【作者】 符史杭；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 兽医， 2010， 博士

【摘要】 全球两栖动物数量正在下降,虎纹蛙已经被收录进IUCN濒危物种红色名录,也已经被确定为国家二级重点保护动物,其濒危程度已经得到共识。海南岛是我国虎纹蛙主要分布区域,历史上虎纹蛙曾广泛分布在全岛各地,近年在野外难见到虎纹蛙的踪迹,以致有学者认为海南虎纹蛙已经消失,了解、把握虎纹蛙资源的现状,采取相应措施有效保护虎纹蛙这个濒危物种已经极为迫切。为了有效保护海南岛虎纹蛙,作者2006年以来多次在岛内进行调查和实验,掌握海南岛虎纹蛙分布、种群数量、非法贸易、农田用途与种群关系、保护执法、食性、食物在胃中停留时间、人工养殖等情况,结合海南的实际情况,建立起一种适合基层使用的执法方法。探讨了海南岛养殖虎纹蛙的最佳模式,将使虎纹蛙资源得到有效的保护。在对虎纹蛙的偷捕痕迹进行记录和实地调查的过程中,证明1991--2007年在海南岛的海口、文昌、琼海、万宁、陵水、三亚、澄迈、定安、屯昌、琼中、临高、儋州、昌江、东方、白沙、乐东等十六市县都有虎纹蛙的分布。2006年3、4、5、8月在海南岛的海口、三亚、琼海、白沙、昌江等五市县采取重捕标志法进行虎纹蛙种群数量研究,根据捕获数量和重捕数量进行计算,结果显示海口市大坡镇227亩的农田生境范围内2006年3月分布约有96只虎纹蛙,2007年3月约有56只虎纹蛙；在桂林洋研究点2006年约有37只虎纹蛙；2007年3月没有发现虎纹蛙的存在；琼海市塔洋镇的研究点2006年4月约有18只虎纹蛙；2007年4月约有9只虎纹蛙；位于昌江县七叉乡的研究点2006、2007年均没有发现虎纹蛙的分布；白沙县青松乡研究点2006、2007年种群数量分别为58只、63只,三亚市高峰乡研究点2006、2007年种群数量分别为21只、11只。目前海南岛虎纹蛙生存的环境主要是正在种植水稻的农田、种植莲藕的农田、种植水芹的农田、农田中的排水沟、撂荒的农田、罗非鱼养殖泥塘、废弃的水塘、种植水芋头的农田等等。表明海南岛还有一定数量的野生虎纹蛙种群,但种群数量下降趋势明显。水田是虎纹蛙的主要栖息地之一,海南岛在发展热带高效农业过程中,实践香蕉下水田、反季节蔬菜生产等新的生产,使水田用途发生相应变化,水田耕作方式也发生变化。通过2006、2007、2008年在海口市东昌农场后湖洋、冯符洋、昌隆洋的研究,发现种植水稻、水芹、水芋头的水田、养殖罗非鱼的泥塘、杂草重生的种植香蕉的水田是虎纹蛙的良好栖息地,种植香蕉、年桔、甘蔗、地瓜、柠檬、青皮冬瓜、番石榴的田块基本没有发现虎纹蛙的分布,表明把水稻田改造为种植水芹、水芋头和养殖罗非鱼的泥塘对虎纹蛙种群的数量没有影响,把水稻田改造为种植香蕉、年桔、甘蔗、地瓜、柠檬、青皮冬瓜、番石榴的田块对虎纹蛙种群数量影响突出,说明在水稻田改种某些作物会对海南岛虎纹蛙种群带来致命的影响,生产中必须慎重考虑改变水田的用途。通过2006-2008年对海南岛虎纹蛙市场贸易情况进行调查,发现海南岛18个市县都存在虎纹蛙贸易现象,大多数市县除每年的1、2月份外,其它的月份均发现存在虎纹蛙贸易现象。非法贸易的前提是获得虎纹蛙,这就引发偷捕虎纹蛙,破坏虎纹蛙资源现象的发生。海南岛存在职业偷捕者,偷捕方法已经发生变化,目前流行的是电击法,其对虎纹蛙种群的破坏力极大。虎纹蛙市场贸易的主要原因是错误的饮食观念导致消费市场的发生、发展；容易捕获；非法贸易的经济利益较大。上述情况表明非法贸易引发的偷捕虎纹蛙行为已经对海南岛虎纹蛙种群造成严重影响。在三年的市场贸易调查过程中检查了2691只虎纹蛙,发现寄生虫病,而没有发现人工养殖中多发的腐皮病、胃肠炎、白内障、红腿病、烂腮病等疾病,没有发现野外虎纹蛙成群死亡的现象,说明目前疾病还没有对海南岛虎纹蛙种群造成明显的影响。对94家养殖场的调查发现2008年海南岛养殖虎纹蛙中疾病的种类有寄生虫病、腐皮病、胃肠炎；2008年海口市、文昌市、三亚市、琼海市在3个养殖周期中都发生过寄生虫病、腐皮病、胃肠炎。养殖中存在对病死虎纹蛙尸体没有进行无害化处理、养殖废水随意排放、亏损出现时停止病蛙治疗向环境放生等现象,对虎纹蛙野生资源将造成一定影响。通过解剖309只虎纹蛙,发现其中的195只虎纹蛙的胃中没有食物残留,114只虎纹蛙的胃中有食物残留,残留食物的成分主要有中腹足目田螺科、蜘蛛目地蛛科、蜈蚣目蜈蚣科、直翅目蝼蛄科、直翅目蟋蟀科、鞘翅目瓢虫科、鞘翅目金龟子科、鳞翅目天蛾科、无尾目蛙科、十足目溪蟹科、直翅目蝗科、莎草目禾本科。其中蝗科23.3%、蝼蛄科22%、瓢虫科11%、溪蟹科9.39%、蟋蟀科9.39%、地蛛科9.39%、金龟子科4.21%、天蛾科4.21%、田螺科2.91%、蜈蚣科2.67%、蛙科1.94%。研究还发现虎纹蛙成蛙个体的大小与虎纹蛙对食物成分的摄食没有显著的差异,虎纹蛙成蛙个体性别与虎纹蛙对食物成分的摄食没有显著的差异。虎纹蛙是被国家林业部批准开展人工养殖的物种之一,其子二代可以上市销售。海南省保护执法中需要鉴定查处没收的虎纹蛙是否为野生虎纹蛙。由于目前已经建立起来的两栖动物鉴定方法不适合在基层执法单位适用,利用野生虎纹蛙食物成分(主要是中腹足目田螺科、蜘蛛目地蛛科、蜈蚣目蜈蚣科、直翅目蝼蛄科、直翅目蟋蟀科、鞘翅目瓢虫科、鞘翅目金龟子科、鳞翅目天蛾科、无尾目蛙科、十足目溪蟹科、直翅目蝗科、莎草目禾本科)与养殖虎纹蛙食物成分(均为膨化颗粒状配合饲料)的差异,可以建立起简便可行的食物成分鉴定方法。从养殖场买来60日龄、90日龄、120日龄、150日龄虎纹蛙,按照相同日龄分组,用混合饲料喂养1周后,绝食24小时,喂人工饲料和蝗虫、蝼蛄、蟋蟀、蜘蛛、田螺、瓢虫、虎纹蛙幼蛙、溪蟹等单种食物,观察排粪情况,用X光透视进行确认。研究发现,蝗虫、蝼蛄、蟋蟀、蜘蛛在同日龄虎纹蛙的胃停留时间基本相同,60日龄虎纹蛙的停留时间为48h±24h、90日龄虎纹蛙的停留时间为48h±12h、120日龄虎纹蛙的停留时间为48h±6h、150日龄虎纹蛙的停留时间为48h±5h。田螺、瓢虫、虎纹蛙幼蛙、溪蟹在相同日龄虎纹蛙的胃中停留时间基本相同,60日龄虎纹蛙的停留时间为96h±24h、90日龄虎纹蛙的停留时间为96h±12h、120日龄虎纹蛙的停留时间为72h±12h、150日龄虎纹蛙的停留时间为48h±12h。蛙膨化型配合饲料在60日龄虎纹蛙的停留时间为24h±12h、90日龄虎纹蛙的停留时间为24h±6h、120日龄虎纹蛙的停留时间为12h±6h、150日龄虎纹蛙的停留时间为12h±2h。说明单种膨化型配合饲料在虎纹蛙胃中停留的时间随虎纹蛙的个体增大变短。研究结果说明在海南岛虎纹蛙执法取证的最佳时间是10小时以内,时间超过120小时将失去取证的意义。根据本研究发现虎纹蛙各种食物在胃中停留时间不同,其中最短时间为10小时,最长时间为120小时的结果,有助执法者准确把握虎纹蛙食性分析时机,提高执法的有效性。从海南省林业局收集到的信息,表明海南省虎纹蛙养殖业开始于1995年,1998年推广到全省,2000、2003、2004、2005、2006、2007年产量分别为超过6000万公斤、200万公斤、150万公斤、558万公斤、648万公斤、518万公斤,2000、2003、2004、2005、2006、2007年产值分别为约108000万元、约8000万元、约6000万元、约3300万元、约3800万元、约3000万元。2000年产量、产值均为养殖史上的最大值。目前虎纹蛙养殖产量处于缓慢恢复的阶段,商品蛙的价格处在低谷徘徊中。