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Abiotic tolerances in different life stages of apple snails Pomacea canaliculata and Pomacea maculata and the implications for distribution.

ABSTRACT In the United States, populations of nonindigenous Pomacea spp. have been reported in at least 10 states, and the rate of spread has been greatest in the southeastern United States, particularly in Florida. Factors impacting successful establishment of molluscs include salinity, pH, and desiccation tolerance. This article reports on median survival and growth of Pomacea canaliculata and Pomacea maculata (hatchlings through adults) in 28-day trials focused on these factors with an ambient condition serving as a control (e.g., 0 = salinity control). Overall hatchlings had the lowest survival, followed by juveniles then adults. Median survival was less than 28 days for all salinity treatments greater than 16. Median survival was 28 days for all pH levels (5.5- 9.5) for juveniles and adults of both species. Median survival in desiccation treatments (>80% relative humidity and wet sand or <60% relative humidity and dry sand) was 28 days for adults of both species and for juvenile P. canaliculata. The closer a treatment was to the control level, the greater the increases in shell length and width. In all treatments, snails experienced a loss in mass; the closer a parameter to the control typically resulted in the greatest loss in mass. The ability of these species to tolerate such diverse conditions will leave many systems vulnerable to invasion.

KEY WORDS: pH, desiccation, salinity, Pomacea canaliculata, Pomacea maculata

INTRODUCTION

New World apple snails (Ampullariidae: Pomacea) are native to South and Central America, with one species Pomacea paludosa (Say, 1829) native to North America. Snails in the genus Pomacea are amphibious, lentic, and lotic, and are generally located in areas of aquatic vegetation. Pomacea spp. are considered to be herbivores although it is not uncommon to observe foraging on carrion or funneling biofilm. The channeled apple snail Pomacea canaliculata (Lamarck, 1819) is considered to be one of the world's worst top 100 invaders (Lowe et al. 2000). The channeled apple snail and the island apple snail Pomacea maculata (Perry, 1810) have been introduced into many countries and have subsequently become established in natural and artificial aquatic systems. Introduced species in the United States include P. canaliculata, P. maculata, Pomacea diffusa (spike-topped apple snail, Blume, 1957), and Pomacea sp. (formerly described as Pomacea haustrum but is pending revision, Reeve, 1856) (Hayes et al. 2009, 2012, Rawlings et al. 2007). In the United States, the average adult size ranges from 40 (P. paludosa) to 120 mm (P. maculata) in length.

In the United States, populations of nonindigenous Pomacea spp. have been reported in at least 10 states, from South Carolina to Texas, and in Arizona, California, and Hawaii. The rate of spread of Pomacea spp. has been greatest in the southeastern United States, particularly in Florida. Nonindigenous Pomacea spp. were probably brought to Florida for human consumption and the aquarium trade but escaped cultivation, and had become established in the wild by 1978 (Thompson 1997). The most prevalent species is Pomacea maculata, found from Miami, in the south, to the western panhandle in the north. Although Pomacea canaliculata (identified by T. Collins, Florida International University, personal communication) is believed to be limited to northeast Florida, their continued spread through this region (Bematis & Warren 2014) suggests that populations may continue to establish through the southeast. There are isolated records in north and south Florida for Pomacea diffusa, whereas Pomacea sp. is believed to be limited to the Loxahatchee National Wildlife Refuge. Importation and interstate transport of all nonindigenous Pomacea (except P. diffusa) is prohibited by the U.S. Department of Agriculture Animal and Plant Health Inspection Service. Some states (e.g., Hawaii, Texas) have specific legislation prohibiting possession of nonindigenous snails; however, Florida does not have any restrictive regulations and the snails are readily available in the pet trade industry.

In the southeast United States, the snails are considered a threat to normal ecosystem functioning because their voracious appetite for aquatic vegetation may lead to trophic shifts (Carlsson et al. 2004). A review of the Florida Agricultural Statistics database shows Florida's aquatic vegetation industry has routinely exceeded $13 million annually, and provides a variety of plant species for restoration activities, ornamental systems, and the pet trade industry. The presence of snails within aquaculture facilities and subsequent transportation of the vegetation have resulted in the introduction of nonindigenous Pomacea in multiple locations throughout Florida (e.g., Lake Josephine, Highlands County, FL; Sweetwater Branch Park, Alachua County, FL). The resulting damage in one system in Tallahassee, FL, was an estimated $500,000, as the majority of the plants were consumed by Pomacea maculata (Bernatis, personal communication). There are a number of anecdotal reports suggesting that introduction of P. maculata has caused displacement of native Pomacea paludosa. This "displacement" has resulted in P. maculata being implicated in the decline of the federally endangered Florida snail kite (Rostrhamus sociabilis plumbeus), which feeds on the smaller native P. paludosa. Cattau et al. (2010) indicate that the young birds are unable to handle the larger size of the nonindigenous snails, although observations indicate Snail Kite populations are increasing in locations with high numbers of P. maculata (Cattau et al. 2016).

Despite their abundance and broad distribution, there is little basic biological research on nonindigenous Pomacea, particularly in terms of physiological tolerances in their introduced ranges (Ramakrishnan 2007). Several abiotic factors are known to influence mollusc distribution, including oxygen, temperature, pH, salinity, [Ca.sup.2+], food availability, and desiccation events (Prosser & Heath 1991, Somero 1995, Jacobsen & Forbes 1997, Berezina 2001, Martin et al. 2001, Estebenet & Martin 2002, Martin & Estebenet 2002, Ansart & Vernon 2003). The presence of a siphon and a lung reduces Pomacea's dependence on dissolved oxygen, potentially increasing habitats suitable for establishment (Burky et al. 1972, Freiberg & Hazelwood 1977, Santos & Mendes 1981, Aldridge 1983, Ito 2002, Seuffert & Martin 2009a, 2009b). Thermal tolerance studies indicate a wide variation among species with Pomacea canaliculata and Pomacea maculata surviving temperatures ranging from -2[degrees]C to 40[degrees]C for at least short periods (10-14 days) (Burky et al. 1972, Albrecht et al. 1999, Martin et al. 2001, Ramakrishnan 2007, Matsukura et al. 2008,2009, Seuffert et al. 2010, Bernatis unpublished data). Byers et al. (2013) attempted to model the future distribution of exotic Pomacea in the southeast United States, with an emphasis on pH. Unfortunately, reliance on limited, third-party databases to predict snail distribution limits was a significant shortcoming of their approach.

