Pseudonaja Textilis Research Articles

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes

BackgroundVenom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris).ResultsAlthough there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis.ConclusionsOur comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Why Australian farmers should not kill venomous snakes

AbstractMany Australians who work outdoors (notably, farmers and graziers) routinely kill venomous snakes. We argue that this attitude is misguided and dangerous. Despite their fearsome reputation, venomous Australian snakes pose little risk to human health (snakes kill an average of less than three people per year in Australia). Also, snakes confer a substantial benefit by consuming agricultural pests such as rodents. We estimate the magnitude of that benefit with data on snake diets, feeding rates and abundances. The most valuable rodent‐controllers are Brownsnakes (genus Pseudonaja), which are rodent‐specialists as adults and are abundant in agroecosystems across much of Australia. We calculate that a free‐living adult Eastern Brownsnake consumes at least 50 mice per year (probably twice that number), and that population densities of Brownsnakes in agricultural areas can exceed 100 per km2. Thus, Brownsnakes remove thousands of mice per square kilometre of farmland per year. That offtake plausibly reduces rodent densities because Brownsnakes take all age classes and both sexes of rodents by hunting in burrows. Tolerating Brownsnakes also would benefit the environment (e.g. less reliance on toxic chemicals) and the health of humans and domestic pets (fewer rodent‐mediated diseases) and counter‐intuitively, might reduce rates of snakebite (because many bites occur when a snake is attacked). In summary, a societal policy of coexisting with highly venomous snakes would confer multiple benefits to Australian farmers.

Suspected snakebite in a tertiary referral centre in Western Sydney: a 10-year case series

ABSTRACT We aimed to determine the epidemiology and clinical management of patients presenting to an Australian tertiary emergency department (ED) with a suspected snake bite. Nepean Hospital covers a geographical area known to harbour venomous elapid snake species. A Retrospective chart review of all patients presenting to Nepean ED from 2011 to 2020 was performed. We searched the electronic medical records (eMR) for triage presentations including search terms of “snakebite”, “bite” “envenomation”, or “bite-snake” and a discharge or independent diagnosis of “snakebite” or “envenomation”. Our review identified 110 patients presenting with snakebite, of whom nine had signs or symptoms indicating envenomation. The most common envenomation syndrome was venom-induced consumption coagulopathy (VICC) and the most commonly suspected culprit snake was the Eastern Brown Snake. Our data broadly agreed with national data on epidemiology and demographics. We noted a satisfactory rate of compliance with local snake bite protocols with evidence of early diagnosis and antivenom administration. We had no cases of anaphylaxis and one case of serum sickness. Our study of a tertiary hospital experience in the management of snake bite demonstrated an appropriate adherence to national guidelines and early clinician recognition of envenomation syndromes. It also demonstrated appropriate antivenom use without obvious time delay.

Why losing Australia's biodiversity matters for human health: insights from the latest State of the Environment assessment.

Why losing Australia's biodiversity matters for human health: insights from the latest State of the Environment assessment.

Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation

AbstractThe prothrombinase complex processes prothrombin to thrombin through sequential cleavage at Arg320 followed by Arg271 when cofactor, factor (f) Va, protease, fXa, and substrate, prothrombin, are all bound to the same membrane surface. In the absence of the membrane or cofactor, cleavage occurs in the opposite order. For the less favorable cleavage site at Arg320 to be cleaved first, it is thought that prothrombin docks on fVa in a way that presents Arg320 and hides Arg271 from the active site of fXa. Based on the crystal structure of the prothrombinase complex from the venom of the Australian eastern brown snake, pseutarin C, we modeled an initial prothrombin docking mode, which involved an interaction with discrete portions of the A1 and A2 domains of fV and the loop connecting the 2 domains, known as the a1-loop. We interrogated the proposed interface by site-directed PEGylation and by swapping the a1-loop in pseutarin C with that of human fV and fVIII and measuring the effect on rate and pathway of thrombin generation. PEGylation of residues within our proposed binding site greatly reduced the rate of thrombin generation, without affecting the pathway, whereas those outside the proposed interface had no effect. PEGylation of residues within the a1-loop also reduced the rate of thrombin generation. The sequence of the a1-loop was found to play a critical role in prothrombin binding and in the presentation of Arg320 for initial cleavage.

