Aspidomorphus, a genus of New Guinea snakes of the Family Elapidae, with notes on related genera

The Australian snakes that have been included in Aspidomorphus differ markedly from that genus, particularly in hemipenial morphology and in the absence of a muscular slip from the quadrate bone to the venom gland; the genus Aspidomorphus is therefore restricted to the New Guniea species. Aspidomorphus (as here restricted) is closely related to Demansia (in the restricted sense of Worrell, essentially D. psammophis, D. torquata , and D. olivacea ), but the Australian species generally referred to Aspidomorphus seem to be related to Glyphodon . The genera Aspidomorphus, Demansia, Rhinhoplocephalus and Drepanodontis form a natural group. Aspidomorphus contains three species, each identifiable by hemipenial features as well as by details of colouration: A. muelleri (= A. mülleri mülleri and A. m. interruptus of Brongersma's revision); A. lineaticollis (= A. mülleri lineaticollis and A. m. lineatus of Brongersma); and A. schlegeli. The last species differs from the others in the form of the maxillary bone and the anterior mandibular dentition; it seems to be confined to northwestern New Guinea and adjacent islands, since specimens from the eastern end of New Guinea that had been referred to schlegeli are actually A. lineaticollis. In all three species some geographical variation can be demonstrated, at least in ventral count, but it is not considered necessary to use trinomials to indicate that geographical variation exists. Pseudonaja textilis is recorded from New Guinea for the first time. (McDowell.)
Examination reveals Demansia ornaticeps is properly referred to Demansia. (Cogger.)     

Venom glands and some associated muscles in sea snakes

The venom glands and related muscles of sea snakes conform in their general structure to those of the terrestrial elapids. The venom gland, however, is smaller in size and the accessory gland is considerably reduced. A similar pattern is found in the Australian elapid Notechis. The musculus compressor glandulae is well developed in the sea snakes and in some species its posterior-medial portion runs uninterruptedly from the origin to the insertion of the muscle. This might be considered as a primitive condition suggesting an early divergence of the sea snakes from an ancestral elapid stock. Three species of sea snakes, Aipysurus eydouxi, Emydocephalus annulatus, and E. ijimae, feed on fish eggs and have very small, but still functioning, venom glands. The reduced accessory gland of the sea snakes is apparently connected with their aquatic environment, as a similar condition is found also in the elapine Boulengerina annulata which lives in large lakes of Central Africa. The similarity in structure of the venom gland between sea snakes and Notechis scutatus may point to a possible phylogenetic relationship between this group of Australian elapids and hydrophiine snakes.     

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.     

Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis.

Aipysurus laevis venom was chromatographed on CM-cellulose and Bio-Rex 70 columns. Three neurotoxic components, toxins Aipysurus laevis a, b and c, were isolated. The toxins a, b and c corresponded to 22, 33 and 21% respectively of the proteins in the original venom, and accounted for almost all the lethal activity of the venom. The three toxins a, b and c were monodisperse on disc electrophoresis at pH4; toxins a and b moved at the same velocity and c a little faster. They were monodisperse also on sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis, giving a molecular weight of 7600. The molecular weight of toxin b estimated by gel filtration was 7000. The amino acid sequence analyses of these toxins revealed that they consisted of 60 amino acid residues and that Aipysurus laevis b was [25-methionine, 28-arginine] Aipysurus laevis a. Aipysurus laevis c was [28-lysine] Aipysurus laevis a, the tryptic peptide sequence relying on homology. The LD50 values of these toxins for 20g mice were 0.076 mug/g body wt. They inhibited the acetylcholine-induced contracture but did not affect the CKl-induced contracture of the isolated muscle.     

