Population genomics reveals moderate genetic differentiation between populations of endangered Forest Musk Deer located in Shaanxi and Sichuan.

BACKGROUND: Many endangered species exist in small, genetically depauperate, or inbred populations, hence promoting genetic differentiation and reducing long-term population viability. Forest Musk Deer (Moschus berezovskii) has been subject to illegal hunting for hundreds of years due to the medical and commercial values of musk, resulting in a significant decline in population size. However, it is still unclear to what extent the genetic exchange and inbreeding levels are between geographically isolated populations. By using whole-genome data, we reconstructed the demographic history, evaluated genetic diversity, and characterized the population genetic structure of Forest Musk Deer from one wild population in Sichuan Province and two captive populations from two ex-situ centers in Shaanxi Province. RESULTS: SNP calling by GATK resulted in a total of 44,008,662 SNPs. Principal component analysis (PCA), phylogenetic tree (NJ tree), ancestral component analysis (ADMIXTURE) and the ABBA-BABA test separated Sichuan and Shaanxi Forest Musk Deer as two genetic clusters, but no obvious genetic differentiation was observed between the two captive populations. The average pairwise FST value between the populations in Sichuan and Shaanxi ranged from 0.05-0.07, suggesting a low to moderate genetic differentiation. The mean heterozygous SNPs rate was 0.14% (0.11%-0.15%) for Forest Musk Deer at the genomic scale, and varied significantly among three populations (Chi-square = 1.22, p < 0.05, Kruskal-Wallis Test), with the Sichuan population having the lowest (0.11%). The nucleotide diversity of three populations varied significantly (p < 0.05, Kruskal-Wallis Test), with the Sichuan population having the lowest genetic θπ (1.69 × 10-3). CONCLUSIONS: Genetic diversity of Forest Musk Deer was moderate at the genomic scale compared with other endangered species. Genetic differentiation between populations in Sichuan and Shaanxi may not only result from historical biogeographical factors but also be associated with contemporary human disturbances. Our findings provide scientific aid for the conservation and management of Forest Musk Deer. They can extend the proposed measures at the genomic level to apply to other musk deer species worldwide.

Regional and seasonal effects on the gastrointestinal parasitism of captive forest musk deer.

Parasite infections can cause adverse effects on health, survival and welfare of forest musk deer. However, few studies have quantified the parasite infection status and evaluated the parasite temporal dynamics and differences between breeding centers for captive forest musk deer. The purpose of this study was to assess seasonal and regional effects on the parasite prevalence, shedding capacity, diversity, aggregation and infracommunity to establish baseline data on captive forest musk deer. The McMaster technique was applied to count parasite eggs or oocysts in 990 fecal samples collected at three breeding centers located in Qinling Mountains and Tibetan Plateau during spring, summer, and winter. Five gastrointestinal parasite groups were found in musk deer, and Eimeria spp. were dominant (mean oocysts per gram=1273.7±256.3). A positive correlation between Eimeria spp. and Strongyloides spp. (r=0.336, p<0.001) based on shedding capacity data was found, as well as a negative correlation between Eimeria spp. and Moniezia spp. (r=-0.375, p=0.003). Both seasonal and regional differences in diversity, prevalence, shedding capacity, aggregation and infracommunity were observed for five parasite groups. The low level of aggregation and high shedding capacity of Eimeria spp. and Strongyloides spp. might reflect the contaminated environment, and indicate that host-parasite relationships are unstable. The high degree of aggregation of Trichuris spp., Ascaris spp., and Moniezia spp. also suggests that some individual hosts had less ability to resist pathogens and greater transmission potential than others. These conclusions suggest that a focus on disease control strategies could improve the health of forest musk deer in captivity.

Determination of ovarian cyclicity and pregnancy using fecal progesterone in forest musk deer (Moschus berezovskii).

The forest musk deer (FMD, Moschus berezovskii) is a threatened species in China. Although crucial for its artificial breeding management and thus protection, to date, gonadal steroidogenic activity data are unavailable in this species. Thus, the objectives of the present study were to characterize ovarian activity throughout the estrous cycle, non-pregnant luteal phase, and gestation in female FMD. These goals were accomplished using an enzyme immunoassay to measure fecal concentrations of estradiol (E2) and progesterone. Fecal samples were collected from female FMD (aged 3-4 years) for one year, including during breeding and non-breeding seasons. Non-pregnant estrous cycles were recorded in females, based on fecal progesterone concentrations, their overall estrous cycle length was (mean±SEM) 24.3±0.5 days, with 21.6±0.5 days in the luteal phase and 2.7±0.3 days in the inter-luteal phase. Fecal progesterone and E2 concentrations were also measured in females that became pregnant and gave birth after gestating approximately 6 months. Two weeks after becoming pregnant, the average fecal progesterone concentration was significantly greater than that during non-pregnancy. The average fecal progesterone concentrations during pregnancy increased 3.2-fold above non-pregnant concentrations, decreasing to non-pregnant concentrations only after parturition. By contrast, average fecal E2 concentrations during gestation and after parturition were not different from average non-pregnant concentration. In addition, no difference was observed between progesterone concentration in the first month after pregnancy and the value during the luteal phase. However, progesterone concentration during the second month of pregnancy was significantly higher than the value during the luteal phase. In conclusion, monitoring fecal progesterone is an effective method for assessing ovarian function in FMD and will be a useful tool for breeding management and development of assisted reproductive techniques, such as artificial insemination, in this species.