Interannual colony exchange among breeding Eastern Brown Pelicans

Las aves marinas coloniales con frecuencia muestra una alta fidelidad interanual en la ubicación de la reproducción, especialmente como adultos. Las especies que usan sistemas cerca de la costa o costales, sin embargo, pueden mostrar comparativamente menor fidelidad que las especies pelágicas. Utilizamos datos de largo plazo de rastreadores GPS para determinar la frecuencia de cambio interanual de colonias por Pelecanus occidentalis carolinensis en la bahía del sur del atlántico, una especie sobre la cual la fidelidad de los adultos es poco entendida. Por medio del uso de patrones comportamentales recursivos, para identificar la ubicación de los nidos, reportamos que el 36% de los intentos de reproducción ocurrieron en colonias diferentes a las colonias utilizadas el año anterior. El intercambio de colonias fue especialmente prevalente en pelicanos hembra y las distancias entre las colonias tuvieron un rango entre 56 y 592 km. Sugerimos que la telemetría a largo plazo puede ser una herramienta confiable para determinar la fidelidad de sitios de reproducción interanual, especialmente cuando es comparada con métodos más tradicionales de anillamiento y reavistamiento.

Fine-scale Weather Patterns Drive Reproductive Success in the Brown Pelican

In the northern Gulf of Mexico, island restoration and creation have been used to mitigate potential negative effects of anthropogenic and environmental stressors to breeding seabirds. The long-term success of such projects can be enhanced when data are available to elucidate how site-specific and larger-scale factors may contribute to reproductive success. Nest-specific daily survival rate (DSR) of Eastern Brown Pelicans (Pelecanus occidentalis carolinensis) during incubation (i.e., pre-hatch; n = 245) and brood-rearing (i.e., post-hatch; n = 185) were measured at two breeding islands in the northern Gulf of Mexico USA in 2017 and 2018 in relation to macro- and micro- scale habitat and environmental measurements. DSR of nests during incubation ranged from 91-99%, and the DSR during brood-rearing exceeded 99% each year. Regional weather variables occurred in top-performing models more often and with more significance compared to microhabitat variables. Results suggest that reproductive success of Brown Pelicans may respond at least in part to weather factors that occur outside of the scope of habitat structure as it is typically incorporated into the restoration or creation of breeding habitat, indicating that climate conditions are likely an important factor in the success of restoration efforts.

PtFVa (Pseudonaja Textilis Venom-Derived Factor Va) Retains Structural Integrity Following Proteolysis by Activated Protein C.

OBJECTIVE: The Australian snake venom ptFV (Pseudonaja textilis venom-derived factor V) variant retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of FVa (mammalian factor Va). APPROACH AND RESULTS: Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic. CONCLUSIONS: Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity.

Evolutionary adaptation of the γ‐carboxyglutamic acid domain in blood coagulation factor X in the Serpentes suborder

The serine protease factor Xa plays an important role in blood coagulation as it, in complex with its cofactor Va, proteolytically activates prothrombin to thrombin, the latter being a key enzyme in coagulation. To perform this vital function, factor Xa undergoes numerous posttranslational modifications including vitamin K-dependent γ-carboxylation of glutamic acid (Glu) residues into so-called Gla residues. A correctly γ-carboxylated GLA domain is essential for interaction with the negatively charged membrane surface of activated platelets or endothelial cells at the site of injury where clot formation is required. Moreover, assembly of factor Xa with the cofactor Va into the prothrombinase enzyme complex occurs on a negatively charged membrane surface only. Interestingly, the venom of some Australian Elapid snakes comprises powerful prothrombin-activating enzyme complexes consisting of factor Xa- and Va-like proteins that are specifically expressed in the venom gland. We have previously demonstrated that this venom enzyme complex is capable of functioning independently of a negatively charged phospholipid membrane. We aim to uncover the putative differential role of the GLA domain between human and snake venom (Pseudonaja textilis, pt) factor Xa. To do so, we exchanged the human factor Xa (hFX) GLA domain for that of the snake venom ortholog (ptFX), thereby generating hFX-wt, hFX-ptGLA, ptFX-wt, ptFX-hGLA. The FX variants were recombinantly expressed and purified employing chromatography techniques. FX activation by either the human extrinsic or intrinsic coagulation tenase complex was analyzed using a peptidyl substrate specific for activated FX. Introduction of the ptGLA domain into hFX modestly improved the catalytic rate of the extrinsic reaction, while for the intrinsic reaction the substrate binding affinity was enhanced. Conversely, using similar conditions no FXa formation could be detected following incubation of ptFX or ptFX-hGLA with the human intrinsic or extrinsic tenase complex. Assessment of the FX-specific plasma clotting activity in which thrombin-dependent fibrin clot formation is monitored following either an extrinsic or intrinsic coagulation trigger revealed that while the extrinsic clotting activity of hFX was drastically reduced when hGLA was exchanged for ptGLA (3% residual activity), its intrinsic activity was unperturbed. Use of human versus rabbit reagents for the extrinsic pathway may explain the different observations. Conversely, exchange of ptGLA for hGLA resulted in an overall substantial increase in the FX-specific clotting activity of ptFX-hGLA. Finally, exchanging the ptGLA domain for the hGLA domain did not abolish the requirement for a negatively charged membrane surface in Va-dependent prothrombin activation. These data indicate that the snake venom GLA domain interacts with the intrinsic tenase complex and contributes to substrate conversion and subsequent thrombin formation, which may point to evolutionary adaptation of this snake venom GLA domain.

