Clonally transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. There are only three known naturally occurring transmissible cancers, and these affect dogs, soft-shell clams, and Tasmanian devils, respectively. The Tasmanian devil transmissible facial cancer was first observed in 1996, and is threatening its host species with extinction. Until now, this disease has been consistently associated with a single aneuploid cancer cell lineage that we refer to as DFT1. Here we describe a second transmissible cancer, DFT2, in five devils located in southern Tasmania in 2014 and 2015. DFT2 causes facial tumors that are grossly indistinguishable but histologically distinct from those caused by DFT1. DFT2 bears no detectable cytogenetic similarity to DFT1 and carries a Y chromosome, which contrasts with the female origin of DFT1. DFT2 shows different alleles to both its hosts and DFT1 at microsatellite, structural variant, and major histocompatibility complex (MHC) loci, confirming that it is a second cancer that can be transmitted between devils as an allogeneic, MHC-discordant graft. These findings indicate that Tasmanian devils have spawned at least two distinct transmissible cancer lineages and suggest that transmissible cancers may arise more frequently in nature than previously considered. The discovery of DFT2 presents important challenges for the conservation of Tasmanian devils and raises the possibility that this species is particularly prone to the emergence of transmissible cancers. More generally, our findings highlight the potential for cancer cells to depart from their hosts and become dangerous transmissible pathogens.

SummaryTransmissible cancers are clonal lineages that spread through populations via contagious cancer cells. Although rare in nature, two facial tumor clones affect Tasmanian devils. Here we perform comparative genetic and functional characterization of these lineages. The two cancers have similar patterns of mutation and show no evidence of exposure to exogenous mutagens or viruses. Genes encoding PDGF receptors have copy number gains and are present on extrachromosomal double minutes. Drug screening indicates causative roles for receptor tyrosine kinases and sensitivity to inhibitors of DNA repair. Y chromosome loss from a male clone infecting a female host suggests immunoediting. These results imply that Tasmanian devils may have inherent susceptibility to transmissible cancers and present a suite of therapeutic compounds for use in conservation.

The devil facial tumor disease (DFTD) is caused by clonal transmissible cancers that have led to a catastrophic decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The first transmissible tumor, now termed devil facial tumor 1 (DFT1), was first discovered in 1996 and has been continually transmitted to new hosts for at least 20 years. In 2015, a second transmissible cancer [devil facial tumor 2 (DFT2)] was discovered in wild devils, and the DFT2 is genetically distinct and independent from the DFT1. Despite the estimated 136,559 base pair substitutions and 14,647 insertions/deletions in the DFT1 genome as compared to two normal devil reference genomes, the allograft tumors are not rejected by the host immune system. Additionally, genome sequencing of two sub-strains of DFT1 detected greater than 15,000 single-base substitutions that were found in only one of the DFT1 sub-strains, demonstrating the transmissible tumors are evolving and that generation of neoantigens is likely ongoing. Recent evidence in human clinical trials suggests that blocking PD-1:PD-L1 interactions promotes antitumor immune responses and is most effective in cancers with a high number of mutations. We hypothesized that DFTD cells could exploit the PD-1:PD-L1 inhibitory pathway to evade antitumor immune responses. We developed recombinant proteins and monoclonal antibodies (mAbs) to provide the first demonstration that PD-1 binds to both PD-L1 and PD-L2 in a non-placental mammal and show that PD-L1 is upregulated in DFTD cells in response to IFN-γ. Immunohistochemistry showed that PD-L1 is rarely expressed in primary tumor masses, but low numbers of PD-L1+ non-tumor cells were detected in the microenvironment of several metastatic tumors. Importantly, in vitro testing suggests that PD-1 binding to PD-L1 and PD-L2 can be blocked by mAbs, which could be critical to understanding how the DFT allografts evade the immune system.

The survival of Atlantic salmon srnolts on exposure to constant concentrations of ammonia has been measured under laboratory conditions. At concentrations of dissolved oxygen close to the air-saturation value, the 24-h LC50 of un-ionised ammonia is 0.15 mg NH, 1 -I in fresh water (hardness 264 mg I-' as CaCO,) and 0.3 mg NH,I-l in 30% sea water; at concentrations of dissolved oxygen of 3.5 mg 1-i in fresh water and 3.1 mg 1 -I in 30% sea water, the 24-h LC50 is 0.09 mg NH, 1-1 and 0.12 mg NH, 1-1 respectively; for fish acclimated for 1 day to a concentration of ammonia close to the 24-h median for un-acclimated fish, the median is increased between 38 and 79%, depending on test conditions.