The paucity of research on the ecophysiology of Pomacea is surprising, given that key elements of invasion success are biological limitations in relation to a novel environment. Therefore, the objectives of this study were to obtain physiological tolerance information for Pomacea canaliculata and Pomacea maculata throughout their different life-history stages using laboratory survival trials. Animals were subjected to varying degrees of salinity, pH, and desiccation, because these may pose major constraints on the survival and dispersal of these species in Florida (Lodge et al. 1987, Jordan & Deaton 1999, Ramakrishnan 2007, Lazzarino et al. 2009). The tolerances of P. maculata and P. canaliculata were investigated at ambient levels, typical to those that they may experience in their natural habitat. The results of this work will serve multiple purposes: (1) provide data for the development of predictive distribution models, (2) aid in the development of management practices for prevention of the spread of snails, and (3) provide data on the basic biology of apple snails.

MATERIALS AND METHODS Collection and Holding

Snails were collected throughout the year. Island apple snails Pomacea maculata were hand collected from lakes in north central Florida (Leon County) and central Florida (Polk County). Channeled apple snails Pomacea canaliculata were collected from a residential retention lake in northeast Florida (Duval County) harboring the only known location of that species. Locations were selected from sites with genetic confirmation data (Rawlings et al. 2007) and confirmed with soft tissue anatomy, shell morphology (large specimens only

>65 mm in length), and egg mass appearance. For the Duval County population, species was genetically confirmed by Timothy Collins (Florida International University) and was the first official population of P. canaliculata in Florida.

The holding and experimental tanks were located in a climate-controlled indoor facility. A 50:50 mix of pond and well water was used in 113-1 tanks to house snails prior to using them in experiments. Tanks were maintained under the following conditions, which were based on observed parameters of collection sites and standard aquaculture protocols: temperature 23-25[degrees]C; dissolved oxygen saturation 80%-90% (via air stones); pH 7.5-8.0; ammonia (nitrate and nitrite) less than 0.0.02 ppm (Swann 1993). Water was continuously filtered through mechanical and chemical media. Snails were fed ad libitum [Hydrilla verticillata, Hydrocotyle sp., Cucumis melo (cantaloupe), and Pyrus spp. (pears)] every 48-72 h. Snails were kept in holding tanks for no more than 2 wk prior to trials. The small size of hatchlings and juveniles required for testing precluded field collection, therefore, these age groups were reared in the laboratory. Egg masses deposited by captive adults during the 1st wk of captivity were hatched, and these hatchlings were used in the trials. Individual snails were used only once in all experiments.

General Procedures

Snail survivorship was tested for in three treatments: pH, salinity, and desiccation tolerance. A 28-day period was used owing to reports of snails surviving lake dry-down events from which standing water was removed for at least 2 wk. Three age groups were used in each treatment: hatchlings (<4 day old and <3.0 mm length), juveniles (10-25 mm length), and adults ([greater than or equal to] 30 mm length). Snails were randomly selected, marked with nail polish by number, and distributed into treatment tanks. The length, width, and mass of each adult and juvenile snail were recorded at the start of the trial (Fig. 1). Mass was measured with an OHAUS Navigator (Ohaus Corporation, NJ) digital scale to the nearest 0.1 g. Measurements of surviving snails were taken posttrial. Mortality rates of 50% were not expected in all conditions (i.e., control conditions); therefore, an examination of growth rates throughout the trials was used to delineate effects of treatments.

Experimental tanks varied in volume for each snail size class: adult tanks were 79.71, juvenile tanks were 39.91, and hatchling tanks were 9.5 1. The experimental design was a replicated, complete randomized block; for example, a replicate adult Pomacea canaliculata salinity block included one tank for each treatment level. Adult treatments had five replicated blocks, whereas juveniles and hatchlings had either five or six replicated blocks, as the number of available juveniles and hatchlings varied at the time of testing. Treatments followed ASTM Standards E 729 and E 1022 (American Society for Testing and Materials 2002), utilizing a continuous mechanical/chemical filtration system. Except for manipulation of chosen parameters, all other water quality parameters were maintained at the same level as the holding tanks (Collection and Holding). All water quality parameters were measured once every 24 h using a YSI 556 and a LaMotte Smart 2 Colorimeter. Mortalities were checked daily and were determined by examining the operculum for resistance and visibility of tissue. When tissue was visible and the snail was not moving, the foot was stimulated for retraction response. If foot retraction did not occur, the animal was classified as deceased and removed from the test tank. Feeding and digestion during environmental perturbations can alter physiological responses and survivability (Legeay & Massabuau 2000, McGaw 2006, McGaw et al. 2009). Therefore, 2 days prior to experiments, snails were starved to clear the gut system of residual food and reduce the impact of specific dynamic action (Wang 2001) and the snails were not fed during the experiments (Yusa et al. 2006b). Furthermore, a preliminary trial (28 days) indicated that all adults and juveniles had at least a 50% survival for 28 days with no food. Hatchlings were not tested for starvation tolerance, as they are known to feed on microalgae. Controlling for microalgae was not possible in experimental tanks, and although macrophytes were not provided in any test condition, there is the possibility snails consumed limited amounts of nutrients provided by microalgae.

Salinity

Salinity tolerance trials were conducted for 0 (control), 8,16, 24, and 32. Artificial seawater (Instant Ocean, Blacksburg, VA) was used and salinities were maintained within [+ or -]2. At the outset of the experiment, all tank salinity levels were at 0 and were increased at a rate of 1/30 min until the desired salinity level was reached; a rate slightly slower than high tide intrusion. This was accomplished through equal volume removal and replacement with higher salinity water; water changes were also made in the 0 condition. Thirty snails were used for all trials and age groups (n = 6 snails/tank). Mean starting length, width, and mass for adults for Pomacea canaliculata were 43.87 mm, 40.31 mm, and 18.99 g, respectively; and for Pomacea maculata were 64.16 mm, 66.26 mm, and 94.54 g, respectively. Mean starting length, width, and mass for juveniles for P. canaliculata were 16.17 mm, 15.16 mm, and 2.03 g, respectively; and for P. maculata were 20.78 mm, 19.81 mm, and 2.55 g, respectively.

pH

The pH tolerance of the snails was investigated over a range of 5.5-9.5 (control = 7.5) at intervals of 1.0 pH unit, as these are commonly occurring pH levels in the southeast United States (Lazzarino et al. 2009). The pH levels were maintained at [+ or -] 0.1 pH units. Commercially available, mollusc-safe, pH adjusters (pH Up and pH Down, Aquarium Pharmaceuticals, PA) were used to regulate pH levels. Test conditions were established in the tanks prior to the introduction of the snails. Thirty adult snails were used for all trials (n = 6 snails/tank), 36 juveniles and hatchlings were tested for each treatment (n = 6 snails/tank). Mean starting length, width, and mass for adults for Pomacea canaliculata were 42.77 mm, 39.75 mm, and 17.49 g, respectively; and for Pomacea maculata were 65.66 mm, 68.02 mm, and 103.16 g, respectively. Mean starting length, width, and mass for juveniles for P. canaliculata were 17.54 mm, 16.29 mm, and 2.29 g, respectively; and for P. maculata were 17.78 mm, 16.59 mm, and 2.79 g, respectively.

