Does Bti (Bacillus thuringiensis var. israelensis) affect Rana temporaria tadpoles?

Biocides based on toxins of Bacillus thuringiensis var. israelensis (Bti) are established as alternatives to conventional chemical insecticides for mosquito control all across the globe since they are regarded ecologically compatible and harmless to non-target species. Since recent studies on amphibian larvae have called this opinion into question, we exposed Rana temporaria tadpoles to single (1 mg/L), tenfold (10 mg/L) and hundredfold (100 mg/L) field concentrations of VectoBac® WG (a water dispersible granule Bti formulation) in the laboratory for eleven days to investigate whether larvae were adversely affected by Bti and its endotoxin proteins. In addition to a negative (water) control, a positive control based on organic rice protein (50 mg/L) was run to check for the nutritional relevance of Bti proteins. There was no Bti-related mortality and a histopathological analysis of tadpole intestines revealed no adverse effects. Analyses of biomarkers for proteotoxicity (stress protein family, Hsp70) and neurotoxicity or metabolic action (b-esterases acetylcholine esterase (AChE) and carboxylesterases) revealed no significant differences between Bti treatments and the negative control. The responses of tadpoles in the protein-supplemented positive control differed from those of the negative control and the Bti treatments. Tadpoles in the positive control had reduced body mass and elevated AChE activity.

Relevance of body size and shell colouration for thermal absorption and heat loss in white garden snails, Theba pisana (Helicidae), from Northern France.

The internal temperature of land snails depends on environmental factors, such as exposure to electromagnetic radiation and airflow as well as biotic factors including shell size, shell colouration and thickness or the resting position of the snail. In controlled field experiments, we quantified heating by thermal absorption of light and airflow-induced heat loss in the white garden snail, Theba pisana, from Normandy, France. Heating experiments revealed a significant positive relation of the internal body temperature with illumination period, shell temperature and air temperature at different times of day. The size of the snails was negatively related with both of the given illumination times: smaller animals heated up stronger than larger ones. The temperature at the surface of the shell significantly depended on the illumination period and the time of day. An AIC-based quality assessment of multiple linear modelling showed that, for explaining both shell surface and internal temperature of the soft body, several factors, i.e., exposure time, daytime, shell size and colouration contributed to the best models, respectively. Similarly, heat loss of the soft body after and during exposure of the snails to sunlight by a constant airflow depended on the initial body temperature, shell size, colouration and ambient air temperature. Our study revealed also the importance of both shell size and colouration for the loss of body temperature under natural conditions: small and banded animals that had heated up to temperatures above 30°C cooled down faster than large and un-banded ones.

Drifting Away of a FET Endpoint: The Heart Rate in Danio rerio Embryos is Extremely Sensitive to Variation in Ambient Temperature.

Beyond the mere detection of presence or absence of heart beat in zebrafish (Danio rerio) embryos in a fish embryo test conducted referring to the OECD TG 236 at 48 hpf (hours post fertilization) onwards, embryo heart rate may serve as an additional and very sensitive endpoint in ecotoxicological studies. But by including heart rate as a sublethal endpoint, care has to be taken of separating effects exerted by a tested compound from those exerted by temperature. Therefore, profound knowledge on the natural variation of zebrafish heart rates at defined temperatures as a basis for the assessment of gained results is mandatorily needed. As such continuous information in D. rerio is lacking from the literature, we designed a study covering a span of 12°C (from 18 to 30°C in steps of 2°C) to quantify the relationship between heart rate and temperature in D. rerio embryos 48 hpf. Conducting a multiple regression analysis, we found a considerably strong relationship between treatment temperature and the log10 of the heart rate, ranging from 82.8 beats per minute at 18°C to 218.0 beats per minute at 30°C. Our results therefore may serve as a reference for heart rates measured under normal conditions to be able to detect potential effects of contaminants in other studies when working under certain temperatures.

Reduction of dioxin-like toxicity in effluents by additional wastewater treatment and related effects in fish.

