Ancient and modern genomes unravel the evolutionary history of the rhinoceros family.

Only five species of the once-diverse Rhinocerotidae remain, making the reconstruction of their evolutionary history a challenge to biologists since Darwin. We sequenced genomes from five rhinoceros species (three extinct and two living), which we compared to existing data from the remaining three living species and a range of outgroups. We identify an early divergence between extant African and Eurasian lineages, resolving a key debate regarding the phylogeny of extant rhinoceroses. This early Miocene (∼16 million years ago [mya]) split post-dates the land bridge formation between the Afro-Arabian and Eurasian landmasses. Our analyses also show that while rhinoceros genomes in general exhibit low levels of genome-wide diversity, heterozygosity is lowest and inbreeding is highest in the modern species. These results suggest that while low genetic diversity is a long-term feature of the family, it has been particularly exacerbated recently, likely reflecting recent anthropogenic-driven population declines.

Epidemiology of Mycobacterium bovis infection in free-ranging rhinoceros in Kruger National Park, South Africa.

Mycobacterium bovis infection, which is a prominent cause of bovine tuberculosis, has been confirmed by mycobacterial culture in African rhinoceros species in Kruger National Park (KNP), South Africa. In this population-based study of the epidemiology of M. bovis in 437 African rhinoceros (Diceros bicornis, Ceratotherium simum), we report an estimated prevalence of 15.4% (95% CI: 10.4 to 21.0%), based on results from mycobacterial culture and an antigen-specific interferon gamma release assay from animals sampled between 2016 and 2020. A significant spatial cluster of cases was detected near the southwestern park border, although infection was widely distributed. Multivariable logistic regression models, including demographic and spatiotemporal variables, showed a significant, increasing probability of M. bovis infection in white rhinoceros based on increased numbers of African buffalo (Syncerus caffer) herds in the vicinity of the rhinoceros sampling location. Since African buffaloes are important maintenance hosts for M. bovis in KNP, spillover of infection from these hosts to white rhinoceros sharing the environment is suspected. There was also a significantly higher proportion of M. bovis infection in black rhinoceros in the early years of the study (2016­2018) than in 2019 and 2020, which coincided with periods of intense drought, although other temporal factors could be implicated. Species of rhinoceros, age, and sex were not identified as risk factors for M. bovis infection. These study findings provide a foundation for further epidemiological investigation of M. bovis, a multihost pathogen, in a complex ecosystem that includes susceptible species that are threatened and endangered.

Historic Sampling of a Vanishing Beast: Population Structure and Diversity in the Black Rhinoceros.

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.

Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum)†.

Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR; Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR; Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G; 11-17 mm), Dominant (D; 18-29 mm), and Pre-ovulatory (P; 30-34 mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNAs previously known via subsequent annotation to all equine (Equus caballus; eca), bovine (Bos taurus; bta), and human (Homo sapiens; hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts.

Evolutionarily Young African Rhinoceros Gammaretroviruses.

