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Â 6 Marmoset Supply and Availability Researchersâ interest in marmosets as animal models has driven a sharp increase in demand for them. Presenters discussed the context of wild marmoset populations, challenges and opportunities for transport- ing marmosets, and innovative approaches to breeding marmosets in captivity. MARMOSETS IN THE WILD Maria AdÃ©lia Borstelmann de Oliveira, professor at the Animal Ecophysiology and Behavior Labor- atory of the Federal Rural University of Pernambuco in Brazil, and Joanna Malukiewicz addressed the status and habits of marmosets in the wild. Brazilâs Wild Marmoset Populations Six marmoset species are part of the genus Callithrix, a relatively young genus under 2.5 million years old (Figure 6-1). Three of theseâC. aurita, C. flaviceps, and C. kuhliiâare endangered. Populations of the other threeâC. jacchus, C. geoffroyi, and C. penicillataâare considered stable. C. jacchus is one of the youngest species and has the lowest genetic diversity, while the oldest, C. aurita, is the most genetically diverse. Brazil is home to an estimated population of more than 10,000 wild mature marmosets across 20,000 square kilometers. Wild marmosets are found mainly in the northeastern Brazilian state of Pernambuco and in the countryâs highly threatened Atlantic forest region (Amora et al. 2013), though they are also found in lowland rain forests, dry forest, dry scrub, and mangroves. Despite being well adapted to these environ- ments, marmosets live in one of the most threatened ecosystems on earth, the remaining 2 percent of which is a fragmented patchwork of small and isolated fragments. In the state of Pernambuco, at least two areas where common marmosets live, are under invasion by the Amazonian squirrel monkey, Saimiri sciureus. Borstelmann de Oliveira described the increased stress and behavioral change of the marmosets in the pres- ence of the larger, and also highly insectivorous, squirrel monkeys, as shown by high levels of fecal cortisol of this population. Marmosets were introduced to Brazilâs South and Southwest regions by humans and are thriving so well they are now considered pests. They are also spreading into Argentina. To protect other wildlife, the Brazilian Center for Primates Conservation8 stresses the importance of controlling and eradicating marmo- sets in areas where they are invasive. Wild marmosets live in groups of 3-15 members. They weigh less than 500gr, are highly reproductive, and highly adaptable. Marmoset species hybridize both naturally and anthropogenically. While they are an invasive pest in some areas, in their native regions they face threats from habitat endangerment, especially in Pernambuco and the Atlantic forest, and competition for resources, especially from the invasive squirrel monkey from the Amazonian basin. Like other primates in Brazil, marmosets are vulnerable to arboviruses. A recent yellow fever outbreak killed nearly ten times as many NHPs as humans (Bicca-Marques et al. 2017). Human aggression and mal- treatment are also leading causes of death of wild marmosets. According to CITES, approximately 1,600 marmosets were legally traded from 2006-2012, although those were mostly captive born. Legal trade is 8 See http://www.icmbio.gov.br/cpb. 34 Prepublication Copy
Marmoset Supply and AvailabilityÂ responsible for removing thousands of primates from the Brazilian wild each year. A recent analysis of CITES permits showed that 90,000 monkeys were legally exported from South American countries to 23 other countries between 1977 and 2013 (de Souza Fialho et al. 2016). FIGURE 6-1 Marmoset genera Callithrix and Mico. SOURCES: Maria AdÃ©lia Borstelmann de Oliveira. Illustrations copyright 2013, Stephen D. Nash/IUCN SSC Primate Specialist Group. Used with permission. Prepublication Copy 35
Care, Use, and Welfare of Marmosets as Animal Models for Gene EditingâBased Biomedical Research Marmoset Research in Brazil Among Latin American countries, Brazil has the strongest legal protections for NHPs. All animal research in Brazil is regulated by the National Council for Animal Experimentation Control, which requires primate researchers to follow the National Research Councilâs 2011 Guide for the Care and Use of Labor- atory Animals. Specific regulations restrict the collection of data and scientific materials in Brazil by for- eigners, establish procedures for the scientific use of animals, and define categories of use and management for wild animals in captivity. However, there remains room for improvement in the translation of these rules into practice both in Brazil and elsewhere; for example, primates under experimental protocols are housed in isolation, and deprived of natural light, despite recommendations emphasizing the importance of social housing. There are more than 800 primates in private facilities,9 including zoos, research