Laboratory assessment of growth and reproduction of Lissachatina fulica (Gastropoda: Achatinidae)

Laboratory assessment of growth and reproduction of Lissachatina fulica (Gastropoda: Achatinidae) Abstract Despite Lissachatina fulica being a pest of agricultural and ornamental plants in the worldwide tropics for more than 100 years, information on its biology is limited. In this study, 360 snails were reared in the laboratory from a colony collected from the wild (non-native) population in Miami-Dade County, Florida, and were monitored for growth (height and mass) and egg production for up to 930 d. Snails averaged 195 d old and 97 mm in height when egg laying was initiated. Though the average snail peristome thickness was lower (0.1 mm) before eggs were first laid than after (0.4 mm), peristome thickness was inconsistent in predicting full reproductive maturity. Egg production averaged 4,698 eggs per snail and egg viability averaged 49% in the first 540 d after hatching. Oviposition without mating was shown to be possible, but occurred rarely (3% of snails) and with lower viability rates and smaller clutch sizes. The ability to oviposit without mating and the high reproductive rates found in this study indicate that eradication efforts addressing new infestations must be complete, because even one snail may start another population. INTRODUCTION Lissachatina fulica (Bowdich, 1822) is a major pest in regions where it is established, probably because it has a diverse diet and is very prolific. The strong reproductive potential of this generalist feeder has helped it to establish populations throughout the worldwide tropics (Raut & Barker, 2002). Despite being a recognized pest species for more than 100 years, information on its biology is limited. Growth rates, age of sexual maturity and fecundity of L. fulica need to be better understood in order to effectively manage and prevent infestations of this species. Growth studies on L. fulica have reported shell heights ranging from 35 to 63 mm after 60 d of age and 83–104 mm after 150 d (Bequaert, 1950; Kondo, 1964; Pawson & Chase, 1984). Because new population centres continue to be discovered in southern Florida, regulatory personnel are interested in distinguishing new infestations from older infestations as they are found (Smith et al., 2013). An estimate of a snail’s age based on shell size could possibly be used to give an estimate of how long snails have inhabited a specific site, since snails become larger over time. Predictability will probably be reduced in later months of life due to the well-known tendency of snail growth rate to decline as snails become older. Nevertheless, establishing the age of younger infestations has value in assessing dispersiveness. The reliability of existing literature on L. fulica is uncertain. For example, sample sizes were not mentioned by Bequaert (1950) or Pawson & Chase (1984), and Kondo (1964) used a sample of only five snails. The diversity of results reported for growth rates in L. fulica could be due to inadequate sample sizes, dissimilar rearing and experimental designs, or genetic differences found in snails in different geographical regions. Lissachatina fulica is hermaphroditic, but the male reproductive system develops first and is capable of copulation before the female reproductive system develops completely (Mead, 1949; Tomiyama, 1993). Only fully mature snails are capable of oviposition. Understanding which snails in a population are capable of producing eggs is important in understanding population growth. There is no known visual method for accurately identifying reproductive maturity in this species. However, the thickness of the peristome has been suggested by Tomiyama (1993) to be a useful tool to assess full sexual maturity. As the snail becomes mature, it allocates less growth towards body mass and shell size (Pawson & Chase, 1984) and more to the thickness of the shell, including the peristome (Tomiyama, 1993). Tomiyama compared peristome thickness and maturity based on the assessment of 52 field-collected snails with thin peristomes (<0.5 mm) and an additional 52 larger snails that had thick peristomes (>0.8 mm). Snails with thick peristomes had oocytes in ovotestes and large albumen glands, which are required for egg laying; by contrast, snails with thin peristomes had no oocytes and smaller albumen glands (Tomiyama, 1993). It would be useful to know if the peristome thickens around the time of the first egg-laying event, as this would help to determine snail age and the duration of infestation. Reproductive success can be measured by determining the age at which egg laying begins, the number of viable eggs laid per clutch and the number of viable eggs laid per year. Literature reports of when oviposition commences range from 5 to 15 months of age (Bequaert, 1950; Ghose, 1959; Pawson & Chase, 1984; Civeyrel & Simberloff, 1996; Raut & Barker, 2002). Egg production also varies, with reports of 160–1,817 eggs produced annually (Kekauoha, 1966; Raut & Barker, 2002) and 10–460 eggs laid per clutch (van der Meer Mohr, 1949; Lange, 1950; Ghose, 1959; Kekauoha, 1966; Pawson & Chase, 1984; Raut & Barker, 2002; Roda et al., 2016). Egg production can vary greatly according to region and parental age (Raut & Barker, 2002), thus more study is needed to narrow the estimate of when oviposition commences in local populations and to estimate egg production rates in snails of known age. Lissachatina fulica is hermaphroditic and the potential for reproduction without mating with another snail would greatly increase its ability to invade and spread to new areas. However, previous reports have been inconsistent in this respect (van der Meer Mohr, 1949; Ghose, 1959; Mead, 1961; Kondo, 1964; Kekauoha, 1966; CLEAPSS, 2006). Ghose (1959) observed egg laying by solitary, unmated snails, but the findings are of limited applicability, because sample sizes were not mentioned and the rate of reproduction without mating was not determined. Sources that mention reproduction without mating have assumed the process to be self-fertilization (van der Meer Mohr, 1949; Ghose, 1959). The purpose of this study was to determine critical aspects of L. fulica biology that affect pest control measures. Specific objectives were to determine (1) growth rates at various densities and diet regimes, (2) the age and size at first oviposition, (3) whether peristome thickness is a reliable indicator of sexual maturity, (4) short-term and long-term reproductive rates and (5) whether this species will reproduce without mating. MATERIAL AND METHODS Rearing methods Experimental snails were reared in the FDACS Florida Biological Control Laboratory (FBCL) in Gainesville, Florida and were F1 generation snails produced by snails that were field collected from the wild population in Miami-Dade County, Florida. Snails were provided a synthetic gypsy moth diet or a diet of organic romaine lettuce. Lettuce was used as a representative of a ‘natural’ diet, more suitable than the synthetic ‘gypsy moth’ diet (Estebenet & Cazzaniga, 1992; Capinera, 2013). The synthetic diet was a wheat germ-based diet used for gypsy moth rearing (Bio-Serv Flemington, NJ) and was selected as a less suitable diet, as it will allow for the growth and reproduction of the species, but has been previously observed (unpublished observations) to produce smaller snails with higher rates of mortality. Use of the less suitable synthetic diet should show how diet affects the onset of reproduction, representing some of the natural variation found in wild snail populations. Calcium needed for shell growth was provided in each experiment as lawn lime suspended in agar (300 g 54% pelletized lime, 25 g agar and 1 l boiling water). Snails were provided with a constant surplus of food and calcium. Cages consisted of 4-l plastic containers with a potting soil substrate (Metro Mix 930, Sun Gro Horticulture, Agawam, MA). The amount of soil provided in a cage increased as the snail grew; this ensured proper habitat for egg laying, but still allowed for easy access to the snail when it was small. All cages were initially filled with 300 ml soil, then cleaned and the soil replaced each month thereafter using 800 ml of soil. The cages were maintained in a photoperiod of 16:8 h L:D; temperature and humidity ranged from 21 to 25 °C and 28–66%, respectively (Raut & Ghose, 1984; Raut & Barker, 2002). Experimental design In order to evaluate the age of sexual maturity, reproductive rates and to determine if mating with another snail is required for egg production, four treatments were compared in a 2 × 2 factorial design: solitary and paired snails were provided either a synthetic diet or a diet of organic romaine lettuce. This experiment was conducted using 4–6 week-old neonates with shell heights (H) of 15–20 mm, as these were old enough to avoid damage to their delicate shells during handling. Also, these snails were young enough to ensure that they were unmated (Ghose, 1959; Tomiyama, 1992). This experiment was conducted with three replicates, each with a different starting date. Replicate 3 ran from 4 May to 30 December, and the other two from 23 January to 20 September and from 17 August to 14 April. Each replicate consisted of 20 cages per treatment, containing snails that hatched on the same date (±24 h) (total n = 360 snails; 60 cages each from the paired and solitary treatments, using lettuce or synthetic diets). H (measured parallel to the axis of coiling from the tip of the spire to the most distant point of the aperture edge) and mass (M) were measured every 30 d for 240 d. H was measured using a