Search

To provide context for the diversification of archosaurs-the group that includes crocodilians, dinosaurs, and birds-we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs.

The Kunjin strain of West Nile virus (WNVKUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNVKUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNVKUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNVKUN and MVEV transmission by FTATM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNVKUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTATM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTATM card system as a sensitive and accurate method to monitor the transmission of WNVKUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a "One Health" issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTATM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities. [ABSTRACT FROM AUTHOR]

The risk of flavivirus infections among the crocodilian species was not recognised until West Nile virus (WNV) was introduced into the Americas. The first outbreaks caused death and substantial economic losses in the alligator farming industry. Several other WNV disease episodes have been reported in crocodilians in other parts of the world, including Australia and Africa. Considering that WNV shares vectors with other flaviviruses, crocodilians are highly likely to also be exposed to flaviviruses other than WNV. A serological survey for flaviviral infections was conducted on saltwater crocodiles (Crocodylus porosus) at farms in the Northern Territory, Australia. Five hundred serum samples, collected from three crocodile farms, were screened using a pan-flavivirus-specific blocking ELISA. The screening revealed that 26% (n = 130/500) of the animals had antibodies to flaviviruses. Of these, 31.5% had neutralising antibodies to WNVKUN (Kunjin strain), while 1.5% had neutralising antibodies to another important flavivirus pathogen, Murray Valley encephalitis virus (MVEV). Of the other flaviviruses tested for, Fitzroy River virus (FRV) was the most frequent (58.5%) in which virus neutralising antibodies were detected. Our data indicate that farmed crocodiles in the Northern Territory are exposed to a range of potentially zoonotic flaviviruses, in addition to WNVKUN. While these flaviviruses do not cause any known diseases in crocodiles, there is a need to investigate whether infected saltwater crocodiles can develop a viremia to sustain the transmission cycle or farmed crocodilians can be used as sentinels to monitor the dynamics of arboviral infections in tropical areas. [ABSTRACT FROM AUTHOR]

Abstract Background Genome elucidation is now in high gear for many organisms, and whilst genetic maps have been developed for a broad array of species, surprisingly, no such maps exist for a crocodilian, or indeed any other non-avian member of the Class Reptilia. Genetic linkage maps are essential tools for the mapping and dissection of complex quantitative trait loci (QTL), and in order to permit systematic genome scans for the identification of genes affecting economically important traits in farmed crocodilians, a comprehensive genetic linage map will be necessary. Results A first-generation genetic linkage map for the saltwater crocodile (Crocodylus porosus) was constructed using 203 microsatellite markers amplified across a two-generation pedigree comprising ten full-sib families from a commercial population at Darwin Crocodile Farm, Northern Territory, Australia. Linkage analyses identified fourteen linkage groups comprising a total of 180 loci, with 23 loci remaining unlinked. Markers were ordered within linkage groups employing a heuristic approach using CRIMAP v3.0 software. The estimated female and male recombination map lengths were 1824.1 and 319.0 centimorgans (cM) respectively, revealing an uncommonly large disparity in recombination map lengths between sexes (ratio of 5.7:1). Conclusion We have generated the first genetic linkage map for a crocodilian, or indeed any other non-avian reptile. The uncommonly large disparity in recombination map lengths confirms previous preliminary evidence of major differences in sex-specific recombination rates in a species that exhibits temperature-dependent sex determination (TSD). However, at this point the reason for this disparity in saltwater crocodiles remains unclear. This map will be a valuable resource for crocodilian researchers, facilitating the systematic genome scans necessary for identifying genes affecting complex traits of economic importance in the crocodile industry. In addition, since many of the markers placed on this genetic map have been evaluated in up to 18 other extant species of crocodilian, this map will be of intrinsic value to comparative mapping efforts aimed at understanding genome content and organization among crocodilians, as well as the molecular evolution of reptilian and other amniote genomes. As researchers continue to work towards elucidation of the crocodilian genome, this first generation map lays the groundwork for more detailed mapping investigations, as well as providing a valuable scaffold for future genome sequence assembly.