laboratories, rehabil- itation centers and sanctuaries in Brazil, used in more than 33 research areas from virology to toxicology to ophthalmology (Pissinatti et al. 2014). In addition to this basic and translational work, researchers are also working to expand their understanding of wild marmosets, by focusing for example, on marmosetsâ taste receptors. Other work is examining the impacts of resource competition between native marmosets and invasive squirrel monkeys. It is clear that a better understanding and consideration of the effects of captivity on marmosets is needed to inform their care and use as animal models. Researchers in Brazil are advancing knowledge in this area by comparing captive and wild marmoset behavior, including studies of gum eating, social and foraging behavior, and sensorial ecology. One study is examining the diet, food competition, and lipid profiles of both captive and wild marmosets; another has concluded that wild marmosets in competition for resources have a higher level of cortisol than captive marmosets or wild marmosets without competition. Investigations by Malukiewicz characterized differences in the microbiomes of captive and wild marmosets (see Chapter 5, Genetic Variation and the Gut Microbiome). Researchers in Brazil and elsewhere are also working to advance conservation efforts for wild mar- moset populations assisted by the legal protections for captive primates in place across various countries in Latin America (Figure 6-2). The demand for marmoset research colonies can add pressures to these wild populations, suggesting a need for careful consideration of how founder marmoset populations are sourced to meet research demands without undermining conservation goals. BREEDING AND TRANSPORTATION Jon Levine is the director of the WNPRC. Saverio âBuddyâ Capuano is the associate director and attending veterinarian for the WNPRC. Together with Erika Sasaki these speakers discussed opportunities to address marmoset supply challenges through innovative approaches to breeding and transportation. Marmoset Supply Challenges It is estimated that research facilities currently house just over 6,000 marmosets worldwide, with about 2,500 of those animals located in Japan; 1,900 in North America; 1,000 in Europe; and 800 in South Amer- ica. An unknown number is located in China. In contrast, macaques are far more widely available for re- search, supported by a strong infrastructure for their breeding and transportation. Macaques are bred by the U.S. National Primate Research Centers (NPRCs), the U.S. National Institutes of Health (NIH), and multi- ple other academic institutions and commercial entities in the United States and abroad. Macaques are also much hardier than marmosets and travel well; each year about 30,000 macaques, compared with only 250 marmosets, are transported for research (Figure 6-3). 9 These facilities do not include the five largest centers of primatology in Brazil, including the Laboratory of Ad- vanced Studies in Primatology (LEAP). 36 Prepublication Copy
Marmoset Supply and AvailabilityÂ PermissionÂ pendingÂ FIGURE 6-2 Legal protections for captive primates in Latin America. The top line refers to the existence of laws specific to wildlife protection (WP); animal welfare (AWP); research laboratories (RESL); zoos; captive facilities (Captive); circuses; primate facilities; the existence of IACUCs; and whether international guidelines for primate hus- bandry or any guideline that specifies regulations on minimum cage size, social environment or health requirements are part of these laws. Scoring was done as follows: - WP: Y, N and if recent (<3 yrs). - AWP: Y, N, w (when wildlife was included), recent, partial if it only included some elements of welfare, or n-w if it explicitly excluded wildlife. - zoos: Y for the presence of specific regulations, N for no regulations at all, and AWP for general animal welfare and protection laws. - captive facilities, they scored Y if specific regulations exist; rescue for regulations specific to rescue centers; zoocriador (zookeeper) for specific mention, N for no regulation, and AWP for when the animal welfare law con- templated captive facilities. - circus: Y for the presence of specific regulations; AWP for animal welfare law; W for when the wildlife law prohibits animals in circuses; N for no measures at all. SOURCES: Maria AdÃ©lia Borstelmann de Oliveira from the forthcoming chapter Primates under Human Care in Developing Countries: Examples from Latin America, by Ferreira et al., in Welfare of Nonhuman Primates, Robinson LM, Weiss A, eds. Springer 2019/2020. In the United States, marmoset demand is quickly outstripping supply. About 1,900 marmosets are spread among 27 research colonies in the United States, where at least 40 primary investigators are working with them. Querying the NIH RePORTER database10 for the text âmarmoset/marmosetsâ showed that each year for the past 10, approximately 50 grants were awarded for marmoset research in the United States a number that is expected to grow. During the discussion an attendee noted that another sign of this increased demand is the sharp increase in references to marmosets in the Society for Neuroscience annual conference programs over the past few years. The most recent conference, for example, featured 61 posters of marmoset research. 10 See https://projectreporter.nih.gov/reporter.cfm. Prepublication Copy 37