Venom and Dufour's glands of the emerald cockroach wasp Ampulex compressa (Insecta,Hymenoptera, Sphecidae): Structural and biochemical aspects

The digger wasp species Ampulex compressa produces its venom in two branched gland tubules. They terminate in a short common duct, which is bifurcated at its proximal end. One leg is linked with the venom reservoir, the other one extends to the ductus venatus. Each venom gland tubule possesses, over its entire length, a cuticle-lined central duct. Around this duct densely packed class 3 gland units each composed of a secretory cell and a canal cell are arranged. The position of their nuclei was demonstrated by DAPI staining. The brush border of the secretory cells surrounds the coiled end-apparatus. Venom is stored in a bladder like reservoir, which is surrounded by a thin reticulated layer of muscle fibres. The reservoir as a whole is lined with class 3 gland units. The tubiform Dufour's gland has a length of about 350 μm (∅ 125 μm) only and is surrounded by a network of pronounced striated muscle fibres. The glandular epithelium is mono-layered belonging to the class 1 type of insect epidermal glands. The gland cells are characterized by conspicuous lipid vesicles. Secretion of material via the gland cuticle into the gland lumen is apparent. Analysis of the polypeptide composition demonstrated that the free gland tubules and the venom reservoir contain numerous proteins ranging from 3.4 to 200 kDa. The polypeptide composition of the Dufour's gland is completely different and contains no lectin-binding glycoproteins, whereas a dominant component of the venom droplets is a glycoprotein of about 80 kDa. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets revealed that all of the major proteinaceous constituents are secreted. The secreted venom contains exclusively proteins present in the soluble contents of the venom gland. The most abundant compound class in the Dufour's gland consisted of n-alkanes followed by monomethyl-branched alkanes and alkadienes. Heptacosane was the most abundant n-alkane. Furthermore, a single volatile compound, 2-methylpentan-3-one, was identified in various concentrations in the lipid extract of the Dufour's gland.     

The mechanism of venom secretion from Duvernoy's gland of the snake Thamnophis sirtalis

The venom glands of snakes of the families Elapidae and Viperidae are thought to have evolved from Duvernoy's gland of colubrid ancestors. In highly venomous snakes elements of the external adductor musculature of the jaw insert fibers directly onto the capsule of the venom gland. These muscles, upon contraction, cause release of contents by increasing intraglandular pressure. In Thamnophis sirtalis, a colubrid, there is no direct connection between Duvernoy's gland and the adductor musculature. The anatomical arrangement of the gland, skull, adductor muscles, and the integument is such that contraction of the muscles may facilitate emptying of the gland. This hypothesis was tested by electrical stimulation of the muscles, which resulted in significantly greater release of secretion than elicited by controls. The results suggest a possible early step in the evolution of a more intimate association between venom glands and adductor musculature in highly venomous snakes.     

Uncovering cryptic diversity in Aspidomorphus (Serpentes: Elapidae): Evidence from mitochondrial and nuclear markers

The Papuan region, comprising New Guinea and nearby islands, has a complex geological history that has fostered high levels of biodiversity and endemism. Unfortunately, much of this diversity remains undocumented. We examine the evolutionary relationships of the venomous snake genus Aspidomorphus (Elapidae: Hydrophiinae), a Papuan endemic, and document extensive cryptic lineage diversification. Between Aspidomorphus species we find 22.2–27.9% corrected cyt-b sequence divergence. Within species we find 17.7–23.7% maximum sequence divergence. These high levels of genetic divergence may have complicated previous phylogenetic studies, which have had difficulty placing Aspidomorphus within the subfamily Hydrophiinae. Compared to previous studies, we increase sampling within Hydrophiinae to include all currently recognized species of Aspidomorphus and increase species representation for the genera Demansia and Toxicocalamus. We confirm monophyly of Aspidomorphus and resolve placement of the genus utilizing a set of seven molecular markers (12S, 16S, cyt-b, ND4, c-mos, MyHC-2, and RAG-1); we find strong support for a sister-group relationship between Aspidomorphus and a Demansia/Toxicocalamus preussi clade. We also use one mitochondrial (cyt-b) and one nuclear marker (SPTBN1) to document deep genetic divergence within all currently recognized species of Aspidomorphus and discuss the Solomon Island Arc as a potential center of divergence in this species. Lastly, we find high levels of concordance between the mtDNA and nuDNA markers used for inter-species phylogenetic reconstruction.     