Desiccation

Desiccation tolerance trials were conducted in two test conditions with an aquatic environment serving as the control (modified from Yusa et al. 2006b). Average Florida relative humidity (RH) for the last 47 y measured from 13 locations across the state ranged from 59.6% to 85.0% (National Oceanographic and Atmospheric Administration 2012). Test conditions were either desiccation (low humidity), defined as less than 60%, RH, or semidesiccation (high humidity) defined as greater than 80% RH. High RH was maintained between 80% and 89%, and low RH ranged from 51 % to 60%. Relative humidity was measured with terrarium hygrometers (Fluker's, Port Allen, LA). Low-humidity tanks had a 5-cm layer of dry sand in the bottom and a desiccant pouch was used if humidity levels exceeded 60%. High-humidity treatments had a 5-cm layer of sand that was kept moist with up to 2 mm of standing water. The control tanks had the same water quality conditions as the holding tanks (Collection and Holding). Snails with closed opercula were tested by gently prying the operculum. If there was resistance, the snail was considered to be alive; if the operculum was easily movable and soft tissue was readily expelled, the snail was deceased. There were 45 adult Pomacea canaliculata in the low- and high-humidity conditions (n = 9/tank) and 40 snails in the control (n = 8/tank); adult Pomacea maculata had 40 snails in each condition (n = 8/tank). There were 60 juveniles (n = 12/tank) and 40 hatchlings (8/tank) used in each treatment for both species. Mean starting length, width, and mass for adults for P. canaliculata were 45.00 mm, 42.34 mm, and 24.64 g, respectively; and for P. maculata were 66.63 mm, 69.31 mm, and 99.69 g, respectively. Mean starting length, width, and mass for juveniles for P. canaliculata were 17.36 mm, 16.07 mm, and 1.87 g, respectively; and for P. maculata were 19.07 mm, 17.34 mm, and 2.20 g, respectively. Transparency of juvenile and some adult P. canaliculata shells provided an opportunity to observe heart rate, which was observed periodically through the study, without moving the snails.

After 28 days, the survival rate of both species was higher than 50% in both treatment conditions. The experiment was, therefore, continued to attempt to ascertain maximal survival time under low- and high-humidity conditions. Snails continued to be checked daily for mortality (i.e., tissue expulsion). Every 2 wk, one of the snails, assumed to be alive, was placed in a tank for "revival." If the snail did not respond, it was classified as dead and the time of death was recorded as 7 days prior (i.e., halfway between the revival episodes). This process continued until a live snail was found, or until no more live snails were present. Live snails were not returned to test conditions, reducing the total number of snails available every 2 wk.

Statistical Analyses

Summary statistics were calculated for all data. Repeated measures analysis of variance was used to test fluctuations in water quality parameters (dissolved oxygen, ammonia, temperature, and pH) and no significant differences were observed. Survival comparisons were made for each species among all treatment levels and between species at each treatment level. Mortality was evaluated as time-to-event data, using standard survival analysis methods (Kleinbaum & Klein 2005, Allison 2010, Daniel & Cross 2013). Median survival time was estimated directly from the survivorship curve, using the Kaplan-Meier procedure. Survival curves were compared using Gehan's generalized Wilcoxon test; follow-up pairwise comparisons, controlling for type-I error, were done in the event that a significant difference was found among curves. Between-species factor comparisons were calculated using the MantelCox log-rank test. In trials where survival exceed 50% for the 28 days, the actual survival rates are provided. Repeated measures analysis of variance was used to evaluate changes in shell TL, TW, and M. All statistical analyses were performed in either SPSS 17.0 (SPSS Inc., Chicago, IL) or SAS 9.2 (SAS Institute, Cary, NC).

RESULTS

Salinity

The overall survival rates for both Pomacea maculata and Pomacea canaliculata were lowest for hatchlings and greatest for adults. For both species and all age groups, the highest survival rates were observed in freshwater (0 = control), whereas the lowest survival rates occurred in salinities of 24 and 32. Survivability at 8 and 16 was dependent on species and age group.

The median survival time for both species of hatchlings varied significantly within species and among treatments (Pomacea canaliculata'. G-W = 140.2, P < 0.001; Pomacea maculata: G-W = 140.0, P < 0.001; Fig. 2). The median survival of 28 days in the control treatment was significantly greater than all other treatments (all curves, both species P < 0.001). In the control treatment, 73% of P. canaliculata and 53% of P. maculata were alive at 28 days. Median survival at 8 was reached at 3.4 days for both species (P. canaliculata = 60%; P. maculata = 73%) with 0% survival by 7 days for both species. No hatchling snails of either species were alive after 2 days in salinities above 8. There were no significant differences between species.

The median survival for both species of juveniles varied significantly within species and among most treatments (Pomacea canaliculata: G-W = 134.8, P < 0.001; Pomacea maculata: G-W = 129.0, P < 0.001; Fig. 2). Survival in the control treatment for both species was 100% after 28 days and declined as salinity increased. Median survival rates for P. canaliculata were 8 = 56% at 28 days, 16 = 50% at 4 days, 24 = 90% at 2.4 days, and 32 = 0% at 2 days. Median survival rates for P. maculata were 8 = 60% at 19.6 days, 16 = 53% at 3.1 days, 24 = 96% at 2.5 days, and 32 = 0% by 2 days. Survival for both species at 0 was significantly greater from all other salinities (all curves P < 0.001). Analyses of all remaining treatments, for both species, were significantly different (all curves P < 0.001). Between species, P. canaliculata survival was significantly greater at 16 ([chi square] = 10.78, P = 0.001).

