Cyanobacterial Genomes from a Brackish Coastal Lagoon Reveal Potential for Novel Biogeochemical Functions and Their Evolution.

Cyanobacteria are recognised for their pivotal roles in aquatic ecosystems, serving as primary producers and major agents in diazotrophic processes. Currently, the primary focus of cyanobacterial research lies in gaining a more detailed understanding of these well-established ecosystem functions. However, their involvement and impact on other crucial biogeochemical cycles remain understudied. This knowledge gap is partially attributed to the challenges associated with culturing cyanobacteria in controlled laboratory conditions and the limited understanding of their specific growth requirements. This can be circumvented partially by the culture-independent methods which can shed light on the genomic potential of cyanobacterial species and answer more profound questions about the evolution of other key biogeochemical functions. In this study, we assembled 83 cyanobacterial genomes from metagenomic data generated from environmental DNA extracted from a brackish water lagoon (Chilika Lake, India). We taxonomically classified these metagenome-assembled genomes (MAGs) and found that about 92.77% of them are novel genomes at the species level. We then annotated these cyanobacterial MAGs for all the encoded functions using KEGG Orthology. Interestingly, we found two previously unreported functions in Cyanobacteria, namely, DNRA (Dissimilatory Nitrate Reduction to Ammonium) and DMSP (Dimethylsulfoniopropionate) synthesis in multiple MAGs using nirBD and dsyB genes as markers. We validated their presence in several publicly available cyanobacterial isolate genomes. Further, we identified incongruities between the evolutionary patterns of species and the marker genes and elucidated the underlying reasons for these discrepancies. This study expands our overall comprehension of the contribution of cyanobacteria to the biogeochemical cycling in coastal brackish ecosystems.

Development of an enzyme immunoassay to measure urinary and faecal 5α-androst-16-en-3-one in pigs.

Androstenone, a volatile steroid that possesses pheromonal activity, is responsible for boar taint, sexual interactions, and reproduction in pigs. A wide range of analytical methods has been developed to quantify and detect androstenone in adipose tissue and blood, which are invasive procedures. Therefore, the present study aimed to develop a non-invasive method to detect and quantify the androstenone. We produced group-specific polyclonal androstenone antibody to standardize and validate an enzyme immunoassay to measure faecal and urinary androstenone in Yorkshire boars and sows. Parallelism was performed to determine the immunoreactivity between faecal and urinary immunoreactive androstenone and respective antibody. In boars, urinary and faecal androstenone concentrations were higher on the day of mounting and copulation with sows. In sows, we also measured faecal progesterone metabolites to confirm the oestrus and mating. Faecal androstenone concentrations were peaked on the day of oestrus and mating in sows. Our results suggest that androstenone could be detected and quantified in faecal and urine samples of boars and sows. •Developed an enzyme immunoassay for measuring 5α-androst-16-en-3-one as a marker of boar taint and sex pheromone in urine and faeces of pigs•Detection of 5α-androst-16-en-3-one using a non-invasive method.

Measures of Adrenal and Gonadal Hormones in Relation to Biological and Management Factors among Captive Red Pandas in Indian Zoos.

Animals in human care are affected by stressors that can ultimately reduce fitness. When reproduction is affected, endangered species conservation programs can be severely compromised. Thus, understanding factors related to stress and reproduction, and related hormones, is important to ensure captive breeding success. Red pandas (Ailurus fulgens) are endangered, and populations in the wild are threatened with extinction. A global captive breeding program has been launched to conserve the species with the goal of reintroduction. However, there is little information on how stressors impact physiological aspects of the species. This study measured fecal glucocorticoid (fGCM), progestagen (fPM), and androgen (fAM) metabolite concentrations in 12 female and 8 male red pandas at 3 zoos in India to determine predictors of adrenal and gonadal steroid activity, and the influence of fGCM on reproduction. Based on the generalized linear mixed model (GLMM), fGCM concentrations were positively correlated with the number of visitors, number of nests and enclosure areas, and negatively related to frequency of feedings, log density, and social time, while fPM concentrations were negatively associated with enclosure areas. A confounder for enclosure areas and number of nests was the fact that these spaces were relatively barren, with limited hiding spaces, compared to the smaller enclosures. By contrast, no significant relationships were found for fAM, perhaps due to the smaller sample size. A negative relationship between fGCM and fPM was observed, indicating increasing adrenal hormones may decrease reproductive function among female red pandas. Results suggest that zoo management should consider increasing feeding frequency, providing larger enclosures with more enrichment and more nests in larger spaces, and regulating visitor numbers to support good welfare and potentially improve reproductive fitness of red pandas in captivity.

