Maternal provisioning of sequestered defensive steroids by the Asian snake <Emphasis Type="Italic">Rhabdophis tigrinus</Emphasis>

Summary.   Rhabdophis tigrinus obtains defensive steroids (bufadienolides) from its diet and sequesters those compounds in specialized structures on its neck known as nuchal glands. Hatchling snakes lacking these steroids must acquire them from toads consumed as prey. Here we show that females provision bufadienolides to their offspring in amounts correlated to the quantity in their own nuchal glands; thus, chemically protected mothers produce defended offspring. Bufadienolides can be provisioned to embryos via deposition in yolk and by transfer across the egg membranes within the oviducts. Maternally provisioned bufadienolides persist in the nuchal glands of juvenile snakes from the time of hatching in late summer until the following spring, when toads of ingestible size become abundant. Therefore, maternal provisioning may provide chemical protection from predators for young R. tigrinus in the absence of dietary sources of bufadienolides.     

Nuchal glands: a novel defensive system in snakes

Of the various chemical defensive adaptations of vertebrates, nuchal glands are among the most unusual. First described in a Japanese natricine snake, Rhabdophis tigrinus, in 1935, these organs are embedded under the skin of the neck region as a series of paired glands that have neither lumina nor ducts. The major chemical components of the glandular fluid are bufadienolides, which are cardiotonic steroids also found in the skin secretion of toads. Here we review early studies of nuchal glands and briefly introduce our recent findings on the sequestration of bufadienolides from consumed toads and the maternal provisioning of those sequestered compounds. We summarize behavioral studies associated with the antipredator function of the nuchal glands, which have been conducted during our more than decade-long collaboration. Results of preliminary analyses on the possible costs of toad-eating and on the ultrastructure of the nuchal glands are also presented. Finally, we discuss the evolutionary origin of the nuchal glands and suggest future directions designed to understand the biological importance of these novel vertebrate organs, which have evolved in a limited number of snake species.     

Amphibian myiasis. Blowfly larvae (Lucilia bufonivora,Diptera: Calliphoridae) coping with the poisonous skin secretion of the common toad (Bufo bufo)

The blowfly Lucilia bufonivora shows high host specificity for toads despite the host’s toxic skin secretion, which consists mainly of bufadienolides. These toxins are effective blockers of the Na⁺, K⁺-ATPase, an enzyme that is essential for many physiological processes in animals. Whereas common toad (Bufo bufo) toxins were identified in the larvae of the fly, few toxins were found in the pupae and empty puparia as trace amounts, while adult flies were entirely free of these toxic compounds. Similar results were obtained when larvae of generalist necrophagous blowflies (L. sericata, Calliphora vicina) fed on toad carcasses. Analysis of the Na⁺, K⁺-ATPase gene revealed no amino acid substitution at positions known to mediate resistance to bufadienolides in other systems. Alternative mechanisms of resistance such as efficient excretion of the compounds may enable the flies to use this poisonous food source.     

The occurrence of defensive alkaloids in non-integumentary tissues of the Brazilian red-belly toad Melanophryniscus simplex (Bufonidae)

The red-belly toads (Melanophryniscus) of southern South America secrete defensive alkaloids from dermal granular glands. To date, all information on Melanophryniscus alkaloids has been obtained by extraction from either skins or whole organisms; however, in other amphibians, tetrodotoxins, samandarines, and bufadienolides have been detected in both skin and other organs, which raise the possibility that lipophilic alkaloids may occur in non-integumentary tissues in Melanophryniscus as well. To test this hypothesis, we studied the distribution of alkaloids in the skin, skeletal muscle, liver, and mature oocytes of the red-belly toad M. simplex from three localities in southern Brazil. Gas chromatography and mass spectrometry of skin extracts from 11 individuals of M. simplex resulted in the detection of 47 alkaloids (including isomers), 9 unclassified and 38 from 12 known structural classes. Each alkaloid that was present in the skin of an individual was also present in the same relative proportion in that individual??s skeletal muscle, liver, and oocytes. The most abundant and widely distributed alkaloids were the pumiliotoxins 251D, 267C, and 323A, 5,8-disubstituted indolizidines 207A and 223D, 5,6,8-trisubstituted indolizidine 231B, 3,5-disubstituted pyrrolizidines cis-223B and cis- and trans-251K, and izidine 211C. We report the first record of piperidines in Melanophryniscus, bringing the total number of alkaloid classes detected in this genus to 16. Alkaloid composition differed significantly among the three study sites. The functional significance of defensive chemicals in non-integumentary tissues is unknown.     