对500名虎纹蛙养殖业从业者的调查发现96.6%的被调查者认为是市场销售价格低影响了养殖户的养殖积极性,原因分析中95.5%的被调查者认为是饲料太贵、81.5%的被调查者认为是饲料老板垄断市场,操纵商品蛙的收购价格。海南岛虎纹蛙养殖业的家庭为投资主体的特点,决定了通过降低投入来提高在海南岛养殖虎纹蛙的经济效益的空间不大。根据海口市和文昌市两个虎纹蛙养殖场虎纹蛙生长的数据,发现在雌雄混合养殖条件下,50、100、150日龄雌蛙的平均体重分别为55.14g、232.76g、386.45g,雄蛙的平均体重分别为30.06g、130.92g、193.15g；在雌雄单性养殖条件下,50、100、150日龄雌蛙的平均体重分别为58.63g、248.22g、415.00g,雄蛙的平均体重分别为30.45g、142.02g、219.15g；在雌雄混合养殖条件下,200、250、300、365日龄种蛙雌蛙的平均体重分别为495.95g、546.79g、576.83g、598.41g,种蛙雄蛙的平均体重分别为234.60g、260.85g、282.47g、396.92g。说明在相同养殖条件下,雌蛙的生长快于雄蛙；在不同的养殖条件下,单性雌蛙养殖的生长最好,可见养殖虎纹蛙的最佳模式为雌蛙单性养殖。通过室内外试验发现,室外环境中水温在20℃±8℃、22℃±9℃、26℃±8℃、30℃±8℃时,孵化出雌性虎纹蛙的比例分别为72%、67%、55%、12%；室内恒温状况下,15℃、20℃、25℃、30℃、35℃实验组的孵出雌蛙率分别为86%、71%、67%、19%、0%,说明相对的低温有效提高虎纹蛙的雌性比例,相对的高温孵出的虎纹蛙中雄蛙比例较高。研究发现,在室外环境的孵化桶水温为20℃±8℃,孵化水体中苯甲酸雌二醇的浓度为0 mg/L、0.01mg/L、0.1mg/L、1mg/L时孵出雌性虎纹蛙占全部孵出蛙的71%、56%、65%、80%。；在室外环境的孵化桶水温为30℃±8℃,孵化水体中苯甲酸雌二醇的浓度为0 mg/L、0.01mg/L、0.1mg/L、1mg/L时孵出雌性虎纹蛙占全部孵出蛙的24%、58%、63%、81%,可见苯甲酸雌二醇明显影响虎纹蛙性别的分化。研究发现在不同温度条件下只要水体中激素浓度相同,孵出雌蛙的比例基本相同,说明在温度和激素条件共同作用下,激素的诱导作用更加明显。更多还原

【Abstract】 The numbers of amphibians are going down in the globe. Tiger Frog (Hoplobatrachus rugulosus) has been listed in the IUCN Red List of threatened species. It is also defined as National Class II protected animal species in China. A common understanding on its endangered status has been reached. Hainan Island is the main distribution region of H. rugulosus in China. In history H. rugulosus had distributed extensively in the whole island. In recent years it is difficult to find the trace of H. rugulosus in wild. Even some scholars think that H. rugulosus has disappeared in Hainan. So it is very urgent to understand and grasp the current situation of H. rugulosus resources and to take measures to protect H. rugulosus effectively.In order to protect H. rugulosus in Hainan Island, the author conducted investigations of many times and experiments in the island, grasped the situation of distribution, number of individual in populations, illegal trade, the relationship between use of paddy field and the population, law enforcement for protection, feeding habits, detention time of food in stomach and artificial breeding in Hainan, established a method fit for basis units to distinguish wild or breeding individual through combing the practical situation in Hainan, and explored the optimal breeding model in Hainan Island by using the technology of applying estradiol benzoate in incubation water to promote number of females. In that H. rugulosus resource will be protected effectively.According to the poaching traces and field studies of tiger frog, it was identified that tiger frogs were distributed in 16 cities or counties in Hianna: Haikou, Wenchang, Qionghai, Wanning, Lingshui, Sanyan, Chenmai, Dingan, Tunchang, Qiongzhong, Lingao, Danzhou, Changjiang, Dongfang, Baisha and Ledong from 1991 and 2007. The populations were studied by using "Labeling and recapturing" method in the 5 cities or counties:Haikou, Sanyan, Qionghai, Baisha and Changjiang on March, April, May and August in 2006. According to the numbers of captured and recaptured individuals and the calculation by the formula of N=Mxn/m(N is the estimated value of the population of tiger frog, M is the captured number of first