Journal of Wildlife and Biodiversity 2(2): 6-14 (2018) by Arak University, Iran (http://jwb.araku.ac.ir/) DOI: 10.22120/jwb.2018.82562.1021 Research Article

Potential use of brush-tailed mice for evolutionary developmental biology studies: Calomyscus elburzensis as an appropriate rodent model

Kordiyeh Hamidi, Jamshid Darvish, potential to be bred all round the year, nearly Maryam M. Matin* short pregnancy period (about 4.5 weeks) and long lifespan in captivity (generally between 4 Department of Biology, Faculty of Science, to 5 years) have the potential to be bred Ferdowsi University of Mashhad, Mashhad, and used in different fields of evolutionary Iran. *email: [email protected] studies. However, low reproductive

Received: 4 March 2018 / Revised: 23 May 2018 / Accepted: 1 output (known up to five till now) and late June 2018 / Published online: 3 June 2018. Ministry of Sciences, sexual maturity (at about four months) as Research and Technology, Arak University, Iran compared with Mus and Rattus, are some Abstract drawbacks associated with Calomyscus, which Rodents (mainly mice and rats) are the most might be improved by gradual genetic changes commonly used animal models for studies of and artificial selection. cancer biology, immunology, genetics, Keywords: Laboratory models, Rodentia, developmental biology and also Goodwin’s brush-tailed mouse, Breeding strain, embryo transfer techniques in domestic and Iran. endangered animal species, and even in humans. Herein, we aimed to evaluate Introduction Goodwin’s brush-tailed mouse (Calomyscus Animal models are mostly used as vital tools in elburzensis) as a suggested model species for medical, immunological, physiological, evolutionary developmental biology (Evo- pathological, developmental, genetic, cancer Devo) studies. Trapping procedure was and neuroscience studies. For more than one performed in Khaje-Morad rocky region, south hundred years, every progress in different fields center of Mashhad, Razavi Khorasan province, of biological studies has been virtually affected Iran between April and September by using animal models (Willis-Owen and Flint 2014. Breeding procedure for 15 adult females 2006). These models still have important roles and 12 adult males was conducted in in our understanding of the function of genes, captivity between 2014 and 2017. Brother × the mechanisms of various diseases, the sister mating in each generation was used effectiveness and toxicity of different chemicals as laboratory rearing method for breeding three and also many other fields (Patten and Hall- generations (F1-F3). The average of pregnancy Porter 2009, Makvand-Hosseini et al. 2014). period and the differences in the sex of Moreover, study of animal models is required newborns in different parturition to preserve endangered and nearly extinct were measured using descriptive statistical species such as the tamarins of Brazil and analyses (independent-sample t-test). We California by applying new reproductive observed that Goodwin’s brush-tailed mice techniques. All in all, use of animal models in were relatively social rodents, and due to their related studies is an essential factor to develop high level of tolerance in harsh conditions, no new techniques which are suitable for hibernation, low cost, small size, the characterizing biological phenomena both in 7 | Journal of Wildlife and Biodiversity 2(2): 6-14 (2018) humans and animals (Willis-Owen and Flint model should be chosen based on its adequacy 2006, Patten and Hall-Porter 2009, Makvand- to the planned procedures (Johnson and Hosseini et al. 2014). Besselsen 2002). The most commonly used model animals are Goodwin's brush-tailed mouse as a new rodent murine species such as house mice (Mus model musculus) and brown rats (Rattus norvegicus), The newly introduced animal model with novel which have made the basis for our current biological characters in comparison with the knowledge in many fields including cancer, previously introduced animal models increase aging, immunology, and genetics as well as the diversity of our available research models many other aspects of science. They are also and supports expanding our fields of study and used to develop and complete the embryo scientific approaches. Herein, we intended to transfer techniques in domestic and endangered consider the possibility of using Goodwin’s animal species, and even in humans (Suckow et brush-tailed mouse (Calomyscus elburzensis al. 2005; Hedrich 2012). Moreover, the other Goodwin, 1938) as a new animal model. rodent species