Highlights • Plasma corticosterone in different sized farmed saltwater crocodiles were measured. • No seasonal effects on CORT were observed. • Farmed saltwater crocodiles are not inherently stressed. Abstract Saltwater crocodiles (Crocodylus porosus) across three size categories (hatchlings, grower and harvest-size) were repeatedly blood sampled on two farms in the Northern Territory, Australia to determine reference plasma corticosterone (CORT; crocodilian stress hormone) levels. The mean CORT values for hatchlings (<1 year old), growers (1–3 years) and harvest-size individuals (2 + years) were 1.65 ± 0.15 ng/ml, 2.73 ± 0.21 ng/ml and 2.19 ± 0.16 ng/ml, respectively. No inter-farm differences within the hatchling or harvest-size crocodiles were detected, but growers on Farm 2 had significantly lower plasma CORT than those on Farm 1. However, the grower growth rate coefficients were the same across both farms so the repeated blood sampling design most likely contributed to the difference in CORT values rather than any management procedures. Plasma corticosterone levels significantly increased with time of day. Substantial variation in plasma CORT was observed at each sampling which is not unprecedented in the literature but requires further elucidation. Irrespective, as CORT values were generally low, our results suggest that the farming environment and husbandry practices, as implemented under the Australian industry Code of Practice, are effective as baseline animal welfare measures although they should be viewed as a foundation for further welfare research and not considered static. [ABSTRACT FROM AUTHOR]

Cutaneous poxvirus infections are common in several crocodilian species and are of importance in crocodile farming due to their potential impact on the tanned hide. To confirm poxvirus infection and understand the impact on saltwater crocodile ( Crocodylus porosus) skin, fourteen animals from different age groups (five hatchlings, five yearlings and four grow-outs) were selected based on a criterion of ten poxvirus-like lesions per animal. One lesion on each animal was extruded for genetic analysis and transmission electron microscopy. Both methods confirmed poxvirus so the remainder of lesions were re-examined every six weeks over a 24 week study period. Each lesion went through four distinct phases: early active, active, expulsion and healing. To understand how these lesions impact on the final skin product, one crocodile from each age group was euthanised and the lesions examined. Using standard skin grading techniques (light-table), the early phase (early active – expulsion) lesions were all translucent and would lead to downgrading of the skin or, at worst, rendering them unsaleable. At the later stages of healing, the translucency reduces. Histological examination of the phases confirm that the basement membrane is not breached by the infection further indicating that poxvirus lesions, given enough time, will eventually have no detrimental effect on skin quality. This is obviously dependent upon no more lesions developing in the interim. [ABSTRACT FROM AUTHOR]

Since 2006, 3 new disease syndromes have emerged in farmed saltwater crocodiles (Crocodylus porosus) in the Northern Territory of Australia. We describe the syndromes through a retrospective study of laboratory findings from 187 diagnostic cases submitted to Berrimah Veterinary Laboratories between 2005 and 2014. The first syndrome was characterized by conjunctivitis and/or pharyngitis (CP), primarily in hatchlings. Herpesviruses were isolated in primary crocodile cell culture, or were detected by PCR directly from conjunctiva or pharyngeal tissue, in 21 of 39 cases of CP (54%), compared with 9 of 64 crocodiles without the syndrome (14%, p < 0.0001). Chlamydiaceae were detected by PCR in conjunctiva or pharyngeal tissue of 55% of 29 CP cases tested, and of these, 81% also contained herpesvirus. The second syndrome occurred in juveniles and growers exhibiting poor growth, and was characterized histologically by systemic lymphoid proliferation and nonsuppurative encephalitis (SLPE). Herpesviruses were isolated or detected by PCR from at least 1 internal organ in 31 of 33 SLPE cases (94%) compared with 5 of 95 crocodiles without the syndrome (5%, p < 0.0001). The third syndrome, characterized by multifocal lymphohistiocytic infiltration of the dermis (LNS), occurred in 6 harvest-sized crocodiles. Herpesviruses were isolated from at least 1 skin lesion in 4 of these 6 cases. Although our study revealed strong associations between herpesvirus and the CP and SLPE syndromes, the precise nature of the role of herpesvirus, along with the pathogenesis and epidemiology of the syndromes, requires further investigation. [ABSTRACT FROM AUTHOR]

