Different disease inoculations cause common responses of the host immune system and prokaryotic component of the microbiome in Acropora palmata.

Reef-building corals contain a complex consortium of organisms, a holobiont, which responds dynamically to disease, making pathogen identification difficult. While coral transcriptomics and microbiome communities have previously been characterized, similarities and differences in their responses to different pathogenic sources has not yet been assessed. In this study, we inoculated four genets of the Caribbean branching coral Acropora palmata with a known coral pathogen (Serratia marcescens) and white band disease. We then characterized the coral's transcriptomic and prokaryotic microbiomes' (prokaryiome) responses to the disease inoculations, as well as how these responses were affected by a short-term heat stress prior to disease inoculation. We found strong commonality in both the transcriptomic and prokaryiomes responses, regardless of disease inoculation. Differences, however, were observed between inoculated corals that either remained healthy or developed active disease signs. Transcriptomic co-expression analysis identified that corals inoculated with disease increased gene expression of immune, wound healing, and fatty acid metabolic processes. Co-abundance analysis of the prokaryiome identified sets of both healthy-and-disease-state bacteria, while co-expression analysis of the prokaryiomes' inferred metagenomic function revealed infected corals' prokaryiomes shifted from free-living to biofilm states, as well as increasing metabolic processes. The short-term heat stress did not increase disease susceptibility for any of the four genets with any of the disease inoculations, and there was only a weak effect captured in the coral hosts' transcriptomic and prokaryiomes response. Genet identity, however, was a major driver of the transcriptomic variance, primarily due to differences in baseline immune gene expression. Despite genotypic differences in baseline gene expression, we have identified a common response for components of the coral holobiont to different disease inoculations. This work has identified genes and prokaryiome members that can be focused on for future coral disease work, specifically, putative disease diagnostic tools.

The assembled and annotated genome of the masked palm civet (Paguma larvata).

BACKGROUND: The masked palm civet (Paguma larvata) acts as an intermediate host of severe acute respiratory syndrome coronavirus (SARS-CoV), which caused SARS, and transfered this virus from bats to humans. Additionally, P. larvata has the potential to carry a variety of zoonotic viruses that may threaten human health. However, genome resources for P. larvata have not been reported to date. FINDINGS: A chromosome-level genome assembly of P. larvata was generated using PacBio sequencing, Illumina sequencing, and Hi-C technology. The genome assembly was 2.44 Gb in size, of which 95.32% could be grouped into 22 pseudochromosomes, with contig N50 and scaffold N50 values of 12.97 Mb and 111.81 Mb, respectively. A total of 21,582 protein-coding genes were predicted, and 95.20% of the predicted genes were functionally annotated. Phylogenetic analysis of 19 animal species confirmed the close genetic relationship between P. larvata and species belonging to the Felidae family. Gene family clustering revealed 119 unique, 243 significantly expanded, and 58 significantly contracted genes in the P. larvata genome. We identified 971 positively selected genes in P. larvata, and one known human viral receptor gene PDGFRA is positively selected in P. larvata, which is required for human cytomegalovirus infection. CONCLUSIONS: This high-quality genome assembly provides a valuable genomic resource for exploring virus-host interactions. It will also provide a reliable reference for studying the genetic bases of the morphologic characteristics, adaptive evolution, and evolutionary history of this species.

Ancestral mitogenome capture of the Southeast Asian banded linsang.

Utilising a reconstructed ancestral mitochondrial genome of a clade to design hybridisation capture baits can provide the opportunity for recovering mitochondrial sequences from all its descendent and even sister lineages. This approach is useful for taxa with no extant close relatives, as is often the case for rare or extinct species, and is a viable approach for the analysis of historical museum specimens. Asiatic linsangs (genus Prionodon) exemplify this situation, being rare Southeast Asian carnivores for which little molecular data is available. Using ancestral capture we recover partial mitochondrial genome sequences for seven banded linsangs (P. linsang) from historical specimens, representing the first intraspecific genetic dataset for this species. We additionally assemble a high quality mitogenome for the banded linsang using shotgun sequencing for time-calibrated phylogenetic analysis. This reveals a deep divergence between the two Asiatic linsang species (P. linsang, P. pardicolor), with an estimated divergence of ~12 million years (Ma). Although our sample size precludes any robust interpretation of the population structure of the banded linsang, we recover two distinct matrilines with an estimated tMRCA of ~1 Ma. Our results can be used as a basis for further investigation of the Asiatic linsangs, and further demonstrate the utility of ancestral capture for studying divergent taxa without close relatives.