Assessing the Potential Impact of Cane Toads on Australian Snakes

Abstract:   Cane toads ( Bufo marinus ) are large, highly toxic anurans that were introduced into Australia in 1937. Anecdotal reports suggest that the invasion of toads into an area is followed by dramatic declines in the abundance of terrestrial native frog-eating predators, but quantitative studies have been restricted to nonpredator taxa or aquatic predators and have generally reported minimal impacts. Will toads substantially affect Australian snakes? Based on geographic distributions and dietary composition, we identified 49 snake taxa as potentially at risk from toads. The impact of these feral prey also depends on the snakes' ability to survive after ingesting toad toxins. Based on decrements in locomotor (swimming) performance after ingesting toxin, we estimate the LD₅₀ of toad toxins for 10 of the at-risk snake species. Most species exhibited a similar low ability to tolerate toad toxins. Based on head widths relative to sizes of toads, we calculate that 7 of the 10 taxa could easily ingest a fatal dose of toxin in a single meal. The exceptions were two colubrid taxa (keelbacks [  Tropidonophis mairii ] and slatey-grey snakes [  Stegonotus cucullatus ]) with much higher resistance to toad toxins (up to 85-fold) and one elapid (swamp snakes [  Hemiaspis signata ]) with low resistance but a small relative head size and thus low maximum prey size. Overall, our analysis suggests that cane toads threaten populations of approximately 30% of terrestrial Australian snake species.     

Differences in stress tolerance and brood size between a non-indigenous and an indigenous gammarid in the northern Baltic Sea

Differences in stress tolerance and reproductive traits may drive the competitive hierarchy between non-indigenous and indigenous species and turn the former ones into successful invaders. In the northern Baltic Sea, the non-indigenous Gammarus tigrinus is a recent invader of littoral ecosystems and now occupies comparable ecological niches as the indigenous G. zaddachi. In laboratory experiments on specimens collected between June and August 2009 around Tvärminne in southern Finland (59°50′N/23°15′E), the tolerances towards heat stress and hypoxia were determined for the two species using lethal time, LT₅₀, as response variable. The brood size of the two species was also studied and some observations were made on maturation of juveniles. Gammarus tigrinus was more resistant to hypoxia and survived at higher temperatures than G. zaddachi. Brood size was also greater in G. tigrinus than in G. zaddachi and G. tigrinus matured at a smaller size and earlier than G. zaddachi. Hence, there are clear competitive advantages for the non-indigenous G. tigrinus compared to the indigenous G. zaddachi, and these may be further strengthened through ongoing environmental changes related to increased eutrophication and a warming climate in the Baltic Sea region.     

Impact of Invasive Cane Toads on Australian Birds

Abstract: The cane toad (Bufo marinus), a large, toxic, American anuran, was introduced to Australia in 1935. Populations of many of Australia's reptiles (snakes, varanid lizards, crocodiles) and carnivorous mammals (dasyurid marsupials) have declined because these predators are killed by the toad's powerful toxins. In contrast to these well‐studied species, little is known about the cane toads impacts on Australian birds. We reviewed published and unpublished data on behavioral interactions between Australian avian predators and cane toads and collated distributional and dietary information to identify avian taxa potentially at risk from cane toad invasion. Cane toads are sympatric with 172 frog‐eating bird species in Australia, and an additional 8 bird species overlap with the predicted future range of the toad. Although many bird species thus are potentially at risk, behavioral observations suggest the risk level is generally low. Despite occasional reports of Australian birds being killed when they ingest cane toads, most birds either ignore toads or survive the predation event. The apparently higher tolerance of Australian birds to toad toxins, compared with Australian reptiles and marsupials, may reflect genetic exchange between Australian birds and Asian populations that encounter other bufonid species regularly and hence have evolved the capacity to recognize or tolerate this toxic prey.     

Chemical studies on Indopacific Ceratosoma nudibranchs illuminate the protective role of their dorsal horn

Summary. Two Ceratosoma species, C. trilobatum and C. gracillimum (Mollusca: Gastropoda: Opistobranchia), collected along the South China Sea coast, have been studied in order to investigate their defensive strategies. Both species contain four typical sponge furanosesquiterpenoids, probably with a dietary origin. The tissue distribution of these compounds has been studied by ¹H-NMR analysis and preliminary bioassays have been carried out in order to evaluate the ecological role of the isolated metabolites. The main sesquiterpene component in both species was (–)-furodysinin (2), which showed significant feeding-deterrent and ichthyotoxic properties in bioassays with freshwater organisms. The work has been performed studying a very limited number of molluscs that prevented the realization of ecologically relevant experiments. However, the almost exclusive presence of compound 2 in the crude extract of the mantle glands concentrated in the dorsal horn of C. gracillimum strongly supports the defensive role of that dorsal protuberance, which probably acts as a defensive lure attracting potential predators to the part of the animal that contains the distasteful chemicals.     

