Reproductive performance of freshwater snail, Helisoma duryi under the effect of bulk and nano zinc oxide.

Nanotechnology has been used to apply nanoparticle essential elements to enhance the ability of animals to absorb these elements and consequently improve their reproductive performance. High concentrations of nanoparticles (NPs) can directly harm a range of aquatic life forms, ultimately contributing to a decline in biodiversity. Helisoma duryi snails are a good model for studying the toxicological effects of bulk zinc oxide (ZnO-BPs) and nano zinc oxide (ZnO-NPs) on freshwater gastropods. This study aimed to compare the toxic effects of ZnO-BPs and ZnO-NPs on H. duryi snails and explore how waterborne and dietary exposure influenced the reproductive performance of this snail. ZnO-BPs and ZnO-NPs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray powder (XRD). This study revealed that the size of ZnO-BPs and ZnO-NPs were 154 nm and 11-31 nm, respectively. The results showed that exposure of adult snails to sub-lethal concentrations of both ZnO forms (bulk and nano) for 24 h/week for 4 weeks markedly changed their reproductive performance in a concentration-dependent manner, where fecundity was negatively affected by high concentrations. It was concluded that dietary exposure to the lowest tested concentration of ZnO-NPs (1 ppm) has a positive effect as the number of eggs and egg masses/snails increased and the incubation period decreased. Also, poly-vitelline eggs (The formation of twins) were observed. ZnO-NPs at low concentrations positively affect the reproductive performance of snails, especially after dietary exposure. The results revealed that 1 ppm ZnO-NPs could be supplementary provided to snails to improve their fertility, reduce the developmental time course, increase hatchability percentage, and produce poly-vitelline eggs.

The opposing roles of lethal and nonlethal effects of parasites on host resource consumption.

Although parasites can kill their hosts, they also commonly cause nonlethal effects on their hosts, such as altered behaviors or feeding rates. Both the lethal and nonlethal effects of parasites can influence host resource consumption. However, few studies have explicitly examined the joint lethal and nonlethal effects of parasites to understand the net impacts of parasitism on host resource consumption. To do this, we adapted equations used in the indirect effects literature to quantify how parasites jointly influence basal resource consumption through nonlethal effects (altered host feeding rate) and lethal effects (increased host mortality). To parametrize these equations and to examine the potential temperature sensitivity of parasite influences, we conducted a fully factorial lab experiment (crossing trematode infection status and a range of temperatures) to quantify feeding rates and survivorship curves of snail hosts. We found that infected snails had significantly higher mortality and ate nearly twice as much as uninfected snails and had significantly higher mortality, resulting in negative lethal effects and positive nonlethal effects of trematodes on host resource consumption. The net effects of parasites on resource consumption were overall positive in this system, but did vary with temperature and experimental duration, highlighting the context dependency of outcomes for the host and ecosystem. Our work demonstrates the importance of jointly investigating lethal and nonlethal effects of parasites and provides a novel framework for doing so.

The Tails of Two Avian Schistosomes: Paired Exposure Study Demonstrates Trichobilharzia stagnicolae Penetrates Human Skin More Readily than a Novel Avian Schistosome from Planorbella.

A novel schistosome from Planorbella snails currently known as avian schistosomatid sp. C (ASC) was recently described as being capable of causing the papules associated with swimmer's itch. We conducted a paired study with 24 human volunteers, exposing each of their forearms to five drops of water containing cercariae of ASC or Trichobilharzia stagnicolae, and examined the skin for papules 1-3 days later. A mixed effects model showed that only the parasite species significantly affected the number of papules, while prior experimental exposure, swimming history, and swimmer's itch experience did not. The total number of papules produced by the two species were very different: ASC produced a total of 2 papules from the 298 cercariae used, compared to 49 papules from 160 T. stagnicolae cercariae, a difference factor of more than 43X, which was comparable to the odds ratio of 45.5 computed using the statistical model. A well-known agent of swimmer's itch, T. stagnicolae, is able to penetrate human skin more frequently than ASC, likely meaning that ASC is only a minor cause of swimmer's itch where T. stagnicolae is present. We also completed limited experiments that compared the cercarial behavior of the two species in vitro and in situ. A known stimulant of schistosome cercarial penetration, α-linolenic acid, did not stimulate ASC cercariae to initiate penetration-associated behaviors as frequently as T. stagnicolae. However, when placed on esophageal tissue of the known vertebrate host for ASC, Canada goose (Branta canadensis), ASC cercariae were observed penetrating the esophageal epithelium quickly, whereas T. stagnicolae cercariae did not exhibit any penetration behaviors.