Eggs-Only Diet: Its Implications for the Toxin Profile Changes and Ecology of the Marbled Sea Snake (Aipysurus eydouxii)

Studies so far have correlated the variation in the composition of snake venoms with the target prey population and snakes diet. Here we present the first example of an alternative evolutionary link between venom composition and dietary adaptation of snakes. We describe a dinucleotide deletion in the only three finger toxin gene expressed in the sea snake Aipysurus eydouxii (Marbled Sea Snake) venom and how it may have been the result of a significant change in dietary habits. The deletion leads to a frame shift and truncation with an accompanying loss of neurotoxicity. Due to the remarkable streamlining of sea snake venoms, a mutation of a single toxin can have dramatic effects on the whole venom, in this case likely explaining the 50- to 100-fold decrease in venom toxicity in comparison to that of other species in the same genus. This is a secondary result of the adaptation of A. eydouxii to a new dietary habit — feeding exclusively on fish eggs and, thus, the snake no longer using its venom for prey capture. This was parallel to greatly atrophied venom glands and loss of effective fangs. It is interesting to note that a potent venom was not maintained for use in defense, thus reinforcing that the primary use of snake venom is for prey capture.Nucleotide sequence data reported here have been deposited in the GenBank database under accession number AY559317.Reviewing Editor: Dr. Martin Kreitman     

An ultrastructural investigation of the striated subumbrellar musculature of the anthomedusan,Pennaria tiarella

The presence of striated subumbrellar musculature in hydromedusae can be related to the development in hydrozoans of a free-swimming life stage. The detailed ultrastructure of the striated subumbrellar musculature of the anthomedusan, Pennaria tiarella is presented. The striated musculature of Pennaria resembles vertebrate striated muscle in filament arrangement (L2 lattice pattern) and M line structure. The striation pattern, out-of-register myofilaments, filament structure as determined by rotational symmetry, Z line structure, types of intercellular junctions, and sarcoplasmic reticulum are more similar to structures found in other invertebrate striated muscle.     

Molecular phylogeny and divergence dates for Australasian elapids and sea snakes (hydrophiinae): evidence from seven genes for rapid evolutionary radiations

One of the most prolific radiations of venomous snakes, the Australo-Melanesian Hydrophiinae includes approximately 100 species of Australasian terrestrial elapids plus all approximately 60 species of viviparous sea snakes. Here, we estimate hydrophiine relationships based on a large data set comprising 5800 bp drawn from seven genes (mitochondrial: ND4, cytb, 12S, 16S; nuclear: rag1, cmos, myh). These data were analysed using parsimony, likelihood and Bayesian methods to better resolve hydrophiine phylogeny and provide a timescale for the terrestrial and marine radiations. Among oviparous forms, Cacophis, Furina and Demansia are basal to other Australian elapids (core oxyuranines). The Melanesian Toxicocalamus and Aspidomorphus group with Demansia, indicating multiple dispersal events between New Guinea and Australia. Oxyuranus and Pseudonaja form a robust clade. The small burrowing taxa form two separate clades, one consisting of Vermicella and Neelaps calanotus, and the other including Simoselaps, Brachyurophis and Neelaps bimaculatus. The viviparous terrestrial elapids form three separate groups: Acanthophis, the Rhinoplocephalus group and the Notechis-Hemiaspis group. True sea snakes (Hydrophiini) are robustly united with the Notechis-Hemiaspis group. Many of the retrieved groupings are consistent with previous molecular and morphological analyses, but the polyphyly of the viviparous and burrowing groups, and of Neelaps, are novel results. Bayesian relaxed clock analyses indicate very recent divergences: the approximately 160 species of the core Australian radiation (including sea snakes) arose within the last 10 Myr, with most inter-generic splits dating to between 10 and 6 Ma. The Hydrophis sea snake lineage is an exceptionally rapid radiation, with > 40 species evolving within the last 5 Myr.     