Background Crocodilians are thought to be hosts to a diverse and divergent complement of endogenous retroviruses (ERVs) but a comprehensive investigation is yet to be performed. The recent sequencing of three crocodilian genomes provides an opportunity for a more detailed and accurate representation of the ERV diversity that is present in these species. Here we investigate the diversity, distribution and evolution of ERVs from the genomes of three key crocodilian species, and outline the key processes driving crocodilian ERV proliferation and evolution. Results ERVs and ERV related sequences make up less than 2% of crocodilian genomes. We recovered and described 45 ERV groups within the three crocodilian genomes, many of which are species specific. We have also revealed a new class of ERV, ERV4, which appears to be common to crocodilians and turtles, and currently has no characterised exogenous counterpart. For the first time, we formally describe the characteristics of this ERV class and its classification relative to other recognised ERV and retroviral classes. This class shares some sequence similarity and sequence characteristics with ERV3, although it is phylogenetically distinct from the other ERV classes. We have also identified two instances of gene capture by crocodilian ERVs, one of which, the capture of a host KIT-ligand mRNA has occurred without the loss of an ERV domain. Conclusions This study indicates that crocodilian ERVs comprise a wide variety of lineages, many of which appear to reflect ancient infections. In particular, ERV4 appears to have a limited host range, with current data suggesting that it is confined to crocodilians and some lineages of turtles. Also of interest are two ERV groups that demonstrate evidence of host gene capture. This study provides a framework to facilitate further studies into non-mammalian vertebrates and highlights the need for further studies into such species. [ABSTRACT FROM AUTHOR]

The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2–6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs. [ABSTRACT FROM AUTHOR]

Major Histocompatibility Complex (MHC) class II genes encode for molecules that aid in the presentation of antigens to helper T cells. MHC characterisation within and between major vertebrate taxa has shed light on the evolutionary mechanisms shaping the diversity within this genomic region, though little characterisation has been performed within the Order Crocodylia. Here we investigate the extent and effect of selective pressures and trans-species polymorphism on MHC class II α and β evolution among 20 extant species of Crocodylia. Selection detection analyses showed that diversifying selection influenced MHC class II β diversity, whilst diversity within MHC class II α is the result of strong purifying selection. Comparison of translated sequences between species revealed the presence of twelve trans-species polymorphisms, some of which appear to be specific to the genera Crocodylus and Caiman. Phylogenetic reconstruction clustered MHC class II α sequences into two major clades representing the families Crocodilidae and Alligatoridae. However, no further subdivision within these clades was evident and, based on the observation that most MHC class II α sequences shared the same trans-species polymorphisms, it is possible that they correspond to the same gene lineage across species. In contrast, phylogenetic analyses of MHC class II β sequences showed a mixture of subclades containing sequences from Crocodilidae and/or Alligatoridae, illustrating orthologous relationships among those genes. Interestingly, two of the subclades containing sequences from both Crocodilidae and Alligatoridae shared specific trans-species polymorphisms, suggesting that they may belong to ancient lineages pre-dating the divergence of these two families from the common ancestor 85–90 million years ago. The results presented herein provide an immunogenetic resource that may be used to further assess MHC diversity and functionality in Crocodylia. [ABSTRACT FROM AUTHOR]