Genetic effects of long-term captive breeding on the endangered pygmy hog.

Long-term captive populations often accumulate genetic changes that are detrimental to their survival in the wild. Periodic genetic evaluation of captive populations is thus necessary to identify deleterious changes and minimize their impact through planned breeding. Pygmy hog (Porcula salvania) is an endangered species with a small population inhabiting the tall sub-Himalayan grasslands of Assam, India. A conservation breeding program of pygmy hog from six founders has produced a multi-generational captive population destined for reintroduction into the wild. However, the impact of conservation breeding on its genetic diversity remained undocumented. Here, we evaluate temporal genetic changes in 39 pygmy hogs from eight consecutive generations of a captive population using genome-wide SNPs, mitochondrial genomes, and MHC sequences, and explore the relationship between genetic diversity and reproductive success. We find that pygmy hog harbors a very low genome-wide heterozygosity (H) compared to other members of the Suidae family. However, within the captive population we find excess heterozygosity and a significant increase in H from the wild-caught founders to the individuals in subsequent generations due to the selective pairing strategy. The MHC and mitochondrial nucleotide diversities were lower in captive generations compared to the founders with a high prevalence of low-frequency MHC haplotypes and more unique mitochondrial genomes. Further, even though no signs of genetic inbreeding were observed from the estimates of individual inbreeding coefficient F and between individuals (FIS) in each generation, the kinship coefficient showed a slightly increasing trend in the recent generations, due to a relatively smaller non-random sample size compared to the entire captive population. Surprisingly, male pygmy hogs that had higher heterozygosity also showed lower breeding success. We briefly discuss the implications of our findings in the context of breeding management and recommend steps to minimize the genetic effects of long-term captive breeding.

Non-Invasive Reproductive Hormone Monitoring in the Endangered Pygmy Hog (Porcula salvania).

The pygmy hog (Porcula salvania), until recently was classified as a critically endangered suid facing the threat of extinction due to habitat degradation. Efforts are being made to protect the pygmy hog from extinction and breed them in captivity under the Pygmy Hog Conservation Programme (PHCP). However, very little information is available on the reproductive physiology of pygmy hogs. Therefore, the present study aims to standardize enzyme immunoassays (EIAs) for monitoring pregnancy and reproductive status using progesterone and testosterone metabolites. A total of 785 fecal samples were collected from five females and two males over a period of one year from the PHCP Research and Breeding Centre, Guwahati, Assam. High-pressure liquid chromatography (HPLC) analysis revealed the presence of immunoreactive progesterone and testosterone metabolites in feces. Mating was observed in all five females, and four of them gave birth successfully. We were able to detect pregnancy using fecal progesterone metabolites. The mean gestation period, based on mating and parturition, was estimated to be 153.25 days from the four females studied. The breeding center recorded 172 births between 1996 and 2000 and found strong seasonal patterns in the birth rate, with most of the births occurring between May and June. In the males, fecal testosterone metabolites were significantly higher in the breeding season than in the non-breeding season. This is the first study on the subject and will help with future breeding programs in other captive breeding centers and with reproductive monitoring of reintroduced populations.

Variation in predicted COVID-19 risk among lemurs and lorises.

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 2 million fatalities since it first emerged in late 2019. As we write, infection rates are at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary target of SARS-CoV-2 is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predict that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results while finding additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.

Non-invasive monitoring of glucocorticoid metabolite concentrations in native Indian, as well as captive and re-wilded tigers in South Africa.

Over the last century, wild tiger (Panthera tigris) numbers have declined from over 100 000 individuals to fewer than 4 000, with animals now confined to less than 5% of their historic range due to habitat loss, persecution, inadequate management, and poaching. In contrast, 15 000-20 000 tigers are estimated to be housed in captivity, experiencing conditions vastly different than their wild counterparts. A total of 280 tigers are currently held at 44 different facilities within South Africa, including zoos, semi-captive 're-wilded' populations, and pets; these animals provide a unique opportunity to measure the impact of extrinsic factors, found in exotic habitats, on the adrenocortical activity of tigers. By monitoring and comparing stress-related faecal glucocorticoid metabolite (fGCM) concentrations of tigers housed at different locations, and free ranging tigers in natural tiger reserves, this project aimed to get a better understanding of the impact of extrinsic factors on adrenocortical function as a measure of stress. The results of this study showed no significant difference in fGCM concentrations between captive, re-wilded, and free-ranging tigers with the exception of one site. Furthermore, factors such as sex and season were not significant drivers of fGCM concentrations. One study group had elevated fGCM concentrations, showing population variation in the stress response. This indicates that populations are able to cope with exotic environments, however, as population-specific differences in the stress response exist, we suggest management protocols be created for each population. This study offered the unique opportunity to see how well tigers are faring outside of their native range and if having re-wilded tigers in exotic locations is a potential welfare-acceptable management option for tiger conservation globally.