Cutaneous Chromatophoromas in Four Species of Australian Elapid Snake

This report documents the clinicopathological features of cutaneous chromatophoromas in four wild-caught, captive Australian elapid snakes: a strap-snouted brown snake (Pseudonaja aspidoryncha), a tiger snake (Notechis scutatus), an Eastern brown snake (Pseudonaja textilis) and a Mengden's brown snake (Pseudonaja mengdeni). All tumours were subclassified as melanophoromas, with three assessed as malignant on the basis of invasive growth or presence of intracoelomic metastases. The chromatophoromas were single or multiple, black or orange pigmented, cutaneous, sometimes ulcerated, plaques or nodules. Microscopically, the neoplastic cells were often spindle shaped with low or variable pigmentation. Neoplastic cells in one tumour were notable for their pleomorphic round cell morphology and high mitotic rate. One snake with late-stage metastasis survived for over 5 years. There are few reports of chromatophoromas in elapid snakes and, to our knowledge, this is the first report of these tumours in Australian elapid snakes.

Demographic analyses of marine and terrestrial snakes (Elapidae) using whole genome sequences.

The question of whether spatial aspects of evolution differ in marine versus terrestrial realms has endured since Ernst Mayr's 1954 essay on marine speciation. Marine systems are often suggested to support larger and more highly connected populations, but quantitative comparisons with terrestrial systems have been lacking. Here, we compared the population histories of marine and terrestrial elapid snakes using the pairwise sequentially Markovian coalescent (PSMC) model to track historical fluctuations in species' effective population sizes (Ne ) from individual whole-genome sequences. To do this we generated a draft genome for the olive sea snake (Aiysurus laevis) and analysed this alongside six published elapid genomes and their sequence reads (marine species Hydrophis curtus, H.melanocephalus and Laticauda laticaudata; terrestrial species Pseudonaja textilis, Naja Naja and Notechis scutatus). Counter to the expectation that marine species should show higher overall Ne and less pronounced fluctuations in Ne , our analyses reveal demographic patterns that are highly variable among species and do not clearly correspond to major ecological divisions. At deeper time intervals, the four marine elapids appear to have experienced relatively stable Ne , while each terrestrial species shows a prominent upturn in Ne starting at ~4 million years ago (Ma) followed by an equally strong decline. However, over the last million years, all seven species show strong and divergent fluctuations. Estimates of Ne in the most recent intervals (~10kya) are lowest in two of four marine species (H.melanocephalus and Laticauda), and do not correspond to contemporary range sizes in marine or terrestrial taxa.

Notes on the stomach contents of a juvenile Sleepy Lizard, Tiliqua rugosa (Gray 1825), killed by an Eastern Brown Snake, Pseudonaja textilis (Duméril, Bibron, and Duméril 1854) in South Australia

Notes on the stomach contents of a juvenile Sleepy Lizard, Tiliqua rugosa (Gray 1825), killed by an Eastern Brown Snake, Pseudonaja textilis (Duméril, Bibron, and Duméril 1854) in South Australia

Observations of an aggregation between two species of Australian elapid snakes: the threatened Little Whip Snake Parasuta flagellum and Common Eastern Brown Snake Pseudonaja textilis

ABSTRACT This short note presents field observations of a pair of adult threatened Little Whip Snakes Parasuta flagellum and a pair of juvenile Common Eastern Brown Snakes Pseudonaja textilis in an aggregation beneath artificially placed Masonite board used in the Turallo Nature Reserve long-term Little Whip Snake monitoring program. The observation represents the first documented record of an interspecific aggregation between these two species. Presumably, there may be thermal or anti-predatory advantages for the Little Whip Snake by aggregating with other snakes, particularly when both species are of a similar size. However, similar behaviours may also have fitness disadvantages including food competition, and potentially predation as the Common Eastern Brown Snake neonates grow and surpass the substantially smaller Little Whip Snake and warrants further experimental research.