High-throughput sequences were generated from DNA and cDNA from four Southern white rhinoceros (Ceratotherium simum simum) located in the Taronga Western Plain Zoo in Australia. Virome analysis identified reads that were similar to Mus caroli endogenous gammaretrovirus (McERV). Previous analysis of perissodactyl genomes did not recover gammaretroviruses. Our analysis, including the screening of the updated white rhinoceros (Ceratotherium simum) and black rhinoceros (Diceros bicornis) draft genomes identified high-copy orthologous gammaretroviral ERVs. Screening of Asian rhinoceros, extinct rhinoceros, domestic horse, and tapir genomes did not identify related gammaretroviral sequences in these species. The newly identified proviral sequences were designated SimumERV and DicerosERV for the white and black rhinoceros retroviruses, respectively. Two long terminal repeat (LTR) variants (LTR-A and LTR-B) were identified in the black rhinoceros, with different copy numbers associated with each (n = 101 and 373, respectively). Only the LTR-A lineage (n = 467) was found in the white rhinoceros. The African and Asian rhinoceros lineages diverged approximately 16 million years ago. Divergence age estimation of the identified proviruses suggests that the exogenous retroviral ancestor of the African rhinoceros ERVs colonized their genomes within the last 8 million years, a result consistent with the absence of these gammaretroviruses from Asian rhinoceros and other perissodactyls. The black rhinoceros germ line was colonized by two lineages of closely related retroviruses and white rhinoceros by one. Phylogenetic analysis indicates a close evolutionary relationship with ERVs of rodents including sympatric African rats, suggesting a possible African origin of the identified rhinoceros gammaretroviruses. IMPORTANCE Rhinoceros genomes were thought to be devoid of gammaretroviruses, as has been determined for other perissodactyls (horses, tapirs, and rhinoceros). While this may be true of most rhinoceros, the African white and black rhinoceros genomes have been colonized by evolutionarily young gammaretroviruses (SimumERV and DicerosERV for the white and black rhinoceros, respectively). These high-copy endogenous retroviruses (ERVs) may have expanded in multiple waves. The closest relative of SimumERV and DicerosERV is found in rodents, including African endemic species. Restriction of the ERVs to African rhinoceros suggests an African origin for the rhinoceros gammaretroviruses.

The hindgut microbiota of coconut rhinoceros beetles (Oryctes rhinoceros) in relation to their geographical populations.

The coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is a palm tree pest capable of rapidly expanding its population in new territories. Previous studies identified a digestive symbiosis between CRB and its gut microbes. However, no research compared the genetic variation of CRBs with their hindgut microbiota on a global scale. This study aims to investigate the genetic divergence of CRB and the compositional variation of CRB's microbiota across different geographical locations, and explore the association between them and their predicted functional profiles and environmental data. The research reveals a distinct and consistent microbial community within local populations, but it varies across different geographical populations. The microbial functional profiles linked to the production of digestive enzymes, including cellulases and ligninases, are nonetheless globally conserved. This suggests that CRBs employ specific mechanisms to select and maintain microbes with functional benefits, contributing to host adaptability, stress tolerance, and fitness. The CRB microbial communities did not appear to recapitulate the genetic variation of their hosts. Rather than depend on obligate symbionts, CRBs seem to establish similar digestive associations with whatever environmentally acquired microbes are available wherever they are, aiding them in successfully establishing after invading a new location.IMPORTANCECoconut rhinoceros beetles (CRBs) are notorious pests on Arecaceae plants, posing destructive threats to countries highly reliant on coconut, oil palm, and date palm as economic crops. In the last century, CRBs have rapidly expanded their presence to territories that were once free of these beetles. The United States, for instance, has officially designated CRBs as invasive and alien pests. Given their remarkable ability to swiftly adapt to new environments, their gut microbes may play a crucial role in this process. While the microbiota of CRBs vary depending on geographical location, these beetles consistently exhibit a functionally identical digestive association with locally acquired microbes. This underscores the significance of CRB-microbe association in shaping the adaptive strategies of this agricultural pest.

Estimation of the forces exerted on the limb long bones of a white rhinoceros (Ceratotherium simum) using musculoskeletal modelling and simulation.