Variation in predicted COVID-19 risk among lemurs and lorises.

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 1.5 million fatalities since it first emerged in late 2019. As we write, infection rates are currently at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary viral target is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predicts that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results and finds additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and Endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.

A practical method for storage, preservation and transportation of anuran urine samples using filter paper for hormone analysis.

Anurans (frogs and toads) expelled urine when handled and it could provide insights into their physiological status. However, storage, preservation and transportation are often challenging. The study aimed to standardize and validate a field method for short-term storage and preserve of anuran urine samples using Whatman filter papers. To examine the efficacy of storage conditions and type of papers, urinary based enzyme immunoassays were used to measure progesterone and testosterone hormone metabolites. High-Performance Liquid Chromatography was performed and revealed immunoreactive progesterone and testosterone metabolites in the urine samples. Urinary hormone metabolites concentration stored in filter paper at room temperature and control samples stored in -20°C for the same period were similar. Whatman grade 50 was found to be more suitable for storage of hormones than grade 3 paper for the experiments performed.•A cheap and simple storage method for storage of anuran urine in field conditions using filter papers.•Anuran urine could be preserved and transported under ambient conditions without significant changes and loss of hormones.•This method would facilitate the endocrine monitoring of anurans in remote areas where limited logistics are available.

Non-Invasive Assessment of Physiological Stress in Captive Asian Elephants.

Asian elephant (Elephas maximus) populations, both in the wild and in captivity, have been continually declining over the decades. The present study examined the physiological stress response of captive Asian elephants in relation to body condition score and different working conditions. A total of 870 dung samples of 37 captive elephants (24 males and 13 females) from four facilities were collected to examine fecal glucocorticoid metabolite concentrations (fGCM). The elephants in forest camps with exposure to natural habitats had a higher body condition score than those in more confined spaces. Wild born elephants and females (except in one case) had higher concentrations of fGCM than captive born elephants and males, respectively. Elephants engaged in the Dussehra festival had elevated fGCM concentrations than their counterparts at Mysore zoo. We recommend a few management practices for the long-term survival of rapidly declining captive elephant populations.

Land Use Change Increases Wildlife Parasite Diversity in Anamalai Hills, Western Ghats, India.

Anthropogenic landscape changes such as land use change and habitat fragmentation are known to alter wildlife diversity. Since host and parasite diversities are strongly connected, landscape changes are also likely to change wildlife parasite diversity with implication for wildlife health. However, research linking anthropogenic landscape change and wildlife parasite diversity is limited, especially comparing effects of land use change and habitat fragmentation, which often cooccur but may affect parasite diversity substantially differently. Here, we assessed how anthropogenic land use change (presence of plantation, livestock foraging and human settlement) and habitat fragmentation may change the gastrointestinal parasite diversity of wild mammalian host species (n = 23) in Anamalai hills, India. We found that presence of plantations, and potentially livestock, significantly increased parasite diversity due possibly to spillover of parasites from livestock to wildlife. However, effect of habitat fragmentation on parasite diversity was not significant. Together, our results showed how human activities may increase wildlife parasite diversity within human-dominated landscape and highlighted the complex pattern of parasite diversity distribution as a result of cooccurrence of multiple anthropogenic landscape changes.

Physiological stress responses of tigers due to anthropogenic disturbance especially tourism in two central Indian tiger reserves.

Tigers continue to face unprecedented threats to their existence due to poaching, habitat loss, habitat fragmentation and anthropogenic disturbances. The present study examines the physiological stress response of tigers due to anthropogenic activities including wildlife tourism in Bandhavgarh Tiger Reserve and Kanha Tiger Reserve using faecal glucocorticoid metabolite (fGCM) measurement. We collected a total of 341 faecal samples from both reserves during tourism and non-tourism periods. Data on various anthropogenic disturbances including tourism activities like number of vehicles and visitors were also collected. We ascertained the species identity and sex of all the samples collected using genetic markers. fGCMs were extracted using a previously reported procedure, and fGCM concentrations were subsequently determined using an established enzyme immunoassay. There was no significant difference in overall mean fGCM concentrations between the two tiger reserves, but within each reserve, concentrations were significantly higher in tigers during the tourism period as compared to the non-tourism period. We also found that the number of tourist vehicles and disturbance level significantly correlated with fGCM concentrations. This study further supports the assumption that unbridled tourism associated with high anthropogenic disturbance can be related to perceived stress and consequently may have an impact on the reproductive fitness of tigers and long-term survival of isolated populations.

