Experimental Primates and Non-Human Primate

ZOOLOGICAL RESEARCH

Volume 35, Issue 6 18 November 2014

CONTENTS

Special Topic for Primates and Animal Models of Human Diseases Review Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress ····················································································································································· ·················Xiao-Liang ZHANG, Wei PANG, Xin-Tian HU, Jia-Li LI, Yong-Gang YAO, Yong-Tang ZHENG (447) Articles Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques (Macaca leonina)·······························································Hong-Yi ZHENG, Ming-Xu ZHANG, Lin-Tao ZHANG, Xiao-Liang ZHANG, Wei PANG, Long-Bao LYU, Yong-Tang ZHENG (465) Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan ··················································································· Jin-Fa LI, Yu-Chao HE, Zhi-Pang HUANG, Shuang-Jin WANG, Zuo-Fu XIANG, Juan-Jun ZHAO, Wen XIAO, Liang-Wei CUI (474) Experimental infection of tree shrews (Tupaia belangeri) with Coxsackie virus A16 ··············· Jian-Ping LI, Yun LIAO, Ying ZHANG, Jing-Jing WANG, Li-Chun WANG, Kai FENG, Qi-Han LI, Long-Ding LIU (485) Isolation and identification of symbiotic bacteria from the skin, mouth, and rectum of wild and captive tree shrews··············Gui LI, Ren LAI, Gang DUAN, Long-Bao LYU, Zhi-Ye ZHANG, Huang LIU, Xun XIANG (492)

Articles Acoustic signal characteristic detection by neurons in ventral nucleus of the lateral lemniscus in mice ······························································································· Hui-Hua LIU, Cai-Fei HUANG, Xin WANG (500) Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) ·············································· Hong-Fa LU, Li-Na DU, Zhi-Qiang LI, Xiao-Yong CHEN, Jun-Xing YANG (510) Genetic diversity and population structure of a Sichuan sika deer (Cervus sichuanicus) population in Tiebu Nature Reserve based on microsatellite variation··············Ya HE, Zheng-Huan WANG, Xiao-Ming WANG (528) Complete mitochondrial genome of yellow meal worm (Tenebrio molitor)············Li-Na LIU, Cheng-Ye WANG (537)

Zoological Research again recognized for Excellence and Impact in Science and Technology Publishing in 2014 (484)

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Zoological Research 35 (6): 447−464 DOI:10.13918/j.issn.2095-8137.2014.6.447

Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress

Xiao-Liang ZHANG1,3, Wei PANG1, Xin-Tian HU1,2, Jia-Li LI1,2, Yong-Gang YAO1,2, Yong-Tang ZHENG1,2,3,*

1. Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China 2. Kunming Primate Research Center of the Chinese Academy of Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China 3. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming Yunnan 650500, China

Abstract: Non-human primates (NHPs) are phylogenetically close to humans, with many similarities in terms of physiology, anatomy, immunology, as well as neurology, all of which make them excellent experimental models for biomedical research. Compared with developed countries in America and Europe, China has relatively rich primate resources and has continually aimed to develop NHPs resources. Currently, China is a leading producer and a major supplier of NHPs on the international market. However, there are some deficiencies in feeding and management that have hampered China’s growth in NHP research and materials. Nonetheless, China has recently established a number of primate animal models for human diseases and achieved marked scientific progress on infectious diseases, cardiovascular diseases, endocrine diseases, reproductive diseases, neurological diseases, and ophthalmic diseases, etc. Advances in these fields via NHP models will undoubtedly further promote the development of China’s life sciences and pharmaceutical industry, and enhance China’s position as a leader in NHP research. This review covers the current status of NHPs in China and other areas, highlighting the latest developments in disease models using NHPs, as well as outlining basic problems and proposing effective countermeasures to better utilize NHP resources and further foster NHP research in China.

Keywords: Non-human primates; Experimental primates; Animal models; Current status

Employing animal models for biomedical research been widely used in lab settings since the 1950s. 1There has been done to speed along research by bypassing has been a corresponding increase in the prominence and ethical restrictions on using humans as experimental importance of using NHP models that have been widely subjects, which averts risks associated with direct used as subjects in infectious diseases (Kaushal et al, research on humans, especially in clinical trials. There 2012; Liu & Zhang, 2010), mental and neurological are likewise numerous advantages to using animal disorders (Perretta, 2009), cardio-cerebrovascular diseases models such as strong controllability of experimental (Sy et al, 2014), and endocrine diseases (Kamath et al, conditions, high repeatability, ease of scale, comparabi- lity of results. However, the effectiveness of animal mod- Received: 11 June 2014; Accepted: 15 August 2014 els is hampered by intrinsic species differences between Foundation items: This work was supported by the National Natural animal models and humans. For example, experimental Science Foundation of China (81172876, 81273251, U1202228, data obtained from rodents, who are phylogenetically 81471620); the National Special Science Research Program of China distant from humans, does not always appropriately (2012CBA01305); the National Science and Technology Major predict the efficacy of a drug treatment or its potential Project (2013ZX10001-002, 2012ZX10001-007) and the Knowledge toxicity in humans (Van Der Worp et al 2010; Xu, 2011). Innovation Program of CAS (KSCX2-EW-R-13, KJZD-EW-L10-02) To bridge the gaps, non-human primates (NHPs) have *Corresponding author, E-mail: [email protected]

Science Press Volume 35 Issue 6

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2011; Pound et al, 2014), to name just a few. utilization of NHP resources in China. The key advantage of using NHPs is that NHPs are phylogenetically the most proximate models to humans, Status of experimental primates abroad bearing many distinctive similarities not found in other There is a disagreement on the total number of animal models. Previous reports found that DNA primate species in existence. According to the 2005 sequence similarities between NHPs and humans can Mammal Species of the World (http: //vertebrates.si.- reach up to 98.77% (Fujiyama et al, 2002) and the edu/msw/mswCFApp/msw/index.cfm), there are 376 pri- concordances rates of gene transcription levels of spleen, mate species. However, the latest statistics from IUCN peripheral blood monocyte cells (PBMCs) and liver Red List of Threatened Species (http: //www.iucnredlist.org) between NHPs and humans can reach as high 91.41%, declare 420 primate species. Occasionally, new primate 84.36%, and 74.29%, respectively (Lu et al, 2008). As a species are described, such as Rhinopithecus strykeri that consequence, NHPs remain the first or the only choice was discovered recently (Geissmann et al, 2011; Long et for investigating major human diseases including al, 2012), make the numbers subject to change. More HIV/AIDS, measles, malaria, hepatitis, but also in the important to the total number of primates are threats study of human cognition and brain diseases (Xu et al, from humans, whose development often interferes with 2013). In addition, NHPs are also used for human cancer primate habitat. As a result, most primate species are research (Xia & Chen, 2011). The data garnered from considered to be endangered. To overcome the threats to such NHP experiments is invaluable as a starting point, NHPs, numerous countries have adopted strict protective because it ensures greater efficiency and surety of future measures on NHP resources, and some have began to pay clinical application, making NHPs the “gold standard” greater detail to utilizing these resources more rationally. for preclinical studies (He et al, 2013). While America has no wild NHPs, it is home to It was half a century ago that the NHP model was primate research institutes including 8 national primate gained traction in Europe and America where most of the research centers (NPRCs) funded by the National scientific research were conducted at the time. They Institutes of Health (NIH) that develop NHP models for began to develop the NHP resources by importing NHPs basic and applied studies of human health: California from other countries, developing husbandry programs National Primate Research Center, New England Primate and exploring the nature of NHPs. As more recent Research Center, Southwest National Primate Research advances in medical research and biotechnology invol- Center, Tulane National Primate Research Center, ving gene modification, gene knockout, epigenetics, Washington National Primate Research Center, precision gene editing and fine regulation of local gene Wisconsin National Primate Research Center, Yerkes expression in experimental primates become more cost- National Primate Research Center, and Oregon National effective and refined, we expect that there will be greater Primate Research Center (Kaiser, 2013). These 8 NPRCs need for NHP resources to meet the demands of many have a total number of 26, 000 NHPs (Hayden, 2008), new applications, which developed countries with representing more than 20 species of NHPs, mostly advanced knowledge of NHPs may struggle to meet. At macaques (largely for practical purposes and ease of this point, countries with indigenous NHPs that can also research). Annually the US federal government invests advance effective protection, sustainable exploitation and about $100 million each year into these NPRCs to utilization of their NHP resources may stand to benefit. financially support ongoing studies of NHPs (http:// Indeed, as its growing life sciences and pharmaceutical www.humanesociety.org/animals_in_research/general_in industries, China has a great opportunity to become a formation_on_animal_research/an_introduction_to_prim leader in NHP resources and even research (Cyranoski, ate_issues.html). As advances in research techniques and 2004; Hao, 2007). methodology using primate models have been created, This review evaluates the ongoing situation of there has been a consummate increase in investment. NHPs in China and other areas as well as the latest From 1999 to 2006, funding through the US federal development of human disease models using NHPs in government for NHPs research increased 7.9-fold, ($15.4 China. We analyze the existing problems for further million to $122 million), while the total number NHPs at developing NHP models and propose targeted counte- the eight NPRCs increased from 2 4 182 to 2 7 914 (http: measures that may allow for better exploitation and //www.all-creatures.org/saen/res-nprcs-8yr.html). Other

Zoological Research www.zoores.ac.cn Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress 449 institutions with strong focuses on NHP research also with some different trends. While the number of NHPs receive federal and state funding, including the New used in experiments in America has grown annually from Iberia Research Center at the University of Louisiana at 2001 to 2010 (Figure 1, data from http: //www.aphis.- Lafayette, the University of Texas MD Anderson Cancer usda.gov), the total amount of NHPs used for research in Center, Alamogordo Primate Facility and Primate the EU remains lower than in America, without stable Foundation of Arizona, the Caribbean Primate Research growth. Within EU, total NHPs used for research in 2003, Center, a squirrel monkey colony of the University of 2005 and 2008 were 10 360, 10 450 and 9 600, South Alabama and a baboon breeding facility of the respectively (https: //www.gov.uk). The most popular University of Oklahoma. Even a few private facilities in NHPs used for research were rhesus macaques (RMs) NHP breeding receive government grants to maintain the and cynomolgus macaques (CMs), with pig-tailed US’s overall capacity for conducting NHP research (http: macaques, squirrel monkeys, African green monkeys, //www.sourcewatch.org/index.php/National_Primate_Re common marmosets, cotton-topped tamarins, baboons, search_Center_System). One further aspect of NHP chimpanzee, among others taking a minor share. No apes research in the US worth noting is that the US is home were used in experiments anywhere in the EU from to the largest colony of Chimpanzees used for research, 2002−2008 according to a survey by the European Com- with around 1 000 currently in government sponsored mission (http: //vertebrates.si.edu/msw/mswCFApp/msw/- labs (Wadman, 2011a). Chimpanzees are the only index.cfm). There is much debate regarding whether great apes known to be captured for biomedical National Institutes of Health (NIH) research chimp- research, even though they are also an endangered anzees be retired (Wadman, 2011b) and US primate species, and given that they are the primates most centre faces scrutiny with stricter criteria (http: //www.- closely related to humans, are used in invasive rese- nature.com/news/us-primate-centre-faces-scrutiny-1.10317). arch, and defined as “inoculation with an infectious In June 2013, NIH announced that it will retire to agent and/or drug testing.” sanctuary nearly all of its research chimpanzees (http: Compared to the US, Europe is the next largest //blogs.nature.com/news/2013/06/nih-retires-most-rese- center of NHP research (Carlsson et al, 2004), though arch-chimpanzees.html).

Figure 1 Number of NHPs used in experiments in America per year from 2001 to 2010

While the US and Europe are the key centers of Laboratory Animal Care (AAALAC) certification. For NHP research, Southeast Asian countries that possess example, Thailand has NHP facilities and primate indigenous NHPs have recently begun to pay more research centers, among which Primate Research Insti- attention to these resources. Southeast Asia has made tute of Thailand (PRIT; http: //www.primate.or.jp/se- rapid advances in its NHP capacity: several NHP mi/Abst_f2nd/suchinda.pdf) possesses 14 species of research institutions and breeding facilities have been set primates, and focuses on primate breeding and producing up in the area, and a few NHP breeding facilities have animal models suitable for diseases research, especially passed the American Association for Accreditation of for tropical diseases research. These advances have made

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 450 ZHANG, et al. the PRIT a key biomedical research center on primates Europe, Japan, and now Korea. In 2013, the number of and an information center for NHPs among Southeast breeding experimental monkeys in China, mainly rhesus Asia. The presence of such facilities and their macaques and cynomolgus macaques, in China there corresponding resources greatly enhances the entire were about 250 000 breeding cynomolgus macaques and region’s NHP research capacity, and complements the 40 000 breeding rhesus macaques (data from The China growth in NHP resources and research being conducted Laboratory Primates Breeding and Development Asso- in China. ciation), with approximately 50 000 to 60 000 experi- mental monkeys born in breeding facilities each year. Status of experimental primates in China Though many NHPs are used for domestic research Compared to US and Europe, NHP resources are projects, a large number are exported abroad. For rich in China. More than 24 species were found. It example more than 65% of the experimental monkey accounts for about 6% of primate species around the imports in the US from Asia are from China, and in world. Yunnan Province in particular possess the most Europe, around 70% of research primate imports are NHP resources, and undertakes a bulk of the earliest and from China. Essentially, though NHP research remains present work on NHPs (Shen & Yang, 2003; Pan & Ben, predominately a Euro-American venture, China is the 1984; Li & Lin, 1983). The anchor institution of this leading supplier of resources necessary to this research. research, Kunming Institute of Zoology (KIZ), Chinese To support China’s shift from an NHP resource Academy of Sciences (CAS), was established in the late producer to an NHP research driver, the Chinese 1950s. KIZ is among the first institutions in China to government has increased investment for experimental start a primate breeding center to conduct basic research primates and new drug development. Over the last and clinical applications on NHPs in taxonomy, ecology, decade, there has been marked progress on research and anatomy, genetics, behavior, evolution, reproductive innovation into experimental primates. Specifically, the biology, neurobiology, immunology, and with a series of Ministry of Science and Technology (MOST) of the laboratories dedicated to animal microbial detection, People's Republic of China has set up an experimental animal nutrition, clinic pathology of animal; KIZ has primate germplasm resources center in Hainan and also produced a series of monographs on NHPs including Suzhou, and in 2011 MOST launched a number of large The Anatomy of the Golden Monkey, The Anatomy of science projects under the platform construction of Macaca mulatta. The primate centre in KIZ hosts promoting the development of primate animal models of currently about 2 800 NHPs, including M. mulatta, M. human major diseases. The General Administration of leonine, M. fascicularis, M. assamensis, M. arctoides, Quality Supervision, Inspection and Quarantine of China Rhinopithecus roxellana, and other species. The facility has likewise set up a state key laboratory for experi- is well-equipped to deal with these NHP resources, as it mental primate animal quarantine in Guangxi Province, has received AAALAC certification, and the centre’s and as we mentioned previously the CAS established the NHP resources and research has received a worldwide Kunming Primate Research Center of Chinese Academy attention (Cyranoski, 2004; Hao, 2007). Alongside KIZ, of Sciences in Yunnan. In addition, the governments are Institute of Medical Biology (IMB), Chinese Academy of providing more support and financial resources to NHP Medicine Science, which was set up in 1958 and before research. In short, these actions will undoubtedly 1980s hosted the greatest number of RMs in China (Shen accelerate the innovation and capacity for NHP research & Yang, 2003), focuses on the research and development in China. Prior to 2005, most high-quality experimental of medical products using experimental primates, NHPs were exported to European, America and other particularly for vaccines. IMB is currently the largest “developed” countries, while the domestic consumption base of the research and production of both attenuated was relatively limited, but thanks to the additional poliovirus oral live vaccine and hepatitis A vaccine in support and development of the Chinese scientific China. community, outsourcing of services related to Prior to China’s development of domestic NHP experimental NHPs have continued to spring up and research into exploitation and utilization of NHP reso- domestic services have exploded in Beijing, Kunming, urces, China was—and remains—a leading producer and Shanghai, Suzhou, Chengdu, Guangzhou, among others, a major supplier country for NHPs to the United States, to improve the development of experimental NHPs in

Zoological Research www.zoores.ac.cn Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress 451

China. models of RMs infected with SIV closely resemble the human disease progression of AIDS. Consequently, Current status and progress on the NHP models of NHPs are the most widely used animal models for HIV/ human diseases in China AIDS research (Lei et al, 2013). Though NHP AIDS Chinese researchers have established numerous models were established using simian retrovirus (SRV) NHP models to model a variety of human diseases, in the early years, NHP AIDS models created with SIV which have made large contributions to the study, preve- better simulate human AIDS. Currently, most NHP AIDS ntion and treatment of human diseases. In China, most models in China for developing vaccines and NHP models for human diseases focus on exploring therapeutics are established via infection of SIV or SHIV, infectious diseases, cardio-cerebrovascular diseases, a simian/human immunodeficiency chimeric virus. endocrine diseases, reproductive diseases, mental and An initial Chinese RM/SIV model established by Li neurological disorders, and ophthalmic diseases. Simi- et al (2007) was accomplished by infecting Chinese RMs larly, some basic research into NHPs by Chinese with SIVmac239. Furthermore, Xia et al (2010, 2011) researchers have greatly extended the usability of NHPs and Li et al (2012) investigated the dynamic and as animal models by exploring transgenic strategies in functional changes of dendritic cell subsets and Treg experimental primates and in 2010 researchers of cells in SIVmac239-infected Chinese RM. The results Kunming Institute of Zoology, CAS created the first confirmed a previous study and showed that disease transgenic monkey in China (Niu et al, 2010). symptoms of SIV-infected Chinese RMs were more

similar to human responses to HIV-1. Feng et al (2007) Primate animal models for infectious diseases Though advances in medicine and medical also set up RM models of SIVmac251 infection using technology have effectively controlled or eradicated several different routes (intravenous, intravaginal and infectious diseases, a variety of new infectious diseases intrarectal routes) and observed differences in disease have continuously emerged, largely as a result of human course after SIVmac251 infection resulting from the activities’ adverse impact on the surrounding environ- different routes; notably, this was the first case of a ment and bringing individuals into closer contact with Chinese NHP AIDS animal model infected with SIV these situations. As a developing country with the worl- through a mucosal route. Later, Cong et al (2011b) d's largest population, China is actually threatened by established a Chinese RM model of SHIV-SF162p3, an many infectious diseases that are exacerbated by HIV-1 subtype B, infected through repeated low-dose population density, intense land-usage, and industrial- intravaginal inoculation, which more closely resembles ization, such as HIV/AIDS tuberculosis (TB), viral the natural transmission of HIV infection in humans. hepatitis, foot-and-mouth disease, avian influenza. This Though HIV-1 subtype C comprises the predominant problem is only magnified on the global scale, especially strains in China (Wu et al, 2006), Cong et al’s model among developing countries that experience the bulk of provides technical reference for establishing further population growth and the increase in population AIDS animal model for evaluating vaccines and densities as a consequence of urbanization and treatment strategies suitable for China's predominant industrialization. Given the nature of infectious diseases epidemic strains, which may change given growing and the larger global trends, developing more ideal and infection rates and variances, especially in Yunnan. targeted animal models is a crucial step for opening up Additionally, researchers have also successfully new strategies against them. established different HIV domestic Chinese RM models (Wang et al, 2011b; Cong et al, 2011a; Yao et al, 2011). HIV/AIDS The characteristics of these models remains to be To our knowledge, no animal models for HIV/AIDS elucidated in future, however, as HIV/AIDS infections research are more suitable than NHPs. HIV-1, for continue to increase globally in general and in China instance, infects only humans and a handful of primate (and Yunnan) in particular, these models may become animals such as the chimpanzee, gibbon and pigtailed increasingly important research models. macaque (Kuang et al, 2009). While HIV-1 can infect While the existing SIV/SHIV-infected macaque these NHPs, for several reasons the disease's progression models are useful for application in AIDS research, is quite different from that in humans, though AIDS genetic differences between SIV/SHIV and HIV-1

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 452 ZHANG, et al. significantly limit the use of SIV/SHIV (Ambrose et al, 2010; Kaushal et al, 2012). In theory, infection with HIV 2007; Van Rompay, 2012). Accordingly, NHP AIDS accelerates the activity of latent MTB and then increases models established with HIV/HSIV infection should be a the incidence of TB and, at the same time, TB seems to better model of human HIV/AIDS infections and speed the HIV replication rate (Enserink, 2001), making pathology; fortunately, researchers at the KIZ are them a potentially lethal complication. Establishing NHP currently establishing northern pig-tailed macaque AIDS models of TB then provides a foundation for the constr- model with HIV/HSIV. If successful, such a novel model uction of NHP models of MTB/HIV co-infection, which may provide an ideal platform for the study, drug may prove more ideal than NHP models of MTB/SIV co- screening and the vaccine evaluation of AIDS. infection (Diedrich et al, 2010) currently in place.

Tuberculosis (TB) Influenza virus Recent studies reveal that mouse models are quite Of the many infectious disease pandemics, infl- questionable, but NHP models avoid many of the pitfalls uenza has been one of the worst (Kilbourne, 2006). This that make rodent models less popular; in particular, NHP is especially poignant for China, as outbreaks of highly models of TB for immunological research and vaccine pathogenic H5N1 avian influenza viruses occurred in test show promising results (Kaushal et al, 2012; Guo & Hong Kong in 1997, killing 6 people (Mounts et al, Ho, 2014). The study of NHP models of TB was later 1999), which was the first worldwide report of avian than other countries in China, despite the ongoing influenza virus directly infecting humans. Following the prevalence of the disease. For TB, there is a certain gap report, avian influenza A infection in humans was also in the types and construction methods of NHP models, reported in Southeast Asia, Europe and North Africa for example, the NHP models of TB latency and (Obenauer et al, 2006), and outbreaks of H7N9 in reactivation have not been done in China. humans in China last year received local and global Currently, there are several reports on the ongoing attention (Gao et al, 2013a, b; Hu et al, 2013; Liu et al, construction of NHP models for TB study in China. For 2013). Since rodent animals are not the natural hosts of instance, Wuhan University began to establish of NHP influenza viruses and show no symptoms of upper- models of TB using fiberoptic bronchoscopes since 2006, respiratory-tract infections (Zak & Sande, 1999), rodent and later delivered Mycobacterium tuberculosis (MTB) models are not good systems for studying human into the lungs of Chinese RMs (Xian et al, 2010; Zhang influenza infections. NHP models are, however, excellent et al, 2011a). This laboratory has become the fifth at mimicking human infections of influenza viruses. standard laboratory for TB vaccine study in the world Foreign researchers have established NHP models (http: //news.sohu.com/20060715/n244270131.shtml), and infected with H1N1, H3N2, H5N1, etc (Baas et al, 2006; models have been used in preclinical testing of candidate Carroll et al, 2008; Cillóniz et al, 2009; Fan et al, 2009; vaccines and drugs. Wang et al (2012) later established Itoh et al, 2009; Kobasa et al, 2007; Laddy et al, 2009; Chinese RM models of TB via bronchoscopic and Rimmelzwaan et al, 2001). Recently, Chen et al (2009) intratracheal instillation into the lungs and found the infected Chinese RMs with the highly pathogenic avian clinical manifestations, disease progression and influenza virus A/Tiger/Harbin/01/2002 (H5N1) intrana- pathological changes of these models closely resembled sally and noted that RMs exhibited symptoms similar to human primary TB and hematogenous disseminated TB. humans, such as fever, loss of appetite, interstitial NHP models of TB have marked advantages in pneumonia, etc. These results indicated that Chinese immunological research and test of drugs as compared RMs could be potentially successful infection models of with non-primate models. Unfortunately, the lack of the H5N1 viruses as they exhibit similar symptoms. To currently available inbred strains of NHPs makes date, this model has some shortcomings, since H5N1 pathological changes varied. The obvious solution is to only reproduced in the respiratory system and that RMs strengthen the system research of pathological features, showed low sensitivity to H5N1 infection. In view of such as lobe fibro-cavitary lesions, etc. Likewise, studies these limitations, Lü et al (2011) infected Chinese RM suggest that there is a high rate of HIV and MTB co- with A/SZ/406H/2006(H5N1) by endotracheal incuba- infection, which is thought to be one of factors tion, and found that this method yielded a more sensitive contributing to the global resurgence of TB (Harrington, NHP model of H5N1 virus infection, and that the virus

Zoological Research www.zoores.ac.cn Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress 453 could replicate in many other organs outside the lung and accordingly foreign researchers have created monkey better resembling severe cases seen in humans. models infected with an HCV/GBV-B chimera (Li et al, Since the influenza virus is primarily transmitted via 2014; Rijnbrand et al, 2005). In future, we should focus air, it is necessary to assess the contributions of small on developing NHP surrogate models for HCV infection. droplet aerosols and larger respiratory droplets to better understand epidemiological vectors for the disease and Enteroviruses public responses to influenza virus outbreaks. Technol- Enteroviruses (EV) affect millions worldwide each ogical hurdles currently make it difficult, such as lack of year. The enterovirus is a genus of RNA viruses assoc- inoculations for influenza viruses in NHPs and the diffi- iated with several human diseases, and among them culty in isolating and quantifying viable influenza viruses Enterovirus 71 (EV71) is highly contagious and causes from dilute aerosols make this currently impractical. foot and mouth disease (HFMD) , which has increasingly been a serious threat for Asian children (Lee & Chang, Hepatitis virus 2010). CMs infected with EV71 via lumbar injection or Compared to other infectious diseases, China has a intravenous route show some symptoms similar to those long history of research into hepatitis. Over 30 decades found in humans, but these CM models do not exhibit ago, Ge et al (1989) performed long-term liver biopsies certain important infection symptoms, such as of M. assamensis, M. Speciosa, Nycticebus Coucang myocarditis and neurogenic pulmonary edema. Likewise, infected with HBV, successfully setting up NHP models most of the EV71 applied to these CM models are host- of HBV infection. Zhang et al (1998) infected RMs with adapted EV71 strains (Hashimoto & Hagiwara, 1982; HGV and established the first RM model of HGV 1983; Hashimoto et al, 1978; Nagata et al, 2004; Nagata infection, while more recently Yang et al (2006) infected et al, 2002), and there can be marked differences Chinese RMs with HEV gene type-IV via intravenous between host-adapted EV71 strains and the epidemic injection. Following Yang’s injection, Chinese RMs strains. Unfortunately, NHPs are not susceptible to exhibited typical acute hepatitis, indicating Chinese RMs infection with epidemic strains of EV71 ( Liu & Zhang, made viable animal models of HEV gene type-IV. 2010). Chinese researchers also successfully established The study of EV71 infection NHP models began Chinese RM models of HEV1 infection, and through relatively late in China, with the first models appearing these models found E2 IgG can serve as a marker of in 2010. Zhang et al (2011b) infected adult Chinese RM HEV infection (Zhang et al, 2003; 2004). with EV71 FY-23 strain via intracerebral, intravenous, Chinese researchers have used HAV, HBV, HEV respiratory and digestive routes and showed similar and HGV to infect NHPs (Ge et al, 1989; Zhang et al, symptoms to human cases but without herpetic lesions, 1998; Zhang et al, 2003; 2004). But there have been no the reason for which may be old-age of these reports of the establishment of domestic NHP models for experimental Chinese RMs. Perhaps neonatal monkeys HCV infection. Chimpanzee models of HCV infection would prove better animal models for the study of EV71 have already been established by foreign researchers, pathogenesis, and accordingly Liu et al (2011) infected though this is expected since chimpanzees are the only neonatal RMs with EV71 FY-23 strain via the respiratory known animal to be susceptible to HCV infection (Bukh tract by tracheoscopy and the results indicated these et al, 2001). However, ethical and financial restrictions neonatal RM models more closely resembled infection of largely limit the use of chimpanzees for such research, human neonates during lactation. Wang et al (2011a) especially in the US and Europe (Akari et al, 2009). likewise infected neonatal RMs with EV71 via nasal GBV-B, a close relative to HCV, provides an effective spray, which further confirmed that a respiratory model and has been used to infect New World monkeys infection route makes models more susceptible to EV71 and the GBV-B infection models which closely resemble infection than the digestive tract infection route, HCV infection in humans (Akari et al, 2009; Bukh et al, providing some valuable information for future studies of 1999). In spite of the value of the GBV-B model, the childhood EV71 transmission. establishment and development of an HCV/GBV-B chimeric virus model has higher application value for a NHP models of cardio-cerebrovascular disease preclinical study of antiviral vaccines and drugs, and Myocardial infarction, a type of cardiovascular

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 454 ZHANG, et al. disease, has high morbidity and mortality, and is com- injecting antiautologous blood slowly and gently into the mon in Europe and America (Yeh et al, 2010). Previous caudate nucleus. This study indicated that the volume, studies indicated that the treatment strategy—proved to site and speed of injection are key factors for establishing be effective in a murine model—applied to NHPs failed a successful model, which is instructive to development to show any efficacy (Norol et al, 2003; Orlic et al, 2001). of the NHP models of intracerebral hemorrhage. Yang et al (2011) recently established RM models of While there has been significant progress in myocardial infarction by ligating the left anterior establishing NHP models of cardio-cerebrovascular descending artery (LAD) and the changes of disease, some shortcomings still exist. The NHPs chosen histopathology and electrocardiogram demonstrated that to be animal models for cardio-cerebrovascular disease this model should be excellent for studying human are usually young and healthy, despite the fact among myocardial infarction, but the main cause of myocardial humans the elderly are at the highest risk for cardio- infarction in human is cardiovascular blockage or spasm cerebrovascular disease (Baccini et al, 2008). Similarly, caused by the long-term effects of several factors the patients with cardio-cerebrovascular disease usually (hypertension, hyperlipidemia, etc.). The pathogenesis of suffer from other health conditions such as hypertension, these RM models created by ligation of the coronary hyperlipidemia or diabetes which may significantly artery existed many differences compared with human, affect cardio-cerebrovascular symptoms. Currently avail- which currently limits drug discovery and development. able studies of NHP models of cardio-cerebrovascular It is necessary that new NHP models should be disease that accompany these additional diseases is quite developed for studing human myocardial infarction in limited, to create viable NHP models of cardio-cerebro- future. vascular disease which is close to clinical nature will be Cerebrovascular disease is the second leading cause our future task. of mortality in China. Ischemic cerebrovascular disease accounts for 80% of cerebrovascular diseases (Feigin et NHP models of diabetes al, 2003). Pan et al (2006) injected polyvinyl alcohol into Diabetes is already quite prevalent in developed a branch of middle cerebral artery to make thrombed, countries, mainly type 1 and type 2, and it is quickly and CT scanning and related behavioral changes showed rising in China now, with an estimated 11% of the significant damage to brain tissue in the area of vascular population currently afflicted to some extent. Although occlusion. These results indicate that NHP models of rodents have been used as the primary model in studying ischemic cerebral infarction were successfully establi- diabetes, rodent models do not adequately mimic human shed and they owned good stability and were suitable in diabetes and make them quite limited for clinical the study of treating acute and chronic diseases. Zhu et al application (Anderson & Bluestone, 2005; Delovitch & (2008) also established CM models of photochemical Singh, 1997). NHPs, due to their similarities with human, thrombosis by focusing the cold lamp-house on the are believed to be the most effective models for human precentral gyrus, and the construction of these models is diabetes. simple and highly reproducible, making them excellent NHP diabetic models are relatively new in China. for the basic study and investigation of treatments for Liu et al (2009) divided the Chinese RM into a 125 cerebral infarction, especially regarding the study of mg/kg STZ group, a 75 mg/kg STZ group and a 50 transplantation of stem cells. Xu et al (2012) recently mg/kg STZ group and then different doses of STZ were generated Chinese RM models of middle cerebral artery injected into corresponding subjects intravenously. The occlusion by injecting auto-blood clots into middle models induced by the dose of 125 mg/kg and 75 mg/kg cerebral artery through the femoral artery with a micro- STZ were not stable, and mortality was high due to the catheter, a method that is easily repeatable, with high toxic effects of STZ. Most subjects in the 50 mg/kg STZ stability and low mortality. Likewise, the extent of group retained a high level of blood glucose and their embolization of the cerebral infarction could be timely pancreas β cells were severely damaged, but their detected. The aforementioned advantages have led to this mortality was quite low, suggesting that 50 mg/kg of technique becoming more common and ideal for the STZ may be optimal for inducing diabetes in Chinese study of cerebral ischemia. Zhu et al (2009) previously RM models. Xu et al (2009) likewise injected STZ at the established CM models of intracerebral hemorrhage by dose of 45mg/kg into the CMs intravenously and found

Zoological Research www.zoores.ac.cn Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress 455 that drinking volume, food intake, urine output increased combined with high-fat diet respectively for 24 months. and body weight was reduced, while fasting glucose was The progression of type 2 diabetes among CMs with increased and the pancreas showed atrophy and fibrosis, high-energy diets showed that similar methods were indicating that the CM diabetic models created with this successful in establishing NHP models of type 2 diabetes, method were viable. Given the serious toxic effects of and that it effectively modeled different stages of type 2 STZ on experimental NHPs, Qiao et al (2009) and Jin et diabetes. These findings also provided data on further al (2010) established RM models of type 1 diabetes by optimizing dietary strategies that could establish even the partial pancreatectomy combined with low-dose STZ. more effective animal models for human type 2 diabetes This strategy is generally safer and more reproducible as research. While type 2 diabetic mouse models have been compared with single high-dose STZ or total made (Kadowaki, 2000), the successful establishment of pancreatectomy. transgenic NHP models of type 2 diabetes should vastly Currently, the most commonly used method to make improve NHP study of type 2 diabete. type 2 diabetic animal models is high fat feeding combined with drugs induction. Typical clinical features NHP models of mental and neurological disorders of type 2 diabetes was found by injection of STZ into To our knowledge, none of animal models can fully CM at a dose of 35 mg/kg after the appearance of recapture the neurological symptoms for neurodegene- hyperlipidemia and obesity based on high-glucose and rative and psychological disorders due to their multiple fat diet feeding, (Lu et al, 2013) and the models showed. origins. While NHPs are close to human in terms of Since pathogenesis of type 2 diabetes induced by STZ is genetics, there are numerous differences in the evolution unnatural as it significantly differs from the pathogenesis and composition in brain. Currently, NHP models of of human type 2 diabetes, theoretically, screening of mental and neurological disorders such as heroin monkeys for spontaneous diabetes would lead to a more addiction, morphine addiction, Parkinson’s disease (PD), natural pathogenesis in NHP models. It will make this depression syndrome, Alzheimer’s disease (AD), spinoc- model more valuable for translational medicine. Wang et erebellar ataxia, spinal cord injury, motor cortex lesion, al (2004) previously screened 3 RMs of spontaneous epilepsy, etc (Chen et al, 2013; Chu et al, 2014; Ding et diabetes from 100 RMs by both the glucose tolerance al, 2008; Liu et al, 1992; Lu et al, 1999; Qiao et al, 2007; and glycosuria tests. These 3 RMs were in middle and Tian & Ma , 2014; Zha et al, 2006; Zhang et al, 1999 ) elderly age, indicating that onset age and the morbidity have been established in China. However, there is a gap rate of spontaneous diabetes in RMs is consistent with in the types of NHP models for mental and neurological that found in humans. After one year of observation, the disorders as compared with similar models established in similarity in clinical symptoms (urinating more and other countries. consuming more aggravated gradually and fasting blood An increased understanding regarding natural and glucose was significantly higher than normal) to humans artificial repair of central nervous system injuries as suggested this method was feasible for screening RM resulted as more researchers have begun focusing on the models of spontaneous diabetes. Unfortunately, the study of nerve regeneration following spinal cord injuries. current number of spontaneous diabetic NHP models is To model these injuries in NHPs, the main strategy is to limited and cannot adequately meet the growing demand inflict spinal cord injury via selective resection of the for effective scientific research. spinal cord with a sharp knife (Tian et al, 2010). Ni et al Given that the morbidity rate of human type 2 (2005) made NHP models of spinal cord hemisection by diabetes increases annually as long as increases in the T-11 laminectomy and then resection of a 1-mm long number of obese people (mainly caused by factors such hemispinal cord, while Zha et al (2006) made the C3-5 as eating patterns, among others), monkey models of exposed via a dorsal midline incision in the neck of RMs type 2 diabetes induced by diet can be accomplished and transected the left hemispinal cord with a knife under more quickly, and compared with models induced by the microscope after C4 laminectomy. The results drugs, more closely resemble the pathogenesis of human showed this method to establish RM models of spinal type 2 diabetes. Zhang et al (2012) recently divided 36 cord injury were simple, effective, stable, accurate and experimental CMs into 4 groups and fed them standard repetitive, even if the method was quite different from basic diet, high-sugar diet, high-fat diet and high-sugar naturally occurring human spinal cord injuries. For

