Comparison of chemosensory behavior and prey trail-following in the varanoid lizards Varanus gouldii and Heloderma suspectum

In the first of three experiments, Gould's monitor lizards Varanus gouldii (n = 8) and Gila monsters Heloderma suspectum (n = 8) were tested to determine whether a postingestion elevation in tongue-flick rate (PETF) occurred. Based on analysis of numbers of tongue-flicks during 20 successive minutes following treatments and controls, significant PETF was detected in V. gouldii. Analysis of numbers of tongue-flicks over 20 successive minutes for H. suspectum indicated that PETF did not occur. A second experiment with refined procedures confirmed the absence of PETF in our H. suspectum. The third experiment was designed to determine whether V. gouldii and H. suspectum were capable of following prey-chemical trails. Varanus gouldii and H. suspectum followed these trails, and did so equally well, whether or not prey were bitten and ingested prior to exposure to chemical trails. © 1996 Wiley-Liss, Inc.     

Differences in primary structure among five phospholipases A2 from Heloderma suspectum

Five increasingly anionic phospholipases A2 (Pa1-Pa5) exist in the venom of the lizard Heloderma suspectum. We recently elucidated the sequence of Pa5, the most abundant and most active variant, towards emulsified phosphatidylcholines. Here we present the primary structures of Pa2, Pa3 (subvariants a and b) and Pa4, based on Edman degradation of tryptic, endoproteinase Arg-C and chymotryptic fragments of the reduced and S-carboxymethylated proteins. Pa1-Pa5, considered collectively, belong to an original class of secretory phospholipases A2 with 141-143 residues, a short hydrophobic N-terminus, 10 half-cystine residues and an extended C-terminus. The only known phospholipase A2 with characteristics close enough to be a member of the same class is that present in the venom from the insect Apis mellifera. More specifically, the sequences of Pa3 and Pa5 are almost identical, and those of Pa2 and Pa4 are also quite similar. Both groups diverge enough to indicate the translation of two mRNA species in the venom gland. The primary structure of Pa3 reveals the existence of subvariants a and b, the sequence of which is identical to that previously defined for Pa5, except that the C-terminal tripeptide GEG in Pa5 is replaced by the dipeptide GE in Pa3a and the tetrapeptide GEGR in Pa3b, Pa4, when compared to Pa5, shows 21 substitutions with a cluster of five modified amino acids in positions 40-44, immediately after the catalytic segment amino acids 30-39, and added changes scattered before the C-terminus. Pa2 differs from Pa4 only by the absence of the Gly142 C-terminal residue. The 15% difference in primary structure observed between the Pa3-Pa5 and Pa2-Pa4 subgroups might be largely responsible for their distinct biological properties.     

The Relationship between Cranial Structure,Biomechanical Performance and Ecological Diversity in Varanoid Lizards

Skull structure is intimately associated with feeding ability in vertebrates, both in terms of specific performance measures and general ecological characteristics. This study quantitatively assessed variation in the shape of the cranium and mandible in varanoid lizards, and its relationship to structural performance (von Mises strain) and interspecific differences in feeding ecology. Geometric morphometric and linear morphometric analyses were used to evaluate morphological differences, and finite element analysis was used to quantify variation in structural performance (strain during simulated biting, shaking and pulling). This data was then integrated with ecological classes compiled from relevant scientific literature on each species in order to establish structure-function relationships. Finite element modelling results showed that variation in cranial morphology resulted in large differences in the magnitudes and locations of strain in biting, shaking and pulling load cases. Gracile species such as Varanus salvadorii displayed high strain levels during shaking, especially in the areas between the orbits. All models exhibit less strain during pull back loading compared to shake loading, even though a larger force was applied (pull =30N, shake = 20N). Relationships were identified between the morphology, performance, and ecology. Species that did not feed on hard prey clustered in the gracile region of cranial morphospace and exhibited significantly higher levels of strain during biting (P = 0.0106). Species that fed on large prey clustered in the elongate area of mandible morphospace. This relationship differs from those that have been identified in other taxonomic groups such as crocodiles and mammals. This difference may be due to a combination of the open ‘space-frame’ structure of the varanoid lizard skull, and the ‘pull back’ behaviour that some species use for processing large prey.     