such as Mongolian gerbils Brush-tailed mice are a group of small rodents (Meriones unguiculatus), Syrian or golden belonging to family Calomyscidae Vorontsov hamsters (Mesocricetus auratus) and Guinea and Potapova, 1979. They are distributed pigs (Cavia porcellus) are also used as animal around Near East and Middle Asia and models. Mongolian gerbil has been used in interestingly, are limited to the Iranian plateau different fields of physiology and medicine and its neighborhoods; they can be usually (e.g. evaluating the efficacy of therapeutic found in semi mountainous areas in desert agents, and the study of epilepsy or defining the regions of Iran, Afghanistan, Pakistan, disease pattern of encephalitic form of Rift Turkmenistan, Azerbaijan and Syria (Musser Valley fever (RVF)) (Somova et al. 2000). and Carleton 2005). They represent an early However, use of this species as animal model split from the rest of the mouse-like rodents has decreased in recent years. Some hamster (such as mice and rats), and also are the sole species (e.g. Mesocricetus auratus, Phodopus member of a clade basal to the radiations of sungorus, Cricetulus griseus) thrive in captivity other Muroidea (Jansa and Weksler 2004). and are easily bred in this condition, so they are Brush-tailed mice have maximum lifespan used in laboratories for behavioral and among muroid rodents. They regularly live over physiological studies (Kuhnen 2002, Fox 2006, four years in captivity; nonetheless there are Mitchell and Tully 2008). Guinea pigs are also several records indicating that they can live as used for medical researches mainly in the fields long as nine years in captivity. Hence, it may of immunology, pathology and physiology suggest that these rodents are very similar in (Fernandez et al. 2006). Experimental models life history traits to much larger rodents such as as well as genetically modified and negative sciurids and hystricognaths, which both of them models of the house mice species Mus can live over ten years in captivity (Musser and musculus are commonly used in evolutionary Carleton 2005). studies. For example, many studies have Calomyscidae contains eight valid species performed on the activities of different genes belonging to a single genus, Calomyscus during dental development (e.g. Nadiri et al. Thomas, 1905. Goodwin’s brush-tailed mouse 2004; Hamidi et al. 2017b). A proper animal has been mainly reported from mountains of model for a research should be selected on the north and northeast of Iran (Musser and basis of the background knowledge of Carleton 2005). In Europe, a species of biological properties, cost and availability, ease Calomyscus is popularly kept as a pet; they are and adaptability to experimental manipulation, labeled C. bailwardi, and probably represent and ecological consequences. The animal either C. mystax or C. elburzensis. However, 8 | Journal of Wildlife and Biodiversity 2(2): 6-14 (2018) they can be rarely found in European pet stores with following dimensions: 40 × 60 × 50 cm3. but commonly presented in dedicated breeders The frame of the cage was made of chewing- (Volf and Volf 2003). In Russia, they have resistant transparent polyvinyl chloride (PVC), been used in laboratories for different sorts of with galvanized wire fencing in some sides of researches and also sold as pets in many pet the cage. The laboratory rearing method was shops. In United Kingdom, they have been organized on brother × sister (full-sib) mating imported for exhibits in zoos (Acron web in each generation. Wild animals were kept and 2000). Herein, assuming the hypothesis of raised for up to three generations (Filial 1-3) in using Goodwin’s brush-tailed mouse as a new captivity. animal model, we aimed to evaluate several Statistical analyses (descriptive analyses) for features of this species, as one of the developmental studies including the duration of representative members of Calomyscidae, pregnancy and the differences in the sex of based on the author’s long term previous and newborns in different parturition have been recent field and lab studies, and also discuss performed using the SPSS 16.0 (SPSS Inc. about the possibilities and limitations which 2007). Animal care was performed in may occur in the way of breeding and compliance with the “Guideline for the care and maintaining the colonies of this exotic old use of laboratory and experimental animals, evolutionary rodent for using as a novel Rodentology Research Group, Ferdowsi laboratory animal model. University of Mashhad” (Darvish 2015).