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 456 ZHANG, et al. epilepsy, models are usually created by the intentional Dopa, a classic therapy, on PD symptoms. But due to application of penicillin, aluminum hydroxide, metrazol MPTP toxicity to the digestive system, digestive system and coriaria lactone. Chen et al (2013) injected kainic disorders of the NHP models induced by MPTP acid into the right intra-hippocampus of RMs by the gradually appeared and would affect immune function stereotacic technique, and the resulting seizure pattern, and nutritional metabolism. Such models are difficult to imaging observation and histopathologic tests showed maintain over long durations, making the future this model closely resembled human temporal lobe development of a long-term stable NHP PD models an epilepsy. Though promising, the long-term stability of urgent issue. this RM models induced by kainic acid requires further Recently, studies have established a link between study. increased endogenous formaldehyde (FA), a methanol Modeling PD has likewise been met with several metabolite, and AD pathology (He et al, 2010; Li et al, successes in China. Liu et al (2006) injected 1-methyl-4- 2008; Tong et al, 2011, 2012). Studies conducted by Hu phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) into right and He’s Labs in China on RM showed chronic feeding side internal carotid artery and then performed 3% methanol ad libitum led to specific damages to the behavioral testing and imaging detection, which showed brain similar to those of Alzheimer’s disease. These were the method to be both safe and simple for establishing of the first studies to show that methanol toxicity inflicted NHP models of PD. However, this method made it all the major hallmarks of AD on rhesus monkey: difficult to control the extent of nigrostriatal damage cognitive decline, tau phosphorylation, and amyloid (Herrero et al, 1991). In order to minimize the plaques formation (Yang et al, 2014). nigrostriatal damage, Yan et al (2014) established a Recent advances in optogenetic technology of have chronic NHP model of PD by intramuscularly injecting been proved as a revolutionary tool for studying neural MPTP. The time of appearance and the severity of each systems. Optogenetics can be used for precisely targeting symptom of this model could be controlled by the daily a specific area of neural systems as well as even in freely dosage and frequency. This model gradually displayed moving mammals. Researchers can then turn different PD symptoms and mimicked the development of human neurons on or off, rapidly and safely, using this PD well. However, the PD symptoms were reversible in technology (Deisseroth, 2010; Liu & Tonegawa, 2010). some MPTP-induced model, being quite different from Currently, research of optogenetic approaches has been the degenerative nature of PD in humans, and limiting done on NHPs by foreign researchers and some progress the study of stem cell therapy for nervous system of this research has been made (Dai et al, 2013), but disorders. Interestingly though, substantia nigra neurons there is lack of such research being done domestically in damage caused by 6-hydroxydopamine (6-OHDA) is China. irreversible, and the extent of nigrostriatal damage can be controlled by the injection amount and the injection sites. NHP models of ophthalmic diseases Accordingly Zhang et al (2006) generated RM models of Ophthalmic diseases present interesting complic- PD by injecting 6-OHDA stereotaxically into the right ations for animal models. Compared with other animals, substantia nigra. In testing potential treatments of PD, the NHP visual system is closer to that of humans than optimal treatment period is a key issue, and one that can any other animal, making experimental data from NHPs be addressed using NHP models of early stage PD. safer and more effective when translated into clinic Accordingly, Ding et al (2008) established NHP early settings. However, both cost restrictions and availability stage models of PD by surgery to make the common of experimental NHPs hamper the development of NHP carotid artery (CCA) of RMs exposed and then injecting models of ophthalmic diseases. Chinese researchers have low-dose MPTP into the exposed CCA. Compared with successfully established several NHP models of late-stage PD NHPs, NHP early stage models may better ophthalmic diseases, such as corneal endothelial injury, mimic the gradual development of human PD. One of the amblyopia, glaucoma and so on (Dai et al, 2005; Zhu et purposes of model development is to find new treatments. al, 2013), but there are some gaps in the types and Yan et al (2014) reported that morphine could alleviate construction methods of NHP models of ophthalmic tremors in a MPTP-induced NHP model, suggesting the diseases between China and other developed countries. therapeutic effects of morphine complement those of L- Zhu et al (2013) established NHP models of corneal

Zoological Research www.zoores.ac.cn Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress 457 endothelial injury by phacoemulsification damaging human metrorrhagia caused by endometritis. corneal endothelial cells. This method proved easy to manipulate and is easily replicable, and it causes negli- Problems, Challenges and prospects gible damage to other tissues, making it a useful While China is a key producer of NHP resources, it technique for constructing models that can accurately still lags behind other countries in its capacity for evaluate the effect of drugs and other factors on the innovative NHP model research. As medical research and endothelial healing. Similarly, Dai et al (2005) biotechnology advances—genetic modification, gene established NHP models of chronic hypertensive glauco- knockout and cloning, etc.—it is likely that a remarkable ma by semiconductor frequency-doubled 532 laser and increase of NHP resources will be developed to meet the argon laser, respectively, aiming at 360° functional trab- needs of more intensive and diverse application. It makes ecular meshwork. The light coagulation times of the for some interesting possibilities in NHP research. Due successful induction of high intraocular pressure in RMs to the economic crises and the subsequent “austerity” in fluctuate greatly, so refining the existing methods of budgets across developed countries in America and photocoagulation should improve modeling efficiency, Europe, funding support for primate research is reduced though this requires greater study to verify. or kept stagnant. Concurrently, growing criticism from animal-rights activists in these countries has brought a NHP models of reproductive diseases great deal of pressure and research institutions have to Reproductive diseases seriously affect human health limit or even abolish primate research (Cyranoski, 2003; and even psychological well-being. Infertility caused by Wadman, 2011b). Given the advances and advantages reproductive diseases affect an average 8-12% of couples provided by using NHP primates, researchers on NHP worldwide (Sellandi et al, 2012), and is quite pronounced models from developed countries are increasingly in developed countries. Reproductive physiology of seeking collaborations with primate research institutes in NHPs is quite similar to human, and the data gathered China and other developing countries in Southeastern from studies on reproductive diseases in human using Asia (Cyranoski, 2004). It is possible to bring NHPs cannot be replaced using any other experimental unprecedented opportunities and challenges to life animals. The progress in the establishment of NHP sciences and biomedical research in China. The key issue models of reproductive diseases, such as polycystic is how to cope with these opportunities and challenges to ovary syndrome (PCOS), oligozoospermia, metrorrhagia further develop NHP resources effectively. and postpartum hemorrhage (PPH), has been made by Development of domestic Chinese NHP resources Chinese researchers. has made significant progress since its inception, Tang et al (2012) gave RMs two cycles of subcut- however, in contrast to countries in America, Europe and aneous injections of propionic acid testosterone (PAT), East Asia (notably Japan and Korea), NHP resources in and then muscle injections of human chorionic gon- China are not widely or effectiely being used. In other adotropin (HCG). The results of this study indicated PAT words, while China has a rich diversity of species, many combined with HCG could successfully induce RM factors such as subspecies and geographical distribution PCOS, which was similar to human PCOS. Sun et al of NHP resources, differences among subspecies in (2006) earlier set up an RM model of oligozoospermia genetics and physiology and the occurrence of induced by orally administered gossypol acetic acid and subspecies hybrid populations in breeding and over the provided a suitable NHP model for the study of infertility course of experiments make the reproducability of in men. Until recently, most studies on PPH still were experimental results poor in working towards etablishing built on the retrospective analysis, with few reports of animal models, evaluating drugs, etc. Nontheless, evaluating or using animal models of PPH. Notably, some common experimental primate resources (espec- Huang (2010) established CM models of PPH with ially exotic species such as Cercopithecus aethiops) are uterine atony induced by medication such as oxytocin, rare or even non-existent in China, which restricting which provided a useable platform for further research. human diseases research and the development of Likewise, You et al (2003) had earlier set up RM models corresponding drugs, but other more general factors such of metrorrhagia induced by endometritis and the as technical skill, experience, and existing resources are symptoms of this model could closely resemble those of also critical. We must realize that Chinese NHP research

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 458 ZHANG, et al. can only be accomplished by bringing important 2008 as well as interference from animal-rights activists experimental primates lacking in China—and researchers have led to a plateau in primate imports from China. On familiar with them—while simultanesouly strengthening the Chinese side, this unexpected lack of growth has led the application existing experimental primates resources to rampant overproduction. Likely one reason for in China. Chinese overproduction is that many NHP breeding Another factor making Chinese NHPs ineffective is facilities in China lack effective communication with a lack of the principle knowledge about NHPs. For their international counterparts, making it difficult to example, compared with some experimental primate project and then match demand. To remedy the situation species such as Indian RMs, common experimental and ensure adequate supply of relevant and in-demand primate species such as Chinese RMs lack sufficient NHP resources, it is vital to establish an information and information regarding the genetic background of these communication platform that can effectively offer NHP primates, and it is also little known about the normal breeding information services and simultaneously carry value of their important biological parameters (Xia et al, out strategic plans for cooperation between NHP 2009). Thankfully, recent genome sequencing and breeding facilities and relevant governing bodies and analysis of CMs and Chinese RMs offers a great deal institutions. more basic data and useful reference points for relevant Two other factors could affect China’s position on NHP model research (Yan et al, 2011). It is necessary to NHP research and supply. First, animal welfare activists complement basic biological data of experimental have had some effects on NHP research. While it is true primates in China and establish subspecies breeding that advocacy of animal welfare helps promote harmony populations whose basic biological data (such as genetic between experimental animals and humans (and conse- backgrounds) are definite. Recently, the Kunming quently decreases the danger of injuries to experimenters Primate Research Center is making efforts to raise a and often offers higher-quality experimental results), northern pig-tailed macaques (NPMs, M. leonina) colony animal welfare has begun to attract extensive attention and Prof. Zheng’s lab has collected many basic which will result in increased legislation on animal biological data of this colony of NPMs (Pang et al, 2013; welfare. China has just recently begun to legislate animal Zhang et al, 2014), in order to promote the standar- welfare. On a cultural level, Chinese citizens’ awareness dization and reliability of experimental primate data. of animal welfare is weak. Awareness of animal welfare Another key issue for NHP research is the demand has placed economic pressure on NHP breeding facilities for specific pathogen-free (SPF) experimental animals. and research institutions. The dual-faced nature of this SPF animals are in great demand right now. While it is dilemma means that effective implementation of available for many traditional models, experimental experimental primate welfare in China is difficult. To primates often carry zoonotic pathogens which not only bring China more in line with international standards pose health risks to experimenters but also partly while continuing to develop China’s domestic research interfere with experimental results. In the US, all 8 capacity, relevant policies should be made for NPRCs funded by grants through NIH can offer SPF strengthening propaganda, supervision, regulation and experimental animals (Zheng, 2012), but to date China support of experimental primate welfare, in such a way has not established SPF core groups of experimental to support the healthy development of the life sciences primates. Presuming that research into drug development and medical industry in China. Second, it is a very and microbe research on diseases such as HIV continues crucial step to develop transgenic NHP resources. Since to grow, if China wants to be a leading player in NHP the birth of the first transgenic mouse, researchers have models, it must establish SPF core groups of experi- established a series of transgenic mice for modeling mental primates and all breeding efforts should be done many types of human diseases. Transgenic mice have only with careful caution and planning. made greater contributions to the study and the treatment In recent years, experimental primate husbandry in of human diseases, but their phylogenetic distance to China has experienced rapid development and the humans hampers the usefulness of their results. product has been increased, but not always in tandem Establishing primate transgenic models should be able to with the demand. As we mentioned earlier, the influence overcome these limitations and better predict the efficacy of the global economic crisis in developed countries in and toxicity of novel treatments in humans. The

Zoological Research www.zoores.ac.cn Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress 459 development of these transgenic primates is still in its In summary, NHPs are ideal experimental models infancy; the first transgenic primate was born in for biomedical research. Compared with developed 2001(Chan et al, 2001), and over the past decade, few countries in America and Europe, China has advantages transgenic NHPs have been born (Chen et al, 2012). The in developing NHPs resources. China is a leading major obstacles in producing transgenic NHPs are producer and a major supplier of NHPs. Although technical in nature. However, thanks to more efficient deficiencies in feeding and management may delay gene-editing techniques involving ZFN, TALEN and China’s growth in NHP research and materials, a number CRISPR/Cas9-based methods, researchers can avoid of primate animal models for human diseases on some shortcomings—notably low efficiency and high infectious diseases, cardiovascular diseases, endocrine toxicity—of previous transgenic methods using retroviral diseases, reproductive diseases, neurological diseases, or lentiviral vectors (Shen, 2013). Chinese researchers and ophthalmic diseases have been established. first applied TALENs and CRISPR/Cas9 to establish Particularly, the site-specific gene mutant RMs and CMs site-specific gene mutant RMs and CMs (Liu et al, 2014b; have successfully been born in China. We believe that Niu et al, 2014). The myriad studies have demonstrated advances in these studies by using NHP models will that using technologies involving TALENs and undoubtedly further promote the development of China's CRISPR/Cas9, multiple and single genetic mutations can life sciences and pharmaceutical industry, and enhance be effectively performed in monkeys (Liu et al, 2014a; China’s position as a leader in NHP research. Niu et al, 2014). In addition to the above-mentioned problems, there Acknowledgments: The opinions expressed in this are still many other key problems with NHP models to review paper reflect personal views of the authors. Due resolve, for example, high cost, difficulties regarding to the limited space of this paper and/or our ignorance how to have a well-defined phenotype characterization, and negligence, we are sorry that we did not include all and a genotype-phenotype correlation for NHPs and how these related works regarding primate studies in China to use cutting-edge molecular imaging system to although we aimed to have a comprehensive review of characterize the NHP brain structure and activity for NHP models of human diseases. We sincerely apologize better understanding mental disease. to all those colleagues whose important work is not cited.

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Zoological Research www.zoores.ac.cn Zoological Research 35 (6): 465−473 DOI:10.13918/j.issn.2095-8137.2014.6.465

Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques (Macaca leonina)

Hong-Yi ZHENG1,2, Ming-Xu ZHANG1,3, Lin-Tao ZHANG1,4, Xiao-Liang ZHANG1,4, Wei PANG1, Long-Bao LYU1,5, Yong-Tang ZHENG1,2,3,5,*

1. Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China 2. School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230026, China 3. Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming Yunnan 650204, China 4. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming Yunnan 650500, China 5. Kunming Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China

Abstract: Pig-tailed macaques (Macaca nemistrina group) have been extensively used as non-human primate animal models for various human diseases in recent years, notably for AIDS research due to their sensitivity to HIV-1. Northern pig-tailed macaques (M. leonina) are distributed in China and other surrounding Southeast Asia countries. Although northern pig-tailed macaques have been bred on a large scale as experimental animals since 2012, the reference value of normal levels of leukocytes is not available. To obtain such information, 62 blood samples from male and female healthy northern pig-tailed macaques at different ages were collected. The normal range of major leukocyte subpopulations, such as T lymphocytes, B lymphocytes, natural killer (NK) cells, monocytes, and the expression levels of activation or differentiation related molecules (CD38, HLA-DR, CCR5, CD21, IgD, CD80 and CD86) on lymphocytes were analyzed by flow cytometry. The counts of B cells decreased with age, but those of CD8+ T cells and NK cells and the frequency of CD38+HLA-DR+CD4+ T cells were positively correlated with age. The counts of leukocyte subpopulations were higher in males than those in females except for CD4+ T cells. Males also showed higher expression levels of IgD and CD21 within B cells. This study provides basic data about the leukocyte subpopulations of northern pig-tailed macaques and compares this species with commonly used Chinese rhesus macaques (M. mulatta), which is meaningful for the biomedical application of northern pig-tailed macaques.

Keywords: Northern pig-tailed macaque; Flow cytometry; Leukocyte subpopulation; Age; Sex

Pig-tailed macaques (Macaca nemistrina group, between the anatomic structure of the genital tract and PTM), rhesus macaques (M. mulatta, RM) and cynom- the menstrual cycle with humans, the feature that they olgus macaques (M. fascicularis, CM), all belonging to breed in all1seasons and have a relatively large body size. the Cercopithecidae family of Old World monkeys, have By contrast, it is difficult to perform colposcopic become more widely used models for AIDS pathogenesis examinations and multiple biopsies on CM as they have in recent years (Lei et al, 2013; Zhu et al, 2010). As their much smaller vaginal canals and cervix diameters than name suggests, PTM are characterized by the short, semi-erect ‘pig-like’ tail and the flat vertex. They Received: 08 May 2014; Accepted: 10 August 2014 diverged from RM and CM approximately 5 million Foundation items: This work was supported by the National Special Science Research Program of China (2012CBA01305); the National years ago, while the divergence between RM and CM Natural Science Foundation of China (81172876, U0832601, was generated at one another 2.4 million years later 81273251, U1202228); the Knowledge Innovation Program of CAS (Baroncelli et al, 2008). (KSCX2-EW-R-13) and the National Science and Technology Major PTM have been widely employed as animal models Project (2013ZX10001-002, 2012ZX10001-007) of sexually transmitted diseases due to the similarities *Corresponding author, E-mail: [email protected]

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PTM. The reproductive physiology of RM is also quite desirable animal model for AIDS studies. different from humans as their constant breeding season Wild northern PTM mainly distribute in southwe- (Patton et al, 2009). More importantly, PTM have the stern Yunnan, China and southeastern Tibet. Because of overriding advantages on their abilities in acute human the scarcity of this primate species, they are under First immunodeficiency virus-1 (HIV-1) infection and Grade State Protection of China. In 2012, Kunming exceptional susceptibility to simian immunodeficiency Primate Research Center, China, first introduced nort- virus (SIV) and simian-human immunodeficiency virus hern PTM from Vietnam for large-scaled breeding for (SHIV). In fact, PTM are the only reported species of laboratory use. However, the limited knowledge of their Old World monkeys that could be infected by HIV-1 immune systems has restricted their applications. In this (Agy et al, 1992; Batten et al, 2006; Bosch et al, 2000). study, we determined the phenotypic characteristics of Although they have been extensively applied in leukocytes in northern PTM ranging from 2 to 11 years biomedical areas, such as cognitive neuroscience, pharm- of age to provide detailed immunological data. acology and infectious etiology, PTM did not obtain a clear taxonomic status until Gippoliti et al (2001) sepa- MATERIALS AND METHODS rated the initial taxonomic three subspecies (nemestrina, Animals and sample collection leonina, pagensis) of the M. nemistrina group at the Cage-bred northern PTM were maintained according species level. Sunda pig-tailed macaques (M. nemestrina) to the regulations approved by the American Association are found in the southern half of the Malay Peninsula for Assessment and Accreditation of Laboratory Animal (only just extending into southernmost Thailand), Borneo, Care (AAALAC) at the Kunming Primate Research Sumatra and Bangka Island. Mentawai macaques (M. Center, Kunming Institute of Zoology, CAS. Ethyle- pagensis) are located in the Mentawai islands. Northern nediamine tetraacetic acid (EDTA) stabilized blood sam- pig-tailed macaques (M. leonina) range from about N8° ples were collected from male (n=15) and female (n=14) in Peninsular Thailand through Burma and Indochina juveniles (mean age=2.4 years (2−3 years)), as well as into Bangladesh, India extending north as far as to the male (n=17) and female (n=16) adults (mean age=6.1 Brahmaputra, and the southernmost of Yunnan, China years (5−11 years)) without apparent external symptoms (Malaivijitnond et al, 2012). (acute infections, trauma and severe diarrhea) for flow Due to the lack of primate models that can be cytometric analysis. For comparison purposes, data from productively infected by HIV-1, especially given that age-appropriate male (n=9) and female (n=8) juvenile HIV-1 infection is more efficiently restricted by tripartite (mean age=2.4 years (2−3 years)), as well as male (n=14) motif protein 5α (TRIM5α), apolipoprotein B mRNA- and female (n=12) adult (mean age=7.5 years (5−10 editing enzyme and catalytic polypeptide-like 3 years)) Chinese rhesus macaques (ChRM) were also (APOBEC3) proteins of macaques than those of humans, collected. Animals’ age and sex information is shown in the pathogenic mechanisms of HIV-1 have not been fully Table 1. All procedures were performed under the elucidated. The early block to HIV-1 infection in monkey guidance of the Ethics Committee of Kunming Institute cells was relieved by interference with TRIM5α of Zoology. expression (Stremlau et al, 2004). In our previous study, we for the first time reported that northern PTM do not Antibodies express TRIM5α but rather TRIM5-CypA fusion gene, in Mouse anti-human CD molecule monoclonal and which the B30.2/ SPRY domain of TRIM5α is substituted polyclonal antibodies that cross-reacted with PTM were by retrotransposed CypA in the 3'-UTR, contributing to used according to standard procedures and appropriate the invalidation of restriction to HIV-1 replication in concentrations in this study. Anti-CD159a PE (clone these animals (Kuang et al, 2009). Recent studies Z199) mAb was obtained from Beckman Coulter. Anti- showed that PTM rather than RM or CM are susceptive CD38 FITC (clone AT-1) mAb was obtained from STE- to siman-tropic HIV-1 (stHIV-1), in which the HIV-1NL4-3 MCELL. Streptomycin-PE-Cy7 was obtained from Biol- vif gene was replaced by the vif genes from SIVmac or egend. Anti-IgD biotin pAb used by conjunction with

HIV-2ROD to suppress the antiretroviral activity of several streptomycin-PE-Cy7 was obtained from Southern Biot- APOBEC3 proteins (Hatziioannou et al, 2009; ech. Anti-CD14 APC (clone M5E2), anti-CD20 PerCP-Cy5. Thippeshappa et al, 2011). Thus northern PTM may is a 5(clone 2H7), anti-CD21 APC (clone B-ly4), anti-CD3

Zoological Research www.zoores.ac.cn Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques (Macaca leonina) 467

Table 1 Study group of northern pig-tailed macaques and Chinese rhesus macaques Northern pig-tailed macaques Chinese rhesus macaques Age groups (years) Males Females Males Females n Age (years) n Age (years) n Age (years) n Age (years) Juvenile (2−3) 15 2.3±0.5 14 2.5±0.5 9 2.3±0.5 8 2.5±0.5 Adult (4−11) 17 6.6±1.6 16 5.4±1.7 14 7.0±1.6 12 8.1±1.5 Total 32 4.6±2.5 30 4.0±2.0 23 5.2±2.6 20 5.9±3.0

APC-Cy7 (clone SP34-2), anti-CD4-PerCP-Cy5.5/ Flow cytometric acquisition was performed on the FITC (clone L200), anti-CD8α PE-Cy7 (clone RPA-T8), BD FACSVerse cytometer driven by the FACSuite anti-CD80 PE (clone HB15e), anti-CD86 PE (clone software (version 1.0.3; BD). The acquired cell number FUN-1), anti-CCR5 PE (clone 3A9) and anti-HLA-DR was at least 100 000 CD3+ T cells or 50 000 CD20+ B APC (clone L243) mAbs were all obtained from BD cells. Analysis of the acquired data was performed using Pharmigen. FlowJo software (version 7.6.1; TreeStar).

Absolute quantification of major leukocyte subpopu- Statistical analysis lations Statistical differences between two groups were The methods of direct cell surface staining for whole determined by the nonparametric Mann-Whitney U rank blood and absolute number analysis were as described sum test. The Spearman’s test was performed for corre- previously (Xia et al, 2009). Briefly, 50 μL of whole lation analysis. Two-tailed P<0.05 was considered statis- blood samples were added in TruCount tube (BD Bio- tically significant. Data were presented as mean±SD. All sciences) and incubated with fluorochrome-conjugated statistical analyses were performed via GraphPad Prism antibodies of CD3, CD4, CD8α, CD20, CD14 and software (version 5.01; GraphPad Software). CD159a mAbs for 30 min on ice. Erythrocytes were lysed with FACS lysing solution (BD Biosciences), and the samples were analyzed with BD FACSVerse RESULTS cytometer directly. The absolute numbers of T cells The absolute number of leukocyte subpopulations in (CD3+CD20-), CD4+ T cells (CD4+CD8a-CD3+), CD8+ T peripheral blood of northern PTM + + cells (CD4-CD8a+CD3+), B cells (CD3-CD20+), NK cells The absolute number of T cells, CD4 T cells, CD8 (CD3-CD8a+CD159a+) and monocytes (CD3-CD20- T cells, B cells, monocytes and NK cells in different CD14+) were calculated using the following formula: groups are listed in Table 2. Numbers of B cells were Cell concentration (cell numbers/μL)=(events in gated negatively correlated with age (r=−0.426, P<0.001). + region (n)×total number of TruCount beads (n))/(number CD8 T cell counts (r=0.336, P=0.008) and NK cell cou- of acquired beads (n)×sample volume (μL)) (Figure 1). nts (r=0.329, P=0.01) were slightly increased with the increasing of age. Adult group (1 117±548/μL) had Phenotype analysis by flow cytometry dramatically less B cells than juvenile group (1 782± As shown in Figure 1, to determine CCR5+ T cells, 741/μL, P<0.001), however, no significant cell count activated (CD38+HLA-DR+) T cells, activated (CD80/ differences of CD8+ T cells and NK cells were found CD86+) B cells, resting (CD21+) B cells and IgD/IgM between adult and juvenile groups. secreting (IgD+) B cells, 100 μL of fresh whole blood In adult groups, compared with females, males used for each analysis were lysed with FACS lysing showed higher counts of T cells (male 3 544±1 341/μL, solution for 10 min at room temperature, followed by female 2 875±1 192/μL; P=0.032), CD8+ T cells (male washing and resuspension with Dulbecco’s phosphate- 1310±56/μL, female 1 046±533/μL; P=0.026), B cells buffered saline (DPBS) with 2% of new-born calf serum (male 1 594±621/μL, female 1 251±789/μL; P=0.016;) and 0.09% sodium azide (staining buffer). The suspe- and NK cells (male 701±617/μL, female 378±303/μL; nding leukocytes were then stained with the relevant P=0.004). However, no significant differences of age or directly conjugated mAbs for 30 min on ice and fixed sex were found in their counts of CD4+ T cells and using PBS containing 4% paraformaldehyde. monocytes.

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 468 ZHENG, et al.

Figure 1 Gating strategies for flow cytometric analysis A: Gating strategies for T cells, B cells, NK cells and monocytes. CD14/side-scatter scattergram used for gating monocytes, TruCount beads and remaining lymphocytes. CD3/CD20 dot polt gated on lymphocytes used to count T and B cells. T cells were further divided into CD4+ and CD8+ T cells. NK cells were defined as CD3-CD8a+CD159a+ phenotype gating on lymphocytes. B: The expression levels of CCR5 were analyzed in each T cell subset. C: CD38+HLA-DR+ cells in each T cell subset were defined as activated cells. D: The expression levels of CD21, CD80/CD86 and IgD were analyzed in B cells.

Table 2 Cell counts (cell numbers/µL) of leukocyte subpopulations in different age and sex groups

Age (years) Sex T cells CD4+ T cells CD8+ T cells B cells NK cells Monocytes

Juvenile M 3 433±1 469 1 886±874 1 209±533 1 751±698 548±411 442±319

(2−3) F 3 524±1 322 1 933±701 1 295±633 1 815±809 421±368 577±330

Adult M 3 642±1 254 1 941±662 1 399±597 1 455±526 836±741 636±412

(4−11) F 2 306±701 1 303±491 828±304 757±281 340±240 414±235

Total M 3 544±1 341 1 915±756 1 310±567 1 594±621 701±617 545±378

F 2 875±1 192 1 597±668 1 046±533 1 251±789 378±303 490±290

All 3 220±1 305 1 761±727 1 182±562 1 428±722 563±510 518±337

Range 1 141−6 635 564−3 794 321−3 001 378−3 282 66−3178 154−1 844

+ Expressions of activation or differentiation related increased progressively with the increaseing of age in CD4 molecules on lymphocytes of northern PTM T cells (r=0.256, P=0.045), and more CD38+HLA-DR+ sub- The expression values of activation or differentiat- set within CD4+ T cells (juvenile 1.3±0.4%, adult 2.0±0.9%; ion related markers on each lymphocyte subset in diffe- P=0.006) as well as CD8+ T cells (juvenile 4.1±1.8%, adult rent groups were shown in Table 3. CD38+HLA-DR+ subset 5.0±1.9%; P=0.02) were observed in adult groups.

Zoological Research www.zoores.ac.cn Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques (Macaca leonina) 469

Figure 2 Comparisons between northern pig-tailed macaques and Chinese rhesus macaques

Table 3 Expression levels (%) of activation or differentiation associated markers in different groups Age CD38+DR+ CD38+DR+ CCR5+ CCR5+ CD21+ IgD+ CD80/CD86+ Sex (years) CD4+ T cells CD8+ T cells CD4+ T cells CD8+ T cells B cells B cells B cells Juvenile M 1.25±0.31 3.85±1.46 3.53±2.15 11.14±6.36 67.99±12.76 72.75±12.93 44.79±12.51 (2−3) F 1.32±0.53 4.32±2.21 3.48±1.98 11.85±4.77 64.67±11.53 65.71±13.97 48.19±9.48 Adult M 2.27±0.92 5.49±2.18 4.07±1.45 12.02±3.73 66.81±12.05 69.97±11.94 41.78±11.11 (4−11) F 1.63±0.81 4.58±1.50 4.01±2.20 14.35±7.19 60.03±14.13 56.12±13.89 60.14±17.42 Total M 1.79±0.86 4.72±2.03 3.82±1.81 11.61±5.07 67.37±12.20 71.27±12.29 43.19±11.69 F 1.49±0.70 4.46±1.84 3.76±2.08 13.19±6.21 62.19±12.98 60.60±14.53 54.57±15.30 All 1.64±0.79 4.59±1.93 3.79±1.93 12.37±5.66 64.86±12.75 66.11±14.35 48.70±14.61 Range 0.49−3.84 1.48−10.70 1.00−8.79 3.42−28.80 33.80−90.50 35.80−93.10 17.00−80.10

Males showed higher frequency of IgD+ B cells ern PTM 3 220±1 305/μL, ChRM 3 998±1 561/μL; (male 71.3±12.3%, female 60.6±14.5%; P=0.006) but P=0.006), less CD8+ T cells (northern PTM 1 182±562/μL, lower frequency of CD80/86+ B cells (male 43.2±11.7%, ChRM 1 634±921/μL; P=0.009), whereas higher freque- female 54.6±15.3%; P=0.004) than females. However, ncies of both CD4+ T cells (northern PTM 55.1±7.3%, no statistically significant age and gender differences in ChRM 47.3±10.1%; P=0.0001) and CD21+ B cells the frequencies of CCR5+CD4+ T cells, CCR5+CD8+ T (northern PTM 69.4±12.8% compared to 54.2±18.8%; cells and CD21+B cells were found. P=0.001) as well as increased CD4/CD8 ratio (northern PTM 1.62±0.61, ChRM 1.38±0.72; P=0.016). Further Comparisons of lymphocyte subpopulations between analysis demonstrated that male northern PTM had less T northern PTM and ChRM cells (northern PTM 3 544±1 341/μL, ChRM As shown in Figure 2, a flow cytometric analysis 4498±1458/μL; P=0.03), less CD8+ T cells (northern was performed to study the differences in lymphocyte PTM 1 310±567/μL, ChRM 1 985±991/μL; P=0.01; subpopulations between PTM and ChRM. Compared northern PTM 36.8±6.9%, ChRM 44.0±13.8%; P=0.013), with ChRM, northern PTM exhibited less T cells (north- but higher frequency of CD4+ T cells (northern PTM

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 470 ZHENG, et al.

54.3±7.2%, ChRM 45.9±11.0%; P=0.003) and CD4/CD8 correlations of the absolute or relative cell numbers of ratio (northern PTM 1.58±0.65, ChRM 1.25±0.75; lymphocyte subpopulations were found in recent study P=0.01) than male ChRM. Whereas, females showed (Klose et al, 2007). Although the results were contr- lower frequency of CD21- B cells (northern PTM adictory in these studies, a widely accepted theory 37.8±13.0%, ChRM 54.6±15.4%; P<0.0001) compared suggests that aging is characterized by the decline in B to female ChRM. cell numbers and an accumulation of CD8+ T cells rather than a loss of CD4+ T cells in peripheral blood (Frasca et DISCUSSION al, 2008; Koch et al, 2007). On the other hand, Studies have suggested that aging is associated with numerous PTM group (M. nemestrina, M. leonine and M. alterations in innate immunity which is the first line of pagensis) have served an important role in the field of defense against pathogens and plays a key role in biomedicine, notably in HIV/AIDS research, in recent regulating the responses of adaptive immunity (Shaw et years (Lei et al, 2013). However, no detailed taxonomic al, 2010). Positive correlations were observed between description of these animals was found in most studies age and CD16+CD56+ NK cell numbers, and it is that use the collectively called “pig-tailed macaques” generally accepted that the CD56dim peripheral NK cell (Agy et al, 1992; Batten et al, 2006; Bosch et al, 2000). population expands with age (Jiao et al, 2009; Mahbub et We previously demonstrated that northern PTM express al, 2011). Tollerud et al (1989) evaluated the influence of TRIM5-CypA fusion gene, resulting in its sensitivity to age, race, and gender on the immune system, and did not HIV-1 and improving the application value of these new observe age related effects for CD14+ cells. However, laboratory animals in HIV/AIDS research (Kuang et al, other recent research reported that the number of 2009). We also provided the reference values of immu- monocytes was significantly higher in the elderly than in noglobulins, complements, C reactive protein (CRP), the young group (Della Bella et al, 2007). hematological and biochemical indexes of north PTM, Because they share greater genetic and physiological and elucidated that gender, age and weight can influence similarities with humans than rodent models, NHP has these indexes (Pang et al, 2013; Zhang et al, 2014). been used for biomedical research for several decades. However, the lack of the immune system characteristics Most of our understanding of the immune system in Old of northern PTM may have restricted its application as World monkeys comes from studies utilizing rhesus AIDS animal models. Here we evaluated the absolute macaques (Messaoudi et al, 2011). However, only few number and the expression of activation or differen- reports focused on the age-related changes in the tiation related markers of major lymphocyte subpop- immune system of PTM, especially northern PTM. ulations in 62 male and female northern PTMs ranging Asquith et al (2012) measured age-related changes in T from 2 to 11 years of age, and compared the lymphocyte cell homeostasis in Indian rhesus macaques (InRM) subpopulations of PTM with those of the most widely ranging from 1 to 30 years old, and indicated that the used ChRM. frequency of CD8+ T cells expands with age while the Serving as a major risk factor of morbidity and frequency of CD4+ T cells remains stable, whereas age- mortality for many infections caused by various patho- related changes of the absolute number of both CD4+ and gens, age influences the immune system in both human CD8+ T cells were not found in InRM (Didier et al, and non-human primates (NHP) (High, 2004). Wiener et 2012). The CD4/CD8 ratio in juvenile InRM is al (1990) found that the absolute number of both CD19+ approximately 2, and starts to decline after the first 2 B cells and CD3+ T cells decreased while the absolute months of life, whereas no further decline is detected number of CD4+ and CD8+ T cells and the CD4/CD8 between 5 and 7 years of age (Dykhuizen et al, 2000). ratio were well maintained with increasing age. The Similarly, B cells in RM are identified based on the following Swedish OCTO immune longitudinal study expression of CD20 and HLA-DR, and show a sharp showed decreased CD4 subset, increased CD8 subset, decrease during aging (Didier et al, 2012). Early studies and a lower CD19+ B cell percentages in aged population indicated that the number of circulating CD56+CD16+ (Ferguson et al, 1995). Fagnoni et al (1996) reported a NK cells in RM reduces with age (Coe & Ershler, 2001). decrease in the absolute number of both CD4+ and CD8+ However, highly different from humans, NK cells in T cells in people older than 60 years. However, no age peripheral blood of macaques express high levels of

Zoological Research www.zoores.ac.cn Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques (Macaca leonina) 471

CD8α and almost all of them express NKG2A, but do ChRM infected with SIV commonly have lower not express CD56, which make them a CD3-CD8α+ plasma viral loads and develop AIDS more slowly than NKG2A+ phenotype (Hong et al, 2013). A recent study of InRM, whereas PTM are susceptible to many lentiviral rhesus macaques ranging from 2 to 24 years old did not infections, including infection with SIV, HIV-1, HIV-2, find any correlation between the circulating NK cells and SHIV and stHIV-1, and their progress to AIDS after SIV age (Didier et al, 2012). Though the frequency of infection are also more rapid than RM (Ling et al, 2002). circulating monocytes increases in InRM older than 5 A comparison of lymphocytes between ChRM and years, the numbers of these cells reduces with aging northern PTM may provide clues to explaining this (Asquith et al, 2012). phenomenon. Previous studies demonstrated that ChRM It has been reported that CD38+HLA-DR+ subset experience significant age-related changes in leukocyte within CD8+ T cells has a strong positive correlation with subpopulations, including decreased lymphocyte HIV-1 disease progression and CD38+HLA-DR+ CD4+ T numbers, CD4+ T cell frequency and CD4/CD8 ratio, cells are highly susceptible to infection with R5-tropic increased CD8+ T cell frequency, but stable monocytes virus because of elevated CCR5 expression (Kestens et (Xia et al, 2009; Zheng et al, 2014). Similarly, northern al, 1992; Meditz et al, 2011). CCR5 plays an essential PTM showed increased numbers of NK cells and CD8+ T role in HIV pathogenesis as a main co-receptor cells, reduced B cells, but stable CD4+ T cells and responsible for viral transmission. CCR5+CD4+ T cells monocytes in our study. In addition, northern PTM also are the target of R5-tropic virus and peripheral blood showed higher CD4+ T cell frequency but fewer CD8+ T CCR5+CD8+ T cells accumulate during HIV infection cells and CD21- B cells than ChRM. (Brenchley et al, 2004). In addition, a loss in the The sex effects on circulating lymphocytes have expression of CD21 and IgD, and increased levels of been reported in both humans and NHP. Uppal et al activation markers, including CD80 and CD86, on circul- (2003) demonstrated that women have a higher number ating B cells are suggested be linked to HIV viremia and frequency of CD4+ T cells, whereas men have a (Moir & Fauci, 2008). Klatt et al (2012) demonstrated higher frequency of CD8+ T cells. However, no that higher pre-infection levels of immune activation differences in circulating B cells, NK cells and CD14+ than RM may contribute to rapid disease progression in cells were found between men and women (Choong et al, PTM. Thus it is also necessary to examine these in both 1995; Tollerud et al, 1989). However, the influence of healthy humans and laboratory animals. The frequency sex on the leukocytes in macaques is less clear. of CCR5+ cells within CD4+ T cells in northern PTM Tryphonas et al (1996) revealed significant gender (3.79±1.93%) was the lowest compared to Sunda PTM differences in the CD4+ T cell percentage (females> (14.54±6.3%) and RM (7.76±4.8%) (Klatt et al, 2012). males) and CD4/CD8 ratio (females>males) of infant The frequency of CD38+HLA-DR+ cells was 4.4±2.5% CM. Recent studies on circulating lymphocytes in InRM for CD4+ T cells and 14.5±7.0% for CD8+ T cells in did not find differences between males and females healthy human as compared with 1.64±0.79% and (Asquith et al, 2012; Didier et al, 2012). However, our 4.59±1.93% for CD4+ and CD8+ T cells, respectively, previous study revealed that female ChRM have higher from northern PTM and 3.03±2.84% for CD8+ T cells counts of CD4+ T cells, CD8+ T cells, B cells and NK from rhesus macaques (Brignolo et al, 2004; Onlamoon cells than males (Xia et al, 2009). In this study, northern et al, 2005). Elevated levels of CD21- B cells are PTM also showed numerous gender differences in believed to account for the poor proliferative responses leukocytes and activation markers, and males may have of B cells. This population is significantly expanded in stronger immune response due to their higher levels of most patients with systemic lupus erythematosus (SLE) CD8+ T cells, B cells and NK cells than females. Howe- and HIV-1 and its normal level is approximately 15% in ver, further work is required to determine the character- healthy people, 45% in CM, 46% in RM and 36% in istics of immune function between males and females. northern PTM (Das et al, 2011; Kling et al, 2011; In summary, the present study not only provided Zandieh et al, 2013). The B cells in the peripheral blood data on the immunological characteristics of the of healthy people have an average 75% IgD+ subset, leukocyte subpopulation, but also demonstrated age and while northern PTM have lower frequency (66%) (Klein sex effects on those in northern PTM. Moreover, the et al, 1998). comparison of northern PTM with ChRM provided clues

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 472 ZHENG, et al. to understanding the big differences between these two Gui Li and Dong-Ti Huang of Kunming Primate species when infected with HIV-1 or SIV. Research Center for their assistance with the exp- Acknowledgments: We thank Mr. Zhen-Fei Hu, eriments.