The incidence and species composition of Gasterophilus (Diptera, Gasterophilidae) causing equine myiasis in northern Xinjiang, China.

A survey was conducted on the detection of the larval Gasterophilus species in 90 equines via necropsy or after administering oral ivermectin in Xinjian, China, from 2008 to 2013. All 90 (100%) equines were infested by larval Gasterophilus, and 3723second instar larvae (L2) and 63,778 third instar larvae (L3) were collected from faecal samples and the digestive tract, a ratio of L2:L3=1:17. Over 84.45% of the animals contained ≤1500 larvae and 7.78% had >2000 larvae. The highest totals of L2 and L 3 larvae in any one animal were 1208 in Mongolian wild ass (Equus hemionus hemionus), 2491 in Przewalski's horse (Equus ferus przewalskii), and 1785 in the domestic horse (Equus ferus caballus). Six species of Gasterophilus were identified, with the following proportions of overall parasite abundance: Gasterophilus pecorum 88.94%, Gasterophilus nigricornis 4.94%, Gasterophilus nasalis 3.93%, Gasterophilus haemorrhoidalis 1.91%, Gasterophilus intestinalis 0.19%, and Gasterophilus inermis 0.087%. A majority of equines (n=32, 35.57%) was infested with five Gasterophilus species, while 29 animals (32.22%) harboured four species, 13 animals (14.44%) had six, 12 animals (13.33%) had three, three (3.33%) had two, and one (1.11%) had only one species. The percentage of Przewalski's horses infested was higher than local domestic horse or Mongolian wild ass.

Oviposition site selection by Gasterophilus pecorum (Diptera: Gasterophilidae) in its habitat in Kalamaili Nature Reserve, Xinjiang, China.

Oviposition site selection is an important aspect of the behavioural ecology of insects. A comparison of the habitats used by a species enhances our understanding of their adaptation to altered environments. We collected data on the oviposition behaviours of Gasterophilus pecorum (Diptera: Gasterophilidae) in its habitat in Kalamaili Nature Reserve (KNR), Xinjiang, China between March and October 2014. We found 91 quadrats were used by G. pecorum for oviposition. Examining 13 ecological factors using the t-test, chi-square test, and principal component analysis showed that G. pecorum's oviposition habitat was preferentially on slopes with inclinations of 10-30° that were semi-sunny, semi-cloudy slopes, in positions high or low on the slopes, with preferences for total plants lower than 10% and Stipa capillata coverage lower than 10% on the low slopes, but Ceratoides latens coverage on the high and intermediate slopes, when the numbers of plant species and families were lower than five. G. pecorum often selected sites at a distance < 2000 m from a water source and average altitude 900-1000 m. The oviposition site selection by G. pecorum may be correlated with the behaviour of Przewalski's horses (Equus ferus przewalskii), and water and food resources may strongly influence oviposition site selection, as Przewalski's horses rest and forage in these areas.

Effects of crowding and sex on fecal cortisol levels of captive forest musk deer.

BACKGROUND: Restricted space and close contact with conspecifics in captivity may be stressful for musk deer, as they are highly territorial and solitary in the wild. So we tested the effects of crowding on stress of forest musk deer (Moschus berezovskii) in heterosexual groups, using fecal cortisol analysis as a non-invasive method. 32 healthy adults during non-breeding seasons were chose as our experimental objects. Group 1 was defined as higher crowding condition, with 10-15 m2/deer (6 enclosures, 10♀ and 6♂); group 2 was defined as lower crowding condition, with 23-33 m2/deer (6 enclosures, 10♀ and 6♂). Every enclosure contained 1 male and 3 female. These patterns had been existed for years. RESULTS: The results showed that females in lower crowding condition (217.1 ± 9.5 ug/g) had significantly higher fecal cortisol levels than those in higher crowding condition (177.2 ± 12.1 ug/g). Interestingly, crowding seemed have no effect on male fecal cortisol levels (148.1 ± 9.1 ug/g and 140.5 ± 13.3 ug/g, respectively). At both groups, cortisol was significantly lower in males than in females. CONCLUSIONS: These results showed that chronic crowding may affect stress status of captive forest musk deer. The captive environment should consider the space need for musk deer.