Genet-specific DNA methylation probabilities detected in a spatial epigenetic analysis of a clonal plant population.

In sessile organisms such as plants, spatial genetic structures of populations show long-lasting patterns. These structures have been analyzed across diverse taxa to understand the processes that determine the genetic makeup of organismal populations. For many sessile organisms that mainly propagate via clonal spread, epigenetic status can vary between clonal individuals in the absence of genetic changes. However, fewer previous studies have explored the epigenetic properties in comparison to the genetic properties of natural plant populations. Here, we report the simultaneous evaluation of the spatial structure of genetic and epigenetic variation in a natural population of the clonal plant Cardamine leucantha. We applied a hierarchical Bayesian model to evaluate the effects of membership of a genet (a group of individuals clonally derived from a single seed) and vegetation cover on the epigenetic variation between ramets (clonal plants that are physiologically independent individuals). We sampled 332 ramets in a 20 m × 20 m study plot that contained 137 genets (identified using eight SSR markers). We detected epigenetic variation in DNA methylation at 24 methylation-sensitive amplified fragment length polymorphism (MS-AFLP) loci. There were significant genet effects at all 24 MS-AFLP loci in the distribution of subepiloci. Vegetation cover had no statistically significant effect on variation in the majority of MS-AFLP loci. The spatial aggregation of epigenetic variation is therefore largely explained by the aggregation of ramets that belong to the same genets. By applying hierarchical Bayesian analyses, we successfully identified a number of genet-specific changes in epigenetic status within a natural plant population in a complex context, where genotypes and environmental factors are unevenly distributed. This finding suggests that it requires further studies on the spatial epigenetic structure of natural populations of diverse organisms, particularly for sessile clonal species.

Phylogeography of the Small Indian Civet and Origin of Introductions to Western Indian Ocean Islands.

The biogeographic dynamics affecting the Indian subcontinent, East and Southeast Asia during the Plio-Pleistocene has generated complex biodiversity patterns. We assessed the molecular biogeography of the small Indian civet (Viverricula indica) through mitogenome and cytochrome b + control region sequencing of 89 historical and modern samples to (1) establish a time-calibrated phylogeography across the species' native range and (2) test introduction scenarios to western Indian Ocean islands. Bayesian phylogenetic analyses identified 3 geographic lineages (East Asia, sister-group to Southeast Asia and the Indian subcontinent + northern Indochina) diverging 3.2-2.3 million years ago (Mya), with no clear signature of past demographic expansion. Within Southeast Asia, Balinese populations separated from the rest 2.6-1.3 Mya. Western Indian Ocean populations were assigned to the Indian subcontinent + northern Indochina lineage and had the lowest mitochondrial diversity. Approximate Bayesian computation did not distinguish between single versus multiple introduction scenarios. The early diversification of the small Indian civet was likely shaped by humid periods in the Late Pliocene-Early Pleistocene that created evergreen rainforest barriers, generating areas of intra-specific endemism in the Indian subcontinent, East, and Southeast Asia. Later, Pleistocene dispersals through drier conditions in South and Southeast Asia were likely, giving rise to the species' current natural distribution. Our molecular data supported the delineation of only 4 subspecies in V. indica, including an endemic Balinese lineage. Our study also highlighted the influence of prefirst millennium AD introductions to western Indian Ocean islands, with Indian and/or Arab traders probably introducing the species for its civet oil.

Classical and Molecular Cytogenetics of the Panther Genet Genetta maculata (Mammalia, Carnivora, Viverridae).