The overall median survival time of both species of adults varied significantly among most treatments, decreasing as salinity increased (Pomacea canaliculata: G-W = 13.03, P < O. 001; Pomacea maculata: G-W = 123.4, P < 0.001; Fig. 2). Survival rates for 28 days in the control (0) treatment for both species were 100% and were significantly greater than 16, 24, and 32 (P < 0.001). Median survival rates for P. canaliculata were 8 = 93% at 28 days, 16 = 56% at 5.7 days, 24 = 90% at 2.4 days, and 32 = 80% at 2.4 days. Median survival rates for P. maculata were 8 = 86% at 28 days, 16 = 60% at 3.6 days, 24 = 86% at 2.4 days, and 32 = 83% at 2.4 days. Survival rates between salinities of 0 and 8 were not significantly different for P. canaliculata but were for P. maculata (G-W = 4.209, P = 0.040). For both species, survival in 16 was significantly less than 0 and 8 but significantly greater than 24 and 32 (all curves P < 0.001). Between species, P. canaliculata had a significantly higher survival rate at 16 ([chi square] = 3.89, P = 0.049).

Juveniles of both species increased in length and width in both 0 and 8, but exhibited a concurrent loss in mass (Table 1); due to high mortality rates, these were the only treatments used in analyses. Measurements for Pomacea canaliculata were compared between 0 and 8; the change in length was significantly greater in 0 than 8 (t = 2.90, P = 0.0056); no other significant changes occurred. No treatment comparisons were made for Pomacea maculata owing to high mortality rates above 0. For between-species comparisons, only 0 was considered and the increases in length and width were significantly greater for P. maculata, but there was not a significant difference between changes in mass.

Changes in adult shell measurements were evaluated in 0 and 8 for each species, and between species (Table 1). No significant differences were found between treatments for either species. Increases in length and width coupled with a decrease in mass were observed for both species in both salinities. In 0, Pomacea canaliculata exhibited a significantly larger increase in length compared with Pomacea maculata and a significantly higher increase in length and width in 8. No significant differences in mass were observed for any treatments.

pH

The overall median survival time for both Pomacea maculata and Pomacea canaliculata was lowest for hatchlings. Adults and juveniles had median survival times of 28 days in all treatments. Significant differences were observed in the number of snails alive in each treatment level.

The median survival for both species of hatchlings was significantly different among treatments; however, this was due to the survival rates at the pH 5.5 (Pomacea canaliculata: G-W = 64.3, P < 0.001; Pomacea maculata, G-W = 72.3, P < 0.001; Fig. 3). Median survival rates for P. canaliculata were 5.5 = 53% at 3.1 days, 6.5 = 55% at 9.3 days, 7.5 = 53% at 10.2 days, 8.5 = 53% at 10.3 days, and 9.5 = 58% at 9.8 days. The survival rates for P. maculata were 5.5 = 91% at 3.0 days, 6.5 = 53% at 8.5 days, 7.5 = 58% at 8.8 days, 8.5 = 64% at 9.7 days, and 9.5 = 50% at 10.0 days. Pairwise post hoc analyses showed only the pH of 5.5 was significantly lower for both species and all treatments. This low rate of survival was strong enough to influence the overall between treatment analyses, as no other significant differences were observed among the remaining pH treatments for either species. Between species, the only significant difference was the greater survival of P. maculata at pH 5.5 ([chi square] = 5.70, P= 0.017).

In all of experiments involving the juvenile snails, at least 50% of the animals were alive at 28 days (Fig. 3). The greatest proportion of surviving snails was observed in the control (7.5) and also 8.5, followed by 9.5, 5.5, and finally 6.5. Survival rates for Pomacea canaliculata were 5.5 = 56%, 6.5 = 56%, 7.5 = 89%, 8.5 = 89%, and 9.5 = 66%, and overall differed significantly (G-W = 13.4, P = 0.010). Significant differences were observed between 7.5 versus 5.5 (G-W = 8.230, P = 0.004), 6.5 (G-W = 9.983, P = 0.002), and 9.5 (G-W = 5.243, P = 0.022), as well as 8.5 versus 5.5 (G-W = 6.840, P = 0.009), 6.5 (G-W = 9.327, P = 0.002), and 9.5 (G-W = 4.081, P = 0.043). Survival rates for Pomacea maculata were 5.5 = 89%, 6.5 = 89%, 7.5 = 78%, 8.5 = 75%, and 9.5 = 89%. No significant differences were observed for P. maculata between treatments (G-W = 5.9, P = 0.253). Between species, survival estimates were significantly different at pH of 5.5 ([chi square] = 9.00, P = 0.003), 6.5 ([chi square] = 10.62, P = 0.001), and 9.5 ([chi square] = 5.66, P = 0.017); in each treatment, P. maculata had the greater survival.

In all of the adult snail treatments, at least 50% of the snails were alive at 28 days (Fig. 3). Survival rates for Pomacea canaliculata were 5.5 = 93%, 6.5 = 93%, 7.5 = 100%, 8.5 = 100%, and 9.5 = 87%. Although a global significance test did not find significant differences (G-W = 7.3, P = 0.120), two pairwise results were significantly different. The rate of survival at pH 9.5 was significantly less than pH 7.5 and 8.5 (both tests G-W = 4.209, P = 0.040). The survival rates for Pomacea maculata were 5.5 = 90%, 6.5 = 97%, 7.5 = 100%, 8.5 = 100%, and 9.5 = 100%. No significant differences were observed among treatments for P. maculata (G-W = 8.8, P = 0.067). Between species, the survival rate of P. maculata at pH 9.5 was significantly greater than P. canaliculata ([chi square] = 4.21, P = 0.040); no other significant differences were observed.

Changes in shell measurements were evaluated across all juvenile treatments. Overall, there was an increase in length and width for all treatments and a decrease in mass (Table 2). For both species and among treatments, the increase in shell length at 5.5 was significantly less than 7.5, 8.5, 9.5, and also for Pomacea canaliculata at 6.5. For both species, the increase in width at 5.5 was significantly lower than at 7.5, and 8.5, and also at 9.5 for Pomacea maculata. At pH 6.5, the increases in length and width for both species were significantly lower compared with pH 7.5 and 8.5, but not 9.5. There were no significant differences in length or width between pH 7.5 and 8.5 for P. canaliculata, although both were significantly greater at 7.5 than 8.5 for P. maculata. The increase in length at 7.5 was significantly greater than 9.5 for P. canaliculata and both species increased significantly more in width at pH 7.5 versus 9.5. The only significant change in mass was observed with P. canaliculata at pH 8.5, which was the greatest decrease among treatments. Between species, the only significant increase in length was at 7.5 for P. maculata. In all treatments P. maculata had a significantly greater increase in width. The decrease in mass was significantly more for P. canaliculata at 8.5.