Materials and methods Results Trapping was performed in Khaje-Morad During present study focused on behavioral and heights (36°08′-37°03′N and 59°13′-59°42′E at biological features of Calomyscus elburzensis, an altitude of 1146 m a.s.l.), south center of we found that this species can be potentially Mashhad, Razavi Khorasan province, Iran considered as a relatively social rodent; between April and September 2014 (Hamidi et Goodwin’s brush-tailed mice were successfully al. 2015). This well drained, barren rocky kept in small colonies and bred in captivity for region is located in cold and dry climatic more than three years. Our observations are condition with generally flat topography and summarized below: some mounds. The typical vegetation of this Feeding area is mainly composed of Mount Atlas They were generally herbivorous. Their natural pistache (Anacardiaceae; Pistacia atlantica diet in north and northeastern Iran was mainly Desf.), shrubby horsetail (Ephedraceae; Pistacia atlantica seeds (also see Hamidi et al. Ephedra sp.) and sun spurge (Euphorbiaceae; 2015, 2017a). During trapping process, they Euphorbia helioscopia L.) as well as some were mainly attracted to the baits resembling lands under cultivation and several industrial their preferred diet such as sunflower seeds and and residential constructions around it (Hamidi gourd seeds. So these seeds as well as hard et al. 2015, 2017a). small fruits such as walnuts, hazelnuts, Fifteen adult females and twelve adult males of chestnuts or almonds were used as their nutrient Goodwin’s brush-tailed mice were captured requirements in captivity. However, when they using custom-made mesh live traps. Captured fed on a high fat diet, they tended to store the rodents were transferred to the animal house fat and became obese, therefore restricted and kept in captivity for breeding programs feeding was necessary. In females, the fat between 2014 and 2017 (Hamidi et al. 2017b; accumulation was associated with some also author’s unpublished data). A single male reproductive difficulties. Our observation and a single female were paired in a separate showed that they had weak willing to use cage. Mating cage was an oblong shaped box commercial feeds (rodent dry pellets). Adults 9 | Journal of Wildlife and Biodiversity 2(2): 6-14 (2018) were daily fed with about 10 g of seeds. Water although special care was essential in was not critically necessary to provide directly separation of semi-aggressive animals. At for the mouse during rearing period because it puberty stage, they often tended to exhibit light could be supplied by providing apple slices and fighting or other weak aggressive behaviors. In lettuce leaves. contrary, young animals had very exploratory actions and engaged in social play for a long Keeping in captivity time; the males were more inquisitive than the Lighting was a very important factor in the females. They were clever animals and breeding of Goodwin’s brush-tailed mice and a extensively used their sense of smell and 12-12 h light-dark cycle was generally used. acoustic abilities to evaluate their surroundings The mice avoided the areas with high level of (their ears are very rounded in shape, devoided light and tended to be hidden in their nests; thus of hair and elongated 17 to 20 mm). Their using nest boxes instead of simple cages is hearing system is highly developed and suggested. Activity was higher during hours of severely sensitized to noise. They were very darkness. They were reasonably cold-tolerant calm animals. However, sudden and extremely animals. Hence, the temperature at 19-23 ˚C loud noises would cause them to escape or was suitable for rearing them. The adequate produce a special voiced sound. humidity range for keeping this species was 25- Goodwin’s brush-tailed mice were able to jump 50%. high and therefore, cages with minimum 50 cm Stress was often occurred when individuals heights were used. The minimum facilities animals were put together later in life or a new included refuges (e.g. nest boxes) and nesting one was introduced into another one’s cage. For materials such as napkins, wadding or evading this tragedy, selecting a paired mating pelletized wood dust. They rarely bite unless scheme and also not regrouping Goodwin’s mishandled and became tame when handled brush-tailed mice during breeding project was gently and frequently. When they were often applied. For breeding, two adults with suddenly excited, they jumped and dart to different sexes were housed together at the age escape from the stimulus agent. of about four months. The female may rarely become a bit aggressive toward the male if kept Reproduction and developmental features together for too long after mating. In some very From what we observed in nature in rare cases, the males were killed after being northeastern parts of Iran, Calomyscus attacked by the female (intersexual aggression). elburzensis were breeding from mid-February We could breed these rodents for up to three to mid-June (generally with two times generations (F1, F2 and F3) in captivity. In pregnancy in this period). Hence, they addition, based on our observation, they could exhibited some appreciable seasonality acts in survive for at least five years in captivity. respect to reproduction and breeding events in nature. It is worth to mention that we were able Behavioral features to breed them successfully while in captivity all Goodwin’s brush-tailed mice were nocturnal year round which resulted to 39 newborns (first especially during the spring and summer, filial: F1) descended from 11 pregnant females however in winter, they were found active in (out of 15 captured females). The duration of the afternoons too. Individuals of C. elburzensis pregnancy was 31.5± 2.1 days on average, and did not generally have hibernation in captivity. females beard up to 5 offspring. Total of 15 They were relatively social rodents and rarely pups were produced by a female belonging to showed pugnacious tendencies in encountering the second filial (F2; raised in captivity), which with their congeneric and hence, could get happened during her six parturitions within together during a short time after housing in about nine months. The breeding intervals for captivity. They tended to form colonial F2 females in captivity were 53.4± 0.6 days on relationships and group housing was possible, 10 | Journal of Wildlife and Biodiversity 2(2): 6-14 (2018) average. In total, there was no significant could be distinguished from the newborn difference in the sex of newborns of F1, F2 and females by their greater anogenital distance and F3 (independent-sample t-test; p < 0.05). larger genital papilla. The female pups had The young mice were unable to feed or care for generally larger size than males. Nipples in the themselves independently for a long period of female pups could be observed about ten days time after birth (altricial). The litters were after birth and testes in the males could be hairless and blind. Their body weight was 2 g, observed in the scrotum at fourth week if the and their body length was about 53 mm on pup was held with its head up. The first average. They were helpless for the first 15 pregnancy in the captive females was occurred days of their life when their eyes opened for the at the age of about four months, and the first time, both male and female contributed duration of postnatal growth and development almost equally to breed their newborns. The was about six months. Significant events litters were weaned no earlier than four weeks happening during the life history of Goodwin’s (mostly up to the postnatal day 35). The mice brush-tailed mouse newborns are listed in Table usually began to explore outside the nest at the 1. age of five weeks (Fig. 1). The newborn males