Heavy animals incur large forces on their limb bones, due to the transmission of body weight and ground reaction forces, and the contractions of the various muscles of the limbs. This is particularly true for rhinoceroses, the heaviest extant animals capable of galloping. Several studies have examined their musculoskeletal system and the forces their bones incur, but no detailed quantification has ever been attempted. Such quantification could help understand better the link between form and function in giant land animals. Here we constructed three-dimensional musculoskeletal models of the forelimb and hindlimb of Ceratotherium simum, the heaviest extant rhino species, and used static optimisation (inverse) simulations to estimate the forces applied on the bones when standing at rest, including magnitudes and directions. Overall, unsurprisingly, the most active muscles were antigravity muscles, which generate moments opposing body weight (thereby incurring the ground reaction force), and thus keep the joints extended, avoiding joint collapse via flexion. Some muscles have an antigravity action around several joints, and thus were found to be highly active, likely specialised in body weight support (ulnaris lateralis; digital flexors). The humerus was subjected to the greatest amount of forces in terms of total magnitude; forces on the humerus furthermore came from a great variety of directions. The radius was mainly subject to high-magnitude compressive joint reaction forces, but to little muscular tension, whereas the opposite pattern was observed for the ulna. The femur had a pattern similar to that of the humerus, and the tibia's pattern was intermediate, being subject to great compression in its caudal side but to great tension in its cranial side (i.e. bending). The fibula was subject to by far the lowest force magnitude. Overall, the forces estimated were consistent with the documented morphofunctional adaptations of C. simum's long bones, which have larger insertion areas for several muscles and a greater robusticity overall than those of lighter rhinos, likely reflecting the intense forces we estimated here. Our estimates of muscle and bone (joint) loading regimes for this giant tetrapod improve the understanding of the links between form and function in supportive tissues and could be extended to other aspects of bone morphology, such as microanatomy.

Genomic identification of Oryctes rhinoceros nudivirus isolates, a biocontrol agent for coconut rhinoceros beetle.

The coconut rhinoceros beetle (Oryctes rhinoceros, CRB) is a serious pest of coconut and oil palms. It is native to South and Southeast Asia and was inadvertently introduced to Samoa in 1909. It has invaded many other Pacific countries throughout the last century. Oryctes rhinoceros nudivirus (OrNV), a natural pathogen of CRB in its native range, was successfully introduced as a classical biocontrol agent and has effectively suppressed invasive CRB populations for decades. However, resurgence of CRB has been recorded, with new invasions detected in several Pacific Island Countries and Territories. Additionally, new populations of CRB are emerging in some invaded areas that have a degree of resistance to the virus isolates commonly released for CRB biocontrol. Here, we designed a fast and reliable tool for distinguishing between different OrNV isolates that can help with the selection process to identify effective isolates for management of new CRB invasions. A comparison of 13 gene/gene region sequences within the OrNV genome of 16 OrNV isolates from native and invaded ranges allowed us to identify unique Single Nucleotide Polymorphisms (SNPs). With these SNPs, we developed an assay using multiplex PCR-amplicon-based nanopore sequencing to distinguish between OrNV isolates. We found that as few as four gene fragments were sufficient to identify 15 out of 20 OrNV isolates. This method can be used as a tool to monitor the establishment and distribution of OrNV isolates selected for release as biocontrol agents in CRB-infected areas.

Kaistella rhinocerotis sp. nov., isolated from the faeces of rhinoceros and reclassification of Chryseobacterium faecale as Kaistella faecalis comb. nov.

Strain Ran72T, a novel Gram-stain-negative, obligately aerobic, non-motile, and rod-shaped bacterium, was isolated from the faeces of the rhinoceros species Ceratotherium simum. The novel bacterial strain grew optimally in Reasoner's 2A medium under the following conditions: 0 % (w/v) NaCl, pH 7.5, and 30 °C. Based on phylogenetic analysis using 16S rRNA gene sequencing, strain Ran72T was found to be most closely related to Chryseobacterium faecale F4T (98.4 %), Kaistella soli DKR-2T (98.0 %), and Kaistella haifensis H38T (97.4 %). A comprehensive genome-level comparison between strain Ran72T with C. faecale F4T, K. soli DKR-2T, and K. haifensis H38T revealed average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values of ≤74.9, ≤19.3, and ≤78.7 %, respectively. The major fatty acids were anteiso-C15 : 0 (22.3 %), with MK-6 being the predominant respiratory quinone. The major polar lipids of strain Ran72T were phosphatidylethanolamine, four unidentified aminolipids, and two unidentified lipids. Based on our chemotaxonomic, genotypic, and phenotype characterizations, strain Ran72T was identified as representing a novel species in the genus Kaistella, for which the name Kaistella rhinocerotis sp. nov. is proposed, with the type strain Ran72T (=KACC 23136T=JCM 36038T). Based on the outcomes of our phylogenomic study, Chryseobacterium faecale should be reclassified under the genus Kaistella as Kaistella faecalis comb. nov.