Changes in adult shell measurements were evaluated across all treatments (Table 2). There were no significant differences in any measurement for Pomacea maculata. There were significant differences among treatments for Pomacea canaliculata in length and width. The increase in length and width at 5.5 was significantly lower compared with 7.5 and 8.5, as was width at 9.5. The increase in length and width at 6.5 was significantly lower than 7.5and 8.5, whereas the increase in length at 7.5 was significantly greater than in 9.5. In all treatments a decrease in mass occurred, but there were no significant differences among treatments. Between species, the increase in length was significantly greater for P. canaliculata than P. maculata at 5.5, as well as width at 5.5,6.5, and 9.5. No significant differences in the changes in mass between species were observed.

Desiccation

The overall survival rates for both Pomacea maculata and Pomacea canaliculata were lowest for hatchlings and greatest for adults. Survival was greatest in the control treatment. This trial was run for the 28-day experimental survival period, and was extended for 1 y for the high- and low-humidity treatments, based on the high rate of survival during the 28 days and anecdotal reports of long-term survival during dry-down events. In high- and low-humidity treatments, snails ceased movement after 48 h and appeared to enter a state of aestivation. Statistics were only calculated for the 28-day trial, due to the change in methods for the extended component of the study. Heart rates for 8 adult and 16 juvenile P. canaliculata were observed. Both age groups ranged between 60 and 66 bpm during the 1st wk dropping to 2-4 bpm by day 21.

Hatchling median survival varied significantly among treatments, with the lowest survival rates in the low- humidity condition (Pomacea canaliculata: G-W = 50.02, P < 0.001; Pomacea maculata G-W = 57.2, P < 0.001; Fig. 4). Median survival rates for P. canaliculata were control = 80% at 28 days, high humidity = 67% at 20.9 days, and low humidity = 50% at 13 days. Each curve for P. canaliculata differed significantly from the others: control versus high humidity (G-W = 23.157, P < 0.001) and low humidity (G-W = 42.569, P < 0.001) and high versus low humidity (G-W = 13.461, P < 0.001). Median survival rates for P. maculata were control = 55% at 27.5 days, high humidity = 60% at 27.3 days, and low humidity = 55% at 3.7 days. The low-humidity treatment had significantly lower survival than the control (G-W = 48.736, P < 0.001) and the high humidity (G-W = 36.096, P < 0.001) treatments. Between species, the only significant difference was between the control ([chi square] = 9.48, P = 0.002) and low humidity ([chi square] = 26.38, P < 0.001); no other significant differences occurred among the remaining treatment comparisons.

In juvenile treatments, survival rates varied significantly with greater survival in the control and high-humidity treatments than the low-humidity treatment (Pomacea canaliculata: G-W = 15.8, P < 0.001; Pomacea maculata: G-W = 115.9, P < 0.001; Fig. 4). Median survival rates, at 28 days, for P. canaliculata were control = 78%, high humidity = 92%, and low humidity = 60%. There was no significant difference in survival between the control and high-humidity treatments. Survival in the lowhumidity treatment was significantly less than the control and the high-humidity treatments (G-W = 4.855, P = 0.028; G-W = 14.911, P < 0.001, respectively). Median survival rates for P. maculata were control = 77% at 28 days, high humidity = 83% at 28 days, and low humidity = 70% at 13.3 days. There was no significant difference in P. maculata survival rates between the control and high-humidity treatments; however, both differed significantly from the low-humidity treatment (G-W = 77.963, P < 0.001; G-W = 84.972, P < 0.001, respectively). Between species, survival in the low-humidity treatment was significantly shorter for P. maculata (%2 = 76.40, P < 0.001). In the extended trial, P. canaliculata survived until days 147 and 98 in high- and low-humidity conditions, respectively, and P. maculata survived in high-humidity conditions until day 133.

The adult median survival for both species in all treatments was 28 days. There were no significant differences in survival curves for Pomacea maculata (G-W = 5.5, P = 0.062; Fig. 4). There was a significant difference in survival for Pomacea canaliculata, in that the high (56% at 28 days) and low (56% at 28 days) humidity treatments were similar to one another, but significantly different from the control treatment (83% at 28 days; G-W = 10.7, P = 0.0058). There were no significant differences in survival for P. maculata: control = 80% at 28 days, high humidity = 62% at 28 days, and low humidity = 57% at 28 days. There were no significant differences in median survival times between species at each treatment level. At the end of 1 y, snails of both species in the high-humidity conditions were alive, whereas in low-humidity conditions, all P. maculata were dead at 153 days and all P. canaliculata at 154 days.

Changes in shell measurements were evaluated across all treatments for juveniles. For both species, the greatest increases in length and width were in the control group and lowest in the low-humidity group, and were significantly different across all treatments (Table 3). The decrease in mass was significantly greater in the control treatment versus high- and low-humidity treatments for Pomacea maculata and versus low-humidity treatment for Pomacea canaliculata. The decrease in mass was significantly greater in the high- versus low-humidity treatments for P. canaliculata. Between species, P. maculata had a significantly greater increase in length in the high-humidity condition and in width for all treatments. There were no significant differences in mass within treatments and between species.

Changes in shell measurements were evaluated across all treatments for adults (Table 3). There were no significant differences for either species between treatments in changes in length or width. The greatest increase in length and width for both species occurred in the control treatment. There was not a significant difference in the change in mass for Pomacea canaliculata; however, the decrease in mass for Pomacea maculata was significantly greater in the control versus the high- and low-humidity treatments. Between species, P. maculata had a significantly greater increase in width in the high-humidity treatment as well as a significantly greater decline in mass in the control treatment.

DISCUSSION

The spread of nonindigenous Pomacea has been rapid throughout Florida, and to a lesser degree the rest of the southeast United States. Although federal restrictions by the United States Department of Agriculture, Animal and Plant Health Inspection Service, prohibits interstate movement and importation of Pomacea maculata and Pomacea canaliculata the continued commercial availability of these snails is in part responsible for the movement and introduction to new locations within Florida (J. Bernatis, personal communication). Natural redistribution has also been observed after periods of high water and flooding (e.g., hurricanes of 2004) causing the temporary connection of multiple aquatic systems. Many locations are connected via the intracoastal waterway and as snails are introduced into one location natural movement occurs through the system (Bernatis & Warren 2014). Understanding the physiological limits of the snail species is, therefore, necessary to predict invasion success and develop adequate management strategies. The survival rates of snails in air, low salinity, and pH were linked to the developmental stage: adult and juvenile snails had greater survivorship, whereas mortality rates were highest for hatchlings.