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Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 Zoological Research 35 (6): 474−484 DOI:10.13918/j.issn.2095-8137.2014.6.474

Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan

Jin-Fa LI1,#, Yu-Chao HE2,#, Zhi-Pang HUANG1, Shuang-Jin WANG1, Zuo-Fu XIANG3, Juan-Jun ZHAO4, Wen XIAO4,*, Liang-Wei CUI1,*

1. Key Laboratory of Forest Disaster Warning and Control in Yunnan Province, Southwest Forestry University, Kunming, Yunnan 650224, China 2. Bureau of Yunling Provincial Nature Reserve, Lanping, Yunnan 671400, China 3. College of Life Science and Technology, Central South University of Forestry and Technology, Changsha, Hunan 410004, China 4. Institute of Eastern-Himalaya Biodiversity Research, Dali University, Dali, Yunnan 671003, China

Abstract: Seasonal variation in environmental factors is vital to the regulation of seasonal reproduction in primates. Consequently, long-term systematic data is necessary to clarify the birth seasonality and pattern of primates in highly seasonal environments. This study indicated that black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha exhibited strict birth seasonality with a pulse model. Infants were born with a certain degree of synchronization. Birth distribution showed three birth peaks, and the birth pattern showed a “V” style in even-numbered years and a gradual increase in odd-numbered years. The beginning date, end date and median birth date were earlier in even-numbered years than those in odd-numbered years. The higher latitude of their habitats, earlier birth date, shorter birth period, fewer birth peaks and stronger birth synchrony might be adaptations for strongly seasonal variation in climate and food resources. After the summer solstice when daylight length began to gradually shorten, R. bieti at Mt. Lasha started to breed during the period with the highest environmental temperature and food availability, which implied that photoperiod may be the proximate factor triggering the onset of estrus and mating. It appears that R. bieti coincided conception and mid-lactation with the peak in staple foods, and weaning with the peak in high quality of foods. Thus, food availability was the ultimate factor regulating reproductive seasonality, and photoperiod was the proximate factor fine-turning the coordination between seasonal breeding and food availability.

Keywords: Rhinopithecus bieti; Birth seasonality; Birth pattern; Birth synchrony

In wild environments, birth seasonality exists in son & Heideman, 1994; Negus & Berger,11972; Andelm- many animals. Primates show reproduction continuum an, 1986; Bronson & Heideman, 1994; Brockman & van from seasonality to un-seasonality like other mammals Schaik, 2005; Tecot, 2010; van Schaik & Pfannes, 2005). (Brockman & van Schaik, 2005). The typical reproduc- In natural environments, seasonal food resources are the tion patterns of primates include seasonality (births most important factor that determine birth pattern, while within a specific period), peaks (birth peaks in certain months) and irregularity (births within the entire bree- Received: 4 March 2014; Accepted: 17April 2014 ding cycle without regularity) (Struhsaker & Leland, Foundation items: This study was supported by the National Natural 1987; Brockman & van Schaik, 2005). In high and Science Foundation of China (31160422, 30960084), China moderate birth seasonality, ≥67% and 33%−67% of infa- Postdoctoral Science Foundation (2013M542379), Program for New nts were found to be born within three months, respecti- Century Excellent Talents in University (NCET-12-1079), and Key vely (van Schaik et al, 1999). External environmental Subject of Wildlife Conservation and Utilization in Yunnan Province factors (e.g. temperature, precipitation, food resources, *Corresponding authors, E-mails: [email protected]; photoperiod, seasonal climate, and food availability) and [email protected] biological rhythms affect reproductive seasonality (Bron- #Authors contributed equally to this work

Science Press Volume 35 Issue 6

Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan 475 precipitation, temperature and daylight cycle function as Lasha group (N26°20′) was found to occur from proximate factors (Wikleski et al, 2000). Animals February 15 to April 7 in 2009−2010, with the median synchronize their critical reproductive period with the birth date on March 27 (Huang et al, 2012), but was from optimal phase of environments to maximize survival February 19 to April 12 in 2011, with the median birth (Negus & Berger, 1972). Photoperiod and temperature in date on March 17 (Wang et al, 2012). It appears, temperate regions, as well as precipitation in tropical therefore, that reproductive patterns are influenced by areas, affect seasonal food resources, and eventually differences in habitat altitude and latitude, with monkeys induce reproductive seasonality (Vivien-Roels & Pevet, in high altitude and latitude areas showing earlier birth 1983). Under some circumstances, however, photoperiod dates but shorter breeding periods, as well as by has no influence on conception, and food availability is differences year to year. Mating behaviors are triggered the only determining factor of reproductive seasonality by various factors, such as photoperiod (Sadlier, 1972), (Huang et al, 2012; Carnegie et al, 2011; Kowalewski & so for some species living in highly seasonal Zunino, 2004). With increases in both latitude and environments, maintaining high energy levels may not altitude, climate and food seasonality change signific- always be necessary for conception (Brockman & van antly. In certain primate species (e.g. Macaca thibetana), Schaik, 2005; Drent & Daan, 1980). the median birth date occurs earlier with the increase in Reproductive seasonality in black-and-white snub- altitude (M. fuscata: Fooden & Aimi, 2003; P. entellus: nosed monkey groups (especially the Xiaochangdu group) Newton, 1987). During the breeding season, an is not induced by photoperiod, but is determined by the individual’s weight gain indicates the accumulation of enriched food resources during the conception period nutrients, which not only guarantees the nutritional req- (Huang et al, 2012; Xiang & Sayers, 2009). Reproduction uirements of mating individuals, infants and nursing of R. beiti is also characterized by birth synchrony, with activities, but also ensures that the critical period of spatial-temporal differences (Huang et al, 2012; Wang et al, infant survival falls into the next autumn with enriched 2012). Reproductive synchrony refers to the way species food resources (Zhao & Deng, 1988). Reproductive maximize food quality and availability with high energy seasonality is more obvious among species living in consumption periods (e.g. nursing and weaning) (van areas with high altitude and latitude (Janson & Verdolin, Schaik & van Noordwijk, 1985). Black-and-white snub- 2005; R. brelichi: Yang et al, 2009; R. roxellana: Qi et al, nosed monkeys, also called snow monkeys (Elliot, 1912), 2008; Ren et al, 2003; Zhang et al, 2000; P. entellus: inhabit areas with high altitude (Long et al, 1994), as well Newton, 1987; C. polykomos: Dasilva, 1989; P. pileatus: as significant seasonal climate changes and food resources Stanford, 1990). In addition, reproductive peaks can be (Kirkpatrick et al, 1998; Xiang & Sayer, 2009; Huang et al, significantly correlated with climate and food seasonality 2012). However, limited short-term data have been unable (e.g. P. entellus: Newton, 1987), while relatively stable to provide a strong interpretation of the reproductive food acquisition (e.g. provision or crop stealing) can seasonality and corresponding environmental factors in the induce reproductive irregularity (Bishop, 1979). breeding of black-and-white snub-nosed monkeys. In this Black-and-white snub-nosed monkeys (Rhinopi- study, reproductive data of the Mt. Lasha black-and-white thecus bieti) are endemic to the Trans-Himalayas, which snub-nosed monkey group were collected to determine the: are bound by the upper rivers of the Yangtze to the east (1) reproductive seasonality; (2) birth pattern (peaks and and the Mekong to the west (N26°14′−N29°20′) (Long et variations); and (3) mechanisms of regulating reproductive al, 1994). From north to south, the altitudes of groups of seasonality. this species decrease, habitat transits from dark coni- ferous into dark coniferous-broadleaf mixed forest, and MATERIALS AND METHODS gradient trends manifest in photoperiod, temperature, precipitation and food availability (Xiang & Sayers, Study areas and animal subjects 2009; Ding & Zhao, 2004; Huang et al, 2012). The birth Our study site is located at Mt. Lasha (N26°20′, period of the Xiaochangdu group (N29°15′) was found to E99°15′) in the Yunling National Reserve, Lanping occur from February 4 to March 14, with the median County, Nujiang Prefecture, Yunnan Province, China. birthdate on February 24 and standard deviation of 6.5 The highest peak is 3 854 m in elevation. Only one days (Xiang & Sayers, 2009). The birth period of the Mt. black-and-white snub-nosed monkey population with

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 476 LI, et al.

130 individuals has been observed at Mt. Lasha, and is and standard deviation (SD) (Caughley, 1977, but see comprised of eleven one-male multi-female units (OMUs) Eisenberg et al, 1981). We then transformed each day and two all-male-units (AMUs) (Huang et al, 2012; into degrees of a circle (360°) for testing birth seasonality Wang et al, 2012). Home range of this population covers by circular statistics, the mean vector length r was an area of 14 km2 and is surrounded by villages and calculated to measure birth season dispersion, ranging from farmland at low altitude (<2 800 m), as well as alpine 0 (uniform) to 1 (clustered) (Batschelet, 1981). The pastures at high altitude (>3 600 m). Its habitat is patchy Rayleigh test (Z=nr2) was used to determine whether the with sporadic pastures, fire grasslands, and charcoal birth data were unevenly distributed throughout the year burning lands (from oak). The vegetation from low to (Batschelet, 1981; Zar, 1999; Huang et al, 2012). high altitude forms zonal distributions of coniferous The I/F data of the Xiaochangdu group obtained forest, mixed broadleaf-conifer forest, broad-leaved from either close tracking recording or field scope deciduous forest and dark coniferous forest (Huang et al, scanning were used to determine birth pattern. When an 2012; Wang et al, 2012). I/F was obtained by both methods at the same time, the Annual average temperature at Mt. Lasha is former had priority (Xiang & Sayers, 2009). 12.42±6.81 °C, with lowest and highest monthly average temperatures of −1.88±7.15 °C (−4.15−1.00 °C, January) RESULTS and 25.84±5.97 °C (15.86−34.06 °C, October), respect- Birth timing ively. An extreme temperature fluctuation occurred from The first R. bieti infant at Mt. Lasha was born on August 2012 to July 2013 (–4.15–34.16 °C). Annual February 20, 2013. The I/F value indicated that the precipitation is 767.60 mm, with the highest proportion breeding season ended on April 14. The numbers of new- falling from March to September (695.60 mm), and can born infants peaked (13) on April 8, and thereafter, be subdivided into precipitation peak (July to September) stabilized on 13. Because the I/F value was rather unstable and sub-peak (March to May). The snow season occurs than the I-value, and the F-value also included adult from November to the following March. females that had given birth in the last year, the I-value Data collection and statistical analyses was the more reliable index reflecting the birth timing. Data collection was scheduled ahead of the monkey The end of the breeding season inferred from the I-value breeding season (from February to April) (Huang et al, was April 8 (Figure 1A). Therefore, in 2013, there were 13 2012; Wang et al, 2012). At panoramic view points (e.g. new-born infants at Mt. Lasha and the breeding season ridges and rocks) opposite monkey activation areas, we was from February 20 to April 8. By referring to the I- recorded numbers of infants and female adults when they values, the birth data of R. bieti at Mt. Lasha from 2009 to are crossing an open area and foraging in the wide-field 2012 was analyzed (Figure 1). Although the beginning of forests using a monocular telescope (Leica, T77). the breeding season was consistent in the two estimations Newborns were characterized by overall white hair based on the I-value and I/F, the latter was overall (expect a hint of dark hair on the head), black tip of the postponed with deviations (−1 to 8 days) (Table 1). tails (one third), as well as red and hairless auricles and extremities. Newborns less than 1.5 months old are Table 1 Breeding seasons of Mt. Lasha monkey group inferred from different indexes carried by adult females. To ensure the accuracy of the Beginning of End of breeding season data, only observations with more than half of adult Year breeding season females of the group were used for further analysis since I/F I Deviations (day) its interbirth interval is two years (Huang et al, 2012; 2009 2-21 4.02 4-03 −1 2010 2-15 4.04 4-04 0 Wang et al, 2012). When multiple observations were 2011 2-19 4.20 4-12 8 carried out in one day, only the most reliable (e.g. best 2012 2-17 4.07 4-06 1 view, most individuals included) was used in statistical 2013 2-20 4.14 4-08 6 analysis. Reproductive seasonality was analyzed by I: Infants; F: Female adults; I/F: Ratio of infants to female adults. considering the numbers of infants and the ratio of infants to female adults (I/F). Birth seasonality We used each 7-day period to calculate all births, to Strict birth seasonality with a pulse type (SD<30 infer the median birth date (Md), mean birth date (Mn) days) was observed in R. bieti at Mt. Lasha. The

Zoological Research www.zoores.ac.cn Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan 477 breeding season lasted 57 days. Reproductive variables exhibited certain synchrony, and showed three peaks on had certain similarities every two years (e.g. beginning February 15−26, March 11−19 and March 20−April 12, phase, ending phase, and Md). The breeding season respectively (Figure 2). The data on infant births using 3- began and ended earlier in even-numbered years than day intervals indicated that the third peak occupied 50% those in odd-numbered years, and Md was postponed by of the entire breeding season, whereas the first and the ten days (Table 2). second peaks accounted for 25% and 23.5%, respectively. The highest birth peak in both odd- and even-numbered Birth pattern years fell in the third peak period, whereas in odd- The birth of new-born infants at Mt. Lasha numbered years, the sub-peak fell in the second peak

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 478 LI, et al.

Figure 1 Values of I/F and I of Rhinopithecus bieti at Mt. Lasha from 2009 to 2013 A: 2013; B: 2012; C: 2011; D: 2010; E: 2009.

Table 2 Indexes of birth seasonality of Rhinopithecus bieti at Mt. Lasha

Year Breeding period (day) F I I/F Mn Md SD (day) R Z

2009 2-21−4-03 (42) 24 11 0.46 3-26 3-21 11.58 0.974 10.44**

2010 2-15−4-04 (49) 27 16 0.59 3-23 3-31 19.48 0.938 14.08**

2011 2-19−4-12 (53) 29 13 0.45 3-24 3-17 17.23 0.960 11.98**

2012 2-17−4-06 (50) 33 15 0.45 3-28 3-28 17.27 0.966 14.00**

2013 2-20−4-08 (48) 34 13 0.38 3-24 3-19 16.90 0.961 12.00**

2009-10 2-15−4-04 (49) − 27 − 3-24 3-27 16.95 0.953 24.51**

2009-11 2-15−4-12 (57) − 40 − 3-25 3-22 16.72 0.955 36.47**

2009-12 2-15−4-12 (57) − 55 − 3-25 3-24 16.73 0.958 50.46**

2009-13 2-15−4-12 (57) − 68 − 3-25 3-22 16.86 0.958 62.43**

Odd-numbered year 2-19−4-12 (53) − 37 − 3-25 3-19 15.81 0.964 34.37**

Even-numbered year 2-15−4-06 (51) − 31 − 3-25 3-29 18.19 0.952 28.07** I: Infants; F: Female adults; Mn: Mean birthdate; Md: Median birthdate; SD: Standard deviation; **: P<0.001.

Zoological Research www.zoores.ac.cn Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan 479

Figure 2 Birth patterns of Rhinopithecus bieti at Mt. Lasha from 2009 to 2013 Disconnected points mean no data was available or fewer infants were recorded than three days earlier. period and in even-numbered years, the sub-peak fell in that of the Xiaochangdu group was double-pulse-type the first peak period (Figure 2). (peak: February 27−March 7, sub-peak: February 6−11) (Figure 3). However, no differences were found in the Birth pattern comparison of the northern and south- percentage of new-born infants between birth peaks of ern monkey groups the two groups (Xiaochangdu vs. Mt. Lasha: 62.2% vs. The Xiaochangdu and Mt. Lasha monkey groups 50%, proportion test: Z=1.20, P=0.11). were 3° apart in altitude. With increasing altitude, the beginning phase, ending phase and Md of the monkeys shifted to an earlier date; moreover, the breeding season DISCUSSION shortened and birth synchrony was strengthened (SD Regulation of birth seasonality decreased by ten days) (Table 3). The birth pattern of R. Reproduction is strongly influenced by biological bieti at Mt. Lasha was triple-pulse-type, whereas, rhythms and external stimulation (e.g., photoperiod,

Table 3 Birth indexes of Rhinopithecus bieti at Xiaochangdu and Mt. Lasha

Populations Breeding season (day) Mn Md SD (day) Altitude (m) Latitude Reference Xiaochangdu 2-04−3-13 (38) 2-28 2-24 6.50 3 500−4 250 N29°15’ Xiang & Sayers, 2009 Mt. Lasha 2-15−4-12 (57) 3-25 3-22 16.86 2 850−3 800 N26°20’ This study Mn: Mean birthdate; Md: Median birthdate; SD: Standard deviation.

Figure 3 Accumulative numbers of Rhinopithecus bieti infants at Xiaochangdu and Mt. Lasha. Data on Xiaochangdu group was collected in 2005 and data on Mt. Lasha group was from 2009 to 2012

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 480 LI, et al.

temperature, precipitation and food abundance) (Amroso and food resources in Xiaochangdu area (Table 4). The & Marshall, 1960; Lancaster & Lee, 1965). Energy mating period of both Xiaochangdu and Mt. Lasha requirements and allocation have been considered the groups was from July to October (Xiang & Sayers, ultimate regulation factors in reproductive patterns. 2009; Huang et al, 2012; Wang et al, 2012; Li, Other factors may play roles in regulating birth seaso- unpublished). The breeding season of black-and-white nality, including temperature and food abundance, to snub-nosed monkeys began after midsummer with meet the requirements of infant survival, nursing and shortening daytime, high temperature, abundant food development (Brockman & van Schaik, 2005; Janson & and they started mating in July. This indicated that Verdolin, 2005). Reproduction in Xiaochangdu and Mt. photoperiod initiated estrus and mating. Huang et al Lasha groups showed strong seasonality. However, (2012) found that low temperature, especially the because Xiaochangdu group’s habitat was 3° higher in lowest temperature during midsummer, triggered repro- altitude than that of the Mt. Lasha group, its reproduction ductive behavior in the Mt. Lasha group. Cozzilino et al was more concentrated over a shorter breeding season (1992) also found that temperature and its variations with an earlier beginning date (Table 3). initiated the beginning of estrus and mating in Macaca More obvious seasonality was found in the climate fuscata.

Table 4 Seasonality of food resources in the Xiaochangdu and Mt. Lasha monkey groups

Xiaochangdu (Xiang et al, 2007; Xiang & Sayer, 2009) Mt. Lasha (Huang et al, 2012; Li, unpublished) Climate Food Climate Food Maximum precipitation from June Usnea thrives, other vegetation buds Maximum precipitation from June Vegetation buds in late-February; to August; in mid-May; to October; Usnea and leaves are in abundant Buds, leaves, flowers and fruits/nuts Monthly average temperature Monthly average temperature from June to September, buds, are in abundance from June to (15.0−18.2 °C) from June to (10.2−12.5 °C); flowers and fruits/nuts are in August; September; abundance from April to May; Monthly average lowest Monthly average lowest Leaves fall in mid-October temperature (12.2−14.7 °C) from Leaves fall in mid-November temperature (3.7−5.4 °C) June to September;

Photoperiod, temperature and precipitation affect conception during the peak in food availability. food abundance, and, in turn, the reproduction period Therefore, complete conception during the peak in food (Bronson, 1988; Sadleier, 1969). Based on birth date availability played an important role in regulating (Xiang & Sayer, 2009) and lactation period (195−204 reproductive seasonality, and food abundance instead of days) of black-and-white snub-nosed monkeys (Ji et al, photoperiod initiated ovulation and conception (Huang et 1998), the conception period of Xiaochangdu group was al, 2012; this study). calculated to be from July 24 to September 2 (median The decrease in both food abundance and tempe- August 13), whereas, the conception period of the Mt. rature during the conception period means that energy Lasha group was from August 6 to September 30 requirements increased. Infants were born during a (median September 9). R. bieti at Xiaochangdu began to period of low temperature and relatively limited food. R. conceive after one month of food abundance peak, and bieti infants at Mt. Lasha began to wean at 13−14 months monkeys at Mt. Lasha did after two months of food peak. old (March−April of the following year) and ended at 18- Although the conception of Xiaochangdu and Mt. Lasha months old (June of the following year) (Li, groups began at different time points, they all terminated unpublished). This “bottle phase” (weaning) fell in the before the end of the food summit. Moreover, the median period with increased growth in “soft” and high nutrition date of conception of the two monkey groups occurred in foods (buds, leaves and flowers). Compared with the Mt. the middle of the last month of the peak in food Lasha area, the peak in food availability at Xiaochangdu availability. The long cold winter with limited food was one month shorter, buds formed 2.5 months later, consumed the energy stores of the monkeys. Breeding and leaves fell one month earlier. Although we lacked individuals recovered, restored energy and completed weaning information on R. bieti infants at Xiaochangdu,

Zoological Research www.zoores.ac.cn Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan 481 according to the relatively short food supply and the fact monkeys; therefore, more obvious seasonality in both that infants at Mt. Lasha rarely foraged independently at climate and food was shown. Although there were diffe- six-months-old (Li, unpublished), we assumed that the rences in birth patterns of two monkey groups, their birth chances of Xiaochangdu infants being weaned during the peaks stabilized during the following 6 to 7 months after same year were quite low, and more likely occurred in the peak in food availability. Thus, the relative stability late-May to early-June of the following year when abun- of the breeding season seems to be an adaptation to dant young vegetation was available. If no appropriate seasonal environments, which was critical for its succe- food is available during weaning, reproduction will ssful reproduction (Table 3). Previous research has sho- ultimately fail and lead to the demise of an entire popula- wn that 100% of wild black-and-white snub-nosed mon- tion, or perhaps even species. Thus, the weaning of R. keys and 96.3% of captive individuals start reproduction bieti during a period of abundant high nutrition food is after mid-summer (Cui et al, 2006), indicating that phot- not only a prerequisite for successful reproduction, but operiod is the triggering factor of their estrus and mating also an adaptive strategy to environmental seasonality. behaviors. Although the stable food supply decreased the Early- and late-lactation period of this species seasonality and synchrony of infants born, a birth peak occurred during relatively low temperature and limited was still observed in captive Rhinopithecus monkeys (R. food periods, whereas, highly energy-consumptive mid- bieti: Cui et al, 2006; R. roxellana: Zhang et al, 2000). lactation overlapped with the peak in food availability. These findings indicate the existence of boilogical Monkeys stored energy during pre-conception and mid- reproductive rhythms that are not influenced by either lactation to alleviate energy requirements during early- photoperiod or temperature (Fooden & Aimi, 2003). This and mid-to-late-lactation. Compared with the Mt. Lasha study indicated that conception and mid-lactation of R. monkeys, Xiaochangdu infants were born one month bieti overlapped with the peak in staple food availability, earlier and weaned 2.5 months later. Their lactation and weaning overlapped with the abundance of high period was prolonged with relatively lower temperatures, quality foods (new suitable vegetation). Therefore, food and higher energy consumption phase (mid-lactation) fell availability was the ultimate factor determining the during the second peak of food resources. We concluded reproductive seasonality of R. bieti, while other enviro- that the high energy requirement of lactation may have nmental factors, such as precipitation, temperature and induced the high infant mortality, which was 54% at photoperiod, functioned as proximate factors for fine- Wuyapuya (N28°35’) (Kirkpatrick et al, 1998), whereas turning coordination between seasonal breeding and food that of the Mt. Lasha group was much more lower (6.7% availability (Wikleski et al, 2000). However, whether in 2012) (Li, unpublished). To avoid energy overdraft of conception or weaning has priority in this regulation of adult females during lactation (especially during winter) reproductive seasonality still needs long-term, systematic and increase infant survival, high frequency of male- observations of monkeys in different habitats. infant-caretaking was found in Xiaochangdu group (Xiang et al, 2010), whereas such behaviors were only Birth synchrony observed at low frequency in the Mt. Lasha group (Wang Restrictions in environmental conditions induce et al, 2012). The short breeding season with early start in seasonal synchrony (Power, 2013). When reproduction the Xiaochangdu group appears to be an adaptive stra- of Northern Plains Gray Langur (Semnopithecus entellus) tegy to high environmental seasonality. Research on M. is not tied with seasonality, synchrony is lost. Obvious fuscata groups at higher altitude showed that infants born reproductive signals (such as menstruation) in females early but with a prolonged developmental period were enable males to control mating, and thereafter decrease able to survive the low temperature and lack of food food competition and increase their fitness. However, the experienced during through winter (Food & Aimi, 2003). more restricted reproduction is by seasonality, the more The reproduction of many high altitude species is significant the reproductive synchrony. Females may affected by photoperiod, with the length of daytime and conceal reproductive signals to confuse paternity and or its variation triggering related neuroendocrine mechan- decrease the chance of adult males initiating infanticide isms (Van Horn, 1980; Bronson & Heideman, 1994; (Power, 2013). In addition, strong reproductive Goldman, 2001). The habitat of Xiaochangdu monkeys seasonality results in greater synchronicity in female was located at a higher altitude than that of Mt. Lasha ovulation and birth (Power, 2013). R. bieti births at

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 482 LI, et al.

Xiaochangdu were more synchronized than those at Mt. OMUs to decrease food competition; however, more Lasha (SD was 6.5 and 16.9 days, respectively). This evidence from long-term food detection and systematic indicated that higher habitat altitudes resulted in stronger research is needed. birth synchronicity, and may also be correlated with Decrease in reproductive rate highly synchronized ovulations in female monkeys. Adult female R. bieti at Mt. Lasha gradually Reproductive synchrony of females, including increased; however, no such trend was found in infants. estrus, birth and length of breeding season, prevents From 2009−2011, all females successfully reproduced. In males from monopolizing mating (Carnegie et al, 2011). 2012, there were 33 adult females (including four The more females share reproductive synchrony, the females that reached maturation in 2012) in the group, more difficult it is for males to control the reproduction but there were only 15 infants. In 2013, the numbers of process (Power, 2013). Breeding patterns, such as adult females and infants were 34 and 13, respectively. polygamy and multi-male-female groups, are determined These results indicate that either some adult females did by reproductive seasonality because even the strongest not reproduce (e.g., in 2012, five females did not male is unable to totally monopolize multiple females in reproduce and in 2013, six did not), or some infants died. synchronized pregnable states (Srivastava & Dunbar, Further research is required to determine if this low 1996). As a result, reproductive synchrony is prone to reproductive rate is caused by individual aging, female induce monkey groups with multiple males and females, sexual competition, habitat saturation, or by infant deaths. whereas polygenous groups are more common with reproductive non-synchrony, which is beneficial in Breeding season determination ensuring paternity (Power, 2013). The Xiaochangdu Black-and-white snub-nosed monkeys inhabit monkeys showed a double peak in births, while the original coniferous forests at high altitude in temperate monkey at Mt. Lasha displayed three peaks. These regions. Due to the steep terrain and harsh climates of phenomena resulted from the strong reproductive their habitats and the nature of monkeys to avoid humans, synchrony (brief breeding season) of the high altitude it is difficult to perform close observation or achieve monkey groups, the rather large group size (Xiaochangdu individual recognition. Under such circumstances, only group was 210 in 2003 and the Mt. Lasha group was 130 partial I/F data was acquired with one observation, and in 2012), and the birth synchrony among different OMUs. only I/F can be used to estimate breeding season (Xiang & Sayers, 2009). However, if all adult females and Annual differences in birth patterns infants could be recognized in multiple observations, The birth peak of the Mt. Lasha group was “V” then the I-value alone is sufficient to infer accurate shaped in even-numbered years, but exhibited an breeding seasonality and birth pattern (this study, Huang increasing trend in odd-numbered years. In odd- et al, 2012). numbered years, the Md shifted ten days earlier than that in even-numbered years. Moreover, the commencement Acknowledgements: We thank the directors and staff from the Administrative Bureau of Yunling National and termination of the breeding season occurred earlier. Reserve, Lanping County, Nujiang Prefecture, Yunan These phenomena may be correlated with annual Province, the residents of Dashanqing village and differences in food resources or strategies adopted by assistants Qing-Sheng Su and Jin-Fu Zhang.

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Research, 33(3): 241-248. (in Chinese) Yang MY, Sun DY, Zinner D, Roos C. 2009. Reproductive parameter in Guizhou snub-nosed monkeys (Rhinopithecus brelichi). American Wikleski M, Hau M, Wingfield JC. 2000. Seasonality of reproduction Journal of Primatology, 71(3): 266-270. in a neotropical rain forest bird. Ecology, 81(9), 2458-2472. Zar JH. 1999. Biostatistical Analysis. 4th ed. New Jersey: Prentice Hall. Xiang ZF, Sayers K. 2009. Seasonality of mating and birth in wild black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Zhang SY, Liang B, Wang LX. 2000. Seasonality of matings and births Xiaochangdu, Tibet. Primates, 50(1): 50-55. in captive Sichuan golden monkeys (Rhinopithecus roxellana). American Journal of Primatology, 51(4): 265-269. Xiang ZF, Huo S, Xiao W. 2010. Male allocare in Rhinopithecus bieti at Xiaochangdu, Tibet: Is it related to energetic stress? Zoological Zhao QK, Deng ZY. 1988. Macaca thibetana, at Mt. Emei, China. II. Research, 31(2): 189-197. Birth seasonality. American Journal of Primatology, 16(3): 261-268.

Zoological Research again recognized for Excellence and Impact in Science and Technology Publishing in 2014

At the September 2014 conference hosted by the Institute of Scientific and Technical Information of China (ISTIC) in Beijing, Zoological Research (ZR) was ranked among the top 300 journals classified as “Outstanding S&T Journals of China” for 2014 due to its high academic value and the leading role it plays in advancing the quality of the S&T journals of China. Few journals in China ever receive this ranking, but this is the second time ZR has now been recognized by this award twice since 2008.

ISTIC also launched a new project to promote scientific communications and the growing internationalization of Chinese S&T journals of China called the “Project of Frontrunner 5000” (F5000). This project plans to highlight the 5000 top articles picked from among the 300 Outstanding S&T Journals of China—four articles published in ZR between 2009-2013 were included in the top 5000.

The Chinese Science Citation Database (CSCD) for 2014 was also released by the National Science Library of the Chinese Academy of Science, ranking ZR among the “Top 300 S&T Journals of China with the Highest Impact Factors.”

All in all, it has been a good year for ZR.

Zoological Research www.zoores.ac.cn Zoological Research 35 (6): 485−491 DOI:10.13918/j.issn.2095-8137.2014.6.485

Experimental infection of tree shrews (Tupaia belangeri) with Coxs- ackie virus A16

Jian-Ping LI, Yun LIAO, Ying ZHANG, Jing-Jing WANG, Li-Chun WANG, Kai FENG, Qi-Han LI*, Long-Ding LIU*

Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical Colleg, Kunming 650118, China

Abstract: Coxsackie virus A16 (CA16) is commonly recognized as one of the main human pathogens of hand-foot-mouth disease (HFMD). The clinical manifestations of HFMD include vesicles of hand, foot and mouth in young children and severe inflammatory CNS lesions. In this study, experimentally CA16 infected tree shrews (Tupaia belangeri) were used to investigate CA16 pathogenesis. The results showed that both the body temperature and the percentages of blood neutrophilic granulocytes / monocytes of CA16 infected tree shrews increased at 4−7 days post infection. Dynamic distributions of CA16 in different tissues and stools were found at different infection stages. Moreover, the pathological changes in CNS and other organs were also observed. These findings indicate that tree shrews can be used as a viable animal model to study CA16 infection.