SUMMARY The hormones of crustaceans may be grouped into those which control effector organs (chromatophores, muscles) and those which control the more gradual sequences of growth, development and reproduction; they have been termed energetic and metabolic hormones respectively. Most of the known crustacean hormones originate in neurosecretory centres in the central nervous system. The sinus gland is an important part of an elaborate neurosecretory system in the eyestalk of stalk‐eyed crustaceans and in the head of sessile‐eyed forms. It is largely composed of the swollen terminations of neurosecretory fibres. Some cellular elements have also been detected, but it is not yet known whether they have a secretory function. It is generally accepted that the greater part of the secretory material in the sinus gland has been transported thither along axon fibres from cell bodies in the central nervous system, though it has been suggested that the staining reactions of the sinus gland indicate also the probability of chemical transformation, if not autochthonous secretion. After‐sinus gland ablation the cut stump of the nerve leading to it continues to accumulate secretory material. An indiscriminate use of the term X organ in the eyestalk has led to confusion. The X organ, originally described in detail by Hanstrom, is characterized by its association with a sensory papilla and by its contained concentric‐layered structures, here called ‘onion bodies’. The term X organ has been used by some authors to denote a group of neurosecretory cell bodies, the fibres of which terminate in the sinus gland. It is suggested that the sensory papilla X organ (Hanstrom's X organ) should be distinguished from the other X organs as it differs from them morphologically and physiologically. The post‐commissure organs comprise enlargements of the epineurium of two post‐commissure nerves, containing the terminations of neurosecretory fibres. They have been shown to contain chromactivating hormones. The pericardial organs comprise epineurial enlargements containing fine‐fibre terminations. They lie in the pericardial spaces and have been shown to contain substances active on the heart. The Y organ, a glandular structure which contains hormones affecting the rate of development, is located in the antennary segment of those forms which have a maxillary excretory organ and in the second maxillary segment of those which have an antennary excretory organ. Various groups of neurosecretory cells have been detected in the brain and in thoracic ganglia. The term neurohaemal organ has been proposed to denote those tissues, through which substances produced in neurosecretory cells gain ready access to the blood. The pigment pattern of any one crustacean species is fairly constant, but patterns differ in the different groups. The pigments are contained in monochromatic, dichromatic, trichromatic and tetrachromatic chromatophores. The chromactivating substances which have so far been separated from tissue extracts seem to act differentially on these chr...

Since 2012, an orthomyxo-like virus has been consistently linked to epizootics in marine farmed Atlantic salmon in Tasmania, Australia. Here we describe the properties of the virus, designated the pilchard orthomyxovirus (POMV), in cell culture and present data verifying its direct role in a disease of Atlantic salmon. In infected cells, viral RNA was detectable in both the nucleus and cytoplasm, consistent with the replication cycle of an orthomyxovirus. Viral replication in vitro was temperature-dependent (within a range of 10-20°C), and yields of virus were typically in excess of 107 TCID50 ml-1. In controlled infection trials, cell culture-derived POMV produced significant morbidity in Atlantic salmon fry, pre-smolt and post-smolt. In all cases, the development of disease was rapid, with moribund fish detected within 5 d of direct exposure to POMV, and maximum cumulative morbidity occurring within 4 wk. The experimentally infected fish developed a characteristic suite of gross and microscopic pathological changes, which were consistent with those observed in Atlantic salmon overtly affected by POMV-associated disease on sea farms. These included necrotic lesions across multiple organs that were directly associated with the presence of the virus. Together, our observations indicate that POMV is an endemic virus likely transmitted from wild fish to farmed Atlantic salmon in Tasmania. The virus is pathogenic to Atlantic salmon in freshwater and marine environments and causes a disease that we have named salmon orthomyxoviral necrosis.

Summary Disease is increasingly being recognised as a risk factor in declining wildlife populations around the globe. However, there are limited protocols to assess disease risks in declining wildlife. Using epidemiological principles, we define a step‐by‐step framework to complete this complex and critical task. As an example, we assessed the potential role of diseases in relation to the decline of the woylie or brush‐tailed bettong (Bettongia penicillata ogilbyi) in Western Australia. Between 1999 and 2006, woylie populations declined by 90%. The wildlife disease risk assessment began with a list of all known or suspected diseases to which the woylie, a species of macropod, is susceptible. This list was assessed against the spatial, temporal and demographic characteristics of the decline. Diseases causing widespread and high mortalities or debilitation leading to predation received high scores. Based on this assessment, priority diseases or pathogens for investigation identified were haemoparasites, gastrointestinal helminths, Neospora caninum, Toxoplasmosis (Toxoplasma gondii), Encephalomyocarditis virus, Macropod Orbiviruses (Wallal virus and Warrego virus), Macropod Herpesviruses (Macropodid herpesvirus 1 and 2) and Salmonella spp.

Tasmanian devils have spawned two transmissible cancer clones, known as devil facial tumour 1 (DFT1) and devil facial tumour 2 (DFT2). DFT1 and DFT2 are transmitted between animals by the transfer of allogeneic contagious cancer cells by biting, and both cause facial tumours. DFT1 and DFT2 tumours are grossly indistinguishable, but can be differentiated using histopathology, cytogenetics or genotyping of polymorphic markers. However, standard diagnostic methods require specialist skills and equipment and entail long processing times. Here, we describe Tasman-PCR: a simple PCR-based diagnostic assay that distinguishes DFT1 and DFT2 by amplification of DNA spanning tumour-specific interchromosomal translocations. We demonstrate the high sensitivity and specificity of this assay by testing DNA from 557 tumours and 818 normal devils. A temporal-spatial screen confirmed the reported geographic ranges of DFT1 and DFT2 and did not provide evidence of additional DFT clones. DFT2 affects disproportionately more males than females, and devils can be co-infected with DFT1 and DFT2.Overall, we present a PCR-based assay that delivers rapid, accurate and high-throughput diagnosis . CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.