Dev-ResNet: automated developmental event detection using deep learning.

Delineating developmental events is central to experimental research using early life stages, permitting widespread identification of changes in event timing between species and environments. Yet, identifying developmental events is incredibly challenging, limiting the scale, reproducibility and throughput of using early life stages in experimental biology. We introduce Dev-ResNet, a small and efficient 3D convolutional neural network capable of detecting developmental events characterised by both spatial and temporal features, such as the onset of cardiac function and radula activity. We demonstrate the efficacy of Dev-ResNet using 10 diverse functional events throughout the embryonic development of the great pond snail, Lymnaea stagnalis. Dev-ResNet was highly effective in detecting the onset of all events, including the identification of thermally induced decoupling of event timings. Dev-ResNet has broad applicability given the ubiquity of bioimaging in developmental biology, and the transferability of deep learning, and so we provide comprehensive scripts and documentation for applying Dev-ResNet to different biological systems.

The Great Pond Snail (Lymnaea stagnalis) as a Model of Aging and Age-Related Memory Impairment: An Overview.

With the increase of life span, normal aging and age-related memory decline are affecting an increasing number of people; however, many aspects of these processes are still not fully understood. Although vertebrate models have provided considerable insights into the molecular and electrophysiological changes associated with brain aging, invertebrates, including the widely recognized molluscan model organism, the great pond snail (Lymnaea stagnalis), have proven to be extremely useful for studying mechanisms of aging at the level of identified individual neurons and well-defined circuits. Its numerically simpler nervous system, well-characterized life cycle, and relatively long life span make it an ideal organism to study age-related changes in the nervous system. Here, we provide an overview of age-related studies on L. stagnalis and showcase this species as a contemporary choice for modeling the molecular, cellular, circuit, and behavioral mechanisms of aging and age-related memory impairment.

Isotopic turnover rates and diet-tissue discrimination depend on feeding habits of freshwater snails.

Estimates of animal diets and trophic structure using stable isotope analysis are strongly affected by diet-tissue discrimination and tissue turnover rates, yet these factors are often unknown for consumers because they must be measured using controlled-feeding studies. Furthermore, these parameters may be influenced by diet quality, growth, and other factors. We measured the effect of dietary protein content on diet-tissue discrimination and tissue turnover in three freshwater snail species. We fed lettuce to individually housed snails (n = 450 per species) for ten weeks, then half were switched to a high-protein diet. Isotopic values of muscle and gonad tissue were assessed at 48 and 80 days post-diet change. Snail discrimination factors varied by diet (low-protein > high-protein) and usually differed among species for both N and C, although species had similar carbon discrimination when fed the low-protein diet. Carbon turnover rates were similar among species for a given tissue type, but nitrogen turnover varied more among species. In addition, diet affected growth of species differently; some species grew larger on high-protein (H. trivolvis) while others grew larger on low-protein diet (Lymnaea spp.). These differences among species in growth influenced turnover rates, which were faster in the species with the highest growth rate following the diet switch from low to high-protein. Thus, growth is one of the main processes that affects tissue turnover, but growth and feeding preference did not affect diet-tissue discrimination, which was greater on low-protein than high-protein diets for all species regardless of growth performance. These results suggest that diet might influence two key parameters of stable isotope analysis differently.

Parental diet affects embryogenesis of the great pond snail (Lymnaea stagnalis) exposed to cadmium, pyraclostrobin, and tributyltin.

Diet quality has a strong impact on life-history traits, but it is not usually considered as a factor in toxicity tests. The purpose of the present study was to evaluate how diets differing in nutritional content affect sensitivity to cadmium, pyraclostrobin, and tributyltin in Lymnaea stagnalis offspring. Three groups were fed a different diet each: lettuce, high-caloric pellets, or a combination of both. Snails fed pellets and both diets had similar growth; however, snails fed lettuce showed lower growth until the fourth month. Egg masses were collected from adult snails fed each diet and exposed to 3 concentrations of either cadmium, pyraclostrobin, or tributyltin. We quantified time to hatch, hatching success, and the developmental stages of embryos. We measured fecundity in adults and total lipids, carbohydrates, and proteins in adult snails and egg masses. Adult snails fed different diets produced a similar number of egg masses, but the number of eggs per egg mass in snails fed pellets was lower than for snails fed the other 2 diets. We found that adult snails fed pellets had a higher lipid content compared to snails fed the other 2 diets. However, egg masses from parental snails fed pellets did not hatch, including those from the controls. Interestingly, egg masses exposed to the lowest concentration of tributyltin had low hatching success. These observations on offspring performance suggest that there are important diet effects that can strongly influence responses that could be diet- and chemical-dependent. Environ Toxicol Chem 2018;37:2428-2438. © 2018 SETAC.