Efficiency of advanced wastewater treatment technologies to reduce micropollutants which mediate dioxin-like toxicity was investigated. Technologies compared included ozonation, powdered activated carbon and granular activated carbon. In addition to chemical analyses in samples of effluents, surface waters, sediments, and fish, (1) dioxin-like potentials were measured in paired samples of effluents, surface waters, and sediments by use of an in vitro biotest (reporter gene assay) and (2) dioxin-like effects were investigated in exposed fish by use of in vivo activity of the mixed-function, monooxygenase enzyme, ethoxyresorufin O-deethylase (EROD) in liver. All advanced technologies studied, based on degradation or adsorption, significantly reduced dioxin-like potentials in samples and resulted in lesser EROD activity in livers of fish. Results of in vitro and in vivo biological responses were not clearly related to quantification of targeted analytes by use of instrumental analyses.

A sublethal imidacloprid concentration alters foraging and competition behaviour of ants.

Neonicotinoid pesticides, such as the widely used compound imidacloprid, are suspected to impair cognitive capacity, behaviour, and fitness of a number of non-target species. We tested whether sublethal imidacloprid concentrations alter the foraging and aggression behaviour of two European ant species. Even though the nestmate-recruitment of Lasius niger was not affected by pesticide exposure, these ants required more time to become active and the number of foraging workers was lower than in sub-colonies not exposed to imidacloprid. In interspecific confrontations, imidacloprid increased the aggressiveness of a usually subordinate species (Lasius flavus) enormously (3.7-fold increase in average number of aggressive encounters), whereas they did not affect a subdominant species (L. niger) that severely (1.2-fold increase in average number of aggressive encounters). The high frequency of aggressive encounters of L. flavus vs. non-exposed L. niger workers, reduced their survival probability significantly down to 60 %. The observed behavioural alterations of the two ant species have the potential to impair their viability and co-occurrence with behaviourally dominate species due to a decreased exploitative competition and a reduced chance to locate and use resources before competitors. As competition is considered key in structuring ant communities, changes in aggressiveness are likely to alter established dominance hierarchies and thereby the dynamic and structure of ant communities.

PIXE-electrophoresis shows starving collembolan reallocates protein-bound metals.

One of multiple functions of metalloproteins is to provide detoxification to excess metal levels in organisms. Here we address the induction and persistence of a range of low to high molecular weight copper- and zinc binding proteins in the collembolan species Tetrodontophora bielanensis exposed to copper- and zinc-enriched food, followed by a period of recovery from metal exposure, in absence and presence of food. After 10 days of feeding copper and zinc contaminated yeast, specimens were either moved to ample of leaf litter material from their woodland stand of origin or starved (no food offered). The molecular weight distribution of metal binding proteins was determined by native polyacryl gel electrophoresis. One gel was stained with Comassie brilliant blue and a duplicate gel dried and scanned for the amount of copper and zinc by particle-induced X-ray emission. Specimens exposed to copper and recovered from it with ample of food had copper bound to two groups of rather low molecular weight proteins (40-50 kDa) and two of intermediate size (70-80 kDa). Most zinc in specimens from the woodland stand was bound to two large proteins of about 104 and 106 kDa. The same proteins were holding some zinc in metal-exposed specimens, but most zinc was found in proteins <40 kDa in size. Specimens recovered from metal exposure in presence of ample of food had the same distribution pattern of zinc binding proteins, whereas starved specimens had zinc as well as copper mainly bound to two proteins of 8 and 10 kDa in size. Thus, the induction and distribution of copper- and zinc-binding proteins depend on exposure conditions, and the presence of low molecular weight binding proteins, characteristic of metallothioneins, was mainly limited to starving conditions.

Antioxidant defence and stress protein induction following heat stress in the Mediterranean snail Xeropicta derbentina.

The Mediterranean snail Xeropicta derbentina (Pulmonata, Hygromiidae), being highly abundant in Southern France, has the need for efficient physiological adaptations to desiccation and over-heating posed by dry and hot environmental conditions. As a consequence of heat, oxidative stress manifests in these organisms, which, in turn, leads to the formation of reactive oxygen species (ROS). In this study, we focused on adaptations at the biochemical level by investigation of antioxidant defences and heat shock protein 70 (Hsp70) induction, both essential mechanisms of the heat stress response. We exposed snails to elevated temperature (25, 38, 40, 43 and 45°C) in the laboratory and measured the activity of the antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPx), determined the Hsp70 level and quantified lipid peroxidation. In general, we found a high constitutive level of CAT activity in all treatments, which may be interpreted as a permanent protection against ROS, i.e. hydrogen peroxide. CAT and GPx showed temperature-dependent activity: CAT activity was significantly increased in response to high temperatures (43 and 45°C), whereas GPx exhibited a significantly increased activity at 40°C, probably in response to high levels of lipid peroxides that occurred in the 38°C treatment. Hsp70 showed a maximum induction at 40°C, followed by a decrease at higher temperatures. Our results reveal that X. derbentina possesses a set of efficient mechanisms to cope with the damaging effects of heat. Furthermore, we demonstrated that, besides the well-documented Hsp70 stress response, antioxidant defence plays a crucial role in the snails' competence to survive extreme temperatures.

