental environment fails to capture natural situations involving an unpredictable environment with various stressors that challenge organismal fitness (Eads et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Integrating both laboratory and field-based investigations through exposure to a (semi)natural environment may reveal novel biological features from study organisms (see Beura et al. 2016; Leung et al. 2018; Lin et al. 2020; Martin et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pedersen and Babayan \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sepp\u0026auml;l\u0026auml; et al. \u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, neonatal immune function differs dramatically between laboratory-maintained mice and so-called \u0026ldquo;\u003cem\u003edirty mice\u003c/em\u003e\u0026rdquo; from the wild or obtained from pet stores (Hamilton et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Accordingly, differential fitness within \u003cem\u003eP. acuta\u003c/em\u003e was also studied using a rewilding approach. Specifically, laboratory-bred \u003cem\u003eP. acuta\u003c/em\u003e from genetically defined populations A and B were exposed temporarily to natural conditions in cages at a field site, to record life history parameters for comparison relative to a \u003cem\u003eP. acuta\u003c/em\u003e (control) group maintained under laboratory conditions. This approach provided increased sensitivity for investigating differential population-level fitness associated with the success and invasiveness of \u003cem\u003eP. acuta\u003c/em\u003e in nature.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection, haplotype characterization, and maintenance of snail populations\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePhysella acuta\u003c/em\u003e snails were field collected at Shady Lakes Commercial Fishing Facility, Albuquerque, New Mexico (35.216 N 106.6 W) in June 2020. The snails were kept individually in 150 ml tanks with artificial spring water at room temperature and fed lettuce, supplemented weekly with shrimp-based feed pellets (Nolan et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Initially, snails were identified as \u003cem\u003eP. acuta\u003c/em\u003e based on morphological features that include a gray body, thread-like tentacles, digitations along the mantle edge, and a sinistral shell exhibiting a \"fawn\" coloration with five distinct whorls and an oval-shaped aperture, (Paraense and Pointier \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). For sequence-based identification and characterization of mito-haplotype of individual snails, genomic DNA was extracted from recently produced snail egg masses using a CTAB-based method (Winnepenninckx et al. \u003cspan citationid=\"CR149\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Amplicons from the mitochondrial \u003cem\u003ecox1\u003c/em\u003e gene sequence, resulting from PCR (TaqGold) using primers LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG \u0026minus;\u0026thinsp;3\u0026rsquo; and HC02198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3\u0026rsquo; (Folmer et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) were sequenced (BigDye 3.1, ABI). Resulting sequences were aligned (Clustal X, Thompson et al. \u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) for phylogenetic analysis (RaxML, Kozlov et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) to determine the mito-haplotype of snails as A or B (Ebbs et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Following mito-haplotype characterization, single snails were allowed to reproduce via selfing (\u003cem\u003eP. acuta\u003c/em\u003e is a simultaneous hermaphrodite), using single egg masses to initiate uniparental populations representing mito-haplotypes A and B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All snails were kept in 20 L tanks as described above. The mito-haplotype and rDNA cassette of 24 snails (representing each population, 13 A and 11 B) were characterized by RNA-seq (see below). All experiments employed reproductively mature (indicated by egg-laying) snails with a shell length\u0026thinsp;\u0026ge;\u0026thinsp;5 mm, experimentally determined as described below.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory fitness\u003c/h2\u003e \u003cp\u003eSnails from each population were evaluated for fitness under laboratory conditions employing three of the seven traits of life history theory listed by Stearns (\u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e1976\u003c/span\u003e, \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e1992\u003c/span\u003e): growth pattern, age (size) at maturity, and fecundity (number of egg masses produced). Growth rates of each \u003cem\u003eP. acuta\u003c/em\u003e population were determined starting with ten newly hatched snails (1 mm shell length) kept in 20 L tanks. The shell length (apex to outside edge of aperture) of each snail was recorded using a ruler (to the closest half mm), twice a week over a 76-day time interval. Three separate trials were performed. Data points were averaged, and curve fitting was used to plot and compare growth rates.