This study provides evidence that juvenile Pomacea maculata and Pomacea canaliculata have the ability to tolerate brackish waters up to 8 for up to 28 days. Further, reproducing populations of P. maculata have been found in several coastal locations (salinity measured up to 6) (Bernatis 2014). The only other Pomacea species with similar tolerances is Pomacea diffusa, surviving up to 2 wk in 8 (Jordan & Deaton 1999). In this study, the hatchlings of both species had less than 50% survival at 8 after 4 days suggesting that this reduced ability to survive higher salinity may limit establishment in brackish waters. The low survival of all ages beyond 16 indicates these snails will not be able to establish themselves in salt marshes or other heavily tidally influenced locations. The short-term tolerance may allow them to spread through tidally influenced locations, naturally or attached to boats, and use these pathways to move into other freshwater systems. The intracoastal waterway stretches from Texas in the Gulf of Mexico up the Atlantic Coast to Virginia. The waterway, which is composed of salt, brackish, and fresh water connections, spans 4,800 km (1,900 km are in Florida). Salinity ranges in Florida for these waters range from less than 1 to greater than 33 (Florida Department of Environmental Protection STORET, 2015), and vary with tidal cycles and freshwater runoff (Livingston 1990). The 54 major water basins in Florida (many of which originate in Alabama and Georgia) all flow into either the Atlantic Ocean or the Gulf of Mexico; 39 of these basins are known to have nonindigenous Pomacea populations. The spread of nonindigenous Pomacea through this network is therefore possible, but continuous, prolonged (>2 days) exposure to saltwater (>16) will likely kill any age Pomacea. Nevertheless, Pomacea egg masses, which are deposited above waterlines (e.g., boat hulls), may easily be transported across salt water to a final freshwater location and the resulting snails becoming established in new areas.

The success and diversity of aquatic organisms is dependent, in part, on the pH (Hall et al. 1980, Herrman et al. 1993, Berezina 2001, Bemvenuti et al. 2003). Molluscs typically have low abundance in acidic habitats with pH less than 6.0, because they are unable to secrete CaC03 to maintain their shells (McKillop & Harrison 1972, Mackie 1987, Hunter 1989, Okland 1992, Berezina 2001). Similar changes were observed in this study for adults and juveniles of both species, but only at a pH of 5.5. Changes in the shell morphology included the spire wearing or breaking off, discoloration (e.g., color pattern disappearance), and brittleness of the outer whorl. Adult and juvenile Pomacea maculata and Pomacea canaliculatet had a very broad tolerance of pH as no difference in survival was observed between 5.5 and 9.5. Survival of hatchlings of both species at pH of 5.5 was significantly lower than at all other pH levels. Sublethal effects of low pH are known to occur in gastropods and include problems with shell formation and regeneration, reproduction, and shell strength (reviewed in Wilbur 1964). A decrease in shell strength increases susceptibility to predation (Stein et al. 1984) or accidental death through crushing (Glass & Darby 2008). Decreases in shell strength under acidic conditions have been reported for Pomacea paludosa at pH less than 6.5 (Glass & Darby 2008). The ability of nonindigenous Pomacea to tolerate and grow in a wide range of pH environments implies that there are abundant potential habitats for colonization. There have been few controlled studies on the effects of pH on freshwater gastropods (Hunter 1989, Glass & Darby 2008). In North America, the majority of research on the potential effect of water pH has been limited to distribution impacts on native P. paludosa (Hurdle 1973, Gleason et al. 1975, Perera et al. 1989) and distribution of nonindigenous species in both North and South America (Martin et al. 2001, Byers et al. 2013). Although few studies have looked specifically at pH tolerance, many have used pH as a factor for gastropod distribution (Harvey & McArdle 1986, Prescott & Curteanu 2004, Watson & Ormerod 2004, Karunaratne et al. 2006). The results of these studies provide a possible explanation for why Pomacea spp. are invasive in one system, but not present in others, as the effects of reduced growth and weakened shells may prevent Pomacea spp. from becoming a threat in lower-pH environments.

Throughout the southeastern United States, aquatic systems are periodically exposed to reduced water levels and dry periods. Current trends in groundwater pumping, combined with natural periods of drought, and altered rainfall patterns have lowered lake and river levels in many basins (Stamey 1996, Bartolino & Cunningham 2003, Light et al. 2006, Haag & Warren 2008, Rugel et al. 2011), such that small lakes have dried out completely (Bernatis, personal communication). The ability of freshwater snails to tolerate aerial exposure for extended periods of time is an evolutionary strength and ecological advantage (Aldridge 1983). Pomacea are among those species that are capable of survival through periods of drought and aerial exposure. In this study, snails of both species and all age groups (except for Pomacea maculata juveniles and hatchlings in the low-humidity condition) survived a minimum of 28 days. In high-humidity conditions, long-term survival of both species ranged from 49 (hatchlings) to 365 days (adults). These results were similar to Ramakrishnan (2007) in which adult Pomacea insularum (now P. maculata) survival increased up to 308 days. The results for Pomacea canaliculata were also similar to Yusa et al. (2006b), with increased survival as size increased. In addition, Pomacea lineata (Little 1968) and Pomacea urceus (Burky et al. 1972) can enter periods of aestivation, thereby, enhancing adult snail survival to 395 and 526 days, respectively. Aestivation has been documented for Pomacea spp. (Meenakshi 1964, Little, 1968, Burky et al. 1972, McMahon, 1976, Aldridge 1983, Darby et al. 2008) and was observed in this study, where heart rates dropped from 64-66 bpm to 2-4 bpm over the course of 28 days for P. canaliculata adults and juveniles. The native Florida apple snail, however, has a lower desiccation tolerance with a 73% mortality rate of adults occurring by 126 days (Darby et al. 2008). Low water level management strategies have been intentionally implemented by agencies (e.g. Florida Fish and Wildlife Conservation Commission) in an effort to control vegetation or fish classed as nuisance species in some systems. Although the Florida Fish and Wildlife Conservation Commission has considered water dry-downs as potential methods for controlling P. maculata, the results of this study provided evidence that this would not be an effective management strategy in systems that are eventually reinundated. These conclusions are supported by numerous studies focused on management of apple snails in wetland crop locations, where the snails have survived long term in conditions where water is drained from fields and the sediment remained moist (Estebenet & Cazzaniga 1990, Litsinger & Estano 1993, Calumpang et al. 1995, Halwart et al. 1998, Wada 2004, Yusa et al. 2006b). Therefore, using the drydown method may in fact harm populations of the less emersion-tolerant native Florida apple snail Pomacea paludosa, where sympatric populations occur (e.g., Lake Tohopekaliga and Lake Okeechobee).