Keywords: Coxsackie virus A16; Infection; Tree shrew

Hand-foot-mouth disease (HFMD) is a pathogenesis animal resources has significantly limited its application. that mainly affects infantile populations. Although most Due to a variety of unique characteristics, e.g., small infection cases show only mild symptoms (Mao et al, adult body size, short reproductive1cycle and life span 2013), there are a small number of individuals suffering (Wang et al, 2013), and especially a close affinity to from neurological symptoms of encephalitis and severe primates, tree shrews (Tupaia belangeri) have been widely heart and lung failure (Tan et al, 2014). From the view of applied in biomedical research, especially in virology the pathogen, HFMD is mainly caused by enterovirus research (Fan et al, 2013). In this study, 2-month-old tree type 71 (EV71) and Coxsackie virus group A type16 shrews were used to investigate GX18 infection and their (CA16) infection (Yang et al, 2014). Currently, EV71 potential as an animal model for CA16 infection was vaccine has completed phase III clinical trial (Chen et al, assessed. 2014; Li et al, 2014; Zhu et al, 2014), whereas a CA16 vaccine is still under development. Due to the lack of MATERIALS AND METHODS systematic analysis on the infection and immunization of Virus and cells CA16 virus, an effective animal model for CA16 The CA16 virus used in this study was an isolated infection is urgently necessary. The conventional animal GX18 strain from an infected child with mild clinical models of CA16 infection, such as mice or rats (Liu et al, symptoms in Guilin, China, 2012. The GX18 strain was 2014), can neither fully reflect the etiological or pathological characteristics of infection and immuni- Received: 11 April 2014; Accepted: 20 May 2014 zation, nor meet the comprehensive assessment require- Foundation items: This work was supported by the National High- ments of vaccine safety and efficacy. Non-human prim- Tech R&D Program (2014ZX09102042), the National Natural ate animal models are of special importance in vaccine Science Foundation of China (81373142) and the Natural Science evaluation, e.g., Zhang et al (2014) used infant rhesus Foundation of Yunnan Province (2012ZA009) monkeys (Macaca mulatta) to explore the efficacy and *Corresponding authors, E-mails: [email protected]; longdingl safety of EV71 vaccine. However, high cost and limited @gmail.com

Science Press Volume 35 Issue 6

486 LI, et al. identified as a CA16 B2 sub-genotype virus by RT-PCR. monkeys showed that virus distributions in blood and The virus grown in Vero cells (cultured by Institute of other organs can be observed via intravenous and Medical Biology) was harvested for freezing at –20 °C intratracheal inoculation routes (Zhang et al, 2011). Ong with a concentration of 106.5 cell culture infectious doses et al (2008) found that infections via intravenous route in

(CCID50)/mL. The Vero cells were maintained in 5% cynomolgus monkeys (Macaca fascicularis) can not yield a MEM containing fetal bovine serum (provided by similar distribution of the virus. Institute of Medical Biology) and grown to a confluent In this study, 12 tree shrews were infected with monolayer in a T-25 vessel or in 96-well plates for viral CA16 (104.5 CCID50) via the respiratory tract by nasal isolation and antibody neutralization assays. spraying. The other 3 tree shrews were mock sprayed by saline as uninfected negative controls. The animals were Virus titration and neutralization assay monitored daily, and their body temperatures were CA16 harvested from cell culture or isolated from measured rectally by a digital stick thermometer (MC- different organs and tissues of infected tree shrews were BOMR, Omron Co.) from day 2 post infection (p.i.). In analyzed by a microtitration assay using a standard parallel, venous blood samples were taken into EDTA protocol (Arita et al, 2006). The neutralization test was coated capillary tubes daily for routine detections of performed according to the standard protocol (WHO, biological indicatorsand viral loads (Veterinary Multi- 1988). A mixture of diluted serum containing anti-CA16 species Hematology System, Hemavet 950FS, Drew antibodies (provided by Institute of Medical Biology) Scientific Co.). Pathogenic and histopathological examinat- and the virus at a titer of 500–1 000 CCID50 in 100 µL of ions were performed on all the tissues and organs of tree PBS (provided by Institute of Medical Biology) was shrews sacrificed by anesthesia at day 4, 7, 10 and 14 p.i.. incubated at 37 °C for 1 h. The cellular pathogenic effect (CPE) of the virus was examined by inoculating the Viral RNA extraction and quantitative RT-PCR mixture onto Vero cells grown in 96-well plates. The Viral RNA was extracted from whole blood (100 µL), plaque assay was performed in 6-well plates containing fresh tissue homogenate (10%, 100 mg) or fecal homo- + 90% confluent Vero monolayer cells. The cells were genate (5%, 100 mg) by TRNzol-A , according to inoculated with samples from blood, throat swabs or feces. manufacturer’s protocols (Tiangen, China). The viral After 1 h incubation at 37 °C, the infection media were RNA was eluted in a final volume of 20 µL. For aspirated off and each well was then covered with 3 mL of quantification, a single-tube, real-time Taqman RT-PCR agar overlay medium and incubated for 2 days at 37 °C in assay was performed using the Taqman 1-step RT-PCR Master Mix in the 7500 Fast Real-time RT-PCR system a CO2 incubator. At the end of the incubation, the cells were fixed with formaldehyde and stained with 0.1% (Applied Biosystems, Foster City, CA, U.S.). The Crystal Violet as a standard protocol (Hung et al, 2010). reaction mixture (20 µL) contained the Taqman Univ- ersal PCR master mix, primers (each at 20 µmol/L), the Tree shrews FAM/TAMRA labeled probe (10 µmol/L) (Takara Co., Fifteen healthy female tree shrews (80±10 g, 2- Ltd. Dalian, China), and RNA (2 µL). The sequences of month-old) were divided into a infection group (n=12) the CA16-specific primers and probe were as following: and a mock-infection control group (n=3). All animal forward primer (5'-ACACTCCATTACCCTGAGGGT procedures were approved by the Office of Laboratory GTA-3'); probe (5'-ATGAGAATCAAACACGTCAG Animal Management of Yunnan Province, China. GGCATGGAT-3'); and reverse primer (5'-ACACTCC Animals were individually caged and fed according to ATTACCCTGAGGGTGTA-3'). The following protocol the guidelines of the Committee of Experimental Anim- was used for all PCR assays: 5 min at 42 °C and 10 s at als at the Institute of Medical Biology, Chinese Academy 95 °C, followed by 40 cycles at 95 °C for 5 s and 60 °C of Medical Sciences (CAMS). Individuals were conf- for 30 s. The standard reference curve was obtained by irmed free of antibodies against CA16 prior to the the measurement of the serially diluted virus RNA experiment via neutralization test. generated by in vitro transcription from a DNA construct that contains the Vp1 region. Viral copies were CA16 infection quantified according to vitro-synthesized RNA by Our previous study on EV71 infection in rhesus spectrophotometry quantification, and the quantity was

Zoological Research www.zoores.ac.cn Experimental infection of tree shrews (Tupaia belangeri) with Coxsackie virus A16 487 expressed as a relative copy number, determined by the mouse IgG antibodies (Sigma, Deisenhofen, Germany) equation: [(μg of RNA/μL)/(molecular weight)] ×Avog- followed by color development with diaminobenzidine adro's number= viral copy number/μL. for the detection of the antigen-antibody reaction.

Histopathological examination and immunohisto- Statistical Analysis chemical analysis All the data were expressed as the mean of three The tissue samples from different organs were fixed samples from all experiments (mean±SE). in 10% formalin in PBS, dehydrated in ethanol gradients and embedded in paraffin before obtaining 4 µm sections for further H-E staining. Histopathological analysis of RESULTS the tissue sections from each organ was performed under Clinical observations of CA16-infected tree shrews a light microscope. For the immunohistochemical analy- No typical papules or vesicles on limbs or mouths sis, tissue samples were embedded in an optimal cutting were found in infected tree shrews, but increased body temperature (OCT) compound (Miles Inc., Elkhart, Ind.) temperatures p.i. (Figure 1A), as well as increased and frozen in liquid nitrogen. The frozen tissues were percentages of neutrophilic granulocytes and monocytes then cut into 4 µm sections, placed on poly-l-lysine- in some animals (Figure 1B, C) from day 5 to 7 p.i. with coated glass slides and fixed in 3.7% paraformaldehyde. the decrease of lymphocyte (Figure 1D) were observed. The endogenous peroxidase activity of the tissues was These findings suggest that similar clinical CA16 inhibited by treating with hydrogen peroxide (2.5%). The infection symptoms as humans (Lo et al, 2011), such as CA16 antigen was detected by mouse anti-CA16 sera fever and inflammatory responses (reflected in blood cell and horseradish peroxidase (HRP)-conjugated anti- analysis), can be found in tree shrews.

Figure 1 Clinical symptoms of CA16 infected tree shrews via respiratory route A: Body temperatures of CA16-infected tree shrews; B: The percentage of neutrophilic granulocytes in leukocytes of CA16-infected tree shrews; C: The percentage of monocytes in leukocytes of CA16-infected tree shrews; D: The percentage of lymphocytes in leukocytes of CA16-infected tree shrews.

Dynamic profiles of virus in blood and stool of CA16- of viral load were observed in the blood (500 copies/100 infected tree shrews µL) at day 6 p.i. and in stool (2 000 copies/100 mg) at We analyzed viral load dynamic profiles in blood day 7 p.i., respectively. These findings indicate that the and stool at different stages of infection. The peak levels viremia of the infected tree shrews develops from day 3

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 488 LI, et al. to day 7 p.i., whereas, the virus shedding in feces is the spleen (356 copies/100 mg), cerebellum (290 detectable from day 3 to day 14 p.i. (Figure 2B). These copies/100 mg), lung (294 copies/ 100 mg), lymph virus dynamic profiles imply that the clinical symptoms node (200 copies/100 mg), parotid gland (155 copies/100 of CA16-infected tree shrews are correlated with the mg) and thigh muscles (290 copies/ 100 mg), whereas no viremia manifestations. detectable viral loads were observed in the same tissues

of non-infected control tree shrews. In general, viral Viral distributions in tissues from CA16-infected tree shrews replication peaked on day 6 or 7 p.i. in the lymph nodes The target organs of CA16 infection in humans and glands (Figure 3B, C), whereas, peaked on day 10 p.i. involve several different tissues (Wang et al, 2004). To in the CNS and spleen, lung and muscles (Figure 3A, D), understand the CA16 distributions in tree shrews, the which might be the major target organs of infection in viral loads in various tissues were determined at different tree shrews. stages of infection. CA16 virus was detectable in the It was reported that in certain cases, CA16 is able to lymph node, lung, spleen, kidney, thigh muscle and brain target CNS and lead to viral encephalitis (Chang et al, from day 7 p.i.. Relatively high viral loads were found in 1999; Goto et al, 2009). In this present study, the total

Figure 2 Dynamic distributions of CA16 in the blood and stool of infected tree shrews via respiratory route A: Viral RNA extracted from blood specimens; B: Viral load in feces.

Figure 3 Viral distributions in tissues of CA16-infected tree shrews A: Viral load in the major organs of infected tree shrews; B: Viral load in glands of infected tree shrews; C: Viral load in the lymph nodes of infected tree shrews; D: Viral load in CNS of infected rhesus monkeys.

Zoological Research www.zoores.ac.cn Experimental infection of tree shrews (Tupaia belangeri) with Coxsackie virus A16 489

distributions of viral loads increased from day 4 to day 10 p.i. (Figure 3B), and higher loads were found specifically in the cerebellum (291 copies/100 mg) and lobus frontalis (294 copies/100 mg) at day 10 p.i., which were consistent with the observation that viral loads peaked in muscles and lungs on day 10 p.i..

Viral antigen detection in tissues from CA16-infected tree shrews To investigate CA16 replications, antigen expres- sions of CA16 in the related tissues were examined immunohistochemically. High expressions of viral antigen in the heart (Figure 4A), spleen (Figure 4B) and lung (Figure 4C) were observed. Viral antigens were also observed in the CNS including cerebellum (Figure 4d). These findings are consistent with the patterns of viral load distributions of widespread infections.

Figure 4 Viral antigen expressions in tissues from CA16 infected tree shrews A: Sample of the heart collected on day 10 p.i.; B: Sample of the spleen collected on day 10 p.i.; C: Samples of the lung collected on day 10 p.i.; D: Samples of the cerebellum collected on day 10 p.i.; Arrows indicate the stained CA16 antigen; Images are shown at 200× magnification.

Pathological changes in tissues from CA16-infected tree shrews In this study, the pathogenic changes of CA16 infected tree shrews were evaluated by systematic pathologic analysis on various tissues, including the liver, kidney, spleen, heart, lung, muscles, spinal cord and Figure 5 Pathological analyses of CA16-infected tree shrews brain. Pathological responses, such as cell damage and The typical features of pathological changes as infiltration of inflammatory inflammatory cell infiltration (Figure 5A−D) were found cells (black arrow) in the lung (A), liver (B), kidney (C) and muscles (D) of in the lung, kidney and muscles, correlating with the CA16 infected tree shrews. Images show the proliferations of neuroglial and presence of high viral loads. The observed neuropath- neuronal lesions in the thalamus (e) andspinal cord (f) of CA16 infected tree ological lesions in the cerebellum indicate that the shrews. Images are shown at 200× magnification.

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 490 LI, et al. pathogenic changes in CNS may contribute to the degen- and mouth were observed, certain clinical features, such eration of neural cells, infiltration and inflammatory cell as fever and lymphocyte-related inflammatory responses aggregation (Figure 5E, F). Moreover, as expected, all could mimic some manifestations in human patients with the observed pathological changes were correlated with HFMD (Mou et al, 2014; Wang et al, 2013). the viral antigen expressions. These results demonstrate The distributions of CA16 virus in tree shrews were that CA16 has a tropism to the CNS and lung and determined by viral loads and pathological changes. The thereafter may induce severe lesions of these tissues. high viral loads found in the lymph nodes, heart, lung, spleen, kidney, thigh muscles and brain imply that the DISCUSSION respiratory route is one of potential natural infection The establishment of an viable animal model plays routes of viral transmissions. The viral load in blood and critical roles in understanding infectious diseases, such the antigen expression in the lymph nodes, lung, muscles as HFMD, and vaccine evaluations. Animal models of and CNS indicate that CA16 can spread throughout the EV71 infection include suckling mice and neonatal whole body of infected animals. Moreover, in this study, rhesus monkeys (Wang et al, 2004; Zhang et al, 2011). high viral loads were detected in cerebellum from day 4 The conventional animal model in the studies of CA16 to day 10 p.i., and neuropathological lesions were obse- infection is still neonatal mice. The mouse model rved via histopathological examinations. These findings provides evidence of the protective immunity of CA16 indicate that CA16 infected tree shrews can eventually vaccine (Dong et al, 2011) but fails to provide convi- display a broad range of viral transmission, including ncing data on the infection pathology and the compreh- CNS. In addition, the viral shedding found in the feces of ensive evaluation of vaccine. According to previous reports, the infected tree shrews indicate that the typical sprea- tree shrews can be widely used as a potential animal model ding route of enterovirus can also be demonstrated in this for many infectious diseases, such as hepatitis C, measles, animal model. atypical pneumonia (SARS), tuberculosis, etc (Han et al, Although this study only demonstrates limited clin- 2011; Xu et al, 2013; Yang et al, 2013). ical manifestations of HFMD, the clinical symptoms and In the present study, experimentally CA16-infected pathological changes found in CA16 infected tree shrews tree shrews have been used to investigate the etiology still reveal a complete process of CA16 infection and str- and pathology of HFMD. In the CA16-infected tree ongly support the application of tree shrews as an animal shrews, although no typical vesicles or papules on limbs model in CA16 infection research and vaccine evaluation.

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Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 Zoological Research 35 (6): 492−499 DOI:10.13918/j.issn.2095-8137.2014.6.492

Isolation and identification of symbiotic bacteria from the skin, mouth, and rectum of wild and captive tree shrews

Gui LI1, Ren LAI1,2,*, Gang DUAN3, Long-Bao LYU1, Zhi-Ye ZHANG1, Huang LIU1, Xun XIANG3

1. Kunming Primate Research Center of the Chinese Academy of Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China 2. Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China 3.Yunnan Agricultural University, Kunming Yunnan 650201, China

Abstract: Endosymbionts influence many aspects of their hosts’ health conditions, including physiology, development, immunity, metabolism, etc. Tree shrews (Tupaia belangeri chinensis) have attracted increasing attention in modeling human diseases and therapeutic responses due to their close relationship with primates. To clarify the situation of symbiotic bacteria from their body surface, oral cavity, and anus, 12 wild and 12 the third generation of captive tree shrews were examined. Based on morphological and cultural characteristics, physiological and biochemical tests, as well as the 16S rDNA full sequence analysis, 12 bacteria strains were isolated and identified from the wild tree shrews: body surface: Bacillus subtilis (detection rate 42%), Pseudomonas aeruginosa (25%), Staphlococcus aureus (33%), S. Epidermidis (75%), Micrococcus luteus (25%), Kurthia gibsonii (17%); oral cavity: Neisseria mucosa (58%), Streptococcus pneumonia (17%); anus: Enterococcus faecalis (17%), Lactococus lactis (33%), Escherichia coli (92%), Salmonella typhosa (17%); whereas, four were indentified from the third generation captive tree shrews: body surface: S. epidermidis (75%); oral cavity: N.mucosa (67%); anus: L. lactis (33%), E. coli (100%). These results indicate that S. epidermidis, N. mucosa, L. lactis and E. coli were major bacteria in tree shrews, whereas, S. aureus, M. luteus, K. gibsonii, E. faecalis and S. typhosa were species-specific flora. This study facilitates the future use of tree shrews as a standard experimental animal and improves our understanding of the relationship between endosymbionts and their hosts.

Keywords: Tree shrew; Microbial; Separation; Identification

Symbiotic bacteria are bacteria living in symbiosis with bacterial diseaseas, etc. Although scientists have carried their hosts. They influence almost every aspect of the out studies on the symbiotic bacteria of healthy wild tress physiological prosess of the host, including the growth shrews (Gao et al, 2009; Wang et al, 1987; Wang et al, and development, physiology and biochemistry, gene 2011; Xing et al, 2012; Zhang et al, 2009), there is still expression, metabolism, immunology, etc. Recent years, no report on symbiotic bacteria from the body surface, tree shrews (Tupaia belangeri chinensis) have been oral cavity and anus of captive tree shrews, especially the rapidly and widely applied in biomedical research as a third generation captive tree shrews. The most commonly novel experimental animal model, especially in studies used method in studying symbiotic bacteria is the meta- on human diseases (Wang et al, 2010; Huang et al, 2013; genomic methods basing on the second generation sequ- Xu et al, 2013). Domestic tree shrews of China include encing tools, which means, the extracted DNA of flora northern tree shrews (Tupaia belangeri) and six will connect with T-vector before Sanger sequencing. 1 suspecies (Simpson, 1945). Currently, laboratory tree shrews are mainly from the field, and therefore their Received: 23 March 2014; Accepted: 25 June 2014 unclarified genetic background, physical condition and Foundation items: This study was supported by the National 863 situations of symbiotic bacteria have brought many Project of China ( 2012AA021801) and the Project of Frontier Study of difficulties into their application in research into the Foundation, CAS (KSCX2-EW-R-11, KSCX2-EW-J-23) development of novel antimicrobial medications, the *Corresponding author, E-mail: [email protected]

Science Press Volume 35 Issue 6

Isolation and identification of symbiotic bacteria from the skin, oral, and rectal of wild and captive tree shrews 493

In this study, the isolated symbiotic bacteria from the classified based on their morphological, cultural, body surface, oral cavity and anus of wild and third physiological and biochemical characteristics, combining generation captive tree shrews were identified based on the results of 16S rDNA whole sequence analysis. The their morphological, cultural, physiological and bioche- flowchart of experimental procedures (approve number: mical characteristics, as well as the results of 16S rDNA SYBW20110416-1) is shown in Figure 1. whole sequence analysis. These findings not only pro- vide basic data in setting microorganism standard of laboratory tree shrews but also are metagenomic refe- rence of studies on the symbiotic bacteria in tree shews.

MATERIALS AND METHODS Experimental animals Adult (12-month-old, body weight=130−150 g), healthy wild (n=12, six males and six females) and the third generation captive tree shrews (n=12, six males Figure 1 Flowchart of experimental procedures and six females) were provided by the Laboratory Bacteria sampling and pure culture Animal Center of Kunming Institute of Zoology, CAS. Next to the alcohol burner inside of the sterile Wild tree shrews were captured from the western suburb operation platform, symbiotic bacteria from the body of Kunming, Yunnan, China, and their symbiotic bacteria surface (sampling a bit of fur by a sterile biceps), (using were sampled on the same day of capture. The rearing sterile cotton swab) and rectum (using sterile cotton environment of captive tree shrews was well ventilated, at swab) of 24 tree shrews were sampled. Body surface and 16−25 ℃ in temperature, 40%−80% in humidity, 12 h/ oral specimens were inoculated onto the Columbia blood 12 h (0800h−2000h lights on) in light/dark cycle. Tree agar bases and LB agar bases, whereas, rectal specimens shrews were fed a grain premixture (main ingredients were inoculated onto the SS agar bases and Mac Conkey include corn meal, wheat flour, fish meal, milk powder, agar bases. Bacterial species can be initially and roughly bone meal, sugar, salt, yeast, dregs of beans and identified according to their growth situations on differ- vitamins), fruits (apples and bananas), terzebrio molitors ent agar bases. Innoculated agar bases were cultured in and cooked eggs. The laborotary animal production and 37 ℃ incubator. The morphology, color, hemolysis and utilization numbers are SCXK (Yunnan) K2012-0001 pigments of the colonies were observed 18−72 h later. and SYXK (Yunnan) K2012-0003, respectively. Single colony (numbered from hs1 to hs12) was picked by a sterile toothpick and placed on a fresh surface for Experimental reagents and facilities futher indentification. Columbia blood agar bases, Salmonella Shigella agar bases and Mac Conkey agar bases were all from OXIOD Morphological identification (UK); Biochemical identification tubes were from Biome- Gram-staining used in this study was ammonium rieux (France); Luria Bertani (LB) agar bases were homemade. oxalate crystal violet staining (Katznelson et al, 1964). The instruments and facitities used in this study incl- Specimens were fixed in 2.5% glutaraldehyde, stained ude PCR instrument (Biorad Mycycler), CO2 incubator (The- with 0.1mol/L tungstophosphoric acid and then observed rmo scientific Series 8000DH), thermostatical rocking plate under a transmission electron microscope. (COS-211C), sterile operation platform (BAKER A2), auto- matic autoclave (THA-3560C), transmission electron micr- Biochemical characteristics identification oscope (HITACHI, H-7650), spectrophotometer (mode 721). Main biochemical indexes of isolated bacteria strain were determined via the biochemical identification tubes. Experimental methods Strains cultured overnight at 30 ℃ were inoculated into Symbiotic bacteria from the body surface, oral the biochemical identification tube via the sterile cavity and rectum of wild and third generation captive inoculating loop.Every strain inoculation were triplicated. tree shrews were sampled, cultured and isolated. The Negative control tubes were void of bacteria. Results isolated bacteria stains were identified and systematically were read within 12−72 h. Positive results went through

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 494 LI, et al. a three-generation continuous inoculation. Indentification www.ncbi. nlm.nih.gov/Blast/). conclusions were obtained by comparing the data against the characterics of other close bacteria strains. Bacteria identification The isolated bacteria stains were identified and syst- Physiological characteristics identification ematically classified based on their morphological, cultu- The physiological characters of bacteria strains ral, physiological and biochemical characteristics, combi- mainly include growth temperature, pH tolerance range ning the results of 16S rDNA whole sequence analysis. and optimal pH, as well as salt endurance. Fresh bacteria fluid (5%) was inoculated onto the RESULTS improved LB liquid agar bases, shaking cultured by Identification results of the isolated symbiotic strains thermostatical rocking plates (180 r/min) at 5, 10, 25, 30, 35, The results of staining microscopy, physiological 37, 40, 42, 45 and 50 ℃, respectively. OD light 600 characteristics and 16S rDNA whole sequence analysis obsorption values were obtained at 24, 48, 72 h, respectively, of the isolated symbiotic bacteria were shown in Table 1. to determine the growth range at different temperatures. High homologies were found by comparing the 16S To obtain the optimal pH value, fresh bacteria fluid rDNA sequences of these isolated strains with the gene (5%) were inoculated onto the improved LB liquid agar sequences of the online registed stains (http: //www.ncbi. bases with pH at 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and nlm.nih.gov). When the results of phylogenetic tree show 10.0, respectively, and shaking cultured at 30 ℃, 180 that an isolated unknown strain has more than 99% p/min. OD light absorption values were obtained at 24, 600 homology with a known strain, then according to its 48, 72 h, respectively, to determine the pH tolerance range. morphology, physiological and biochemical characte- Fresh bacteria fluid were inoculated onto the improved ristics, this isolated unknown strain can be identified. LB liquid agar bases with different NaCl concentrations (0%−10%) and cultured at 30 ℃ for 48 h, then their OD600 Physiological and biochemical characteristics of the light absorption values were obtained to determine their salt isolated bacterial strains endurance range and the optimal salt concentration. The physiological and biochemical characteristics of the isolated bacterial strains, from hs1 to hs12, were 16S rDNA sequence analysis shown in Table 2 to Table 13, respectively. The unindentified bacteria strains were under pure cultures and small scale amplifications. Universal primers Detected bacteria from the wild (Table 14) and third generation captive tree shrews (Table 15) were designed based on the high conservativeness of 16S In this study, 12 bacteria species were identified from rDNA sequence in prokaryotes and produced by Sangon the wild tree shrews, body surface: B. subtilis (42%), P. Biotech (Shanghai, China) (forward: 5'-AGAGTTTGA aeruginosa (25%), S. aureus (33%), S. epidermidis TCCTGGCTCAG-3'; reverse: 5'-AAG GAGGTGATCC (75%), M. luteus (25%) and K. gibsonii (17%); oral AAGCCGCA-3'). Bateria DNA were templates and the cavity: N. mucosa (58%), S. pneumoniae, (17%); anus: E. PCR products were larger than 1 400 bp. The reaction faecalis (17%), L. lactis (33%), E. coli (92%) and S. solution (50 µL) included 2.5 ng templated DNA, 10 µL typhosa (17%). Four bacteria specises were identified forward primer, 10 µL reverse primer, 0.25 µL from the third generation captive tree shrews, body TaKaRa (5 U/µL), 5.0 µL 10× PCR Buffer (Mg2+ Free), surface: S. epidermidis (75%); oral cavity: N. mucosa 4.0 µL MgCl dNTP mixture and 35.75 µL sterile 2 (67%); rectum: L. lactis (33%) and E. coli (100%). S. distilled water. PCR reactions were pre-denaturation at epidermidis, N. mucosa, L. lactis and E. coli were the 94 ℃ for 4 min, denaturation at 94 ℃ for l min, major symbiotic bacteria strains of tree shews, whereas, S. renaturation at 52 ℃ for l min, extension at 72 ℃ for 2 aureus, M. luteus, K. gibsonii, E. faecalis and S. typhosa min, incubate at 72 ℃ for 10 min after 30 cycles and are species-specific bacteria strains of tree shrews. then kept at 8 ℃ . PCR products were detected by 1% agarose gel electrophoresis and then sequenced by DISCUSSION Genscript (Nanjing, China). 16S rRNA sequences were obtained via Chromas Symbiotic bacteria affect many aspects of the host’s and DNASTAR.Lasergene.v7.1, and then went through physiological conditions, including the growth and the phylogenetic analysis via Blast (NCBI) (http: development, gene expression, metabolism, biochemical

Zoological Research www.zoores.ac.cn Isolation and identification of symbiotic bacteria from the skin, oral, and rectal of wild and captive tree shrews 495

Table 1 Results of staining microscopy, physiological characteristics and electrophoregram of the isolated symbiotic bacteria pH Microscopy Temperature (℃) NaCl agar base (%) tolerence range Bacteria Electrophoregram Gram’s Brood Growth Growth Morphology Flagellum Capsule Optimal Optimal stain body range range

Rhabditiform; paired B. subtilis + or in chains; round Y 5−50 25−30 6.5−9.5 0.6−1.2 1.0 or squre ends

Brevibacterium; median sized; blunt E. coli − N 5−40 20−30 6.5−9.5 0.1−1.0 0.6 ends; scattered or paired

E. faecalis + Globular or oval N Y N 10−45 37 4.2−4.6 0.1−6.5 4.0

Thallus slender and in various sizes; P. − shape in rod or N Y 25−42 25−30 4.2−4.6 0.1−6.5 4.0 aeruginosa linear; paired or in short chains

K. gibsonii + Blunt ends; scattered N Y 25−42 25−30 4.2−4.6 0−6.0 4.0

Small thallua, N.mucosa − reniformed; N Y 25−38 30 7.0−7.5 0−6.0 4.0 in dual rank

S.epidermidis + N N N 20−45 37 6.0−9.0 1.0−10.0 5.0

S. Globular or slightly + N N N 25−40 37 4.5−9.8 1.0−8.5 3.0 epidermidis oval; aciniformed

L. lactis + Globular or oval N N 5−40 30 6.5−9.8 0.1−7.5 2.0

Cellular spherical; M. luteus + paired, in quadruplet N 5−40 25−37 5.5−9.0 0.1−7.5 2.5 or clustered

S. Globular or oval; + N N N 5−40 37 6.5−8.5 0.1−8.5 4.0 pneumoniae paired or in chains

Brevibacterium; S. typhosa − N N 5−40 35 6.5−9.5 0.1−1.0 0.6 blunt ends; scattered

+: Positive in Gram’s stain; −: Negative in Gram’s stain; Y: Presence; N: Absence.

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 496 LI, et al.

Table 2 Physiological and biochemical characteristics of Table 5 Physiological and biochemical characteristics of hs1 hs4 Characteristics Results Characteristics Results Characteristics Results Characteristics Results Catalase + Nitrate-reducing + Maltose − Glutin + Carbamide − Amylohydrolysis + Mannite − Mushroom sugar − Gelatin liquefaction + Citrate + Sucrose − Xylose − Sucrose + D-Xylose + Galactose − Acetamide +

D-mannitol + D-Mannose +⁄− Carbamide + H2S − Sorbierite − Maltose − Arabinose − Nitrate-reducing + D-raffinose − L-pectinose + Fructose − Oxidase + +: Positive or capable of utilizing; −: negative or incapable of utilizing; +/−: Indole − Arginine hydrolysis + Subtle differences between strains of one genus. Glucose; acid-producing + Citrate + Glucose; aerogenesis − Table 3 Physiological and biochemical characteristics of +: Positive or capable of utilizing; − : Negative or incapable of utilizing. hs2 Table 6 Physiological and biochemical characteristics of Characteristics Results Characteristics Results hs5 Mannite + V-P test − Characteristics Results Characteristics Results Lactose + Phaseomannite − Arabinose − Mannite − Esculiw + D+ (−) glycogen − V-P test − Seminose − Maltose + Salicin − Esculin hydrolysis − Indole − Glucose + Methyl red + Rhamnose − Fucose − Raffinose + Mushroom sugar + Lactose − A-methyl-D-glucoside − Arabinose + Peroxidase + Citrate − Tyrosine − Methyl red − Nitrate-reducing − Xylose + Indole + Fructose − Acetate − Carbamide − Melibiose + Sucrose − Sorbose −

Phenylalanine − Oxidase − Raffinose − Phaseomannite − Casein protein H S − − 2 breakdown Maltose − Β-galactosidase − Fructose + Amylohydrolysis + Galactose − Melibiose −

Sorbierite + Gelatin liquefaction − Sorbierite − Glycerol − +: Positive or capable of utilizing; −: Negative or incapable of utilizing. Glucose; acid-producing − Citrate − Glucose; aerogenesis Table 4 Physiological and biochemical characteristics of Urease − Gelatin liquefaction − hs3 Amylase − Oxidase − Characteristics Results Characteristics Results Melampyrite − Catalase + Lactose + Hemolytic + +: Positive or capable of utilizing; −: Negative or incapable of utilizing. Maltose + Catalase − Table 7 Physiological and biochemical characteristics of Mannite + Sorbierite + hs6 Glucose + 10% gail + Characteristics Results Characteristics Results Raffinose − 40% gail + Xylose − Salicin + Sucrose + Nitrate + Arabinose − B-Galactose glucoside enzyme + Glucose + Nitrite + Rhamnose − Oxidase − Peroxidase + Oxidase +

Amylase − Arginine hydrolysis + Maltose: acid-producing + Indole − Aerogenesis Esculin hydrolysis + Sucrose + Nitrate-reducing + Lactose − +: Positive or capable of utilizing; −: Negative or incapable of utilizing. +: Positive or capable of utilizing; −: Negative or incapable of utilizing.

Zoological Research www.zoores.ac.cn Isolation and identification of symbiotic bacteria from the skin, oral, and rectal of wild and captive tree shrews 497

Table 8 Physiological and biochemical characteristics of Table 12 Physiological and biochemical characteristics of hs7 hs11 Characteristics Results Characteristics Results Characteristics Results Characteristics Results Robiocina + Sucrose + Starch + Synanthrin − Plasma-coagulase + lactose + Mannite − Lactose −

Oxidase + maltose + 50% gall + Maltose + Carbamide + glucose + Sucrose + Arabinose + Methyl red + arginine + Glucose − Sorbierite + Mannite + V-P test Weak + Raffinose − Gelatin + nitrate + +: Positive or capable of utilizing; −: Negative or incapable of utilizing. Liquefaction +: Positive or capable of utilizing; −: Negative or incapable of utilizing. Table 13 Physiological and biochemical characteristics of hs12 Table 9 Physiological and biochemical characteristics of hs8 Characteristics Results Characteristics Results

Characteristics Results Characteristics Results Arabinose − Sucrose − Mannite: acid-producing + Ornithine + Gelatin liquefaction + Glucose + Aerogenesis + V-P test + Mannite − Maltose + Carbamide − D-tartrate − Urease − Sucrose + Lactose − A-methyl-D-glucoside − Plasma-coagulase − Lactose + V-P test − Melampyrite − H2S − Maltose + Sorbierite − Methyl red + M．R + Fructose + Glucose: acid- + producing Phenylalanine − +: Positive or capable of utilizing; − : Negative or incapable of utilizing. Glucose: aerogenesis + Table 10 Physiological and biochemical characteristics of Fructose − Glutin − hs9 Indole − Citrate + +: Positive or capable of utilizing; −: Negative or incapable of utilizing. Characteristics Results Characteristics Results Arginine hydrolase + Maltose + changes and immunity. Therefore, it is important to Glucose − Melibiose − understand the classifications of symbiotic bacteria.

Amylase − Galactose + The symbiotic bacteria hosted in tree shrews are Indole − Lactose + affected by the living environment of microorganisms Methyl red + Ribose + The symbiotic bacteria hosted in tree shrews are Oxidase − Fructose + diversified and complicated, and are greatly affected by

Glutin − Melizitose − the living environment of microorganisms. In this study, 12 bacteria strains were isolated and identified from the Catalase − Raffinose − wild tree shrews. B. subtilis widely exsits in earth and +: Positive or capable of utilizing; − : Negative or incapable of utilizing. soiled organisms, and multiplies quickly in hay infusion. Table 11 Physiological and biochemical characteristics of In this study, B. subtilis was identified only from the hs10 body surface of wild tree shrews, with detectable rate at Characteristics Results Characteristics Results 50%, indicating it is a commonly hosted bacterium in Mannite − Lactose − wild tree shrews. P. aeruginosa, which was indentified

Gelatin hydrolysate + Inorganic nitrogen agar + from wild tree shrews with detectable rate at 25% in this

Glycerol − Glucose − study, is one of the most commonly found bacteria in earth. S. aureus is highly pathogenic and exsits in air, Oxidase + Arginine − water, dirt and excretions, whereas, S. epidermidis breeds Catalase + Nitrate − on body surfaces and is a normal flora. The detectable Esculin hydrolysis + Citrate − rates of these two Staphlococcus bacteris, which were +: Positive or capable of utilizing; −: Negative or incapable of utilizing. 33% and 75%, respectively, are probably due to their

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 498 LI, et al.

Table 14 Detected bacteria of 12 wild tree shrews Sampling area Numbers of the detected bacteria (n) Detectable rate Genus Species Body surface Oral Rectal ♀ ♂ (%) Bacillus B. subtilis ● ○ ○ 3 2 42 Escherichia E. coli ○ ○ ● 6 5 92 Enterococcus E. faecalis ○ ○ ● 1 1 17 Pseudomonas P. aeruginosa ● ○ ○ 1 2 25 Kurthia K. gibsonii ● ○ ○ 1 1 17 Kurthia N. mucosa ○ ● ○ 3 4 58 Staphlococcus S. aureus ● ○ ○ 2 2 33 Staphlococcus S. epidermidis ● ○ ○ 5 4 75 Lactococus L. lactis ○ ○ ● 2 2 33 Micrococcus M. luteus ● ○ ○ 1 2 25 Streptococcus S. pneumoniae ○ ● ○ 1 1 17 Salmonella S. typhosa ○ ○ ● 1 1 17 ●: Positive; ○: Negative.