time, n is the captured number of the second time, m is the captured number of the second time with the label of the first time), it was estimated that there were 96 individuals on March in 2006 and 56 individuals on March in 2007 in 227 mu cropland in Dapo town in Haikou city. In Guilingyang study spot there were 37 individuals on April in 2006 and 0 individuals on April in 2007.In Tayang town in Qionghai city there were 18 individuals on April in 2006 and 9 individuals on April in 2007. In the valley of Mt. Bawanling in Qicha town in Changjiang city there was no frog both in 2006 and in 2007. In Qingsong town in Baisha county there were 58 individuals in 2006 and 63 individuals in 2007. In Gaofeng town in Sanya city there were 21 individuals in 2006 and 11 individuals in 2007. It was found that the major habitats of tiger frogs were paddy field, lotus field, water celery field, ditch in cropland, discarded cropland, muddy pond for tilapia fish, discarded pond, taro field and so on. It was indicated that there were certain numbers of wild population of tiger frog, but the number changed dramatically. It needs more study on the factors impacting on the wild populations, so as to protect wild tiger frogs.Paddy field is one of the major habitats of tiger frogs. During the development process of high efficient agriculture in Hainan Island, the new practice of banana planting in water field and the production of reverse season vegetables caused the change of land use. The plough way of paddy field was also changed. The study on use of paddy field and the number distribution of tiger frog was conducted in Fengfuyang since 2006 and extended to nearby Houfuyang and Changlong in 2008 in Dongchang Farm in Haikou city. It was found that on March in 2006 there were tiger frogs in paddy field, sweet potato field, water celery field, banana field, taro water field and muddy pond for tilapia fish, but not in tangerine field, sugar cane filed and taro dry field in Fengfuyang in Dongchang Farm. On March in 2007 there were tiger frogs in paddy field, sweet potato field, water celery field, banana field, taro water field and muddy pond for tilapia fish, but not in tangerine field, sugar cane filed and taro dry field in Fengfuyang in Dongchang Farm. On March in 2008 there were tiger frogs in sweet potato field, water celery field, taro water field and muddy pond for tilapia fish, but not in wax gourd field, banana field, tangerine field, sugar cane filed and taro dry field in Fengfuyang in Dongchang Farm. There were tiger frogs in paddy field, banana field, water celery field and taro water field, but not in sweet potato field, sugar cane field and taro dry field in Houhuyang in Dongchang Farm. There were tiger frogs in sweet potato field, water celery field, banana field and taro water field and muddy pond for tilapia fish, but not in tangerine field, sugar cane field and taro dry field in Changlongyang in Dongchang Farm. On March in 2009 there were tiger frogs in sweet potato field, water celery field, banana field, taro water field and muddy pond for tilapia fish, but not in tangerine field, sugar cane filed and taro dry field in Fengfuyang in Dongchang Farm. There were tiger