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhysella acuta\u003c/em\u003e is a simultaneous hermaphrodite that preferentially reproduces by outcrossing in the presence of other snails, or by selfing in isolation. The latter may be associated with a delay in reproduction, a so-called lag time, (Tsitrone et al. \u003cspan citationid=\"CR137\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Newly hatched snails (shell length\u0026thinsp;~\u0026thinsp;1 mm) were kept separately (n\u0026thinsp;=\u0026thinsp;5) or as pairs (2 x 5) of the same population (A or B) in separate containers (150 ml). The number of days was recorded until an egg mass was first produced, and the shell length of single or paired snails in that container was measured (to the closest half mm) to determine the minimum size at which snails were reproductively mature. Three trials were performed.\u003c/p\u003e \u003cp\u003eFecundity was measured as the average number of total egg masses produced over a 108-day interval by individual snails from \u003cem\u003eP. acuta\u003c/em\u003e populations A and B. Ten newly hatched snails (1 mm) from each population were separated into individual 150 ml containers. Twice a week, egg masses produced by each snail were enumerated and removed. Three trials were performed.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhysella acuta\u003c/b\u003e \u003cb\u003efitness in rewilding experiments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCohorts of snails from each population were evaluated simultaneously for fitness under field and laboratory conditions, employing two of the seven traits of life history theory as listed by Stearns (\u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e1976\u003c/span\u003e, \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e1992\u003c/span\u003e): survival (number of live snails after experimental exposures), and fecundity, considering both population-level- and realized fecundity (see definitions below).\u003c/p\u003e \u003cp\u003ePopulations of A and B \u003cem\u003eP. acuta\u003c/em\u003e snails were rewilded by placing genetically- characterized, laboratory-bred \u003cem\u003eP. acuta\u003c/em\u003e snails temporarily under natural conditions. This rewilding approach examines how organisms function under actual natural environmental conditions, supplementing observations made under controlled laboratory conditions (Leung et al. 2018; Lin et al. 2020; Pedersen and Babayan \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe relative fitness of populations A and B in the field and the laboratory was investigated using groups of adult siblings of each type of snail (n\u0026thinsp;=\u0026thinsp;20). Snails were housed in flow-through cages (20 L) with two 12 x 15 cm screens with 1 mm mesh size. Snails were exposed to both experimental conditions (laboratory and field) for either one- or two-week intervals. Two field sites were used: one experiment was done at Shady Lakes (the original snail-collection site), and the others at the Rio Grande Nature State Park (35.128 N 106.685 W). \u003cem\u003ePhysella acuta\u003c/em\u003e occurs naturally at both locations. In the laboratory, the flow-through cages holding \u003cem\u003eP. acuta\u003c/em\u003e snails were kept in a 150 L artificial pond containing snail-conditioned water. Every second day, all snails were fed lettuce and fitness parameters were recorded. Survival was determined by the number of individual \u003cem\u003eP. acuta\u003c/em\u003e snails that were actively moving or firmly adhering to substrates in the cages. Inactive snails were examined to confirm death, and empty shells were removed from the cages. Fecundity was tracked by enumerating and removing all egg masses in each cage. Population fecundity was determined as total egg masses produced by the snail population in a cage, starting at n\u0026thinsp;=\u0026thinsp;20, and irrespective of the demise of individual snails, during the complete one- or two-week exposure interval. Data recorded after the first week of two-week exposure intervals and results from the one-week exposures were combined for analysis. Realized fecundity was calculated every second day from the number of egg masses divided by the number of living snails in a cage. Four one-week trials and three two-week trials were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWater chemistry for rewilding experiments\u003c/h2\u003e \u003cp\u003eTo ascertain variations between the environmental conditions experienced by field and laboratory populations of \u003cem\u003eP. acuta\u003c/em\u003e snails, several environmental parameters were recorded. These included water temperature, nitrite and nitrate levels, general hardness (calcium and magnesium content), German carbonate hardness, and pH, which were monitored every second day throughout all trials using the freshwater master test kit from API.