Background complexity can mitigate poor camouflage.

Avoiding detection through camouflage is often key to survival. However, an animal's appearance is not the only factor affecting conspicuousness: background complexity also alters detectability. This has been experimentally demonstrated for both artificially patterned backgrounds in the laboratory and natural backgrounds in the wild, but only for targets that already match the background well. Do habitats of high visual complexity provide concealment to even relatively poorly camouflaged animals? Using artificial prey which differed in their degrees of background matching to tree bark, we were able to determine their survival, under bird predation, with respect to the natural complexity of the background. The latter was quantified using low-level vision metrics of feature congestion (or 'visual clutter') adapted for bird vision. Higher background orientation clutter (edges with varying orientation) reduced the detectability of all but the poorest background-matching camouflaged treatments; higher background luminance clutter (varying achromatic lightness) reduced average mortality for all treatments. Our results suggest that poorer camouflage can be mitigated by more complex backgrounds, with implications for both camouflage evolution and habitat preferences.

The perils of flawed science in wildlife trade literature.

Despite broad scientific consensus that sustainable use of wildlife can enhance conservation efforts, ethical concerns have led some community groups to oppose use of wild animals. Voicing those concerns is legitimate, but underlying philosophical bias should not influence science-based analysis and interpretation. We argue that philosophical biases are common in the scientific literature on trade in wildlife. The critically important case of bias surrounding the use of reptile leathers for luxury fashion illustrates the problem. Based on analysis of official seizures of fashion products made from wildlife, a recent study inferred that criminal activity (as inferred by noncompliance with regulations) was common and increasing and, hence, that authorities needed to adopt more stringent restrictions on the trade. In fact, the conclusions of that study are artifacts of pseudoreplication (e.g., multiple counts of single violations) and biased sampling (e.g., focus on companies with high rates of error) and run directly opposite to actual patterns in the data. As a proportion of overall trade, rates of noncompliance are exceptionally low (<0.4%), are declining, and result primarily from paper-work errors rather than criminal intent (e.g., such errors are more frequent for goods shipped by government authorities than by the commercial fashion industry). The recommendation by the study authors to prohibit the international trade in wildlife-based fashion products is imperiling a sustainable trade that can benefit biodiversity and people's livelihoods by providing financial incentives for conservation of species and habitats. This example offers a warning of the dangers of basing research on the wildlife trade on ethical or philosophical positions rather than objective evaluations of evidence.

Los Peligros de la Ciencia Errada en la Literatura sobre el Comercio de Fauna Resumen A pesar del amplio consenso científico de que el uso sustentable de la fauna puede mejorar los esfuerzos de conservación, las preocupaciones éticas han llevado a algunos grupos comunitarios a oponerse al uso de fauna silvestre. Es legítimo otorgarles una voz a estas preocupaciones, pero el sesgo filosófico subyacente no debería influenciar el análisis basado en la ciencia y su interpretación. Discutimos que los sesgos filosóficos son comunes en la literatura científica sobre la fauna. El caso críticamente importante del sesgo alrededor del uso de pieles de reptiles para artículos lujosos de moda ilustra este problema. Con base en el análisis de incautaciones oficiales de productos de moda hechos de fauna, Sosnowski y Petrossian (2020) infirieron que la actividad criminal (deducida como el no cumplimiento de las regulaciones) era común y se encontraba en crecimiento y por lo tanto, que las autoridades necesitaban adoptar restricciones más estrictas para el mercado. De hecho, las conclusiones de Sosnowski y Petrossian (2020) son artefactos de pseudoreplicación (es decir, conteos múltiples de violaciones únicas) y un muestreo sesgado (es decir, enfocado en compañías con tasas altas de error) y van directamente en contra de los patrones actuales en los datos. Como una proporción del mercado en general, las tasas de no cumplimiento son excepcionalmente bajas (<0.4%), están declinando y son el resultado principal de los errores cometidos en el papeleo y no de la intención criminal (es decir, dichos errores son más frecuentes para los bienes enviados por las autoridades del gobierno que para los envíos realizados por la industria de la moda comercial). La recomendación hecha por Sosnowski y Petrossian (2020) de prohibir el mercado internacional de productos de moda hechos con productos animales está poniendo en peligro al mercado sustentable que puede beneficiar a la biodiversidad y al sustento de las personas al proporcionar incentivos financieros para la conservación de especies y hábitats. Este ejemplo ofrece una advertencia sobre los peligros de basar la investigación del mercado de fauna en posiciones éticas o filosóficas en lugar de evaluaciones objetivas de la evidencia.

