Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom.

1. An arginine ester hydrolase was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. Molecular weight was determined to be ca 29 kDa, with an isoelectric point of 4.4. 5. The enzyme was stable to heat treatment (95 degrees C, 10 min) and to pH changes over the range 2-11. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), beta-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.     

Isolation and characterization of six microsatellites in the Mexican beaded lizard Heloderma horridum

Six polymorphic microsatellite loci were isolated and characterized in the threatened Mexican beaded lizard, Heloderma horridum. The number of alleles per locus ranged from three to 12, with observed heterozygosity estimates ranging from 0.00 to 0.77, and expected heterozygosity estimates ranging from 0.00 to 0.73. These microsatellites will provide a valuable tool for the investigation of the genetic variation and structure of both wild and captive H. horridum populations.     

Isolation and characterization of horridum toxin with arginine ester hydrolase activity from Heloderma horridum (beaded lizard) venom

A hemorrhagic toxin with lethal and arginine ester hydrolytic activities was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel, and Q-Sepharose column chromatography. The hemorrhagic toxin was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis and immunodiffusion. Its molecular weight is approximately 31,000 with an isoelectric point of 3.9. Hemorrhagic, lethal, and benzoyl-L-arginine ethyl ester hydrolytic activities of this preparation were inhibited by diisopropyl fluorophosphate (DFP), N-bromosuccinimide, and beta-mercaptoethanol, suggesting that serine, tryptophan, and disulfide bonds are involved in these activities. Also there was an increase in creatine kinase activity in mice serum which is an indicator that the toxin is involved in muscle damage. This protein was stable to heat and pH ranges between 2 and 11. The Michaelis constant (Km), for benzoyl-L-arginine ethyl ester, and inhibition constant (Ki), for DFP, were found to be 6.9 X 10(-3) and 1.93 X 10(-4) M, respectively.     

Parentage assignment and extra-group paternity in a cooperative breeder: the Seychelles warbler (Acrocephalus sechellensis)

We describe the development and initial application of a semiautomated parentage testing system in the Seychelles warbler (Acrocephalus sechellensis). This system used fluorescently labelled primers for 14 polymorphic microsatellite loci in two multiplex loading groups to genotype efficiently over 96% of the warbler population on Cousin island. When used in conjunction with the program CERVUS, this system provided sufficient power to assign maternity and paternity within the Seychelles warbler, despite the complications associated with its cooperative breeding system and a relatively low level of genetic variation. Parentage analyses showed that subordinate 'helper' females as well as the dominant 'primary' females laid eggs in communal nests, indicating that the Seychelles warbler has an intermediate level of female reproductive skew, in between the alternative extremes of helper-at-the-nest and joint nesting systems. Forty-four per cent of helpers bred successfully, accounting for 15% of all offspring. Forty per cent of young resulted from extra-group paternity.     

Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wiegmann)

A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)     

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)⁵-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides.     

Parentage assignment of the mud crab (Scylla paramamosain) based on microsatellite markers

In this study, microsatellite markers were employed to identify the parentage relationship in Scylla paramamosain. The exclusion probability of loci was found to be related with the level of their heterozygosity. When no parent information or only one parent information was available, the exclusion probability ranged from 22.0% to 56.6% and from 41.2% to 73.1%, with the combined exclusion probability for ten loci being 97.0% and 99.8%, respectively. The cumulative assignment success rate was 100% when no parent information was available using seven most informative microsatellite markers. Moreover, the power of the seven microsatellite markers for parentage assignment was tested by a double-blind test, which indicated that 95% of the progeny can be correctly assigned to their parents. This study provided a microsatellite-based approach for parentage assignment in S. paramamosain that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this important crab species.     