The need for a socioecological harm reduction approach to reduce illegal wildlife trade.

The burgeoning illegal trade in succulents in southern Africa presents a critical conservation and social development challenge. Drawing parallels with the trajectory of the response to rhinoceros poaching, we considered the consequences of conservation law enforcement measures, particularly the militarization of antipoaching efforts. The response to rhinoceros poaching not only resulted in so-called green militarization, but also led to extrajudicial killings, human rights abuses, and the disproportionate targeting of low-level poachers. The nature of wildlife trade prohibition is complex and often contested, and many actors operating in illegal wildlife trades dispute the label of illegal for socioeconomic, cultural, historical, or political reasons. This contestation is crucial when considering Indigenous cultural and medicinal values of succulents, with Indigenous Peoples and local communities questioning the criminalization of traditional plant harvesting practices. As the illegal trade in succulents continues to grow, it is imperative for conservationists to consider a nuanced approach. We call for a socioecological harm reduction approach that emphasizes community engagement, sustainable use, and codesigned interventions. Such an approach could help balance the scales of ecological conservation and human dignity in the face of growing wildlife trade challenges.

La necesidad de una estrategia socioecológica de reducción de daño para disminuir el mercado ilegal de fauna Resumen El emergente mercado ilegal de suculentas en el sur de África representa un reto importante para la conservación y el desarrollo social. Partimos de las similitudes con la trayectoria de la respuesta a la caza furtiva de rinocerontes para considerar las consecuencias de la aplicación de las leyes de conservación, en particular la militarización de los esfuerzos contra la caza furtiva. La respuesta a la caza furtiva no sólo derivó en la llamada militarización verde, sino también llevó a ejecuciones extrajudiciales, abuso de los derechos humanos y a la selección desproporcionada de cazadores de bajo nivel. La naturaleza de la prohibición del mercado de fauna es compleja y con frecuencia se impugna, y muchos actores que operan en los mercados ilegales disputan la etiqueta ilegal por razones socioeconómicas, culturales, históricas o políticas. Esta impugnación es crucial cuando consideramos los valores culturales y medicinales que los indígenas dan a las suculentas, sobre todo cuando los pueblos indígenas y las comunidades locales cuestionan la criminalización de las prácticas tradicionales de recolección de plantas. Conforme el mercado ilegal de suculentas sigue creciendo, es imperativo que los conservacionistas consideren una estrategia con matices. Pedimos una estrategia socioecológica de reducción de daños que resalte la participación comunitaria, el uso sustentable y las intervenciones con co­diseño. Dicha estrategia podría ayudar a equlibrar la balanza de la conservación ecológica y la dignidad humana de cara al incremento de retos en el mercado de fauna.

Post-mortem interval determinations using insects collected from illegally hunted and dehorned rhinoceros in the Republic of South Africa from 2014 to 2021.

Wildlife forensic science is a growing research field globally with application in criminal cases of illegal hunting requiring an estimate of time of death based on insect fauna. The techniques and procedures of forensic entomology acquired over the last 40 years, used in legal cases relating to human remains, can be adapted to decomposing wildlife. Research on carrion utilising the rate of development of insect immatures provides a biological clock from which a minimum post-mortem interval (minPMI) can be derived. The following study concerns 19 rhinoceros that were illegally killed and dehorned in the Republic of South Africa between 2014 and 2021. The paper details 74 samples of insect evidence collected from these rhinoceros remains from which an accurate estimate of their PMI was calculated. The specimens comprised 18 species from 12 families belonging to three insect orders. Many Dipteran and Coleopteran species were found on and around each carcass. The species of fly larvae (family Calliphoridae) used in each case to estimate the PMI are as follows: Chrysomya marginalis (Wiedemann) (13 cases), Chrysomya chloropyga (Wiedemann) (2 cases), Chrysomya albiceps (Wiedemann) (1 case) and Chrysomya megacephala (Fabricius) (1 case). Two species of Coleoptera from the family Dermestidae and Silphidae involved Dermestes maculatus (DeGeer) and Thanatophilus micans (Fabricius), respectively, also were involved in one PMI estimation each. The paper highlights opportunities for improving our global understanding of gaps in procedures and training related to wildlife criminal cases.