Fisher Scientific 15-077-958 caliper, and M using a Mettler Toledo ML1502e balance. In some cases, the spire of the shell was damaged, reducing H; these specimens were removed from analyses. Snails were monitored for egg production for up to 930 d. Data were analysed using R statistical computing software (R Core Team, 2014), the lawstat package (v. 2.4.1.tar.gz) and the dunn.test package. A two-way ANOVA was used to determine the effects of diet and density on H and M 240 d after hatching. H and M were analysed within each diet and density treatment, using the two-tailed Student’s t-test. To further test diet quality, a two-way ANOVA was used to test whether diet and density affected mean percent survival. Pattern of growth The pattern of growth (H) for snails from the reproduction studies described above was analysed with polynomial regressions. After the growth studies were terminated at 240 d, additional measurements were continued on a subset of the snails. Snails from the first replicate were monitored for long-term growth for 600 d (total n = 60 snails; 10 cages each from the paired and solitary treatments, and the lettuce and synthetic diets). First oviposition When snails in the growth studies first produced eggs, they were measured for H. After 240 d, many snails in replicate 3 had not deposited eggs, which was in strong contrast to the previous two replicates. For this replicate, monitoring was extended until 360 d after hatching. However, only data from the first 300 d were used in the reproductive rate analysis of these snails. A two-way ANOVA was used to determine the effects of diet and density on (1) age and (2) H of the adults at the first egg-laying event. Student’s t-test was used to compare mean age and H of the adults at the first egg-laying event within each diet treatment and within each density treatment. Peristome thickness was measured using a Marathon CO 030150 caliper every 30 d, to determine whether the peristome thickened around the time eggs were first laid. Peristome thickness was plotted against the time at which the measurements were taken relative to the first oviposition (i.e. negative values for time before first oviposition, positive values for time after first oviposition). A repeated-measures ANOVA was used to analyse peristome thicknesses before and after first oviposition. Short-term reproduction As egg laying commenced, the eggs in each clutch were counted. Clutches laid between hatching and 241 d by paired snails were randomly chosen to test egg viability, including 91 clutches from paired snails offered lettuce and 86 clutches from paired snails offered the synthetic diet. All clutches laid by solitary snails were tested for viability (n = 5 clutches from snails fed lettuce and 2 from snails fed synthetic diet). For determination of egg viability, eggs were buried under a thin layer of moist soil in a 15.2-cm diameter petri dish. Live hatchlings were counted after 3 weeks and clutch viability was calculated. Cages were checked for eggs every 3–5 d. The total number of eggs found in each cage (n = 59 cages for lettuce and 58 for synthetic diet paired treatments; n = 60 cages for lettuce and 56 for synthetic diet solitary treatments), between hatching and 241 d, was divided by the number of snails in each cage to determine the mean number of eggs produced per snail. The analysis did not include eggs produced after 300 d by late-developing snails in replicate 3. A two-way ANOVA was used to test whether diet and density affected the total number of eggs laid per snail, the percent viability of eggs laid and clutch size from cages monitored for 240 d (or 300 d). In order to address the unequal variance assumption violation detected by Levene’s test, a Mann-Whitney rank sum test was used to test the significance of differences in the total number of eggs laid per snail between diet treatments. Student’s t-test was used to test the significance of differences found between diet treatments in the percent viability of eggs laid and clutch size. The same tests were run for the total number of eggs laid per snail, for the percent viability of eggs laid and for clutch size between density treatments. Long-term reproduction As with growth, egg production monitoring continued on a subset of snails from the first replicate. However, egg production was monitored for longer than growth, for a total of 930 d after hatching. The data reported only include surviving snails from the lettuce treatment (n = 16 snails), because only one snail survived to 930 d in the synthetic diet treatment. All eggs that were laid were counted and 128 clutches were chosen to test egg viability, as described above. The numbers of eggs found in each of the eight paired-snail cages were counted every 30 d after hatching and then divided by the number of snails in each cage to find the number of eggs laid per month per snail. Snails that did not produce eggs that month were included in the analysis. Self-fertilization extended In addition to the eggs produced by snails in the reproduction experiment described above, we were able to assess egg production from additional solitary and paired snails. Added to the above experiment were egg-production data from 134 solitary snails (total n = 246 including the previous replicates), of which 65 were offered lettuce and 69 the synthetic diet, and data from 132 snails raised in pairs (total n = 366, including the previous replicates). These snails were reared similarly, but 116 were kept in a greenhouse with ambient lighting and humidity, with temperature ranging from 23 to 26 °C. After some died or were culled at 240 d, 143 solitary and 92 paired snails were still being monitored until 540 d after hatching. Dull, soft, nonviable eggs were frequently found in solitary snail cages. Egg clutches with only dull eggs were not considered in fecundity data. Otherwise, clutches (including eight clutches from snails fed the synthetic diet and seven clutches from snails fed lettuce) were tested for viability as described above. RESULTS Growth was affected by diet and density after 240 d, while survival was not. From the average of solitary and paired snails, the lettuce diet produced snails with greater H (F1,341 = 200.06, P < 0.01) and M (F1,346 = 231.39, P < 0.01), as compared with the synthetic diet (Table 1). In comparing the solitary snail treatment with the paired snail treatment, regardless of diet, the solitary snail treatment produced snails with greater mean H (F1,341 = 181.81, P < 0.01) and M (F1,346 = 127.25, P < 0.01) (Table 1). No interaction effect was found on H (F1,341 = 2.57, P = 0.11), but an interaction between diet and density was noted with respect to M (F1,346 = 5.13, P = 0.02). Diet (F1,8 = 2.52, P = 0.15) and density (F1,8 = 0.33, P = 0.58) did not significantly affect survival and no interaction effect was found (F1,8 = 0.91, P = 0.37). Table 1. Means (±SD), ranges and sample sizes (number of snails, n) for height and mass of Lissachatina fulica when offered lettuce or synthetic diet and reared alone or in pairs for 240 d after hatching. Density  Diet  Height (mm)  Mass (g)      Mean  Range  n  Mean  Range  n  Paired  Lettuce  107 ± 8 aB  75–121  117  139 ± 32 aB  53–264  118    Synthetic diet  96 ± 9 bB  73–133  113  92 ± 28 bB  43–170  116  Solitary  Lettuce  121 ± 8 aA  103–134  60  190 ± 41 aA  115–278  60    Synthetic diet  107 ± 9 bA  90–121  55  125 ± 35 bA  76–192  56  Density  Diet  Height (mm)  Mass (g)      Mean  Range  n  Mean  Range  n  Paired  Lettuce  107 ± 8 aB  75–121  117  139 ± 32 aB  53–264  118    Synthetic diet  96 ± 9 bB  73–133  113  92 ± 28 bB  43–170  116  Solitary  Lettuce  121 ± 8 aA  103–134  60  190 ± 41 aA  115–278  60    Synthetic diet  107 ± 9 bA  90–121  55  125 ± 35 bA  76–192  56  Means in a column, within each density, followed by the same lowercase letters are not significantly different (P > 0.05; two-tailed t-test). Means in a column, within each diet, followed by the same uppercase letters are not significantly different (P > 0.05; two-tailed t-test). The growth plots of paired and solitary snails produced negative exponential curves (Fig. 1). According to the H/growth regression for paired snails (Fig. 1A), for these experimental conditions, a snail of H 20 mm is 41 d old. Similarly, 34 and 85 mm snails are 60 and 150 d, respectively. The trend for greater H among solitary snails than paired snails further supports a density effect on growth. Data from long-term growth studies show that snails, on average, did not continue to grow after 240 d (Fig. 1). Figure 1. View largeDownload slide Growth of Lissachatina fulica reared on a diet of romaine lettuce or on a synthetic diet. Polynomial regressions were fitted on individual measurements for shell height. A. Paired snails regardless of diet. B. Paired snails. C. Solitary snails. Figure 1. View largeDownload slide Growth of Lissachatina fulica reared on a diet of romaine lettuce or on a synthetic diet. Polynomial regressions were fitted on individual measurements for shell height. A. Paired snails regardless of diet. B. Paired snails. C. Solitary snails. First oviposition The minimum time from hatch to egg laying was 124 d (Table 2). The number of days from hatch until the first egg-laying event did not differ between diet treatments (F1,117 = 0.01, P = 0.93), or between paired and single treatments (F1,117 = 0.26, P = 0.61) and there was no interaction effect (F1,117 = 0.06, P = 0.81) (Table 2). Diet affected snail H when oviposition commenced (F1,228 = 42.53, P < 0.01); however, density did not (F1,228 = 1.28, P = 0.26) and no interaction was found between diet