Economics, life history and international trade data for seven turtle species in Indonesian and Malaysian farms.

We collected data on the trade of seven turtle and tortoise species endemic to Indonesia and Malaysia (Amyda cartilaginea, Batagur borneoensis, Cuora amboinensis, Carettochelys insculpta, Heosemys annandalii, Heosemys grandis, and Heosemys spinosa). The data on those species included: operations costs of three breeding farms and one export facility; species life-history traits; and species international legal trade and confiscation data. We collected data for the facilities (one in Malaysia and three in Indonesia) using site visits and a semi-structured questionnaire. We conducted a literature review to compile relevant information on species' life-history traits to estimate breeding viability. We downloaded species-specific data on international trade from the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Trade Database for the exporting countries (Malaysia and Indonesia) for 2000-2015. We compared legal trade with confiscation data obtained from CITES. The data in this article can provide insights into the operations of turtle breeding farms in Southeast Asia. These data can be used as a reference for the inspection of breeding farms and for legislative bodies to determine whether captive breeding for select turtle species is feasible.

Phylogenomics, biogeography and taxonomic revision of New Guinean pythons (Pythonidae, Leiopython) harvested for international trade.

The large and enigmatic New Guinean pythons in the genus Leiopython are harvested from the wild to supply the international trade in pets. Six species are currently recognized (albertisii, biakensis, fredparkeri, huonensis, meridionalis, montanus) but the taxonomy of this group has been controversial. We combined analysis of 421 nuclear loci and complete mitochondrial genomes with morphological data to construct a detailed phylogeny of this group, understand their biogeographic patterns and establish the systematic diversity of this genus. Our molecular genetic data support two major clades, corresponding to L. albertisii and L. fredparkeri, but offer no support for the other four species. Our morphological data also only support two species. We therefore recognize L. albertisii and L. fredparkeri as valid species and place L. biakensis, L. meridionalis, L. huonensis and L. montanus into synonymy. We found that L. albertisii and L. fredparkeri are sympatric in western New Guinea; an atypical pattern compared to other Papuan species complexes in which the distributions of sister taxa are partitioned to the north and south of the island's central mountain range. For the purpose of conservation management, overestimation of species diversity within Leiopython has resulted in the unnecessary allocation of resources that could have been expended elsewhere. We strongly caution against revising the taxonomy of geographically widespread species groups when little or no molecular genetic data and only small morphological samples are available.

Behavioral and corticosterone responses to carbon dioxide exposure in reptiles.

The use of carbon dioxide (CO2) exposure as a means of animal euthanasia has received considerable attention in mammals and birds but remains virtually untested in reptiles. We measured the behavioral responses of four squamate reptile species (Homalopsis buccata, Malayopython reticulatus, Python bivitattus, and Varanus salvator) to exposure to 99.5% CO2 for durations of 15, 30, or 90 minutes. We also examined alterations in plasma corticosterone levels of M. reticulatus and V. salvator before and after 15 minutes of CO2 exposure relative to control individuals. The four reptile taxa showed consistent behavioral responses to CO2 exposure characterized by gaping and minor movements. The time taken to lose responsiveness to stimuli and cessation of movements varied between 240-4260 seconds (4-71 minutes), with considerable intra- and inter-specific variation. Duration of CO2 exposure influenced the likelihood of recovery, which also varied among species (e.g., from 0-100% recovery after 30-min exposure). Plasma corticosterone concentrations increased after CO2 exposure in both V. salvator (18%) and M. reticulatus (14%), but only significantly in the former species. Based on our results, CO2 appears to be a mild stressor for reptiles, but the relatively minor responses to CO2 suggest it may not cause considerable distress or pain. However, our results are preliminary, and further testing is required to understand optimal CO2 delivery mechanisms and interspecific responses to CO2 exposure before endorsing this method for reptile euthanasia.