Exploring Serum Ferritin's Connection to the Acute Phase Response in Zoo-Managed African Rhinoceroses.

Despite serum ferritin's potential as an iron status indicator, its concentrations vary significantly throughout a black rhinoceros's (Diceros bicornis) life, sometimes irrespective of iron load. We explored acute phase response-related biomarkers, serum amyloid A (SAA) and ceruloplasmin (Cp), to better understand the mechanisms influencing serum ferritin changes in managed black rhinoceroses. The objective was to evaluate the relationships between circulating levels of ferritin, SAA, and Cp in black and white rhinoceroses (Ceratotherium simum). We analyzed banked serum samples collected serially from 11 black (n = 222) and 7 white (n = 134), rhinoceroses (rhinos) and classified samples based on SAA values: clinically healthy (< 1 mg/L), subclinical (1-7 mg/L), or clinically abnormal (> 7 mg/L). In black rhinos, serum ferritin was not different between health status categories (p = 0.5292), nor was it correlated with SAA (p = 0.4164). However, Cp activity was significantly lower in clinically healthy sera (p < 0.0001) and had a moderate positive association with SAA (r = 0.477, p < 0.0001). Among the white rhino samples, only five had SAA values greater than 1 mg/L, limiting the assessment of ferritin and Cp activity in the health context. Minor, yet significant, relationships were observed between serum ferritin and Cp activity, negative in black rhinos (r = -0.206; p = 0.0022) and positive in white rhinos (r = 0.289, p = 0.0008). Cp activity may aid in diagnosing illness in black and white rhinos, based on values observed in sera collected near the time of death. However, acute inflammatory processes do not appear to be one of the primary drivers of the high ferritin concentrations detected in some black rhinos.

A Portable Fluid Administration System for Prolonged Intravenous Fluid Administration in Subadult and Adult White Rhinoceroses (Ceratotherium simum).

While translocations of white rhinoceroses have become an important conservation tool, dehydration during long-distance transports has been identified as a welfare concern. Intravenous (iv) fluid administration might therefore be useful to mitigate dehydration; however, special requirements need to be met to make iv fluid administration suitable for large, wild rhinoceroses during transport. Requirements include a portable and robust system that is capable of delivering high flow rates, is easy to set up, and remains patent and operating for long periods of time while allowing the animals to freely stand or lay down in the transport crates. Due to the lack of suitable fluid administration systems, we developed a custom-made system consisting of 8 L drip bags, a three-part, 4.4-m-long, large bore and partially coiled administration set, and a robust, battery-operated infusion pump, which allowed us to successfully administer iv fluids at a maintenance rate of 1-2 mL/kg/h to eight rhinoceroses for 24 h during a mock transport. While iv fluid administration in transported rhinoceroses is time intensive and the large amount of drip bags required during lengthy transports might pose a limitation, the developed system may be useful for the long-distance transport of small groups of rhinoceroses. Furthermore, this system would be of value for injured or sick rhinoceroses, which require parenteral fluid therapy when commercially available infusion pumps cannot provide the large fluid volumes needed.

Bacteroides rhinocerotis sp. nov., isolated from the fresh feces of rhinoceros in Beijing Zoo.