The effect of rearing environment on memory formation.

Lymnaea stagnalis is a well-studied model system for determining how changes in the environment influence associative learning and memory formation. For example, some wild strains of L. stagnalis, collected from separate geographic locations, show superior memory-forming abilities compared with others. Here, we studied memory formation in two laboratory-bred L. stagnalis strains, derived from the same original population in The Netherlands. The two strains were reared in two different laboratories at the University of Calgary (C-strain) and at Brock University (B-strain) for many years and we found that they differed in their memory-forming ability. Specifically, the C-strain required only two training sessions to form long-term memory (LTM) whereas the B-strain required four sessions to form LTM. Additionally, the LTM formed by the B-strain persisted for a shorter amount of time than the memory formed by the C-strain. Thus, despite being derived from the same original population, the C- and B-strains have developed different memory-forming abilities. Next, we raised the two strains from embryos away from home (i.e. in the other laboratory) over two generations and assessed their memory-forming abilities. The B-strain reared and maintained at the University of Calgary demonstrated improved memory-forming ability within a single generation, while the C-strain reared at Brock University retained their normal LTM-forming ability across two subsequent generations. This suggests that local environmental factors may contribute to the behavioural divergence observed between these two laboratory-bred strains.

Diet quality affects chemical tolerance in the freshwater snail Lymnaea stagnalis.

Organisms generally select high-quality diets to obtain maximal energy while devoting the least amount of time and energy. Diets, however, can vary in natural systems. In ecotoxicological testing, the effect of diet type on organismal responses to toxicants has not been explored despite the potential for dietary effects to influence toxicological endpoints. We first evaluated diet quality using growth rate and sensitivity to the fungicide pyraclostrobin of Lymnaea stagnalis fed lettuce (common laboratory diet), turtle pellets (high nutrient composition), and a combination diet of both food items. We also measured the macronutrient content of snails raised on the multiple diets to determine how diet may have impacted energy allocation patterns. Finally, we evaluated whether snails discernibly preferred a particular diet. Snails fed high-nutrient and combination diets grew larger overall than snails fed a lettuce-only diet. Snails fed the high-nutrient and combination diets, both juvenile and adult, were significantly more tolerant to pyraclostrobin than snails fed lettuce. When measured for macronutrient content, snails raised on high-nutrient and combination diets had significantly higher carbohydrate content than snails fed lettuce. Despite the strong effects of diet type, snails did not exhibit a clear diet choice in preference trials. Dietary composition clearly influences growth rate, sensitivity, and macronutrient content of Lymnaea stagnalis. These results suggest that the nutritional environment has potentially strong impacts on toxicant sensitivity. Environ Toxicol Chem 2018;37:1158-1167. © 2017 SETAC.

Transgenerational endpoints provide increased sensitivity and insight into multigenerational responses of Lymnaea stagnalis exposed to cadmium.

Ecotoxicology provides data to inform environmental management. Many testing protocols do not consider offspring fitness and toxicant sensitivity. Cadmium (Cd) is a well-studied and ubiquitous toxicant but little is known about the effects on offspring of exposed parents (transgenerational effects). This study had three objectives: to identify endpoints related to offspring performance; to determine whether parental effects would manifest as a change in Cd tolerance in offspring and how parental exposure duration influenced the manifestation of parental effects. Adult snails were exposed to Cd 0, 25, 50, 100, 200 and 400 µg Cd/L for eight weeks. There were effects on adult endpoints (e.g., growth, reproduction) but only at the highest concentrations (>100 µg/L). Alternatively, we observed significant transgenerational effects at all Cd concentrations. Surprisingly, we found increased Cd tolerance in hatchlings from all parental Cd exposure concentrations even though eggs and hatchlings were in Cd-free conditions for 6 weeks. Explicit consideration of offspring performance adds value to current toxicity testing protocols. Parental exposure duration has important implications for offspring effects and that contaminant concentrations that are not directly toxic to parents can cause transgenerational changes in resistance that have significant implications for toxicity testing and adaptive responses.