Recording from Snail Motor Nerves to Investigate Central Pattern Generation.

Feeding in pond snails has long been a model system for central pattern generation and its modulation. The pattern is generated by a small set of neurons in the buccal ganglia, which innervate the buccal mass, esophagus, and salivary glands. In this exercise, students observe feeding behavior and then record and quantify rhythmic motor activity and its response to feeding stimulants and neuromodulators. In a standard three-hour class period, students do a dissection, record from several nerves, and perform experimental manipulations such as adding feeding stimulants, serotonin, or dopamine to the preparation. Depending on the course goals, data can be presented qualitatively or cyclic measurements and spike-rate analysis can be done. This exercise leads to discussion of neural circuitry and intrinsic properties that support pattern generation for rhythmic activities such as feeding, locomotion, and respiration.

Evidence of a Putative Novel Species of Avian Schistosome Infecting Planorbella trivolvis.

Freshwater gastropods of the genera Lymnaea Lamarck, 1799, Physa Draparnaud, 1801, Gyraulus Charpentier, 1837, Radix Montfort, 1810, and Stagnicola Jeffreys, 1830 are considered suitable intermediate hosts for avian schistosomes. A large trematode biodiversity survey performed across 3 yr on 6 lakes in Alberta confirmed 3 already-reported snail hosts for 7 North American avian schistosomes; however, the cytochrome c oxidase subunit 1 (COI) nucleotide sequence from 1 cercarial sample (from a single specimen of Planorbella trivolvis) was distinct from all other COI schistosome sequences. As part of a simultaneous, comparable study of P. trivolvis by us in Michigan, we collected another cercarial type from 6 lakes that was 99% similar (COI) to the aforementioned cercarial type. Phylogenetic analyses of the COI and 28S rDNA genes recovered the former cercaria in a clade of avian schistosomes. In Michigan, the feces of a Canada goose (Branta canadensis Linnaeus, 1758) had a miracidium with an identical COI nucleotide sequence. Preliminary swimmer's itch and cercarial emergence studies were performed to determine if the cercariae could cause swimmer's itch and to study the emergence pattern as compared with species of Trichobilharzia Skrjabin and Zakharow, 1920.

Handling times and saturating transmission functions in a snail-worm symbiosis.

All dynamic species interaction models contain an assumption that describes how contact rates scale with population density. Choosing an appropriate contact-density function is important, because different functions have different implications for population dynamics and stability. However, this choice can be challenging, because there are many possible functions, and most are phenomenological and thus difficult to relate to underlying ecological processes. Using one such phenomenological function, we described a nonlinear relationship between field transmission rates and host density in a common snail-oligochaete symbiosis. We then used a well-known contact function from predator-prey models, the Holling Type II functional response, to describe and predict host snail contact rates in the laboratory. The Holling Type II functional response accurately described both the nonlinear contact-density relationship and the average contact duration that we observed. Therefore, we suggest that contact rates saturate with host density in this system because each snail contact requires a non-instantaneous handling time, and additional possible contacts do not occur during that handling time. Handling times and nonlinear contact rates might also explain the nonlinear relationship between symbiont transmission and snail density that we observed in the field, which could be confirmed by future work that controls for other potential sources of seasonal variation in transmission rates. Because most animal contacts are not instantaneous, the Holling Type II functional response might be broadly relevant to diverse host-symbiont systems.

GABA-like immunoreactivity in Biomphalaria: Colocalization with tyrosine hydroxylase-like immunoreactivity in the feeding motor systems of panpulmonate snails.