Genets (Genetta) are a genus of African mammalian carnivorans with 14 currently recognized species, although taxonomic uncertainties remain, particularly regarding the number of species within the large-spotted genet complex. This study presents the first banded karyotype and molecular cytogenetic analysis of a genetically identified panther genet, Genetta maculata, the most common and widespread taxon of the large-spotted genet complex, with a wide distribution in sub-Saharan Africa. Sampled in Gauteng Province, South Africa, it could be assigned to the subspecies G. m. letabae on geographic grounds and had a similar karyotype (2n = 52, FNa = 96) to those published for a panther genet from Ethiopia and for the West African large-spotted genet G. pardina. Notably, the specimen had a different autosomal morphology (2 acrocentric chromosomes) from that previously attributed to letabae (a single acrocentric chromosome), but the latter assignment was uncertain because the studied individuals were captive born and assigned based solely on a presumed origin in the former Transvaal Province of South Africa. Fluorescence in situ hybridization with a telomere repeat probe revealed the presence of telomeric sequences in the centromeres of most chromosomes, the so-called interstitial telomeric sites (ITSs). Since genets seem to have a unique, highly rearranged karyotype among feliforms and relatively low interspecific karyotypic variation, and considering the known instability of ITSs, we suggest that the large amount of ITSs found here might be due to evolutionarily recent extensive genomic rearrangements. This study provides cytogenetic information that contributes to our understanding of chromosomal variation and genomic rearrangements in genets, and valuable baseline data for future studies of karyotype evolution in carnivores in general and viverrids in particular.

The complete mitochondrial genome of the small Indian civet, Viverricula indica taivana - the first complete representation of the genus Viverricula.

The complete mitochondrial genome of Viverricula indica taivana, exclusive of tandem repeats within the control region, is 16,583 bp in length, with a total base composition of: 33.18% A, 28.93% T, 24.88% C, and 13.00% G in H-strand. The genome contains 37 genes, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region.

The complete mitochondrial genome of the Servaline Genet, Genetta servalina, the first representative from the family Viverridae (Mammalia, Carnivora).

Here I report the complete mitochondrial genome of the Servaline Genet, Genetta servalina, as sequenced from overlapping PCR products. The genome is 16,938 base pairs in length and contains the 37 genes found in a typical mammalian genome: 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The control region of G. servalina includes both RS2 and RS3 tandem repeats. The overall base composition on the L-strand is A: 32.8%, C: 25.5%, G: 13.5%, and T: 28.2%.

The complete mitochondrial genome of the Spotted Linsang, Prionodon pardicolor, the first representative from the family Prionodontidae (Mammalia, Carnivora).

The complete mitochondrial genome of the Spotted Linsang, Prionodon pardicolor, was sequenced using overlapping PCR products. The genome is 16,718 base pairs in length and contains the 37 genes found in a typical mammalian genome: 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The overall base composition on the L-strand is A: 32.4%, C: 25.0%, G: 13.9%, T: 28.7%. The control region of P. pardicolor includes both RS2 and RS3 tandem repeats. Phylogenetic analyses support a sister relationship with the Felidae.

The complete mitochondrial genome of the masked palm civet (Paguma larvata, Mammalia, Carnivora).

The complete mitochondrial genome of the masked palm civet (Paguma larvata, Mammalia, Carnivora) is a circular molecule of 16 710 bp in length, containing 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes, and a control region. The features of the mitochondrial genome of the masked palm civet are similar to the other mammals. The phylogenetic analysis shows that all species from the family Viverridae cluster together, in which P. larvata exhibits the closest relationship with Genetta servalina.

Utility of hair shafts from study skins for mitochondrial DNA analysis.

The condition of mtDNA in hair shafts preserved in a museum was examined using 30 study skins of masked palm civets, Paguma larvata (Viverridae), collected between 1924 and 2011. Comparisons of extracts from fresh and burnt alum-fixed hair shafts showed that burnt alum, which is commonly used in taxidermy, had no harmful effect on the amount of total DNA and lengths of the mtDNA fragments. Burnt alum-fixed hair shafts had a tendency to develop a small degree of melanin hindering PCR amplification compared with fresh hair shafts, although that observation was not supported statistically (Wilcoxon signed-rank test, Z = -0.183, P = 0.855). However, the amount of total DNA decreased after preparation of specimen in an exponential relation (regression of log DNA amount with year, regression analysis, F = 7.065, P = 0.013). Nevertheless, the oldest specimen, collected in 1924, yielded 1341.5 ng of DNA per 100 hair shafts, which was sufficient for PCR amplification. In addition, the mtDNA fragment length and amount of melanin in the hair shaft were not significantly correlated with the passage of time after preparation (F = 0.244, P = 0.625 for mtDNA fragment length; F = 0.039, P = 0.845 for the amount of melanin). Therefore, hair shafts prepared and preserved by chemical treatment in museums are good sources of mtDNA and useful for genetic analysis.