Embryonic exposure to model naphthenic acids delays growth and hatching in the pond snail Lymnaea stagnalis.

Naphthenic acids (NAs), a class of structurally diverse carboxylic acids with often complex ring structures and large aliphatic tail groups, are important by-products of many petrochemical processes including the oil sands mining activity of Northern Alberta. While it is evident that NAs have both acute and chronic harmful effects on many organisms, many aspects of their toxicity remain to be clarified. Particularly, while substantive data sets have been collected on NA toxicity in aquatic prokaryote and vertebrate model systems, to date, nothing is known about the toxic effects of these compounds on the embryonic development of aquatic invertebrate taxa, including freshwater mollusks. This study examines under laboratory conditions the toxicity of NAs extracted from oil sands process water (OSPW) and the low-molecular weight model NAs cyclohexylsuccinic acid (CHSA), cyclohexanebutyric acid (CHBA), and 4-tert-butylcyclohexane carboxylic acid (4-TBCA) on embryonic development of the snail Lymnaea stagnalis, a common freshwater gastropod with a broad Palearctic distribution. Evidence is provided for concentration-dependent teratogenic effects of both OSPW-derived and model NAs with remarkably similar nominal threshold concentrations between 15 and 20 mg/L and 28d EC50 of 31 mg/L. In addition, the data provide evidence for substantial toxicokinetic differences between CHSA, CHBA and 4-TBCA. Together, our study introduces Lymnaea stagnalis embryonic development as an effective model to assay NA-toxicity and identifies molecular architecture as a potentially important toxicokinetic parameter in the toxicity of low-molecular weight NA in embryonic development of aquatic gastropods.

Natural selection on immune defense: A field experiment.

Predicting the evolution of phenotypic traits requires an understanding of natural selection on them. Despite its indispensability in the fight against parasites, selection on host immune defense has remained understudied. Theory predicts immune traits to be under stabilizing selection due to associated trade-offs with other fitness-related traits. Empirical studies, however, report mainly positive directional selection. This discrepancy could be caused by low phenotypic variation in the examined individuals and/or variation in host resource level that confounds trade-offs in empirical studies. In a field experiment where we maintained Lymnaea stagnalis snails individually in cages in a lake, we investigated phenotypic selection on two immune defense traits, phenoloxidase (PO)-like activity and antibacterial activity, in hemolymph. We used a diverse laboratory population and manipulated snail resource level by limiting their food supply. For six weeks, we followed immune activity, growth, and two fitness components, survival and fecundity of snails. We found that PO-like activity and growth were under stabilizing selection, while antibacterial activity was under positive directional selection. Selection on immune traits was mainly driven by variation in survival. The form of selection on immune defense apparently depends on the particular trait, possibly due to its importance for countering the present parasite community.

Juveniles of Lymnaea 'smart' snails do not perseverate and have the capacity to form LTM.

Previously, it was concluded that the nervous systems of juvenile snails were not capable of mediating long-term memory (LTM). However, exposure and training of those juvenile snails in the presence of a predator cue significantly altered their ability to learn and form LTM. In addition, there are some strains of Lymnaea which have been identified as 'smart'. These snails form LTM significantly better than the lab-bred strain. Here, we show that juveniles of two smart snail strains not only are capable of associative learning but also have the capacity to form LTM following a single 0.5 h training session. We also show that freshly collected 'wild' 'average' juveniles are also not able to form LTM. Thus, the smart snail phenotype in these strains is expressed in juveniles.

Training Lymnaea in the presence of a predator scent results in a long-lasting ability to form enhanced long-term memory.