Effects of Prey Movement and Prey Odor on Feeding in Garter Snakes

The role of prey movement in feeding behavior was investigated in 10 garter snakes (Thamnophis sirtalis) repeatedly presented with paired stationary and continuously rotating sections of earthworm (Lumbricus terrestris). Additionally, prey odor intensity and source were varied and the performance of a tongueless snake was compared to normal animals. Experiment 1 showed that garter snakes will selectively attack rotating over nonmoving sections of earthworm across a wide range of speeds (1–2048 rpm) with an optimum between 16 and 256 rpm. However, blocking the odor from the sections and presentation of speeds greater than 500 rpm decreased response to moving sections. Experiment 2 showed that at 22–32 rpm moving sections were selected over stationary sections when odor from both was blocked. Experiment 3 assessed the effects of varying ambient odor conditions upon selection of artificial moving and stationary prey. Ambient earthworm odor resulted in a sustained high rate of tongue-flicking while, with no odor present, snakes showed a gradually increasing rate of tongue-flicking that declined within a few minutes. Experiments 4 and 5 studied the effects of tongue removal upon the selection of moving and nonmoving prey. Gross changes in the feeding sequence were noted. A long-term tongue-less adult fed by opening her mouth and thrashing about her cage when presented with earthworm odor and only preferred moving prey at 32 rpm; a control showed the normal stalk-and-strike sequence. The tongueless snake was less attracted to the moving earthworm at a distance than were normal snakes and the use of vision seemed less integrated rather than compensably improved. The results are discussed in reference to the critical flicker-fusion frequency, klepto-parasitism, and escape tactics of prey.     

Chemical, immunological and biological properties of peptides like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide extracted from the venom of two lizards (Heloderma horridum and Heloderma suspectum)

Having previously isolated helodermin, the major peptide like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide, from the venom of the lizard Heloderma suspectum, we decided on a systematic exploration of all (VIP-PHI)-like peptides present in the venom of another lizard of the Helodermatidae family: Heloderma horridum. Six (VIP-PHI)-like peptides (PHH1 to 6) were purified to homogeneity from the venom of the lizard H. horridum with PHH3 and PHH4 representing two minor forms. All peptides cross-reacted in radioimmunoassays for helodermin and PHI but not for VIP. They yielded four fragments (T1 to T4) after trypsin digestion. T1, T2 and T3 showed the same retention time by reverse-phase HPLC and the same amino acid composition; the differences were confined to T4, the C-terminal sequence. PHH5 and PHH6 were found to be identical to synthetic helospectins I and II respectively. PHH1 and PHH3 probably resulted from a secondary modification of PHH5, while PHH2 and PHH4 derived from PHH6. Thus, the VIP-like peptides, previously called helospectins, are in fact typical of H. horridum venom. We confirmed that helodermin is the major (VIP-PHI)-like peptide of the venom of H. suspectum and observed its absence in H. horridum venom. Also, we found that positions 8 and 9 of helodermin are occupied by two Glu residues instead of two Gln as previously published. Helospectin-like material was also present in H. suspectum venom but in very small amount. In both venoms all VIP-like peptides were equally potent and efficient when tested for (a) their ability to occupy VIP as well as secretin receptors in rat pancreatic membranes and VIP receptors in rat liver membranes, and (b) the ensuing activation of adenylate cyclase in both membrane preparations.     

The functional anatomy of the shoulder of the savannah monitor lizard (Varanus exanthematicus)

The excursions of the scapulocoracoid and forelimb and the activity of 18 shoulder muscles were studied by simultaneous cineradiography and electromyography in Savannah Monitor lizards (Varanus exanthematicus) walking on a treadmill at speeds of 0.7–1.1 km/hour. During the propulsive phase, the humerus moves anteroposteriorly 40–55° and rotates a total of 30–40°. Simultaneously, the coracoid translates posteriorly along the tongue-and-groove coracosternal joint by a distance equivalent to about 40% the length of the coracoid. Biceps brachii, coraco-brachialis brevis and longus, the middle and posterior parts of the latissimus dorsi and pectoralis, serratus anterior, serratus anterior superficialis, subscapularis, supracoracoideus, and triceps usually become active during the late swing phase and continue activity throughout most or all of propulsion. The anterior part of the latissimus dorsi is active during the transition from propulsive to swing phases. Brachialis, deltoideus scapularis, levator scapulae, the anterior part of pectoralis, scapulo-humeralis posterior, and subcoracoideus are active primarily during the swing phase; they are occasionally active during propulsion. Deltoideus clavicularis, scapulo-humeralis posterior, sternocoracoideus, and the posterior part of the trapezius are biphasic, with activity in both the propulsive and swing phases. A number of shoulder muscles in Varanus exanthematicus and Didelphis virginiana (the Virginia opossum) are similar in attachments, in activity patterns with respect to phases of the step cycle, and in apparent actions. These similarities are interpreted as a pattern inherited from the ancestors of higher tetrapods. The sliding coracosternal joint permits an increase in step length without demanding greater excursion at the shoulder and elbow joints.     