Shell colouration and parasite tolerance in two helicoid snail species.

The polymorphism of shell colouration in helicoid snails is a well-known phenomenon attributed to different factors such as predation and climatic effects. Another aspect contributing to this polymorphism could be the interplay of melanin production and phenoloxidase-related immunity. Therefore, in this study we aimed at answering the questions whether there is a differential sensitivity of different snail shell colour morphs to nematode infection, and whether this can be related to differences in phenoloxidase (PO) activity levels using the two helicoid, polymorphic snail species Cepaea hortensis and Cernuella virgata. Snails of both species were artificially infected with the parasitic nematode Phasmarhabditis hermaphrodita, and analysed for mortality and PO activity levels. We found C. virgata to be more severely affected by P. hermaphrodita infection than C. hortensis, and the dark C. virgata morphs to be more resistant to lethal effects of this infection than pale morphs. However, these differences in sensitivity to the parasite could not clearly be related to different PO activity levels.

Invertebrates as indicators for chemical stress in sewage-influenced stream systems: toxic and endocrine effects in gammarids and reactions at the community level in two tributaries of Lake Constance, Schussen and Argen.

The present study investigates the impact of releases from waste water treatment plants and storm water overflow basins on gammarids and other macrozoobenthos. The study relates to a recent upgrading of a waste water treatment plant (Langwiese) at the Schussen river, an important tributary to Lake Constance. Samples were taken at different sites at the Schussen river upstream and downstream of a storm water overflow basin and the waste water treatment plant Langwiese and, in parallel, at the Argen river, a less polluted reference stream. We assessed the influence of water quality on the distribution of macrozoobenthos and on the health of gammarid populations by a variety of ecotoxicological methods including biomarkers prior to the expansion of the waste water treatment plant. Through histopathological studies, the impact of parasites on host tissue health was evaluated. Analyses of heat shock protein (hsp70) levels allowed us to draw conclusions about the proteotoxicity-related stress status of the organisms. Furthermore, gammarid populations from all sites were investigated in respect to sex ratio, parasitism rate, and fecundity. Macrozoobenthos community integrity was determined by means of the saprobic index and the abundance as well as by the number of taxa. In gammarids, the sex ratio was significantly shifted towards females, fecundity was significantly decreased, and the hsp70 level was significantly increased downstream of the waste water treatment plant Langwiese, compared to the upstream sampling site. Similarly, these effects could be detected downstream of three small storm water overflow basins. In the macrozoobenthos communities, the abundance of taxa, the number of taxa, the number of ephemeroptera, plecoptera, and trichoptera taxa (EPT-taxa), and the number of sensitive taxa decreased downstream of the storm water overflow basin Mariatal as well as downstream of the waste water treatment plant Langwiese. Our study showed, that waste water treatment plants and storm water overflow basins affected macroinvertebrate communities and the health of gammarids.

Thermal Impact and the Relevance of Body Size and Activity on the Oxygen Consumption of a Terrestrial Snail, Theba pisana (Helicidae) at High Ambient Temperatures.

Metabolism, mainly driven by oxygen consumption, plays a key role in life, as it is one of the main ways to respond to extreme temperatures through internal processes. Theba pisana, a widespread Mediterranean land snail, is exposed to a wide range of ambient temperature. In this species the oxygen consumption was tested as a response variable by multiple regression modelling on the "explanatory" variables shell-free mass, temperature, and relative humidity. Our results show that the oxygen consumption of T. pisana can be well described (73.1%) by these three parameters. In the temperature range from 23 °C to 35 °C the oxygen consumption decreased with increasing temperature. Relative humidity, in the range of 67% to 100%, had the opposite effect: if it increases, oxygen consumption will increase as well. Metabolism is proportional to an individual's mass to the power of the allometric scaling exponent α, which is between 0.62 and 0.77 in the mentioned temperature range. CT scans of shells and gravimetry revealed the shell-free mass to be calculated by multiplying the shell diameter to the third power by 0.2105. Data were compared to metabolic scaling exponents for other snails reported in the literature.