Table 15 Detected bacteria of 12 third generation captive tree shrews Sampling area Numbers of the detected bacteria (n) Genus Species Detectable rate (%) Body surface Oral Rectal ♀ ♂ Escherichia E. coli ○ ○ ● 6 6 100 Neisseria N. mucosa ○ ● ○ 4 4 67 Staphlococcus S. epidermidis ● ○ ○ 5 4 75 Lactococus L. lactis ○ ○ ● 2 2 33 ●: Positive; ○: Negative. wide distrbutions in natural environments. M. luteus can water, food, vegetable, dirt and animal excretions. We be found in air, earth, water, as well as on the body only found Salmonella typhosa in two wild tree shrews, surface of animals and plants. This conditioned pathogen indicating it is a rare strain in tree shrews. may cause local or severe infections in tissues. K. gibsonii aften grows in animal excretions or meat The influences of artificial environments to the sym- products and so far, there are no reports of K. gibsonii as biotic bacteria hosted in tree shrews a pathogen. In this study, 17% K. gibsonii were detected The succeful artifical reproductions of tree shrews from the body surface of wild tree shrews. The detectable require several aspects the artifical environments, rate of oral N. mucosa was 67%. It is mainly hosted in including the facilities, nutritions, as well as the proper the oral mucosa of mammals and is a non-pathogenic managing and reproducing methods (Li et al, 2009; Jiang bacterium. S. pneumoniae exists in natural environments, et al, 2011; Zhao et al, 2013). The long-term and frequent animal excretions and the pharynx nasalis of healthy application of sterilization measures determined that less human beings and was detected in two wild tree shrews. bacteria exist in artifical environments than in wild. E. faecalis was only found in wild tree shrew rectums. L. In this study, four bacteria strains were isolated and lactis can be found in diary and plantation products. Our identified from the third generation of captive tree shre- results show that its detectable rates in both wild and ws, suggesting that the living environments are signif- third generation tree shrew were both 33%, indicating its icantly correlated with the species of microorganisms. probiotic advantages in tree shrews. E. coli are widely As a novel animal to study human diseases and from distributed in natural environments and mostly are non- the angle of evolution, to understand the similarities and panthogenic. Although the detectable rates of Escherichia differences of symbiotic bacteria from the same boby coli were extremely high in this study (92%−100%), we part of human and tree shrewsis necessary. Now, Human consider them as intestinal tract normal flora, because all Microbiome Project (HMP) has carried out detailed the experimental tree shrews were healthy and did not investigations on the bacteria flora of human skin, oral show any clinical disorders. S. typhosa is one of the cavities and intestinal tracts to determine the factors bacteria that can easily cause infections and exists in influencing the growth of symbiotic bacteria, such as almost every natural environment, including the air, gender, race, geography distribution, diet and body weight.

Zoological Research www.zoores.ac.cn Isolation and identification of symbiotic bacteria from the skin, oral, and rectal of wild and captive tree shrews 499

CONCLUSIONS jejuni and Shigella exsit in the intestinel tract of healthy The tree shrews used in this study were all in good adult tree shrews. So far, no Brucella spp., Leptospira healthy condition at time of sampling and 30 days after spp., Mycobacterium tuberculosis or Yersinia enteroc- sampling. Therefore, we assume that the indentified olitica have been found in tree shrews, suggesting that bacteria are major parasitic bacteria of tree shrews. The bacteria strains may function species-specifically in high detectable rate indicates that E.coli is the major different parts of different animals. normal parasitic bacteria, which is consistant with the In recent years, the advantages of tree shrews as a study of Gao et al (2009). In other conventional novel animal model have become apparent, because they laboratory animals, Staphlococcus bacteria is rare; are close to primates, they have been widely applied in however, in both wild and third generation captive tree studies on virus (Li et al, 2011; Wang et al, 2012b), neuronal shrews, its detectable rates were high (75%). Our results peptide Y (Dong et al, 2011), disbetes (Wu et al, 2013), also show a high occurrence of pathogens in wild tree depression (Wang et al, 2012a), cerebral ischemia (He et al, shrews (17%−33%), whereas no pathogens were found 2011), etc. Therefore, it is nessassary and to set standards to in the third generation captive tree shrews. normalize tree shrews as a laboratory animal model. As a novel laboratory animal, the applications of Based on the enriched tree shrew resources of Yunnan tree shrews are still lack of national standards (Shen et al, Province, our lab for the first time has successfully isolated 2011). For example, for the conventional animal model and identified the symbiotic bacteria of the third generation rhesus monkeys (Macaca mulatta), it is clearly pointed captive tree shrews. These findings not only provide basic out that laboratory rhesus monkeys have to be clean of data in setting the microorganism standard of laboratory tree Salmonella spp. Pathogenic dermal fungi and Cam- shrews but also are metagenomic reference of studies on the pylobaceter jejuni. Wang et al (1987) reported that C. symbiotic bacteria in tree shews.

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Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 Zoological Research 35 (6): 500−509 DOI:10.13918/j.issn.2095-8137.2014.6.500

Acoustic signal characteristic detection by neurons in ventral nucleus of the lateral lemniscus in mice

Hui-Hua LIU, Cai-Fei HUANG, Xin WANG*

College of Life Sciences and Hubei Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, China

Abstract: Under free field conditions, we used single unit extracellular recording to study the detection of acoustic signals by neurons in the ventral nucleus of the lateral lemniscus (VNLL) in Kunming mouse (Mus musculus). The results indicate two types of firing patterns in VNLL neurons: onset and sustained. The first spike latency (FSL) of onset neurons was shorter than that of sustained neurons. With increasing sound intensity, the FSL of onset neurons remained stable and that of sustained neurons was shortened, indicating that onset neurons are characterized by precise timing. By comparing the values of Q10 and Q30 of the frequency tuning curve, no differences between onset and sustained neurons were found, suggesting that firing pattern and frequency tuning are not correlated. Among the three types of rate-intensity function (RIF) found in VNLL neurons, the proportion of monotonic RIF is the largest, followed by saturated RIF, and non-monotonic RIF. The dynamic range (DR) in onset neurons was shorter than in sustained neurons, indicating different capabilities in intensity tuning of different firing patterns and that these differences are correlated with the type of RIF. Our results also show that the best frequency of VNLL neurons was negatively correlated with depth, supporting the view point that the VNLL has frequency topologic organization.

Keywords: Ventral nucleus of the lateral lemniscus; Firing pattern; Frequency tuning; Intensity tuning; Mouse

The ventral nucleus of the lateral lemniscus (VNLL) acoustic signal characteristics are closely correlated. For is the largest nucleus of the lateral lemniscus (LL) (Batra example, the first spike latency (FSL) of onset neurons is & Fitzpatrick, 2002). As a station in the auditory short and with increasing sound intensity,1 the FSL shor- ascending pathway, the VNLL plays a vital role in tens insignificantly, whereas those of the sustained acoustic signal processing by receiving afferent neurons are opposite (Zhang & Kelly, 2006). The projections from the contralateral ventral cochlear minimal threshold (MT) of onset neurons is also higher nucleus (VCN), ipsilateral trapezoid body (TB), superior than that of sustained neurons (Aitkin et al, 1970). olivary complex (SOC) and contralateral dorsal cochlear Regarding firing tuning in VNLL neurons, the value of nucleus (DCN), and casts efferent projections into the Q10 (Q10=best frequency/the band width at 10 dB above inferior colliculus (IC) (Benson & Cant, 2008; Schofield threshold) is used to assess the sharpening of the & Cant, 1997; Kelly et al, 2009; Huffman & Covey, frequency tuning cures (FTC). Studies on different

1995; Prather, 2013). animals show that the value of Q10 in onset neurons is Electrophysiological studies on the VNLL indicate smaller than in sustained neurons (Aitkin et al, 1970; that VNLL neurons can detect, process and encode the Covey & Casseday, 1991). Regarding intensity tuning in characteristics of acoustic signals and have various VNLL neurons, the dynamic range (DR, according to the responding reactions. The firing patterns of VNLL neurons include onset firing (instantaneous firing to the Received: 15 March 2014; Accepted: 01 June 2014 onset of acoustic signals) and sustained firing (sustained Foundation items: This study was supported by the National Natural firing during the whole interval of acoustic signals) Science Foundation of China (31000493) and the Central China Normal University Independent Scientific Research Project Fund for (Zhang & Kelly, 2006). Recently, it was reported that the Youth Scholars (11A01025) firing pattern and ability of VNLL neurons to detect *Corresponding author. E-mail: [email protected] Science Press Volume 35 Issue 6

Acoustic signal characteristic detection by ventral nucleus neurons in the lateral lemniscus in mice 501 rate-intensity function, RIF, of neurons, DR is usually set During the experiment, 0.6% mebumal sodium solution at 10% higher than the smallest firing spikes and 10% was used to keep the animal anesthetized and paralyzed. lower than the highest firing spikes) is used to assess By referencing previous studies (Cheng et al, 2013; intensity tuning abilities. The study on Dutch-belted Mei et al, 2012), a homemade bipolar tungsten electrode rabbits (Oryctolagus cuniculus) indicates that the DR of made of two insulated tungsten wire electrodes (FHC lnc, onset neurons is smaller than that of sustained neurons Bowdoin, ME, USA) was used to record neuronal responses (Batra & Fitzpatrick, 1999). of the VNLL and to confirm the recording site after We assume that the firing pattern, intensity and experimentation. The electrode was <10 μm in diameter, frequency of VNLL neurons have some sort of conne- 1−5 MΩ in impedance and the two poles were 30−80 μm ction. Despite being an animal model for acoustic studies, apart. detection characteristics and frequency topologic organ- ization of VNLL neurons to acoustic signals in Kunming Acoustic signal system and neuronal response recor- mice (Mus musculus) remain unclear. Here, using single ding unit extracellular recording, we observed the firing Under free field conditions, the speaker was placed patterns of VNLL neurons and compared FSL, frequency at 0° in the pitching position, and 60° in the contralateral and intensity tuning characteristics under different firing meridional direction (opposite to the animal’s external patterns. Our results are helpful in more fully auditory canal opening). Acoustic signals were produced understanding correlations between VNLL neuron firing by the Tucker-Davis Technology System 3 (Alachua, patterns and acoustic signal detection characteristics, and USA). Parameters, such as frequency, intensity and exploring the physiological functions of VNLL neurons duration were controlled by computer and calibrated by a and the effects of ascending projections on the IC. sound meter (2610, B&K, Denmark) and 1/4-inch microphone (4936, B&K, Denmark). Acoustic signals were tone bursts with a duration of 40 ms, rise and fall of MATERIALS AND ANIMALS 5.0 ms, and a frequency of 2 stimuli/s. The bipolar Animal surgery and electrode preparation tungsten electrode was vertically inserted into the VNLL Kunming mice (n=54, 18−28 g) with normal via a hydraulic drive (Kopf 640, USA). Tone burst was hearing were obtained from the Laboratory Animal adopted to locate acoustic signal sensitive neurons. The Center, Institute of Hubei Preventive Medicine, China. responding characteristics of the neurons of the dorsal Animal surgical procedures are referenced in previous nucleus of the lateral lemniscus (DNLL) (unpublished studies (Wang et al 2007; Tang et al, 2007). All experi- data) were used as parallel comparisons to improve ment procedures were performed strictly in accordance accuracy when locating the nucleus. Neuronal responses with the guidelines published in the NIH Guide for the were amplified by bioelectric amplifier (ISO-DAM, WPI, care and use of Laboratory Animals. Anesthesia was USA) via the bipolar tungsten electrode, displayed on the induced by injection of mebumal sodium (45−50 mg/kg, oscilloscope (PM3084, FLUKE, USA), monitored by the bw). After the mouse was immobilized in a stereotaxic monitoring apparatus (AM9, GRASS, USA) and then apparatus, the VNLL was exposed (4.16−4.48 mm stored in a computer. The depth, best frequency (BF) and posterior to the bregma, 1.10−1.95 mm lateral to the MT of the acoustic signal sensitive neuron were recorded. midline, 2.85−4.10 mm in dorsal ventral depth) by Each tone burst stimulus was repeated 32 times and the removing skull and dura over the VNLL (Paxinos & induced neuronal firings were profiled into a peri- Franklin, 2001). An electrode was inserted into the small stimulus time histogram (PSTH). Under the BF, starting hole (200−500 μm in diameter) over the VNLL. After from the MT, firing times with the intensity of acoustic surgery, the mouse was transferred onto a shake proof signal increasing every 10 dB segment were recorded to bench in the anechoic shielded chamber and its head was infer the RIF. Meanwhile, the highest and lowest immobilized on a homemade bracket with the aid of a tack frequencies of the neuron were found and their FTCs (1.8 cm in length) stuck on the skull. The animal’s eyes, were inferred. ears and the center of the speaker were at the same level. Background noise inside the anechoic shielded chamber Electrical burning and histological examination was 29 dB SPL and the temperature was set at 25−28 ℃. At the end of the experiment, the recording site in

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 502 LIU, et al. the anesthetic mouse was burned for 10 s by a 0.1 mA FSL was 5.78±2.79 ms (2−18.5 ms). Linear regression direct current for precise location later. Current stimula- analysis showed linear relationships between BF and tion was generated by a stimulator and introduced to the dorsal ventral depth (R=−0.36, P<0.001) (Figure 1A). mouse via a direct current stimulation isolator (Model We found that neurons sensitive to high frequencies are SEN-7203, Nihon Kohden Co, Shinjuku, Tokyo, Japan) located in the dorsal VNLL. From dorsal to ventral, the and the bipolar tungsten electrode. After a 2 h perfusion BF of VNLL neurons decreased, indicating frequency with 200 mL saline water and 4% paraformaldehyde, the topological organization along the dorsal-ventral-axis of brain was isolated and fixed in 4% paraformaldehyde for the VNLL. No linear relationships were found among 24 h. Regular paraffin sections (40 μm) were prepared FSL, MT and dorsal ventral depth (R=0.1, P>0.05; with a vibration slicing machine (VT 1000S, Leica CO, R=0.06, P>0.05) (Figure 1B, C). USA) and under Nissl’s staining (toluidine blue) for immunohistochemistry examination. The burned location Temporal firing pattern of VNLL neurons on the section was observed under a light microscope As one of the basic features of central auditory (Leica, DM4000B, Germany) and photographed. neurons, the temporal firing pattern reflects firing changes in neurons before and after stimulation. With Statistical analysis acoustic signals at BF and MT+20 dB, the firing patterns All statistical analyses were completed using SPSS of 98 VNLL neurons were recorded. Following the 13.0 and all figures were generated with Sigmaplot 10.0. research of Zhang & Kelly (2006), our observed firing Data are expressed as mean±SD. One-way ANOVA and patterns were categorized into two types: onset and t-tests were used to compare differences. sustained. The onset type can be sub-categorized into: (1) phasic: only one or two spikes to the onset of the aco- RESULTS ustic signals; (2) phasic burst: several spikes to the Characteristics of VNLL neurons onset of the acoustic signals; and (3) double burst: fixed For the 98 VNLL neurons we recorded in this study, intervals can be found between the spikes to the onset of the dorsal ventral depth was 3 368±156.2 μm (2 844−4 the acoustic signal. Likewise, the sustained type can also 090 μm); BF was 19.89±11.12 kHz (2.6−39 kHz); MT be sub-categorized into: (1) onset plus sustained: significant was 52.66±13.11 dB SPL (13.83−71.61 dB SPL); and response can be found to the onset of the acoustic signals,

Figure 1 Correlations between recording depth and BF (A), FSL (B) and MT (C) in VNLL neurons and Nissl-stained coronal section of the VNLL and surrounding structures (D) n: Numbers of neurons; R: correlation coefficient.

Zoological Research www.zoores.ac.cn Acoustic signal characteristic detection by ventral nucleus neurons in the lateral lemniscus in mice 503 then decreases and lasts through the entire duration; Among them, phasic burst had the highest percentage, (2) tonic: a fixed firing rate lasts through the entire followed by tonic, phasic and onset plus sustained, duration; and (3) primary-like: the firing rate to the onset respectively; primary-like and double burst had the of the acoustic signals quickly reaches maximum, then smallest percentages. Although differences were found decreases to a certain level. The representative post- among the percentages of various firing patterns, the stimulus time histograms (PSTH) of these six firing quantities of onset (52/98, 53.06%) and sustained (46/98, patterns and neuron numbers are shown in Figure 2. 46.94%) patterns were comparable.

Figure 2 Types of onset (A-1, A-2, A-3) and sustained (B-1, B-2, B-3) patterns of recorded VNLL neurons n: Numbers of neurons; Recording depth (μm), MT (dB SPL) and BF (kHz) of these neuron are 3118, 53.83, 16.2 (A-1); 3739, 64.28, 35 (A-2); 3970, 66.79, 10.6 (A-3); 2908, 38.14, 13.7 (B-1); 4090, 54.22, 3.4 (B-2); 3042, 3.14, 17.8 (B-3), respectively.

Under intensity at MT+20 dB, the FSL of onset divided into six types: (1) V-shape: with increasing VNLL neurons was 5.47±1.86 ms (2−11 ms), with intensity, the verges of high and low frequency are 67.31% of the FSL within the range of 2.5−5 ms; the broadening; (2) U-shape: the changes in FTC are FSL of sustained VNLL neurons was 7.1±3.13 ms independent of intensities [the V- and U-shape can be (2−19.5 ms) with a relatively wide distribution (Figure differentiated by bandwidth level intolerance (BLI, 3A, C). The FSL of onset VNLL neurons was shorter BLI=BWmax/BW10), if BLI<3, it is U-shape, if BLI≥3, than that of sustained neurons (P<0.001, unpaired t-test). it is V-shape]; (3) Lower-tail-upper-sharp (LTUS): the When the intensity of the acoustic signal increased every low frequency verge is characterized with a long tail and 10 dB segment to MT+50 dB, no significant changes in the high frequency verge is very sharp; (4) Upper-tail- the FSL of onset VNLL neurons were observed (one-way lower-sharp (UTLS): the high frequency verge is charac- ANOVA, P>0.05), whereas, the FSL of sustained terized with a long tail and the low frequency verge is neurons decreased (one-way ANOVA, P<0.001) (Figure very sharp [in LTUS and UTLS, the SlopeMT+30−MT+10≥30%, 3B, D). otherwise, if SlopeMT+30−MT+10<30%, the FTC is V- or U- Frequency tuning of VNLL neurons shape]; (5) W-shape: two peaks can be found in FTC and Frequency is a basic parameter of acoustic signals. the two frequency tuning segments are partitioned by an Auditory neurons analyze and encode external acoustic auditory completely insensitive area only under low stimulation, which play an important role in the behavior intensity, but not under high intensity; and (6) Double V- of humans and other animals. FTC are commonly used in shape: two peaks can be found in FTC and the two assessing the frequency tuning of auditory neurons. frequency tuning segments are partitioned by an auditory According to Suga (1997) and our previous data, the completely insensitive area under both low and high FTC of the 71 VNLL neurons recorded here were intensities (Figure 4).

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 504 LIU, et al.

Figure 3 Distribution of FSL in onset neurons (A) and sustained neurons (C) and FSL-intensity function in onset neurons (B) and sustained neurons (D) Abscissa of B and D were normalized by each neuron’s MT as the control of tone intensity; numbers above the vertical bars in B and D are the numbers of neurons calculated statistically.

Figure 4 Types of frequency tuning curves of recorded VNLL neurons n: Numbers of neurons; Recording depth (μm), MT (dB SPL) and BF (kHz) of these neuron are 3114, 32.22, 16.8 (A); 3925, 49.63, 11.2 (B); 3001, 61.66, 25.5 (C); 3513, 55.81, 11.5 (D); 3009, 58.03, 40.3 (E); 3179, 67.09, 51.8 (F), respectively.

Among the 71 neurons, 38 were onset and 33 sustained neurons was 3.43±3.83 (0.8−19.26) (P>0.05, were sustained. The MT of the onset neurons was unpaired t-test), whereas, the value of Q30 of the FTC 55.33±10.83 dB SPL (27.09−69.34 dB SPL) and that of onset neurons was 0.94±0.52 (0.26−2.45) and that of the sustained neurons was 48.83±12.85 dB SPL of the sustained neurons was 0.96±0.26 (0.13−1.99) (13.83−73.54 dB SPL) (P<0.05, unpaired t-test) (P>0.05, unpaired t-test) (Figure 5 B, C), indicating

(Figure 5 A). The value of Q10 of the FTC of onset that the firing patterns and frequency tuning of VNLL neurons was 2.49±1.67 (0.65−7.01) and that of the neurons are irrelevant.

Zoological Research www.zoores.ac.cn Acoustic signal characteristic detection by ventral nucleus neurons in the lateral lemniscus in mice 505

Figure 5 Differences in the MT (A), Q10 value (B) and Q30 value (C) between onset neurons and sustained neurons *: P<0.05.

Intensity tuning of VNLL neurons 8.16% (4/49), respectively; of sustained neurons the Intensity reflects the amplitude of acoustic vibration. percentages were 61.9% (26/42), 21.43% (9/42) and Within certain range, most of the auditory neurons 16.67% (7/42), respectively (Figure 7A). Therefore, in increase spiking with increasing intensity. Under onset and sustained neurons, the distribution patterns of relatively high intensity, different neurons show different RIF from high to low were monotonic, saturated and changes in firing rate function. With an acoustic signal at non-monotonic, respectively. The DR of the onset BF, 40 ms, and intensity increases every 10 dB segment, neurons was smaller than that of sustained neurons the firing numbers of VNLL neurons changed and (P<0.01, unpaired t-test). In neurons with monotonic and showed various characteristics of intensity tuning. Accor- saturated RIF, the DR of the onset neurons was smaller ding to previous work and our own data (Tang et al, 2007; than sustained neurons (P<0.01; P<0.05, unpaired t-test); Zhou & Jen, 2000), under increasing intensity, if the in neurons with non-monotonic RIF, no differences were firing numbers keep increasing, neurons are recognized found between onset and sustained neurons (P>0.05, as monotonic RIF; if the firing numbers do not change or unpaired t-test) (Figure 7B). decrease to <25%, neurons are recognized as saturated RIF; and if the firing numbers decrease to≥25%, neurons DISCUSSION are recognized as non-monotonic RIF. The representative Firing patterns and temporal features of VNLL curves of these three types of RIF and numbers of neurons neurons are shown in Figure 6. The monotonic RIF had The percentages of onset and sustained patterns the highest percentages, followed by saturated and then were comparable, 53.06% (52/98) and 46.94% (46/98), non-monotonic RIF. respectively, consistent with previous findings on big Of the 91 VNLL neurons we recorded in this study, brown bats (Eptesicus serotinus) (Covey & Casseday, 49 were onset and 42 were sustained. The percentages of 1991), rats (Zhang & Kelly, 2006) and rabbits (Batral & monotonic, saturated and non-monotonic RIF of onset Fitzpatrick, 1999). The 2.04% (2/98) double-burst neurons were 48.98% (24/49), 42.86% (21/49) and pattern we found has only been reported in rats (Zhang &

Figure 6 Types of rate-intensity function of recorded VNLL neurons n: Numbers of neurons; Recording depth (μm), MT (dB SPL) and BF (kHz) of these neuron are 3403, 50.52, 33 (A); 3259, 27.09, 24.8 (B); 3211, 38.89, 19.8 (C), respectively.

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 506 LIU, et al.

Figure 7 Differences in percentage of RIF (A) and DR (B) between onset neurons and sustained neurons M: monotonic; Sat: saturated; NM: non-monotonic. *: P<0.05; **: P<0.01.

Kelly, 2006) and its biological meaning in acoustic signal the intensity, the shorter the FSL, and the quicker the processing remains unclear. signal can be recognized. However, correlations between The FSL of onset neurons was shorter than FSL and intensity are unclear. Pharmacological studies sustained neurons. With the increase in intensity, the FSL show that neitherγ-aminobutyric acid (GABA) nor glyc- of onset neurons remained stable (Figure 3B), indicating ine (Gly) affects correlations between FSL and intensity in a feature of precise timing of onset neurons. This specific AC and IC neurons (Zhang et al, 2005), indicating that feature of onset VNLL neurons may be attributable to these correlations may be encoded by auditory neurons projections of octopus cells in the cochlear nucleus (CN) below the IC, but this requires further exploration. (Adams et al, 1997; Pollak et al, 2011). The octopus cell have large, irregularly shaped cell bodies with three to Frequency tuning and frequency topologic organiza- five thick primary dendrites emerging from only one side tion of VNLL neurons of the cell body. The sizable low voltage activated Although the percentages of six different types of potassium channels on its membranes endow the cell FTC are consistent with previous studies on rats (Zhang with a low impedance and fast time constant, which & Kelly, 2006) and rabbits (Batra & Fitzpatrick, 1999), bestow its projections to the VNLL with temporal the degrees of sharpening are quite different among advantages (Johnston et al, 2010; Bal & Ocrtel, 2007). different species. For example, in this study, the average

The precise timing of onset VNLL neurons is not only value of Q10 of the FTC of VNLL neurons in Kunming beneficial in the encoding of acoustic signal onset, but mice was 2.9, but was 7.1 (Zhang & Kelly, 2006) in rats also affects the process of IC neurons to temporal and 9.1 (Covey & Casseday, 1991) in bats, respectively, information via its ascending projections. For example, indicating that different species are characterized with studies on rats (Nayagam et al, 2005), Mexican free- different capabilities of frequency tuning. The sharp- tailed bats (Tadarida brasilensis mexicana) (Xie et al, ening of FTC and GABAergic inhibitory projection are 2007) and big brown bats (Voytenko & Galazyuk, 2008) correlated with the formation of the inhibitory area of show that an inhibitory postsynaptic potential (IPSP) can frequency tuning (Chen & Jen, 2000). The FTC of VNLL be observed ahead of the excitations of some IC neurons, neurons in mice have smaller Q10 and low degrees of which means an IPSP occurs before depolarization. It is sharpening. Because the VNLL in mice receives less assumed that because of the temporal advantage of inhibitory projections from the ipsilateral trapezoid body, VNLL neurons possess, the inhibitory projection from but has a wide frequency receptive range to excitatory VNLL neurons will reach the IC ahead of the excitatory projections from the contralateral cochlear nucleus, projection, thereafter, an early IPSP is initiated. perhaps the FTC of VNLL neurons is not characterized The FSL of sustained VNLL neurons decreased by significant sharpening. with increasing intensity (Figure 3D). This phenomenon No differences were found in the Q10 of the FTC in is not specific to sustained VNLL neurons (Zhang & onset and sustained neurons (Figure 5). However, studies

Kelly, 2006) and has also been observed in the CN on big brown bats and cats show that the Q10 in onset (Goldberg & Brownell, 1973), SOC (Sanes & Rubel, neurons is smaller than sustained neurons (Covey & 1988), IC (Tan et al, 2008) and auditory cortical (AC) Casseday, 1991; Aitkin et al, 1970). Based on these (Heil, 1997). Therefore, it is assumed that FSL encodes discrepancies, it is assumed that different species have the intensity of the onset of the acoustic signal. The larger different neural circuits corresponding to specific behaviors.

Zoological Research www.zoores.ac.cn Acoustic signal characteristic detection by ventral nucleus neurons in the lateral lemniscus in mice 507

For example, because the frequency tuning of the onset input tuning (Chen & Jen, 2000; Andrew & Ellen, 2009 VNLL neurons in Mexican free-tailed bats affects the reor- Wu et al, 2006; Tang et al, 2008). The low level of non- ganization of IC neurons to some special communication monotonic RIF in VNLL neurons may be consistent with signals, they show certain corresponding firing patterns and less inhibitory inputs in the VNLL. Previous histoc- features of frequency tuning (Pollak et al, 2011). hemical studies show that excitatory projections in the Frequency topological organization of VNLL neu- VNLL are mainly from the CN (Suneja et al, 1995), rons remains controversial. When the IC of rats was whereas low levels of inhibitory projections are mainly infected with retrograde tracing agents, low frequency from the TB (Yavuzoglu et al, 2010). neurons in the VNLL distributed along the band, whereas, The intensity tuning of onset and sustained neurons high frequency neurons were evenly distributed (Kelly et were compared and values of DR were used as an index al, 1998). Electrophysiological experiments on bats to assess the sensitivity of neurons to acoustic signal (Covey & Casseday, 1991) and cats (Aitkin et al, 1970) intensity. The smaller the DR, the narrower the intensity show that low frequency neurons are distributed dorsally tuning and the higher the sensitivity, meaning that of the VNLL, whereas, high frequency neurons are variation in intensity can induce significant changes in distributed ventrally of the VNLL. Although differences the neuronal spiking of humans and other animals. The exist in the distribution patterns of different species, the smaller DR in onset neurons compared to sustained logical distribution of low and high frequency neurons neurons indicates different sensitivity in these two types supports the view of frequency topologic organization in of neurons and implies that the intensity encoded by the VNLL. However, an electrophysiological experiment neuronal impulses is affected by neuronal firing patterns. on rats (Nayagam et al, 2006) and the study on infecting Although previous studies have reported similar retrograde tracing agents in the IC of cats (Glendenning phenomena in the VNLL (Batra & Fitzpatrick, 1999), & Hutson, 1998) and the primary auditory field (AI) of DNLL (unpublished data) and IC (Bal et al, 2002), if Mongolian gerbils (Budinger et al, 2013) indicate a lack differences in DR can be observed among neurons with of topologic organization in the VNLL. Possible reasons different firing patterns then correlations with RIF of these discrepancies are differences in animal models remain unclear. The DR of two neuron types with three and experimental methods. In this study, the BF and different RIF was compared. In monotonic and dorsal ventral depth of VNLL neurons were negatively saturated RIF, the DR of onset neurons was smaller correlated (Figure 1A). Neurons with high frequencies than sustained neurons, but in non-monotonic RIF, no were located dorsally of the VNLL and from dorsal to differences were found (Figure 7B), indicating that DR ventral, the BF of neurons decreased, indicating is correlated with RIF type and in general, onset frequency topologic organization along the dorsal- neurons are more sensitive than sustained neurons in ventral-axis of the VNLL. intensity tuning. The firing patterns of VNLL neurons are affected by Intensity tuning of VNLL neurons multiple factors and truthfully reflect their ability in inte- In the 91 VNLL neurons recorded here, the nsity tuning, The firing patterns of certain VNLL neurons percentage of non-monotonic RIF (12.09%) was lower change with changes in acoustic signals (Zhang & Kelly, than that in the advanced auditory nuclei of mice, such as 2006) indicating that their firing pattern and intensity are the IC (Tang et al, 2007) and AC (Qi et al, 2013). The regulated by different signal pathways and integrations of occurrence of non-monotonic type is due to inhibitory excitatory and inhibitory inputs within the time window.

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Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 Zoological Research 35 (6): 510−527 DOI:10.13918/j.issn.2095-8137.2014.6.510

Morphological analysis of the Chinese Cipangopaludina species (Gast- ropoda; Caenogastropoda: Viviparidae)

Hong-Fa LU1,†, Li-Na DU2,†, Zhi-Qiang LI1,*, Xiao-Yong CHEN3,*, Jun-Xing YANG3,*

1. Zhejiang Normal University, Xiaoshan Campus, Hangzhou 311231, China 2. Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China 3. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China

Abstract: Viviparidae are widely distributed around the globe, but there are considerable gaps in the taxonomic record. To date, 18 species of the viviparid genus Cipangopaludina have been recorded in China, but there is substantial disagreement on the validity of this taxonomy. In this study, we described the shell and internal traits of these species to better discuss the validity of related species. We found that C. ampulliformis is synonym of C. lecythis, and C. wingatei is synonym of C. chinensis, while C. ampullacea and C. fluminalis are subspecies of C. lecythis and C. chinensis, respectively. C. dianchiensis should be paled in the genus Margarya, while C. menglaensis and C. yunnanensis belong to genus Mekongia. Totally, this leaves 11 species and 2 subspecies recorded in China. Based on whether these specimens’ spiral whorl depth was longer than aperture depth, these species or subspecies can be further divided into two groups, viz. chinensis group and cathayensis group, which can be determined from one another via the ratio of spiral depth and aperture depth, vas deferens and number of secondary branches of vas deferens. Additionally, Principal Component Analysis indicated that body whorl depth, shell width, aperture width and aperture length were main variables during species of Cipangopaludina. A key to all valid Chinese Cipangopaludina species were given.