A Gram-negative strain, anaerobic, non-motile, non-spore-forming, rod-shaped bacterial strain named as NGMCC 1.200684 T was isolated from the fresh feces of rhinoceros in Beijing Zoo. Based on 16S rRNA gene sequences, phylogenetic analysis indicated that strain NGMCC 1.200684 T belonged to the genus Bacteroides and was most strongly related to the type strain of Bacteroides uniformis ATCC 8492 T (96.88%). The G + C content of the genomic DNA was determined to be 46.62%. Between strains NGMCC 1.200684 T and B. uniformis ATCC 8492 T, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were 93.89 and 67.60%, respectively. Strain NGMCC 1.200684 T can produce acid from fermentation of several substrates, including glucose, mannitol, lactose, saccharose, maltose, salicin, xylose, cellobiose, mannose, raffinose, sorbitol, trehalose, D-galactose, and maltotriose. The major cellular fatty acids (> 10%) were identified as anteiso-C15:0, iso-C15:0, iso-C14:0, and iso-C17:0 3-OH. The polar lipid profiles of strain NGMCC 1.200684 T were determined to contain diphosphatidyl glycerol, phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids, and two unknown amino-phospholipids. Based on phenotypic, phylogenetic, and chemotaxonomic characteristics, a novel species of the genus Bacteroides, Bacteroides rhinocerotis sp. nov. is proposed. The type strain is NGMCC 1.200684 T (= CGMCC 1.18013 T = JCM 35702 T).

Synthesis and Characterization of Curcumin-Chitosan Loaded Gold Nanoparticles by Oryctes rhinoceros' Chitin for Cosmeceutical Application.

A breakthrough in cosmeceuticals by utilizing insects as major ingredients in cosmetic products is gaining popularity. Therefore, the interest in rare sources of ingredients, for instance, from the Oryctes rhinoceros beetle, can bring huge benefits in terms of turning pests into wealth. In this study, curcumin was chosen as the active ingredient loaded into chitosan-gold nanoparticles (CCG-NP). Curcumin is unstable and has poor absorption, a high rate of metabolism, and high sensitivity to light. These are all factors that contribute to the low bioavailability of any substance to reach the target cells. Therefore, chitosan extracted from O. rhinoceros could be used as a drug carrier to overcome these limitations. In order to overcome these limitations, CCG-NPs were synthesized and characterized. Chitosan was isolated from O. rhinoceros and CCG-NPs were successfully synthesized at 70 °C for 60 min under optimal conditions of a reactant ratio of 2:0.5 (0.5 mM HAuCl4: 0.1% curcumin). Characterizations of CCG-NP involved FTIR analysis, zeta potential, morphological properties determination by FE-SEM, particle size analysis, crystallinity study by XRD, and elemental analysis by EDX. The shape of the CCG-NP was round, its size was 128.27 d.nm, and the value of the zeta potential was 20.2 ± 3.81 mV. The IC50 value for cell viability is 58%, indicating a mild toxicity trait. To conclude, CCG-NP is a stable, spherical, nano-sized, non-toxic, and homogeneous solution.

Postoccupancy evaluation of staff, visitors, and three species of animals in a zoo setting.

Postoccupancy evaluation (POE) was used to assess newly constructed zoo exhibits from the perspective of three user groups: zoo staff, zoo visitors, and the animals. Staff represents a generally understudied user group in zoo-based POEs. We asked staff to rate the animals' space, the visitors' space, and the staff's space at previous and new exhibits. We also compared zoo visitors' ratings of the animals' behavior and environments, overall exhibit impressions, and the time visitors spent viewing previous and new exhibits. Lastly, we compared activity and space use of a Komodo dragon (Varanus komodoensis), two red pandas (Ailurus fulgens), and one rhinoceros (Rhinoceros unicornis) in their previous and new exhibits. Staff rated animal, visitor, and staff areas higher at the new exhibits compared to the previous exhibits. Visitors also rated several factors higher and spent more time at the new exhibits. The most naturalistic exhibit received the most favorable ratings in all categories and animal activity increased visitor stay time. We found that red pandas were less active in their new exhibit, and the Komodo dragon and rhino showed no difference in activity. The red pandas and the Komodo dragon used more available space in their new exhibits; however, we recommend using Electivity index to examine resource preferences for these species, whose enclosure use has been less studied than large mammals. We emphasize the importance of including staff in zoo-based POE, make other recommendations for future POE studies, and discuss various factors that could have influenced our results.

A high-quality de novo genome assembly based on nanopore sequencing of a wild-caught coconut rhinoceros beetle (Oryctes rhinoceros).