Evolution of venom delivery structures in madtom catfishes (Siluriformes: Ictaluridae)

The use of venom to subdue prey or deter predators has evolved multiple times in numerous animal lineages. Catfishes represent one of the most easily recognized, but least studied groups of venomous fishes. Venom glands surround spines on the dorsal and pectoral fins that serve as venom delivery structures. Species of madtom catfishes in the genus Noturus were found to each have one of four venom delivery morphologies: (1) smooth spine with no venom gland; (2) smooth spine with venom gland associated with shaft of spine; (3) serrated spine with venom gland associated with shaft of spine; and (4) serrated spine with venom gland associated with shaft of spine and posterior serrations. Analyses accounting for the phylogenetic history of Noturus species suggest that a serrated pectoral spine with a venom gland is the ancestral condition for the genus. The presence of serrations and a venom gland have been largely conserved among Noturus species, but sting morphology has changed at least five times within the genus. Four of these changes have resulted in a loss of morphological complexity, including the loss of posterior serrations, loss of venom glands associated with the posterior serrations, and one complete loss of the venom gland. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 115–129.     

Physiological and Biochemical Analysis of Factors in the Female Venom Gland and Larval Salivary Secretions of the Ectoparasitoid Wasp Eupelmus orientalis

The stinging adult female and the biting newly-hatched larva of the solitary ectoparasitoid wasp Eupelmus orientalis can both cause permanent paralysis and stop the development of Callosobruchus maculatus host larvae. These two processes of host envenomation appeared to be independent and complementary in primary parasitism or in hyperparasitism of a distantly related hymenopteran host species. In contrast, the development of larvae as hyperparasites on members of their own species or genus depended completely on the prior injection of female venom. The venoms of the female and the first instar larva had similar effects on the cellular metabolism of the primary hosts. Protein synthesis was blocked in C. maculatus hosts envenomated by a female or a first instar larva of E. orientalis, but the absence of DNA breakdown indicated that these paralysed hosts were alive and quiescent. The venomous secretions injected by adult females and first instar larvae of E. orientalis had distinct electrophoretic profiles. The immunoreactive features of proteins from female venom and larval secretions were also examined. There is evidence for antigenic conservation between some venom proteins of E. orientalis and Apis mellifera. Lastly, the hyaluronidase, phospholipase and lipase activities in the female venom gland and in larval-derived secretions of E. orientalis were assayed. No lipase activity was detected. Phospholipase activity was found in both the female venom and the larval secretions of E. orientalis, whereas hyaluronidase was specific to the female venom.     

Ventilation rates in a prey species of fish (Dascyllus aruanus) treated with the venom fractions of the olive sea snake,Aipysurus laevis

A prey species of fish (Dascyllus aruanus) was envenomated with venom fractions of Aipysurus laevis. Fraction group ventilation profiles were compared to a model of ventilation changes possessing three divisions for a medium venom dose to examine differences in fraction profiles with whole venom. All groups, including controls, showed an increase in ventilation in division 1 due to subject manipulation. Fractions 1, 3, 5, 6 and a fraction recombination showed significant increases in ventilation rates from the control in division 2. Fractions 2 and 4 were shown to be significantly lower in ventilation rates during this division. Fractions 1, 3, 5 and 6 are suspected of having neurotoxic characteristics with fraction 6 being the major neurotoxin in A. laevis venom. Significant differences were seen between the experimental group ventilation profiles.     

Chemical composition of the poison apparatus secretions of the African weaver ant, Oecophylla longinoda, and their role in behaviour

ABSTRACT. Major workers of Oecophylla longinoda emit venom from the tip of the abdomen, as it is brought immediately above the head. The sources of the venom are (a) the poison gland, which contains formic acid, and (b) Dufour's gland, which contains hydrocarbons, including n-undecane and other n-alkanes, 4-tridecene, 8-heptadecene and 4, 7-heptadecadiene. The venom elicits a 'mass attack' response in other major workers. Formic acid and n-undecane presented together experimentally also evoke this response, the mixture being considerably more effective than either compound tested separately. These compounds act in combination with the mandibular gland secretions to form a complex alarm/defence system.     

