OBJECTIVE: The black rhinoceros (Diceros bicornis) is an endangered mammal for which a captive breeding program is part of the conservation effort. Black rhinos in zoo\'s often suffer from chronic infections and heamochromatosis. Furthermore, breeding is hampered by low male fertility. To aid a research project studying these topics, we sequenced and assembled the genome of a captive male black rhino using ONT sequencing data only.
METHODS: This work produced over 100 Gb whole genome sequencing reads from whole blood. These were assembled into a 2.47 Gb draft genome consisting of 834 contigs with an N50 of 29.53 Mb. The genome annotation was lifted over from an available genome annotation for black rhino, which resulted in the retrieval of over 99% of gene features. This new genome assembly will be a valuable resource in for conservation genetic research in this species.

The black rhinoceros (Diceros bicornis L.) is a critically endangered species historically distributed across sub-Saharan Africa. Hunting and habitat disturbance have diminished both its numbers and distribution since the 19th century, but a poaching crisis in the late 20th century drove them to the brink of extinction. Genetic and genomic assessments can greatly increase our knowledge of the species and inform management strategies. However, when a species has been severely reduced, with the extirpation and artificial admixture of several populations, it is extremely challenging to obtain an accurate understanding of historic population structure and evolutionary history from extant samples. Therefore, we generated and analyzed whole genomes from 63 black rhinoceros museum specimens collected between 1775 and 1981. Results showed that the black rhinoceros could be genetically structured into six major historic populations (Central Africa, East Africa, Northwestern Africa, Northeastern Africa, Ruvuma, and Southern Africa) within which were nested four further subpopulations (Maasailand, southwestern, eastern rift, and northern rift), largely mirroring geography, with a punctuated north-south cline. However, we detected varying degrees of admixture among groups and found that several geographical barriers, most prominently the Zambezi River, drove population discontinuities. Genomic diversity was high in the middle of the range and decayed toward the periphery. This comprehensive historic portrait also allowed us to ascertain the ancestry of 20 resequenced genomes from extant populations. Lastly, using insights gained from this unique temporal data set, we suggest management strategies, some of which require urgent implementation, for the conservation of the remaining black rhinoceros diversity.

The black rhinoceros (rhino) (Diceros bicornis) is critically endangered, with poaching being one of several threats to the species\' survival. Many reserves across several countries, including Namibia, South Africa, and Zimbabwe, now dehorn their rhinos in an attempt to reduce poaching. Historical data collected by the Namibian Ministry of Environment, Forestry, and Tourism and Save the Rhino Trust were used to investigate whether dehorning has an effect on age at first reproduction (AFR), inter-calving interval (ICI), birth sex ratios, calf survival, cause of death, and lifespan. These were assessed in four Namibian sub-populations (hereafter referred to as \'populations\') of black rhino (denoted A, B, C, and D) which have undergone varying levels of dehorning. No significant difference was found in any of the variables between dehorned and horned individuals. Population was a significant predictor of AFR (LRT = 7.433, p = 0.024) and ICI (LRT = 13.281, p = 0.004), although pairwise comparisons only found populations A and B to be significantly different (AFR: z = -2.736, p = 0.017, ICI: z = -3.705, p = 0.001). Additionally, a significantly higher number of males than females were born in population D (p = 0.021, CI = 0.544, 0.960). The main cause of death across all individuals was poaching, although there was no significant difference in the proportion of rhinos poached between dehorned and horned individuals (X 2 = 0.638, p = 0.424, n = 265). No evidence was found to suggest that dehorning has any effect on AFR, ICI, birth sex ratios, calf survival, or lifespan in the black rhino, which is reassuring in the continued use of dehorning as an anti-poaching technique in this species.

Conservation management interventions for the critically endangered black rhinoceros (Diceros bicornis) require immobilization, which offer opportunities for semen collection and cryopreservation to establish genetic reservoirs. In free-ranging rhinoceroses, a combination of the potent opioid etorphine and the tranquilizer azaperone is routinely used for chemical immobilization but is associated with muscle rigidity and severe cardiopulmonary changes. Additionally, azaperone inhibits semen emission. Seven free-ranging, male, sexually mature black rhinoceroses were immobilized with an alternative protocol consisting of 4.5 mg etorphine, 5 mg medetomidine, 50 mg midazolam and 2,500 IU hyaluronidase delivered remotely by darting from a helicopter. During the immobilization, electro-ejaculation was performed with a portable electro-ejaculator, and a species-specific rectal probe. Animals were observed for muscle tremors. Longitudinal changes in respiratory rate, heart rate and peripheral oxyhemoglobin saturation, measured at 5 min intervals, were assessed using a general mixed model. Non-invasive oscillometric blood pressure and arterial blood gas variables were measured at first handling and before reversal and compared using the Wilcoxon rank sum test. All animals were successfully immobilized, showed no muscle tremors, presented with normal heart rates and lactate concentration (<5 mmol/L), recovered uneventfully, but experienced acidemia, hypoxemia and hypercapnia. Induction time and total time in recumbency were 4.2 ± 0.41 and 38.4 ± 6.9 min, respectively. Electro-stimulation commenced after 11.7 ± 3.98 min and completed after 24.3 ± 6.65 min. Semen-rich fractions were successfully collected from six animals. Our observations indicate that etorphine-medetomidine-midazolam provides a promising immobilization protocol for free-ranging black rhinoceroses, that allows for successful electro-ejaculation.