and density (F1,228 = 0.235, P = 0.63) (Table 2). Eight solitary virgin snails laid large clutches, five of which proved to be viable, attesting that oviposition without mating with another snail is possible. Snails offered lettuce were larger at the first oviposition than were snails offered the synthetic diet, but only among paired snails (t-test, t222 = 6.18, P < 0.01), not among solitary snails (t-test, t6 = 2.27, P = 0.06). Table 2. Means (±SD), ranges and sample sizes (number of cages) for the number of days from hatching until Lissachatina fulica laid eggs, according to diet and density treatments. Density  Diet  Prereproductive period (d)  Height (mm)      Mean  Range  n (cages)  Mean  Range  n (snails)  Paired  Lettuce  194 ± 57 aA  127–339  56  102 ± 11 aA  78–124  112    Synthetic diet  195 ± 56 aA  124–339  57  92 ± 12bA  73–122  112  Solitary  Lettuce  201 ± 59 aA  164–314  6  107 ± 8 aA  100–121  6    Synthetic diet  214 ± 49 aA  179–249  2  93 ± 5 aA  90–97  2  Density  Diet  Prereproductive period (d)  Height (mm)      Mean  Range  n (cages)  Mean  Range  n (snails)  Paired  Lettuce  194 ± 57 aA  127–339  56  102 ± 11 aA  78–124  112    Synthetic diet  195 ± 56 aA  124–339  57  92 ± 12bA  73–122  112  Solitary  Lettuce  201 ± 59 aA  164–314  6  107 ± 8 aA  100–121  6    Synthetic diet  214 ± 49 aA  179–249  2  93 ± 5 aA  90–97  2  Means (±SD), medians, ranges, sample sizes (number of snails) and height of L. fulica at the first egg-laying event are given. Only eight solitary virgin snails laid large, viable-looking clutches. Means in a column, within each density, followed by the same lowercase letters are not significantly different (P > 0.05; two-tailed t-test). Means in a column, within each diet, followed by the same uppercase letters are not significantly different (P > 0.05; two-tailed t-test). Peristome thickness increased as snails oviposited for the first time (F1,932 = 1021, P < 0.01) (Fig. 2). Despite the significant relationship between peristome thickness and the onset of oviposition, outliers were present indicating peristome thicknesses <0.1 mm after eggs were first laid. Figure 2. View largeDownload slide Individual measurements of Lissachatina fulica peristome thickness taken before (–) and after (+) first oviposition. First oviposition occurred on day 0. Figure 2. View largeDownload slide Individual measurements of Lissachatina fulica peristome thickness taken before (–) and after (+) first oviposition. First oviposition occurred on day 0. Egg production The number of eggs laid per snail (F1,229 = 9.11, P < 0.01; F1,229 = 236.70, P < 0.01), egg viability (F1,180 = 8.71, P < 0.01; F1,180 = 13.65, P < 0.01) and clutch size (F1,476 = 24.31, P < 0.01; F1,476 = 25.92, P < 0.01) were affected by diet and density, respectively (Table 3). The interaction between diet and density was significant for the number of eggs laid per snail (F1,229 = 9.88, P < 0.01), but not egg viability (F1,180 = 2.75, P = 0.10) or clutch size (F1,476 = 0.48, P = 0.49). The average total number of eggs laid per snail over the course of the experiment from paired snails was higher from the lettuce diet than the synthetic diet (U = 2168, P = 0.01) (Table 3). Also, paired snails offered lettuce laid eggs with higher viability (t-test, t175 = 3.45, P < 0.01) and also laid larger clutches (Student’s t-test, t471 = 5.14, P < 0.01) than paired snails offered the synthetic diet. Paired snails laid far more eggs per snail than solitary snails offered lettuce (U = 3332.5, P < 0.01) and synthetic diet (U = 3074, P < 0.01). In lettuce treatments, egg production without mating also resulted in eggs with lower percent viability (t-test, t84 = 4.36, P < 0.01) and smaller clutch sizes (t-test, t267 = 3.97, P < 0.01) than sexual reproduction. Two clutches were laid by solitary snails on the synthetic diet. These clutches had fewer eggs than clutches laid by paired snails on the synthetic diet (t-test, t209 = 3.01, P < 0.01), but egg viability did not differ (t-test, t86 = 0.55, P = 0.58). Table 3. Means (±SD), ranges and sample sizes (n) for total number of eggs laid per snail, percent egg viability and clutch sizes among diet treatments for eggs laid before 241 d for Lissachatina fulica. Density  Diet  Eggs/snail  Viability (%)  Eggs/clutch      Meana  Range  n (cages)  Meanb  Range  n (clutches)  Meanb  Range  n (clutches)  Paired  Lettuce  738 ± 526 aA  0–1863  59  68 ± 28 aA  0–100  91  322 ± 151 aA  13–1033  264    Synthetic diet  486 ± 294 bA  0–1034  58  52 ± 33 bA  0–99  86  261 ± 91 bA  28–584  209  Solitary  Lettuce  5 ± 18 aB  0–109  60  13 ± 22 aB  0–50  5  53 ± 32 aB  34–109  5    Synthetic diet  2 ± 14 aB  0–103  56  40 ± 33 aA  16–63  2  67 ± 52 aB  30–103  2  Density  Diet  Eggs/snail  Viability (%)  Eggs/clutch      Meana  Range  n (cages)  Meanb  Range  n (clutches)  Meanb  Range  n (clutches)  Paired  Lettuce  738 ± 526 aA  0–1863  59  68 ± 28 aA  0–100  91  322 ± 151 aA  13–1033  264    Synthetic diet  486 ± 294 bA  0–1034  58  52 ± 33 bA  0–99  86  261 ± 91 bA  28–584  209  Solitary  Lettuce  5 ± 18 aB  0–109  60  13 ± 22 aB  0–50  5  53 ± 32 aB  34–109  5    Synthetic diet  2 ± 14 aB  0–103  56  40 ± 33 aA  16–63  2  67 ± 52 aB  30–103  2  aMeans in a column, within each density, followed by the same lowercase letters are not significantly different (P > 0.05; Mann-Whitney rank sum test). Means in a column, within each diet, followed by the same uppercase letters are not significantly different (P > 0.05; Mann-Whitney rank sum test). bMeans in a column, within each density, followed by the same lowercase letters are not significantly different (P > 0.05; two-tailed t-test). Means in a column, within each diet, followed by the same uppercase letters are not significantly different (P > 0.05; two-tailed t-test). Egg laying continued while monitoring long-term paired snails and the mean number of eggs laid per month per snail and percent egg viability are shown in Figure 3. Figure 3. View largeDownload slide The mean total Lissachatina fulica eggs laid per snail over time and the mean percent of eggs that were viable. Error bars indicate standard error. Figure 3. View largeDownload slide The mean total Lissachatina fulica eggs laid per snail over time and the mean percent of eggs that were viable. Error bars indicate standard error. Self-fertilization extended In the original experiment with 120 solitary snails, 116 snails survived and were monitored from hatching to between 240 and 300 d. Of these, eight solitary virgin snails (7%) laid one clutch each. Of the total 246 solitary snails monitored for 240 d, seven snails (3%) laid one clutch each (Table 4). When eggs from one clutch laid by a virgin snail were reared to adulthood, they produced viable offspring. Table 4. Total eggs produced per snail, mean (±SD) egg viability and mean (±SD) clutch size for eggs produced 0–240 d and 0–540 d for solitary and paired Lissachatina fulica, regardless of diet. Duration (d)  Treatment  Snails monitored  Cages Monitored  Cages with eggs (%)  Eggs/snail  Viability (%)  Eggs/clutch  240  Paired  366  183  67  843  52 ± 35  330 ± 160    Solitary  246  246  3  1.24  25 ± 27  44 ± 31  540  Paired  92  46  98  4698  49 ± 36  332 ± 162    Solitary  143  143  4  10.18  27 ± 31  132 ± 136  Duration (d)  Treatment  Snails monitored  Cages Monitored  Cages with eggs (%)  Eggs/snail  Viability (%)  Eggs/clutch  240  Paired  366  183  67  843  52 ± 35  330 ± 160    Solitary  246  246  3  1.24  25 ± 27  44 ± 31  540  Paired  92  46  98  4698  49 ± 36  332 ± 162    Solitary  143  143  4  10.18  27 ± 31  132 ± 136  Six of the 143 solitary snails (4%) monitored for 540 d laid eggs that appeared viable. One snail laid five egg clutches, while the remainder laid just one clutch each. Egg viability ranged from 0% to 98% per clutch (Table 4). DISCUSSION Diet can significantly affect both H (Upatham, Kruatrachue & Baidikul, 1988) and M (Egonmwan, 2007; Ejidike, 2007) in Achatinidae. Upatham et al. (1988) found that a combination of a synthetic diet and lettuce promoted increases in both H and M as compared with lettuce alone. This may indicate that lettuce is lacking in a beneficial nutrient or that snails benefit from dietary changes. The synthetic diet used in their study may have provided a missing nutrient, whereas our wheat germ-based synthetic diet did not. Upatham et al. (1988) did not test their synthetic diet on its own to see if it could support growth. They also did not specify the type of lettuce used for rearing. Iceberg lettuce, for example, would likely be less nutritious than romaine (Kim et al., 2016). The growth and reproduction results of this study support the hypothesis that the synthetic diet was a less suitable diet. By including snails offered a less suitable diet, we attempted to represent the variation in growth and reproduction found in wild snail populations. The growth equation developed in this study does well at estimating juvenile age based on size. However, as a snail becomes larger it is more difficult to estimate its age due to the slower growth rate. Thus, size is likely a reliable indicator of age only in the range of H less than 95 mm. The use of this information in modelling population growth is limited due to the absence of field data and is beyond the scope of this study. The observed patterns of Lissachatina fulica growth are similar to those reported in the literature (Bequaert, 1950; Kondo, 1964; Pawson & Chase, 1984), although Pawson & Chase (1984) reported larger juvenile H and Kondo (1964) reported higher adult H than those found in the present study (Fig. 4). The observed reduction in