Arne R. Rasmussen

A Fast Preparation of Skeletal Materials Using Enzyme Maceration

Abstract:  The current study investigates the removal of soft tissues from mice and rats by the use of three different proteases and one lipase from Novozymes A/S. The results demonstrate the enzyme maceration to be remarkably fast (1–3 h) compared to the traditional warm‐water procedure, which requires up to several days. In addition, the enzyme maceration eliminates the odor problem associated with the traditional procedure. It is shown that stirring of the enzyme maceration bath is the main factor which determines the speed of the maceration. For mice, the time required for enzyme maceration can vary from 1 to 8 h depending on the stirring speed. The method investigated here allows preparation of skeletal material in an essentially odorless way within a matter of hours, making the method useful in particular for forensic science, private conservation workshops, and educational purposes.

Danger in the reef: Proteome, toxicity, and neutralization of the venom of the olive sea snake, Aipysurus laevis.

Four specimens of the olive sea snake, Aipysurus laevis, were collected off the coast of Western Australia, and the venom proteome was characterized and quantitatively estimated by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses. A. laevis venom is remarkably simple and consists of phospholipases A2 (71.2%), three-finger toxins (3FTx; 25.3%), cysteine-rich secretory proteins (CRISP; 2.5%), and traces of a complement control module protein (CCM; 0.2%). Using a Toxicity Score, the most lethal components were determined to be short neurotoxins. Whole venom had an intravenous LD50 of 0.07 mg/kg in mice and showed a high phospholipase A2 activity, but no proteinase activity in vitro. Preclinical assessment of neutralization and ELISA immunoprofiling showed that BioCSL Sea Snake Antivenom was effective in cross-neutralizing A. laevis venom with an ED50 of 821 μg venom per mL antivenom, with a binding preference towards short neurotoxins, due to the high degree of conservation between short neurotoxins from A. laevis and Enhydrina schistosa venom. Our results point towards the possibility of developing recombinant antibodies or synthetic inhibitors against A. laevis venom due to its simplicity.

Recent rapid speciation and ecomorph divergence in Indo‐Australian sea snakes

The viviparous sea snakes (Hydrophiinae) are a young radiation of at least 62 species that display spectacular morphological diversity and high levels of local sympatry. To shed light on the mechanisms underlying sea snake diversification, we investigated recent speciation and eco‐morphological differentiation in a clade of four nominal species with overlapping ranges in Southeast Asia and Australia. Analyses of morphology and stomach contents identified the presence of two distinct ecomorphs: a ‘macrocephalic’ ecomorph that reaches >2 m in length, has a large head and feeds on crevice‐dwelling eels and gobies; and a ‘microcephalic’ ecomorph that rarely exceeds 1 m in length, has a small head and narrow fore‐body and hunts snake eels in burrows. Mitochondrial sequences show a lack of reciprocal monophyly between ecomorphs and among putative species. However, individual assignment based on newly developed microsatellites separated co‐distributed specimens into four significantly differentiated clusters corresponding to morphological species designations, indicating limited recent gene flow and progress towards speciation. A coalescent species tree (based on mitochondrial and nuclear sequences) and isolation‐migration model (mitochondrial and microsatellite markers) suggest between one and three transitions between ecomorphs within the last approximately 1.2 million to approximately 840 000 years. In particular, the macrocephalic ‘eastern’ population of Hydrophis cyanocinctus and microcephalic H. melanocephalus appear to have diverged very recently and rapidly, resulting in major phenotypic differences and restriction of gene flow in sympatry. These results highlight the viviparous sea snakes as a promising system for speciation studies in the marine environment.

An analysis of Hydrophis ornatus (Gray), H. lamberti Smith, and H. inornatus (Gray) (Hydrophiidae, Serpentes) based on samples from various localities, with remarks on feeding and breeding biology of H. ornatus

Hydrophis ornatus (Gray) is described from Phuket Island, Andaman Sea, Siam Gulf, and the Philippine Islands, with remarks on feeding and breeding biology. Hydrophis lamberti Smith, is recognised as a distinct species and described from type locality (Bight of Bangkok), Siam Gulf, and the Philippine Islands. The type specimen of Hydrophis inornatus was reexamined. The results showed certain similarities between H. ornatus and the type specimen of H. inornatus, however, in the present paper the type specimen is maintained as a distinct species. Position of internal organs, in relation to number of vertebrae and ventral scales, is used as a new method for identifying sea snakes. Furthermore the number of vertebrae appeared to be a useful character to distinguish between H. ornatus and H. lamberti.

Molecules and Morphology Reveal Overlooked Populations of Two Presumed Extinct Australian Sea Snakes (Aipysurus: Hydrophiinae)

The critically endangered leaf-scaled (Aipysurus foliosquamaI) and short-nosed (A. apraefrontalis) sea snakes are currently recognised only from Ashmore and Hibernia reefs ~600km off the northwest Australian coast. Steep population declines in both species were documented over 15 years and neither has been sighted on dedicated surveys of Ashmore and Hibernia since 2001. We examine specimens of these species that were collected from coastal northwest Australian habitats up until 2010 (A.foliosquama) and 2012 (A. apraefrontalis) and were either overlooked or treated as vagrants in conservation assessments. Morphological variation and mitochondrial sequence data confirm the assignment of these coastal specimens to A. foliosquama (Barrow Island, and offshore from Port Hedland) and A.apraefrontalis (Exmouth Gulf, and offshore from Roebourne and Broome). Collection dates, and molecular and morphological variation between coastal and offshore specimens, suggest that the coastal specimens are not vagrants as previously suspected, but instead represent separate breeding populations. The newly recognised populations present another chance for leaf-scaled and short-nosed sea snakes, but coastal habitats in northwest Australia are widely threatened by infrastructure developments and sea snakes are presently omitted from environmental impact assessments for industry. Further studies are urgently needed to assess these species’ remaining distributions, population structure, and extent of occurrence in protected areas.