Immune responsiveness, the ability of an organism to effectively respond immunologically following antigenic exposure, is an essential component of life history, as organisms require effective immune functionality in order to grow, survive and reproduce. However, immune status is also associated with concomitant trade-offs in these physiological functions. Herein we demonstrate the validation of phytohæmagglutinin (PHA) injection in saltwater crocodiles, Crocodylus porosus, to assess cellular immune responsiveness. Following injection of 2 mg mL[sup -1] PHA into the hind toe webbing, we observed a peak swelling response 12h after injection, with PHA inducing increased thickness compared with webs injected with phosphate-buffered saline (PBS) (F[sub 5,518] = 145.13, P < 0.001). Subsequent injections increased responsiveness relative to the primary injection response (F[sub 5,290] = 2.92, P = 0.029), suggesting that PHA exposure induced immunological memory, a tenet of acquired immunity. Histological examination revealed that PHA-injected toe webs displayed increased numbers of leukocytes (granulocytes, macrophages, and lymphocytes) relative to PBS-injected webs, with peak leukocytic infiltrate observed 12 h after injection. We suggest the use of PHA injection in crocodilians as a measure of cellular immune responsiveness in agricultural (production and animal welfare), ecological, and toxicological contexts. [ABSTRACT FROM AUTHOR]

Background: Genome elucidation is now in high gear for many organisms, and whilst genetic maps have been developed for a broad array of species, surprisingly, no such maps exist for a crocodilian, or indeed any other non-avian member of the Class Reptilia. Genetic linkage maps are essential tools for the mapping and dissection of complex quantitative trait loci (QTL), and in order to permit systematic genome scans for the identification of genes affecting economically important traits in farmed crocodilians, a comprehensive genetic linage map will be necessary. Results: A first-generation genetic linkage map for the saltwater crocodile (Crocodylus porosus) was constructed using 203 microsatellite markers amplified across a two-generation pedigree comprising ten full-sib families from a commercial population at Darwin Crocodile Farm, Northern Territory, Australia. Linkage analyses identified fourteen linkage groups comprising a total of 180 loci, with 23 loci remaining unlinked. Markers were ordered within linkage groups employing a heuristic approach using CRIMAP v3.0 software. The estimated female and male recombination map lengths were 1824.1 and 319.0 centimorgans (cM) respectively, revealing an uncommonly large disparity in recombination map lengths between sexes (ratio of 5.7:1). Conclusion: We have generated the first genetic linkage map for a crocodilian, or indeed any other non-avian reptile. The uncommonly large disparity in recombination map lengths confirms previous preliminary evidence of major differences in sex-specific recombination rates in a species that exhibits temperature-dependent sex determination (TSD). However, at this point the reason for this disparity in saltwater crocodiles remains unclear. This map will be a valuable resource for crocodilian researchers, facilitating the systematic genome scans necessary for identifying genes affecting complex traits of economic importance in the crocodile industry. In addition, since many of the markers placed on this genetic map have been evaluated in up to 18 other extant species of crocodilian, this map will be of intrinsic value to comparative mapping efforts aimed at understanding genome content and organization among crocodilians, as well as the molecular evolution of reptilian and other amniote genomes. As researchers continue to work towards elucidation of the crocodilian genome, this first generation map lays the groundwork for more detailed mapping investigations, as well as providing a valuable scaffold for future genome sequence assembly. [ABSTRACT FROM AUTHOR]

In genetic improvement programs, candidates for breeders are ranked by the profitability of their offspring, expressed as a weighted sum of the genetic gain from selection. In this paper, we estimate the economic values of a genetic improvement program for Australian farmed saltwater crocodiles. A bioeconomic profit function for a representative breeding pair is used to determine the optimal slaughter age following genetic improvement in each selection objective. The results indicate that estimated farm profitability increases by nearly $A111 for a 1-week reduction in juvenile slaughter age, $A78 for a 1 per cent increase in the proportion of first-grade skins produced, and $A33 for an increase in the number of viable hatchlings per clutch. The implications of the analysis for the Australian crocodile industry and the limitations of the research are explored. [ABSTRACT FROM AUTHOR]

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described., Organismic and Evolutionary Biology

West Nile virus, Kunjin strain (WNVKUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNVKUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 105 (n = 10) or 104 (n = 11) TCID50-doses of WNVKUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2–17 dpi and in-contact animals 11–21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNVKUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNVKUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies. [ABSTRACT FROM AUTHOR]