frogs in banana field, water celery field and taro water field, but not in sweet potato field, sugar cane field and taro dry field in Houhuyang in Dongchang Farm. There were tiger frogs in banana field, water celery field, taro water field and muddy pond for tilapia fish, but not in sweet potato field, tangerine field, sugar cane field, taro dry field, lemon field and guava field in Changlongyang in Dongchang Farm.It was also found that paddy field, water celery field, taro water field, muddy pond for tilapia fish and banana water field with overgrown weeds were good habitats of tiger frogs. It was indicated that the changes of paddy field into water celery field, taro water field or muddy pond for tilapia fish did not affect the individual number of tiger frog’s population. The changes of paddy field into banana field, tangerine field, sugar cane field, sweet potato field, lemon field, wax gourd field or guava field affected the individual number of tiger frog’s population apparently. So it must be careful to change the use of paddy field.A survey of the market trading of H. rugulosus in Hainan was conducted during 2006—2008. The results showed that trades of H. rugulosus were found in 18 cities or counties in Hainan lasting 10 months in each year excepting for January and February. There were professional poachers in Hainan. The ways of poaching had changed. The illegal trades were because of a consuming market arising from wrong dietetic idea, easy capture and great benefits from the illegal trades. For protection of H. rugulosus the suggestions are propounded that strengthening enforcement of the law, promoting the public awareness through environmental education activities, conducting scientific researches on H. rugulosus, enhancing the monitoring of the species and complementing the natural population through artificial breeding of the species.During the 3 years’market investigation 2691 individuals were checked, parasitic disease was found, while skin rot disease, gastroenteritis, cataract, red-leg disease, gill rotten disease and so on which often occuerred in artificial breeding were not found. Through dissecting 309 individuals of tiger frog, it was found that 195 individuals had no food residue and 114 individuals had food residue in their stomachs. The chief composition of the food residue included animal species belonging to Mesogastropoda Viviparidae, Araneida Atypidae, Scolopendromorpha Scolopendridae, Orthoptera Gryllotalpidae, Orthoptera Gryllidae, Coleoptera Coccinellidae, Coleoptera Scarabeidae, Lepidoptera Sphingidae, Anura Ranidae, Decapoda Potamidae, Orthoptera Acrididae and plant species belonging to Cyperales Gramineae, in which Acrididae accounted for 23.3%, Gryllotalpidae 22%, Coccinellidae 11%, Potamidae 9.39%, Gryllidae 9.39%, Atypidae 9.39%, Scarabeidae 4.21%, Sphingidae 4.21%, Viviparidae 2.91%, Scolopendridae 2.67%and Ranidae 1.94%. It was also found that the body size of tiger frogs was not significantly different with the composition of feeding food, and the sex of adult tiger frog was not significantly different with the composition of feeding food.Tiger frogs, bought from breeding farm with 60 days,90 days,120 days and150 