BACKGROUND: An optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetle's mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation. RESULTS: High-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.1%). These quality metrics place our assembly ahead of the published Coleopteran genomes, including that of an insect model, the red flour beetle (Tribolium castaneum). The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes, with only 2.8% missing BUSCOs, and the expected number of non-coding RNAs. The number and structure of paralogous genes in a gene family like Sigma GST is lower than in another scarab beetle (Onthophagus taurus), but higher than in the red flour beetle (Tribolium castaneum), which suggests expansion of this GST class in Scarabaeidae. The quality of our gene models was also confirmed with the correct placement of O. rhinoceros among other members of the rhinoceros beetles (subfamily Dynastinae) in a phylogeny based on the sequences of 95 protein-coding genes in 373 beetle species from all major lineages of Coleoptera. Finally, we provide a list of 30 candidate dsRNA targets whose orthologs have been experimentally validated as highly effective targets for RNAi-based control of several beetles. CONCLUSIONS: The genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

Joining forces toward proactive elephant and rhinoceros conservation.

Proactive approaches that anticipate the long-term effects of current and future conservation threats could increase the effectiveness and efficiency of biodiversity conservation. However, such approaches can be obstructed by a lack of knowledge of habitat requirements for wildlife. To aggregate and assess the suitability of current information available on habitat requirements needed for proactive conservation, we conducted a systematic review of the literature on elephant and rhinoceros habitat requirements and synthesized data by combining a vote counting assessment with bibliometric and term maps. We contextualized these numeric and terminological results with a narrative review. We mapped current methods, results, terminology, and collaborations of 693 studies. Quantitative evidence for factors that influence the suitability of an area for elephants and rhinoceros was biased toward African savanna elephants and ecological variables. Less than one third of holistic approaches considered equal amounts of ecological and anthropogenic variables in their assessments. There was a general lack of quantitative evidence for direct proxies of anthropogenic variables that were expected to play an important role based on qualitative evidence and policy documents. However, there was evidence for a segregation in conceptual frameworks among countries and species and between science versus policy literature. There was also evidence of unused potential for collaborations among southern hemisphere researchers. Our results indicated that the success of proactive conservation interventions can be increased if ecological and anthropogenic dimensions are integrated into holistic habitat assessments and holistic carrying capacities and quantitative evidence for anthropogenic variables is improved. To avoid wasting limited resources, it is necessary to form inclusive collaborations within and across networks of researchers studying different species across regional and continental borders and in the science-policy realm.

Colaboración de Fuerzas hacia la Conservación Proactiva de Elefantes y Rinocerontes Resumen Los enfoques proactivos que anticipan los efectos a largo plazo de las amenazas a la conservación actuales y futuras podrían incrementar la efectividad y la eficiencia de la conservación de la biodiversidad. Sin embargo, dichos enfoques pueden ser frenados por una falta de conocimiento de los requerimientos de hábitat para la vida silvestre. Para sumar y evaluar la idoneidad de la información disponible sobre los requerimientos del hábitat necesitados para la conservación proactiva realizamos una revisión sistemática de la literatura sobre los requerimientos de hábitat de los elefantes y rinocerontes y sintetizamos los datos combinándolos en una evaluación de conteo de votos con mapas bibliométricos y de términos. Contextualizamos estos resultados numéricos y terminológicos con una revisión narrativa. Mapeamos los métodos, resultados, terminologías y colaboraciones actuales de 693 estudios. La evidencia cuantitativa de los factores que influyen sobre la idoneidad de un área para los elefantes y los rinocerontes estuvo sesgada hacia los elefantes de la sabana africana y las variables ecológicas. Menos de un tercio de las estrategias holísticas consideró cantidades iguales de variables ecológicas y antropogénicas en sus evaluaciones. Hubo una carencia generalizada de evidencias cuantitativas para los indicadores directos de las variables antropogénicas que se esperaba tendrían un papel importante con base en la evidencia cualitativa y los documentos de las políticas. Sin embargo, hubo evidencias de una segregación en el marco conceptual entre los países y las especies y entre la ciencia versus la literatura política. También hubo evidencias de un potencial sin explotar para las colaboraciones entre los investigadores del hemisferio sur. Nuestros resultados indicaron que el éxito de las intervenciones de conservación proactiva puede incrementarse si las dimensiones ecológicas y antropogénicas se integran a las evaluaciones holísticas del hábitat y si se mejoran las capacidades de carga holísticas y las evidencias cuantitativas de las variables antropogénicas. Para evitar gastar los recursos limitados, es necesario formar colaboraciones inclusivas dentro y a lo largo de las redes de investigadores que están estudiando a diferentes especies en las fronteras regionales y continentales y dentro del ámbito de la ciencia política.