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA and RNA Extraction from\u003c/b\u003e \u003cb\u003ePhysella acuta\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing each rewilding experiment, RNA was extracted from each surviving \u003cem\u003eP. acuta\u003c/em\u003e snail from populations A and B, and from both field and laboratory exposures. Individual whole snails were placed in 1.5 ml tubes, disrupted with a pestle, and mixed with 1 ml Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). To accommodate the screening of all field-exposed snails for naturally incurred parasites (see below), the Trizol protocol was modified (according to the manufacturer\u0026rsquo;s instructions) to also extract DNA in addition to RNA. The quality of the extracted nucleic acids was evaluated spectrophotometrically (NanoDrop 6000, Thermo Scientific).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eEvaluation of rewilded snails for trematode parasites\u003c/h2\u003e \u003cp\u003eField exposure may lead to trematode parasite infection in \u003cem\u003eP. acuta\u003c/em\u003e snails. The exclusion of infected snails from this study is important because these parasites impact snail host neurophysiology and general fitness (e.g. De Jong-Brink \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Half of the rewilded snails served as sentinel snails to determine the risk of parasite infections for field-exposed \u003cem\u003eP. acuta\u003c/em\u003e. After a rewilding trial, these snails were maintained in 20 L tanks in the laboratory and checked after 3- and 6 weeks for the shedding of cercaria as an indication of a digenean trematode infection.\u003c/p\u003e \u003cp\u003eDNA was extracted from rewilded snails (see above) for PCR screening against potential trematode parasite infections. Genbank entries of \u003cem\u003e28S\u003c/em\u003e-derived sequences from digenean trematodes that employ \u003cem\u003eP. acuta\u003c/em\u003e as a host were aligned (Clustal X) and conserved regions were used to design primers (5\u0026rsquo; to 3\u0026rsquo;) DT_FP_28S: CACTTATCAAGTGTTGTGC and DT_RP_28S: CTACACCACAGACTATTGG. The PCR reactions used TaqGold (as above) with 4 \u0026micro;l of the DNA extracted from rewilded \u003cem\u003eP. acuta\u003c/em\u003e as the template in a 50\u0026micro;l reaction, Tm 55\u0026deg;C for a total of 40 cycles with a final extension at 72\u0026deg;C for 5 min. Presence of amplicons was checked by agarose gel electrophoresis.\u003c/p\u003e \u003cp\u003eFinally, \u003cem\u003e18S\u003c/em\u003e, \u003cem\u003e28S\u003c/em\u003e, \u003cem\u003e16S\u003c/em\u003e, and \u003cem\u003ecox1\u003c/em\u003e sequences of digenean trematodes known to infect \u003cem\u003eP. acuta\u003c/em\u003e were retrieved from GenBank, see table (Online Resource 1) and blasted against raw RNA-seq reads collected from rewilded snails, available from this study.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq\u003c/h2\u003e \u003cp\u003eIndividual snail RNA samples of 24 snails (7 field-exposed, 6 laboratory-kept snails from population A; 5 field-exposed, 6 laboratory-kept snails from population B) from a one-week rewilding experiment performed at the Shady Lakes field site were used for commercial library preparation and Illumina NovaSeq RNA-Seq sequencing (Novogene). Raw RNA-Seq paired-end (PE150) reads were trimmed with Trimmomatic (Bolger et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and adapters were removed using fastp (Chen et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) employing Galaxy (Pond et al. 2009). The resulting RNA-seq data were used to set up a local blastable database (Sequenceserver, Priyam et al. 2019). The transcriptomic data were examined for parasite-derived sequences to screen for parasite-infected snails (described above).\u003c/p\u003e \u003cp\u003eRNA-seq data was used to assemble the mitogenomes for each individual snail (see Bunk\u0026oacute;czi et al. 2011; Forni et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Smith \u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) employing MitoBIM (Hahn et al. 2014) with the \u003cem\u003eP. acuta\u003c/em\u003e A and B mitogenomes (GenBank NC_023253, JQ390526; Nolan et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) as references. Additionally, BBDUK (Guzman and D\u0026rsquo;Orso \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) was used to collect RNAseq raw reads similar to these reference mitogenomes for assembly using MEGAHIT (Li et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Any discrepancies between the resulting assemblies were reconciled using ras paired-end sequence data. Automated mitogenome annotation (MITOS; Bernt et al. 2013; Donath et al. 2019) was checked using criteria established by Fourdrilis et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) through Snapgene (Dotmatics). The nuclear rDNA cassette of each of the 24 snails was reconstructed manually from RNA-seq data by iterative blasting, initiated with \u003cem\u003eP. acuta\u003c/em\u003e ITS sequences (GenBank KF316326-9). Multiple sequence alignments (Clustal X) were checked by eye to compare the mitogenomes and rDNA cassettes of each snail population among individuals and between populations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eGrowth patterns, reproductive output, and size at maturity (single snails) of populations A and B within the laboratory were compared using a one-tailed t-test. The age at maturity of A and B snails (both kept single and in pairs) was compared using a Kruskal-Wallis test. Differences between abiotic conditions in the field and laboratory for \u003cem\u003eP. acuta\u003c/em\u003e were determined using one-tailed t-tests for water temperature, nitrite and nitrate levels, degree of general hardness, degree of carbonate hardness, and pH. Various statistical tests were applied to assess the life history parameters studied in the rewilding experiments between A and B snail populations under laboratory and field conditions. One-tailed t-tests were utilized for realized fecundity, chi-squared tests for population-level fecundity, and a rank test (via the R package 'survival'; accessed through \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://astatsa.com/LogRankTest/\u003c/span\u003e\u003cspan address=\"https://astatsa.com/LogRankTest/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Vasavada \u003cspan citationid=\"CR143\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for comparing survival in rewilding experiments.\u003c/p\u003e \u003cp\u003eAll abiotic environmental parameters were plotted as bell curves to identify extreme conditions experienced by snails in the laboratory and during rewilding over one- and two-week trials. Extreme high and low values were defined by values differing by greater than one standard deviation from the mean. The range of each abiotic parameter recorded during a specific trial was plotted onto the bell curve to check for potential associations between environmental variation and recorded fitness (survival and realized fecundity).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003ePhysella acuta\u003c/b\u003e \u003cb\u003efrom the field and laboratory populations\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe initial morphology-based identification of field-collected snails as Physidae was further developed by identification as \u003cem\u003ePhysella acuta\u003c/em\u003e by phylogenetic analysis of \u003cem\u003ecox1\u003c/em\u003e sequences amplified by PCR from genomic DNA extracted from embryos in single egg masses produced by individual snails via selfing. Of 23 \u003cem\u003eP. acuta\u003c/em\u003e collected in the same location, ten snails exhibited mito-haplotype A and 13 mito-haplotype B (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Two separate laboratory-maintained populations representing each mito-haplotype (A or B) were initiated with progeny produced by selfing from one individual of these field-collected \u003cem\u003eP. acuta\u003c/em\u003e snails (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRNA-seq data collected from 24 third-generation snails of the inbred laboratory populations were used to assemble the rDNA cassette for individual snails (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The sequences (7,023 bp); 13 from population A and 11 from B were identical within the populations but differed by two single nucleotide polymorphisms (0.03%) between populations A and B. BLAST searches showed that the rDNA sequences had the highest similarity to previous GenBank entries for \u003cem\u003eP. acuta\u003c/em\u003e. Assembly and analysis of full-length mitogenomes showed identical sequences within the populations, while showing a 9.3% in sequence content between populations A and B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Specific genes within these mito-haplotypes displayed different levels of sequence identity, ranging from 95% for \u003cem\u003ecox1\u003c/em\u003e to 72% for \u003cem\u003enad4L\u003c/em\u003e, see table (Online Resource 2). Furthermore, the mitogenome of population A was most similar to the previously characterized mitogenome of type A \u003cem\u003eP. acuta\u003c/em\u003e. Likewise, the mitogenome of population B was most similar to the previously described mitogenome of \u003cem\u003eP. acuta\u003c/em\u003e type B (NC_023253, JQ3905261; Nolan et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFitness in the laboratory\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePhysella acuta\u003c/em\u003e snail populations A and B showed only minor differences in fitness parameters when maintained under controlled and consistent environmental conditions in the laboratory. The average growth rate of individual snails (both by size measurements and fitted curve comparisons) and the average total number of egg masses per snail over time were not significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). The age of snails at maturity (first production of egg masses), either kept as individuals or in pairs, showed some variation but was not significantly different between populations A and B (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Within each population, however, there was a lag time in reproduction between single and paired snails. On average, single snails from population A first produced an egg mass 8 days later than paired snails; this difference was 14 days for population B. In both instances, the time differences due to the observed lag time were not statistically significant (ANOVA, A snails p\u0026thinsp;=\u0026thinsp;0.880; B snails p\u0026thinsp;=\u0026thinsp;0.585). Regarding size, individually kept \u003cem\u003eP. acuta\u003c/em\u003e snails first reached maturity at 6.5 mm for population A and 7 mm for population B (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). When