Table 1. The most significant events in the life history of Calomyscus elburzensis newborns

Postnatal Significant event(s) day (PN) Dark body without hair; closed eyes; ears separated from the head and with visible PN0 blood vessels; enlarged moist bulge of olfactory organ around the nose covered with long whiskers PN5 Emergence of the hair (body fur) PN10 Appearance of six nipple glands in female pups PN15 Eyes opening; enable to walk; grasp objects Changing in the grayish color of dorsal fur near the head to the brownish (grey hairs PN20 change to brown initially from the proximal part of the back of the body to the distal part); emergence of the tail tuft as some dark color assembled hairs PN35 Weaning from milk and start feeding on seeds; beginning to explore outside the nest PN40 Separation of pups from mum; each was kept in a separate cage

Figure 1. Life history of Calomyscus elburzensis newborn pups: A, B, C, D, E, F, G and H represent postnatal days 0, 5, 10, 15, 20, 30, 35 and 40, respectively. Mum is seen near her pups in F and G.

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Discussion have been evolved later. For example, As new laboratory techniques develop and Mesocricetus species were evolved during the available data related to biology, life style and late Miocene about 7-12 Ma ago (Kowalski reproduction characters of many organisms 2001) and the evolutionary time of species increase, new animal models will be thought to be on the line leading to Mus, is introduced and old ones may be ignored. Some considered as 10.4 Ma ago (Jacobs and Flynn laboratory rodents e.g. the house mouse, have 2005). Comparative evolutionary been very well characterized genetically and developmental biology (Evo-Devo) studies have undergone genetic manipulations and focusing on the similarity in dental morphology artificial selections to produce animals with of Calomyscus species (with considering uniformly heritable phenotypes (e.g. Hedrich Calomyscidae as a basal group) with many 2012, Kuramoto 2012). other muroid rodents (such as members of Different breeding systems, selective breeding, Murinae, Acomyinae, Dendromurinae and and genetic-engineering techniques have been Cricetomyinae) (Jansa and Weksler 2004, used to produce strains and stocks of rodents Wessels 2009) can be very helpful to for particular experimental purposes: inbred understand the way of convergent (or maybe strains, transgenic strains, recombinant inbred parallel) evolution of these rodents. In other strains, hybrid strains, and outbred stocks. A words, studies on changes in the gene strain is a group in which all members are expression during pre- and postnatal closely identical from the point of genetic traits. development of Calomyscus species which are In laboratory mice and rats, this is bred in the lab would add excellent data in this accomplished through inbreeding. Full-sib regard. inbreeding for 20 generations will result in Furthermore, because of the long life history of more than 98% genetic homogeneity, at which brush-tailed mice and different climatic impacts point the members of the stock are isogenic and on their habitat and distribution range, they can the stock is considered an inbred strain remarkably suffer harsh conditions. Goodwin’s (Hedrich and Bullock 2004; Hedrich 2012). brush-tailed mice may be considered as an ideal Many inbred strains of mice are widely used in selection for specific laboratory experiments different types of research. A few inbred strains especially for evolutionary studies. Some of rats, Guinea pigs, Syrian hamsters, and advantages of these animals include no gerbils have also been developed. By having hibernation, low cost, small size, easier this kind of isogenic populations, it is possible handling, nearly short pregnancy period (about to conduct researches on the roles of genes, or 4.5 weeks), long lifespan in captivity (generally carry out experiments that do not require between 4 to 5 years) and karyological genetic variations. By contrast, outbred variations (Graphodatsky et al. 2000, Grzimek populations are used when genetic variations 2004, Musser and Carleton 2005, Hamidi et al. among members are required or identical 2015). They can be bred and used in different genotypes are unnecessary. These populations fields of scientific studies such as basic are usually referred to as stocks rather than biological research, educational and applied strains and are used primarily when genetic research, veterinary medicine, breeding heterogeneity is desired (Krinke 2000). programms, etc. Moreover, their relatively slow Brush-tailed mice are considered as the only growth rate makes them suitable for chronic existing members of the subfamily studies (e.g. their usefulness in prenatal Myocricetodontinae Lavocat, 1961 which protocols which last between 4 to 5 weeks vs. occurred since early Miocene (about 13.7-18 about 3 weeks in mice). Numerous experiments Ma) (Wessels 2009), and they are also called can be designed on Calomyscidae members living fossils. Other common rodent models based on the karyological variations observed 12 | Journal of Wildlife and Biodiversity 2(2): 6-14 (2018)