Incubation temperature has long-term effects on behaviour of young Pine snakes (Pituophis melanoleucus)

Summary Eggs of pine snakes (Pituophis melanoleucus) were incubated at constant temperatures of 21°, 23°, 26°, 28°, 30° and 32° C to determine behavioural differences as a function of incubation temperature. For all behavioural and physiological measures hatchlings from medium incubation temperatures (26°, 28°) performed tests better than those hatchlings from eggs incubated at low temperatures (21°, 23°). For some behavioural tests hatchlings from eggs incubated at high temperature (30°, 32°) performed less well than medium temperature hatchlings. These differences were not due to ambient temperatures or age of testing since these were held constant. Some of the behavioural differences persisted for 24 weeks.     

Modeling amphibian energetics, habitat suitability, and movements of western toads, Anaxyrus (=Bufo) boreas, across present and future landscapes

Effective conservation of amphibian populations requires the prediction of how amphibians use and move through a landscape. Amphibians are closely coupled to their physical environment. Thus an approach that uses the physiological attributes of amphibians, together with knowledge of their natural history, should be helpful. We used Niche Mapper™ to model the known movements and habitat use patterns of a population of Western toads (Anaxyrus (=Bufo) boreas) occupying forested habitats in southeastern Idaho. Niche Mapper uses first principles of environmental biophysics to combine features of topography, climate, land cover, and animal features to model microclimates and animal physiology and behavior across landscapes. Niche Mapper reproduced core body temperatures (T_c) and evaporation rates of live toads with average errors of 1.6 ± 0.4 °C and 0.8 ± 0.2 g/h, respectively. For four different habitat types, it reproduced similar mid-summer daily temperature patterns as those measured in the field and calculated evaporation rates (g/h) with an average error rate of 7.2 ± 5.5%. Sensitivity analyses indicate these errors do not significantly affect estimates of food consumption or activity. Using Niche Mapper we predicted the daily habitats used by free-ranging toads; our accuracy for female toads was greater than for male toads (74.2 ± 6.8% and 53.6 ± 15.8%, respectively), reflecting the stronger patterns of habitat selection among females. Using these changing to construct a cost surface, we also reconstructed movement paths that were consistent with field observations. The effect of climate warming on toads depends on the interaction of temperature and atmospheric moisture. If climate change occurs as predicted, results from Niche Mapper suggests that climate warming will increase the physiological cost of landscapes thereby limiting the activity for toads in different habitats.     

Effects of incubation temperature on behavior of hatchling pine snakes: implications for reptilian distribution

Summary Incubation temperatures in vertebrates affect incubation periods, and in some reptiles incubation temperature determines sex ratios and some limited behavior. Here I present evidence that incubation temperature in pine snakes (Pituophis melanoleucus) affects not only incubation periods and posthatching behavior in the laboratory, but also the behavior of hatchlings required for successful emergence and survival. These behavioral differences have evolutionary implications for selection of hatchlings from particular temperature nests. With increasing temperature, incubation periods decreased in the laboratory. In addition, incubation temperature affected hatching and emergence times as well as movement speed and foraging ability. Hatchlings from medium temperature conditions emerged from nests in the field in less time than hatchlings incubated at high or low temperatures, and hatchlings from low temperatures moved slower and were less able to capture and eat mice in the laboratory than hatchlings incubated at medium or high temperatures. Taken together, these laboratory and field experiments suggest that hatchlings from low temperature nests, compared to those from higher temperature nests, would be less able to emerge, find food, and locate hibernation sites prior to the onset of cold temperatures in the New Jersey Pine Barrens. These results suggest that incubation temperature affects a whole range of behavior and that distributional ranges of reptiles and other poikilotherms could be affected by summer temperatures (via incubation period and subsequent behavior) as well as ambient winter temperatures.     

Antipredator activity and endogenous biosynthesis of defensive secretion in larval and pupal Delphastus catalinae (Horn) (Coleoptera: Coccinellidae)

Delphastus catalinae (Horn) is a predatory ladybird beetle (Coccinellidae) commonly used as a biocontrol agent against greenhouse infestation by whiteflies. It belongs to the basal subfamily Microweisinae, a group for which chemical defenses have not been previously investigated. The larval and pupal stages of D. catalinae possess minute secretory hairs that produce droplets containing compounds of both isoprenoid and polyketide origin. Bioassays with the predatory ant Crematogaster lineolata showed both the larval and pupal secretions to be deterrent. Moreover, isolated secretion components, from both classes of compounds, displayed antipredator activity against the ant. Experiments with D. catalinae larvae fed isotopically labeled glucose showed ¹³C-incorporation into both categories of compounds within the pupal secretion, demonstrating that these antipredator compounds, which differ from the typical nitrogenous defensive molecules of coccinellids, are biosynthesized endogenously. This suggests that the wide use of alkaloids by more derived coccinellids may have arisen after their divergence from the more basal Microweisinae.     