External and internal shell formation in the ramshorn snail Marisa cornuarietis are extremes in a continuum of gradual variation in development.

BACKGROUND: Toxic substances like heavy metals can inhibit and disrupt the normal embryonic development of organisms. Exposure to platinum during embryogenesis has been shown to lead to a "one fell swoop" internalization of the shell in the ramshorn snail Marisa cornuarietis, an event which has been discussed to be possibly indicative of processes in evolution which may result in dramatic changes in body plans. RESULTS: Whereas at usual cultivation temperature, 26°C, platinum inhibits the growth of both shell gland and mantle edge during embryogenesis leading to an internalization of the mantle and, thus, also of the shell, higher temperatures induce a re-start of the differential growth of the mantle edge and the shell gland after a period of inactivity. Here, developing embryos exhibit a broad spectrum of shell forms: in some individuals only the ventral part of the visceral sac is covered while others develop almost "normal" shells. Histological studies and scanning electron microscopy images revealed platinum to inhibit the differential growth of the shell gland and the mantle edge, and elevated temperature (28 - 30°C) to mitigate this platinum effect with varying efficiency. CONCLUSION: We could show that the formation of internal, external, and intermediate shells is realized within the continuum of a developmental gradient defined by the degree of differential growth of the embryonic mantle edge and shell gland. The artificially induced internal and intermediate shells are first external and then partly internalized, similar to internal shells found in other molluscan groups.

LogD-based modelling and ΔlogD as a proxy for pH-dependent action of ionizable chemicals reveal the relevance of both neutral and ionic species for fish embryotoxicity and possess great potential for practical application in the regulation of chemicals.

Depending on the ambient pH, ionizable substances are present in varying proportions in their neutral or charged form. The extent to which these two chemical species contribute to the pH-dependant toxicity of ionizable chemicals and whether intracellular ion trapping has a decisive influence in this context is controversially discussed. Against this background, we determined the acute toxicity of 24 ionizable substances at up to 4 different pH values on the embryonic development of the zebrafish, Danio rerio, and supplemented this dataset with additional data from the literature. The LC50 for some substances (diclofenac, propranolol, fluoxetine) differed by a factor of even >103 between pH5 and pH9. To simulate the toxicity of 12 acids and 12 bases, six models to calculate a pH-dependant logD value as a proxy for the uptake of potentially toxic molecules were created based on different premises for the trans-membrane passage and toxic action of neutral and ionic species, and their abilities to explain the real LC50 data set were assessed. Using this approach, we were able to show that both neutral and charged species are almost certainly taken up into cells according to their logD-based distribution, and that both species exert toxicity. Since two of the models that assume all intracellular molecules to be neutral overestimated the real toxicity, it must be concluded, that the toxic effect of a single charged intracellularly present molecule is, on the average, lower than that of a single neutral molecule. Furthermore, it was possible to attribute differences in toxicity at different pH values for these 24 ionizable substances to the respective deltas in logD at these pH levels with high accuracy, enabling particularly a full logD-based model on the basis of logPow as a membrane passage descriptor to be used for predicting potential toxicities in worst-case scenarios from existing experimental studies, as stipulated in the process of registration of chemicals and the definition of Environmental Quality Standards (EQS).

Thermobiological effects of temperature-induced color variations in Aglais urticae (Lepidoptera, Nymphalidae).

Coloration of animals is important for camouflage, for social behavior, or for physiological fitness. This study investigates the color variation in adults of Aglais urticae obtained on subjecting some pre-imaginal stages to different temperature conditions and their thermobiological consequences. To investigate the evolutionary-ecological interactions of temperature and pigmentation in butterflies, caterpillars, and pupae of the small tortoiseshell, Aglais urticae (Lepidoptera, Nymphalidae), larvae from Central Europe and Scandinavia were reared at temperatures between 7 and 34°C in the laboratory or in the field. After emergence, the intensity of pigmentation of the imagines and their increase in body temperature under defined full-spectrum light irradiation were quantified by image analysis and thermal imaging. At constant conditions, ambient rearing temperature and pigmentation intensity of imagines were negatively and linearly correlated in Central European butterflies, regardless of whether the pupal stage alone or, additionally, the last period of the larval stage was exposed to these conditions: low temperatures induced darker coloration and high temperatures led to lighter individuals. A thermal pulse of a few days alone at the beginning of pupal dormancy led to a similar, albeit weakened, effect. Caterpillars of the Scandinavian subspecies A. urticae polaris, whose pupal dormancy took place under Central European field conditions, developed into strongly pigmented imagines. The thermobiological relevance of more intense pigmentation was shown by significantly higher absorption of light, and thus stronger increased body temperature after 5 min of defined illumination, but this difference ceased after 15 min. Our results show that phenotypic plasticity in wing coloration is adaptive since temperature-induced developmental changes provide thermobiological benefit in adult butterflies. We propose that, in subpolar latitudes, darker coloration likely has a selection advantage favoring individuals with reaction norms gradually shifted to stronger pigmented phenotypes, possibly leading to the establishment of a pigmentation cline.