Care, Use, and Welfare of Marmosets as Animal Models for Gene EditingâBased Biomedical Research FIGURE 6-3 U.S. imports of macaques (left axis, blue bars) and marmosets (right axis, white bars), 2010-2017. SOURCES: Figure developed by Saverio Capuano based on statistics provided by Robert Mullan, U.S. Centers for Disease Control and Prevention. The increased demand has come somewhat as a surprise. In fact, the WNPRC had until fairly recently been using birth control drugs to keep its marmoset population in check and avoid overtaxing the centerâs resources and infrastructure to care for the animals. The WNPRC has also given some marmosets to other institutions to start their own research colonies, though the number of marmosets available for transfer has not been sufficient to meet the current demand. Japan is seeing a similar gap between marmoset demand and availability. Marmoset research in Japan began in 1978, when leading scientist Tatsuji Nomura identified marmosets as an ideal NHP model. In 1996, Nomuraâs breeding colony was transferred to CLEA Japan, Inc. Until around 2000, researchers in Japan showed little interest in marmosets, preferring to work with macaques, leading CLEA to reduce its marmoset breeding program to address persistent oversupply. Today CLEAâs 1,000 marmosets are insuf- ficient to meet the demand and the organization is actively trying to grow this population. Importing marmosets to Japan is not an option. In a frustrating catch-22, only Air France is allowed to transport marmosets to Japan, but in Japan it is prohibited to import marmosets from France. Another challenge is that marmosets in Japan all come from the same initial colony (imported from the United Kingdom) and so are interrelated, which makes the lack of genetic diversity a serious issue for the research- ers working with them. Drivers of Research Demand All primates are considered desirable models for high priority research areas across the globe, includ- ing cognition, neurodevelopmental disorders, neurodegenerative diseases, aging, behavior, reproductive biology, physiology, and metabolic diseases. For this work, and as other speakers mentioned, marmosets present with specific advantages: they are easier to work with and maintain; they require less space; they have a short generation time and short lifespan; and they have multiple births. Current shortages notwith- standing, marmosets are also relatively inexpensive to acquire and to maintain compared to other primate species. As discussed in Chapter 2 (Current Use and Key Advantages), marmoset demand is also on the rise because they are well suited for emerging transgenic and gene editing techniques. One WNPRC study is using CRISPR/Cas9 gene editing approaches to develop a marmoset model for familial Parkinsonâs disease, 38 Prepublication Copy
Marmoset Supply and AvailabilityÂ a process extending over multiple generations of animals that involves much trial and error. Another WNPRC study is using nanoparticles to genetically edit marmoset neurons for neurogenetic diseases re- search (Furtado et al. 2018). NIH researchers are studying somatic cell genomic editing in marmosets. These methods and others have contributed to the development of marmoset models for MPTP-induced Parkinsonâs and spinal injury and the establishment of marmoset embryonic stem cell lines. In Japan, much of the increased demand for marmosets has been propelled by two large-scale scien- tific initiatives: the 2008 Strategic Research Program for Brain Science and the 2014 Brain/MINDS project (Okano et al. 2016). Both use marmosets as a primary model organism. Approaches to Increase Marmoset Supply Panelists discussed three possible solutions to improve researchersâ access to marmosets: a consor- tium of marmoset PIs, a coordinated strategic plan to increase the marmoset population, and an embryo exchange. The first two are already underway, and Sasaki proposed the third in her presentation. Marmoset PI Consortium: Recently, PIs who use marmosets held an informal meeting to discuss cur- rent research, marmoset management issues, and goals for the development of marmoset models. The PIs agreed to create a consortium to facilitate efforts to share information and trade animals. Consortium mem- bers discussed several options for improving marmoset supplies. These include the establishment of cen- tralized breeding facilities, a