Noninvasive paternity assignment in Gombe chimpanzees

The relative success of chimpanzee male mating strategies, the role of male dominance rank and the success of inbreeding avoidance behaviour can only be assessed when paternities are known. We report the probable paternities of 14 chimpanzees included in a long‐term behavioural study of chimpanzees (Pan troglodytes schweinfurthii) at Gombe National Park, Tanzania. DNA samples were collected noninvasively from shed hair and faeces and genotyped using 13–16 microsatellite loci characterized in humans. All 14 offspring could be assigned to fathers within the community. While there is a positive relationship between male rank and reproductive success, we demonstrate that a range of male mating strategies (possessiveness, opportunistic mating and consortships) can lead to paternity across all male ranks. Several adult females were at risk of breeding with close male relatives. Most successfully avoided close inbreeding but in one case a high‐ranking male in the community mated with his mother and produced an offspring. In contrast to recent data on chimpanzees (P. t. verus) from the Taï forest, Côte d’Ivoire, no evidence of extra‐group paternity was observed in our study. Reanalysis of Taï data using a likelihood approach casts doubt on the occurrence of extra‐group paternity in that community as well.     

First evidence of multiple paternity in the blue shark (Prionace glauca)

Multiple paternity (MP) is defined as the behaviour in which females successfully mate with multiple males leading to offspring from different sires within the same litter. MP seems to be frequent and an evolutionary advantage in elasmobranchs. Here the authors report for the first time the occurrence of MP in the cosmopolitan blue shark Prionace glauca L. The evidence, gathered via microsatellite genotyping of pregnant females and their embryos, suggests that MP is very frequent in this species. Knowledge of MP in P. glauca should help describe more precisely its reproductive biology and contribute to the management of its populations.     

Finding fathers: spatio-temporal analysis of paternity assignment in grey seals (Halichoerus grypus)

Molecular studies of pinniped breeding systems exhibit a broad range of agreement and disagreement with observational indices of male breeding success. Grey seal studies have reported considerable discrepancies between genetic and behavioural paternity measures that have been interpreted as evidence of previously unidentified male strategies and/or tactics. Therefore, these studies have the power to fundamentally alter our perceptions of mating systems. However, other pinniped studies exhibit no such disagreements, and one possible explanation for disparities may be sampling biases in space and time. Therefore, it is essential that potential sampling biases are examined to evaluate the likelihood of previously unidentified male strategies. We examined paternities assigned at the North Rona grey seal colony between 1999 and 2002 in relation to concurrent detailed behavioural and locational data for males and females. We found that (i) for females observed in sexual interaction(s) during their oestrus period, it was highly probable that one of the interacting males fathered their next pup; (ii) over 80% of assigned paternities agreed with observations of the in-colony behaviour and spatio-temporal proximity of the males and females involved; and (iii) a minority of females exhibit mate choice and seek sires outside their local male's home range, although evidence suggests that these females mate on the colony rather than at sea. In conclusion, nearly all paternities assigned agreed with expectation based upon detailed knowledge of the spatio-temporal patterns of individuals during the breeding season. We found little evidence of unidentified male strategies at North Rona, Scotland, whereas further examination of mechanisms of female choice may be productive.     

Patterns of multiple paternity in fruits of Mimulus ringens (Phrymaceae)

Multiply sired fruits provide unambiguous evidence that pollen from two or more donors was deposited on a stigma and successfully fertilized ovules. Such multiple paternity within fruits can have important consequences for both parental and offspring fitness, but little is known about the frequency of multiple paternity or the mechanisms causing it. In this study we quantify the extent of multiple paternity in replicate experimental arrays of Mimulus ringens (square-stem monkeyflower) and use observations of pollinator behavior to infer mechanisms generating multiply sired fruits. In each array, floral displays were trimmed to two, four, eight, or 16 flowers per plant to span the range of display sizes observed in nature. In our sample of 204 fruits, more than 95% had two or more outcross pollen donors. The number of sires per fruit averaged 4.63 ± 0.10 (mean ± 1 SE), including selfs, and did not vary significantly with floral display treatment. Patterns of bumble bee foraging, combined with limited pollen carryover, suggest that observed levels of multiple paternity cannot be fully explained by single probes that deposited mixed pollen loads. Multiple probes to flowers, each delivering pollen from 1-3 different sires, are more likely to have caused the observed patterns. These sequential visits may reduce the potential for pollen competition and female choice based on pollen tube growth rate.     