days old, were divided into groups by the age of days. They were fed with mixed diet for 1 week, then hunger stricken for 24-hours, and then fed artificial food, locusts, mole crickets, crickets, spiders, snails, ladybugs, tiger frog, crabs and other single type of food. Defecating conditions were observed and excrements were examined by X-ray. It was found that locust, mole cricket, cricket and spider stayed for basically similar time in the stomach of the frogs with same age of days. The detention time of these types of food was 48h±24h for the group of 60 days’ old of frog,48h±12h for the 90 days’,48h±6h for the120 days, and 48h±5h for 150 days. Snails, ladybugs, young tiger frogs and river crabs stayed for basically similar time in the stomach of the frogs with same age of days. The detention time of these types of food was 96h±24h for 60 days old of frog,96h±12h for 90 days,72h±12h for 120 days, and 48h±12h for 150 days.The detention time of the expanded mixed feed for frogs was 24h±12h for 60 days old of frog,24h±6h for 90 days,12h±6h for 120 day, and 12h±2h for 150 days. It is indicated that the detention time of single type of the expanded mixed feed for frogs deceased with the increase of frog size.The results showed that the optimal time is 10 hours in Hainan for collection of evidence for law enforcement. When the time is more than 120 hours, it lost the meaning of evidence. The results of this study could help to make sure accurate time for analysis of tiger frog’s feeding in law enforcement, and to enhance the effectiveness of law enforcement. The information collected from the Forestry Bureau of Hainan Province showed that tiger frog breeding in Hainan Province began since 1995 and extended to the whole province in 1998. The annual production was more than 60 million,2 million,1.5 million, 5.58 million,6.48 million and 5.18 million kg in 2000,2003,2004,2005,2006 and 2007 respectively, and the output value was about 1.08 billion,80 million,60 million,33 million, 38 million and 30 million yuan in 2000,2003,2004,2005,2006 and 2007 respectively. In 2000 tiger frog breeding got the maximum annual production and the maximum annual output value in the history. Currently the production of tiger frog aquaculture is in a slow recovery phase and the price of marking frogs is hovering in the low valley bottom. The diseases found in tiger frog breeding farm were parasitic disease, skin fester disease and gastroenteritis from the survey of 94 farms in Hainan Island in 2008. Parasitic disease, skin fester disease and gastroenteritis were found in all the three breeding cycles in Haikou City, Wenchang City, Sanya City, and Qionghai City in 2008. It is found from the survey of 500 breeders of tiger frog that 96.6% of respondents believe that the low marketing price of tiger frog impacts the enthusiasm of farmers.95.5% of respondents believe that the feed is too expensive and 81.5% of the respondents think the feed suppliers monopolize the price of feed and manipulate the marketing price of frog. The characteristics of single family as main investor in tiger frog breeding in Hainan determined the little room to increase economic benefits through lowering the