Successful establishment of a species depends on many factors, habitat and biological compatibility among them. Given their physiological tolerances, combined with their unique biological traits (i.e., water and air breathing, rapid growth rates and generalist feeding behavior), few obstacles will block colonization of these invasive gastropods. Ultimately, the risk of spread and establishment throughout the United States is great. The results of this study indicate that factors normally prohibitive to freshwater species may not prevent the spread and establishment of Pomacea maculata and Pomacea canaliculata. Nor will traditional methods of control/eradication (i.e., water dry-downs) be adequate for control or eradication of P. maculata and P. canaliculata from all systems. There has been some work on biological controls that may help stabilize populations in some larger systems. Yusa et al. (2006a) looked at numerous potential predators for P. canaliculata, and many of the test predators or congeners occur in Florida. Other strategies, such as hand removal of eggs and live snails, are successful in some situations (e.g., urban ponds), and may be more effective than drawdowns or chemical applications (Bernatis & Warren 2014). Unfortunately, the ability of these species to tolerate such diverse conditions will leave many systems vulnerable to invasion.

ACKNOWLEDGMENTS

We would like to thank the anonymous reviewers for their comments and suggestions. J. L. Bernatis would also like to thank Drs. Steve Johnson, Mark Brenner, Jim Williams, Ken Langeland, and Mr. Gary Warren for their support, comments and suggestions during this project.

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JENNIFER L. BERNATIS, (1) * IAIN J. MCGAW (2) AND CHAD L. CROSS (3)

(1) Florida Fish and Wildlife Conservation Commission, 7386 Northwest 71st Street, Gainesville, FL 32653; (2) Department of Ocean Sciences, Memorial University, O Marine Lab Road, St. John's, Newfoundland, Canada A1C 5S7;3Nevada State College, 1125 Nevada State Drive, Henderson, NV 89002

* Corresponding author. E-mail: jennifer.bernatis@myfwc.com

DOI: 10.2983/035.035.0424

TABLE 1.
Salinity trial differences in shell length, width, and mass for
treatments with 28-day median survival for adults and juveniles.

                                             Adults

Treatment                         L             W             M

0
  P. canaliculata (30; 30)   2.70 (1.04)   2.43 (0.93)   -1.97 (0.99)
  P. maculata (30; 30)       1.04 (1.31)   1.35 (4.56)   -6.24 (5.71)
Between species                             t = -2.21
                                           P = 0.0281
8
  P. canaliculata (28; 17)   2.69 (0.67)   2.43 (0.57)   -1.29(1.21)
  P. maculata (26; *)        0.44 (0.62)   0.44 (0.74)   -6.68 (3.64)
Between species               t = -2.15     t = -3.3
                             P = 0.0328    P = 0.0012

                                            Juveniles

Treatment                         L             W             M

0
  P. canaliculata (30; 30)   2.69 (0.54)   2.55 (0.50)   -0.15 (0.11)
  P. maculata (30; 30)       2.97 (0.61)   3.05 (0.59)   -0.17 (0.11)
Between species               t = 2.82      t = 4.43
                             P = 0.0053    P = 0.0001
8
  P. canaliculata (28; 17)   2.16 (0.49)   2.00 (0.45)   -0.18 (0.10)
  P. maculata (26; *)
Between species

The mean differences from starting measures of length (L) and width
(W) are given in millimeters. The mean difference from starting mass
(M) is given in grams. Standard deviation is in parentheses. The
total number of snails used in each analyses is provided below
species name (Adults; Juveniles). Only significant results are given.

* Median survival did not reach 28.

TABLE 2.
pH trial differences in shell length, width, and mass for treatments
with 28-day median survival for adults and juveniles.

                                              Adults

Treatment                          L             W             M

5.5
  P. canalkulata (28; 21)     0.75 (0.73)   0.92 (0.65)   -1.65 (1.22)
  P. maculata (27; 32)        0.31 (0.61)   0.43 (0.67)   -4.57 (3.12)
Between species                t = 2.45      t = 2.52
                              P = 0.0147    P = 0.0121
6.5
  P. canciliculata (28; 20)   1.47 (0.78)   1.34 (0.74)   -1.22(1.14)
  P. maculata (29; 32)        0.25 (0.65)   0.46 (0.65)   -5.21 (3.59)
Between species                              t = 2.52
                                            P = 0.0121
7.5
  P. canaliculata (30; 32)    2.87 (0.93)   2.62 (0.67)   -1.91 (1.28)
  P. maculata (30; 28)        0.22 (0.44)   0.36 (0.63)   -8.1 (4.95)
Between species

8.5
  P. canaliculata (30; 34)    2.61 (0.87)   2.44 (0.79)   -1.49(1.29)
  P. maculata (30; 27)        0.86 (0.87)   1.01 (0.91)   -7.16(6.48)
Between species

9.5
  P. canaliculata (29; 27)    2.07 (0.77)   1.87 (0.60)   -1.21 (0.82)
  P. maculata (30; 32)        0.90 (1.01)   1.07 (1.05)   -6.71 (5.08)
Between species                              t = 2.09
                                            P = 0.0372
Within species
  P. canaliculata
    5.5 versus 6.5

    5.5 versus 7.5             t = -5.08     t = -4.02
                              P < 0.0001    P < 0.0001
    5.5 versus 8.5             t = -4.47     t = -4.02
                              P < 0.0001    P < 0.0001
    5.5 versus 9.5                           t = -1.99
                                            P = 0.0472
    6.5 versus 7.5             t = -3.64     t = -3.38
                              P = 0.0003    P = 0.0003
    6.5 versus 8.5             t = -3.03     t = -3.38
                              P = 0.0026    P = 0.0008
    7.5 versus 8.5

    7.5 versus 9.5             t = 2.04
                              P = 0.0420
    8.5 versus 9.5