centralized pedigree tracking system and animal locator, and a quarantine facility for imported animals. The consortium also hopes to improve overall communication, especially with South American col- leagues closest to wild marmoset populations, and identify best practices for marmoset transportation and care. Members plan to publish a white paper to enhance the visibility of marmoset supply challenges, and to convene again in 2019. NIH Strategic Plan: The NPRC directors have been working closely to address the problem of mar- moset availability. The group has identified several urgent needs: to accurately monitor supply and demand of marmosets, to develop a plan to adjust breeding targets, to expand facilities and increase resources, to distribute expertise and core services more efficiently, and to address the concerns that have been raised about marmoset genetic diversity. The NIH and the Office of Research Infrastructure Programs have taken several concrete steps to address these needs and strategically plan for improving the environment for NHP research more broadly. As a first step, a needs assessment was conducted that recommends developing and expanding colonies, identifying the new techniques driving the research, and helping the NPRCs and other facilities meet the increased demand. The first phase of this effort focused mainly on macaques, but many of its findings apply to marmosets, as well. The second part of this effort convened an expert panel discussion including program officers from nearly all NIH institutes. The panel was charged to forecast the future of NHPs in biomedical research, assess existing resources and infrastructure and the potential need for expansion, and identify key chal- lenges. Challenges identified include funding, space, and infrastructure limitations; a lack of standardized data collection and reporting; and needs related to personnel training. Among several recommendations emerging from the discussion, the one most pertinent to marmoset research is that the NIH should prioritize, fund, and establish a breeding production site for marmosets as part of a federally distributed system for animal production (NIH 2018). Embryo Transfer: In Japan, a limited marmoset supply combined with a lack of genetic diversity and restrictions on importing have driven a focus on developing techniques to transfer marmoset embryos, ra- ther than live animals. Sasakiâs team has developed nonsurgical techniques to remove fertilized embryos, freeze them, and implant them in a surrogate female for gestation, resulting in successful healthy births (Thompson et al. 2014). If permitted under CITES, fertilized marmoset embryos (including wildtype embryos and/or genet- ically modified ones) could be transported among research facilities within and between countries for im- plantation in surrogate marmosets at the receiving facility. Embryo transfers have a higher success rate than Prepublication Copy 39
Care, Use, and Welfare of Marmosets as Animal Models for Gene EditingâBased Biomedical Research insemination after cryopreservation, and this approach is easier from a technical perspective compared with other procedures. Embryo transfers also entail a complete genetic exchange, whereas sperm transfers result in a transfer of only half a set of genes. REFERENCES Amora TD, BeltrÃ£o-Mendes R, Ferrari SF. 2013. Use of alternative plant resources by common marmosets (Callithrix jacchus) in the semi-arid caatinga scrub forests of northeastern Brazil. Am J Primatol Apr;75(4):333-341. Bicca-Marques JC, Calegaro-Marques C, Rylands A, et al. 2017. Yellow fever threatens Atlantic Forest primates. Science Advances 3.e1600946/tab-e. de Souza Fialho M, Ludwig G, Valenca-Montenegro MM. 2016. Legal international trade in live neotropical primates originating from South America. Primate Conservation 30:1-6. Furtado D, BjÃ¶rnmalm M, Ayton S, et al. 2018. Overcoming the blood brain barrier: The role of nanomaterials in treating neurological diseases. Adv Mater 30(46):e1801362. NIH (National Institutes of Health). 2018. Nonhuman Primate Evaluation and Analysis, Part 2. Report of the Expert Panel Forum on Challenges in Assessing Nonhuman Primate Needs and Resources for Biomedical Research. Available at: https://orip.nih.gov/about-orip/research-highlights/nonhuman-primate-evaluation-and-analysis-pa rt-2-report-expert-panel. Okano H, Sasaki E, Yamamori T, et al. 2016. Brain/MINDS: A Japanese national brain project for marmoset neuro- science. Neuron 92(3):582-590. Pissinatti L, Pissinatti A, Burity CHF, et al. 2007. OcorrÃªncia de acantocephala em Leontopithecus (Lesson, 1840) cativos: Aspectos clÃnico-patolÃ³gicos. CallitrichidaeâPrimates. Arq Bras Med Vet Zootec 59:1473-1477. Thomson JA, Kalishman J, Hearn JP. 1994. Nonsurgical uterine stage preimplantation embryo collection from the common marmoset. J Med Primatol 23(6):333-336. 40 Prepublication Copy