Species delimitation and systematics of the green pythons (Morelia viridis complex) of melanesia and Australia.

Molecular data sets and the increasing use of integrative systematics is revealing cryptic diversity in a range of taxa - particularly in remote and poorly sampled landscapes like the island of New Guinea. Green pythons (Morelia viridis complex) are one of the most conspicuous elements of this island's fauna, with large numbers taken from the wild to supply international demand for exotic pets. We test hypotheses about species boundaries in green pythons from across New Guinea and Australia with mitochondrial genomes, 389 nuclear exons, and comprehensive assessment of morphological variation. Strong genetic structuring of green python populations and species delimitation methods confirm the presence of two species, broadly occurring north and south of New Guinea's central mountains. Our data also support three subspecies within the northern species. Subtle but consistent morphological divergence among the putative taxa is concordant with patterns of molecular divergence. Our extensive sampling identifies several zones of hitherto unknown biogeographical significance on the island of New Guinea. We revise the taxonomy of the group, discuss the relevance of our findings in the context of Papuan biogeography and the implications of our systematic changes for the conservation management of these taxa.

Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor.

Cane toads beneath bird rookeries: utilization of a natural disturbance by an invasive species.

Many invasive species exploit anthropogenically disturbed habitats, but most of those taxa evolved long before humans. Presumably, then, an ability to use natural (non-anthropogenic) disturbances pre-adapted invaders to a world later degraded by people. Studies on invasive species in naturally disturbed habitats thus can clarify the ancestral niche of invaders. In the Australian tropics, metallic starlings Aplonis metallica nest communally in emergent rainforest trees during the wet-season, and invasive cane toads Rhinella marina join other predators (mammals, birds, reptiles, and other anurans) to exploit the food resources beneath those trees. Compared to conspecifics found along nearby roads through the forest, cane toads beneath bird-nesting trees occur at higher densities, and are smaller in body size. The sex ratio is female-biased, and recapture records suggest that females may be philopatric at these sites (whereas recaptures were rare for both sexes found along the roads). Some toads were found under the same trees in successive wet-seasons. Spooling showed that distances moved per night were similar along the road versus under the trees, but toads under trees showed lower net displacements. Diets also differed (based upon scat analysis), with tree toads feeding more on beetles and less on ants. These nutrient-rich hotspots are exploited primarily by adult females and juvenile toads, whereas adult males congregate at breeding sites. By magnifying pre-existing intraspecific divergences in habitat use, bird rookeries may enhance population viability of cane toads by enabling critical age and sex classes to exploit food-rich patches that are rarely used by adult males.

Communally Nesting Migratory Birds Create Ecological Hot-Spots in Tropical Australia.

Large numbers of metallic starlings (Aplonis metallica) migrate annually from New Guinea to the rainforests of tropical Australia, where they nest communally in single emergent trees (up to 1,000 birds). These aggregations create dense and species-rich faunal "hot-spots", attracting a diverse assemblage of local consumers that utilise this seasonal resource. The starlings nested primarily in poison-dart trees (Antiaris toxicaria) near the rainforest-woodland boundary. Surveys underneath these colonies revealed that bird-derived nutrients massively increased densities of soil invertebrates and mammals (primarily wild pigs) beneath trees, year-round. Flying invertebrates, nocturnal birds, reptiles, and amphibians congregated beneath the trees when starlings were nesting (the wet-season). Diurnal birds (primarily cockatoos and bush turkeys) aggregated beneath the trees during the dry-season to utilise residual nutrients when the starlings were not nesting. The abundance of several taxa was considerably higher (to > 1000-fold) under colony trees than under nearby trees. The system strikingly resembles utilisation of bird nesting colonies by predators in other parts of the world but this spectacular system has never been described, emphasizing the continuing need for detailed natural-history studies in tropical Australia.