Morphologic analysis of the venom gland of Apis mellifera L. (Hymenoptera: Apidae) in populations of Mato Grosso do Sul, Brazil

In Apis mellifera L. the venom gland (also called acid gland) is composed of secretory cells that surround a channel that opens into a reservoir devoid of musculature. This gland can present apical branching. In this study the frequency of branched venom glands in Africanized honeybee workers (A. mellifera) from eleven localities in the state of Mato Grosso do Sul was recorded. The relations among the length of the main duct, the length of the duct from the reservoir to the beginning of branching, the length of the branched segment (when present) and the total length of the gland were also analyzed. The frequency of branched glands varied from 50% to 83% in the workers, indicating that this characteristic is primitive in those bees. The results of the Analysis of Discriminant Functions indicated significant differences in the morphometrical segments of the venom gland (Wilks Lambda = 0.092; F (40, 55) = 3.43; P < 0.001), and permitted a differentiation of the populations studied. Using the Mantel test we verified that there does not exist a significant correlation between the morphologic characteristics and the geographical distance between the localities evaluated (Mantel r = -0.006, P = 0.48). The high frequency of workers with large venom gland in all the apiaries considered makes viable the development of a selection program in order to obtain bees with longer venom glands, aimed at the commercial production of venom by the beekeepers of those localities of Mato Grosso do Sul.     

Venom gland of the digger wasp Liris niger: morphology, ultrastructure, age-related changes and biochemical aspects

Females of the parasitoid digger wasp species Liris niger hunt crickets as food for their future brood. The wasps paralyse the prey by injecting their venom directly into the CNS. The venom is produced in a gland consisting of two ramified glandular tubules terminating in a common reservoir. The reservoir contents enter the sting bulb via a ductus venatus. Secretory units of dermal gland type III line the two free gland tubules, the afferent ducts to the reservoir and the cap region within the reservoir. Secretion products of tubules reach the reservoir through the cuticle-lined central funnel. Secretory cells in the distal and middle parts of the tubules contain extensive rough endoplasmic reticulum and numerous electron-dense vesicles, whereas secretory cells of the afferent ducts and the cap region of the reservoir lack electron-dense vesicles and the endoplasmic reticulum is poorly developed. The secretory apparatus undergoes age-related changes. The secretory units in the venom gland tubules and inside the reservoir complete differentiation 1 day after imaginal ecdysis. After 30 days, massive autolytic processes occur in the secretory cells and in the epithelial cells of the reservoir. Analysis of the polypeptide composition demonstrates that the venom reservoir contains numerous proteins ranging from 3.4 to 200 kDa. A dominant component is a glycoprotein of about 90 kDa. In contrast the polypeptide composition of Dufour's gland is completely different and contains no glycoproteins. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets reveals that all of the major proteinaceous constituents become secreted. Thus the secreted venom contains exclusively proteins present in the soluble contents of the venom gland.     

Immunocytochemical localization and secretion process of the toxin CSTX-1 in the venom gland of the wandering spider Cupiennius salei (Araneae: Ctenidae)

Fluorescein and horseradish peroxidase-labeled monoclonal antibodies were used to localize the predominant toxic peptide CSTX-1 in the venom gland of the spider Cupiennius salei. There was no polarity of CSTX-1 expression in repleted glands, whereas the glands of previously milked spiders showed a decreasing immunofluorescent response from the distal to the proximal portion. Detailed investigation revealed a new structure in the venom-secreting epithelium, which is postulated to be an evolutionary adaptation to increasing gland volume. CSTX-1 was found to be synthesized and stored as a fully active toxin within complex units, composed of long interdigitating cells running perpendicular to the muscular sheath and extending into the central lumen of the gland. These venom-producing units were found in all sectors of the gland, including the transitional region between the main gland and the venom duct. The venom is liberated from the venom-producing units into the glandular lumen following the contraction of the surrounding muscle layer. Free nuclei or other cellular fragments, which would have provided evidence for a holocrine secretion process, were not found in the glandular lumen or in the crude venom obtained by electrical stimulation. The fine regulation of the spider's venom injection process is postulated to be the function of the bulbous ampulla, situated in the anterior third of the venom duct.     

Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.