The birth of two eastern black rhinoceros (Diceros bicornis michaeli) calves (0.2) at the Blank Park Zoo in Des Moines, IA offered an opportunity to collect normative data for the first six months of calf development. Both calves successfully stood within 76 minutes of birth. Nursing first occurred at approximately 130 minutes. First defecation occurred within 1-2 days. Body weight increased by approximately 1.2 kg/day. Activity budgets for the first six months of the calves\' lives revealed that they spent a substantial proportion of their time resting, followed by standing, walking, and eating, respectively. Nursing duration increased gradually over the study period. Calves spent the majority of their time throughout the first six months in close proximity to the cow. Interactions with inanimate features of the environment began early, and no clear developmental pattern was evident in the data. These data can serve as the initial entry in a public record of developmental information for this species and as a reference point for other zoos, assisting them in monitoring the health and proper development of black rhinoceros calves and maximizing their welfare.

The two black rhinoceros subspecies (Diceros bicornis bicornis and D. b. minor) in South African conservation areas are managed as separate metapopulations. Since infection with Babesia bicornis can be fatal in black rhinoceroses, occurrence of this and other piroplasms in the two metapopulations was determined to assess possible risk. Blood specimens were collected from 156 black rhinoceroses: 80 from D. b. bicornis and 76 from D. b. minor. DNA was extracted; the V4 hypervariable region of the parasite 18S rRNA gene was amplified and subjected to the Reverse Line Blot (RLB) hybridization assay. There was a significant difference in occurrence of piroplasms: 18/80 (23%) in D. b. bicornis and 39/76 (51%) in D. b. minor. Theileria bicornis occurred in significantly more of the D. b. minor population (36/76; 47%) than the D. b. bicornis population (1/80; 1%); with B. bicornis the difference was not significant: D. b. bicornis 5/80 (6%) and D. b. minor 9/76 (11%). Three individuals were infected with Theileria equi. Results were confirmed using molecular characterization of the near full-length parasite 18S rRNA gene of 13 selected specimens. We identified four (Tb1, Tb2, Tb3 and Tb4) 18S rDNA sequence types for T. bicornis, two for B. bicornis (Bb1 and Bb2) and one for T. equi (Teq1). We furthermore identified T. bicornis haplotypes H1, H3 and H4 in 10 rhinoceroses; H3 was the most common haplotype identified. Rhinoceroses inhabiting more arid areas are apparently free of T. bicornis and B. bicornis, probably due to the absence or scarcity of vectors. When individuals are relocated for metapopulation management purposes, appropriate prophylactic action should be taken to minimise the risk of babesiosis, which could be fatal.

Upsurge of antibiotic resistance in wildlife poses unprecedented threat to wildlife conservation. Surveillance of antibiotic resistance at the human-wildlife interface is therefore needed. We evaluated differences in antibiotic resistance of Escherichia coli isolates from human and the endangered black rhinoceros in Lambwe Valley, Kenya. We used standard microbiological techniques to carry out susceptibility assays using eight antibiotics of clinical and veterinary importance. Standard PCR method was used to characterize antibiotic resistance genes. There was no difference in resistance between E. coli isolates from human and those from rhinoceros (U = 25, p = 0.462). However, higher resistance in isolates from humans was noted for cotrimoxazole (p = 0.000, OR = 0.101), ceftriaxone (p = 0.005, OR = 0.113) and amoxicillin/clavulanic acid (p = 0.017, OR = 0.258), whereas isolates from rhinoceros showed higher gentamicin resistance (p = 0.001, OR = 10.154). Multi-drug resistance phenotype was 69.0% in humans and 43.3% in rhinoceros. Isolates from both species contained blaTEM, tetA, tetB, dfrA1 and sul1 genes. Resistance profiles in the two species suggest potential for cross-transfer of resistance genes or exposure to comparable selective pressure and call for a multi-sectorial action plan on surveillance of antibiotic resistance at the human-wildlife interface. Genome-wide studies are needed to explicate the direction of transfer of genes that confer antibiotic resistance at the human-wildlife interface.