Comparative chromosome painting in Carnivora and Pholidota.

The order of Carnivora has been very well characterized with over 50 species analyzed by chromosome painting and with painting probe sets made for 9 Carnivora species. Representatives of almost all families have been studied with few exceptions (Otariidae, Odobenidae, Nandiniidae, Prionodontidae). The patterns of chromosome evolution in Carnivora are discussed here. Overall, many Carnivora species retained karyotypes that only slightly differ from the ancestral carnivore karyotype. However, there are at least 3 families in which the ancestral carnivore karyotype has been severely rearranged - Canidae, Ursidae and Mephitidae. Here we report chromosome painting of yet another Carnivora species with a highly rearranged karyotype, Genetta pardina. Recurrent rearrangements make it difficult to define the ancestral chromosomal arrangement in several instances. Only 2 species of pangolins (Pholidota), a sister order of Carnivora, have been studied by chromosome painting. Future use of whole-genome sequencing data is discussed in the context of solving the questions that are beyond resolution of conventional banding techniques and chromosome painting.

Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus.

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Novel sylvatic rabies virus variant in endangered golden palm civet, Sri Lanka.

Information is scarce about sylvatic rabies virus in Asia and about rabies in palm civets. We report a novel sylvatic rabies virus variant detected in a golden palm civet in Sri Lanka. Evolutionary analysis suggests the virus diverged from canine rabies viruses in Sri Lanka in ≈1933 (range 1886-1963).

Origins and founder effects on the Japanese masked palm civet Paguma larvata (Viverridae, Carnivora), revealed from a comparison with its molecular phylogeography in Taiwan.

The source areas of the Japanese populations of the masked palm civet Paguma larvata (Viverridae, Carnivora), an alien species in Japan, have not been identified. In the present study, to reveal their origins and genetic features, we determined the full mitochondrial DNA cytochrome b sequences (1,140 base-pairs) of a total of 206 individuals of P. larvata from the Honshu and Shikoku islands of Japan (186 animals) and Taiwan (20 animals), and investigated their molecular phylogeography and the genetic relationships between populations in these countries. We found that each animal from Japan exhibited one of four haplotypes (JA1, JA2, JA4, and JA5), and that JA1 and JA4 were more frequent in eastern Honshu and Shikoku-central Honshu, respectively. By contrast, six haplotypes consisting of four new types (TW1, TW2, TW3, and TW4) and the previously reported two types (JA1 and JA4) were identified from 20 animals from native populations in Taiwan. Within Taiwan, one haplotype set (JA1, TW1, and TW2) was distributed in the western region, while a second (JA4, TW3, and TW4) was found in the eastern region; these regions are separated by high mountain ranges. Our comparison of haplotype distributions strongly demonstrated that the eastern Japanese populations originated from animals of western Taiwan, and that the western Japanese populations originated from those of eastern Taiwan. In addition, the lower genetic variability and particular distribution patterns of haplotypes in Japan showed founder effects, which may have resulted from multiple introductions of P. larvata to Japan from Taiwan.

Identification of a forensic case using microscopy and forensically informative nucleotide sequencing (FINS): a case study of small Indian civet (Viverricula indica).