kept as pairs, \u003cem\u003eP. acuta\u003c/em\u003e of both populations first produced an egg mass at 5 mm in size. These differences in size, nor the other fitness parameters tested under this section, were not statistically significant at \u0026#120572;= 0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRewilding experiments\u003c/h2\u003e \u003cp\u003eRelative to the laboratory maintenance, the abiotic environmental conditions at the field site were significantly different (p-values\u0026thinsp;\u0026le;\u0026thinsp;0.05). Water temperature, pH and water hardness (general hardness, German carbonate hardness) had higher values in the field (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). Ammonia levels were higher in the laboratory (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), whereas nitrates and nitrites were not detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, g). Temperature and pH were more variable in the field, and water hardness and ammonia levels were more so in the laboratory. Parasite infections were not observed in sentinel snails during rewilding experiments, nor were infections detected from experimental snails, either by PCR or analyses of RNA-seq data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe population-level fecundity of rewilded snails in populations A and B was three times higher than laboratory-maintained snails of the same populations at one- and two-week intervals. When comparing A and B laboratory-maintained snails, the total and average numbers of egg masses in 7 trials over one week did not differ significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In one of these trials, however, population B produced 112 egg masses, significantly more than the 77 from population A (p\u0026thinsp;=\u0026thinsp;0.011). Under rewilding conditions, the average number of egg masses per one-week trial did not differ (p\u0026thinsp;=\u0026thinsp;0.123). However, population A snails produced more egg masses in total over all seven trials than snails from population B (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with population A significantly outperforming B in 3 of 7 trials (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). More pronounced differences were recorded from the three trials of two-week time intervals. Under laboratory conditions, population A produced more egg masses than population B, both for the combined total egg masses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the average number over three trials (p\u0026thinsp;=\u0026thinsp;0.003), with population A significantly outperforming B in 2 of 3 trials (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). These trends were more prominent under field conditions, with both greater total (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and average (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) numbers of egg masses recorded from rewilded snails of population A versus B. Population A generated significantly more total egg masses than B in 2 of 3 trials, p\u0026thinsp;\u0026le;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCumulative population-level fecundity (total number of egg masses) of 20 \u003cem\u003ePhysella acuta\u003c/em\u003e snails from population A and B, laboratory maintained or rewilded, over 7 one- and 3 two-week trials\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eONE WEEK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLABORATORY A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLABORATORY B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eREWILDED A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eREWILDED B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e139\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e117\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e203\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e135\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTWO WEEKS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e324\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e176\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIAL 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e218\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eComparison of survival in the laboratory during one- and two-week intervals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) revealed minimal snail mortality with modestly higher survival rates for A snails compared to B snails (trials 1, 2, 3, and 5), except for trial 4 (two dead A snails versus no mortality of B snails), and equal numbers of population A and B survived in trials 6 and 7. These laboratory trials showed no significant differences in survival rates. In contrast, rewilded snails displayed significant differences in survival, with A snails outperforming B snails in trials 1, 6, and 7. Trials 2, 3, and 4 each showed a single mortality for population A, with all B snails surviving (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, no significant differences). Notably, no rewilded snails from A and B populations died in trial 5.