Keywords: Cipangopaludina; Gastropoda; Bellamyinae; Anatomy; Taxonomy; Chinese

The freshwater gastropod Viviparidae is currently diverse genus mainly distributed in China, Japan, Korea, divided into three subfamilies (Bouchet & Rocroi, 2005): Thailand, Vietnam, Laos, India, Burma and Malaya Viviparinae Gray, 1847, Bellamyinae Rohrbach, 1837 (Brandt, 1974; 1Liu et al, 1993) that is comprising of 35 and Lioplacinae Gill, 1863, which together comprise species and subspecies (Global Names Index; http:// approximately 150 species and 31 genera recognized to gni.Globalnames.org), there has been substantial debate date (Franke et al, 2007). Typically, viviparid species on the taxonomy of Cipangopaludina. Hannibal (1912) inhabit lakes, ponds, and lentic rivers of temperate to had previously proposed Cipangopaludina as a subgenus tropical region (Strong et al, 2008; Ying et al, 2013), of Idiopoma, based on invasive specimens collected in explaining their wide global distribution across every California which were identified as Paludina malleata continent save for South America and Antartica (Brown, 1994). In China, the existing taxonomy for Viviparidae Received: 02 April 2014; Accepted: 20 July 2014 includes approximately 61 recognized species in 9 Foundation items: This research was funded by the National Natural genera (Du et al, 2011; Liu, 1991; Wang & Xie, 2005; Science Foundation of China (31301865); the Natural Science Foun- Yen, 1943): Viviparus Montfort, 1810, Bellamya Jousse- dation of Zhejiang Province of China (LY12C19006) and the Collec- aume, 1886, Filopaudina Habe, 1964, Mekongia Crosse tion and Preparation of Display Specimens at Kunming Natural et Fischer, 1876, Angulyagra Benson, 1836, Margarya History Museum of Zoology (KSZD–EW–TZ–005) Nevill, 1877, Rivularia Heude, 1890 and Trochotaia *Corresponding authors, E-mail: [email protected]; chenxy@mail. Brandt, 1974, Cipangopaludina Hannibal, 1912. The last kiz.ac.cn; [email protected] genus, Cipangopaludina is particularly interesting as a †Authors contributed equally to this work

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Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 511

Reeve. Meanwhile, Annandale (1920) proposed Lecyth- elevation and demotion of species to subspecies and vice oconcha as a distinct genus for Paludina lecythis Benson versa, the existing literature names 18 potential species while Prashad (1928) argued that Lecythoconcha was of Cipangopaludina distributed in China: C. ampullacea, invalid and adopted the name Cipangopaludina. C. ampulliformis, C. aubryana, C. cathayensis, C. Even within China, there is no clear verdict on the chinensis (synopsis C. leucostoma and C. diminuta), C. validity of each species or how many are even distributed dianchiensis, C fluminalis, C. haasi (synopsis C. in the area. Heude (1890) listed 9 species from China, longispira), C. hainanensis (synopsis C. compacta), C. viz. C. chinensis (Benson (non Gray)), C. lecythoides latissima, C. lecythoides, C. lecythis, C. menglaensis, C. (Heude), C. diminuta (Heude), C. longispira (Heude), C. patris, C. ussuriensis, C. ventricosa, C. wingatei and C. fluminalis (Heude), C. leucostoma (Heude), C. cath- yunnanensis. Similarly, the disparate results of the ayensis (Heude), C. ventricosa (Heude) and C. aubryana previous studies on the topic also highlight how difficult (Heude). Later, comprehensive work by Kobelt (1909) and complex the identification of Cipangopaludina is, revised Viviparidae, including practically all the forms of largely due to intraspecific variations in shell shape. This the family known at the time, which was one species and has left the taxonomic position and phylogenetic 12 subspecies in China, with most of species being relationships of these species largely unknown. In this designated varieties of the species C. chinensis (Gray): study, we opted to perform a fresh check on the cathayensis (Heude), compacta (Nevill), ventricosa morphology and anatomy of Chinese Cipangopaludina (Heude), longispira (Heude), fluminalis (Heude), species currently being held at repositories in Beijing and hainanensis (Kobelt), diminuta (Heude), leucostoma Kunming in order to arrive at a more complete species (Heude), aubryana (Heude), patris (Kobelt), wingatei list. Morphometric analyses of shell variations were (Smith), lecythoides (Benson) and its form latissima conducted on Cipangopaludina species through a (Dautzenberg et H. Fischer). Several decades later, Yen Principal Component Analysis. (1943) revised species of Chinese Viviparidae, and pointed out that leucostoma and diminuta were synon- MATERIALS AND METHODS yms of chinensis and that lecythoides (Heude, non Sample collection and identification Benson) distributed in Zhoushan Island, compacta (Ko- Four hundred and ninety-six, including 11 Cipang- belt, non Nevill) distributed in Hainan, and lecythis opaludina species were collected from Jilin, Jiangxi, crassior (Annandale) distributed in western Yunnan were Zhejiang, Guangxi and Yunnan Provinces. These specim- all synonyms of patris (Kobelt), and longispira (Heude) ens were preserved in Kunming Institute of Zoology, was argued to be synonymous with haasi. Liu (1991) Chinese Academy of Sciences (KIZ). Additionally, 145 later clarified the distribution and taxonomy of Cipangopaludina specimens, including 18 Cipangop- Cipangopaludina and raised 15 of the subspecies to the aludina species or its synonyms listed in the existing species level, viz. C. chinensis, C. cathayensis, C. literature preserved in the Invertebrate Museum of the fluminalis, C. aubryana, C. latissima, C. lecythoides, C. Institute of Zoology, Chinese Academy of Sciences, ussuriensis (Gerstfeldt), C. yunnanensis, C. dianchiensis, Beijing (IOZ) were measured. A morphological analysis C. ampullacea, C. haasi, C. lecythis, C. longispira, C. was conducted on these specimens and the validity of the ventricosa and C. menglaensis. Finally, Wang & Xie currently identified species was determined. Specimens (2005) evaluated the extinction risk of Cipangopaludina were identified mainly based on original description, species, listing 14 distinct species: Cipangopaludina type specimens, Liu et al (1979, 1993), Kobelt (1909) hainanensis, C. leucostoma, C. menglaensis, C. latissima, and Prashad (1928). C. dianchiensis, C. lecythis C. lecythoides, C. haasi, C. ampullacea, C. ventricosa, C. ampulliformis, C. Characters selected for morphometric analysis aubryana, C. longispira and C. ussuriensis. Curiously, To select characters for use in morphometric anal- these latest works by Liu (1991) and Wang & Xie (2005) ysis, we reviewed the available taxonomic descriptions made no mention of two species, C. patris and C. from literature (Liu et al, 1979). Five shell characters wingatei, and ignored some of the earlier work done by were measured with callipers accurate to 0.1 mm: Shell Yen (1943). depth (D), the maximum dimension parallel to the axis of Despite discrepancies and arguments as well as the coiling; Shell width (W) the maximum dimension

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 512 LU, et al. perpendicular to D; Length of the aperture (LA), the Indian Museum, 6: 401. maximum dimension from the junction of the outer lip Viviparus (Cipangopaludina), Clench & Fuller, with the penultimate whorl to the anterior edge of the 1965, Occasional papers on Mollusks, Museum of Com- aperture (oblique to coiling axis); width of the aperture parative Zoology, 2: 385–412. (WA), the maximum dimension perpendicular to LA; Bellamya (Cipangopaludina), Smith, 2000, Nautilus, depth of the body whorl (BW), the dimension from the 114(2): 31–37. lower margin of the aperture to the upper suture Diagnosis: Shell medium to large (up to 70 mm), delimiting the first whorl; and N, the number of whorls. oval, brown or greenish in colour. Apex pointed or Embryonic shells were measured to 0.1 mm using obtused. Suture narrow. Umbilicus, big or narrow or an ocular micrometer. Anatomy was studied using a closed. Sculpture lacking except growth lines and axial microscope with drawing apparatus. Radulae and embry- undulations. Aperture subcircular, inner lip whitish blue onic shells were studied via scanning electron micro- and outer lip black in colour, easily broken. Young scopy (SEM). The radulae were cleaned enzymatically specimens (from brood pouch) with up to 4 whorls; with proteinase K, as described by Holznagel (1998), protoconch smooth, three primary rows of chaetae on last then sonicated and mounted on aluminium specimen whorl and other whorls have two rows of chaetae. Male’s stubs with adhesive pads. Embryonic shells were clea- testis with two or three main branches. ned mechanically, sonicated, and mounted on adhesive Cipangopaludina chinensis was the only species carbon-coated pads. Both radulae and embryonic shells given a detailed description, while diagnosis charac- were coated with gold-palladium and studied on AMR- terstics are listed for all other species. All morphometric AY 1000B scanning electron microscope at 30 kV. The and meristic characters of Cipangopaludina species are description of shell features and visceral hump follows given in Table 1. the general terminology suggested by Simone (2004). Cipangopaludina ampullacea (Charpentier, 1863)

(Figure 1A) Allometric shell growth Paludina ampullacea Charpentier: Küster, 1852, Syste- Due to ontogenetic effect of allometric growth have matisches Conchylien–Cabinet. 2 ed.p. 23, pl. 4, fig 2–3. usually caused confusion in morphometric analyses, Material: 25 ex. (2 female and 3 male were anat- these individuals within the range of allometric growth omy). KIZ 000110–000117, collected from Qiubei Cou- were eventually excluded before multivariate analyses nty, Yunnan in August 2005 by Cui GH and Du LN; KIZ (Armbruster, 1995; Chiu et al, 2002; Valovirta & Vaisa- 000453–000470, collected from Beihai, Tengchong nen, 1986). However, in the present-day study, it is County, Yunnan in April 2006 by Chen XY. difficult for us to obtain snails of all size classes from Diagnosis: shell larger, up to 53 mm, brown– each population per species and examine the allometric greenish, with 5 whorls. Apex obtused. Spiral whorl shell growth. Under this circumstance, the consequence depth is less than aperture length. Body whorl inflated, of Chiu et al (2002) was followed that excluded shell width about 80.1%–90.6% shell depth. Body whorl individuals with aperture lengths of less than 1.0 cm. depth is less than shell width. Spiral whorls inflated and Morphometric data was explored through a Principal round, form a flat shoulder. Osphradium ridge-like, Component Analysis by SPSS 10.0 (SPSS for Windows, shorter or equal with length from anterior tip of Chicago, IL, USA). osphradium to mantle border. The base of gill filaments wide and apex narrow and round, curve to left. Kidney RESULTS arch-shape, the dorsal length of kidney right plane about Systematics Cipangopaludina Hannibal, 1912 1.5 times than ventral left plane and posterior plane, and Idiopoma (Cipangopaludina) Hannibal 1912, Proc- left plane and posterior plane very curve (Figure 2C-II). eedings of the Malacological Society of London 10: 194 Vas deferens with 2 main branches and the first anterior (type species, Paludina malleata Reeve, original desig- branch with 4 parallel secondary branches. Vas deferens nation) opens in last 1/4 of testis. Pouch filled with less than 44– Lecythoconcha Annandale 1920, Records of the 48 embryos in various stages of development. Embryos Indian Museum. 19: 111 (type species, Paludina lecythis with 4 whorls, body whorl with 3 chaetae and other Benson, monotypic); Annandale 1921, Records of the whorls have 2 rows of chaetae.

Zoological Research www.zoores.ac.cn Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 513

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 514 LU, et al.

Figure 1 Shell of Cipangopaludina A: C. lecythis ampullacea, KIZ 000453; B: C. lecythis, KIZ000284; C: C. leucostoma, KIZ 000270; D: C. chinensis, IOZ–FG166277; E: C. chinensis fluminalis, KIZ 000443; F: C. haasi, KIZ 000101; G: C. dianchiensis, KIZ 000536; H: C. hainanensis, IOZ–FG168161; I: C. latissima, IOZ–FG84656; J: C. lecythoides, IOZ–FG166538; K, L: C. menglaensis, IOZ–FG0142 (Holotype); M: C. yunnanensis, IOZ–FG0152 (Holotype); N: C. aubryana, IOZ–FG 170556; O: C. ussuriensis, IOZ–FG84819; P: C. ventricosa, KIZ000227. Scale bars=1 cm.

Figure 2 Anatomy and genital system of Cipangopaludina A: Shell removed showing external features of animal; B: Male with mantle cavity opened mid–dorsally; C: Two type of kidney, I triple shape and II arch shape. Abbreviations: aa — anterior aorta; ag — albumen gland; an — anal papilla; au —auricle; bp — brood pouch; cm—columellar muscle; ct — ctendium; df — dorsal fold of buccal mass; eg — egg-shell gland; es — esophagus; ey — eye; ft — foot; int — intestine; jw — jaw; kd — kidney; l — liver; me — mantle edge; mo — mouth; ne — nephrostome; op — operculum; pa — posterior aorta; pe—penis; pg—prostate gland; re—rectum; sn—snout; te—tentacle; ur—ureter; ve— ventricle; vd—vas deferens.

Distribution: Southwest of China, Yunnan, Sichuan shorter or equal with length from anterior tip of provinces and Macao (Liu, 1991). osphradium to mantle border, vs. longer in C. ventricosa, Remarks: Cipangopaludina ampullacea can be C. patris and C. aubryana; shell larger, apex eroded vs. distinguished from C. chinensis, C. fluminalis, C. haasi, shell medium and apex pointed in C. yunnanensis; C. menglaensis, C. dianchiensis and C. ussuriensis by umbilicus small or closed vs. big and round in C. spiral depth shorter than aperture depth. Cipangop- latissima; apex obtused vs. pointed in C. cathayensis and aludina ampullacea can be distinguished from other C. lecythoides. Cipangopaludina ampullacea can be species by the following characters: osphradium length distinguished from C. lecythis by apex eroded vs. pointed;

Zoological Research www.zoores.ac.cn Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 515 the upper angle of aperture acute vs. straight. However, Embryos with 4 whorls, body whorl with 3 chaeta and the anatomy of C. ampullacea and C. lecythis are not other whorls have 2 rows of chaetae. obviously different, so it is suitable to treat C. Distribution: Sichuan, Hunan, Guangdong, Guizhou ampullacea as a subspecies of C. lecythis. and Yunnan Provinces, China (Liu, 1991). Remarks: Cipangopaludina aubryana can be disti- Cipangopaludina aubryana (Heude, 1890) (Figure 1N) nguished from C. chinensis, C. haasi, C. fluminalis, C. Paludina aubryana Heude, 1890, Memoires conce- dianchiensis, C. ussuriensis and C. menglaensis by spiral mant L’Histoire naturelle e L’empire chinois par des depth less than aperture length and shell medium. peres de la compagnie de Jesus, 175, pl. 39. Cipangopaludin aubryana can be distinguished from Vivipara (chinensis) aubryana Heude, Kobelt, 1909, other species by the following characteristics: kidney Abbildungen Nach de Natur mit Beschreibngen, p 118, pl. triple-shape vs. arch-shape in C. ampullacea, C. patris 20, fig 6–7. and C. ventricosa; acute spiral whorl and without Viviparus chinensis aubryana (Heude), Yen, 1939, shoulder (vs. spiral whorl inflated and form shoulder in C. Diese Arbeit erscheint gleichzeitig in den Abhandlungen lecythoides and C. yunnanensis); shell medium and body der Senckenbergischen Naturforschenden Gesellschaft whorl not very inflated vs. shell larger and body whorl Abhandlung, 444, p. 35, taf. 3. very inflated in C. latissima; apex pointed, not eroded vs. Viviparus chinensis aubryana (Heude), Yen, 1942, apex eroded, with only 4 whorls in C. hainanensis; vas Proceedings of the Malacological Society of London, 190, deferens opens in last 1/3 of testis vs. 1/4 in C. lecythis 200, pl. 13, fig 32. and C. l. ampullacea. Cipangopaludina chinensis aubrayana (Heude), Pradshad, 1928, Memoirs of the Indian Museum, Calc- Cipangopaludina cathayensis (Heude, 1890) (Figure 1C) utta, 8: 168. Paludina catayensis Heude, 1890, Memoires conce- Cipangopaludina lecythoides aubryana (Heude), mant L’Histoire naturelle e L’empire chinois par des Yen, 1943, Nautilus, 56(4): 124–130. peres de la compagnie de Jesus, 175, pl. 39. Cipangopaludina aubryana (Heude), Liu, 1991, Vivipara chinensis cathayensis Heude, Kobelt, 1909, Proceeding of Tenth International Malacological Con- Abbildungen Nach de Natur mit Beschreibngen, p 112, pl. gress, 590. 18, fig 5–6. Materials: 20 ex. (4 females and 4 males were anat- Cipangopaludina chinensis cathayensis (Heude): omy). IOZ–FG166735, IOZ–FG166737, from Wang Co- Prashad, 1928, Memoirs of the Indian Museum, Calcutta, unty, Sichuan Province in June 1964; IOZ–FG170556, 8: 168; Yen, 1943, Nautilus, 56(4): 124–130. IOZ–FG170553, IOZ–FG170551, IOZ–FG 170549, from Cipangopaludina cathayensis (Heude), Liu, 1991, Hanshou County, Hunan province in March 1963; KIZ Proceeding of Tenth International Malacological Cong- 000087–100, from Pu’er City, Yunnan in April 2011 by ress, 590. Zheng LP. Materials: 34 ex. (2 males and 2 females were Diagnosis: shell medium, brown green, with 5 who- anatomy) KIZ000016–17, collected from Lake Xingyun, rls. Body whorl inflated, but other whorls not, without Yunnan in July 2008 by Du LN; KIZ000422–434, shoulders. Apex pointed. Spiral whorl depth less than collected from Lake Dianchi, Yunnan in September 2006 aperture depth. Osphradium ridge-like, longer than by Du LN, Jiang YE and Cui GH; KIZ 000076 collected length from anterior tip of osphradium to mantle border. from Debao County, Guangxi in August 2009 by Yu GH The base of gill filaments wide and apex narrow and and Li Y; KIZ000190–196, collected from Liaoyuan City, pointed, does not curve to right. Kidney triple-shape, the Jilin in October 2012 by Du LN. dorsal length of kidney right plane about 1.2 times than Diagnosis: shell shin, medium, up to 60 mm, with 6 ventral left plane, 2 times than posterior plane, the left inflated whorls, spiral whorl with shoulder. Apex pointed. plane and posterior plane do not curve much (Figure 2C- Spiral whorl depth less than aperture depth. Osphradium I). Vas deferens with 3 main branches and the first length similar with length from anterior tip of osphr- anterior branch with 4 parallel secondary branches. Vas adium to mantle border, anterior 2/3 part of osphradium deferens opens in last 1/3 of testis. Pouch filled with less ridge-like and posterior 1/3 part inflated. Gill filaments than 10 embryos in various stages of development. same with C. chinensis. Kidney triple-shape. Vas defer-

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 516 LU, et al. ens with 3 main branches and the first anterior branch Congress, 590. with 3–4 parallel secondary branches. Vas deferens opens Material: 40 ex. (1 males and 2 females were in last 1/3 of testis. Female pouch filled with more than anatomy). IOZ–FG166277, Shangsi Co. Guangxi Prov- 60 embryos in various stages of development. Embryos ince in 19th April 1974; IOZ84521, Zhaotong City, Yun- with 3 whorls, body whorl with 3 chaeta and other nan Province in May 1932; IOZ166773, IOZ166777, whorls have 2 rows of chaetae. Qionghai Lake, Xichang County, Sichuan Province in 4th Distribution: Jilin, Shanxi, Hebei, Shandong, Anhui, July 1964; IOZ166733, Luofang village, Guangdong Jiangsu, Zhejiang, Hunan, Sichuan, Guizhou and Yunnan Province in March 1982; KIZ000164–165, Lake Erhai, provinces (Liu, 1991). Yunnan, collected by Du LN, Jiang YE and David Remarks: In the original description, this species Aldridge in 14 April 2006; KIZ 000294–310, Lake north was spelt catayensis, but later reviewers all spelt it mountain, Jilin City, Jilin Province, collected by Du LN cathayensis, according to the International Code of on 13 October 2012; KIZ 000132–133, KIZ 000149–150, Zoological Nomenclature (1999). Catayensis is then West Lake, Dali City, Yunnan, collected by Du LN in 13 treated as an inadvertent error, and thus this spelling is April 2006, Jiang YE and Aldridge D; KIZ 000157–158, incorrect. Cipangopaludina cathayensis can be Lake Xinyun, Jiangchuan County, Yunnan, collected by distinguished from C. chinensis, C. haasi, C. fluminalis, Du LN, Jiang YE and Cui GH in September 2009; KIZ C. dianchiensis and C. menglaensis by spiral depth less 000013015, KIZ 000038040, KIZ 0000363–366, KIZ than aperture depth. Cipangopaludina cathayensis can be 000421, Lake Dianchi, Kunming City, Yunnan, collected distinguished from other species by the following by Du LN and Aldridge D. in March 2006. characteristics: anterior 2/3 of osphradium ridge-like and posterior 1/3 inflated; female’s pouch with more than 60 Description embryos, and embryo’s have 3 whorls. Shell: large (up to 70 mm), oval, brown or greenish in colour, with 6–7 whorls. Apex pointed. Suture narrow. Cipangopaludina chinensis (Gray, 1834) (Figure 1D) Umbilicus narrow or closed. Sculpture lacking except Paludina chinensis Gray: in Griffith & Pidgeon, growth lines and axial undulations. Aperture subcircular, 1834, Animal Kingdom, pl. 1, fig 5. inner lip whitish blue and outer lip black in colour, easily Paludina chinensis Gray: Gray in Griffith & brown. Young specimens (from brood pouch) with up to Pidgeon, 1834, Animal Kingdom, 599. 4 whorls; protoconch smooth, three primary rows of Vivipara chinensis (Gray), Kobelt, 1909, Abbildun- chaetae on last whorl and other whorls have two rows of gen Nach de Natur mit Beschreibngen, p111, pl 23, fig 1. chaetae. Cipangopaludina chinensis (Gray): Prashad, 1928, Operculum: Occupying entire shell aperture. Centre Memoirs of the Indian Museum, Calcutta, 8: 168. is brown and outer parts greenish. Nucleus sub-central, Viviparus chinensis chinensis (Gray): Yen, 1939, located closer to inner margin. Sculptured with conce- Diese Arbeit erscheint gleichzeitig in den Abhandlungen ntric growth lines. Inner surface glossy; scar sub-circular, der Senckenbergischen Naturforschenden Gesellschaft close to inner margin, occupying about 2/3 of opercula Abhandlung, 444, 35. Taf. 3. area. Viviparus chinensis (Gray): Yen, 1942, Proceedings External morphology (Figure 2B): head and foot of the Malacological Society of London, 190, 200, pl. 13, black. Snout cylindrical, anterior margin flat. Length of fig 32. tentacles about 1.3 times longer than snout length, base Cipangopaludina chinensis leucostoma (Heude): at side of snout base. Ommatophore short, located Yen, 1943, Nautilus, 56(4): 124–130. between basal and middle third of outer surface of each Cipangopaludina chinensis diminuta (Heude): Yen, tentacle. Foot large, sole simple. Opercular pad larger 1943, Nautilus, 56(4): 124–130. than its base in dorsal foot surface. Columellar muscle Bellamya (Cipangopaludina) chinensis (Gray): thick. Smith, 2000, Nautilus, 114(2): 31–37. Radula: Central tooth with wide rounded major Cipangopaludina chinensis (Heude): Prashad, 1928, denticles and 4 smaller triangular denticles on each side. Memoirs of the Indian Museum, Calcutta, 8: 168; Liu, Lateral tooth with tongue-shaped major denticles and 3 1991, Proceeding of Tenth International Malacological minor denticles on each side. Inner marginal tooth with

Zoological Research www.zoores.ac.cn Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 517 tongue-shape major denticles and 3 minor denticles and posterior plane. Nephrostome is a round, small pore, marginal teeth with 10 almost equal-sized denticles. located in centre basally inferior region of ventral surface Mantle organs (Figure 2A-B): Mantle border simple of kidney. Ureter runs between rectum and oviduct (in and thick. Osphradium ridge-like, very close to ctenid- females) or right pallial cavity edge (in males). Ureter ium, about same length from anterior tip of osphradium pore just posterior to anus (or just posterior to female to mantle border. Ctenidium long and narrow, about pore). same length as pallial cavity, anterior end at mantle Genital system. border. Gill filaments very tall and narrow, arched Male (Figure 2A): Testis compact, semi-lunar, on towards right and apex slightly pointed, close to food right of mantle cavity. Extends to upper end of mantle groove. cavity, where apex close to pericardial cavity also Hypobranchial gland lies on left of gill margin. connected by thin fold of membrane with lower surface Right margin of mantle cavity filled by oviduct in of digestive gland. Testis flattened laterally, right surface females. Ureter runs along mantle cavity right margin in abuts columellar mussel while left surface abuts right males or edging oviduct in females. Rectum on dorsal wall of ureter. Vas deferens very narrow, running on and left sides of ureter. At right end of mantle border, in columellar and inferior ventral margin of testis, with 2 females, presenting three openings, most posterior and main branches, and in the anterior branch with 5–6 smaller is ureter pore, anus most anterior, and female parallel secondary branches, the branches with many pore larger, located between anus and ureter pore. very narrow branches from different portions of testis. Alimentary canal: Oval mouth, bounded by fleshy Vas deferens opens in last 1/3 of testis and runs to lips, ventral at anterior end of snout. Jaw plates ridge-like prostate gland in mantle cavity along mantle cavity floor (Figure 2A). Esophagus with pair of dorsal, longitudinal for about 3/4 of its length; left 1/4 length of vas deferens folds, about 1.5 times of buccal mass length. Esophagus abruptly narrows and is surrounded by very thick, runs along the hypobranchial gland to posterior of muscular walls. Vas deferens runs all along the right columellar muscle, and then turns right and passes cephalic tentacle, its inner surface relatively broad, with upwards in floor of pericardial chamber to reach some inner, longitudinal folds. Vas deferens opens in digestive gland in upper part of visceral hump where it tentacle tip as broad papilla with rounded tip. Right curves round to open into stomach. Stomach located tentacle possesses deep concavity located at right from halfway posterior to pallial cavity, posterior half genital papilla. Papilla can retract into its concavity. immersed in digestive gland. Esophagus is inserted in Female. Ovary grey, located in same position as posterior gastric extremity, and as gradually enlarges testis. Ovary in close contact with posterior wall of curves towards left. After this curve, stomach abruptly cardiac region of stomach and along course of hepatic expands becoming almost as wide as whorl, posterior artery. Oviduct very narrow; from albumen gland it runs narrowing, without clear separation with intestine. to posterior end of columella, and then turns left, making Intestine forms loop overlying pericardial cavity and a loop. Oviduct increases in size and runs to capsule when it reaches digestive gland turns sharply forwards to gland along brood pouch. Brood pouch located in dorsal continue as rectum. Rectum passes forwards on right of of oviduct. Albumen gland tongue-shaped, slightly mantle cavity to open at anus lying just behind mantle curved, capsule gland on dorsal and posterior surface and edge. opens to brood pouch. Walls of brood pouch thin, semi- Circulatory and excretory systems: Heart located at transparent, smooth. Pouch filled with about 45–84 anterior ventral of kidney. Auricle connected with embryos in various stages of development. ctenidial vein just posterior to gill. Ventricle posteriorally Ecology and habitat: it lives in slow-moving water connected to auricle by a thin tube. The size of ventricle such as lakes, pond, irrigation canals, ditches and slow is same as the auricle. Kidney triangular (Figure 2 C-I), moving stream (Pace, 1973). It is a benthic grazer and with four almost plane surfaces: 1) posterior surface filter feeder, feeding on benthic and epiphytic diatom towards pericardium; 2) left with pallial cavity; 3) (Plinski et al, 1978), often found on sandy to muddy ventral right with ureter; 4) dorsal with mantle. The substrates. dorsal length of the kidney’s left plane is similar to the Distribution: original range in China, Taiwan, Korea, ventral right plane, and about two times longer than and Japan, but has also been introduced in freshwater

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 518 LU, et al. ponds and lakes in Canada, the north-eastern USA, and Distribution: only known to live in Lake Dianchi, Europe (Soes et al, 2011). Yunnan. Remarks: Although Yen (1943) mentioned that C. Remark: Morphology characteristics, such as shell leucostoma (Heude) and C. diminuta (Heude) are thickness, body whorl with clear ridges, female’s pouch synonymous with C. chinensis (Gray), the authors with few (6–8) embryos and embryo shell with ridges thought these two species are synonymous with C. patris can be distinguished clearly from other species of genus (Kobelt) according to apex obtuse and less than 6 whorls. Cipangopaludina. Additionally, phylogenetic analysis Additionally, Cipangopaludina wingatei (Smith) could indicated that C. dianchiensis clustered with species of be synonymous with C. chinensis according to shell size, Margarya in Lake Dianchi (Du et al, 2013). The morp- spiral depth longer than length of aperture, and number hology and molecular results indicated that C. dianch- of whorls. Cipangopaludina chinensis, C. dianchiensis, iensis should be placed into the genus Margarya. C. fluminalis, C. haasi, C. ussuriensis and C. menglaensis share the characteristic that spiral depth is Cipangopaludina fluminalis (Heude, 1890) (Figure 1E) longer than length of the aperture. However, C. chinensis Paludina fluminalis Heude, 1890, Memoires can be distinguished from C. dianchiensis and C. concemant L’Histoire naturelle e L’empire chinois par ussuriensis by its shell smooth rather than having a shell des peres de la compagnie de Jesus, 175, pl. 39. with a ridge; female’s pouch has more than 80 embryos Vivipara chinensis fluminalis Heude, Kobelt, 1909, vs. less than 10 embryos in C. dianchiensis; embryo with Abbildungen Nach de Natur mit Beschreibngen, p 116, pl. three primary rows of chaetae on last whorl vs. 3 ridges 19, fig. 4–5. in C. dianchiensis. Cipangopaludina chinensis can be Viviparus chinensis fluminalis (Heude), Yen, 1939, distinguished from C. fluminalis by the following Diese Arbeit erscheint gleichzeitig in den Abhandlungen characters: shell thin vs. thick, umbilicus close vs. big der Senckenbergischen Naturforschenden Gesellschaft and deeply. Abhandlung, 444, p. 35, taf. 3; Yen, 1942, Proceedings of the Malacological Society of London, 190, 200, pl. 13, Cipangopaludina dianchiensis Zhang, 1990 (Figure 1F) fig 32. Cipangopaludina dianchiensis Zhang, 1990, Acta Cipangopaludina chinensis fluminalis (Heude), Zootaxonomica Sinica, 15(1): 25–27. Prashad, 1928, Memoirs of the Indian Museum, Calcutta, Materials: 34 ex. (2 males and 3 females were 8: 168. anatomy). KIZ 000233, collected in November 2002 by Cipangopaludina lecythoides fluminalis (Heude): Cui GH; KIZ 000001–KIZ 000004, collected in June Yen, 1943, Nautilus, 56(4): 124–130. 2008 by Du LN, Jiang YE and Barclay H; KIZ 000005– Cipangopaludina fluminalis (Heude), Liu, 1991, KIZ 000012, collected in September 2005 by Du LN and Proceeding of Tenth International Malacological Aldridge D; KIZ 000212–KIZ 000217, KIZ 000287–KIZ Congress, 590. 000293; KIZ 000311–KIZ 000316, collected in January Materials: 21 ex (1 male and 1 female were 2006 by Du LN and Yuan C; KIZ 000373, KIZ 000231 anatomy). KIZ 000181 collected from Jinping County, collected in June 2006 by Du LN. All specimens were Honghe prefecture, Yunnan in April, 2011 by Jiang WS; from Lake Dianchi. KIZ 000204–000211 collected from West Lake, Dali Diagnosis: shell thick. Body whorl with 7–12 ridges prefecture, Yunnan in April 2006 by Du LN, Jiang YE (3–4 ridges clearly). Apex pointed. Osphradium ridge– and Aldridge D; KIZ 000384 collected from Songming like, shorter than length from anterior tip of osphradium County, Kunming prefecture, Yunnan in December 2005 to mantle border. Gill filaments and jaw plate are the by Du LN; KIZ 000442–000451 collected from same as C. chinensis. The size of ventricle is the same as Songming County, Kunming prefecture, Yunnan in the auricle. Vas deferens has 3 main branches and the December 2004 by Du LN. first anterior branch has 4 “V” shaped secondary Diagnosis: shell is thick, larger, up to 60 mm, with 7 branches. Vas deferens opens in last 1/3 of testis. Pouch slightly inflated whorls. Apex pointed. Spiral whorl filled with about 6–8 embryos in various stages of depth longer than aperture depth. Umbilicus big and deep. development. Embryos with 5 whorls, body whorl with 3 Osphradium length similar with length from anterior tip ridges and other whorls with 1 ridge. of osphradium to mantle border. Gill filaments same as C.

Zoological Research www.zoores.ac.cn Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 519 chinensis. Kidney triple shaped, the dorsal length of bungen neuer oder wenig gekannter Conchylien heraus- kidney left plane about same length as ventral right plane, gegeben, II. p.133, pl 2, fig 1; Küster, 1852, In Abbildu- 1.5 times longer than posterior plane. Vas deferens with 3 ngen nach der Natur mit Beschreibungen. Systematisches main branches and the first anterior branch with 5–6 Conchylien-Cabinet 2ed, p. 23, pl. 5, fig 1, 2; Reeve, parallel main secondary branches. Vas deferens opens in 1862, Conchologia iconica, or, illustrations of the shell last 1/3of testis. Pouch filled with 12 embryos in various of molluscous animals, pl. 4, fig 21; Dautzenberg et H. stages of development. Embryos with 4 whorls, body wh- Fischer, 1905, Journal de Conchylologie, p417–418. orl with 3 chaeta and other whorls have 2 rows of chaetae. Paludina (Vivipara) lecythoides Benson: Mabille et Distribution: Yangtze River (Yen, 1943; Liu, 1991). Le Mesle, 1866, Journal de Conchyliologie, p. 134; Remarks: Cipangopaludina fluminalis is very Fischer, 1891, Catalogue et Distribution géographique similar with C. chinensis except the shell thick rather des Mollusques terrestres, fluviatiles et marins d’une than thin; umbilicus big and deep rather than small or partie de l’Indo-Chine (Siam, Laos, Cambodge, Conchi- closed. The anatomy characteristics were relatively nchine, Annam, Tonkin), p. 177. similar with little difference, making it is possible to treat Vivipara (chinensis var.) lecythoides (Benson), C. fluminalis as subspecies of C. chinensis. Kobelt, 1909, Abbildungen Nach de Natur mit Beschrei- bngen, p 119, pl. 23, fig 8. Cipangopaludina latissima (Dautzenberg et H. Fischer, Cipangopaludina chinensis lecythoides (Benson), 1905) (Figure 1I) Prashad, 1928, Memoirs of the Indian Museum, Calcutta, Paludina lecythoides latissima Dautzenberg et H. 8: 168. Fischer, 1905, Journal de Conchylologie, p418, pl. 10, Cipangopaludina lecythoides (Benson), Yen, 1943, fig 17; Kobelt, 1909, Abbildungen Nach de Natur mit Nautilus, 56(4): 124–130; Liu et al, 1991, Proceeding of Beschreibngen, p 144, pl. 29, fig 1. Tenth International Malacological Congress, 590. Cipangopaludina lecythoides latissima (Dautzen- Materials: 1 ex. KIZ 000507, collected from Hang- berg et H. Fischer), Pradshad, 1928, Memoirs of the zhou City, Zhejiang Province in April 2013 by Lu HF; Indian Museum, Calcutta, 8: 168. Diagnosis: shell depth 55 mm, with 6 inflated whorls. Cipangopaludina latissima (Dautzenber et H. Fis- Shell dark brown, with clearly growth lines. Apex pointed. cher): Yen, 1943, Nautilus, 56(4): 124–130; Liu et al, Spiral whorl depth shorter than aperture depth. Umbilicus 1991, Proceeding of Tenth International Malacological small, nearly closed. Vas deferens has 2 main branches and Congress, 590. the first anterior branch with 3 parallel secondary branches. Materials: 15 shell specimens. IOZ–FG84653–84667, Vas deferens opens in last 1/3 of testis. collected from Mengzi County, Yunnan in March 1927. Distribution: Zhejiang Province. Diagnosis: shell is large, round, up to 53 mm, with 6 Remarks: Although Wang & Xie (2005) mentioned whorls. Apex pointed. Body whorl very inflated, shell that this species is distributed in Yunnan, it was not width is about 85%–95% of shell depth. Due to the body collected during field excursions undertaken between whorl inflated, the upper of body whorl has a flat 2003–2008 by authors Du LN and Chen XY. Generally, shoulder. Aperture ellipse. Umbilicus very big, round. the validity of this species is unclear, as it is difficult to Anatomy characteristics unknown. distinguish C. lecythoides and C. cathayensis by shell Distribution: only known to be from Mengzi County, morphology alone. Further anatomic characteristics are Yunnan. required to determine the validity of this species. Remarks: Cipangopaludina latissima can be distin- guished from other species of Cipangopaludina by the Cipangopaludina lecythis (Benson, 1836) (Figure 1B) body whorl special inflated, and the upper of body whorl Paludina lecythis Benson, 1836, Journal of Asiatic with a flat shoulder. Society of Bengal, 5: 745. Paludina chinensis lecythis Benson: Nevill, 1885, Cipangopaludina lecythoides (Benson, 1842) (Figure 1J) Hand list of Mollusca in the Indian Museum: Gastropoda. Paludina lecythoides Benson, 1842, The Annals and Prosobranchia–Neurobranchia, p. 20. Magazine of Natural History, Zoology, Botany and Geo- Paludina ampullacea Reeve (not Charpentier), 1862, logy 9, 488; Philippi, 1846, Abbildungen und Beschrei- Conchologia iconica, or, illustrations of the shell of

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 520 LU, et al. molluscous animals, 12. 168. Vivipara lecythis Benson, Kobelt, 1909, Abbildungen Cipangopaludina lecythoides (Heude, non Benson): Nach de Natur mit Beschreibngen, p 148, pl. 30, fig 1–2. Yen, 1943, Nautilus, 56(4): 124–130. Cipangopaludina chinensis lecythis (Benson): Prashad, Cipangopaludina compacta (Kobelt, non Nevill): 1928, Memoirs of the Indian Museum, Calcutta, 8: 168. Yen, 1943, Nautilus, 56(4): 124–130. Cipangopaludina lecythis (Benson): Yen, 1943, Cipangopaludina lecythis crassior Annandale: Yen, Nautilus, 56(4): 124–130; Liu et al, 1991, Proceeding of 1943, Nautilus, 56(4): 124–130. Tenth International Malacological Congress, 590. Materials: 28 ex (2 females and 2 males were Materials: 25 ex. (1 female and 2 males). KIZ anatomy). KIZ 000268–000279, collected from Yuanji- 000018–000030 collected from Songming County, Kun- ang County, Yunnan in January 2010 by Du LN; KIZ ming City, Yunnan in April 2006 by Du LN and Yuan C; 000369–000372, collected from Jinning County, Yunnan KIZ 000044 collected from Lake Dianchi, Yunnan in in August 2006 by Du LN; KIZ 000391–000402, April 2006 by Du LN and Yang J; KIZ 000144 collected collected from Yuanjiang County, Yunnan in January from Lake Erhai in April 2006 by Du LN, Yang YE and 2013 by Du LN. Aldridge D; KIZ 000151–156 collected from Lake Diagnosis: shell medium, up to 50 mm, brownish, Xingyun, Yunnan in September by Du LN, Jiang YE and with 6 whorls. Apex obtuse. Whorls inflated, especially Cui GH; KIZ 000224 collected from Lake Xingyun, last two whorls, upper three whorls slightly inflated and Yunnan in April by Du LN; KIZ 000284–286 collected always eroded. Spiral whorl depth shorter or equal with from Lake Dianchi, Yunnan in March 2006 by Du LN. aperture depth. Aperture ellipse. Umbilicus closed. The Diagnosis: shell larger, up to 60 mm, brown gree- shape of the kidney is similar with C. ampullacea. Osph- nish, with 7 whorls. Apex pointed. Spiral whorl depth sh- radium ridge-like, longer than length from anterior tip of orter than aperture depth. Whorls inflated, with a shoul- osphradium to mantle border. Gill filaments same with C. der. Aperture ellipse, the upper of aperture form a strai- chinensis. Vas deferens with 3 main branches and the ght shoulder. Umbilicus small or closed. Osphradium first anterior branch with 1 “V” shape and 3 parallel sec- ridge-like, shorter than length from anterior tip of ondary branches. Vas deferens opens in last 1/3 of testis. osphradium to mantle border. Gill filaments same with C. The loop of oviduct exceeds the posterior margin of col- chinensis. Vas deferens with 2 main branches and the umellar muscle. Pouch filled with 96–109 embryos in first anterior branch with 4 parallel secondary branches. various stages of development. Embryos with 3 whorls, Vas deferens opens in last 1/4 of testis. Pouch filled with body whorl with 3 chaetae and other whorls with 2 chaetae. about 37 embryos in various stages of development. Distribution: Yunnan, Guangdong and Zhejiang pro- Embryos with 3–4 whorls, body whorl with 2 chaetae vinces, China. and other whorls with 1 chaetae. Remarks: Cipangopaludina patris can be disting- Distribution: Yunnan, China. Burma (Kobelt, 1909). uished from C. chinensis, C. haasi, C. dianchiensis, C. Remarks: Cipangopaludina lecythis can be distin- ussuriensis and C. menglaensis by a spiral depth shorter guished from other species of this genus by its special than aperture length and a medium shell. Cipang- aperture shape, the upper of aperture straight and form a opaludina patris can be distinguished from other species flat shoulder. Kobelt (1909) list C. lecythis ampulliformis by the following characteristics: larger shell, up to 50 (Souleyet) as a subspecies of C. lecythis, which are mm (vs. medium, up to 26 mm in C. yunnanensis); the distributed in both Burma and Yunnan, China. According kidney arch-shape is similar with C. ampullacea, (vs. to Kobelt (1909) it is difficult to distinguish between triple-shape in C. cathayensis, C. aubryana and C. these two species, so we opted to treat C. lecythis ampu- lecythoides); medium shell and body whorl are not very lliformis as synonymous with C. lecythis. inflated (vs. shell larger and body whorl very inflated in C. latissima); shell with 6 whorls (vs. only with 4 whorls Cipangopaludina patris (Kobelt, 1906) (Figure 1C) in C. hainanensis); the upper of aperture without straight Paludina patris Kobelt, 1909, Abbildungen Nach de shoulder (vs. with straight shoulder in C. lecythis). Natur mit Beschreibngen, p 118, pl. 23, fig 4–5. Cipangopaludina chinensis patris (Kobelt), Pras- Cipangopaludina haasi (Prashad, 1928) (Figure 1F) had, 1928, Memoirs of the Indian Museum, Calcutta, 8: Cipangopaludina chinensis haasi Prashad, 1928,