in this family (e.g. Graphodatsky et al. 2000). It Acknowledgment would also be possible to breed different strains This research was funded by a grant (no. of Goodwin’s brush-tailed mice in captivity 3/29121) from Ferdowsi University of which result in producing enough numbers of Mashhad. The authors wish to thank H. genetically identical samples for genetic Mozaffari and A. Hamidi for their kind studies. supports during this survey, also Dr. V.G. On the other hand, Goodwin’s brush-tailed Malikov and Dr. M. Nassirkhani for their mice have lower reproductive outputs (known critical review of the first draft of the up to five till now), a relatively longer period of manuscript. The authors wish to dedicate this growth and development (between five to seven paper to the late Dr. M. Tarahhomi for his kind months) and later sexual maturity (at about four heart and great soul. months) (Meyer and Malikov 1996, author’s unpublished data) than the members of family References Muridae (e.g. mice and rats). These characters Acorn Web, 2000. "About Hamsters; may hamper the studies which require several Calomyscus bailwardi" (On-line). Accessed generations of offspring during a reasonable February 24, 2004 at period of time. These limitations can be http://www.petwebsite.com/mouse.htm. improved by gradual genetic changes resulted Darvish J. 2015. Methodology in animal through genetic manipulation, genetic biosystematics. Jehad Daneshgahi Mashhad improvement of valuable traits, and artificial (JDM) Press, Mashhad, Iran, 336 p. selection. Therefore, Goodwin’s brush-tailed Fernandez M.L., Volek J.S. 2006. Guinea pigs: mice can be introduced as an alternative ideal a suitable animal model to study lipoprotein rodent model for Evo-Devo studies. metabolism, atherosclerosis and

Conclusion inflammation. Nutrition and Metabolism The long history of Goodwin’s brush-tailed 3(1): 17. mice, and the basal situation of Calomyscidae Graphodatsky A.S., Sablina O.V., Meyer M.N., among those radiations representing Muroidea Malikov V.G., Isakova E.A., Trifonov in different molecular trees can provide a great V.A., Polyakov A.V., Lushnikova T.P., opportunity for scientists to have an animal Vorobieva N.V., Serdyukova N.A., model with the potential of more suitability for Perelman P.L., Borodin P.M., Benda P., comparative phylogenetic studies and Frynta D., Leikepova L., Munclinger P., especially in the field of Evo-Devo biology. Pialek J., Sadlova J., Zima J. 2000. Chance of finding genetic markers which are Comparative cytogenetics of hamsters of closely linked to major genes affecting some the genus Calomyscus. Cytogenetic and important quantitative traits will be increased Genome Research 88: 296-304. by completing our knowledge about the Hamidi, K., Darvish J., M. Matin M., Malikov genome of this species. However, it is necessary to continue comprehensive research V.G. 2015. A field behavioral study of on biology, ecology and behavior of this exotic Calomyscus elburzensis (Rodentia: animal. In conclusion, beside some limitations Calomyscidae); the effects of sexual and which may be improved by genetic seasonal variations in the trapping success. manipulations and artificial selection, brush- Acta Zoologica Bulgarica 67(4): 521-528. tailed mice have a bright future perspective Hamidi, K., Darvish J., M. Matin M. 2017a. among other animal models for using in Evo- Habitat and ecological survey of two Devo studies. Calomyscidae species; Goodwin’s brush-

tailed mouse (Calomyscus elburzensis) and 13 | Journal of Wildlife and Biodiversity 2(2): 6-14 (2018)

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