Iron overload disorder (IOD) affects many wildlife species cared for ex situ. Two of the four rhinoceros species in human care, Sumatran rhinoceros (Dicerorhinus sumatrensis) and black rhinoceros (Diceros bicornis), are susceptible, whereas the other two, white rhinoceros (Ceratotherium simum) and greater one-horned (GOH) rhinoceros (Rhinoceros unicornis), are relatively resistant to IOD. Complex interrelationships exist between mammalian hosts, their indigenous gut microbiota, metabolome, physical condition, and iron availability. The goal of this study was to gain insight into these relationships within the family Rhinocerotidae. Specific objectives were to (1) characterize the gut microbiome and metabolome of four rhinoceros species; (2) compare the microbiome and metabolome of IOD-susceptible and IOD-resistant rhinoceros species; and (3) identify variation in the microbiome and metabolome associated with compromised health or disease in IOD-susceptible rhinoceroses. Fecal samples were collected from 31 rhinoceroses (Sumatran rhinoceros, n = 3; black rhinoceros, n = 6; GOH rhinoceros, n = 9; white rhinoceros, n = 13) located at five facilities, and matched fecal aliquots were processed for microbiome and metabolome analyses using 16S rRNA gene sequencing and nuclear magnetic resonance spectroscopy, respectively. Despite the phylogenetic disparity and dissimilar zoo diets of the hosts, the structure of the fecal microbiota of the two IOD-susceptible rhinoceros species were more closely related to each other than to those of the two IOD-resistant species (Bray-Curtis dissimilarity; IOD-susceptible vs. IOD-resistant p-value < 0.001). In addition, IOD-susceptible rhinoceroses exhibited less microbial diversity than their IOD-resistant relatives (Shannon diversity; p-value < 0.001) which could have health implications. Of note, the black rhinoceros was distinct among the four rhinoceros species with the most divergent fecal metabolome; interestingly, it contained higher concentrations of short chain fatty acids. Neither age nor sex were associated with differences in microbial community composition (p = 0.253 and 0.488, respectively) or fecal metabolomic profile (p = 0.634 and 0.332, respectively). Differences in the distal gut microbiomes between IOD-resistant and IOD-susceptible rhinoceroses support hypotheses that gut microbes play a role in host iron acquisition, and further studies and experiments to test these hypotheses are warranted.

The proportion of females calving (PFC) each year has been employed as an indicator of population reproductive performance in ungulates, especially for species that breed annually, because it requires less detailed population data than inter-birthing intervals and age at first reproduction. For asynchronous breeders with inter-birthing intervals longer than a year such as megaherbivores, however, it is unclear how much annual variation in PFC is expected and whether false signals of density feedback or environmental influence might result from analyzing PFC data. We used census data from a well studied, closed, expanding population of black rhinoceros (Diceros bicornis) to study annual variation in PFC over 22 years. Our analysis of PFC data yielded no false signals of density feedback but weak evidence for an unexpected influence of rainfall. The PFC data exhibited considerable variation, which we attribute to autocorrelation in the time series of PFC data, \'demographic-founding effects\', changes in stage structure, and demographic stochasticity, some of which the modelling of PFC appears to confuse with an influence of rainfall. We expect such variation to be common in introduced populations and to persist for some years, complicating the interpretation of PFC, though moving averages of PFC can help if employed cautiously. While our analysis does not undermine the possible utility of PFC, the analysis and interpretation of PFC values require care.

  Iron overload disorder (IOD) can lead to organ dysfunction and may exacerbate other diseases in the critically endangered black rhinoceros ( Diceros bicornis). It is important to develop methods for monitoring the progression of iron storage (hemosiderosis), diagnosing the disease, and evaluating treatments in this species. Traditionally, an equine enzyme immunoassay (EIA) was used to measure rhinoceros ferritin, a serum protein correlated to iron stores. The goal of this study was to validate a rhinoceros-specific assay and investigate factors potentially associated with ferritin concentrations in black rhinoceros. A ferritin EIA developed for Sumatran rhinoceros was validated for black rhinoceros via Western blot analysis of liver ferritin and confirmed parallelism of serum samples to the EIA standard curve and used to analyze serum samples ( n = 943) collected from 36 black rhinoceros (<1-33 yr) at 14 U.S. institutions. Mean (±SEM) serum ferritin concentration was 6,738 ± 518 ng/ml (range: 85-168,451 ng/ml). Concentrations differed among individuals with eastern black rhinoceros (7,444 ± 1,130 ng/ml) having a higher mean ferritin than southern black rhinoceros (6,317 ± 505 ng/ml; P < 0.05) and higher mean values in wild-born (11,110 ± 1,111 ng/ml) than captive-born individuals (3,487 ± 293 ng/ml; P < 0.05). Ferritin concentrations did not differ between young rhinoceros (<5 yr old; 2,163 ± 254 ng/ml) and adults (7,623 ± 610 ng/ml) and were not correlated with age ( r = 0.143) or time in captivity ( r = 0.146, wild born; r = 0.104, all animals). Ferritin concentration was not impacted by sex (female: 2,086 ± 190 ng/ml; male: 8,684 ± 717 ng/ml), date, month, or season of collection ( P > 0.05). Data indicate ferritin concentrations are variable and not necessarily associated with IOD; ferritin is not recommended for diagnosing or monitoring IOD in black rhinoceros.