Pseudonajide peptide derived from snake venom alters cell envelope integrity interfering on biofilm formation in Staphylococcus epidermidis

BackgroundThe increase in bacterial resistance phenotype cases is a global health problem. New strategies must be explored by the scientific community in order to create new treatment alternatives. Animal venoms are a good source for antimicrobial peptides (AMPs), which are excellent candidates for new antimicrobial drug development. Cathelicidin-related antimicrobial peptides (CRAMPs) from snake venoms have been studied as a model for the design of new antimicrobial pharmaceuticals against bacterial infections.ResultsIn this study we present an 11 amino acid-long peptide, named pseudonajide, which is derived from a Pseudonaja textilis venom peptide and has antimicrobial and antibiofilm activity against Staphylococcus epidermidis. Pseudonajide was selected based on the sequence alignments of various snake venom peptides that displayed activity against bacteria. Antibiofilm activity assays with pseudonajide concentrations ranging from 3.12 to 100 μM showed that the lowest concentration to inhibit biofilm formation was 25 μM. Microscopy analysis demonstrated that pseudonajide interacts with the bacterial cell envelope, disrupting the cell walls and membranes, leading to morphological defects in prokaryotes.ConclusionsOur results suggest that pseudonajide’s positives charges interact with negatively charged cell wall components of S. epidermidis, leading to cell damage and inhibiting biofilm formation.

Pets in peril: The relative susceptibility of cats and dogs to procoagulant snake venoms

Snakebite is a common occurrence for pet cats and dogs worldwide and can be fatal. In Australia the eastern brown snake (Pseudonaja textilis) is responsible for an estimated 76% of reported snakebite cases to domestic pets nationally each year, with the primary pathology being venom-induced consumptive coagulopathy. While only 31% of dogs survive P. textilis bites without antivenom, cats are twice as likely to survive bites (66%). Even with antivenom treatment, cats have a significantly higher survival rate. The reason behind this disparity is unclear. Using a coagulation analyser (Stago STA R Max), we tested the relative procoagulant effects of P. textilis venom—as well as 10 additional procoagulant venoms found around the world—on cat and dog plasma in vitro, as well as on human plasma for comparison. All venoms acted faster upon dog plasma than cat or human, indicating that dogs would likely enter coagulopathic states sooner, and are thus more vulnerable to procoagulant snake venoms. The spontaneous clotting time (recalcified plasma with no venom added) was also substantially faster in dogs than in cats, suggesting that the naturally faster clotting blood of dogs predisposes them to being more vulnerable to procoagulant snake venoms. This is consistent with clinical records showing more rapid onset of symptoms and lethal effects in dogs than cats. Several behavioural differences between cats and dogs are also highly likely to disproportionately negatively affect prognosis in dogs. Thus, compared to cats, dogs require earlier snakebite first-aid and antivenom to prevent the onset of lethal venom effects.

An in vivo examination of the differences between rapid cardiovascular collapse and prolonged hypotension induced by snake venom

We investigated the cardiovascular effects of venoms from seven medically important species of snakes: Australian Eastern Brown snake (Pseudonaja textilis), Sri Lankan Russell’s viper (Daboia russelii), Javanese Russell’s viper (D. siamensis), Gaboon viper (Bitis gabonica), Uracoan rattlesnake (Crotalus vegrandis), Carpet viper (Echis ocellatus) and Puff adder (Bitis arietans), and identified two distinct patterns of effects: i.e. rapid cardiovascular collapse and prolonged hypotension. P. textilis (5 µg/kg, i.v.) and E. ocellatus (50 µg/kg, i.v.) venoms induced rapid (i.e. within 2 min) cardiovascular collapse in anaesthetised rats. P. textilis (20 mg/kg, i.m.) caused collapse within 10 min. D. russelii (100 µg/kg, i.v.) and D. siamensis (100 µg/kg, i.v.) venoms caused ‘prolonged hypotension’, characterised by a persistent decrease in blood pressure with recovery. D. russelii venom (50 mg/kg and 100 mg/kg, i.m.) also caused prolonged hypotension. A priming dose of P. textilis venom (2 µg/kg, i.v.) prevented collapse by E. ocellatus venom (50 µg/kg, i.v.), but had no significant effect on subsequent addition of D. russelii venom (1 mg/kg, i.v). Two priming doses (1 µg/kg, i.v.) of E. ocellatus venom prevented collapse by E. ocellatus venom (50 µg/kg, i.v.). B. gabonica, C. vegrandis and B. arietans (all at 200 µg/kg, i.v.) induced mild transient hypotension. Artificial respiration prevented D. russelii venom induced prolonged hypotension but not rapid cardiovascular collapse from E. ocellatus venom. D. russelii venom (0.001–1 μg/ml) caused concentration-dependent relaxation (EC50 = 82.2 ± 15.3 ng/ml, Rmax = 91 ± 1%) in pre-contracted mesenteric arteries. In contrast, E. ocellatus venom (1 µg/ml) only produced a maximum relaxant effect of 27 ± 14%, suggesting that rapid cardiovascular collapse is unlikely to be due to peripheral vasodilation. The prevention of rapid cardiovascular collapse, by ‘priming’ doses of venom, supports a role for depletable endogenous mediators in this phenomenon.