cost in tiger frog breeding. It has influenced to certain extent the wildlife resources of tiger frog since no sound processing for dead bodies of tiger frog with diseases, disposition of wastewater without control, release of patient frogs to surrounding environment without treating when economic loss appearing and so on in the breeding.According to the growth data from two frog farms in Haikou City and Wenchang City, it was found that under the condition of male and female mixed-breeding the average body weight of female individual was 22.15,55.14 90.57,126.48,163.07,199.38,232.76, 294.95 and 386.45g and that of male individual was 12.48,30.06,50.36,73.15,94.37, 114.59,130.92,160.11 and 193.15g for the frogs with the age of 40,50,60,70,80,90,100, 120 and150 days respectively. Under the condition of male and female separated-breeding (single sex breeding) the average body weight of female individual was 23.70,58.63,95.73, 133.85,173.30,212.13,248.22,318.56 and 415.00g and that of male individual was 12.67, 30.45,52.74,75.61,98.73,121.14,142.02,177.81 and 219.15g for the frogs with the same order of age above mentioned. Under the condition of male and female mixed-breeding the average body weight of adult female breeding frog was 495.95,546.79,576.83 and 598.41g and that of adult male breeding frog was234.60g,260.85g,282.47g and 396.92g for the groups with age of 200,250,300 and 365 days respectively. It indicated that under the same breeding conditions female frogs grew faster than males. Under different breeding conditions the best growth of female individual appeared in the single sex breeding case. So the best breeding model was single sex breeding of female.Through outdoor and indoor experiments it was found that under the outdoor environment conditions with the water temperature at 20℃±8℃,22℃±9℃,26℃±8℃and 30℃±8℃, the hatching rate of female tiger frog was 72%,67%,55% and 12% respectively. Under the indoor conditions with constant room temperature at 15℃,20℃, 25℃,30℃and 35℃, the hatching rate of female tiger frog was 86%,71%,67%,19% and 0% relatively. It was indicated that relatively low temperature could effectively increase the proportion of female tiger frogs, while relatively high temperature hatched a higher proportion of males. Under the outdoor environment condition with water temperature in breeding pond at 20℃±8℃and the concentration of estradiol in incubation water at 0 mg/L,0.01mg/L, 0.1mg/L and 1mg/L, the female frog accounted for 71%,56%,65%and 80%of all hatched frogs respectively. Under the outdoor environment condition with water temperature in breeding pond at 30℃±8℃and the concentration of estradiol in incubation water at 0 mg/L, 0.01mg/L, 0.1mg/L and 1mg/ L, the female frog accounted for 24%,58%,63% and 81% of all hatched frogs respectively. It was shown that estradiol benzoate apparently affected the sexual differentiation of tiger frogs. The results showed that as long as the same concentrations of hormone existed in water under different temperatures, the proportions of the female frog hatched were similar. It was indicated that under the combined action of temperature and hormone, the effect of hormone induction was more apparent.更多还原