Alterations of endocrine pancreas B cells in a sahelian reptile (Varanus exanthematicus) during starvation

During the long starvation period (November to June) of the lizard (Varanus exanthematicus), pancreatic B cells undergo profound modification. The degeneration of beta granules observed in electron microscopy appears correlated with the diminution of the immunoreactive insulin-like content of the pancreas. The analogy between the phenomena observed here and those reported in animals treated with alloxan is discussed.     

Somatostatin-like immunoreactivity in pancreatic extracts of a sahelian lizard (Varanus exanthematicus) during starvation

In the Varanus exanthematicus, the pancreatic complex comprises the true pancreas as well as an intrasplenic islet comparable to a Brockmann's body. Somatostatin content and concentration were estimated by radioimmunoassay in acetic acid extracts of both organs. Relatively large amounts of somatostatin (SLI) are present in the pancreas (2.17 +/- 0.07 micrograms) without any difference in distribution between the cranial (CP) and mediocaudal (MCP) regions. The intrasplenic islet contains as much SLI material as the whole pancreas (3.38 +/- 0.85 microgram); thus, this primitive organ presents a very high hormonal concentration (109.34 +/- 40.30 ng/mg wt). Serial dilutions of the extracts gave parallel immunoassay displacement curves and gel-filtration revealed two immunoreactive peaks: the most important one was found in the synthetic tetradecapeptide fraction, the other one near the void volume fraction. These results show an immunological similarity between the SLI substance of the pancreatic complex and the synthetic somatostatin; however, a molecular heterogeneity must not be excluded. The results are discussed from a phylogenetic point of view.     

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal’s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.     

Hysteresis of heart rate and heat exchange of fasting and postprandial savannah monitor lizards (Varanus exanthematicus)

Reptiles are ectothermic, but regulate body temperatures (T(b)) by behavioural and physiological means. Body temperature has profound effects on virtually all physiological functions. It is well known that heating occurs faster than cooling, which seems to correlate with changes in cutaneous perfusion. Increased cutaneous perfusion, and hence elevated cardiac output, during heating is reflected in an increased heart rate (f(H)), and f(H), at a given T(b), is normally higher during heating compared to cooling ('hysteresis of heart rate'). Digestion is associated with an increased metabolic rate. This is associated with an elevated f(H) and many species of reptiles also exhibited a behavioural selection of higher T(b) during digestion. Here, we examine whether digestion affects the rate of heating and cooling as well as the hysteresis of heart rate in savannah monitor lizards (Varanus exanthematicus). Fasting lizards were studied after 5 days of food deprivation while digesting lizards were studied approximately 24 h after ingesting dead mice that equalled 10% of their body mass. Heart rate was measured while T(b) increased from 28 to 38 degrees C under a heat lamp and while T(b) decreased during a subsequent cooling phase. The lizards exhibited hysteresis of heart rate, and heating occurred faster than cooling. Feeding led to an increased f(H) (approximately 20 min(-1) irrespective of T(b)), but did not affect the rate of temperature change during heating or cooling. Therefore, it is likely that the increased blood flows during digestion are distributed exclusively to visceral organs and that the thermal conductance remains unaffected by the elevated metabolic rate during digestion.     

Morphology of the nasal capsule of Heloderma suspectum with comments on the systematic position of helodermatids (Squamata: Helodermatidae)

A serially sectioned embryonic head of Heloderma suspectum formed the basis for a three-dimensional reconstruction of the cartilaginous nasal capsule and its membrane bones. Further, the soft parts of the nasal capsule were analysed microscopically. The embryonic nasal capsule is described and compared to the morphological conditions in other lizards, especially the varanids, which many recent authors believe to be their closest relatives.
The results support the close affinities between the helodermatids and the varanids. Both groups have a well developed recessus lateralis in a cartilaginous capsule, entered by the nasal concha. Further, in both groups a fragmentation of the cartilago paraseptalis occurs in a comparable location and form. Heloderma has no double lacrimal duct, in contrast to previous accounts, but the two canaliculi at the origin of the duct fuse noteworthy far ahead (in other squamates they fuse in front of the bulbus oculi). In the varanids the two branches of the lacrimal duct remain separate and open separately.
Some unique features of Heloderma are presented as well, above all the presence of a second concha.     