growth rates after 150 d is consistent with other studies showing that growth slows around the time maturity is reached (Bequaert, 1950; Kondo, 1964; Pawson & Chase, 1984; Tomiyama, 1992, 1993), but does not stop once snails became sexually mature (Tomiyama, 1996). Figure 4. View largeDownload slide Growth of Lissachatina fulica. A–D. Shell height. E, F. Snail mass. Figure 4. View largeDownload slide Growth of Lissachatina fulica. A–D. Shell height. E, F. Snail mass. It is not clear why solitary snails grew significantly larger than the paired snails, although density generally affects animal growth in this manner (Lazaridou-Dimitriadou et al., 1998; Dupont-Nivet et al., 2000; Mangal, Paterson & Fenton, 2010). Possibly, there was a cage effect and the paired snails were crowded. Alternatively, there may have been competition for food, although food availability always appeared adequate. Chemical exudates have been postulated to inhibit growth in aquatic snails (Mangal et al., 2010; Garr et al., 2011). Finally, the absence of sexual reproduction in solitary snails may have led to their larger growth. The earliest initiation of egg laying found in the present study is about a month earlier than in other reports (Bequaert, 1950; Civeyrel & Simberloff, 1996; Raut & Barker, 2002). It is unknown why snails in replicate 3 took longer to lay eggs than those in the previous replicates. All replicates were kept in the same windowless room, under the same environmental conditions, but during different times of the year. It is possible the snails perceived the change in seasons, which affected their development. Alternatively, the differences may simply indicate a natural variation in development often found in this species (Ghose, 1959; Raut & Barker, 2002; Venette & Larson, 2004). Peristome thickness may suggest full reproductive maturity, as originally reported by Tomiyama (1993); however, peristomes were much thinner in the present study. Perhaps this reflects a difference between snails reared in a laboratory and field-collected snails. The presence of outliers in this study demonstrates that the peristome does not consistently thicken as the snail develops into a mature state. Thus, peristome thickness is not a definitive measure of maturity. On average, egg production rates were much higher in these studies than in many others (van der Meer Mohr, 1949; Lange, 1950; Ghose, 1959; Kekauoha, 1966; Pawson & Chase, 1984; Raut & Barker, 2002; Roda et al., 2016). The highest annual egg production previously reported was 1,817 eggs and was based on the total eggs laid by laboratory snails from July to January (Kekauoha, 1966). This 7-month period represented the reproductive season in the field in Hawaii (Kekauoha, 1966). In contrast, snails in our study laid 2,838 eggs from July to January. It is also possible that other reported egg production levels only included viable eggs. Viability in our study was lower on average than the 82% and 93% average viability reported in other studies (van der Meer Mohr, 1949; Kekauoha, 1966). Taking viability into consideration, with an average viability of 60%, snails in our long-term study laid a mean of 2,572 viable eggs during their first year of egg production. This is still higher than has previously been reported. All our studies were done in the laboratory, where egg production can be higher than in the field (Lange, 1950). The higher egg production seen in our study is a result of more eggs being laid per clutch than in other laboratory studies (van der Meer Mohr, 1949; Lange, 1950; Ghose, 1959; Kekauoha, 1966; Pawson & Chase, 1984). Reductions in egg production a year after egg laying began are consistent with previous reports (Kekauoha, 1966; Pawson & Chase, 1984; Raut & Barker, 2002). However, it is important to note that egg production continued throughout the course of our experiment. The reproductive potential of this species is a significant aspect when considering eradication. Snails that each produce more than 100 eggs per month even after 2 years, as in this study, present considerable obstacles to population elimination. Oviposition without mating with another snail was shown to occur, as was demonstrated by Ghose (1959). In addition, the present study was able to compare this method of reproduction with sexual reproduction, showing that reproduction without mating occurs rarely and with lower viability rates and smaller clutch sizes. In some cases, egg production seemed to be delayed in virgin snails, occurring after 240 d. This is consistent with other studies in which snail species prioritize sexual reproduction, delaying self-fertilization and thus reducing inbreeding and its associated risks (Escobar et al., 2011). Because apparent self-fertilization in L. fulica can be delayed, it may occur more often than is observed in short-term studies; as many as 3% of solitary snails can produce viable eggs. The results of this study have shed light on some aspects of the basic biology of L. fulica that partly account for its success as an invasive species and have considerable implications for eradication efforts. (1) Predicting infestation age may be difficult based strictly on juvenile size and such estimates may be altered by ecological factors, such as density. (2) Peristome thickness is not a reliable indicator of sexual maturity, which therefore cannot be precisely determined without dissection. (3) High reproductive rates accentuate the need for rapid control efforts to address new infestations. (4) The ability of a single unmated snail to lay viable eggs means that every snail must be removed during eradication efforts, because even one can start another population. ACKNOWLEDGEMENTS We thank Cory Penca, Amy Howe, Shannen Leahy, Shweta Sharma and everyone in the DPI lab who helped with counting eggs, feeding snails and colony maintenance. This research was funded by the United States Department of Agriculture, Animal and Plant Health Inspection Service and the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, under the Giant African Land Snail Response Program. This research was approved by the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, for publication as contribution 1304. REFERENCES Bequaert, J.C. 1950. Studies on the Achatinidae, a group of African land snails. Bulletin of the Museum of Comparative Zoology , 105: 1– 216. Capinera, J. 2013. Cuban brown snail, Zachrysia provisoria (Gastropoda): damage potential and control. Crop Protection , 52: 57– 63. Google Scholar CrossRef Search ADS   Civeyrel, L. & Simberloff, D. 1996. A tale of two snails: is the cure worse than the disease? Biodiversity and Conservation , 5: 1231– 1252. Google Scholar CrossRef Search ADS   CLEAPSS. 2006. Giant African land snails. Brunel University. Available at: www.cleapss.org.uk/attachments/article/0/L197.pdf. Accessed 30 January 2016. Dupont-Nivet, M., Coste, V., Coinon, P., Bonnet, J.C. & Blanc, J.M. 2000. Rearing density effect on the production performance of the edible snail Helix aspersa Muller in indoor rearing. Annales de Zootechnie , 49: 447– 456. Google Scholar CrossRef Search ADS   Egonmwan, R.I. 2007. Food utilisation in a laboratory colony of the giant African land snail, Archachatina marginata (Swainson) (Pulmonata: Achatinidae). Journal of Zoology , 31: 265– 270. Ejidike, B.N. 2007. Influence of articial diet on captive rearing of African giant land snail Archachatina marginata Pulmonata: Stylommatophora. Journal of Animal and Veterinary Advances , 6: 1028– 1030. Escobar, J.S., Auld, J.R., Correa, A.C., Alonso, J.M., Bony, Y.K., Coutellec, M.A., Koene, J.M., Pointier, J.P., Jarne, P. & David, P. 2011. Patterns of mating-system evolution in hermaphroditic animals: correlations among selfing rate, inbreeding depression, and the timing of reproduction. Evolution , 65: 1233– 1253. Google Scholar CrossRef Search ADS PubMed  Estebenet, A.L. & Cazzaniga, N.J. 1992. Growth and demography of Pomacea canaliculata (Gastropoda: Ampullariidae) under laboratory conditions. Malacological Review , 25: 1– 12. Garr, A.L., Lopez, H., Pierce, R. & Davis, M. 2011. The effect of stocking density and diet on the growth and survival of cultured Florida apple snails. Pomacea paludosa. Aquaculture , 311: 139– 145. Google Scholar CrossRef Search ADS   Ghose, K.C. 1959. Observations on the mating and oviposition of two land Plumonates, Achatina fulica Bowdich and Macrochlamys indica Godwin-Austen. Bombay Natural History Society , 56: 183– 187. Kekauoha, W. 1966. Life history and population studies of Achatina fulica. Nautilus , 80: 39– 46. Kim, M.J., Moon, Y., Tou, J.C., Mou, B. & Waterland, N.L. 2016. Nutritional value, bioactive compounds and health benefits of lettuce (Lactuca sativa L.). Journal of Food Composition and Analysis , 49: 19– 34. Google Scholar CrossRef Search ADS   Kondo, Y. 1964. Growth rates in Achatina fulica Bowdich. Nautilus , 78: 6– 15. Lange, W.H. 1950. Life history and feeding habits of the giant African snail on Saipan. Pacific Science , 4: 323– 335. Lazaridou-Dimitriadou, M., Alpoyanni, E., Baka, M., Brouziotis, T., Kifonidis, T., Mihaloudi, E., Sioula, D. & Vellis, G. 1998. Growth, mortality and fecundity in successive generations of Helix aspersa Muller cultured indoors and crowding effects on fast-, medium- and slow-growing snails of the same clutch. Journal of Molluscan Studies , 64: 67– 74. Google Scholar CrossRef Search ADS   Mangal, T.D., Paterson, S. & Fenton, A. 2010. Effects of snail density on growth, reproduction and survival of Biomphalaria alexandrina exposed to Schistosoma mansoni. Journal of Parasitology Research , 2010: 1– 6. Google Scholar