Hair Cortisol in Sheltered Cows and Its Association with Other Welfare Indicators.

India, the country with the largest population of dairy cows in the world, has a policy of retiring abandoned and non-lactating cows in shelters, but the level of provision for their welfare in these shelters is unclear. Cows in 54 shelters across India were assessed for historic evidence of physiological stress, through determination of hair cortisol in 540 samples from 10 cows in each shelter by enzyme immunoassay. Animal-based and shelter resource-based welfare measures were recorded and correlations with the hair cortisol investigated by multivariable analysis. High hair cortisol concentrations were associated with dung in the lying area of the cowshed, a low dry bulb temperature there and little cow access to yards, as shelter-based variables. At a cow level, high hair cortisol concentrations were associated with dirty flanks, hock joint ulceration, carpal joint injuries, body lesions, dehydration, an empty rumen, old age, and low levels of body hair loss. Hair cortisol level promises to be an effective biomarker of stress in cows when conducting studies under field conditions.

Comparison of tigers' fecal glucocorticoids level in two extreme habitats.

Application of different antibodies and extraction methods results in a wide range of steroid metabolite concentrations obtained during noninvasive hormones monitoring. It makes regional comparisons of steroid concentration very difficult. We compared three methods for extraction of glucocorticoids metabolites in tiger feces to examine correct stress level in Bengal and Amur tigers in India and Russia respectively. The results obtained with three different extraction methods correlate with each other positively and significantly. The highest concentration of fecal glucocorticoids metabolites (FGCM) was found after the extraction of wet feces samples with 90% methanol. The level of FGCM was significantly higher in Bengal tigers in India than in Amur tigers in Russian Far East. The reasons might be related to tigers' density or anthropogenic pressure.

Non-invasive monitoring of reproductive and stress hormones in the endangered red panda (Ailurus fulgens fulgens).

The red panda (Ailurus fulgens fulgens) is classified as endangered due to its declining population, habitat fragmentation and poaching. Efforts are being made to breed them in captivity as part of nationwide conservation breeding program. This study aimed to standardize Enzyme immunoassays (EIAs) to monitor reproductive (Progesterone metabolite, Testosterone) and stress hormone (Cortisol) in red panda. For this purpose, we collected 1471 faecal samples from four females and one male over a period of one year from Padmaja Naidu Himalayan Zoological Park, Darjeeling, India. HPLC confirmed the presence of immunoreactive 5α-pregnan-3α-ol-20-one, testosterone and cortisol metabolites in faecal samples. Using 5α-pregnan-3α-ol-20-one EIA, we were able to monitor reproduction and detect pregnancy in one of the females, which successfully conceived and delivered during the study period. We were also able to monitor testosterone and cortisol in faecal samples of the red panda. Faecal testosterone levels were found in higher concentration in breeding season than in non-breeding season. Faecal cortisol concentrations showed a negative relationship with ambient temperature and peaked during winter months in all animals. Standardization of EIAs and faecal hormone monitoring would facilitate red panda conservation breeding programs in India and elsewhere.

Genetic Diversity and Structure among Isolated Populations of the Endangered Gees Golden Langur in Assam, India.

Gee's golden langur (Trachypithecus geei) is an endangered colobine primate, endemic to the semi-evergreen and mixed-deciduous forests of Indo-Bhutan border. During the last few decades, extensive fragmentation has caused severe population decline and local extinction of golden langur from several fragments. However, no studies are available on the impact of habitat fragmentation and the genetic diversity of golden langur in the fragmented habitats. The present study aimed to estimate the genetic diversity in the Indian population of golden langur. We sequenced and analyzed around 500 bases of the mitochondrial DNA (mtDNA) hypervariable region-I from 59 fecal samples of wild langur collected from nine forest fragments. Overall, genetic diversity was high (h = 0.934, π = 0.0244) and comparable with other colobines. Populations in smaller fragments showed lower nucleotide diversity compared to the larger forest fragments. The median-joining network of haplotypes revealed a genetic structure that corresponded with the geographical distribution. The Aie and Champabati Rivers were found to be a barrier to gene flow between golden langur populations. In addition, it also established that T. geei is monophyletic but revealed possible hybridization with capped langur, T. pileatus, in the wild. It is hoped that these findings would result in a more scientific approach towards managing the fragmented populations of this enigmatic species.