Lymnaea exposed to crayfish effluent (CE) gain an enhanced ability to form long-term memory (LTM). We test the hypothesis that a single CE exposure and operant conditioning training leads to long lasting changes in the capability of snails to form LTM when tested in pond water four weeks later. We trained both juvenile and adult snails with a single 0.5 h training session in CE and show that LTM was present 24 h later. Snails trained in a similar manner in just pond water show no LTM. We then asked if such training in CE conferred enhanced memory forming capabilities on these snails four weeks later. That is, would LTM be formed in these snails four weeks later following a single 0.5 h training session in pond water? We found that both adult and juvenile snails previously trained in CE one month previously had enhanced LTM formation abilities. The injection of a DNA methylation blocker, 5-AZA, prior to training in adult snails blocked enhanced LTM formation four weeks later. Finally, this enhanced LTM forming ability was not passed on to the next generation of snails.

How a single gene twists a snail.

The gastropod Lymnaea has unique features, that is, chirality, sinistrality, or dextrality, is displayed externally as well as internally, and is hereditary, being determined by a single-locus that functions maternally at the very early embryonic stage. Both sinistral and dextral snails exist in nature with the dextral one being dominant. Thus, the genus Lymnaea is an ideal target for studying chiromorphogenesis. This article gives a brief overview of the current state of research on chiromorphogenesis of Lymnaea (L.) stagnalis, mainly focusing on our own studies. Breeding experiments were performed and embryonic development was closely observed for the both chiralities. By fluorescently labeling filamentous actin and microtubules, cytoskeletal dynamics of spiral cleavages for the sinistral and dextral embryos were shown not to be mirror images of each other at the critical third-cleavage. The spiral deformation and spindle inclination were uniquely observed only in the dominant dextral embryos, and they were shown to be strongly linked to the gene determining the direction of chirality. Based on these findings, we created fertile snails of situs inversus by micromanipulation at the third-cleavage. Surprisingly, the arrangement of the blastomere regulates asymmetric expression of nodal-Pitx genes in later development. The expression patterns display interesting similarity and dissimilarity with those of the vertebrates. Thus, study of L. stagnalis has given an insight into "how a single gene twists a snail."

Effects of chronic waterborne nickel exposure on growth, ion homeostasis, acid-base balance, and nickel uptake in the freshwater pulmonate snail, Lymnaea stagnalis.

The freshwater pulmonate snail, Lymnaea stagnalis, is the most sensitive aquatic organism tested to date for Ni. We undertook a series of experiments to investigate the underlying mechanism(s) for this observed hypersensitivity. Consistent with previous experiments, juvenile snail growth in a 21-day exposure was reduced by 48% relative to the control when exposed to 1.3 µg l(-1) Ni (EC20 less than the lowest concentration tested). Ca(2+) homeostasis was significantly disrupted by Ni exposure as demonstrated by reductions in net Ca(2+) uptake, and reductions in Ca(2+) concentrations in the hemolymph and soft tissues. We also observed reduced soft tissue [Mg(2+)]. Snails underwent a significant alkalosis with hemolymph pH increasing from 8.1 to 8.3 and hemolymph TCO2 increasing from 19 to 22 mM in control versus Ni-exposed snails, respectively. Unlike in previous studies with Co and Pb, snail feeding rates were found to be unaffected by Ni at the end of the exposure. Snails accumulated Ni in the soft tissue in a concentration-dependent manner, and Ni uptake experiments with (63)Ni revealed a biphasic uptake profile - a saturable high affinity component at low exposure concentrations (36-189 nM) and a linear component at the high exposure concentrations (189-1,897 nM). The high affinity transport system had an apparent Km of 89 nM Ni(2+) and Vmax of 2.4 nmol g(-1)h(-1). This equates to a logK of 7.1, significantly higher than logK's (2.6-5.2) for any other aquatic organisms evaluated to date, which will have implications for Biotic Ligand Model development. Finally, pharmacological inhibitors that block Ca(2+) uptake pathways in snails did not inhibit Ni uptake, suggesting that the uptake of Ni does not occur via Ca(2+) uptake pathways. As with Cu and Pb, the exact mechanism for the significant disruption in Ca(2+) homeostasis and reduction in juvenile snail growth remains unknown.

[Endogenous biorhythms of the specific growth rate in individual development of Lymnaea stagnalis (Lymnaeidae, Gastropoda)].