Mitochondrial and pedigree analysis in Przewalski's horse populations: implications for genetic management and reintroductions.

BACKGROUND AND AIMS: Przewalski's horses have been imported from the western zoos to China since 1985. Yet the genetic diversity in China's populations has not been studied, thus lacking of such knowledge inevitably affects this population's management. The aim of this study was to assess genetic diversity in Chinese population of Przewalski's horses via mitochondrial DNA (mtDNA) control region and pedigree analysis. MATERIALS AND METHODS: Two captive and one reintroduced populations were examined based on mitochondrial DNA control region variation via fecal sampling from 2010 to 2012, together with pedigree analysis. RESULTS: Amplification success rates of fecal mtDNA were as high as 96.2% (93.8%-100%), and were higher for sample in winter than in summer and autumn. Two haplotypes were identified and shared among three populations, but the proportion of individuals with each haplotype varied among the three populations (F(ST) = 0.10874, p = 0.00978). Haplotype diversity in the released population (0.153) was much lower than that in the two captive populations (0.4011 and 0.4966), in accordance with the direction of increase in probability of identity at the dam lines. CONCLUSION: Future concerns in Przewalski's horse population management should emphasize on strict reproduction control to minimize inbreeding in captivity, followed by long-term genetic diversity guidelines and non-invasive monitoring in the reintroduction programmes.

Effects of crowding and sex on fecal cortisol levels of captive forest musk deer

BACKGROUND: Restricted space and close contact with conspecifics in captivity may be stressful for musk deer, as they are highly territorial and solitary in the wild. So we tested the effects of crowding on stress of forest musk deer (Moschus berezovskii) in heterosexual groups, using fecal cortisol analysis as a non-invasive method. 32 healthy adults during non-breeding seasons were chose as our experimental objects. Group 1 was defined as higher crowding condition, with 10-15 m²/deer (6 enclosures, 10♀ and 6♂); group 2 was defined as lower crowding condition, with 23-33 m²/deer (6 enclosures, 1010♀ and 6♂). Every enclosure contained 1 male and 3 female. These patterns had been existed for years. RESULTS: The results showed that females in lower crowding condition (217.1 ± 9.5 ug/g) had significantly higher fecal cortisol levels than those in higher crowding condition (177.2 ± 12.1 ug/g). Interestingly, crowding seemed have no effect on male fecal cortisol levels (148.1 ± 9.1 ug/g and 140.5 ± 13.3 ug/g, respectively). At both groups, cortisol was significantly lower in males than in females. CONCLUSIONS: These results showed that chronic crowding may affect stress status of captive forest musk deer. The captive environment should consider the space need for musk deer.

Cuticular structures on antennae of the bot fly, Portschinskia magnifica (Diptera: Oestridae).

Portschinskia magnifica Pleske, 1926, is an endangered subcutaneous parasitic fly of voles that mimics bumblebees in appearance. Although the importance of antennae in Diptera has been repeatedly stressed, there is still a lack of morphology information of this group, let alone this species. Antennae of adult P. magnifica were studied with a stereoscopic microscope and scanning electron microscope. Six subtypes of antennal sensilla were observed on the funiculus and arista (one subtype of trichoid sensilla, one subtype of basiconic sensilla, and four subtypes of coeloconic sensilla). Sensilla on the antennal funiculus from one sample of both male and female flies were also mapped. Female P. magnifica bear a larger number of sensilla than males, and more sensilla were discovered on the posterior surface than on the anterior surface. However, what make this species special are the distinct structures and new morphological characteristics discovered in the antenna. The antennal funiculus of P. magnifica is completely enveloped by an antennal pedicel. A large number of branched or unbranched trichoid and basiconic sensilla are identified on the antennal funiculus. These two make the most numerous types of sensilla distributed all over the funicular surface. P. magnifica has the most coeloconic sensilla subtypes in previously studied oestrid flies. A total of four subtypes of coeloconic sensilla are found, with subtype I and subtype II on the proximal and middle part of the antennal funiculus and subtype III and subtype IV on the antennal arista. Two large bristles that resemble mechanoreceptors on the proximal two antennal segments located close to the arista are a unique feature. This has never been described in the oestrid funiculus. Interestingly, these bristles can appear as multi-tipped short structures in some individuals. P. magnifica lacks sensory pits that are usually abundant in other oestrid flies. The size, number, and distribution of sensilla on the male and female antennal funiculus are provided here, armed with a discussion of their presumable sensilla functions and evolutionary trends.