A Complete Embryonic Developmental Table of Microhyla Fissipes (Amphibia, Anura, Microhylidae)
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Asian Herpetological Research 2017, 8(2): 108–117 ORIGINAL ARTICLE DOI: 10.16373/j.cnki.ahr.170006 A Complete Embryonic Developmental Table of Microhyla fissipes (Amphibia, Anura, Microhylidae) Shouhong WANG1,2, Lanying ZHAO1,2, Lusha LIU1, Dengwei YANG1, Janak Raj KHATIWADA1,2, Bin WANG1 and Jianping JIANG1* 1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2 University of Chinese Academy of Sciences, Beijing 100049, China Abstract Access to embryonic developmental stages is essential basic work for understanding how organisms develop. In this study, seven egg clutches (range 209–564 eggs) of ornamented pygmy frog Microhyla fissipes (Amphibia, Anura, Microhylidae) were obtained from seven breeding pairs in laboratory. One egg clutch of them was observed for the embryonic development, and the staging table of normal development was constructed based on morphological and physiological characteristics. Forty-five developmental stages were defined for M. fissipes, and two major developmental periods were designated: 1) early embryonic development period (stages 1–28), from fertilization to operculum completion stage, lasted for 82.6 hours at water temperature (WT) 23–25oC; 2) larval development period (stages 29–45), from operculum completion to tail complete absorption stage, took 38 days at WT 22–26.5oC, showing that the embryos of this species develop rapidly. In addition, the tadpoles were transparent, which is similar to those in field. These characteristics suggest that M. fissipes would be a good model to study developmental biology, adaptive mechanisms from aquatic to terrestrial phases, environmental toxicology, and human disease. Keywords Microhylidae, embryonic stage, external characters, morphogenesis 1. Introduction characteristics of tadpole development provides the basis for ensuring the same tadpole materials. Among vertebrates, amphibians are known for their Most early staging tables were not comprehensive or unique developmental cycle, especially metamorphosis, undocumented the full embryonic period, as they only which has important implications in their life history focused on either early embryonic or larval development (Werner, 1986). Accordingly, embryonic development separately (Pollister and Moore, 1937; Shumway, (including early embryonic and larval development) 1940; Taylor and Kollros, 1946). However, a more is considered as a critical factor for understanding comprehensive staging table of embryonic development phylogeny, comparative embryology, and molecular for anurans with notes on identification was first reported mechanisms of development in amphibians (Hurney by Gosner (1960). Since then, more staging table studies of anuran embryonic development have been reported, et al., 2015). In particular, since the larvae of anurans which can provide comprehensive and systematic details are morphologically distinct from the adults, it could for embryo developmental research of other species be problematic in confirming the tadpoles through their (Nieuwkoop and Faber, 1967; Iwasawa and Futagami, adults. In addition, the morphological characteristics 1992; Shimizu and Ota, 2003; Xiong et al., 2010; Grenat of tadpoles are unidentical at different developmental et al., 2011; Mohammad Ridzuan, 2013; Narzary and stages (Mohammad Ridzuan, 2013). Information, thus, on Bordoloi, 2013; Aminutes et al., 2015). Nevertheless, * such developmental descriptions are not yet available for Corresponding author: Prof. Jianping JIANG, from Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China, with his all groups of anurans. research focusing on taxonomy, systematics and evolution of amphibians To date, there are at least 100 known developmental and reptiles. E-mail: [email protected] tables for anurans, but few of them are for microhylids Received: 11 April 2017 Accepted: 7 June 2017 (Rao, 1917; Mohanty-Hejmadi et al., 1980; Padhye No. 2 Shouhong WANG et al. Embryonic Development of Microhyla fissipes 109 and Ghate, 1989; Liu et al., 1995; Geng et al., 1995; Each male and female was given a single dose LHRHa Shimizu and Ota, 2003; Fabrezi et al., 2012; Narzary and with 0.3 μg/g body weight (Kouba et al., 2009). After Bordoloi, 2013). The absence of developmental data for hormone injection, the brooders were released into their microhylid frogs is one of the limitations in interpreting respective containers and raised at 28oC. The next day, 7 their evolutionary history (Fabrezi et al., 2012). egg clutches were obtained and the number of eggs was Microhyla fissipes (Anura: Microhylidae) is a small- counted. sized, widespread species which distributes in lowland 2.3 Observation of embryonic development scrub