On the basis of data on the growth of freshwater gastropods Lymnaea stagnalis, the dependence of the specific growth rate on age in the late postlarval ontogeny is calculated. The presence of two endogenous biorhythms of the specific growth rate with periods of 10.3 weeks and 6.5 weeks was revealed using singular spectrum analysis. Local maxima of both biorhythms in different individuals fall on the same age, and their periods are approximately the same in all animals studied and remain unchanged during individual postlarval development. The biorhythm with a period of 10.3 weeks is decaying, its amplitude is reduced from 3.2 year(-1) (at the age of 10 weeks) to 0.8 year(-1) (at the time of death of the shellfish). The biorhythm with a period of 6.5 weeks is non-decaying and has an average amplitude of 0.8 year(-1).

Growth inhibition in early life-stage tests predicts full life-cycle toxicity effects of lead in the freshwater pulmonate snail, Lymnaea stagnalis.

The freshwater pulmonate snail, Lymnaea stagnalis, is the most sensitive freshwater organism tested to date for several metals (Co, Cu, Pb, Ni) based on 28 d early life-stage (ELS) tests in which growth was the most sensitive endpoint. The United States Environmental Protection Agency (USEPA) has expressed concern that growth in 28 d ELS tests with mollusks may overpredict toxicity because of the potential for recovery in a full life-cycle (LC) test. Consequently, the USEPA only accepts the survival endpoint for these tests in establishing water quality criteria (WQC). To address this concern, the current study aimed to test the sensitivity of L. stagnalis to Pb in a 56 d full LC test evaluating survival, growth, reproductive and embryonic growth endpoints and compare the estimated effect levels to those established using the 28 d ELS test design. The most sensitive endpoints in this study were 28 d growth and 56 d egg mass production, both with a NOEC of <1.0 µg L(-1) and a LOEC of 1.0 µg L(-1), showing that the ELS growth endpoint is predictive of the 56 d reproduction endpoint. Snails exposed to 1.0 and 2.7 µg L(-1) Pb showed full and partial recovery from growth inhibition between 28 and 56 d. While this recovery supports the USEPA's concern about the 28 d growth endpoint; considering the reproductive lifespan of L. stagnalis and the recovery dose-response, we conclude that the 28 d growth endpoint will be within a factor of 3 of full LC endpoints. This is consistent with the level of precision previously determined for fish ELS tests, which the USEPA accepts for WQC derivation, and suggests that tests using 28 d ELS growth endpoint for L. stagnalis may be acceptable for inclusion in WQC derivation.

Neuronal control of pedal sole cilia in the pond snail Lymnaea stagnalis appressa.

5-HT (serotonin) is a ubiquitous neurotransmitter that produces ciliary beating in gastropods when applied topically, but ciliary beating caused by gastropod serotonergic neurons has been described in only three neuron pairs. We extend these results to the North American Lymnaea stagnalis appressa, which is a different species from the European Lymnaea stagnalis. We describe a non-serotonergic neuron pair, PeV1, which accelerates pedal sole mucociliary transport and a serotonergic neuron pair, PeD7, which slows mucociliary transport. We compare and discuss development and identified neurons in L. s. appressa and in L. stagnalis, which have homologs to L. s. appressa PeD7 and PeV1 neurons. In addition to PeD7 and PeV1 neurons, we test neurons immunoreactive to Tritonia pedal peptide antibodies with negative results for mucociliary transport. In characterizing PeD7 and PeV1 neurons, we find that PeV1 does not excite PeD7. In semi-intact preparations, a strong increase in PeD7 neuron activity occurs during tactile stimulation, but V1 neurons are inhibited during tactile stimulation. Following tactile stimulation, PeV1 neurons show strong activity. This suggests a distinct difference in function of the two neuron pairs, which both have their axons overlying pedal sole ciliary cells. Application of 5-HT to the pedal sole initiates mucociliary transport in 1.4-1.9 s with a time course similar to that seen when stimulating a PeV1 neuron. This result appears to be through a 5-HT(1A)-like receptor on the pedal sole. We describe a possible external source of 5-HT on the pedal sole from 5-HT immunoreactive granules that are released with mucus.