Zoological Research www.zoores.ac.cn Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 521

Memoirs of the Indian Museum, Calcutta, 168. Distribution: Hunan, Jiangxi, Sichuan, Zhejiang and Paludina longispira Heude, 1890, Memoires Yunnan Provinces, China. concemant L’Histoire naturelle e L’empire chinois par Remarks: Cipangopaludina haasi, C. dianchiensis, des peres de la compagnie de Jesus, 175, pl. 39. C. chinensis, C. ussuriensis and C. menglaensis share the Vivipara chinensis longispira Heude, Kobelt, 1909, characteristic that spiral depth is longer than length of the Abbildungen Nach de Natur mit Beschreibngen, p 115, pl. aperture. However, C. haasi could be distinguished from C. 19, fig 3. dianchiensis and C. ussuriensis by shell smooth vs. shell Viviparus chinensis longispira (Heude), Yen, 1939, with ridge; female’s pouch with more than 128–188 Diese Arbeit erscheint gleichzeitig in den Abhandlungen embryos vs. less than 10 embryos in C. dianchiensis; der Senckenbergischen Naturforschenden Gesellschaft embryo with three primary rows of chaetae on last whorl Abhandlung, 444, p. 35, taf. 3. vs. 3 ridges in C. dianchiensis. Cipangopaludina haasi Viviparus chinensis longispira (Heude), Yen, 1942, could be distinguished from C. chinensis by the following Proceedings of the Malacological Society of London, 190, characteristics: body whorl not inflated vs. inflated; body 200, pl. 13, fig 32. whorl depth same or shorter than body width vs. obviously Cipangopaludina haasi (Prashad): Yen, 1943, Naut- longer. Cipangopaludina haasi could be distinguished ilus, 56(4): 124–130. from C. menglaensis by a larger shell, up to 65 mm vs. Cipangopaludina longispira (Heude), Liu, 1991, Proc- smaller, up to 30 mm; body whorl not inflated vs. inflated. eeding Tenth International Malacological Congress, 590. Cipangopaludina hainanensis (Möllendorff, 1909) Materials: 44 ex (2 females and 2 males were (Figure 1H) anatomy). KIZ 000197–000198, KIZ 000280–000282, Vivipara (chinensis var.) hainanensis Möllendorff, collected from Black dragon spring, Songming Town, in Kobelt, 1909, Abbildungen Nach de Natur mit Kunming, Yunnan in December 2004 by Cui GH and Beschreibngen, p 117, pl. 19, fig. 6–7. Chen XY; KIZ 000122–131, collected from Xiayu Cipangopaludina chinensis hainanensis (Möllend- Village, Zhangshu City, Jiangxi Province in February orff): Prashad, 1928, Memoirs of the Indian Museum, 2006 by Yu GH; KIZ 00101–00108, collected from Lake Calcutta, 168. Yilong, Yunnan in February 2008 by Du LN and Jiang Cipangopaludina hainanensis (Möllendorff): Yen, YE; KIZ 000477–000497, collected from Hangzhou City, 1943, Nautilus, 56(4): 124–130. Zhejiang Province in April 2013 by Lu HF. Materials: 5 ex. (without specimens were anatomy). Diagnosis: shell larger, up to 65 mm, greenish, with IOZ–FG 168660 collected from Ledong County, Hainan 7 whorls. Apex pointed. Suture shallow. Spiral whorl Island in December 1989; IOZ–FG 168160–168163 depth higher than aperture depth. Shell width less than collected from Chang Jiang County, Hainan Island in body whorl depth. Whorls slightly inflated, do not form December 1989. shoulder. Umbilicus small. Aperture ellipse. Osphradium Diagnosis: shell medium, up to 40mm. Apex eroded, ridge-like, longer than the length from anterior tip of left 4–5 inflated whorls. Suture deeply. Umbilicus big osphradium to mantle border. Gill filaments same as C. and deep. chinensis. Esophagus has pair of dorsal, longitudinal Distribution: only known from Hainan Island. folds, about 2 times of buccal mass length. Stomach Remarks: this species was evaluated as being elliptical, spoon-shape. Heart located anterior ventral of extinct by Wang and Xie (2005). Existing specimens can kidney. Ventricle is about 2 or 3 times larger than auricle. be distinguished from other species by medium shell, Kidney triple-shape. Nephrostome round, small pore, apex eroded, with 4–5 inflated whorls and umbilicus big located in posterior 1/5 of ventral surface of kidney. Vas and deep. deferens with 2 main branches and the first anterior branch has 8 to 9 parallel secondary branches. Vas Cipangopaludina menglaensis Zhang, Liu et Wang, deferens opens in last 1/4 of testis. Female’s pouch 1981 (Figure 1L) length is about 4 times longer than egg gland. Pouch Cipangopaludina menglaensis Zhang, Liu et Wang, filled with 128–188 embryos in various stages of 1981, Acta Zootaxonomica Sinica, 6(1): 40. development. Embryos with 4 whorls, body whorl with 3 Materials: 10 ex. IOZ–FG 0142 (holotype), IOZ– chaetae and other whorls with 2 chaetae. FG 00143–151 (Paratypes) from Manzhazai Village,

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 522 LU, et al.

Pu’er City, Yunnan in March 1965 by Zhang WZ and Liu Cipangopaludina chinensis ventricosa (Heude), YY. Pradshad, 1928, Memoirs of the Indian Museum, Calc- Diagnosis: shell dark brown, small, up to 30 mm, utta, 8: 168. with 6 whorls. Apex pointed. Suture deeply. Central Cipangopaludina ventricosa (Heude), Yen, 1943, tooth with wide rounded major denticles and 3 or 4 Nautilus, 56(4): 124–130; Liu, 1991, Proceeding Tenth smaller triangular denticles on each side. Lateral tooth International Malacological Congress, 590. with tongue-shaped major denticles and 4 or 5 minor Materials: 24 ex. (1 female and 2 males were denticles on each side. Inner marginal tooth with tongue- anatomy). KIZ 000031 collected from Yangguang Cou- shape major denticles and 3 or 4 minor denticles and nty, Yunnan in February 2006 by Du LN; KIZ 000036– marginal teeth with 20 almost equal sized denticles. 38, KIZ 000174, KIZ 000227–228, KIZ 000230 collec- Distribution: only known from type locality. ted from Lake Dianchi, Yunnan in March 2006 by Du Remarks: Without anatomized specimens, the anat- LN; KIZ 000143 collected from Lake Erhai, Yunnan in omy characteristics remain unknown. However, this April 2006 by Du LN, Jing YE and Aldridge A; KIZ species can be distinguished from other species of 000175–180 collected from Jiuzai dragon spring, Yunnan Cipangopaludina by a smaller shell, up to 30 mm and in March 2006 by Du LN; KIZ 000226 collected from marginal teeth with 20 almost equal sized denticles. Lake Xingyun, Yunnan in April 2006 by Du LN; KIZ 000283 collected from Black dragon spring, Songming Cipangopaludina ussuriensis (Gerstfeldt, 1859) (Figure County, Yunnan in December 2004 by Cui GH and Chen 1O) XY; KIZ 000361–362 collected from Black dragon Paludina ussuriensis Gerstfeldt, 1859, Memoires spring, Songming County, Yunnan in December 2005 by des savants Strangers, 9: 507, fig 1–4; Reeve, 1862, Du LN; KIZ 000437–441 collected from Black dragon Conchologia iconica, or, illustrations of the shell of spring, Songming County, Yunnan in April 2006 by Du molluscous animals, pl. 8, fig 1–4; LN. Vivipara ussuriensis (Gerstfeldt), Kobelt, 1909, Diagnosis: shell larger, medium thick, up to 65 mm, Abbildungen Nach de Natur mit Beschreibngen, p108, pl. with 7 whorls. Body whorl inflated. Aperture inflated 18, fig 1–4. and round, but the upper angle does not forms a straight Cipangopaludina ussuriensis (Gerstfeldt), Yen, shoulder. Apex pointed. Umbilicus big and deep. 1943, Nautilus, 56(4): 124–130; Liu et al, 1991, Proce- Osphradium ridge-like, longer than length from anterior eding Tenth International Malacological Congress, 590. tip of osphradium to mantle border. Gill filaments same Material: 1 ex. IOZ–FG84819, collected from with C. chinensis. The dorsal length of kidney left plane Khanka Lake, Heilongjiang Province in 1960. about same length with ventral right plane, 1.5 times than Diagnosis: shell larger, up to 54 mm, brown–gree- posterior plane. Vas deferens with 3 main branches and nish, with 6 whorls and two or three brown band. Apex the first anterior branch with 1 “V” shape and 3 parallel obtuse. Spiral depth longer than aperture depth. Shell secondary branches. Vas deferens opens in last 1/4 of width is about 78.5% shell depth. Body whorl with 4 testis. Pouch filled with 33 embryos in various stages of clear ridges, other whorls with 2 ridges. Umbilious small. development. Embryos with 3 whorls, body whorl with 3 Distribution: Lower Amur River. chaetae and other whorls with 2 chaetae. Remarks: Cipangopaludina ussuriensis can be Distribution: Yunnan, Sichuan and Guizhou Provi- distinguished from other species of this genus by the nces, China (Liu, 1991). following character: apex obtuse, body whorl with 4 Remarks: Cipangopaludina ventricosa is similar to ridges and other whorl with 2 ridges. C. cathayensis and C. ampullacea with a slightly larger Cipangopaludina ventricosa (Heude, 1890) (Figure 1P) shell, apex pointed and whorls inflated. However, C. Paludina ventricosa Heude, 1890, Memoires conc- ventricosa can be distinguished from C. cathayensis by emant L’Histoire naturelle e L’empire chinois par des following characteristics: shell with 7 whorls vs. 6; peres de la compagnie de Jesus, 175, pl. 39. aperture round vs. ellipse; Osphradium ridge-like, longer Vivpara chinensis ventricosa (Heude): Kobelt, 1909, than length from anterior tip of osphradium to mantle Abbildungen Nach de Natur mit Beschreibngen, p 114, pl. border vs, anterior 2/3 part of osphradium ridge-like and 19, fig 1–2. posterior 1/3 part inflated, osphradium length similar

Zoological Research www.zoores.ac.cn Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 523 with length from anterior tip of osphradium to mantle aperture and body whorl depth. The pot of C. latissima border; Vas deferens opens in last 1/4of testis vs. 1/3. located in the right of region II, it indicated that this Cipangopaludina ventricosa can be distinguished from C. species has a taller body whorl and shell width, but has a ampullacea by the following characteristics: upper shorter spiral whorl. To the contrary, C. haasi looks aperture does not form straight shoulder vs. having a slimmer due to taller spiral whorl and smaller shell width. straight shoulder; umbilicus big and deep vs. closed; Osphradium longer than length from anterior tip of osphradium to mantle border vs. shorter; Vas deferens opens in last 1/4 of testis vs. 1/3.

Cipangopaludina yunnanensis Zhang, Liu et Wang, 1981 (Figure 1M) Cipangopaludina menglaensis Zhang, Liu et Wang, 1981, Acta Zootaxonomica Sinica, 6(1): 40–41. Materials: 48 ex. IOZ–FG 0152 (holotype), IOZ– FG164354–164400 (Paratypes) from Maliping village, Pu’er City, Yunnan in May 1957 by Zhang WZ and Liu

YY. st nd Diagnosis: shell greenish, small, up to 26 mm, with Figure 3 Scatterplots of scores on 1 and 2 priniciple 5 whorls. Apex obtuse. Shell surface with conspicuous components of Chinese Cipangopaludina growth lines and irregular malleation. Central tooth with wide rounded major denticles and 3 or 4 smaller trian- Table 2 Loadings of the first two principal components for gular denticles on each side. Lateral tooth with tongue- 5 shell characteristics of Chinese Cipangopaludina species shaped major denticles and 3 or 5 minor denticles on PCA 1 PCA 2 each side. Inner marginal tooth with tongue-shape major W/D 0.832* 0.322 denticles and 3 or 4 minor denticles and marginal teeth LA/D 0.571 0.513 with 13 almost equal-sized denticles. WA/D 0.397 0.914* Distribution: only known from type locality. BW/D 0.949* 0.288 Remarks: Without anatomized specimens, the WA/LA 0.078 0.858* anatomy characters remain unknown. However, it can be BW/LA 0.856* 0.001 distinguished from other species of Cipangopaludina by BW/WA 0.727* –0.682 a smaller shell, up to 30 mm and marginal teeth with 13 Prp. Total 55.9 29.5 almost equal-sized denticles. * loadings> 0.80.

Morphometric analysis by PCA Additionally, C. haasi, located in left bottom of The five shell characteristics are shown in Table 1. region III, has an oval aperture. Cipangopaludina The first two factors PCA accounted for 85.4% of the hainanensis and C. cathayensis located in the top of total variance (Table 2). The first factor (55.9% of the region I and II, they have a bigger WA and relatively variation of the selected variables) separated the ratios smaller LA, indicating these two species have a round W/D, BW/D, BW/LA and BW/WA (factor loadings >80). aperture. The second factor (29.5% of the variation of the selected Key to species of Chinese Cipangopaludina variables) separated the WA/D and WA/LA. A scatter 1 Shell with brown color band and ridge map was made by averaging the first and second PCA ...... C. ussuriensis factors per species (Figure 3). In PCA 1 axis (X-axis), W – Shell without brown color band or ridge ...... 2 and BW are positively, D and LA negatively related to 2 Apex pointed...... 3 X-axis, respectively. In PCA 2 axis (Y-axis), WA is – Apex obtuse or eroded ...... 11 positive, D and LA negatively related to Y-axis, 3 The upper of body whorl form a flat shoulder respectively. These variances are related to the shape of ...... C. latissima

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 524 LU, et al.

– The upper of body whorl without a flat shoulder ...... 4 these, as the shell of Cipangopaludina, Margarya, and 4 Shell with 5 whorls, spiral whorl not inflated Trochotaia tend to be larger, while other four genera ...... C. aubryana species are more mid-sized. Similarly, species of – Shell with 6–7 whorls, and spiral whorl inflated...... 5 Filopaludina and Mekongia have a color band, and 5 Vas deferens opens in last 1/3 of testis ...... 6 Angulyagra has ridges. For more detailed markers, – Vas deferens opens in last 1/4 of testis ...... 10 Cipangopaludina can be distinguished from Trochotaia 6 The first anterior branch of vas deferens with more by shell round or oval vs. depressed pyramidally, last than 5 secondary branches...... 7 whorl with a sharp keel, and from Margarya by shell

– The first anterior branch of vas deferens with less than smooth vs. shell with ridges, nodules or spines; embryo 3 secondary branches...... 9 with chaetae vs. ridges or nodules. Based on these 7 Shell width shorter than body whorl depth; the first criteria, we opted to treat Cipangopaludina as a valid anterior branch of vas deferens with 8 secondary genus. branches...... C. haasi Generally, the diagnostic characteristics of

– Shell width longer than body whorl depth; the first ant- Cipangopaludina are shell median to large, sculpture erior branch of vas deferens with 5–6 secondary bra- lacking except growth lines and axial undulations. nches...... 8 Aperture subcircular. Young specimens (from brood 8 Shell thin; umbilicus small, nearly closed pouch) with up to 3–4 whorls; protoconch smooth, three ...... C. chinensis primary rows of chaetae on last whorl and other whorls

– Shell thick; umbilicus big and deeply .....C. c. fluminalis have two rows of chaetae. However, C. menglaensis and 9 Vas deferens with 3 main branches...... C. cathayensis C. yunnanensis are small, up to 30 mm, smooth and solid, – Vas deferens with 2 main branches ...... C. lecythoides suggesting that C. menglaensis and C. yunnanensis 10 Vas deferens with 2 main branches; kidney triple belong to the genus Mekongia. It is also possible that C. shape...... C. lecythis dianchiensis could belong to the genus Margarya due to – Vas deferens with 3 main branches; kidney arch shape several characteristics: shell thick with ridges, female ...... C. ventricosa pouch with few big embryos and embryos shell with

11 Shell medium, with 4–5 whorls...... C. hainanensis ridges (not chaetae). Similarly, Cipangopaludina – Shell larger, with 6 whorls ...... 12 ampullacea and C. lecythis, C. fluminalis and C. 12 Vas deferens with 2 main branches, opens in last 1/4 of chinensis can be distinguished by shell characteristics, testis...... C. l. ampullacea but they do not appear to possess any obviously – Vas deferens with 3 main branches, opens in last 1/3 distinctive anatomy, placing C. ampullacea and C. of testis ...... C. leucostoma fluminalis as a subspecies of C. lecythis and C. chinensis, respectively. Our analysis also placed C. leucostoma DISCUSSION (Heude) and C. diminuta (Heude) as synonymous with C. patris (Kobelt) according to apex obtuse and less than 6 Alongside numerous difficulties in determining the whorls. However, according to the International Code of validity of numerous Cipangopaludina—lack of anatomy Zoological Nomenclature (ICZN, 1999), C. leucostoma samples, discrepancies in the literature, or ambiguous should be valid due to its publication earlier than C. morphological data—the genus itself is also problematic. patris. Finally, Cipangopaludina wingatei and C. Smith (2000) treated Cipangopaludina as subgenus of ampulliformis were found to be synonyms of C. Bellamya, while Sengupta et al (2009) indicated that chinensis and C. lecythis, respectively. species of Bellamya from Asia could not belong to this Totally, our analysis of 18 Cipangopaludina species genus. In China, there are currently seven recognized resulted in a revised taxonomy that includes 11 species genera of Bellamyinae recorded, viz. Margarya Nevill, and 2 subspecies recorded in China, viz. C. aubryana, C. 1877, Cipangopaludina Hannibal, 1912, Bellamya cathayensis, C. chinensis, C. haasi, C. hainanensis, C. Jousseaume, 1886, Mekongia Crosse et Fischer, 1876, latissima, C. lecythis, C. lecythoides, C. leucostoma, C. Filopaludina Habe, 1964, Angulyagra Benson, 1836 and ussuriensis, C. ventricosa and C. chinensis fluminalis, C. Trochotaia Brandt, 1974 (Liu et al, 1979, 1993; Du et al, lecythis ampullacea. Based on whether or not the spiral 2011). Generally speaking, it is possible to distinguish whorl depth was longer than aperture depth, Chinese

Zoological Research www.zoores.ac.cn Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 525

Cipangopaludina species could be divided to two groups, anterior 2/3 part of osphradium ridge-like and posterior the chinensis group with spiral whorl depth longer than 1/3 part inflated. Cipangopaludina leucostoma can be aperture depth that includes C. chinensis, C. haasi, C. distinguished from C. l. ampullacea and C. ventricosa by ussuriensis and C. c. fluminalis, and the other the vas deferens opening in last 1/3 vs. 1/4, and C. ventricosa cathayensis group with spiral whorl depth being shorter can be distinguished from C. l. ampullacea by vas than aperture depth, including C. aubryana, C. catha- deferens with 3 branches vs. 2 branches. yensis, C. hainanensis, C. latissima, C. lecythis, C. lecyt- Similar to our study, most viviparids are primarily hoides, C. leucostoma, C. ventricosa and C. l. ampul- delineated by their shells, though a few observations on lacea. Several anatomical characteristics can also help the anatomy have also been made. Vail (1977) for distinguish between the two groups. For example, the example mentioned that the reproductive system was a chinensis group can be distinguished from cathayensis useful characteristics to delineate viviparid subfamilies group by vas deferens: chinensis group species with and genera by anatomical studies of mature male and more than 5 (C. haasi with 8) parallel secondary bran- female Campeloma geniculum (Conrad, 1834), Lioplax ches in anterior branch, while cathayensis group species pilsbryi Walker, 1905 and Viviparus georgianus (Lea, always with 4 secondary branches. The number of 1834). Simone (2004) and Rao (1925) also reported secondary branches of vas deferens are in connection intergeneric differences in the central nervous system, with size of body whorl. The shell of chinensis group stomach lining and form of the ctenidial filaments in the species was taller, with body whorl depth being the same five Asian genera. Du et al (2011) later reported a or longer than body width, making the vas deferens difference in kidney shape and the number of embryos longer and with more secondary branches, especially in present in the uterus of four Chinese viviparids, C. haasi. However, the body whorl of cathayensis group Angulygra, Cipangopaludina, Margarya and Trochotaia. species is inflated, with body whorl depth shorter than In this study, the size of osphradium, location of vas body width, resulting in vas deferens being short and fat, deferens opens and the number of secondary branches, and the secondary branch with many tiny branches, kidney shape were useful anatomical characteristics to looking rather similar to a lush tree. identify interspecies of Cipangopaludina. The allometric Within each group, there are further defining growth or environmental effects could result in characteristics of individual species. In chinensis group, intraspecific variations in shell morphology and C. ussuriensis can be easily distinguished from other operculum (Chiu et al, 2002), but these anatomical species by body whorl with 4 ridges and colour band. characteristics could be used to identify young specimen. Cipangopaludina haasi can be distinguished from other Given the difficulties in identifying species solely species by body whorl not inflated, vas deferens with 8 by shell characters, further description of such species secondary branches in anterior branch and vas deferens anatomical characteristics of both young and mature opens in last 1/4 of testis. Cipangopaludina chinensis individuals may prove useful in further refining the fluminalis, however, is quite similar with C. chinensis, taxonomy and identifying species and determining their except for the shell thick vs. thin; umbilicus big and validity. Unfortunately, until further studies are deeply vs. small or closed. In cathayensis group, C. undertaken that more accurately characterize the latissima could be distinguished from other species by its anatomy or even underlying genetic differences, there markedly enlarged aperture while C. hainanensis can be will remain a variety of unanswered or unanswerable distinguished from other species by apex eroded, left 4 questions. To date, the monophyly of Cipangopaludina is whorls. According to kidney shape, the other species still unresolved. A recent study by Du et al (2013) on the could be divided to two subgroups. Triple shape, phylogeny of Margarya based on combined COⅠ and including C. cathayensis, C. aubryana and C. lecythis, 16S rRNA sequences indicated that Cipangopaludina is and arch shape, including C. l. ampullacea, C. not a monophyly, since species cluster with Margarya; leucostoma and C. ventricosa. Cipangopaludina lecythis however, the specimens of Cipangopaludina and can be distinguished from C. cathayensis and C. Margarya are from Lake Dianchi. Likely further studies aubryana by vas deferens with 2 branches and opens in into the phylogeny and ecology of the Cipangopaludina last 1/4 of testis vs. 3 branches and 1/3, and C. catha- in different environments and distributions will further yensis can be distinguished from C. aubryana by the enhance our understanding of these organisms.

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 526 LU, et al.

Acknowledgments: We thank Jiang YE, Cui CAS for generous helping when first author check GH, Zheng LP, Jiang WS, Yuan C, Li Y and Yu GH of specimens at IOZ. We specially thank Liu YY for Kunming Institute of Zoology, CAS and Aldridge D of showing how to identify these species. We thank the Cambridge University for collecting these specimens. We anonymous reviewers for their helpful comments on the greatly thank Meng K and Jia LJ of Institute of Zoology, manuscript.

References

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Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 Zoological Research 35 (6): 528−536 DOI:10.13918/j.issn.2095-8137.2014.6.528

Genetic diversity and population structure of a Sichuan sika deer (Cervus sichuanicus) population in Tiebu Nature Reserve based on microsatellite variation

Ya HE 1,2, Zheng-Huan WANG1, Xiao-Ming WANG1,2,*

1. School of Life Sciences, East China Normal University, Shanghai 200062, China 2. Shanghai Science & Technology Museum, 2000 Century Avenue, Shanghai 200127, China

Abstract: Cervus sichuanicus is a species of sika deer (Cervus nippon Group). To date, research has mainly focused on quantity surveying and behavior studies, with genetic information on this species currently deficient. To provide scientific evidence to assist in the protection of this species, we collected Sichuan sika deer fecal samples from the Sichuan Tiebu Nature Reserve (TNR) and extracted DNA from those samples. Microsatellite loci of bovine were used for PCR amplification. After GeneScan, the genotype data were used to analyze the genetic diversity and population structure of the Sichuan sika deer in TNR. Results showed that the average expected heterozygosity of the Sichuan sika deer population in TNR was 0.562, equivalent to the average expected heterozygosity of endangered animals, such as Procapra przewalskii. Furthermore, 8 of 9 microsatellite loci significantly deviated from the Hardy-Weinberg equilibrium and two groups existed within the Sichuan sika deer TNR population. This genetic structure may be caused by a group of Manchurian sika deer (Cervus hortulorum) released in TNR.

Keywords: Sichuan sika deer; Microsatellite; Genetic diversity; Population structure

The Sichuan sika deer (Cervus sichuanicus) (Guo et relationship of sika deer using mitochondrial DNA, al, 1978) is a species of the Cervus nippon group (Groves which also had a small number (16) of Sichuan sika deer and Grubb, 2011). It is an endemic species in China and samples. These studies focused on the differences among is listed as a classⅠ endangered species, as well as an species and genetic structure of sika deer in China. Due endangered species by the International Union for to the limited samples, however, the above data do not Conservation of Nature and Natural Resources (IUCN). reflect genetic information in one species or population. Currently, the Sichuan sika deer is distributed in three Moreover, using traditional capture methods makes it unconnected areas located in northwest Sichuan Province difficult to obtain abundant samples to study genetic and southwest Gansu Province. Compared to other spec- diversity and population structure within Sichuan sika ies in China, Sichuan has the largest sika deer population deer populations. 1 in the wild, consisting of about 850 individuals (Guo, The Tiebu Nature Reserve (TNR) in the Zoige 2000). administrative district of northwest Sichuan has the In recent years, most research has focused on largest sika deer population. According to monitoring macroscopic aspects such as social behavior (Guo et al, data from TNR, there were about 1 000 Sichuan sika deer 1991), distribution and quantity surveying (Guo, 2000), activity rhythms (Liu et al, 2004), and food habits (Guo, Received: 25 March 2014; Accepted: 01 August 2014 2001). At the genetic level, Wu et al (2008) chose 16 Foundation items: The work was supported by the National Natural microsatellite loci to analyze the genetic diversity of Sika Science Foundation of China (30870308 and 31071944) and the deer in China, including 24 samples of Sichuan sika deer. Shanghai Rising-Star Program (10QA1402200) Lü et al (2006) and Liu et al (2003) studied the genetic *Corresponding author, E-mail: [email protected]

Science Press Volume 35 Issue 6

Genetic diversity and population structure of a Sichuan sika deer (Cervus sichuanicus) population in Tiebu Nature Reserve based on microsatellite variation 529 in the reserve in 2012. TNR has been chosen as the main from those samples, nine Bovinae microsatellite loci area to study Sichuan sika deer due to large population were amplified and the products were used for gene size and better protection situation (Li, 2010; Ning et al, scanning. We then used the genotype data to analyze 2008; Qi et al, 2010). To date, however, no genetic genetic diversity and population structure. The results of information studies on TNR sika deer have been this study provide a scientific basis for the protection of conducted and the genetic diversity and possible the Sichuan sika deer. population substructures remain unknown. Previous studies have shown that the geographical distribution MATERIALS AND METHODS pattern of the population is related to geographical isolation (Boyce et al, 1999; Gavin et al, 1999; Scandura Study area and sampling collection et al, 1998). However, it is unclear whether the rivers, Fecal samples were collected in seven areas in TNR 2 roads, villages, and crop protection fences across the (E102°46′–103°14′, N33°58′–34°16′, about 206 922 km ), reserve affect gene flow or generate genetic structures as shown in Figure 1. To avoid cross-contamination within the sika deer population. Therefore, we when sampling, we only collected one pellet from each hypothesized that geographic and artificial factors may fecal heap. We collected five hides, one tissue sample, affect the gene flow of sika deer, and thus established and 149 fecal samples. The hide samples were collected seven sampling areas in TNR to obtain sika deer fecal from dead individuals in the wild. The only tissue sample samples. Overall, we collected five hides, one tissue came from an individual that had been killed by poachers. sample, and 149 fecal samples. After extracting DNA All samples were stored in 95% ethanol.

Figure 1 Distribution of sampling areas in Tiebu Nature Reserve

DNA extraction and amplification dified fluorescent groups. The PCR mixture contained DNA was extracted using an E.Z.N.A HP Tissue 1.5 μL genomic DNA (100 ng/μL), 1 μL of each pri- DNA Maxi Kit and a Stool DNA kit (OMEGA) mer (10 pmol/μL), 0.5 μL BSA (20 mg/μL), and 7.5 μL according to the manufacturer’s protocols. The former of Premix Ex Taq (Takara), with deionized water used to primers of nine Bovinae microsatellite loci (IDVGA29, make the sample volume up to 15 μL. The PCR amp- BM4107, RT1, TGLA53, RM188, RT13, BM6506, lification was carried out using an initial denaturation at BL42, CSSM019, ETH225, and CERVID14) were mo- 94 C for 3 min, followed by 35 cycles at 94 C for 30 s,

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 530 HE, et al. annealing for 30 s, primer extension at 72 C for 45 s, control to ensure that PCR was performed correctly and a final extension at 72 C for 10 min. The and to avoid contamination. The PCR products were products were preserved at 4 C. One tissue sample sent to Shanghai MAP Biotechnology Co., Ltd was used as positive control and water as negative (Shanghai, China) for GeneScan.

Table 1 Information on the nine microsatellite bovine loci used in this research Annealing Sources of Locus Repeat motif Primer sequence Size range (bp) temperature (℃) primers

CCC ACA AGG TTA TCT ATC TCC AG Konfortov et al IDVGA29 (AC)n 137−157 60 CCA AGA AGG TCC AAA GCA TCC AC (1996)

AGC CCC TGC TAT TGT GTG AG Bishop et al BM4107 (AC)n (TC)n (TG)n 162−172 60 ATA GGC TTT GCA TTG TTC AGG (1994)

TGC CTT CTT TCA TCC AAC AA Wilson & White RT1 (GT)n 222−240 55 CAT CTT CCC ATC CTC TTT AC (1998)

CAG CAG ACA GCT GCA AGA GTT AGC Hoffmann et al TGLA53 (AC)n 176−216 50 CTT TCA GAA ATA GTT TGC ATT CAT GCA G (2004)

GGG TTC ACA AAG AGC TGG AC Barendse et al RM188 (AC)n 141−151 50-52 GCA CTA TTG GGC TGG TGA TT (1994)

GCC CAG TGT TAG GAA AGA AG Bishop et al RT13 (GT)n 293−324 60 CAT CCC AGA ACA GGA GTG AG (1994)

GCA CGT GGT AAA GAG ATG GC Moore et al BM6506 (AC)n 196−212 59−63 AGC AAC TTG AGC ATG GCA C (1994)

CAA GGT CAA GTC CAA ATG CC Bishop et al BL42 (AC)n 246−258 58−60 GCA TTT TTG TGT TAA TTT CAT GC (1994)

TTG TCA GCA ACT TCT TGT ATC TTT Wilson & White CSSM019 (AC)n 138−156 52−58 TGT TTT AAG CCA CCC AAT TAT TTG (1998)

GAT CAC CTT GCC ACT ATT TCC T Steffen et al ETH225 (AC)n 140−193 60 ACA TGA CAG CCA GCT GCT ACT (1993)

TCT CTT GCG TCT CCT GCA TTG AC De Woody et al CERVID14 (AC)n 214−231 61−65 AAT GGC ACC CAC TCC AGT ATT CTT C (1995)

Species identification and population assessment (Park, 2001) to assess the population number: we We amplified all samples using two pairs of primers allowed one genetic mismatch at one allele for one locus of the mitochondrial control region and compared the to be considered as two samples belonging to the same sequences in GenBank to ensure the samples in our individual (Bellemain et al, 2005). To detect the validity research were from sika deer. We used GeneMapper v. 4 of the above principle, we randomly selected 50 fecal (Applied Biosystems) to obtain the microsatellite samples (accounting for a third of the total number) and fragment sizes and identified the heterozygotes and amplified the nine loci twice. At the same time, we homozygotes. We did two repeats if the genotypes of the amplified another two loci (ETH225 and CERVID14) two repetitive PCRs were different; for example, if there of the selected 50 samples. We tested whether the was a heterozygote in one repeat but a homozygote in the influence of population number may be caused by the other repeat. If there was a heterozygote in three of four repeat amplification and increasing loci. repeats, we considered this site to be a heterozygous locus; the same for a homozygote locus. The probability Genetic diversity and population structure of identity (PI) and the PI between siblings were We computed the expected heterozygosity (HE), calculated in Gimlet 1.3.3 (Valière, 2002; Liu & Yao, observed heterozygosity (HO), and polymorphism infor- 2013) using genotype data of the nine microsatellite loci. mation content (PIC) using Mstools. Arlequin 3.1 (Exc- We used the following principle when using Mstools offier et al, 2005) and Fstat 2.9.3.2 (Goudet, 2001) were

Zoological Research www.zoores.ac.cn Genetic diversity and population structure of a Sichuan sika deer (Cervus sichuanicus) population in Tiebu Nature Reserve based on microsatellite variation 531 used to test the Hardy-Weinberg equilibrium and Fis deer individuals from 143 face samples. After the second separately. The fixation index (FST) (Weir & Cockerham, amplification of the randomly selected samples, the 1984) among sampling areas was tested in Arlequin 3.1, population number did not change. The additional two using 1 000 random iterations to calculate confidence loci (ETH225 and CERVID14) increased the population intervals (CI). Software structure 2.3 (Pritchard et al, by one individual, accounting for 3% of the total number. 2000) was used to estimate the potential populations (K). The distribution of the 76 individuals in the samplings The admixture model was used and run three times using areas is shown in Table 3. 10 replications of K ranging from 1 to 7. For those runs the following conditions were adopted: a burn-in period at 10 000 following 10 000 000 replicates of the MCMC. The ΔK calculated as per Evanno et al (2005) was used to estimate optimal K. The formulas were: ΔK=m|L(K+1)−2L(K)−L(K−1)|/s[L(K)]. We referred to the structure output lnP(D) as L(K). m was the mean value and s was the standard deviation. Software CLUMPP Windows.1.1.2 (Jakobsson & Rosenberg, 2007) and destruct (Rosenberg, 2004) were used to visualize the individual coefficients of membership in the subpopulation calculated by structure 2.3.