\u003c/p\u003e \u003cp\u003eDuring either one- or two-week intervals, the realized fecundity of both populations was similar in 5 of 7 trials under laboratory conditions. Significant differences were evident in trial 3, with B snails outperforming A snails (p\u0026thinsp;=\u0026thinsp;0.048, marginally significant), and in trial 4, with greater realized fecundity for A snails compared to B snails (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Rewilding in the field showed that the realized fecundity of A snails was similar to B snails, with minor differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), except in trials 3 and 4, where A snails had significantly higher realized fecundity compared to B snails (p values \u0026lt; \u0026#120572;).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of environmental variation did not indicate any association between differential fitness and the abiotic parameters tested, for example see graph (Online Resource 4), otherwise not shown.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe snail \u003cem\u003ePhysella acuta\u003c/em\u003e is globally invasive (Vinarski \u003cspan citationid=\"CR145\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and considered to be an exceptionally efficient invader (Bousset et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It is important to recognize that this trait is not universal to all individuals of this snail species. Population studies using \u003cem\u003ecox1\u003c/em\u003e as a marker showed that the global distribution of \u003cem\u003eP. acuta\u003c/em\u003e only involves a distinct genetic lineage of this species, termed population A (Ebbs et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Another lineage, \u003cem\u003eP. acuta\u003c/em\u003e 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. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nolan et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Vinarski \u003cspan citationid=\"CR145\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral 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 \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) 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 \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pozzi and Dowling \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vogt \u003cspan citationid=\"CR147\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), or the emergence of new gene alleles, including advantageous immune traits. Indications of the latter are provided by the presence in \u003cem\u003eP. acuta\u003c/em\u003e 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. \u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eComparisons 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 \u003cem\u003eP. acuta\u003c/em\u003e, and invasiveness in general. The side-by-side presence of both populations in New Mexico (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, also see Ebbs et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nolan et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) suggests the absence of conditions that constrain expansion of population B beyond the native range.\u003c/p\u003e \u003cp\u003eTo prevent confounding effects from working with a field-collected mixture of morphologically indistinguishable snails from both \u003cem\u003eP. acuta\u003c/em\u003e populations, this study established laboratory-maintained populations of genetically characterized \u003cem\u003eP. acuta\u003c/em\u003e 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 \u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e1976\u003c/span\u003e, \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The \u003cem\u003ecox1\u003c/em\u003e marker divided field-collected physid snails that were morphologically indistinguishable into two clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) that separated the \u003cem\u003ecox1\u003c/em\u003e sequences from the original A- and B-type \u003cem\u003eP. acuta\u003c/em\u003e (Nolan et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe selfing reproduction mode of \u003cem\u003eP. acuta\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Analyses of RNA-seq data confirmed that this approach generated two laboratory-maintained populations of \u003cem\u003eP. acuta\u003c/em\u003e, 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). 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\u0026thinsp;~\u0026thinsp;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. (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These results underscore the considerable mitogenome sequence variability in \u003cem\u003eP. acuta\u003c/em\u003e, 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 \u003cem\u003eP. acuta\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe availability of laboratory-maintained populations of genetically defined \u003cem\u003eP. acuta\u003c/em\u003e enabled the study of possible association of population-specific geographical distribution patterns (and invasive potentials) with differential population-level fitness. Accordingly, these two \u003cem\u003eP. acuta\u003c/em\u003e 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 \u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e1976\u003c/span\u003e, \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Each parameter differed by small values that were not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, 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 \u003cem\u003eP. acuta\u003c/em\u003e population (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). \u003cem\u003ePhysella acuta\u003c/em\u003e prefers reproduction by outcrossing (mating with another partner), but in the absence of a mating partner, it will ultimately engage in selfing (No\u0026euml;l et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tsitrone et al. \u003cspan citationid=\"CR137\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). This lag time was shorter by ~\u0026thinsp;1 week (although not significant) in population A snails compared to population B snails.