Non-invasive assessment of fecal glucocorticoid, progesterone, and androgen metabolites and microbiome in free-ranging southern white rhinoceros (Ceratotherium simum simum) in South Africa.

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). Fecal microbial composition was assessed via next generation sequencing. EIAs with antibodies raised against progesterone (P4; mouse monoclonal - CL425 clone), testosterone (T; rabbit polyclonal), corticosterone (B; sheep polyclonal) were utilized. Pregnant females had large quantities of fecal progesterone metabolites (FPMs) detected by CL425 EIA. Pregnant females also had native P4 and 11α-hydroxydihydroprogesterone (11αOHDHP4; 4-pregnen-11α-ol-3,20-dione) detected by UPC2-MS/MS but these concentrations were 1000-fold less than the concentrations of FPMs detected by the CL425 EIA. By contrast, non-pregnant females had FPM concentrations detected by CL425 EIA which were similar to native P4 and 11αOHDHP4 concentrations detected by UPC2-MS/MS. Mean fecal androgen metabolite (FAM) concentrations detected by the T EIA were similar between males and females. 11-ketoandrostenedione (11KA4) detected by UPC2-MS/MS was higher in females than males. However, there was no difference between males and females in the concentration of fecal glucocorticoid metabolites (FGMs) detected by the B EIA. Bacteroidia, followed by Clostridia, was the most abundant classes of fecal microbes. The unfiltered microbiome of females was more diverse than that of males. The core fecal microbiome of young rhinoceros had a higher observed species richness (Shannon diversity index, and Simpson diversity index) than that of old rhinoceros. In the alpha male, immobilization was associated with an increase in FGMs detected by 11-deoxycortisol (S) detected by UPC2-MS/MS coupled with decreased abundance of Spirochaetia. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC2-MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC2-MS/MS and the fecal microbiome in wild white rhinoceros.

Identification and Characterization of Genes for the Allo A Lectins in Japanese Rhinoceros Beetle (Trypoxylus dichotomus [Allomyrina dichotoma]).

The Japanese rhinoceros beetle (Trypoxylus dichotomus [Allomyrina dichotoma]) produces the lectins allo A-I and allo A-II, which have strong N-acetyllactosamine (Galß1-4GlcNAc)-binding activity. It has been suggested that the two lectins are formed from three subunits (α, ß, and γ), with allo A-I comprising α and γ subunits and allo A-II comprising ß and γ subunits. Here, we determined the cDNA sequences of these subunits using both conventional polymerase chain reaction (PCR)-cloning-sequencing and transcriptome-sequencing analyses. For the α and ß subunits, one gene (locus) for each was predicted, whereas for the γ subunit, two types of cDNA sequences were obtained, which we named γ1 and γ2. These two types probably have distinct loci. Average nucleotide sequence identities among the subunits ranged from 87.6% (between α and γ1) to 92.6% (between γ1 and γ2), suggesting that they form a gene family. Although no homology was found between the sequences of allo A and other known lectin proteins in a protein database search, some unknown proteins containing the DUF3421 domain were identified. Those DUF3421 domain-encoding proteins are upregulated in the insect larval midgut. Thus, we infer that allo A genes also play an important role in larvae and that their lectin activity may have been obtained collaterally.