Comparative study on reproductive behavior of three Thai indigenous apple snails Pila celebensis (Quoy & Gaimard, 1834), P. pesmei (Morlet, 1889) และ P. virescens (Deshayes, 1824)

Species of the Thai indigenous apple snail genus Pila Röding, 1798 are undergoing population decline. Accordingly, their conservation is needed, but the lack of knowledge of their reproductive biology is a significant obstacle to implementing conservation measures. Therefore, this study analyzed the mating and egg-laying behavior of three of the five Thai indigenous apple snails: P. celebensis, P. pesmei, and P. virescens using continuous video recording under laboratory conditions. In addition, the egg-laying behavior of P. virescens in a seminatural habitat was observed in the rainy season of 2023. Moreover, their egg, egg mass, incubation time, and hatchling characteristics were examined. The mating processes were found to be similar among the three species with 15 mating behaviors identified in all three species and two unique behaviors identified only in P. celebensis: ‘mucus secretion’ and ‘mucus deglutition’. Three behaviors were identified for the first time in apple snails: ‘shell pushing’, relating to coercive copulation; and ‘jerking’ and ‘retracting’, which are mate-rejection behavior. The egg-laying processes are different among the three species. Whereas P. celebensis lays eggs on the aquarium wall well above water, P. pesmei lays eggs in a shallow cavity burrowed into the soil, and P. virescens presents variation of oviposition locations, laying eggs at variable distances on the aquarium wall under laboratory conditions, and on the ground or floating objects in a seminatural habitat. The egg and egg mass characteristics differ between P. celebensis and the other two species. That is, P. celebensis produces large polygonal eggs (8.70 ± 0.96 mm) of an orange color, whereas P. pesmei and P. virescens produce smaller spherical eggs of a white color. However, P. virescens eggs (5.24 ± 0.28 mm) are larger than those of P. pesmei (4.37 ± 0.40 mm). The egg mass of P. celebensis includes two components: 21–50 eggs and coating scales, while those of P. pesmei and P. virescens contain 336–485 and 146–399 eggs, respectively, with no coating scales. Due to their largest size, P. celebensis eggs have the longest average incubation time of 24 days, whereas P. pesmei and P. virescens have slightly shorter average incubation times of 21 days and 18 days, respectively. Hatchlings of P. celebensis are the largest among the three species with an average size (shell height) of 7.54 ± 0.72 mm compared to 3.57 ± 0.16 mm and 3.93 ± 0.28 mm for P. pesmei and P. virescens hatchlings respectively. Overall, this study provides a broad understanding of the reproductive biology of the Thai indigenous apple snails that can be applied to implement their conservation. Moreover, ethograms of the mating and egg-laying behavior of apple snails were created for the first time. These ethograms will facilitate future comparative and evolutionary studies on the reproductive strategy of the family Ampullariidae.