Two pathways for venom toxin entry consequent to injection of an Australian elapid snake venom

Here we test and refute the hypothesis that venom toxins from an Australian elapid, the Eastern Brown snake (Pseudonaja textilis, PTx), solely require lymphatic transport to enter the circulation. Studies were made using anaesthetised non-recovery rats in which a marker dye (India ink) or highly potent PTx venom was injected into the hind paw. The studies required a means of inhibiting lymphatic function, as achieved by cooling of the test hind limb to low temperatures (~3 °C). Maintained entry of a non-lethal dose (0.15 mg/kg) and respiratory arrest consequent to injection of a lethal dose (1 mg/kg) of PTx venom at these low temperatures indicate that venom including toxin components enter the circulation directly via the vascular system, a process facilitated by, but not dependent on, lymphatic transport. Notably, the venom had a direct effect on vascular permeability markedly increasing this to allow extravasation of plasma albumin (MWt ~60 kDa).

Nephelometry vs turbidimetry in transferrin measurement

Immunoturbidimetry studies the phenomenon of immunoprecipitation of antigens and antibodies in solution, where there is the formation of large, polymeric insoluble immunocomplexes that increase the turbidity of the solution. We used immunoturbidimetry to investigate the interaction between commercial snake antivenoms and snake venoms, as well as cross-reactivity between different snake venoms.Serial dilutions of commercial snake antivenoms (100 μl) in water were placed in the wells of a microtitre plate and 100 μl of a venom solution (50 μg/ml in water) was added. Absorbance readings were taken at 340 nm every minute on a BioTek ELx808 plate reader at 37 °C. Limits imposed were a 30 minute cut-off and 0.004 as the lowest significant maximum increase. Reactions with rabbit antibodies were carried out similarly, except that antibody dilutions were in PBS.Mixing venom and antivenom/antibodies resulted in an immediate increase in turbidity, which either reached a maximum or continued to increase until a 30 minute cut-off. There was a peak in absorbance readings for most Australian snake venoms mixed with the corresponding commercial antivenom, except for Pseudonaja textilis venom and brown snake antivenom. There was cross-reactivity between Naja naja venom from Sri Lanka and tiger snake antivenom indicated by turbidity when they were mixed. Mixing rabbit anti-snake antibodies with snake venoms resulted in increasing turbidity, but there was not a peak suggesting the antibodies were not sufficiently concentrated. The absorbance reading at pre-determined concentrations of rabbit antibodies mixed with different venoms was able to quantify the cross-reactivity between venoms. Indian antivenoms from two manufacturers were tested against four Sri Lankan snake venoms (Daboia russelli, N. naja, Echis carinatus and Bungarus caeruleus) and showed limited formation of immunocomplexes with antivenom from one manufacturer.The turbidity test provides an easy and rapid way to compare and characterise interactions between antivenoms and snake venoms.

A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro

Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host’s blood and protect a parasite’s gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.

Observations supporting parental care by a viviparous reptile: aggressive behaviour against predators demonstrated by Cunningham’s skinks

Most reptiles exhibit no parental care and aggressive behaviour towards heterospecific predators has rarely been recorded in the natural environment. Several species of the subfamily Egerniinae are amongst the most highly social of all squamate reptiles, exhibiting stable social aggregations and high levels of long-term social and genetic monogamy. We have examined Cunningham’s skinks, Egernia cunninghami, over a three-year period during late January and early February (total 32 days) in the alpine region of New South Wales using video and thermal imaging. Four birthing sessions were witnessed during our field studies of social aggregations of skinks. Our observations monitored skink encounters, in the presence of offspring, with an eastern brown snake, Pseudonaja textilis (two separate encounters, one recorded by video/imaging) and 12 encounters with the Australian magpie, Gymnorhina tibicen. All events were associated with aggressive chasing and/or attack by adult skinks. The first snake encounter involved the active targeting of a recently born juvenile with the mother of the juvenile attacking the snake (running towards the snake, biting and remaining attached for several seconds). The second encounter (the following year) comprised two adult skinks attacking and biting a snake, Pseudonaja textilis. All magpie encounters resulted in chases by adult skinks.