Jungle Giants: Assessing Sustainable Harvesting in a Difficult-to-Survey Species (Python reticulatus).

Sustainability of wildlife harvests is critical but difficult to assess. Evaluations of sustainability typically combine modelling with the measurement of underlying abundances. For many taxa harvested in developing countries, however, abundances are near-impossible to survey and a lack of detailed ecological information impedes the reliability of models. In such cases, repeated surveys of the attributes of harvested individuals may provide more robust information on sustainability. If the numbers, sizes and other demographic attributes of animals taken for the commercial trade do not change over biologically significant time intervals (decades), there is a prima facie case that the harvest is indeed sustainable. Here, we report the results of examinations of > 4,200 reticulated pythons (Python reticulatus) taken for the commercial leather industry in northern and southern Sumatra, Indonesia. The numbers, mean body sizes, clutch sizes, sizes at maturity and proportion of giant specimens have not decreased between our first surveys (1995) and repeat surveys (2015). Thus, despite assumptions to the contrary, the harvest appears to be sustainable. We use our data to inform the design of future monitoring programs for this species. Our study underpins the need for robust science to inform wildlife trade policy and decision-making, and urges wildlife managers to assess sustainability of difficult-to-survey terrestrial wildlife by drawing inferences directly from the harvest itself.

Extinction in a hyperdiverse endemic Hawaiian land snail family and implications for the underestimation of invertebrate extinction.

The International Union for Conservation of Nature (IUCN) Red List includes 832 species listed as extinct since 1600, a minuscule fraction of total biodiversity. This extinction rate is of the same order of magnitude as the background rate and has been used to downplay the biodiversity crisis. Invertebrates comprise 99% of biodiversity, yet the status of a negligible number has been assessed. We assessed extinction in the Hawaiian land snail family Amastridae (325 species, IUCN lists 33 as extinct). We did not use the stringent IUCN criteria, by which most invertebrates would be considered data deficient, but a more realistic approach comparing historical collections with modern surveys and expert knowledge. Of the 325 Amastridae species, 43 were originally described as fossil or subfossil and were assumed to be extinct. Of the remaining 282, we evaluated 88 as extinct and 15 as extant and determined that 179 species had insufficient evidence of extinction (though most are probably extinct). Results of statistical assessment of extinction probabilities were consistent with our expert evaluations of levels of extinction. Modeling various extinction scenarios yielded extinction rates of 0.4-14.0% of the amastrid fauna per decade. The true rate of amastrid extinction has not been constant; generally, it has increased over time. We estimated a realistic average extinction rate as approximately 5%/decade since the first half of the nineteenth century. In general, oceanic island biotas are especially susceptible to extinction and global rate generalizations do not reflect this. Our approach could be used for other invertebrates, especially those with restricted ranges (e.g., islands), and such an approach may be the only way to evaluate invertebrates rapidly enough to keep up with ongoing extinction.

Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis.

The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the prothrombinase complex-the molecular engine of blood coagulation.

Thrombin inhibition by serpins disrupts exosite II.