Saltwater crocodilepox virus (SwCRV), belonging to the genus Crocodylidpoxvirus, are large DNA viruses posing an economic risk to Australian saltwater crocodile (Crocodylus porosus) farms by extending production times. Although poxvirus-like particles and sequences have been confirmed, their infection dynamics, inter-farm genetic variability and evolutionary relationships remain largely unknown. In this study, a poxvirus infection dynamics study was conducted on two C. porosus farms. One farm (Farm 2) showed twice the infection rate, and more concerningly, an increase in the number of early- to late-stage poxvirus lesions as crocodiles approached harvest size, reflecting the extended production periods observed on this farm. To determine if there was a genetic basis for this difference, 14 complete SwCRV genomes were isolated from lesions sourced from five Australian farms. They encompassed all the conserved genes when compared to the two previously reported SwCRV genomes and fell within three major clades. Farm 2′s SwCRV sequences were distributed across all three clades, highlighting the likely mode of inter-farm transmission. Twenty-four recombination events were detected, with one recombination event resulting in consistent fragmentation of the P4c gene in the majority of the Farm 2 SwCRV isolates. Further investigation into the evolution of poxvirus infection in farmed crocodiles may offer valuable insights in evolution of this viral family and afford the opportunity to obtain crucial information into natural viral selection processes in an in vivo setting. [ABSTRACT FROM AUTHOR]

Agricultural production of the saltwater crocodile ( Crocodylus porosus ) is an emergent industry in northern Australia with many of the factors affecting production remaining unknown. In this study, we sought to expand upon our previous findings of reference corticosterone and immune function by reporting baseline sex hormone levels [testosterone (TEST) and estradiol (ESTR)] and their association with growth. This was achieved by sampling 253 hatchling crocodiles repeatedly at 3, 6, and 9 months of age. Sampling age had a significant effect on both TEST ( p < 0.001) and ESTR ( p < 0.001) suggesting climatic/abiotic factors have an influence even in prepubescent crocodiles. Stress, as measured by plasma corticosterone, had no detectable effect on plasma ESTR or TEST levels. Unexpectedly however, TEST was higher in slower-growing crocodiles, which is contrary to what has been reported for the American alligator. ESTR was not associated with growth. [ABSTRACT FROM AUTHOR]

Highlights • The impact of known crocodilian skin pathogens including viral, bacterial, fungal and helminth taxa are reviewed. • Best husbandry practices to minimise these pathogens are discussed. • Husbandry improvements, such as heating, have already seen a reduced incidence in many of these pathogens. • Viral pathogens are the largest risk to crocodilian skin quality and require more than improved husbandry practices. Abstract The control of pathogens that target crocodilian skin is essential to the long-term success and sustainability of intensive farming operations worldwide. To understand the impact these pathogens may have on the skin, a brief overview of skin histology is given. A review of the known viral, bacterial, fungal and helminth taxa associated with skin conditions in commercially significant crocodilian species is presented. Best management practices are discussed, with an emphasis on addressing extrinsic factors that influence transmission and pathogenicity. It is argued that, in the past, reduced immune function arising from inadequate thermal regulation was the leading cause of skin disease in captive crocodilians. Consequently, innovations such as temperature control, coupled with the adoption of more stringent hygiene standards, have greatly reduced the prevalence of many infectious skin conditions in intensively farmed populations. However, despite improvements in animal husbandry and disease management, viral pathogens such as West Nile virus, herpesvirus and poxvirus continue to afflict crocodilians in modern captive production systems. [ABSTRACT FROM AUTHOR]