CrossRef Search ADS   Mead, A.R. 1949. The giant snails. Atlantic Monthly , 184: 38– 42. Mead, A.R. 1961. The giant African snail: a problem in economic malacology . University of Chicago Press, Chicago. Pawson, P.A. & Chase, R. 1984. The life cycle and reproductive activity of Achatina fulica (Bowdich) in laboratory culture. Journal of Molluscan Studies , 50: 85– 91. Google Scholar CrossRef Search ADS   R Core Team. 2014. R: a language and environment for statistical computing . R Foundation for Statistical Computing, Vienna. www.R-project.org/. Accessed 30 January 2016. Raut, S.K. & Barker, G.M. 2002. Achatina fulica Bowdich and other Achatinidae as pests in tropical agriculture. In: Molluscs as crop pests  ( G.M. Barker, ed.), pp. 55– 114. CAB International, Wallingford, UK. Google Scholar CrossRef Search ADS   Raut, S.K. & Ghose, K.C. 1984. Pestiferous land snails of India. In: Zoological Survey of India , 11: pp. 1– 151. Bani Press, Calcutta. Roda, A., Nachman, G., Weihman, S., Yong Cong, M. & Zimmerman, F. 2016. Reproductive ecology of the giant African snail in south Florida: implications for eradication programs. PloS One , 11: e0165408. Google Scholar CrossRef Search ADS PubMed  Smith, T.R., White-Mclean, J., Dickens, K., Howe, A.C. & Fox, A. 2013. Efficacy of four molluscicides against the giant African snail, Lissachatina fulica (Gastropoda: Pulmonata: Achitinidae). Florida Entomologist , 96: 396– 402. Google Scholar CrossRef Search ADS   Tomiyama, K. 1992. Homing behaviour of the giant African snail, Achatina fulica (Ferussac) (Gastropoda; Pulmonata). Journal of Ethology , 10: 139– 147. Google Scholar CrossRef Search ADS   Tomiyama, K. 1993. Growth and maturation pattern in the African giant snail, Achatina fulica (Ferussac) (Stylommatophora: Achatinidae). Venus , 52: 87– 100. Tomiyama, K. 1996. Mate-choice criteria in a protandrous simultaneously hermaphroditic land snail Achatina fulica (Ferussac) (Stylommatophora: Achatinidae). Journal of Molluscan Studies , 62: 101– 111. Google Scholar CrossRef Search ADS   Upatham, E.S., Kruatrachue, M. & Baidikul, V. 1988. Cultivation of the giant African snail, Achatina fulica. Journal of the Science Society of Thailand , 14: 25– 40. Google Scholar CrossRef Search ADS   van der Meer Mohr, J.C. 1949. On the reproductive capacity of the African or giant snail, Achatina fulica (Fer). Treubia , 20: 1– 10. Venette, R.C. & Larson, M. 2004. Mini risk assessment giant African snail, Achatina fulica Bowdich [Gastropoda: Achatinidae] . University of Minnesota. Published by Oxford University Press on behalf of The Malacological Society of London 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molluscan Studies Oxford University Press

Laboratory assessment of growth and reproduction of Lissachatina fulica (Gastropoda: Achatinidae)

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Published by Oxford University Press on behalf of The Malacological Society of London 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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Abstract

Abstract Despite Lissachatina fulica being a pest of agricultural and ornamental plants in the worldwide tropics for more than 100 years, information on its biology is limited. In this study, 360 snails were reared in the laboratory from a colony collected from the wild (non-native) population in Miami-Dade County, Florida, and were monitored for growth (height and mass) and egg production for up to 930 d. Snails averaged 195 d old and 97 mm in height when egg laying was initiated. Though the average snail peristome thickness was lower (0.1 mm) before eggs were first laid than after (0.4 mm), peristome thickness was inconsistent in predicting full reproductive maturity. Egg production averaged 4,698 eggs per snail and egg viability averaged 49% in the first 540 d after hatching. Oviposition without mating was shown to be possible, but occurred rarely (3% of snails) and with lower viability rates and smaller clutch sizes. The ability to oviposit without mating and the high reproductive rates found in this study indicate that eradication efforts addressing new infestations must be complete, because even one snail may start another population. INTRODUCTION Lissachatina fulica (Bowdich, 1822) is a major pest in regions where it is established, probably because it has a diverse diet and is very prolific. The strong reproductive potential of this generalist feeder has helped it to establish populations throughout the worldwide tropics (Raut & Barker, 2002). Despite being a recognized pest species for more than 100 years, information on its biology is limited. Growth rates, age of sexual maturity and fecundity of L. fulica need to be better understood in order to effectively manage and prevent infestations of this species. Growth studies on L. fulica have reported shell heights ranging from 35 to 63 mm after 60 d of age and 83–104 mm after 150 d (Bequaert, 1950; Kondo, 1964; Pawson & Chase, 1984). Because new population centres continue to be discovered in southern Florida, regulatory personnel are interested in distinguishing new infestations from older infestations as they are found (Smith et al., 2013). An estimate of a snail’s age based on shell size could possibly be used to give an estimate of how long snails have inhabited a specific site, since snails become larger over time. Predictability will probably be reduced in later months of life due to the well-known tendency of snail growth rate to decline as snails become older. Nevertheless, establishing the age of younger infestations has value in assessing dispersiveness. The reliability of existing literature on L. fulica is uncertain. For example, sample sizes were not mentioned by Bequaert (1950) or Pawson & Chase (1984), and Kondo (1964) used a sample of only five snails. The diversity of results reported for growth rates in L. fulica could be due to inadequate sample sizes, dissimilar rearing and experimental designs, or genetic differences found in snails in different geographical regions. Lissachatina fulica is hermaphroditic, but the male reproductive system develops first and is capable of copulation before the female reproductive system develops completely (Mead, 1949; Tomiyama, 1993). Only fully mature snails are capable of oviposition. Understanding which snails in a population are capable of producing eggs is important in understanding population growth. There is no known visual method for accurately identifying reproductive maturity in this species. However, the thickness of the peristome has been suggested by Tomiyama (1993) to be a useful tool to assess full sexual maturity. As the snail becomes mature, it allocates less growth towards body mass and shell size (Pawson & Chase, 1984) and more to the thickness of the shell, including the peristome (Tomiyama, 1993). Tomiyama compared peristome thickness and maturity based on the assessment of 52 field-collected snails with thin peristomes (<0.5 mm) and an additional 52 larger snails that had thick peristomes (>0.8 mm). Snails with thick peristomes had oocytes in ovotestes and large albumen glands, which are required for egg laying; by contrast, snails with thin peristomes had no oocytes and smaller albumen glands (Tomiyama, 1993). It would be useful to know if the peristome thickens around the time of the first egg-laying event, as this would help to determine snail age and the duration of infestation. Reproductive success can be measured by determining the age at which egg laying begins, the number of viable eggs laid per clutch and the number of viable eggs laid per year. Literature reports of when oviposition commences range from 5 to 15 months of age (Bequaert, 1950; Ghose, 1959; Pawson & Chase, 1984; Civeyrel & Simberloff, 1996; Raut & Barker, 2002). Egg production also varies, with reports of 160–1,817 eggs produced annually (Kekauoha, 1966; Raut & Barker, 2002) and 10–460 eggs laid per clutch (van der Meer Mohr, 1949; Lange, 1950; Ghose, 1959; Kekauoha, 1966; Pawson & Chase, 1984; Raut & Barker, 2002; Roda et al., 2016). Egg production can vary greatly according to region and parental age (Raut & Barker, 2002), thus more study is needed to narrow the estimate of when oviposition commences in local populations and to estimate egg production rates in snails of known age. Lissachatina fulica is hermaphroditic and the potential for reproduction without mating with another snail would greatly increase its ability to invade and spread to new areas. However, previous reports have been inconsistent in this respect (van der Meer Mohr, 1949; Ghose, 1959; Mead, 1961; Kondo, 1964; Kekauoha, 1966; CLEAPSS, 2006). Ghose (1959) observed egg laying by solitary, unmated snails, but the findings are of limited applicability, because sample sizes were not mentioned and the rate of reproduction without mating was not determined. Sources that mention reproduction without mating have assumed the process to be self-fertilization (van der Meer Mohr, 1949; Ghose, 1959). The purpose of this study was to determine critical aspects of L. fulica biology that affect pest control measures. Specific objectives were to determine (1) growth rates at various densities and diet regimes, (2) the age and size at first oviposition, (3) whether peristome thickness is a reliable indicator of sexual maturity, (4) short-term and long-term reproductive rates and (5) whether this species will reproduce without mating. MATERIAL AND METHODS Rearing methods Experimental snails were reared in the FDACS Florida Biological Control Laboratory (FBCL) in Gainesville, Florida and were F1 generation snails produced by snails