Figure 2 Multi-loci PI (biased and unbiased) and PI (sibs) for RESULTS sika deer in increasing order of single-locus values

Species identification and population assessment Genetic diversity and population structure Comparing the obtained sequences of mitochondrial In the Sichuan sika deer population in TNR, the hypervariable region I from our research to the mean number of alleles / locus was 6.56 and the HE was corresponding sequence in GenBank, all samples were 0.562. The PIC values were higher than or close to 0.5, obtained from sika deer. Thereinto, 143 of 155 samples except for RT13 (0.129). All detected loci deviated from belonged to the haplotypes of Sichuan sika deer and the the Hardy-Weinberg equilibrium significantly (Figure 2) remainder belonged to the haplotypes of Manchurian and the P value was 0. In addition to the locus RT13, the sika deer (Cervus hortulorum). Fis values of the remaining eight loci were negative. The

Most of the Sichuan sika deer samples could be values of HE ranged from 0.4292 to 0.6865 in the seven amplified at all the nine microsatellite loci, few samples sampling areas and all HO values were higher than those of

(11) had a loss at one locus. Analyzing the united PI HE (Table 3). The FST among the seven sampling areas values for nine loci, we found that the discrimination ranged from –0.04781 to 0.05243. The highest FST power of nine loci was close to 100% (Figure 2). (0.05243) appeared between Area Ⅳ and Ⅵ but the lowest According to the rule of Bellemain, we assessed 76 sika (–0.04781) appeared between Area Ⅰ and Ⅲ (Table 4).

Table 2 Microsatellite diversity indices of Sichuan sika deer in Tiebu Nature Reserve

Locus No. of alleles HE HO PIC Fis IDVGA29 5 0.587 0.722 0.516 –0.230 BM4107 7 0.560 0.654 0.495 –0.169 RT1 6 0.633 0.951 0.558 –0.506 CSSM019 6 0.635 0.926 0.560 –0.462 TGLA53 8 0.643 0.899 0.571 –0.401 BL42 9 0.607 0.913 0.523 –0.509 RM188 6 0.656 0.738 0.607 –0.125 BM6506 8 0.604 0.974 0.519 –0.620 RT13 4 0.134 0.026 0.129 0.810 Mean 6.56 0.562 0.756 0.496 –0.347

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 532 HE, et al.

Table 3 Indices of genetic diversity of Sichuan sika deer in different sampling areas

Sample area Sample size Loci typed HE HO Ⅰ 10 9 0.4292 0.7160 Ⅱ 13 9 0.5499 0.8034 Ⅲ 7 9 0.4831 0.7778 Ⅳ 8 9 0.6804 0.7284 Ⅴ 20 9 0.6865 0.7444 Ⅵ 11 9 0.4350 0.7063 Ⅶ 7 9 0.5368 0.8571

Table 4 F-statistics (FST, below diagonal) and significant differences among different areas Sample area Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ Ⅶ Ⅰ + – + + – + Ⅱ –0.00493 – – – + – Ⅲ –0.04781 –0.03753 – – – – Ⅳ 0.03553 –0.0052 –0.00225 – + – Ⅴ 0.05177 0.00789 0.01646 –0.01778 + – Ⅵ –0.02823 –0.00142 –0.0334 0.03287 0.05243 + Ⅶ 0.01389 –0.02825 –0.03837 –0.01869 0.01487 –0.00278 + denotes significant difference, – in contrast (95% confidence interval).

Population structure Structure analysis showed that when K=2, the ΔK DISCUSSION exhibited an obvious apex (Figure 3). The FST between subpopulations was 0.535. Individual coefficients of Population assessment In recent years, non-invasive sampling methods membership in subpopulations (Figure 4) showed that have been widely used in genetic studies of endangered when K=2, the genetic amount of cluster 1 (Yellow) was animals (He et al, 2010; Wang et al, 2012). Because of larger and distributed in all sampling areas. In contrast, the the endangered status and fugacious feature of sika deer, genetic amount of cluster 2 (Blue) was less than cluster 1, non-invasive sampling methods (obtaining fecal samples) and had a relatively smaller distribution area. Most of the have become an important means of population blue genotype was distributed in Areas Ⅳ and Ⅴ. assessment and individual identification. In this study, the stability of repeat amplification of nine microsatellite loci had no effect on assessing the population number, that is, our genotype data were reliable. After adding two loci, there was a 3% error between previous and post assessment results, which is much higher than 2% (Bellemain et al, 2005). Bellemain et al (2005) attributed such errors in population assessment to pseudogenes, pollution and missing data.

Figure 3 Value of ΔK as a function of K based on 10 runs Genetic diversity and differentiation Genetic diversity helps populations adapt to changing environments. With more variation, it is more likely that some individuals in a population will possess variations of alleles that are suited for the environment. The population will continue for more generations Figure 4 Individual coefficients of membership in each because of the success of these individuals (Khater et al,

sampling area 2011). In our research, the HE of Sichuan sika deer

Zoological Research www.zoores.ac.cn Genetic diversity and population structure of a Sichuan sika deer (Cervus sichuanicus) population in Tiebu Nature Reserve based on microsatellite variation 533 population was 0.562, close to the mean HE (0.559) of showing negative value in some regions. This indicated sika deer in China, but much higher than the mean HE for that the genetic variation among the seven sampling Sichuan sika deer (0.477) and slightly lower than the areas mainly came from inside the sampling area, and the mean HE for Manchurian sika deer (0.584) and south geographical barriers and human factors do not affect China sika deer (Jiangxi, 0.585; Zhejiang, 0.589) (Wu et gene flow in Sichuan sika deer in TNR. In addition, due al, 2008). Compared to other Ungulata, such as red deer to the relatively closed geographical position of TNR,

(Cervus elaphus) (HE=0.78, Hajji et al, 2007; HE=0.804, very few little sika deer were distributed in the periphery

Pérez-Espona et al, 2008; HE=0.62−0.85, Zachos et al, of the protected areas (Guo, 2000). Gene flow through 2007), forest musk deer (Moschus berezovskii) immigration and emigration could not significantly affect

(HE=0.8−0.9, Zhou et al, 2005), and Mongolian wild ass the Sichuan sika deer populations to deviate from the

(Equus hemionus) (HE=0.83, Kaczensky et al, 2011), Hardy Weinberg equilibrium.

Sichuan sika deer had a relatively lower HE and was Microsatellite polymorphism studies of sika deer in equivalent to Przewalski's Gazelle (Procapra przewalskii) China found that 16 microsatellite loci significantly

(HE=0.552, Yang & Jiang, 2011) and milu (Elphurus deviated from the Hardy Weinberg equilibrium, but HEs davidianus) (HE=0.46−0.54, Zeng et al, 2007). The HEs were higher than HOs (Wu et al, 2008). In the present values were different in each sampling area. Area Ⅰ had study, HOs were significantly higher than the HEs. The the lowest HE (0.4292), while Area V had the highest Fis values of eight microsatellite loci were negative, (0.6865). Polymorphism information content (PIC) is a showing heterozygosis excess. When a population measure of variation between microsatellite loci. It is experiences a reduction in its effective size, it generally generally considered that a PIC value greater than 0.5 is develops heterozygosity excess at selectively neutral loci for highly polymorphic loci, a PIC value of less than 0.5 (Cornuet & Luikart, 1996). Xu et al (2009) used and greater than 0.25 is for moderately polymorphic loci, microsatellites to detect the population structure of and a PIC of less than 0.25 is for low polymorphic loci Himalayan marmots and found heterozygosity excess (Niu et al, 2001). In this study, the PIC values of six of due to historic population decline. Wu et al (2008) tested the nine microsatellite loci were higher than 0.5, bottlenecks in four Chinese sika deer species and found indicating highly polymorphic loci, while two were that Sichuan sika deer had not experienced a population moderately polymorphic loci and one was a low bottleneck in recent history. Therefore, the polymorphic locus. Heterozygosity and PIC information heterozygosity excess of Sichuan sika deer in TNR could indicated Sichuan sika deer in TNR had abundant genetic not be caused by population reduction. diversity. Hartl & Clark (1997) considered that population Population structure deviation from the Hardy Weinberg equilibrium was A population composed of individuals from two mainly due to small populations, not random mating, different source populations will tend to have an excess gene mutation, migration or other factors. The present of heterozygotes (Milkman, 1975). In testing population study showed that nine microsatellite loci of the Sichuan structure in the present study, we found that when K=2, sika deer population in TNR deviated significantly from ΔK exhibited a significant apex, indicating that two the Hardy Weinberg equilibrium. The Sichuan sika deer subpopulations existed within the Sichuan sika deer population numbers in TNR grew steadily from 650 in population. Considering that the Sichuan sika deer 2000 (Guo, 2000) to 1 000 in 2012 (monitoring data from population had heterozygote excess and two subpo- TNR). As a result, the deviation from the Hardy pulations, we speculated that the sika deer in TNR might Weinberg equilibrium is unlikely attributed to a have two different sources populations. According to Wu reduction in the population. The coefficient of genetic (2004) and TNR staff, a captive-bred Manchurian sika differentiation (FST), on the other hand, is an important deer group was bred and released in TNR in the late indicator of genetic differentiation among subpopul- 1960s. The mitochondrial data showed that haplotypes of ations. According to Wright (1978), FSTs values bet- Manchurian sika deer (unpublished data) do exist. ween 0−0.5 suggest no differentiation between sub- Previous research has shown that introduced species groups. The FSTs values in our seven sampling areas were often hybridize with local sibling species (Avise et al, all less than 0.5, ranging from 0.04781 to 0.05243, and 1974; Gutiérrez, 1993; Perry & Goudet, 2001) and

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 534 HE, et al. panmictic admixtures can develop rapidly over a short indicate the genotypes of Sichuan sika deer. period of time (Echelle & Connor, 1989; Echelle, 1987; In the past, researchers have defined the Sichuan 1997). Hybridization abounds in Cervidae, and sika deer and Manchurian sika deer as two subspecies of introduced sika deer have been shown to hybridize with sika deer. Groves & Grubb (2011) compared antecedent native red deer (Senn & Pemberton, 2009). Therefore, research on the morphology and molecular data of sika there is a strong possibility that hybridization occurred deer and suggested the Sichuan and Manchurian sika between the two sika deer species in our research. The deer were two independent species in the Cervus nippon released Manchurian sika deer explained the existence of group. If hybridization and introgression occurred, it may subpopulations in the Sichuan sika deer population in lead to a local genetic extinction of a population (Avise et TNR. We speculate that the Manchurian sika deer al, 1997; Templeton, 1986). From the perspective of cons- genotype is the minority – the blue parts in the individual ervation biology, how genes flow between two species coefficients of membership, which were mainly mixed by human influence is a focus for future research. distributed in Areas Ⅳ and Ⅴ– because the number of introduced Manchurian sika deer is not clear and of the Acknowledgments: We wish to express our thanks to 155 sika deer fecal samples examined, only 12 belonged the staff at Tiebu Nature Reserve and Mr L. Zuo at Zoige to the haplotypes of Manchurian sika deer. The Sichuan Forestry Administration for providing support during our sika deer population were in larger quantities than that of sampling period. Special thanks to Miss J.F. Yang for our Manchurian sika deer. So, the yellow parts in Figure 4 sampling guide.

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Zoological Research www.zoores.ac.cn Zoological Research 35 (6): 537−545 DOI:10.13918/j.issn.2095-8137.2014.6.537

Complete mitochondrial genome of yellow meal worm (Tenebrio molitor)

Li-Na LIU1, Cheng-Ye WANG2,*

1. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China 2. Key Laboratory of Cultivating and Utilization of Resource Insects of State Forestry Administration, Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming 650224, China

Abstract: The yellow meal worm (Tenebrio molitor L.) is an important resource insect typically used as animal feed additive. It is also widely used for biological research. The first complete mitochondrial genome of T. molitor was determined for the first time by long PCR and conserved primer walking approaches. The results showed that the entire mitogenome of T. molitor was 15 785 bp long, with 72.35% A+T content [deposited in GenBank with accession number KF418153]. The gene order and orientation were the same as the most common type suggested as ancestral for insects. Two protein-coding genes used atypical start codons (CTA in ND2 and AAT in COX1), and the remaining 11 protein-coding genes started with a typical insect initiation codon ATN. All tRNAs showed standard clover-leaf structure, except for tRNASer (AGN), which lacked a dihydrouridine (DHU) arm. The newly added T. molitor mitogenome could provide information for future studies on yellow meal worm.

Keywords: Tenebrionidae; Mitogenome; Evolution; Resource insect; Yellow meal worm

Mitochondrial genes have been widely used in important resource insect, which has great value as an studies of phylogenetics, phylogeography and molecular animal feed additive because of its high protein content. diagnostics (Kocher et al, 1989; Simmons & Weller, It has also been used in numerous experimental studies 2001; Wolstenholme, 1992). The full-length mitochon- (Gomez et al, 2013; Simon et al, 2013). Its relatively drial genome (mitogenome) has become an important large body-size and ease of rearing make it attractive for tool for studies of genome architecture, population gene- biological research. An increasing number of researchers tics, primer design, and molecular evolution (Kim et al, have used this beetle as a model system for studies in 2009; Lee et al, 2009; Liao et al, 2010; Yukuhiro et al, biology, biochemistry, evolution, immunology and 2002). Furthermore, mitogenome sequences provide im- physiology (Dobson et al, 2012; Goptar et al, 2013; Ohde portant information for pest control. For example, insec- et al, 2013; Prokkola et al, 2013; Yan et al, 2013; Zhu et ticide resistance in arthropod pest Tetranychus urticae is al, 2013). However, the genetic background of this beetle, controlled by mtDNA (Van Leeuwen et al, 2008). in both nuclei and organelles, has not yet been determ- Tenebrionidae is a large family of Coleoptera and ined, and information is urgently needed. 1 more than 20 000 species have been described worldwide In this study, we sequenced the whole mitogenome (Bouchard et al, 2011). Despite this large taxonomic of T. molitor for the first time and analyzed its nucleotide diversity, information about the Tenebrionidae mito- composition and major characteristics. The entire genome is still limited. To date, only two complete mit- mitogenome was found to be 15 785 bp long and harbo- ogenomes are available in GenBank (NC_003081: Trib- red 13 protein-coding genes (PCGs), 22 tRNA genes, and olium castaneum and NC_013554: Adelium sp. NCS- 2009). Due to the importance of mitogenomes in popul- Received: 27 March 2014; Accepted: 25 July 2014 ation studies and pest control, studies on the mitogenome Foundation item: This work was supported by grant from the Science of tenebrionids are required. and Technology Committee of Yunnan Province (2011FB141) The yellow meal worm (Tenebrio molitor L.) is an *Corresponding author, E-mail: [email protected]

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2 rRNA genes. The gene arrangement in the T. molitor Gene identification and tRNA structures mitogenome was identical to the ancestral insect mitog- Sequences from overlapping fragments were assem- enome arrangement (Boore et al, 1998). The mitogenome bled with the neighboring fragments using the SeqMan of T. molitor is an important addition to the continuing program included in the Lasergene software package efforts in developing T. molitor as a model system for (DNAStar Inc., Madison, Wisc.). Comparison of the biological studies. DNA or amino acid sequences with homologous regions of known full-length insect mitogenome sequences MATERIALS AND METHODS indicated 13 protein-coding genes (PCGs) and two rRNA genes based on MEGA 5.0 software (Tamura et al, 2011). Sample and DNA extraction The nucleotide sequences of the PCGs were translated on An adult T. molitor was collected in Kunming, the basis of the invertebrate mtDNA genetic code. Yunnan Province, China, on July 29, 2012. The adult Transfer RNA gene analysis was conducted based on insect was one of the descendants from our inbred line, tRNAscan-SE software v.1.21 (Lowe & Eddy, 1997), and which has been established and maintained in our the folding of the predicted tRNA sequences were further laboratory since May 2008. All members of this inbred confirmed by visual inspection. Finally, the T. molitor line have one common ancestral mitochondrial geno- mitogenome sequence was deposited in GenBank with me background. After collection, the fresh materials accession number KF418153. were immediately preserved in 100% ethanol and stored in a −20 °C refrigerator before genomic DNA Sequence analysis extraction. Total genomic DNA was extracted with the The A+T-content of the whole genome was calcul- WizardTM Genomic DNA Purification Kit, in accor- ated based on the EditSeq program included in the dance with the manufacturer's instructions (Promega, Lasergene software package. The nucleotide composition USA). at each codon position of the PCGs and codon usage were calculated based on MEGA 5.0. Gene overlaps and PCR amplification and sequencing intergenic-space sequences were hand-counted. To sequence the complete mitogenome, long PCR Nucleotide composition skew was calculated for the primers and some short PCR primers were designed by PCGs and the whole genome base on the EditSeq prog- multiple sequence alignments of all available complete ram included in the Lasergene software package (DN- Coleoptera mitogenomes using ClustalX1.8 (Thompson AStar Inc., Madison, Wisc.), using the following formula et al, 1997) and Primer Premier 5.0 software. Primer proposed by Perna & Kocher (1995): GC-skew=(G– sequence information can be obtained from the authors C)/(G+C) and AT-skew=(A–T)/(A+T), where C, G, A and upon request. Long PCRs were performed using TaK- T are the frequencies of the four bases. aRa LA Taq polymerase with the following cycling parameters: initial denaturation for 5 min at 95 °C, followed by 30 cycles at 95 °C for 50 s, 50 °C for 50 s, RESULTS AND DISCUSSION 68 °C for 2 min and 30 s; and a final extension step of General features of Tenebrio molitor mitogenome 68 °C for 10 min. Short fragments were amplified The complete mitogenome of T. molitor was 15 785 with TaKaRa Taq polymerase: initial denaturation for bp in length, and consisted of 2 rRNAs (srRNA and 5 min at 94°C, followed by 35 cycles at 94 °C for 1 lrRNA), 13 PCGs (ATP6, ATP8, COI-III, ND1-6, ND4L, min, 45−53 °C for 1 min, 72 °C for 2 min, and a final CytB), and 22 tRNAs (Figure 1). Like many other insect extension step of 72 °C for 10 min. The PCR mitogenomes, the major strand (J strand) carried most of products were detected via electrophoresis in 1.5% the genes (9 PCGs and 14 tRNAs), whereas the agarose gel, and purified using the QIAquick PCR remaining genes were on the minor strand (N strand) (4 Purification Kit (QIAGEN, USA). Sequencing PCGs, 8 tRNAs and 2 rRNA genes) (Figure 1). Gene reaction was performed using a BigDye Terminator order and orientation were the same as the most common v3.1 Cycle Sequencing Kit (Applied Biosystems, type suggested as ancestral for insects [18, 23] (Boore et Foster City, CA). Electrophoresis of purified al, 1998; Taanman, 1999). sequencing products was performed on an ABI-3730 All genes were closely assembled and only nine DNA Analyzer (Applied Biosystems). intergenic spacers were observed. These intergenic

Zoological Research www.zoores.ac.cn Complete mitochondrial genome of yellow meal worm (Tenebrio molitor) 539

Figure 1 Circular map of the Tenebrio molitor mitogenome COⅠ, COⅡ and COⅢ refer to the cytochrome C oxidase subunits; CytB refers to cytochrome B; ATP6 and ATP8 refer to subunits 6 and 8 of F0 ATPase; ND1-6 refer to components of NADH dehydrogenase. tRNAs are denoted as one-letter symbols consistent with the IUPAC-IUB single letter amino acid codes. Gene names not underlined indicate a clockwise transcriptional direction, whereas underlines indicate a counter-clockwise transcriptional direction. L* and S* denote tRNALeu(UUR) and tRNASer(UCN), respectively. spacers were 53 bp in total, with individual sizes ranged codon for COⅠ, COⅡ, ND5 and CytB, and TA was from 1 to 22 bp. In addition, there were 36 bp overl- deemed as the stop codon for ATP6, ND4, ND4L and apping sequences totally in 15 overlaps between different ND6 in Coreana raphaelis (Kim et al, 2006); similarly, a genes, ranging from 1−7 bp (Table 1). single T was considered as the stop codon for COⅠ, CO Comparative analysis with two other published Ⅱ and ND4, and TA residue as the stop codon for ATP6 Tenebrionidae mitogenomes of Tribolium castaneum and in Hyphantria cunea (Liao et al, 2010). Incomplete stop Adelium sp. NCS-2009 exhibited highly conserved geno- codons also exist in vertebrate mitochondrial genomes such me architectures including gene order, nucleotide compo- as in human mitochondria (Andrews et al, 1999). It has sition, codon usage, and amino acid composition (Friedr- been demonstrated that incomplete stop codons can produce ich & Muqim, 2003; Sheffield et al, 2009). functional stop codons in polycistronic transcription cleavage and polyadenylation processes (Ojala et al, 1981). Protein-coding genes (PCGs) Eleven of the 13 PCGs of T. molitor used standard Transfer RNA and ribosomal RNA genes ATN (Met) start codon, while the remaining two PCGs Twenty-two tRNA genes (one specific for each used atypical start codons (CTA in ND2 and AAT in amino acid and two for leucine and serine) were identi- COX1) (Table 1). ATP6, ATP8, ND4L, ND6, and CytB fied within the T. molitor mitogenome. The 22 tRNA genes had the common stop codon TAA; ND1 and ND3 genes were interspersed throughout the coding region had the stop codon TAG; while ND2, COX1, COX2, and ranged from 60 to 70 bp in length (Table 1). All COX3, ND5, and ND4 terminated with incomplete stop tRNAs, except tRNASer (AGN), were folded into typical codon T or TA (Table 1). Similar cases have been found cloverleaf secondary structures (Figure 2). The unusual in other insect mitogenomes (Yin et al, 2012). For tRNASer (AGN) lacked a dihydrouridine (DHU) arm, example, a single T residue was deemed as the stop which was replaced by a simple loop (Figure 2). Another

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6 540 LIU &WANG unusual feature was the use of TCT as the tRNASer (AGN) et al, 2011; Liao et al, 2010; Salvato et al, 2008; anticodon instead of GCT, which is used in most other Wolstenholme, 1992; Yang et al, 2011). arthropods. This incomplete tRNASer structure has also Twenty-two non-Watson-Crick base pairs were been detected in other insect groups (Kim et al, 2006; Li observed in the T. molitor mt tRNAs, including 17 G-U

Table 1 Summary of Tenebrio molitor mitogenome Gene Direction Nucleotide number Size (bp) Anticodon Start codon Stop codon tRNAIle F 1–64 64 29–31 GAT – – tRNAGln R 62–130 69 98–100 TTG – – tRNAMet F 131–198 68 160–162 CAT – – ND2 F 204–1206 1003 – CTA(M) T(AA)# tRNATrp F 1207–1272 66 1240–1242 TCA – – tRNACys R 1295–1355 61 1324–1326 GCA – – tRNATyr R 1356–1421 66 1388–1390 GTA – – COX1 F 1423–2956 1534 – AAT(M) T(AA)# tRNALeu(UUR) F 2957–3021 65 2986–2988 TAA – – COX2 F 3022–3709 688 – – – tRNALys F 3710–3779 70 3740–3742 CTT – – tRNAAsp F 3779–3843 65 3809–3811 GTC – – ATP8 F 3844–3999 156 – ATA(M) TAA ATP6 F 3993–4664 672 – ATG(M) TAA COX3 F 4664–5448 785 – ATG(M) TA(A)# tRNAGly F 5448–5509 62 5478–5480 TCC − − ND3 F 5510–5863 354 – ATT(M) TAG tRNAAla F 5862–5927 66 5891–5893 TGC – – tRNAArg F 5927–5990 64 5957–5959 TCG – – tRNAAsn F 5990–6054 65 6021–6023 GTT – – tRNASer(AGN) F 6055–6114 60 6077–6079 TCT – – tRNAGlu F 6116–6177 62 6146–6148 TTC – – tRNAPhe R 6176–6239 64 6206–6208 GAA – – ND5 R 6240–7953 1714 – ATA(M) T(AA)# tRNAHis R 7951–8013 63 7981–7983 GTG - - ND4 R 8014–9346 1333 – ATG(M) T(AA)# ND4L R 9340–9627 288 – ATG(M) TAA tRNAThr F 9630−9692 63 9660−9662 TGT – – tRNAPro R 9693−9758 66 9726−9728 TGG – – ND6 F 9761−10258 498 – ATT(M) TAA CytB F 10258−11394 1137 – ATG(M) TAA tRNASer(UCN) F 11393−11458 66 11423−11425TGA − − ND1 R 11476−12429 954 − ATA(M) TAG tRNALeu(CUN) R 12427−12491 65 12460−12462TAG – – lrRNA R 12491–13771 1281 − – – tRNAVal R 13774−13842 69 13810-13812 TAC – – srRNA R 13844−14615 772 – – – A+T-rich region R 14616−15785 1170 – – – tRNA abbreviations follow the IUPAC-IUB three letter code. #: TAA stop codon is completed by the addition of 3' A residues to the mRNA.

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Figure 2 Predicted secondary clover-leaf structure for the 22 tRNA genes of the Tenebrio molitor mitogenome The tRNAs are labeled with abbreviations of their corresponding amino acids. Point (●) indicate Watson-Crick base-pairing. Arms of tRNAs (clockwise from top) are amino acid acceptor (AA) arm, TψC (T) arm, anticodon (AC) arm and dihydrouridine (DHU) arm.

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pairs, which form a weak bond in the tRNAs. The (Andrews et al, 1999; Kim et al, 2009). The A+T-rich remaining five were atypical pairings: one mismatch in region in insect mitogenomes is equivalent to the control tRNALeu (CUN) (U-U), one in tRNALeu (UUR) (U-U), two region of vertebrate mitogenomes, which harbor the in tRNATyr (2 U-U), and one in tRNATrp (A-G) (Figure 2). origin sites for transcription and replication (Andrews et These mismatches found in tRNAs can be corrected al, 1999; Yukuhiro et al, 2002; Zhu et al, 2013). through RNA-editing mechanisms (Lavrov et al, 2000). The lrRNA and srRNA genes of the T. molitor Nucleotide composition and codon usage mitogenome were 1 281 and 772 bp in length, respec- The A+T content of the whole T. molitor mitogen- tively. As observed in other insects (Boore et al, 1998; ome coding region was 72.35%, showing an obvious AT Kim et al, 2009), these two genes were located between mutation bias (Eyre-Walker, 1997), as observed in two tRNALeu (CUN) and tRNAVal as well as between tRNAVal other published Tenebrionidae species (Friedrich & and the A+T-rich region, respectively (Figure 1). The Muqim, 2003; Sheffield et al, 2009). The AT-skew value A+T contents of the lrRNA and srRNA genes were of the major strand was 0.204, indicating the occurrences 78.92% and 77.33%, respectively. of As in the major strands were greater than those of Ts. The mean value of the A+T content of the 13 PCGs was A+T-rich region 69.27%, with a strong A+T bias. The A+T content at the The 1 170 bp A+T-rich region of the T. molitor third codon position (76.30%) was higher than the first mitogenome was located between the srRNA and tRNAIle (64.75%) and second position (66.77%), indicating that genes (Table 1). Like the two other Tenebrionidae the third codon position was most susceptible to AT mitogenomes, the A+T-rich region also exhibited the mutation bias (Eyre-Walker, 1997). highest A+T content (85.38%) in the T. molitor The relative synonymous codon usage (RSCU) in mitogenome. Unlike some insect species (Kim et al, the T. molitor mitochondrial PCGs was investigated and 2006; Yin et al, 2012), the region did not have large the results are summarized in Table 2. The four most tandem repetitive sequences; however, it did have some frequently used codons were ATT (Ile), ATA (Met), TTT microsatellite-like repeats (for example, (T)15, (AT)7, (Phe), and TTA (Leu), accounting for 25.7% of all

(A)20, and (G)8). The poly-T stretch (15 bp) has been codons in the T. molitor mitogenome. These four codons suggested to function as a possible recognition site for were composed of A or T nucleotides, indicating that the initiation of replication of the mtDNA minor strand their biased usage resulted from strong AT mutation bias

Table 2 Codon number and relative synonymous codon usage in Tenebrio molitor mitochondrial protein coding genesa) Codon Count RSCU Codon Count RSCU Codon Count RSCU Codon Count RSCU UUU(F) 227 1.34 UCU(S) 99 2.28 UAU(Y) 113 1.47 UGU(C) 25 1.61 UUC(F) 113 0.66 UCC(S) 23 0.53 UAC(Y) 41 0.53 UGC(C) 6 0.39 UUA(L) 198 2.29 UCA(S) 101 2.33 UAA(*) 0 0 UGA(W) 74 1.61 UUG(L) 73 0.84 UCG(S) 11 0.25 UAG(*) 0 0 UGG(W) 18 0.39 CUU(L) 90 1.04 CCU(P) 46 1.39 CAU(H) 31 0.87 CGU(R) 26 1.82 CUC(L) 9 0.1 CCC(P) 18 0.55 CAC(H) 40 1.13 CGC(R) 2 0.14 CUA(L) 131 1.51 CCA(P) 61 1.85 CAA(Q) 50 1.54 CGA(R) 26 1.82 CUG(L) 18 0.21 CCG(P) 7 0.21 CAG(Q) 15 0.46 CGG(R) 3 0.21 AUU(I) 274 1.52 ACU(T) 53 1.07 AAU(N) 116 1.21 AGU(S) 26 0.6 AUC(I) 86 0.48 ACC(T) 40 0.8 AAC(N) 75 0.79 AGC(S) 6 0.14 AUA(M) 252 1.67 ACA(T) 98 1.97 AAA(K) 78 1.46 AGA(S) 66 1.52 AUG(M) 49 0.33 ACG(T) 8 0.16 AAG(K) 29 0.54 AGG(S) 15 0.35 GUU(V) 91 1.69 GCU(A) 48 1.22 GAU(D) 42 1.2 GGU(G) 50 0.94 GUC(V) 14 0.26 GCC(A) 24 0.61 GAC(D) 28 0.8 GGC(G) 20 0.38 GUA(V) 92 1.7 GCA(A) 81 2.05 GAA(E) 53 1.43 GGA(G) 102 1.92 GUG(V) 19 0.35 GCG(A) 5 0.13 GAG(E) 21 0.57 GGG(G) 40 0.75 a) RSCU, relative synonymous codon usage. Letters in brackets are the single-letter amino acid codes. Total number of codons, excluding the stop codons (*)=3696.

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(Powell & Moriyama, 1997; Rao et al, 2011). study intoxication response. However, analyzing the The total number of non-stop codons (CDs) of transcriptome of T. molitor is time-consuming work due PCGs was 3 696. Among all amino acids encoded by the to the lack of a sequenced genome (Oppert et al, 2012). A 13 PCGs, Leu (14.04%), Ile (9.74%), Ser (9.38%), and known T. molitor mitogenome sequence could accelerate Phe (9.20%) were the four most abundant amino acids, contig assembly and annotation, as well as help to study three of which were encoded by the AT-rich codons (see informative rearrangements in mitochondrial genomes above), suggesting that the AT bias affected amino acid (Timmermans & Vogler, 2012) and perform high composition (Foster et al, 1997; Min & Hickey, 2007). resolution phylogenetic analysis of Coleoptera (Kim et al, 2009).

SUMMARY Acknowledgements: We thank Dr. Zhong-Bao ZHAO Recently, T. molitor has been used as a model to for helpful suggestions during manuscript preparation.

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Comin 牌检测试剂盒 简介 苏州科铭生物技术有限公司自主研发、生产和销售 Comin 牌检测试剂盒，目前拥有 26 个系列 200 种常用 测定指标 330 种检测试剂盒，其中辅酶Ⅰ（NAD）系列（15 种）、辅酶Ⅱ（NADP）系列（16 种）、维生素 C 系列（7 种）、脂肪酸系列（15 种）、淀粉系列（13 种）、蔗糖系列（17 种）、P450 系列（10 种）、土壤系 列（14 种）和转氢酶系列（2 种）是市场上独家拥有的试剂盒。详情请登录本公司网站查询，或者致电本公司 申请纸质版本产品手册。 试剂盒质量和免责声明 1. 本公司保证用户严格按照试剂盒所附说明书进行保存和操作测定常见样品时能够得到重复性好的可靠数 据。 2. 本公司仅对试剂盒质量本身负责。对于因为样品本身、试剂盒保存不当和操作错误等本公司不能控制的因 素造成的测定失败不负责任。 3. 建议用户认真阅读说明书后，严格按照说明书，选择预计差异大的两个样品进行预测定。如果存在问题请 及时与本公司联系，并且如实准确全面地反映问题，以便技术人员弄清问题的真实原因，调整测定方案， 及时解决问题，协助用户顺利完成实验。 4. 如果因为生产人员失误或者快递野蛮运输造成缺失试剂或者漏液，请及时与本公司联系，以便补发相应的 试剂。 5. 经过沟通确认是原试剂盒质量存在问题，本公司负责提供新试剂盒或者免单。 6. 本公司保留随时改进试剂盒的权利，恕不一一通知。为了保证测定效果，请用户严格按照当时所购买试剂 盒的说明书进行保存和操作。 提高研究性价比的措施 1. 集中精力做好研究方案。测定失败不仅浪费了样品、经费和精力，而且耽误结题和申请新课题，进一步影 响职称晋升和研究生按时毕业。因此，测定失败是最大的浪费。可见，研究者集中精力做好研究方案，而 把没有足够把握的测定工作外包，是明智的选择。 2. 把测定工作部分或者全部外包给可靠的专业公司，如购买试剂盒或者代测。研究者按照研究方案，做好样 品处理，把测定工作外包，可以显著提高测定效率，避免测定失败。 3. 降低外包费用的方法。 （1）查阅文献确认样品含有拟测定指标，避免误测和漏测。最好测定一组相互紧密关联-的指标，以得到 明确结论，提高论文发表档次。 （2）缩短取样间隔以免漏掉指标变化显著的样品。分两步测定：先大间隔选取样品进行测定，以找出可能 的关键点样品；然后围绕可能关键点附近，小间隔选取样品，找到变化关键点，从而在保证不漏掉关键点的前 提下显著减少测定的总样品数量，降低测定费用。

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Zoological Research Editorial Board

EDITOR-IN-CHIEF： Yong-Gang YAO Kunming Institute of Zoology, CAS, China

ASSOCIATE EDITORS-IN-CHIEF： Bing-Yu MAO Kunming Institute of Zoology, CAS, China Ying-Xiang WANG Kunming Institute of Zoology, CAS, China Yun ZHANG Kunming Institute of Zoology, CAS, China Yong-Tang ZHENG Kunming Institute of Zoology, CAS, China

MEMBERS: Jing CHE Kunming Institute of Zoology, CAS, China Biao CHEN Capital Medical University, China Ce-Shi CHEN Kunming Institute of Zoology, CAS, China Gong CHEN Pennsylvania State University, USA Jiong CHEN Ningbo University, China Xiao-Yong CHEN Kunming Institute of Zoology, CAS, China Michael H. Ferkin University of Memphis, USA Nigel W. Fraser University of Pennsylvania, USA Colin P. Groves Australian National University, Australia Wen-Zhe HO Wuhan University, China David Irwin University of Toronto, Canada Nina G. Jablonski Pennsylvania State University, USA Prithwiraj Jha Raiganj Surendranath Mahavidyalaya, India Xiang JI Nanjing Normal University, China Xue-Long JIANG Kunming Institute of Zoology, CAS, China Le KANG Institute of Zoology, CAS, China Ren LAI Kunming Institute of Zoology, CAS, China Bin LIANG Kunming Institute of Zoology, CAS, China Wei LIANG Hainan Normal University, China Si-Min LIN Taiwan Normal University, China Huan-Zhang LIU Institute of Hydrobiology, CAS, China Jie MA Harvard University, USA Masaharu Motokawa Kyoto University Museum, Japan Victor Benno Meyer-Rochow University of Oulu, Finland Monica Mwale South African Institute for Aquatic Biodiversity, South Africa Neena Singla Punjab Agricultural University, India Bing SU Kunming Institute of Zoology, CAS, China Wen WANG Kunming Institute of Zoology, CAS, China Fu-Wen WEI Institute of Zoology, CAS, China Jian-Fan WEN Kunming Institute of Zoology, CAS, China Richard Winterbottom Royal Ontario Museum, Canada Jun-Hong XIA Sun Yat-sen University, China Lin XU Kunming Institute of Zoology, CAS, China Jian YANG Columbia University, USA Xiao-Jun YANG Kunming Institute of Zoology, CAS, China Hong-Shi YU University of Melbourne, Australia Li YU Yunnan University, China Lin ZENG Academy of Military Medical Science, China Xiao-Mao ZENG Chengdu Institute of Biology, CAS, China Ya-Ping ZHANG Chinese Academy of Sciences, China