forest, grassland, agricultural land, pastureland and 2.3.1 Management of embryos and tadpoles Only one urban areas of Southeast Asia, Mainland and Taiwan of egg clutch was transferred to glass petri dishes (diameter: China (Matsui et al., 2005; Frost et al., 2017). Females 160 mm) for observation of embryonic development. spawn clutches of 243–453 eggs (diameter: 0.8–1.0 mm) After hatching, every 60 tadpoles was transferred to a and the embryos develop fast, with hatchlings taking 20- container (420 × 300 × 230 mm3) with 150 mm depth de- 30 days to metamorphose (Fei et al., 2009). Furthermore, chlorinated tap water and half of the water was replaced the tadpoles are transparent which provide facilities for every two days. Tadpoles, when their yolks were entirely observing the developmental physiological processes, absorbed, were fed with the solution of boiled chicken such as heartbeat and gill-circulation (Fei et al., 2009; Liu egg yolk once a day. Water temperature was measured et al., 2016). These characteristics suggest that M. fissipes o could be an ideal model for researching the adaptive to the nearest 0.1 C with a thermometer around 4:30 pm mechanism from aquatic to terrestrial phases, development every day. biology, environmental toxicology, and human disease 2.3.2 Definition of embryonic development stages A (Liu et al., 2016). To compensate previous studies which stereo microscope (JSZ8T, Jiang Nan Yong Xin, Nanjing, only reported the early embryonic or postembryonic China) with Mshot Image Analysis system (Mc50-N) development of M. fissipes (Geng et al., 1995; Liu was used to observe the embryonic developmental et al., 1995; Xu and Xu, 2012), a complete developmental morphological characteristics, photograph embryos and staging table with live photographs, presenting all the tadpoles of each development stage, and measure their morphological characters, was accomplished. This study total length (TOL). Embryonic developmental stages were is imperative for further researches of this species and defined based on their morphological and physiological provides important information to understand biodiversity characteristics following published methods (Shumway, of anuran development and the reciprocal influences 1940; Gosner, 1960; Shimizu and Ota, 2003). between larval and adult body plans. 2.4 Data analysis Total number of laying eggs, fertilization ratio (total fertilized eggs / total released 2. Materials and Methods eggs), hatching ratio (total hatched eggs / total fertilized eggs), and abnormality ratio (total deformed embryos 2.1 Animals Seven pairs of adult M. fissipes, collected / total hatchlings) of each group were calculated. from Shuangliu, Chengdu, China (30.5825o N, 103.8438o Polynomial regression was used to explore the E) in June 2015, were used in this work. These pairs relationship between TOL and embryonic development. were labeled from I to VII, and housed in separate plastic Descriptive statistics was present with Mean ± SD. containers (220 ×125 × 135 mm3). These containers contained 20 mm deep de-chlorinated water and were 3. Results decorated with small stones and mosses, making water- land area 3:2. Animal procedures were approved by the 3.1 Development parameters Clutch sizes (n = 7) Animal Care and Use Committee of Chengdu Institute of ranged from 209 to 564 eggs (337.86 ± 127.24). The Biology. mean fertilization ratio was 0.95 ± 0.02 (n = 6) and 2.2 Induced breeding with LHRHa Embryos were hatching ratio was 0.92 ± 0.05 (n = 4), while deformity produced by natural matings between hormonally- ratio was 0.05 ± 0.01 (n = 4) (Table 1). Fertilized egg induced male and female frogs. Ovulation was induced diameter was 0.976 ± 0.023 (n = 6, range 0.942–1.001 by intraperitoneal injections of 0.1 ml Luteinizing mm) and TOL varied considerable during the whole Hormone Releasing Hormone analogue hormones embryonic development (Table 2). Besides, a hump- (LHRHa) at 17:00 on 9 June, 2015, which was diluted shape relationship was presented between TOL and to a concentration of 3μg/ml in sterile 0.65% NaCl. development, with reaching maximum at stage 40 (Figure 110 Asian Herpetological Research Vol. 8 All animal hemispheres rotated for the upper location after fertilization. Stage 2. Late cleavage (lasted 22 minutes): A stage that was from fertilized egg to prior to the appearance of the first cleavage groove (Figure 3, 2a). Stage 3. First cleavage (lasted 13 minutes): A stage characterized by appearance of the first cleavage (meridional), producing two cells of equal sizes, and cleavage groove was from animal hemisphere to vegetal hemisphere. Stage 4. Second cleavage (lasted 15 minutes): Four equal sizes cells were formed with the second cleavage (meridional). Stage 5. Third cleavage (lasted 15 minutes): This stage was a latitudinal cleavage