that were field collected from the wild population in Miami-Dade County, Florida. Snails were provided a synthetic gypsy moth diet or a diet of organic romaine lettuce. Lettuce was used as a representative of a ‘natural’ diet, more suitable than the synthetic ‘gypsy moth’ diet (Estebenet & Cazzaniga, 1992; Capinera, 2013). The synthetic diet was a wheat germ-based diet used for gypsy moth rearing (Bio-Serv Flemington, NJ) and was selected as a less suitable diet, as it will allow for the growth and reproduction of the species, but has been previously observed (unpublished observations) to produce smaller snails with higher rates of mortality. Use of the less suitable synthetic diet should show how diet affects the onset of reproduction, representing some of the natural variation found in wild snail populations. Calcium needed for shell growth was provided in each experiment as lawn lime suspended in agar (300 g 54% pelletized lime, 25 g agar and 1 l boiling water). Snails were provided with a constant surplus of food and calcium. Cages consisted of 4-l plastic containers with a potting soil substrate (Metro Mix 930, Sun Gro Horticulture, Agawam, MA). The amount of soil provided in a cage increased as the snail grew; this ensured proper habitat for egg laying, but still allowed for easy access to the snail when it was small. All cages were initially filled with 300 ml soil, then cleaned and the soil replaced each month thereafter using 800 ml of soil. The cages were maintained in a photoperiod of 16:8 h L:D; temperature and humidity ranged from 21 to 25 °C and 28–66%, respectively (Raut & Ghose, 1984; Raut & Barker, 2002). Experimental design In order to evaluate the age of sexual maturity, reproductive rates and to determine if mating with another snail is required for egg production, four treatments were compared in a 2 × 2 factorial design: solitary and paired snails were provided either a synthetic diet or a diet of organic romaine lettuce. This experiment was conducted using 4–6 week-old neonates with shell heights (H) of 15–20 mm, as these were old enough to avoid damage to their delicate shells during handling. Also, these snails were young enough to ensure that they were unmated (Ghose, 1959; Tomiyama, 1992). This experiment was conducted with three replicates, each with a different starting date. Replicate 3 ran from 4 May to 30 December, and the other two from 23 January to 20 September and from 17 August to 14 April. Each replicate consisted of 20 cages per treatment, containing snails that hatched on the same date (±24 h) (total n = 360 snails; 60 cages each from the paired and solitary treatments, using lettuce or synthetic diets). H (measured parallel to the axis of coiling from the tip of the spire to the most distant point of the aperture edge) and mass (M) were measured every 30 d for 240 d. H was measured using a Fisher Scientific 15-077-958 caliper, and M using a Mettler Toledo ML1502e balance. In some cases, the spire of the shell was damaged, reducing H; these specimens were removed from analyses. Snails were monitored for egg production for up to 930 d. Data were analysed using R statistical computing software (R Core Team, 2014), the lawstat package (v. 2.4.1.tar.gz) and the dunn.test package. A two-way ANOVA was used to determine the effects of diet and density on H and M 240 d after hatching. H and M were analysed within each diet and density treatment, using the two-tailed Student’s t-test. To further test diet quality, a two-way ANOVA was used to test whether diet and density affected mean percent survival. Pattern of growth The pattern of growth (H) for snails from the reproduction studies described above was analysed with polynomial regressions. After the growth studies were terminated at 240 d, additional measurements were continued on a subset of the snails. Snails from the first replicate were monitored for long-term growth for 600 d (total n = 60 snails; 10 cages each from the paired and solitary treatments, and the lettuce and synthetic diets). First oviposition When snails in the growth studies first produced eggs, they were measured for H. After 240 d, many snails in replicate 3 had not deposited eggs, which was in strong contrast to the previous two replicates. For this replicate, monitoring was extended until 360 d after hatching. However, only data from the first 300 d were used in the reproductive rate analysis of these snails. A two-way ANOVA was used to determine the effects of diet and density on (1) age and (2) H of the adults at the first egg-laying event. Student’s t-test was used to compare mean age and H of the adults at the first egg-laying event within each diet treatment and within each density treatment. Peristome thickness was measured using a Marathon CO 030150 caliper every 30 d, to determine whether the peristome thickened around the time eggs were first laid. Peristome thickness was plotted against the time at which the measurements were taken relative to the first oviposition (i.e. negative values for time before first oviposition, positive values for time after first oviposition). A repeated-measures ANOVA was used to analyse peristome thicknesses before and after first oviposition. Short-term reproduction As egg laying commenced, the eggs in each clutch were counted. Clutches laid between hatching and 241 d by paired snails were randomly chosen to test egg viability, including 91 clutches from paired snails offered lettuce and 86 clutches from paired snails offered the synthetic diet. All clutches laid by solitary snails were tested for viability (n = 5 clutches from snails fed lettuce and 2 from snails fed synthetic diet). For determination of egg viability, eggs were buried under a thin layer of moist soil in a 15.2-cm diameter petri dish. Live hatchlings were counted after 3 weeks and clutch viability was calculated. Cages were checked for eggs every 3–5 d. The total number of eggs found in each cage (n = 59 cages for lettuce and 58 for synthetic diet paired treatments; n = 60 cages for lettuce and 56 for synthetic diet solitary treatments), between hatching and 241 d, was divided by the number of snails in each cage to determine the mean number of eggs produced per snail. The analysis did not include eggs produced after 300 d by late-developing snails in replicate 3. A two-way ANOVA was used to test whether diet and density affected the total number of eggs laid per snail, the percent viability of eggs laid and clutch size from cages monitored for 240 d (or 300 d). In order to address the unequal variance assumption violation detected by Levene’s test, a Mann-Whitney rank sum test was used to test the significance of differences in the total number of eggs laid per snail between diet treatments. Student’s t-test was used to test the significance of differences found between diet treatments in the percent viability of eggs laid and clutch size. The same tests were run for the total number of eggs laid per snail, for the percent viability of eggs laid and for clutch size between density treatments. Long-term reproduction As with growth, egg production monitoring continued on a subset of snails from the first replicate. However, egg production was monitored for longer than growth, for a total of 930 d after hatching. The data reported only include surviving snails from the lettuce treatment (n = 16 snails), because only one snail survived to 930 d in the synthetic diet treatment. All eggs that were laid were counted and 128 clutches were chosen to test egg viability, as described above. The numbers of eggs found in each of the eight paired-snail cages were counted every 30 d after hatching and then divided by the number of snails in each cage to find the number of eggs laid per month per snail. Snails that did not produce eggs that month were included in the analysis. Self-fertilization extended In addition to the eggs produced by snails in the reproduction experiment described above, we were able to assess egg production from additional solitary and paired snails. Added to the above experiment were egg-production data from 134 solitary snails (total n = 246 including the previous replicates), of which 65 were offered lettuce and 69 the synthetic diet, and data from 132 snails raised in pairs (total n = 366, including the previous replicates). These snails were reared similarly, but 116 were kept in a greenhouse with ambient lighting and humidity, with temperature ranging from 23 to 26 °C. After some died or were culled at 240 d, 143 solitary and 92 paired snails were still being monitored until 540 d after hatching. Dull, soft, nonviable eggs were frequently found in solitary snail cages. Egg clutches with only dull eggs were not considered in fecundity data. Otherwise, clutches (including eight clutches from snails fed the synthetic diet and seven clutches from snails fed lettuce) were tested for viability as described above. RESULTS Growth was affected by diet and density after 240 d, while survival was not. From the average of solitary and paired snails, the lettuce diet produced snails with greater H (F1,341 = 200.06, P < 0.01) and M (F1,346 = 231.39, P < 0.01), as compared with the synthetic diet (Table 1). In comparing the solitary snail treatment with the paired snail treatment, regardless of diet, the solitary snail treatment produced snails with greater mean H (F1,341 = 181.81, P < 0.01) and M (F1,346 = 127.25, P < 0.01) (Table 1). No interaction effect was found on H (F1,341 = 2.57, P = 0.11), but an interaction between diet and density was noted with respect to M (F1,346 = 5.13, P = 0.02). Diet (F1,8 = 2.52, P = 0.15) and density (F1,8 = 0.33, P = 0.58) did not significantly affect survival and no interaction effect was found (F1,8 = 0.91, P = 0.37). Table 1. Means (±SD), ranges and sample sizes (number of snails, n) for height and mass of Lissachatina fulica when offered lettuce or synthetic diet and reared alone or in pairs for 240 d after hatching. Density  Diet  Height (mm)  Mass (g)      Mean  Range  n  Mean  Range  n  Paired  Lettuce  107 ± 8 aB  75–121  117  139 ± 32 aB  53–264  118    Synthetic diet  96 ± 9 bB  73–133  113  92 ± 28 bB  43–170  116  Solitary  Lettuce  121 ± 8 aA  103–134  60  190 ± 41 aA  115–278  60    Synthetic diet  107 ± 9 bA  90–121  55  125 ± 35 bA  76–192  56  Density  Diet  Height (mm)  Mass (g)      Mean  Range  n  Mean  Range  n  Paired  Lettuce  107 ± 8 aB  75–121  117  139 ± 32 aB  53–264  118    Synthetic diet  96 ± 9 bB  73–133  113  92 ± 28 bB  43–170  116  Solitary  Lettuce  121 ± 8 aA  103–134  60  190 ± 41 aA  115–278  60    Synthetic diet  107 ± 9 bA  90–121  55  125 ± 35 bA  76–192  56  Means in a column, within each density, followed by the same lowercase letters are not significantly different (P > 0.05; two-tailed t-test). Means in a column, within each diet, followed by the same uppercase letters are not significantly different (P > 0.05; two-tailed t-test). The growth plots of paired and solitary snails produced negative exponential curves (Fig. 1). According to the H/growth regression for paired snails (Fig. 1A), for these experimental conditions, a snail of H 20 mm is 41 d old. Similarly, 34 and 85 mm snails are 60 and 150 d, respectively. The trend for greater H among solitary snails than paired snails further supports a density effect on growth. Data from long-term growth studies show that snails, on average, did not continue to grow after 240 d (Fig. 1). Figure 1. View largeDownload slide Growth of Lissachatina fulica reared on a diet of romaine lettuce or on a synthetic diet. Polynomial regressions were fitted on individual measurements for shell height. A. Paired snails regardless of diet. B. Paired snails. C. Solitary snails. Figure 1. View largeDownload slide Growth of Lissachatina fulica reared on a diet of romaine lettuce or on a synthetic diet. Polynomial regressions were fitted on individual measurements for shell height. A. Paired snails regardless of diet. B. Paired snails. C. Solitary snails. First oviposition The minimum time from hatch to egg laying was 124 d (Table 2). The number of days from hatch until the first egg-laying event did not differ between diet treatments (F1,117 = 0.01, P = 0.93), or between paired and single treatments (F1,117 = 0.26, P = 0.61) and there was no interaction effect (F1,117 = 0.06, P = 0.81) (Table 2). Diet affected snail H when oviposition commenced (F1,228 = 42.53, P < 0.01); however, density did not (F1,228 = 1.28, P = 0.26) and no interaction was found between diet and density (F1,228 = 0.235, P = 0.63) (Table 2). Eight solitary virgin snails laid large clutches, five of which proved to be viable, attesting that oviposition without mating with another snail is possible. Snails offered lettuce were larger at the first oviposition than were snails offered the synthetic diet, but only among paired snails (t-test, t222 = 6.18, P < 0.01), not among solitary snails (t-test, t6 = 2.27, P = 0.06). Table 2. Means (±SD), ranges and sample sizes (number of cages) for the number of days from hatching until Lissachatina fulica laid eggs, according to diet and density treatments. Density  Diet  Prereproductive period (d)  Height (mm)      Mean  Range  n (cages)  Mean  Range  n (snails)  Paired  Lettuce  194 ± 57 aA  127–339  56  102 ± 11 aA  78–124  112    Synthetic diet  195 ± 56 aA  124–339  57  92 ± 12bA  73–122  112  Solitary  Lettuce  201 ± 59 aA  164–314  6  107 ± 8 aA  100–121  6    Synthetic diet  214 ± 49 aA  179–249  2  93 ± 5 aA  90–97  2  Density  Diet  Prereproductive period (d)  Height (mm)      Mean  Range  n (cages)  Mean  Range  n (snails)  Paired  Lettuce  194 ± 57 aA  127–339  56  102 ± 11 aA  78–124  112    Synthetic diet  195 ± 56 aA  124–339  57  92 ± 12bA  73–122  112  Solitary  Lettuce  201 ± 59 aA  164–314  6  107 ± 8 aA  100–121  6    Synthetic diet  214 ± 49 aA  179–249  2  93 ± 5 aA  90–97  2  Means (±SD), medians, ranges, sample sizes (number of snails) and height of L. fulica at the first egg-laying event are given. Only eight solitary virgin snails laid large, viable-looking clutches. Means in a column, within each density, followed by the same lowercase letters are not significantly different (P > 0.05; two-tailed t-test). Means in a column, within each diet, followed by the same uppercase letters are not significantly different (P > 0.05; two-tailed t-test). Peristome thickness increased as snails oviposited for the first time (F1,932 = 1021, P < 0.01) (Fig. 2). Despite the significant relationship between peristome thickness and the onset of oviposition, outliers were present indicating peristome thicknesses <0.1 mm after eggs were first laid. Figure 2. View largeDownload slide Individual measurements of Lissachatina fulica peristome thickness taken before (–) and after (+) first oviposition. First oviposition occurred on day 0. Figure 2. View largeDownload slide Individual measurements of Lissachatina fulica peristome thickness taken before (–) and after (+) first oviposition. First oviposition occurred on day 0. Egg production The number of eggs laid per snail (F1,229 = 9.11, P < 0.01; F1,229 = 236.70, P < 0.01), egg viability (F1,180 = 8.71, P < 0.01; F1,180 = 13.65, P < 0.01) and clutch size (F1,476 = 24.31, P < 0.01; F1,476 = 25.92, P < 0.01) were affected by diet and density, respectively (Table 3). The interaction between diet and density was significant for the number of eggs laid per snail (F1,229 = 9.88, P < 0.01), but not egg viability (F1,180 = 2.75, P = 0.10) or clutch size (F1,476 = 0.48, P = 0.49). The average total number of eggs laid per snail over the course of the experiment from paired snails was higher from the lettuce diet than the synthetic diet (U = 2168, P = 0.01) (Table 3). Also, paired snails offered lettuce laid eggs with higher viability (t-test, t175 = 3.45, P < 0.01) and also laid larger clutches (Student’s t-test, t471 = 5.14, P < 0.01) than paired snails offered the synthetic diet. Paired snails laid far more eggs per snail than solitary snails offered lettuce (U = 3332.5, P < 0.01) and synthetic diet (U = 3074, P < 0.01). In lettuce treatments, egg production without mating also resulted in eggs with lower percent viability (t-test, t84 = 4.36, P < 0.01) and smaller clutch sizes (t-test, t267 = 3.97, P < 0.01) than sexual reproduction. Two clutches were laid by solitary snails on the synthetic diet. These clutches had fewer eggs than clutches laid by paired snails on the synthetic diet (t-test, t209 = 3.01, P < 0.01), but egg viability did not differ (t-test, t86 = 0.55, P = 0.58). Table 3. Means (±SD), ranges and sample sizes (n) for total number of eggs laid per snail, percent egg viability and clutch sizes among diet treatments for eggs laid before 241 d for Lissachatina fulica. Density  Diet  Eggs/snail  Viability (%)  Eggs/clutch      Meana  Range  n (cages)  Meanb  Range  n (clutches)  Meanb  Range  n (clutches)  Paired  Lettuce  738 ± 526 aA  0–1863  59  68 ± 28 aA  0–100  91  322 ± 151 aA  13–1033  264    Synthetic diet  486 ± 294 bA  0–1034  58  52 ± 33 bA  0–99  86  261 ± 91 bA  28–584  209  Solitary  Lettuce  5 ± 18 aB  0–109  60  13 ± 22 aB  0–50  5  53 ± 32 aB  34–109  5    Synthetic diet  2 ± 14 aB  0–103  56  40 ± 33 aA  16–63  2  67 ± 52 aB  30–103  2  Density  Diet  Eggs/snail  Viability (%)  Eggs/clutch      Meana  Range  n (cages)  Meanb  Range  n (clutches)  Meanb  Range  n (clutches)  Paired  Lettuce  738 ± 526 aA  0–1863  59  68 ± 28 aA  0–100  91  322 ± 151 aA  13–1033  264    Synthetic diet  486 ± 294 bA  0–1034  58  52 ± 33 bA  0–99  86  261 ± 91 bA  28–584  209  Solitary  Lettuce  5 ± 18 aB  0–109  60  13 ± 22 aB  0–50  5  53 ± 32 aB  34–109  5    Synthetic diet  2 ± 14 aB  0–103  56  40 ± 33 aA  16–63  2  67 ± 52 aB  30–103  2  aMeans in a column, within each density, followed by the same lowercase letters are not significantly different (P > 0.05; Mann-Whitney rank sum test). Means in a column, within each diet, followed by the same uppercase letters are not significantly different (P > 0.05; Mann-Whitney rank sum test). bMeans in a column, within each density, followed by the same lowercase letters are not significantly different (P > 0.05; two-tailed t-test). Means in a column, within each diet, followed by the same uppercase letters are not significantly different (P > 0.05; two-tailed t-test). Egg laying continued while monitoring long-term paired snails and the mean number of eggs laid per month per snail and percent egg viability are shown in Figure 3. Figure 3. View largeDownload slide The mean total Lissachatina fulica eggs laid per snail over time and the mean percent of eggs that were viable. Error bars indicate standard error. Figure 3. View largeDownload slide The mean total Lissachatina fulica eggs laid per snail over time and the mean percent of eggs that were viable. Error bars indicate standard error. Self-fertilization extended In the original experiment with 120 solitary snails, 116 snails survived and were monitored from hatching to between 240 and 300 d. Of these, eight solitary virgin snails (7%) laid one clutch each. Of the total 246 solitary snails monitored for 240 d, seven snails (3%) laid one clutch each (Table 4). When eggs from one clutch laid by a virgin snail were reared to adulthood, they produced viable offspring. Table 4. Total