Prevalence of Gastrointestinal Parasites in Civets of Fragmented Rainforest Patches in Anamalai Hills, Western Ghats, India.

: Parasitism, driven by anthropogenic habitat modifications, is being increasingly recognized as a major threat to wildlife. Unfortunately, even baseline parasite data for most wildlife species are lacking in India, including the civets, which are particularly vulnerable due to their proximity to human habitations. Civet fecal samples were collected from 10 forest fragments that vary in size and disturbance level in Anamalai Hills, Western Ghats, India. These samples were screened for the presence of gastrointestinal parasites using fecal floatation and fecal sedimentation techniques. From a total of 180 civet fecal samples, 15 gastrointestinal parasite taxa were recovered, and these species are also known to infect domesticated animals. Additionally, small, disturbed forest fragments recorded higher mean gastrointestinal parasite taxa and greater prevalence when compared to large, undisturbed forest fragments, indicating a potential relationship between anthropogenic activities and gastrointestinal parasitism of civets in the Anamalai Hills.

Non-invasive assessment of fecal progestagens and pregnancy detection in Himalayan musk deer (Moschus chrysogaster).

The Himalayan musk deer (Moschus chrysogaster), an endangered species, is facing threat of extinction globally due to severe hunting for its musk, and efforts are under way in India to breed them in captivity. However, no information is available on the reproductive cycles of the species. In this study, we aimed to standardize an enzyme immunoassay (EIA) procedure for monitoring pregnancy using fecal samples. We collected fecal samples for 12 months from five captive females maintained at the Musk Deer Research Centre, Bageshwar, Uttarakhand, India. Three of these females were observed mating and gave birth, whereas two were seen mating but did not give birth. The gestation periods for the three females were 183, 185, and 199 days, respectively. High-pressure liquid chromatography revealed the presence of immunoreactive pregnanediol-3-glucuronide (PdG), progesterone, and 5α-pregnan-3α-ol-20-one (5-alpha-pregnane) metabolites in the fecal samples. We used EIAs against progesterone, PdG, and 5-alpha-pregnane to monitor pregnancy. We found PdG EIA to be a highly accurate and sensitive assay compared with the other two assays in detecting pregnancy. We conclude that PdG EIA can be used to diagnose and monitor pregnancy in Himalayan musk deer using fecal steroid analysis, at an early stage of 3 months after mating. This study would help in conservation breeding of musk deer in captivity and in monitoring the reproductive status of the species in the wild.

Non-Invasive Pregnancy Diagnosis in Big Cats using the PGFM (13,14-dihydro-15-keto-PGF2α) Assay.

Non-invasive monitoring of hormones using feces has become a vital tool for reproductive management and reliable pregnancy diagnosis in big cats. Previous studies described the PGF2α metabolite (PGFM) as an indicator of pregnancy in various feline species. The present study aimed to standardize pregnancy detection in big cats like the tiger (Panthera tigris), jaguar (Panthera onca) and lion (Panthera leo) using fecal samples. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM in feces. An EIA developed against 9α,11α-dihydroxy-15-oxo-prost-5-en-1-oic acid-BSA was used to assay PGFM in fecal samples of the Bengal tiger, Asiatic lion and jaguar. The PGFM levels increased after 9 weeks of pregnancy and remained elevated until parturition. All animals showed elevated levels of PGFM in the last trimester of pregnancy, thus making PGFM a reliable tool for pregnancy diagnosis during this period that can be useful in captive breeding programs in these species.

Pre-Historic and Recent Vicariance Events Shape Genetic Structure and Diversity in Endangered Lion-Tailed Macaque in the Western Ghats: Implications for Conservation.

Genetic isolation of populations is a potent force that helps shape the course of evolution. However, small populations in isolation, especially in fragmented landscapes, are known to lose genetic variability, suffer from inbreeding depression and become genetically differentiated among themselves. In this study, we assessed the genetic diversity of lion-tailed macaques (Macaca silenus) inhabiting the fragmented landscape of Anamalai hills and examined the genetic structure of the species across its distributional range in the Western Ghats. We sequenced around 900 bases of DNA covering two mitochondrial regions-hypervariable region-I and partial mitochondrial cytochrome b-from individuals sampled both from wild and captivity, constructed and dated phylogenetic trees. We found that the lion-tailed macaque troops in the isolated forest patches in Anamalai hills have depleted mitochondrial DNA diversity compared to troops in larger and continuous forests. Our results also revealed an ancient divergence in the lion-tailed macaque into two distinct populations across the Palghat gap, dating to 2.11 million years ago. In light of our findings, we make a few suggestions on the management of wild and captive populations.