Effects of the Antidepressant Amitriptyline on Juvenile Brown Trout and Their Modulation by Microplastics.

Pharmaceuticals such as antidepressants are designed to be bioactive at low concentrations. According to their mode of action, they can also influence non-target organisms due to the phylogenetic conservation of molecular targets. In addition to the pollution by environmental chemicals, the topic of microplastics (MP) in the aquatic environment came into the focus of scientific and public interest. The aim of the present study was to investigate the influence of the antidepressant amitriptyline in the presence and absence of irregularly shaped polystyrene MP as well as the effects of MP alone on juvenile brown trout (Salmo trutta f. fario). Fish were exposed to different concentrations of amitriptyline (nominal concentrations between 1 and 1000 µg/L) and two concentrations of MP (104 and 105 particles/L; <50 µm) for three weeks. Tissue cortisol concentration, oxidative stress, and the activity of two carboxylesterases and of acetylcholinesterase were assessed. Furthermore, the swimming behavior was analyzed in situations with different stress levels. Exposure to amitriptyline altered the behavior and increased the activity of acetylcholinesterase. Moreover, nominal amitriptyline concentrations above 300 µg/L caused severe acute adverse effects in fish. MP alone did not affect any of the investigated endpoints. Co-exposure caused largely similar effects such as the exposure to solely amitriptyline. However, the effect of amitriptyline on the swimming behavior during the experiment was alleviated by the higher MP concentration.

Heart rate as an early warning parameter and proxy for subsequent mortality in Danio rerio embryos exposed to ionisable substances.

Environmental risk assessments of organic chemicals usually do not consider pH as a key factor. Hence, most substances are tested at a single pH only, which may underestimate the toxicity of ionisable substances with a pKa in the range of 4-10. Thus, the ability to consider the pH-dependent toxicity would be crucial for a more realistic assessment. Moreover, there is a tendency in acute toxicity tests to focus on mortality only, while little attention is paid to sublethal endpoints. We used Danio rerio embryos exposed to ten ionisable substances (the acids diclofenac, ibuprofen, naproxen and triclosan and the bases citalopram, fluoxetine, metoprolol, propranolol, tramadol and tetracaine) at four external pH levels, investigating the endpoints mortality (LC50) and heart rate (EC20). Dose-response curves were fitted with an ensemble-model to determine the true uncertainty and variation around the mean endpoints. The ensemble considers eight (heart rate) or twelve (mortality) individual models for binominal and Poisson distributed data, respectively, selected based on the Akaike Information Criterion (AIC). In case of equally good models, the mean endpoint of all models in the ensemble was calculated, resulting in more robust ECx estimates with lower 'standard errors' as compared to randomly selected individual models. We detected a high correlation between mortality (LC50) at 96 hpf and reduced heart rate (EC20) at 48 hpf for all compounds and all external pH levels (r = 0.98). Moreover, the observed pH-dependent effects were strongly associated with log D and thus, likely driven by differences in uptake (toxicokinetic) rather than internal (toxicodynamic) processes. Prospectively, the a priori consideration of pH-dependent effects of ionisable substances might make testing at different pH levels redundant, while the endpoint of mortality might even be replaced by a reliable sublethal proxy that would reduce the exposure, accelerating the evaluation process.

Modeling the oxygen uptake, transport and consumption in an estivating terrestrial snail, Xeropicta derbentina, by the Colburn analogy.