Similar Odor Discrimination Behavior in Head-Restrained and Freely Moving Mice

A major challenge in neuroscience is relating neuronal activity to animal behavior. In olfaction limited techniques are available for these correlation studies in freely moving animals. To solve this problem, we developed an olfactory behavioral assay in head-restrained mice where we can monitor behavioral responses with high temporal precision. Mice were trained on a go/no-go operant conditioning paradigm to discriminate simple monomolecular odorants, as well as complex odorants such as binary mixtures of monomolecular odorants or natural odorants. Mice learned to discriminate both simple and complex odors in a few hundred trials with high accuracy. We then compared the discrimination performance of head-restrained mice to the performance observed in freely moving mice. Discrimination accuracies were comparable in both behavioral paradigms. In addition, discrimination times were measured while the animals performed well. In both tasks, mice discriminated simple odors in a few hundred milliseconds and took additional time to discriminate the complex mixtures. In conclusion, mice showed similar and efficient discrimination behavior while head-restrained compared with freely moving mice. Therefore, the head-restrained paradigm offers a relevant approach to monitor neuronal activity while animals are actively engaged in olfactory discrimination behaviors.     

Spared Piriform Cortical Single-Unit Odor Processing and Odor Discrimination in the Tg2576 Mouse Model of Alzheimer's Disease

Alzheimer''s disease is a neurodegenerative disorder that is the most common cause of dementia in the elderly today. One of the earliest reported signs of Alzheimer''s disease is olfactory dysfunction, which may manifest in a variety of ways. The present study sought to address this issue by investigating odor coding in the anterior piriform cortex, the primary cortical region involved in higher order olfactory function, and how it relates to performance on olfactory behavioral tasks. An olfactory habituation task was performed on cohorts of transgenic and age-matched wild-type mice at 3, 6 and 12 months of age. These animals were then anesthetized and acute, single-unit electrophysiology was performed in the anterior piriform cortex. In addition, in a separate group of animals, a longitudinal odor discrimination task was conducted from 3–12 months of age. Results showed that while odor habituation was impaired at all ages, Tg2576 performed comparably to age-matched wild-type mice on the olfactory discrimination task. The behavioral data mirrored intact anterior piriform cortex single-unit odor responses and receptive fields in Tg2576, which were comparable to wild-type at all age groups. The present results suggest that odor processing in the olfactory cortex and basic odor discrimination is especially robust in the face of amyloid β precursor protein (AβPP) over-expression and advancing amyloid β (Aβ) pathology. Odor identification deficits known to emerge early in Alzheimer''s disease progression, therefore, may reflect impairments in linking the odor percept to associated labels in cortical regions upstream of the primary olfactory pathway, rather than in the basic odor processing itself.     

Sex Chromosomes and Karyotype of the (Nearly) Mythical Creature,the Gila Monster,Heloderma suspectum (Squamata: Helodermatidae)

A wide variety of sex determination systems exist among squamate reptiles. They can therefore serve as an important model for studies of evolutionary transitions among particular sex determination systems. However, we still have only a limited knowledge of sex determination in certain important lineages of squamates. In this respect, one of the most understudied groups is the family Helodermatidae (Anguimorpha) encompassing the only two venomous species of lizards which are potentially lethal to human beings. We uncovered homomorphic ZZ/ZW sex chromosomes in the Gila monster (Heloderma suspectum) with a highly heterochromatic W chromosome. The sex chromosomes are morphologically similar to the ZZ/ZW sex chromosomes of monitor lizards (Varanidae). If the sex chromosomes of helodermatids and varanids are homologous, female heterogamety may be ancestral for the whole Anguimorpha group. Moreover, we found that the karyotype of the Gila monster consists of 2n = 36 chromosomes (14 larger metacentric chromosomes and 22 acrocentric microchromosomes). 2n = 36 is the widely distributed chromosomal number among squamates. In his pioneering works representing the only previous cytogenetic examination of the family Helodermatidae, Matthey reported the karyotype as 2n = 38 and suggested a different chromosomal morphology for this species. We believe that this was probably erroneously. We also discovered a strong accumulation of telomeric sequences on several pairs of microchromosomes in the Gila monster, which is a trait documented relatively rarely in vertebrates. These new data fill an important gap in our understanding of the sex determination and karyotype evolution of squamates.     

