Rapid authenticity testing of artificially bred green turtles (Chelonia mydas) using microsatellite and mitochondrial DNA markers

Introduction

Sea turtles are umbrella species of the marine ecosystem and flagship species of marine conservation (Bouchard & Bjorndal, 2000; Hamann et al., 2010). However, the global population and distribution of the sea turtle have sharply reduced due to long-term threats, such as over-exploitation, illegal trade, marine pollution, and climate change (Chan et al., 2007; Hawkes et al., 2009; Lam et al., 2011). All five sea turtle species distributed in China have been included in the list of Chinese National Fist-Level Key Protected Wild Animals and the Appendix I of Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES, 2019), indicating that sea turtles are strictly protected and the international trade of these species is prohibited except when the purpose of the import is not commercial.

The fast-growing demand for sea turtle displays from Chinese aquariums and private individuals has led to a large-scale illegal trade of live turtles, seriously threatening the survival of wild sea turtles (Lin et al., 2021). The Chinese government issued the “Sea Turtle Conservation Action Plan (2019–2033)” in 2018 and the plan recommended “strengthening research on the technology of artificial breeding of sea turtles and promoting the use of artificial breeding sea turtles for aquarium display, publicity, and education to reduce human demand for sea turtles in the wild” (Chinese Ministry of Agriculture and Rural Affairs, 2019). Therefore, the number of institutions for artificial breeding of sea turtles is expected to gradually increase in China. However, artificial breeding of sea turtles is still fairly difficult and only a few successful cases have been reported worldwide, such as the Cayman Islands, UK (Barbanti et al., 2019), Miami Seaquarium, FL, USA (Sizemore, 2002), Sea Life Park, Hawaii, USA (Wood & Wood, 1980), and Ishigaki Island in Japan (Shimizu et al., 2007). In China, only the Huidong Turtle National Nature Reserve in Guangdong and Haichang Whale Shark Aquarium in Shandong have successfully bred turtles using wild parent turtles under captive conditions (Gao, Ye & Chen, 2015; Li, 2016). Due to inefficient breeding methods, some facilities illegally purchased wild turtle eggs from the South China Sea or Southeast Asia, and then sold the incubated turtles to aquariums or private organizations by marketing them as artificially bred turtles (Shanker & Pilcher, 2003; Lam et al., 2011). It is well-known that turtle farms are usually the biggest purchasers of wild turtles in China (Shi et al., 2007), and the origins of many turtles in captivity breeding and trade are false or unclear (Parham & Shi, 2001; Parham et al., 2001). Therefore, to prevent the illegal use of wild populations, it is necessary to develop reliable methods to distinguish the authenticity of artificially bred sea turtles in forensic enforcement and population management.

DNA analysis methods have been widely used in wildlife forensic enforcement. Among these methods, the nuclear microsatellite gene is widely used in molecular marker-assisted breeding and parentage test of captive animals; it has the advantages of high variability and polymorphism, as well as codominance (Schlötterer & Pemberton, 1994). The nuclear microsatellite gene can be stably passed from one generation to the other, that is, half of the genetic material of the offspring is contributed by the father and half by the mother. Therefore, on a specific microsatellite locus, two alleles of the offspring should be found in the genotype of the father or the mother, otherwise, they do not have a parent–offspring relationship (Huang & Wang, 2004; Zi-Tuo & Liu, 2014; Zhang et al., 2018). Mitochondrial sequences have the characteristics of maternal inheritance and the offspring will have the same haplotype of the mitochondrial gene as the mother. Sea turtles are highly loyal to their birthplace and female adults return to their hatchling beach to lay their eggs. Thus, turtles from the same birthplace share unique haplotypes of the mitochondrial gene (Bowen et al., 1992; Nishizawa et al., 2011). Therefore, the mitochondrial gene can be used to identify the birthplace of sea turtles, as well as the mother-child relationship between a baby turtle and an adult female. The combination of microsatellite and mitochondrial DNA markers could be used to rapidly exclude the parentage relationship and also trace the source of illegal trade or capture in forensic enforcement for the authenticity of artificially bred animals.

In 2019, a turtle farm in southern China claimed that six captive adult green turtles (four females and two males) had successfully been reproduced and more than 400 baby turtles hatched between 2015 and 2017. Although the farm has the breeding license, the success of artificial reproduction could not be verified, since it is not possible to provide evidence of sea turtle breeding, mating, and egg laying. To verify this, we collected skin samples from 19 adult turtles, including the above six potential parents, and 16 juvenile turtles randomly selected. The mitochondrial D-loop gene and two microsatellite loci were jointly sequenced and analyzed to detect whether a parent–offspring relationship existed between adult and juvenile turtles and to infer the birthplaces of these turtles.