To determine reference levels for on-farm stressors on immune responsiveness and growth rate, 253 hatchling crocodiles from 11 known breeding pairs were repeatedly measured and blood sampled during their first year. Plasma corticosterone (CORT) was used to quantify baseline stress levels in captive animals and were found to be lower (mean 1.83 ± SE 0.16 ng/mL) than previously reported in saltwater crocodile hatchlings. Two tests of immune function were also conducted. Innate constitutive immunity was assessed using bacterial killing assays (BKA) against two bacterial species: Escherichia coli and Providencia rettgeri , whereby the latter causes considerable economic loss to industry from septicaemic mortalities. Although the bactericidal capabilities were different at approximately 4 months old (32 ± 3% for E. coli and 16 ± 4% for P. rettgeri ), the differences had disappeared by approximately 9 months old (58 ± 2% and 68 ± 6%, respectively). To assess immune responsiveness to a novel antigen, the inflammatory swelling response caused by phytohaemagglutinin (PHA) injection was assessed but was only significantly different between Samplings 1 and 3 (5% LSD). There were no significant clutch effects for CORT or PHA but there were for both BKA traits. CORT was not significantly associated with growth (head length) or the immune parameters except for P. rettgeri BKA where higher CORT levels were associated with better bactericidal capability. As such, these results suggest that the crocodiles in this study are not stressed, therefore endorsing the management strategies adopted within the Australian industry Code of Practice. [ABSTRACT FROM AUTHOR]

Knowledge of endogenous retroviruses (ERVs) in crocodilians (Crocodylia) is limited, and their distribution among extant species is unclear. Here we analyzed the phylogenetic relationships of these retroelements in 20 species of crocodilians by studying the pro-pol gene. The results showed that crocodilian ERVs (CERVs) cluster into two major clades (CERV 1 and CERV 2). CERV 1 clustered as a sister group of the genus Gammaretrovirus, while CERV 2 clustered distantly with respect to all known ERVs. Interestingly, CERV 1 was found only in crocodiles (Crocodylidae). The data generated here could assist future studies aimed at identifying orthologous and paralogous ERVs among crocodilians. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are 18–24 nucleotide regulatory RNAs. They are involved in the regulation of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Data suggests a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to birds, the closest extant relatives of crocodilians. Understanding gene regulation in the saltwater crocodile in the context of relatively slow genomic change thus holds potential for the investigation of genomics, evolution, and adaptation. Utilizing eleven tissue types and sixteen small RNA libraries, we report 644 miRNAs in the saltwater crocodile with >78% of miRNAs being novel to crocodilians. We also identified potential targets for the miRNAs and analyzed the relationship of the miRNA repertoire to transposable elements (TEs). Results suggest an increased association of DNA transposons with miRNAs when compared to retrotransposons. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile. In addition, the data suggests a supporting role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation. Image 1 • We identified the miRNA (microRNA) repertoire is several tissues of the saltwater crocodile, Crocodylus porosus. • We identified patterns of differential expression among tissues. • We overlapped miRNA loci with transposable element loci in the genome assembly. • Transposable elements give rise to nearly half of expressed miRNAs with a bias toward arising from DNA transposons. [ABSTRACT FROM AUTHOR]

Up to 10 males were reported to sire clutches of crocodilian eggs but review of the underlying study designs raised questions of potential upward bias of inferred sire numbers. To test this premise, different scenarios were explored using a published dataset of 16 known single-sire saltwater crocodile pairs and their offspring which were originally confirmed using a 11 loci microsatellite panel in CERVUS. Varying the number of microsatellites, omitting one or both parental genotypes and using different parentage analysis techniques revealed that total allele number, rather than number of loci, determined inferred sire accuracy in two opposing ways. Using the single-locus minimum method and GERUD, which both require prior knowledge of family groupings (i.e., nests), fewer alleles (and loci) accurately inferred only one father. In contrast, CERVUS and COLONY required all 11 loci (65 alleles) and both parental genotypes to (a) assign correct family groups and (b) infer the correct sire number, except in one family where two sires were equally assigned based on their number of homozygous loci. When less genotype information was provided, CERVUS and COLONY inferred up to six and seven sires, respectively. Given this data is from confirmed single-sire matings, and yet up to seven sires could be inferred, the significance of inappropriate study design is clearly demonstrated. Consideration should be carefully given to genotype data, particularly those collected specifically for population diversity studies, which are also used to infer multiple paternity because the underlying data collection assumptions are not equivalent between the two outcomes., Competing Interests: The author declares no conflict of interest., (© 2022 The Author. Ecology and Evolution published by John Wiley & Sons Ltd.)