An Adaptive Difference in the Relationship between Foraging Mode and Responses to Prey Chemicals in two Congeneric Scincid Lizards

Sensory abilities must allow efficient detection of prey, but the senses used and their relative importance may vary with hunting methods. In lizards, ambush foragers locate prey visually and active foragers use a combination of vision and vomerolfaction, the chemical sense associated with the vomeronasal system. Active foragers, but not ambush foragers, discriminate between prey chemicals and other chemical stimuli sampled by tongue-flicking. In active foragers, features of the tongue that may improve chemical sampling, such as elongation and forking are more pronounced and density of vomeronasal chemoreceptors is greater, than in ambush foragers. Foraging mode is fixed in most lizard families, and correlated evolution has been demonstrated among foraging mode, discrimination of prey chemicals, and lingual-vomeronasal morphology by interfamilial comparisons. Here I present information on a rare case of an intrageneric difference in foraging mode in the genus Mabuya . Laboratory experiments on the discrimination of prey chemicals showed that the active forager M . striata sparsa exhibits prey chemical discrimination, but the ambush forager M . acutilabris does not. The active forager also has a slightly more elongated tongue with deeper notching at the tip than the ambush forager, which might be a response to a change in foraging behavior or a reflection of unrelated differences in head shape. These findings confirm predictions based on correlated evolution between the hunting method and use of the chemical sense to locate food. They further show that chemosensory behavior is adjusted to change in foraging mode more rapidly than was previously known and suggest that behavioral changes may occur more rapidly than associated modifications of chemosensory morphology.     

Concentration and Membrane Fluidity Dependence of Odor Discrimination in the Turtle Olfactory System

In the present study, we examined the concentration dependenceof odor discrimination in turtle olfactory bulbar responsesusing the cross-adaptation technique. In the odorant pairs withdiverse molecular structures, the degree of discrimination wasunchanged or only slightly decreased with an increase in odorantconcentrations, suggesting that odorants are well discriminatedeven at high concentrations. In the odorant pairs with closelyrelated molecular structures, the degree of discrimination wasdecreased with an increase in odorant concentrations. An increasein the temperature of turtle olfactory epithelium also decreasedthe ability to discriminate these odorants. There was a goodcorrelation between changes in the odor discriminating abilityinduced by an increase in odor concentrations and those inducedby a temperature increase. The liposomes were made of lipidsextracted from the turtle olfactory epithelia and changes oftheir membrane fluidity induced by adsorption of odorants weremonitored with DPH. There was a good correlation between a decreasein odor discriminating ability and the membrane fluidity changesinduced by odorants. We suggest that decreases in odor discriminatingability induced either by an increase in odor concentrationor by a temperature increase are ultimately caused by changesin the membrane fluidity. Chem. Senses 22: 553–563, 1997.     

Purification and characterization of five variants of phospholipase A2 and complete primary structure of the main phospholipase A2 variant in Heloderma suspectum (Gila monster) venom

1. Five increasingly anionic variants (Pa1-Pa5) of Ca2+-dependent phospholipase A2 were purified to homogeneity from the venom of the lizard Heloderma suspectum (Gila monster). The purification procedure was based on semi-preparative reverse-phase HPLC followed by anion-exchange HPLC and analytical reverse-phase HPLC. 2. Their Mr were 17,000-18,000, as deduced by SDS/PAGE. Specific activities tested by the capacity to hydrolyze phosphatidylcholines at pH 8.5 decreased as follows: Pa3 greater than Pa5 greater than Pa4 greater than Pa1 greater than Pa2. These activities showed the same optimum pH (9.0), were mainly of the phospholipase A2 type and were lost upon p-bromophenacyl bromide treatment. 3. All five phospholipases efficiently stimulated amylase release from dispersed rat pancreatic acini at pH 7.4, their potency decreasing as follows: Pa2 greater than Pa1 approximately equal to Pa4 greater than Pa3 approximately equal to Pa5. No deleterious effect was apparent based on the lack of lactate dehydrogenase release. 4. The five variants, Pa1-Pa5, differed significantly in amino acid composition and this, together with distinct antigenic properties of Pa2 and Pa5, establishes the subheterogeneity of this new type of phospholipase A2, despite the fact that the N-terminal amino acid sequence (31 residues) of Pa1-Pa5 was exactly the same. 5. The full sequence of the major variant, Pa5, showed that this 142-amino-acid protein exhibited greater similarity to the bee venom enzyme than to any class I or class II secretory phospholipase A2 from snake venom and mammalian pancreas. While Pa5 displayed the highly conserved region between Asp30 and Cys39 (the essential active site of all phospholipases A2), its salient original points included 10 half-cystine residues only, an incomplete N-terminal sequence, large changes in the putative calcium loop, several alterations after the active site and a C-terminal extension never seen in other phospholipases A2, with the only exception being bee venom.