Materials & Methods

Results

Discussion

Although this turtle farm claimed that the six adult green turtles bred all the 400 juvenile turtles in captivity, no parentage relationship could be identified between the randomly selected 16 juvenile turtles and the six adult green turtles, or even to the other 13 adults. Thus, it is evident that this farm is dishonest, is illegally farming and trading wild sea turtles, and marketing them as artificially bred turtles. The more surprising fact is that the farm has a breeding license issued by the local government. The legal license facilitates the laundering of illegal business in this case, similar to other cases found in China (Lin et al., 2021). These sea turtles in captivity may have various illegal overseas sources, their birthplace was mainly the Sulu Sea, followed by the Marshall Islands, as well as Paracel Islands and Wanan Island in the South China Sea (Fig. 1). Thus, there is a great possibility that this farm purchased wild turtle eggs or hatchlings from the above birthplaces through illegal trade chains. Further investigation is warranted to uncover the detailed source and illegal trade chain by the law enforcement staff.

The huge demand for sea turtles has made China a major market and destination of illegal trade for a long time, and most traded sea turtles are mainly from fishermen who captured turtles directly or purchased from dealers in southeast Asian countries (Lam et al., 2011; Gomez & Krishnasamy, 2019; Lin et al., 2021). Wild sea turtle populations in the South China Sea and the Coral Triangle area suffer the most, which has been revealed by mix-stock analysis of sea turtle samples from the Hainan Islands, China (Gaillard et al., 2020), Brunei Bay and Mantanani, Malaysia (Joseph et al., 2014; Joseph et al., 2016). The results of the present study are consistent with their findings and provide further evidence on the gravity of illegal turtle fishing and trade in these areas. Our data show juveniles and adults are generally collected from the Sulu Sea, but that hatchlings are more heavily harvested from the Paracel Islands than in adults. However, adults are being collected from distant rookeries as the Marshall Islands made up over 20% of their stock contribution. It could be that adult turtles are migrating to the feeding grounds in the Sulu Sea and are being collected there, however, previous analysis of foraging grounds in the Sulu Sea did not find stock contributions from the Marshall Islands (Jensen, Pilcher & Fitzsimmons, 2016; Joseph et al., 2016) suggesting these animals were collected from a rookery not found in the Sulu Sea. Therefore, regarding the current sea turtle illegal trade, stricter and more efficient law enforcement is crucial (Lin et al., 2021), and simultaneous monitoring of market trends and trade routes must occur (Lam et al., 2011), multinational cooperation is also required to properly protect green turtles in the South China Sea.

To the best of our knowledge, the present study is the first attempt to use nuclear microsatellite gene combined with mitochondrial gene markers for authenticity testing of artificially bred green turtles in forensic enforcement. The trinucleotide (B103) and tetranucleotide (D1) microsatellite loci used in this study have high genetic diversity and are sufficient to ensure the accuracy of the discrimination results, while the D-loop fragments in mitochondrial DNA are mostly used to identify the origin of sea turtles (Gaos et al., 2017; Hill et al., 2018). Their combination is reliable to rapidly distinguish the authenticity of artificial breeding and also accurately trace the source of the illegal trade or capture of sea turtles. Meanwhile, the testing is fast and cheap, as the samples can be tested within 48 h and each sample costs about 200 RMB on average. However, due to limited conditions, only 16 juvenile and 19 adult turtles were sampled in this study, which could be successfully identified using two microsatellite and one mitochondrial loci. But for testing a larger number of samples, it is recommended to increase the number of microsatellite loci and use the multiplex PCR method adopted in this study to ensure the accuracy of detection. Microsatellite loci, such as A6, B123, C102, Cm3, Cm58, etc. used by Wright et al. (2012), could be selected to satisfy the identification of more samples. As such, we believe that this method can serve as a good reference for identifying artificially bred green turtles in farms or as part of illegal trade and help obtain reliable evidence of turtle origins in future law enforcement.

Conclusions and conservation implications

The turtle farm investigated in the present study is illegally trading wild sea turtles and marketing them as artificially bred turtles. This method provides reliable evidence for this conclusion and also helps reveal the possible illegal trade chain. As the number of facilities for artificial breeding of sea turtles is expected to gradually increase with the encouragement of artificial breeding of sea turtles, this method could help distinguish the authenticity of artificial breeding in law enforcement and assess its impact on sea turtle populations in future. Moreover, international cooperation between China and Southeast Asian countries should be strengthened to combat the illegal trade of sea turtles and protect their wild populations in the South China Sea and Coral Triangle areas.

Supplemental Information