Parentage assignment detects frequent and large-scale dispersal in water voles

Estimating the rate and scale of dispersal is essential for predicting the dynamics of fragmented populations, yet empirical estimates are typically imprecise and often negatively biased. We maximized detection of dispersal events between small, subdivided populations of water voles (Arvicola terrestris) using a novel method that combined direct capture-mark-recapture with microsatellite genotyping to identify parents and offspring in different populations and hence infer dispersal. We validated the method using individuals known from trapping data to have dispersed between populations. Local populations were linked by high rates of juvenile dispersal but much lower levels of adult dispersal. In the spring breeding population, 19% of females and 33% of males had left their natal population of the previous year. The average interpopulation dispersal distance was 1.8 km (range 0.3-5.2 km). Overall, patterns of dispersal fitted a negative exponential function. Information from genotyping increased the estimated rate and scale of dispersal by three- and twofold, respectively, and hence represents a powerful tool to provide more realistic estimates of dispersal parameters.     

Microsatellites indicate a high frequency of multiple paternity in Apodemus (Rodentia)

Microsatellites were employed to estimate frequency of multiple paternity litters of two species of mice (genus Apodemus): striped field mouse (A. agrarius), and wood mouse (A. sylvaticus). Ten pregnant females of A. agrarius and six of A. sylvaticus were collected from natural populations in the northern Ukraine and analysed with 11 and nine microsatellite loci, respectively. Multiple paternity was indicated in eight of 10 litters in A. agrarius and in three of six litters in A. sylvaticus. Multiple paternity was documented at several loci (ranging from two to 10). In two cases (A. agrarius), three males were estimated to have fathered the litter.     

Manipulation of egg production reveals costs of reproduction in the tree lizard (Urosaurus ornatus)

Life-history theory predicts that the allocation of energy to current reproduction is associated with a decrement in future fecundity, future survival, or both. I treated this notion as the cost hypothesis, and tested the assumption that current reproduction exacts a cost in future survival and fecundity. Surgical manipulations of egg production were applied to natural populations of the tree lizard, Urosaurus ornatus, in south western New Mexico by yolkectomy surgery in two different years. I reduced the number of eggs produced in the first clutch during vitellogenesis by approximately 50% in yolkectomized females relative to controls. Subsequent survival, fecundity, and growth of females were followed for two or three years, depending on the cohort. Treated females in both cohorts showed significantly higher growth and survivorship throughout the experiment than in controls. After 2 years, yolkectomized females had grown an additional 2 mm (snout-vent length) compared to controls, enough for them to add on average an additional egg to their next clutch. This demonstrated a cost in terms of future fecundity through a reduction in growth and an increase in mortality in these lizards.     

Synchronous polyandry and multiple paternity in the frog Crinia georgiana (Anura: Myobatrachidae)

Multiple paternity has rarely been reported in anuran amphibians, with only three previous documented examples. For the Australian frog Crinia georgiana, we observed synchronous polyandry in an average of 44% of matings observed at four field sites. This suggests matings involving more than one male are common in this species. One to eight males were observed in amplectant groups with second males amplexed ventrally. Genetic analyses, using allozyme electrophoresis, of offspring from two matings indicated that at least two of three possible males fathered offspring. Third males were unlikely to have shared paternity, explained by their inappropriate position during amplexus. Multiple paternity may be more common in frogs than has been reported. Copyright 1999 The Association for the Study of Animal Behaviour.     

Geographic variation in multiple paternity within natural populations of the guppy (Poecilia reticulata)

Mating can increase an individual''s risk of mortality by predation. In response to predation hazards, males in some species court females less often, but alternatively engage in coerced copulations more frequently and females become less selective. Such predator-mediated shifts in mating tactics may result in higher levels of multiple inseminations in females and, thus, in greater frequencies of females with broods of mixed paternity. We tested this hypothesis using two polymorphic microsatellite loci to estimate conservatively multiple paternity in broods of female guppies (Poecilia reticulata) originating from ten natural populations that have evolved under different fish predation regimes in Trinidad. The frequency of broods that were multiply sired was significantly greater on average in populations experiencing high predation pressure compared to populations experiencing a relatively low predation risk. These results suggest that the intensity of male sperm competition covaries geographically with predation pressure in this species and that the local risk of predation mediates the opportunity for sexual selection within populations.     