The simpler nervous systems of certain invertebrates provide opportunities to examine colocalized classical neurotransmitters in the context of identified neurons and well defined neural circuits. This study examined the distribution of γ-aminobutyric acid-like immunoreactivity (GABAli) in the nervous system of the panpulmonates Biomphalaria glabrata and Biomphalaria alexandrina, major intermediate hosts for intestinal schistosomiasis. GABAli neurons were localized in the cerebral, pedal, and buccal ganglia of each species. With the exception of a projection to the base of the tentacle, GABAli fibers were confined to the CNS. As GABAli was previously reported to be colocalized with markers for dopamine (DA) in five neurons in the feeding network of the euopisthobranch gastropod Aplysia californica (Díaz-Ríos, Oyola, & Miller, 2002), double-labeling protocols were used to compare the distribution of GABAli with tyrosine hydroxylase immunoreactivity (THli). As in Aplysia, GABAli-THli colocalization was limited to five neurons, all of which were located in the buccal ganglion. Five GABAli-THli cells were also observed in the buccal ganglia of two other intensively studied panpulmonate species, Lymnaea stagnalis and Helisoma trivolvis. These findings indicate that colocalization of the classical neurotransmitters GABA and DA in feeding central pattern generator (CPG) interneurons preceded the divergence of euopisthobranch and panpulmonate taxa. These observations also support the hypothesis that heterogastropod feeding CPG networks exhibit a common universal design.

Modulation of growth cone filopodial length by carbon monoxide.

Carbon monoxide (CO) is physiologically produced via heme degradation by heme oxygenase enzymes. Whereas CO has been identified as an important physiological signaling molecule, the roles it plays in neuronal development and regeneration are poorly understood. During these events, growth cones guide axons through a rich cellular environment to locate target cells and establish synaptic connections. Previously, we have shown that another gaseous signaling molecule, nitric oxide (NO), has potent effects on growth cone motility. With NO and CO sharing similar cellular targets, we wanted to determine whether CO affected growth cone motility as well. We assessed how CO affected growth cone filopodial length and determined the signaling pathway by which this effect was mediated. Using two well-characterized neurons from the freshwater snail, Helisoma trivolvis, it was found that the CO donor, carbon monoxide releasing molecule-2 (CORM-2), increased filopodial length. CO utilized a signaling pathway that involved the activation of soluble guanylyl cyclase, protein kinase G, and ryanodine receptors. While increases in filopodial length often occur from robust increases in intracellular calcium levels, the timing in which CO increased filopodial length corresponded with low basal calcium levels in growth cones. Taken together with findings of a heme oxygenase-like protein in the Helisoma nervous system, these results provide evidence for CO as a modulator of growth cone motility and implicate CO as a neuromodulatory signal during neuronal development and/or regeneration. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 677-690, 2017.

Host and parasite thermal acclimation responses depend on the stage of infection.

Global climate change is expected to alter patterns of temperature variability, which could influence species interactions including parasitism. Species interactions can be difficult to predict in variable-temperature environments because of thermal acclimation responses, i.e. physiological changes that allow organisms to adjust to a new temperature following a temperature shift. The goal of this study was to determine how thermal acclimation influences host resistance to infection and to test for parasite acclimation responses, which might differ from host responses in important ways. We tested predictions of three, non-mutually exclusive hypotheses regarding thermal acclimation effects on infection of green frog tadpoles (Lithobates clamitans) by the trematode parasite Ribeiroia ondatrae with fully replicated controlled-temperature experiments. Trematodes or tadpoles were independently acclimated to a range of 'acclimation temperatures' prior to shifting them to new 'performance temperatures' for experimental infections. Trematodes that were acclimated to intermediate temperatures (19-22 °C) had greater encystment success across temperatures than either cold- or warm-acclimated trematodes. However, host acclimation responses varied depending on the stage of infection (encystment vs. clearance): warm- (22-28 °C) and cold-acclimated (13-19 °C) tadpoles had fewer parasites encyst at warm and cold performance temperatures, respectively, whereas intermediate-acclimated tadpoles (19-25 °C) cleared the greatest proportion of parasites in the week following exposure. These results suggest that tadpoles use different immune mechanisms to resist different stages of trematode infection, and that each set of mechanisms has unique responses to temperature variability. Our results highlight the importance of considering thermal responses of both parasites and hosts when predicting disease patterns in variable-temperature environments.

Population-specific toxicity of six insecticides to the trematode Echinoparyphium sp.