  P. maculata
    5.5 versus 7.5

    5.5 versus 8.5

    5.5 versus 9.5

    6.5 versus 7.5

    6.5 versus 8.5

    7.5 versus 8.5

    7.5 versus 9.5

    8.5 versus 9.5

                                             Juveniles

Treatment                          L             W             M

5.5
  P. canalkulata (28; 21)     1.81 (0.79)   1.64 (0.55)   -0.19(0.15)
  P. maculata (27; 32)        1.82 (0.85)   2.01 (0.29)   -0.14(0.15)
Between species                t = 8.85      t = 2.28
                              P = 0.0001    P = 0.0228
6.5
  P. canciliculata (28; 20)   2.23 (0.53)   1.87 (0.38)   -0.12(0.17)
  P. maculata (29; 32)        2.17 (0.32)   2.01 (0.35)   -0.16(0.15)
Between species                              t = 2.37
                                            P = 0.0181
7.5
  P. canaliculata (30; 32)    2.75 (0.81)   2.31 (0.65)   -0.15 (0.14)
  P. maculata (30; 28)        3.67 (1.33)   3.74 (0.98)   -0.18 (0.78)
Between species                              t = 9.77
                                            P = 0.0001
8.5
  P. canaliculata (30; 34)    2.74 (0.91)   2.30 (0.80)   -0.30 (0.50)
  P. maculata (30; 27)        2.98 (1.02)   2.95 (0.87)   -0.19(0.19)
Between species                              t = 4.47       t = 2.62
                                            P = 0.0001     P = 0.0090
9.5
  P. canaliculata (29; 27)    2.45 (0.93)   2.07 (0.76)   -0.17(0.11)
  P. maculata (30; 32)        2.61 (0.95)   2.49 (0.77)   -0.16(0.15)
Between species                              t = 2.71
                                            P = 0.0068
Within species
  P. canaliculata
    5.5 versus 6.5             t = -2.18
                              P = 0.0297
    5.5 versus 7.5             t = -5.4      t = -3.97
                              P < 0.0001    P < 0.0001
    5.5 versus 8.5             t = =1.81     t = -3.37      t = 2.32
                              P < 0.0001    P = 0.0008     P = 0.0209
    5.5 versus 9.5             t = -3.44
                              P = 0.0006
    6.5 versus 7.5             t = -2.94     t = -3.06
                              P = 0.0034    P = 0.0023
    6.5 versus 8.5             t = -2.35     t = -2.47      t = 3.90
                              P = 0.0193    P = 0.0137     P = 0.0001
    7.5 versus 8.5                                          t = 3.43
                                                           P = 0.0007
    7.5 versus 9.5             t = 1.98      t = 2.33
                              P = 0.0477    P = 0.0202
    8.5 versus 9.5                                         t = -2.65
                                                           P = 0.0083
  P. maculata
    5.5 versus 7.5             t = -9.47    t = -11.59
                               < 0.0001     P < 0.0001
    5.5 versus 8.5             t = -5.10     t = -5.50
                              P < 0.0001    P < 0.0001
    5.5 versus 9.5             t = -3.49     t = -2.29
                              P = 0.0005    P = 0.0223
    6.5 versus 7.5             t = -8.05    t = -10.57
                              P < 0.0001    P < 0.0001
    6.5 versus 8.5             t = -3.69     t = -4.49
                              P = 0.0002    P < 0.0001
    7.5 versus 8.5             t = 4.14      t = 5.78
                              P < 0.0001    P < 0.0001
    7.5 versus 9.5             t = 6.15      t = 9.41
                              P < 0.0001    P < 0.0001
    8.5 versus 9.5                           t = 3.35
                                            P = 0.0009

The mean differences from starting measures of length (L) and width
(W) are given in millimeters. The mean difference from starting mass
(M) is given in grams. SD is in parentheses. The total number of
snails used in each analyses is provided below species name (Adults;
Juveniles). Between species statistical results are provided within
each treatment. Within species and between treatments statistical
results are provided. Only significant results are given.

TABLE 3.
Desiccation trial differences in shell length, width, and mass for
treatments (high = high humidity, low = low humidity) with 28-day
median survival for adults and juveniles.

                                              Adults

Treatment                          L             W             DM

Control
  P. canaliculatei (33; 47)   1.79 (1.22)   1.60 (1.12)   -1.66 (0.90)
  P. maculata (32; 46)        0.74 (1.22)   0.89 (4.56)   -9.92 (5.83)
Between species                                             t = 4.92
                                                           P < 0.0001
High
  P. canaliculate (25; 55)    0.79 (0.47)   0.79 (0.48)   -2.06(1.36)
  P. maculata (26; 50)        0.13 (0.23)   0.15 (0.25)   -5.18(3.09)
Between species                              t = 2.14
                                            P = 0.0325
Low
  P. canaliculate (25; 36)    0.31 (0.17)   0.27 (0.25)   -2.04 (0.82)
  P. maculata (30; 60)        0.1 (0.02)    0.01 (0.02)   -5.77 (3.75)
Between species

Within species
  P. canaliculate
    Control versus high

    Control versus low

    High versus low

  P. maculata
    Control versus high                                    t = -4.40
                                                           P < 0.0001
    Control versus low                                     t 1 u> bo
                                                           P = 0.0001
    High versus low

                                             Juveniles

Treatment                          L             W             M

Control
  P. canaliculatei (33; 47)   3.39 (0.71)   3.03 (0.73)   -0.20 (0.13)
  P. maculata (32; 46)        3.24 (0.66)   3.36 (0.68)   -0.22 (0.13)
Between species                              t = -2.74
                                            P < 0.0001
High
  P. canaliculate (25; 55)    1.35 (0.50)   1.10 (0.39)   -0.17(0.12)
  P. maculata (26; 50)        1.84 (0.65)   1.82 (0.67)   -0.14(0.07)
Between species                t = -4.48     t = -5.22
                              P < 0.0001    P < 0.0001
Low
  P. canaliculate (25; 36)    0.65 (0.46)   0.54 (0.41)   -0.09 (0.05)
  P. maculata (30; 60)        0.89 (0.39)   0.81 (0.69)   -0.13 (1.32)
Between species                              t = 2.15
                                            P = 0.0320
Within species
  P. canaliculate
    Control versus high        t = 18.74     t = 16.22
                              P < 0.0001    P < 0.0001
    Control versus low         r = 23.16     t = 19.60     t = -3.92
                              P < 0.0001    P < 0.0001     P = 0.0001
    High versus low            t = 6.93      t = 5.53      t = -2.23
                              P < 0.0001    P < 0.0001     P = 0.0264
  P. maculata
    Control versus high        t = 11.97     t = 13.56     t = -2.51
                              P < 0.0001    P < 0.0001     P = 0.0123
    Control versus low         t = 23.27     t = 23.11     t = -3.13
                              P < 0.0001    P < 0.0001     P = 0.0018
    High versus low            t = 11.04     t = 9.19
                              P < 0.0001    P < 0.0001

The mean differences from starting measures of length (L) and width
(W) are given in millimeters. The mean difference from starting mass
(M) is given in grams. SD is in parentheses. The total number of
snails used in each analyses is provided below species name (Adults;
Juveniles). Between species statistical results are provided within
each treatment. Within species and between treatments statistical
results are provided. Only significant results are given.


----------

Please note: Some tables or figures were omitted from this article.
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Article Details
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Author:Bernatis, Jennifer L.; McGaw, Iain J.; Cross, Chad L.
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2016
Words:11307
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