Thrombin uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite I and the exposure of exosite II. The physiological inhibitors of thrombin are all serpins, whose mechanism involves significant conformational change in both serpin and protease. It has been shown that the formation of the thrombin-serpin final complex disorders the active site and exosite I of thrombin, but exosite II is thought to remain functional. It has also been hypothesized that thrombin contains a receptor-binding site that is exposed upon final complex formation. The position of this cryptic site may depend on the regions of thrombin unfolded by serpin complexation. Here we investigate the conformation of thrombin in its final complex with serpins and find that in addition to exosite I, exosite II is also disordered, as reflected by a loss of affinity for the γ'-peptide of fibrinogen and for heparin and by susceptibility to limited proteolysis. This disordering of exosite II occurs for all tested natural thrombin-inhibiting serpins. Our data suggest a novel framework for understanding serpin function, especially with respect to thrombin inhibition, where serpins functionally "rezymogenize" proteases to ensure complete loss of activity and cofactor binding.

NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation.

The serine protease thrombin is generated from its zymogen prothrombin at the end of the coagulation cascade. Thrombin functions as the effector enzyme of blood clotting by cleaving several procoagulant targets, but also plays a key role in attenuating the hemostatic response by activating protein C. These activities all depend on the engagement of exosites on thrombin, either through direct interaction with a substrate, as with fibrinogen, or by binding to cofactors such as thrombomodulin. How thrombin specificity is controlled is of central importance to understanding normal hemostasis and how dysregulation causes bleeding or thrombosis. The binding of ligands to thrombin via exosite I and the coordination of Na(+) have been associated with changes in thrombin conformation and activity. This phenomenon has become known as thrombin allostery, although direct evidence of conformational change, identification of the regions involved, and the functional consequences remain unclear. Here we investigate the conformational and dynamic effects of thrombin ligation at the active site, exosite I and the Na(+)-binding site in solution, using modern multidimensional NMR techniques. We obtained full resonance assignments for thrombin in seven differently liganded states, including fully unliganded apo thrombin, and have created a detailed map of residues that change environment, conformation, or dynamic state in response to each relevant single or multiple ligation event. These studies reveal that apo thrombin exists in a highly dynamic zymogen-like state, and relies on ligation to achieve a fully active conformation. Conformational plasticity confers upon thrombin the ability to be at once selective and promiscuous.

Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-A structure of the ternary complex.

Factor (f) IXa is a critical enzyme for the formation of stable blood clots, and its deficiency results in hemophilia. The enzyme functions at the confluence of the intrinsic and extrinsic pathways by binding to fVIIIa and rapidly generating fXa. In spite of its importance, little is known about how fIXa recognizes its cofactor, its substrate, or its only known inhibitor, antithrombin (AT). However, it is clear that fIXa requires extensive exosite interactions to present substrates for efficient cleavage. Here we describe the 1.7-A crystal structure of fIXa in its recognition (Michaelis) complex with heparin-activated AT. It represents the highest resolution structure of both proteins and allows us to address several outstanding issues. The structure reveals why the heparin-induced conformational change in AT is required to permit simultaneous active-site and exosite interactions with fIXa and the nature of these interactions. The reactive center loop of AT has evolved to specifically inhibit fIXa, with a P2 Gly so as not to clash with Tyr99 on fIXa, a P4 Ile to fit snugly into the S4 pocket, and a C-terminal extension to exploit a unique wall-like feature of the active-site cleft. Arg150 is at the center of the exosite interface, interacting with AT residues on beta-sheet C. A surprising crystal contact is observed between the heparin pentasaccharide and fIXa, revealing a plausible mode of binding that would allow longer heparin chains to bridge the complex.

Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization.

Repeating intermolecular protein association by means of beta-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer's, Huntington's and Parkinson's and the prion encephalopathies. A family of proteins, known as the serpins, also forms large stable multimers by ordered beta-sheet linkages leading to intracellular accretion and disease. These 'serpinopathies' include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in alpha(1)-antitrypsin (alpha(1)AT), and thrombosis caused by mutations in antithrombin. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the beta-sheet expansion disorders collectively known as the conformational diseases. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel beta-strands inserting in the centre of the principal beta-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible beta-sheet expansion.

Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation.

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted approximately 20 A, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.

Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation.

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10,000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.