eggs produced per snail, mean (±SD) egg viability and mean (±SD) clutch size for eggs produced 0–240 d and 0–540 d for solitary and paired Lissachatina fulica, regardless of diet. Duration (d)  Treatment  Snails monitored  Cages Monitored  Cages with eggs (%)  Eggs/snail  Viability (%)  Eggs/clutch  240  Paired  366  183  67  843  52 ± 35  330 ± 160    Solitary  246  246  3  1.24  25 ± 27  44 ± 31  540  Paired  92  46  98  4698  49 ± 36  332 ± 162    Solitary  143  143  4  10.18  27 ± 31  132 ± 136  Duration (d)  Treatment  Snails monitored  Cages Monitored  Cages with eggs (%)  Eggs/snail  Viability (%)  Eggs/clutch  240  Paired  366  183  67  843  52 ± 35  330 ± 160    Solitary  246  246  3  1.24  25 ± 27  44 ± 31  540  Paired  92  46  98  4698  49 ± 36  332 ± 162    Solitary  143  143  4  10.18  27 ± 31  132 ± 136  Six of the 143 solitary snails (4%) monitored for 540 d laid eggs that appeared viable. One snail laid five egg clutches, while the remainder laid just one clutch each. Egg viability ranged from 0% to 98% per clutch (Table 4). DISCUSSION Diet can significantly affect both H (Upatham, Kruatrachue & Baidikul, 1988) and M (Egonmwan, 2007; Ejidike, 2007) in Achatinidae. Upatham et al. (1988) found that a combination of a synthetic diet and lettuce promoted increases in both H and M as compared with lettuce alone. This may indicate that lettuce is lacking in a beneficial nutrient or that snails benefit from dietary changes. The synthetic diet used in their study may have provided a missing nutrient, whereas our wheat germ-based synthetic diet did not. Upatham et al. (1988) did not test their synthetic diet on its own to see if it could support growth. They also did not specify the type of lettuce used for rearing. Iceberg lettuce, for example, would likely be less nutritious than romaine (Kim et al., 2016). The growth and reproduction results of this study support the hypothesis that the synthetic diet was a less suitable diet. By including snails offered a less suitable diet, we attempted to represent the variation in growth and reproduction found in wild snail populations. The growth equation developed in this study does well at estimating juvenile age based on size. However, as a snail becomes larger it is more difficult to estimate its age due to the slower growth rate. Thus, size is likely a reliable indicator of age only in the range of H less than 95 mm. The use of this information in modelling population growth is limited due to the absence of field data and is beyond the scope of this study. The observed patterns of Lissachatina fulica growth are similar to those reported in the literature (Bequaert, 1950; Kondo, 1964; Pawson & Chase, 1984), although Pawson & Chase (1984) reported larger juvenile H and Kondo (1964) reported higher adult H than those found in the present study (Fig. 4). The observed reduction in growth rates after 150 d is consistent with other studies showing that growth slows around the time maturity is reached (Bequaert, 1950; Kondo, 1964; Pawson & Chase, 1984; Tomiyama, 1992, 1993), but does not stop once snails became sexually mature (Tomiyama, 1996). Figure 4. View largeDownload slide Growth of Lissachatina fulica. A–D. Shell height. E, F. Snail mass. Figure 4. View largeDownload slide Growth of Lissachatina fulica. A–D. Shell height. E, F. Snail mass. It is not clear why solitary snails grew significantly larger than the paired snails, although density generally affects animal growth in this manner (Lazaridou-Dimitriadou et al., 1998; Dupont-Nivet et al., 2000; Mangal, Paterson & Fenton, 2010). Possibly, there was a cage effect and the paired snails were crowded. Alternatively, there may have been competition for food, although food availability always appeared adequate. Chemical exudates have been postulated to inhibit growth in aquatic snails (Mangal et al., 2010; Garr et al., 2011). Finally, the absence of sexual reproduction in solitary snails may have led to their larger growth. The earliest initiation of egg laying found in the present study is about a month earlier than in other reports (Bequaert, 1950; Civeyrel & Simberloff, 1996; Raut & Barker, 2002). It is unknown why snails in replicate 3 took longer to lay eggs than those in the previous replicates. All replicates were kept in the same windowless room, under the same environmental conditions, but during different times of the year. It is possible the snails perceived the change in seasons, which affected their development. Alternatively, the differences may simply indicate a natural variation in development often found in this species (Ghose, 1959; Raut & Barker, 2002; Venette & Larson, 2004). Peristome thickness may suggest full reproductive maturity, as originally reported by Tomiyama (1993); however, peristomes were much thinner in the present study. Perhaps this reflects a difference between snails reared in a laboratory and field-collected snails. The presence of outliers in this study demonstrates that the peristome does not consistently thicken as the snail develops into a mature state. Thus, peristome thickness is not a definitive measure of maturity. On average, egg production rates were much higher in these studies than in many others (van der Meer Mohr, 1949; Lange, 1950; Ghose, 1959; Kekauoha, 1966; Pawson & Chase, 1984; Raut & Barker, 2002; Roda et al., 2016). The highest annual egg production previously reported was 1,817 eggs and was based on the total eggs laid by laboratory snails from July to January (Kekauoha, 1966). This 7-month period represented the reproductive season in the field in Hawaii (Kekauoha, 1966). In contrast, snails in our study laid 2,838 eggs from July to January. It is also possible that other reported egg production levels only included viable eggs. Viability in our study was lower on average than the 82% and 93% average viability reported in other studies (van der Meer Mohr, 1949; Kekauoha, 1966). Taking viability into consideration, with an average viability of 60%, snails in our long-term study laid a mean of 2,572 viable eggs during their first year of egg production. This is still higher than has previously been reported. All our studies were done in the laboratory, where egg production can be higher than in the field (Lange, 1950). The higher egg production seen in our study is a result of more eggs being laid per clutch than in other laboratory studies (van der Meer Mohr, 1949; Lange, 1950; Ghose, 1959; Kekauoha, 1966; Pawson & Chase, 1984). Reductions in egg production a year after egg laying began are consistent with previous reports (Kekauoha, 1966; Pawson & Chase, 1984; Raut & Barker, 2002). However, it is important to note that egg production continued throughout the course of our experiment. The reproductive potential of this species is a significant aspect when considering eradication. Snails that each produce more than 100 eggs per month even after 2 years, as in this study, present considerable obstacles to population elimination. Oviposition without mating with another snail was shown to occur, as was demonstrated by Ghose (1959). In addition, the present study was able to compare this method of reproduction with sexual reproduction, showing that reproduction without mating occurs rarely and with lower viability rates and smaller clutch sizes. In some cases, egg production seemed to be delayed in virgin snails, occurring after 240 d. This is consistent with other studies in which snail species prioritize sexual reproduction, delaying self-fertilization and thus reducing inbreeding and its associated risks (Escobar et al., 2011). Because apparent self-fertilization in L. fulica can be delayed, it may occur more often than is observed in short-term studies; as many as 3% of solitary snails can produce viable eggs. The results of this study have shed light on some aspects of the basic biology of L. fulica that partly account for its success as an invasive species and have considerable implications for eradication efforts. (1) Predicting infestation age may be difficult based strictly on juvenile size and such estimates may be altered by ecological factors, such as density. (2) Peristome thickness is not a reliable indicator of sexual maturity, which therefore cannot be precisely determined without dissection. (3) High reproductive rates accentuate the need for rapid control efforts to address new infestations. (4) The ability of a single unmated snail to lay viable eggs means that every snail must be removed during eradication efforts, because even one can start another population. ACKNOWLEDGEMENTS We thank Cory Penca, Amy Howe, Shannen Leahy, Shweta Sharma and everyone in the DPI lab who helped with counting eggs, feeding snails and colony maintenance. This research was funded by the United States Department of Agriculture, Animal and Plant Health Inspection Service and the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, under the Giant African Land Snail Response Program. This research was approved by the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, for publication as contribution 1304. REFERENCES Bequaert, J.C. 1950. Studies on the Achatinidae, a group of African land snails. Bulletin of the Museum of Comparative Zoology , 105: 1– 216. Capinera, J. 2013. 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Mini risk assessment giant African snail, Achatina fulica Bowdich [Gastropoda: Achatinidae] . University of Minnesota. Published by Oxford University Press on behalf of The Malacological Society of London 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Published: Feb 1, 2018

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