Enzymatic maceration of bone: a gentler technique than boiling

This proof of concept study investigates the removal of soft tissue from human ribs with the use of two common methods: boiling with a laundry detergent and using enzymes. Six individuals were autopsied, and one rib from each individual was removed for testing. Each rib was cut into pieces and afterwards macerated by one of the two methods. DNA extraction was performed to see the effect of the macerations on DNA preservation. Furthermore, the bone pieces were examined in a stereomicroscope to assess for any bone damage. The results demonstrated that both methods removed all flesh/soft tissue from the bones. The DNA analysis showed that DNA was preserved on all the pieces of bones which were examined. Finally, the investigation suggests that enzyme maceration could be gentler on the bones, as the edges appeared less frayed. The enzyme maceration was also a quicker method; it took three hours compared with the traditional method which took about 24 hours. However, a more standardised study should be performed to confirm this.

Independent Innovation in the Evolution of Paddle-Shaped Tails in Viviparous Sea Snakes (Elapidae: Hydrophiinae)

The viviparous sea snakes (Hydrophiinae) comprise ~90% of living marine reptiles and display many physical and behavioral adaptations for breathing, diving, and achieving osmotic balance in marine habitats. Among the most important innovations found in marine snakes are their paddle-shaped (dorsoventrally expanded) tails, which provide propulsive thrust in the dense aquatic medium. Here, we reconstruct the evolution of caudal paddles in viviparous sea snakes using a dated molecular phylogeny for all major lineages and computed tomography of internal osteological structures. Bayesian ancestral state reconstructions show that extremely large caudal paddles supported by elongated vertebral processes are unlikely to have been present in the most recent common ancestor of extant sea snakes. Instead, these characters appear to have been acquired independently in two highly marine lineages of relatively recent origin. Both the Aipysurus and Hydrophis lineages have elongated neural spines that support the dorsal edge of their large paddles. However, whereas in the Aipysurus lineage the ventral edge of the paddle is supported by elongated haemapophyses, this support is provided by elongated and ventrally directed pleurapophyses in the Hydrophis lineage. Three semi-marine lineages (Hydrelaps, Ephalophis, and Parahydrophis) form the sister group to the Hydrophis clade and have small paddles with poorly developed dorsal and ventral supports, consistent with their amphibious lifestyle. Overall, our results suggest that not only are the viviparous hydrophiines the only lineage of marine snakes to have acquired extremely large, skeletally supported caudal paddles but also that this innovation has occurred twice in the group in the past ~2-6 million years.

Rediscovery of the rare sea snake Hydrophis parviceps Smith 1935 : identification and conservation status

Smiths small-headed sea snake, Hydrophis parviceps, was originally described in 1935 from a single type specimen collected in southern Vietnam. Since this time there has been only one further record for the species—a specimen collected near the type locality in 1960 that has since been lost. Through field surveys in southern Vietnam in 2001, we obtained three additional specimens of this poorly known sea snake from fisheries bycatch. In the current paper, we present morphological and molecular data for the three new specimens and the holotype, and redescribe H. parviceps based on data synthesized for all five specimens known to date. We also discuss the conservation status of the species based on its vulnerability to fisheries bycatch mortality and seemingly low abundance within a very restricted geographic distribution.

The taxonomic status of the sea snake Hydrophis czeblukovi (Kharin, 1984) from north-west Australian waters

Hydrophis czeblukovi, a species only known from north-west Australian waters, is redescribed using external and internal morphological characters and is compared with its congeners. Hydrophis geometricus Smith, 1986 is synonymized with H. czeblukovi.

Trophic specialization drives morphological evolution in sea snakes

Viviparous sea snakes are the most rapidly speciating reptiles known, yet the ecological factors underlying this radiation are poorly understood. Here, we reconstructed dated trees for 75% of sea snake species and quantified body shape (forebody relative to hindbody girth), maximum body length and trophic diversity to examine how dietary specialization has influenced morphological diversification in this rapid radiation. We show that sea snake body shape and size are strongly correlated with the proportion of burrowing prey in the diet. Specialist predators of burrowing eels have convergently evolved a ‘microcephalic’ morphotype with dramatically reduced forebody relative to hindbody girth and intermediate body length. By comparison, snakes that predominantly feed on burrowing gobies are generally short-bodied and small-headed, but there is no evidence of convergent evolution. The eel specialists also exhibit faster rates of size and shape evolution compared to all other sea snakes, including those that feed on gobies. Our results suggest that trophic specialization to particular burrowing prey (eels) has invoked strong selective pressures that manifest as predictable and rapid morphological changes. Further studies are needed to examine the genetic and developmental mechanisms underlying these dramatic morphological changes and assess their role in sea snake speciation.