\u003c/p\u003e \u003cp\u003eWhereas 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 \u003cem\u003eP.acuta\u003c/em\u003e snails.\u003c/p\u003e \u003cp\u003eSeveral 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. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Flies and Wild Comparative Immunology Consortium 2020; Martin et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pedersen and Babayan \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For example, mice and oysters (\u003cem\u003eCrassostrea hongkongensis\u003c/em\u003e) 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. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Oysters in locations with different levels of ocean acidification show distinct immune responses to \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e infection (Dang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMindful 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 \u003cem\u003eP. acuta\u003c/em\u003e 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 \u003cem\u003eP. acuta\u003c/em\u003e (e.g., Camargo and Alonso \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; De Castro-Catal\u0026agrave; et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Prieto-Amador et al. \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; 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 \u003cem\u003eP. acuta\u003c/em\u003e (Spyra, \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). 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, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), as exemplified by the natural presence in the field of digenean trematodes, specialized parasites of \u003cem\u003eP. acuta\u003c/em\u003e (Kraus et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). 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 \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Hall et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) was excluded from this study.\u003c/p\u003e \u003cp\u003eThe rewilding approach not only tested the impact of different environmental conditions on the fitness of \u003cem\u003eP. acuta\u003c/em\u003e 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 \u003cem\u003eP. acuta\u003c/em\u003e that was not provided by the initial laboratory-based study of fitness in individual snails.\u003c/p\u003e \u003cp\u003eRewilding 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 \u003cem\u003eP. acuta\u003c/em\u003e was similar with a small (not significant) advantage for population A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). With rewilding, certainly combined with an increased 2-week experimental time interval, population A snails displayed significantly higher fitness than population B \u003cem\u003eP. acuta\u003c/em\u003e. First, the survival rate of both populations was usually similar in these same trials, except for dramatic crashes with \u0026ge;\u0026thinsp;55% mortality in snails of population B kept under rewilding conditions that did not similarly impact population A \u003cem\u003eP. acuta\u003c/em\u003e (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 \u003cem\u003eP. acuta\u003c/em\u003e was marginally significant.\u003c/p\u003e \u003cp\u003eThis 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 \u003cem\u003eP. acuta\u003c/em\u003e. Although such modest differences may become biologically meaningful, similar fitness levels may explain why both populations of \u003cem\u003eP. acuta\u003c/em\u003e persist side-by-side in the environmental conditions that prevail in the native range of the species \u003cem\u003eP. acuta\u003c/em\u003e. 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 \u003cem\u003eP. acuta\u003c/em\u003e 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. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Godwin et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sepulveda and Moeller \u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, no correlation was revealed between any particular environmental parameter and the observed fitness of \u003cem\u003eP. acuta\u003c/em\u003e, as shown in the graph (Online Resource 3). \u003cem\u003ePhysella acuta\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eDefinition 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 \u003cem\u003eP. acuta\u003c/em\u003e, 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. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Papier et al. \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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 \u003cem\u003eP. acuta\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003e \u003cb\u003eEthics declarations\u003c/b\u003e \u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eConflicts of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003ePermits\u003c/h2\u003e \u003cp\u003eThe Energy, Minerals and Natural Resources Department, State Park Division, New Mexico provided permits for field research.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eKevin A. McQuirk acknowledges graduate research scholarships from the Biology Department and the Graduate and Professional Student Association of the University of New Mexico. Maria G. Castillo was supported by NIH 5SC2AI133645.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eKevin A McQuirk, Maria G Castillo and Coen M Adema contributed to the study conception and design. Material preparation, data collection and analysis were performed by Kevin A McQuirk, Julie DeCore, Maria G Castillo, and Coen M Adema. The first draft of the manuscript was written by Kevin A McQuirk and Coen M Adema and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003e \u003cb\u003eAcknowledgments\u003c/b\u003e.\u003c/h2\u003e \u003cp\u003eWe thank the superintendents of the Rio Grande Nature Park in Albuquerque, New Mexico for access to the Discovery Pond. Field research permits were provided by the Energy, Minerals and Natural Resources Department, State Park Division, New Mexico. Maria G. Castillo was supported by NIH 5SC2AI133645. Kevin A. McQuirk acknowledges graduate research scholarships from the Biology Department and the Graduate and Professional Student Association of the University of New Mexico.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll sequence data are available from GenBank under accessions: OQ561510- 21; OQ918702; OQ923605; OR003928-36; OR026032; OR208260-61; OR220884\u0026ndash;903.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlonso-Trujillo M, Mu\u0026ntilde;iz-Gonz\u0026aacute;lez AB, Mart\u0026iacute;nez-Guitarte JL (2020) Endosulfan exposure alters transcription of genes involved in the detoxification and stress responses in \u003cem\u003ePhysella acuta\u003c/em\u003e. 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Trends Genet 9:407. https://doi.org/10.1016/0168-9525(93)90102-n\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Physella acuta, realized fecundity, fitness, invasiveness, mitogenome, hermaphrodite","lastPublishedDoi":"10.21203/rs.3.rs-3994352/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3994352/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe freshwater snail \u003cem\u003ePhysella acuta\u003c/em\u003e is globally invasive. Within this species, however, \u003cem\u003ecox1\u003c/em\u003e haplotype markers distinguished a globally invasive population (A) from a non-invasive population (B) restricted to North America, the native range of \u003cem\u003eP. acuta\u003c/em\u003e. This study investigated whether invasiveness is associated with differential population fitness. Field-collected \u003cem\u003eP. acuta\u003c/em\u003e were genetically characterized to establish laboratory populations representing mito-haplotypes A and B. While the nuclear rDNA cassette (7,023 nt) differed only by 0.03% between populations A and B, the mitogenome haplotypes differed in size (14,383 vs 14,333 bp) and sequence content (~\u0026thinsp;9%). Under controlled laboratory conditions, growth rate, age at maturity, size at maturity, and reproductive output did not show fitness differences between populations A and B (3 trials). Population fitness was also studied using a rewilding approach. Survival and fecundity of A and B snails were evaluated during one- or two-week intervals among cohorts of 20 laboratory-bred \u003cem\u003eP. acuta\u003c/em\u003e adult snails in flow-through cages in the laboratory or exposed to natural field conditions. Only modest differences in fitness parameters were indicated under laboratory conditions, providing no clear association of population fitness with global distribution patterns. Under field conditions, however, population level fecundity differed with population A having a 3-fold greater fitness than population B in 5 of 7 trials (survival in 3 trials; realized fecundity in 2 trials). Whereas laboratory-based studies indicated only minor differences, the rewilding approach showed significant differential fitness between \u003cem\u003eP. acuta\u003c/em\u003e populations A and B that differ in invasiveness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Rewilding shows differential fitness of Physella acuta snail populations with different invasive potential","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-02 06:55:53","doi":"10.21203/rs.3.rs-3994352/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57ed45e4-4b28-4d1e-b098-a3af51dcaf3d","owner":[],"postedDate":"April 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-07T11:31:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-02 06:55:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3994352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3994352","identity":"rs-3994352","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}