First insights into the chemical defensive system of the erotylid beetle, Tritoma bipustulata

The present study provides the first insights into the chemical defensive system of the erotylid beetle, Tritoma bipustulata, and furthermore reports the previously hardly known ability of abdominal reflex bleeding in this coleopteran family. The defensive chemistry of the secretion of pronotal glands, abdominal reflex blood as well as of the haemolymph were analysed by GC-MS. The different secretions were dominated by aromatic compounds; in addition, we detected alkenes, ketones, organic acids as well as a single sesquiterpene. The majority of these detected compounds had strong antimicrobial properties in microbiological assays with entomopathogenic micro-organisms. In feeding bioassays with ants, only benzyl alcohol, benzothiazole, indole and 3-methylindole, detected in the abdominal reflex blood, were significantly deterrent.     

Experimental evidence for the costs and hygienic significance of the antibiotic metapleural gland secretion in leaf-cutting ants

The paired exocrine metapleural glands present in the large majority of ant species produce compounds with antibiotic properties. In the leaf-cutting ant, Acromyrmex octospinosus, the secretion consists of more than 20 different compounds and it has generally been assumed that the glands serve as a general defence against various infectious microbes of fungal and bacterial origin. We present results illuminating the direct costs and benefits of these metapleural gland defences in A. octospinosus. We show that major workers of this leaf-cutting ant experience a significant reduction in their respiration rate when the metapleural glands are experimentally closed, indicating that metapleural gland secretion incurs a substantial cost and that the production of compounds from these glands is terminated when the ants are incapable of secreting them. In another set of experiments, we show that the ability to secrete antibiotic compounds from the metapleural glands is of significant importance when ants are exposed to a general but potentially virulent insect pathogen, Metarhizium anisopliae. Infection with this fungus is lethal within a few days when ants have their metapleural glands experimentally closed, but relatively harmless when the metapleural glands are functional. These findings support experimentally the view that the metapleural glands play an important hygienic role in leaf-cutting ants.     

Effects of incubation temperature on hatchling pine snakes: implications for survival

Incubation temperature in ectothermic vertebrates affects incubation periods, and in some reptiles it affects sex ratios and behavior. I present evidence that incubation temperature affects emergence and post-hatching behavior of pine snakes (Pituophis melanoleucus) that could influence survival in the weeks before hibernation. Hatchlings incubated at low temperatures remained in the nest longer, had fewer alternate nest openings, and fewer underground tunnels to hide in than did hatchlings from warmer temperatures. These conditions could render hatchlings from low-temperature nests more vulnerable to predation because, if a nest is opened, they are not inside tunnels where they would be protected. Hatchlings from nests incubated at low temperatures took longer to find shade during a thermoregulation test, and were less likely to move about in search of other cover than were those from higher-incubation-temperature artificial nests. Similarly, hatchlings from nests with low incubation temperatures were less responsive to a predatory stimulus and had a longer latency to strike than other hatchlings. Taken together, hatchlings from nests with low incubation temperatures might be less able to avoid predators and find shade than those from nests incubated at higher temperatures, and thus could be expected to have lower survival in nature. Received: 21 July 1997 / Accepted after revision: 15 February 1998     

Use of defensive glands during mating in a cockroach (Diploptera punctata)

Summary. The female of the cockroach, Diploptera punctata, mates immediately after the last molt, when she is still teneral. In this condition, her quinone-producing defensive glands are empty and she is potentially vulnerable to predation. By mating with fully sclerotized males whose glands are functional, she may benefit, indirectly, from the protection provided by the male's own glandular discharges. However, while males do indeed eject their defensive spray when disturbed while mating, and as a rule tend to remain coupled to the female when disturbed, they spray only when they themselves are the target of the offense. Copulating males do not activate their defenses in response to disturbance of the female.     