The present work gives insight into the internal heat management of the respiratory system in the terrestrial snail Xeropicta derbentina, which has to cope with extreme climate conditions in its habitat. A realistic model of the lung´s vein system was constructed and the active diffusive surface of capillaries and main vein was calculated and confirmed by geometrical measurements. We here present a model that is able to validate the measured oxygen consumption by the use of the Colburn analogy between mass and momentum transfer. By combining basic diffusion laws with the momentum transfer, i.e. wall shear stress, at the inner wall of the lung capillaries and the main vein, the progression of the oxygen mass fraction in the hemolymph can be visualized.

Growth-Promoting Gold Nanoparticles Decrease Stress Responses in Arabidopsis Seedlings.

The global economic success of man-made nanoscale materials has led to a higher production rate and diversification of emission sources in the environment. For these reasons, novel nanosafety approaches to assess the environmental impact of engineered nanomaterials are required. While studying the potential toxicity of metal nanoparticles (NPs), we realized that gold nanoparticles (AuNPs) have a growth-promoting rather than a stress-inducing effect. In this study we established stable short- and long-term exposition systems for testing plant responses to NPs. Exposure of plants to moderate concentrations of AuNPs resulted in enhanced growth of the plants with longer primary roots, more and longer lateral roots and increased rosette diameter, and reduced oxidative stress responses elicited by the immune-stimulatory PAMP flg22. Our data did not reveal any detrimental effects of AuNPs on plants but clearly showed positive effects on growth, presumably by their protective influence on oxidative stress responses. Differential transcriptomics and proteomics analyses revealed that oxidative stress responses are downregulated whereas growth-promoting genes/proteins are upregulated. These omics datasets after AuNP exposure can now be exploited to study the underlying molecular mechanisms of AuNP-induced growth-promotion.

Experimental simulation of environmental warming selects against pigmented morphs of land snails.

In terrestrial snails, thermal selection acts on shell coloration. However, the biological relevance of small differences in the intensity of shell pigmentation and the associated thermodynamic, physiological, and evolutionary consequences for snail diversity within the course of environmental warming are still insufficiently understood. To relate temperature-driven internal heating, protein and membrane integrity impairment, escape behavior, place of residence selection, water loss, and mortality, we used experimentally warmed open-top chambers and field observations with a total of >11,000 naturally or experimentally colored individuals of the highly polymorphic species Theba pisana (O.F. MÜller, 1774). We show that solar radiation in their natural Mediterranean habitat in Southern France poses intensifying thermal stress on increasingly pigmented snails that cannot be compensated for by behavioral responses. Individuals of all morphs acted neither jointly nor actively competed in climbing behavior, but acted similarly regardless of neighbor pigmentation intensity. Consequently, dark morphs progressively suffered from high internal temperatures, oxidative stress, and a breakdown of the chaperone system. Concomitant with increasing water loss, mortality increased with more intense pigmentation under simulated global warming conditions. In parallel with an increase in mean ambient temperature of 1.34°C over the past 30 years, the mortality rate of pigmented individuals in the field is, currently, about 50% higher than that of white morphs. A further increase of 1.12°C, as experimentally simulated in our study, would elevate this rate by another 26%. For 34 T. pisana populations from locations that are up to 2.7°C warmer than our experimental site, we show that both the frequency of pigmented morphs and overall pigmentation intensity decrease with an increase in average summer temperatures. We therefore predict a continuing strong decline in the frequency of pigmented morphs and a decrease in overall pigmentation intensity with ongoing global change in areas with strong solar radiation.

Turning snails into slugs: induced body plan changes and formation of an internal shell.

The archetypal body plan of conchiferan molluscs is characterized by an external calcareous shell, though internalization of shells has evolved independently in a number of molluscan clades, including gastropod families. In gastropods, the developmental process of torsion is regarded as a hallmark that is associated with a new anatomical configuration. This configuration is present in extant prosobranch gastropod species, which predominantly bear external shells. Here, we show that short-term exposure to platinum during development uncouples at least two of the processes associated with torsion of the freshwater snail Marisa cornuarietis. That is, the anus of the treated snails is located anteriorly, but the gill and the designated mantle tissue remains in a posterior location, thus preventing the formation of an external shell. In contrast to the prosobranchian archetype, platinum treatment results in the formation of a posterior gill and a cone-shaped internal shell, which persists across the lifetime. This first finding of artificially induced snail-slug conversion was also seen in the pulmonate snail Planorbarius corneus and demonstrates that selective alteration of embryonic key processes can result in fundamental changes of an existing body plan and-if altered regulation is inherited-may give rise to a new one.