Ultrastructural features of skin pigmentation in the lizard Heloderma suspectum with emphasis on xanto‐melanophores

The morphological origin of the dark and pink‐orange areas in the skin of the venomous lizard Heloderma suspectum is not known. Histology and electron microscopy show that dark‐grey areas of the skin derived from dermal chromatophores localized in specific areas present underneath the epidermis. A dynamic chromatophoric unit in the dermis is absent. In the darkest areas of the skin, the accumulation of melanosomes in cells of the beta‐layer contributes to increase the black intensity. In the orange‐pink areas, the superficial dermis contains xantophores storing numerous carotenoid vesicles, rare or absent lamellated pterinosomes and a variable number of melanosomes. These xanto‐melanophores predominate over the remaining chromatophores and form a continuous stratum underneath the epidermis. Beneath this lipoid‐rich stratum, iridophores are infrequent and do not form a continuous layer in the dermis. In the paler areas of the skin, melanophores are sparse in both superficial and deeper part of the dermis where irregularly oriented bundles of collagen fibrils are present. The prevalent xanto‐melanophores localized in the pink‐orange areas of the skin contribute to an effective sunlight protection in desert conditions in addition to the darker regions occupied by melanophores.     

Three-dimensionally preserved integument reveals hydrodynamic adaptations in the extinct marine lizard Ectenosaurus (Reptilia, Mosasauridae)

The physical properties of water and the environment it presents to its inhabitants provide stringent constraints and selection pressures affecting aquatic adaptation and evolution. Mosasaurs (a group of secondarily aquatic reptiles that occupied a broad array of predatory niches in the Cretaceous marine ecosystems about 98-65 million years ago) have traditionally been considered as anguilliform locomotors capable only of generating short bursts of speed during brief ambush pursuits. Here we report on an exceptionally preserved, long-snouted mosasaur (Ectenosaurus clidastoides) from the Santonian (Upper Cretaceous) part of the Smoky Hill Chalk Member of the Niobrara Formation in western Kansas, USA, that contains phosphatized remains of the integument displaying both depth and structure. The small, ovoid neck and/or anterior trunk scales exhibit a longitudinal central keel, and are obliquely arrayed into an alternating pattern where neighboring scales overlap one another. Supportive sculpturing in the form of two parallel, longitudinal ridges on the inner scale surface and a complex system of multiple, superimposed layers of straight, cross-woven helical fiber bundles in the underlying dermis, may have served to minimize surface deformation and frictional drag during locomotion. Additional parallel fiber bundles oriented at acute angles to the long axis of the animal presumably provided stiffness in the lateral plane. These features suggest that the anterior torso of Ectenosaurus was held somewhat rigid during swimming, thereby limiting propulsive movements to the posterior body and tail.     

Microsatellite inheritance and multiple paternity in the deep-sea octopus Graneledone boreopacifica (Mollusca: Cephalopoda)

Abstract. Octopodids are a globally distributed group of marine molluscs. Despite this, our knowledge of their reproductive biology rests heavily on inference, as all phases of copulation, beginning with sperm transfer, occur within the mantle cavity. Male octopuses insert a spermatophore into the female's oviduct, which is predicted to release a sperm-filled sac that either bursts to release sperm for storage or to itself be stored in a gland in the middle of the oviduct. To test whether female octopuses use sperm from multiple males to fertilize their eggs, as may be predicted from anatomy and anecdotal accounts, we apply microsatellite analysis to a partial clutch of Graneledone boreopacifica collected at 1600-m depth to test for multiple paternity. At least two genetically distinct sires contributed sperm to the hatchlings analyzed, demonstrating for the first time multiple paternity in octopodids.     

Albumin Mexico (Al Me ) in the Guatemalan highlands

The examination of the sera of two samples of Guatemalans (total n = 386) revealed the presence of albumin Mexico (Al^Me) among unrelated individuals from the western highlands near the Mexican border. The frequency in the putatively less admixed sample, from Quiché, was 0.008. This is the first report of albumin polymorphism among American Indians south of Mexico. The presence of the Al^Me allele among Indians of the Guatemalan highlands argues for a zone of gene flow between contiguous groups of Mexico and Guatemala. The failure to detect albumin polymorphism among lowland populations of the Yucatan peninsula may likewise be significant, but more testing of samples is required before definitive statements can be made with respect to contacts among and within highland and lowland groups.