Organization of the serotonergic innervation of the feeding (buccal) musculature during the maturation of the pond snail Lymnaea stagnalis: a morphological and biochemical study.

The serotonergic innervation of the buccal musculature responsible for feeding (radula protraction) was investigated during the maturation of the pond snail, Lymnaea stagnalis L., applying light and electron microscopic immunohistochemistry and biochemical approaches. According to epifluorescence and laser confocal microscopy, the first 5-HT-like-immunoreactive (5-HTLIR) processes appeared on the surface of the musculature at the postmetamorphic E80% embryonic stage. Until hatching, the innervation continued to increase in density, showing axon arborizations with projections into the deeper muscle levels. An adult-like pattern of 5-HTLIR innervation appeared at P2-P3 juvenile stages. At the ultrastructural level, close (16-20 nm) but mostly unspecialized neuromuscular contacts were formed by both unlabeled and 5-HTLIR axon profiles from the E80% embryonic stage. Labeled processes were also found located relatively far from the muscle cells. An HPLC assay showed a gradual increase of the 5-HT level in the buccal mass during development. The buccal mass was characterized by a single-component high-affinity 5-HT uptake system, and 5-HT release could be evoked by 100 mM K(+) and blocked in Ca(2+) -free medium. It is suggested that 5-HT plays a wide modulatory role in the peripheral feeding system and is also involved in the functional maturation of the muscle system.

Multi-linear regression analysis, preliminary biotic ligand modeling, and cross species comparison of the effects of water chemistry on chronic lead toxicity in invertebrates.

The current study examined the chronic toxicity of lead (Pb) to three invertebrate species: the cladoceran Ceriodaphnia dubia, the snail Lymnaea stagnalis and the rotifer Philodina rapida. The test media consisted of natural waters from across North America, varying in pertinent water chemistry parameters including dissolved organic carbon (DOC), calcium, pH and total CO(2). Chronic toxicity was assessed using reproductive endpoints for C. dubia and P. rapida while growth was assessed for L. stagnalis, with chronic toxicity varying markedly according to water chemistry. A multi-linear regression (MLR) approach was used to identify the relative importance of individual water chemistry components in predicting chronic Pb toxicity for each species. DOC was an integral component of MLR models for C. dubia and L. stagnalis, but surprisingly had no predictive impact on chronic Pb toxicity for P. rapida. Furthermore, sodium and total CO(2) were also identified as important factors affecting C. dubia toxicity; no other factors were predictive for L. stagnalis. The Pb toxicity of P. rapida was predicted by calcium and pH. The predictive power of the C. dubia and L. stagnalis MLR models was generally similar to that of the current C. dubia BLM, with R(2) values of 0.55 and 0.82 for the respective MLR models, compared to 0.45 and 0.79 for the respective BLMs. In contrast the BLM poorly predicted P. rapida toxicity (R(2)=0.19), as compared to the MLR (R(2)=0.92). The cross species variability in the effects of water chemistry, especially with respect to rotifers, suggests that cross species modeling of invertebrate chronic Pb toxicity using a C. dubia model may not always be appropriate.

Mechanisms of waterborne Cu toxicity to the pond snail Lymnaea stagnalis: physiology and Cu bioavailability.

We examined the mechanisms of toxicity of waterborne Cu to the freshwater pond snail Lymnaea stagnalis. The snail is one of the most sensitive species to acute Cu exposure (96 h LC(50), LC(20): 24.9, 18.0 µgl(-1)); they are not protected by the water quality criteria of the US EPA. Tissue Na and Ca were also reduced by Cu in the acute exposure. In contrast, during 28 d chronic exposures to Cu in the presence of food, which resulted in higher DOC concentrations, there was no significant mortality but an inhibition of growth, which may reflect a re-allocation of resources to detoxification. Cu detoxification was evidenced in chronic exposure by increases in metallothionein-like protein concentrations and Cu binding to metal-rich granules, decreases in thiobarbituric acid-reactive substances, and changes in the subcellular distribution in the soft tissues. Our results demonstrated that apart from external Cu bioavailability, compartmentalization of metals within the cells can alter toxicity of Cu to the snails.