The complete genome sequence of molluscum contagiosum virus 1 (MOCV1) isolate NT2017 was sequenced from a tissue sample from an Australian woman. The genome consisted of 185,655 bp encoding 169 predicted open reading frames. Phylogenetically, isolate NT2017 was most closely related to an MOCV1 strain from Slovenia., (Copyright © 2021 Sarker et al.)

Crocodilepox virus is a large dsDNA virus belonging to the genus Crocodylidpoxvirus, which infects a wide range of host species in the order Crocodylia worldwide. Here, we present genome sequences for a novel saltwater crocodilepox virus, with two subtypes (SwCRV-1 and -2), isolated from the Australian saltwater crocodile. Affected belly skins of juvenile saltwater crocodiles were used to sequence complete viral genomes, and perform electron microscopic analysis that visualized immature and mature virions. Analysis of the SwCRV genomes showed a high degree of sequence similarity to CRV (84.53% and 83.70%, respectively), with the novel SwCRV-1 and -2 complete genome sequences missing 5 and 6 genes respectively when compared to CRV, but containing 45 and 44 predicted unique genes. Similar to CRV, SwCRV also lacks the genes involved in virulence and host range, however, considering the presence of numerous hypothetical and or unique genes in the SwCRV genomes, it is completely reasonable that the genes encoding these functions are present but not recognized. Phylogenetic analysis suggested a monophyletic relationship between SwCRV and CRV, however, SwCRV is quite distinct from other chordopoxvirus genomes. These are the first SwCRV complete genome sequences isolated from saltwater crocodile skin lesions.

Agricultural production of the saltwater crocodile (Crocodylus porosus) is an emergent industry in northern Australia with many of the factors affecting production remaining unknown. In this study, we sought to expand upon our previous findings of reference corticosterone and immune function by reporting baseline sex hormone levels [testosterone (TEST) and estradiol (ESTR)] and their association with growth. This was achieved by sampling 253 hatchling crocodiles repeatedly at 3, 6, and 9months of age. Sampling age had a significant effect on both TEST (p<0.001) and ESTR (p<0.001) suggesting climatic/abiotic factors have an influence even in prepubescent crocodiles. Stress, as measured by plasma corticosterone, had no detectable effect on plasma ESTR or TEST levels. Unexpectedly however, TEST was higher in slower-growing crocodiles, which is contrary to what has been reported for the American alligator. ESTR was not associated with growth., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To determine reference levels for on-farm stressors on immune responsiveness and growth rate, 253 hatchling crocodiles from 11 known breeding pairs were repeatedly measured and blood sampled during their first year. Plasma corticosterone (CORT) was used to quantify baseline stress levels in captive animals and were found to be lower (mean 1.83±SE 0.16 ng/mL) than previously reported in saltwater crocodile hatchlings. Two tests of immune function were also conducted. Innate constitutive immunity was assessed using bacterial killing assays (BKA) against two bacterial species: Escherichia coli and Providencia rettgeri, whereby the latter causes considerable economic loss to industry from septicaemic mortalities. Although the bactericidal capabilities were different at approximately 4 months old (32±3% for E. coli and 16±4% for P. rettgeri), the differences had disappeared by approximately 9 months old (58±2% and 68±6%, respectively). To assess immune responsiveness to a novel antigen, the inflammatory swelling response caused by phytohaemagglutinin (PHA) injection was assessed but was only significantly different between Samplings 1 and 3 (5% LSD). There were no significant clutch effects for CORT or PHA but there were for both BKA traits. CORT was not significantly associated with growth (head length) or the immune parameters except for P. rettgeri BKA where higher CORT levels were associated with better bactericidal capability. As such, these results suggest that the crocodiles in this study are not stressed, therefore endorsing the management strategies adopted within the Australian industry Code of Practice., (Copyright © 2015 Elsevier Inc. All rights reserved.)