Behavioural and electrophysiological responses of the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), to male- and female-borne sex attractants

In the olive fruit fly, Bactrocera oleae, females attract males by producing 1,7-dioxaspiro[5.5]undecane (olean), the main component of the sex pheromone secreted by rectal glands. It has been recently demonstrated that males are able to produce (Z)-9-tricosene (muscalure) in rectal glands, a compound that selectively attracts females. In this study, a male grooming reaction that may transfer the male-borne compounds from rectal to urotergal glands was observed, suggesting that urotergal glands could be involved in B. oleae sexual communication. GC/MS, EAG, GC/EAD analyses and behavioural assays were carried out to compare the role of male rectal and urotergal glands during courtship. In both male glands, olean and muscalure amounts were age dependent. Extracts of rectal glands contained higher amounts of olean and/or muscalure than urotergal ones. Extracts of rectal and urotergal glands of males and females elicited EAG responses in both sexes. GC/EAD showed that female EAG response to male rectal extracts was mainly due to olean and muscalure. Synthetic compounds evoked EAG dose-dependent responses in both sexes, and the EAG response to muscalure was higher as compared to olean. Rectal and urotergal glands from old males were able to attract females, while urotergal glands from young males attracted only males. Overall, our results add knowledge to the mating system of B. oleae, giving first evidences on the electrophysiological activity of muscalure towards both sexes, as well as on the involvement of male urotergal glands in the chemical sexual communication of this pest.     

Stereochemistry affects sesquiterpene lactone bioactivity against an herbivorous grasshopper

Sesquiterpene lactones are defensive compounds which protect plants against a variety of herbivores and other natural enemies. Sesquiterpene lactones from higher plants can be divided into two groups based on the stereochemistry of their lactone ring junction, either cis-fused or trans-fused. It is unclear whether and how this variation affects potentially important ecological interactions. To investigate whether stereochemical variation in sesquiterpene lactone ring junctions can influence resistance to herbivorous insects, we performed controlled feeding trials with two pairs of diastereomeric sesquiterpene lactones and examined the deterrent effect of each compound on feeding by the polyphagous grasshopper Schistocerca americana (Drury). Sesquiterpene lactone stereochemistry and concentration significantly influenced feeding behavior with grasshoppers consuming less of the trans-fused compounds than the cis-fused compounds. To our knowledge, this is the first demonstration that sesquiterpene lactone ring junction stereochemistry influences the feeding behavior of herbivores. Because this stereochemical trait polymorphism is widely distributed in nature, it could have substantial consequences for the ecology and evolution of large groups of plants, particularly the Asteraceae.     

Ecophysiology of Limonium axillare and Avicennia marina from the coastline of Arabian Gulf-Qatar

Attention is being focused on the coastline from Doha to Ras Laffan in Qatar since higher activities in the development of land and establishment of roads, highways and new buildings and houses is not coupled by serious studies on habitat destruction, fragmentation or disturbances. Ecophysiological study was carried out to investigate the adaptation of two halophytes (Limonium axillare and Avicennia marina) in this area, with special emphasis on the ultrastructure of salt glands found in the leaves. Soils in these locations accumulated much Na⁺ and Cl^? as compared to other cations like K⁺, Ca²⁺ and Mg²⁺. Both plants accumulated higher concentrations of Na⁺ , Cl^?, and Ca²⁺ and lower concentrations of K⁺ and Mg²⁺. Organic compounds found in leaves of these plants under their natural habitats including proline, soluble sugars and nitrogen, and photosynthetic pigments were determined. Scanning electron micrographs of the surface of leaves showed that salt glands of these plants are well developed. It is urgently required that exact vegetation maps, and monitoring exercises will be conducted, in order to document exactly the state of the vegetation in Qatar. Only this will allow the environment authorities to bring forward suggestions for vegetation and ecosystem management to the decision makers.     

The ecological impact of invasive cane toads on tropical snakes: field data do not support laboratory-based predictions

Predicting which species will be affected by an invasive taxon is critical to developing conservation priorities, but this is a difficult task. A previous study on the impact of invasive cane toads (Bufo marinus) on Australian snakes attempted to predict vulnerability a priori based on the assumptions that any snake species that eats frogs, and is vulnerable to toad toxins, may be at risk from the toad invasion. We used time-series analyses to evaluate the accuracy of that prediction, based on >3600 standardized nocturnal surveys over a 138-month period on 12 species of snakes and lizards on a floodplain in the Australian wet-dry tropics, bracketing the arrival of cane toads at this site. Contrary to prediction, encounter rates with most species were unaffected by toad arrival, and some taxa predicted to be vulnerable to toads increased rather than declined (e.g., death adder Acanthophis praelongus; Children's python Antaresia childreni). Indirect positive effects of toad invasion (perhaps mediated by toad-induced mortality of predatory varanid lizards) and stochastic weather events outweighed effects of toad invasion for most snake species. Our study casts doubt on the ability of a priori desktop studies, or short-term field surveys, to predict or document the ecological impact of invasive species.