The exhibits obtained in wildlife offence cases quite often present a challenging situation for the forensic expert. The selection of proper approach for analysis is vital for a successful analysis. A generalised forensic analysis approach should proceed from the use of non-destructive techniques (morphological and microscopic examination) to partially destructive and finally destructive techniques (DNA analysis). The findings of non-destructive techniques may sometime be inconclusive but they definitely help in steering further forensic analysis in a proper direction. We describe a recent case where a very small dried skin piece (<0.05 mg) with just one small trimmed guard hair (0.4 cm) on it was received for species identification. The single guard hair was examined microscopically to get an indication of the type of species. We also describe the extraction procedure with a lower amount of sample, using an automated extraction method (Qiagen Biorobot EZ1) and PCR amplification of three mitochondrial genes (16s rRNA, 12s rRNA and cytochrome b) for species identification. Microscopic examination of the single hair indicated a viverrid species but the initial DNA analysis with 16s rRNA (through NCBI BLAST) showed the highest homology (93%) with a hyaenid species (Hyaena hyaena). However, further DNA analysis based on 12s rRNA and cytochrome b gene proved that the species was indeed a viverrid i.e. Viverricula indica (small Indian civet). The highest homology shown with a Hyaenid species by the 16s rRNA sequence from the case sample was due to lack of a 16s rRNA sequence for Viverricula indica in the NCBI data base. The case highlights the importance of morphological and microscopic examinations in wildlife offence cases. With respect to DNA extraction technology we found that automatic extraction method of Biorobot EZ1 (Qiagen) is quite useful with less amount of sample (much below recommended amount).

Analyses of sweet receptor gene (Tas1r2) and preference for sweet stimuli in species of Carnivora.

The extent to which taste receptor specificity correlates with, or even predicts, diet choice is not known. We recently reported that the insensitivity to sweeteners shown by species of Felidae can be explained by their lacking of a functional Tas1r2 gene. To broaden our understanding of the relationship between the structure of the sweet receptors and preference for sugars and artificial sweeteners, we measured responses to 12 sweeteners in 6 species of Carnivora and sequenced the coding regions of Tas1r2 in these same or closely related species. The lion showed no preference for any of the 12 sweet compounds tested, and it possesses the pseudogenized Tas1r2. All other species preferred some of the natural sugars, and their Tas1r2 sequences, having complete open reading frames, predict functional sweet receptors. In addition to preferring natural sugars, the lesser panda also preferred 3 (neotame, sucralose, and aspartame) of the 6 artificial sweeteners. Heretofore, it had been reported that among vertebrates, only Old World simians could taste aspartame. The observation that the lesser panda highly preferred aspartame could be an example of evolutionary convergence in the identification of sweet stimuli.

Phylogenetic relationships of the Asian palm civets (Hemigalinae & Paradoxurinae, Viverridae, Carnivora).

The Viverridae (Mammalia, Carnivora), one of the least studied groups of carnivorans, include two subfamilies of Asian palm civets: Hemigalinae and Paradoxurinae. The relationships between and within these two subfamilies have never been thoroughly tested using an extensive molecular sample set. In this study, we gathered sequences of four genes (two mitochondrial: Cytochrome b and ND2 and two nuclear: beta-fibrinogen intron 7 and IRBP exon 1) for eight of the eleven extant species representing these two subfamilies. The results showed that: (1) the Asian palm civets (Hemigalinae and Paradoxurinae) have a single origin and form the sister-group of the (Genettinae+Viverrinae) clade, (2) the Hemigalinae (including the otter civet Cynogale bennettii) are monophyletic, (3) the Paradoxurinae are monophyletic and (4) the small-toothed palm civet (Arctogalidia trivirgata) is an early offshoot within the Paradoxurinae. Using a relaxed molecular clock analysis, the differentiation of the (Hemigalinae+Paradoxurinae) was inferred to occur in the Late Oligocene/Early Miocene.

Isolation and characterization of polymorphic microsatellite markers for the masked palm civet (Paguma larvata).

The masked palm civet (Paguma larvata) has been suspected to be the host of a SARS-like CoV virus that causes severe acute respiratory syndrome in humans. In China, the palm civet lives wild and is farmed, but even though the species is a potential carrier of the virus, its geographic distribution and genetic diversity have never been studied. We report the isolation and characterization of six polymorphic microsatellite markers for P. larvata. To characterize each locus, two farmed masked palm civet populations from Shanxi and Guangxi provinces in China were genotyped. The number of alleles per locus ranged from 3 to 15, and the observed heterozygosity for these populations was 47.1 and 68.7%, respectively.