The ubiquitous use of pesticides has increased concerns over their direct and indirect effects on disease dynamics. While studies examining the effects of pesticides on host-parasite interactions have largely focused on how pesticides influence the host, few studies have considered the effects of pesticides on parasites. We investigated the toxicity of six common insecticides at six environmentally-relevant concentrations to cercariae of the trematode Echinoparyphium from two populations. All six insecticides reduced the survival of cercariae (overall difference between mortality in control vs pesticide exposure = 86·2 ± 8·7%) but not in a predictable dose-dependent manner. These results suggest that Echinoparyphium are sensitive to a broad range of insecticides commonly used in the USA. The lack of a clear dose-dependent response in Echinoparyphium highlights the potential limitations of toxicity assays in predicting pesticide toxicity to parasites. Finally, population-level variation in cercarial susceptibility to pesticides underscores the importance of accounting for population variation as overlooking this variation can limit our ability to predict toxicity in nature. Collectively, this work demonstrates that consideration of pesticide toxicity to parasites is important to understanding how pesticides ultimately shape disease dynamics in nature.

Development of Helisoma trivolvis pond snails as biological samplers for biomonitoring of current-use pesticides.

Nontarget aquatic organisms residing in wetlands are commonly exposed to current-use pesticides through spray drift and runoff. However, it is frequently challenging to measure exposure because of rapid dissipation of pesticides from water and reduced bioavailability. The authors' hypothesis is that freshwater snails can serve as bioindicators of pesticide exposure based on their capacity to passively accumulate tissue residues. Helisoma trivolvis snails were evaluated as biomonitors of pesticide exposure using a fungicide formulation that contains pyraclostrobin and metconazole and is frequently applied to crops surrounding depressional wetlands. Exposure-response studies indicate that H. trivolvis are tolerant of pyraclostrobin and metconazole at concentrations >10 times those lethal to many aquatic species, with a median lethal concentration based on pyraclostrobin of 441 µg/L (95% confidence interval of 359-555 µg/L). Bioconcentration factors ranged from 137 mL/g to 211 mL/g and from 39 mL/g to 59 mL/g for pyraclostrobin and metconazole, respectively. Elimination studies suggested one-compartmental elimination and snail tissue half-lives (t50 ) of approximately 15 h and 5 h for pyraclostrobin and metconazole, respectively. Modeling derived toxicokinetic parameters in the context of an environmentally relevant pulsed exposure suggests that residues can be measured in snails long after water concentrations fall below detection limits. With high fungicide tolerance, rapid accumulation, and slow elimination, H. trivolvis may be viable for biomonitoring of pyraclostrobin and should be investigated for other pesticides. Environ Toxicol Chem 2016;35:2320-2329. © 2016 SETAC.

Dispersal of a defensive symbiont depends on contact between hosts, host health, and host size.

Symbiont dispersal is necessary for the maintenance of defense mutualisms in space and time, and the distribution of symbionts among hosts should be intricately tied to symbiont dispersal behaviors. However, we know surprisingly little about how most defensive symbionts find and choose advantageous hosts or what cues trigger symbionts to disperse from their current hosts. In a series of six experiments, we explored the dispersal ecology of an oligochaete worm (Chaetogaster limnaei) that protects snail hosts from infection by larval trematode parasites. Specifically, we determined the factors that affected net symbiont dispersal from a current "donor" host to a new "receiver" host. Symbionts rarely dispersed unless hosts directly came in contact with one another. However, symbionts overcame their reluctance to disperse across the open environment if the donor host died. When hosts came in direct contact, net symbiont dispersal varied with both host size and trematode infection status, whereas symbiont density did not influence the probability of symbiont dispersal. Together, these experiments show that symbiont dispersal is not a constant, random process, as is often assumed in symbiont dispersal models, but rather the probability of dispersal varies with ecological conditions and among individual hosts. The observed heterogeneity in dispersal rates among hosts may help to explain symbiont aggregation among snail hosts in nature.

Cell-specific regulation of neuronal activity by endogenous production of nitric oxide.

Nitric oxide (NO) is a key regulator of neuronal excitability in the nervous system. While most studies have investigated its role as an intercellular messenger/modulator, less is known about potential physiological roles played by NO within NO-producing neurons. We showed previously that intrinsic production of NO within B5 neurons of the pond snail Helisoma trivolvis increased neuronal excitability by acting on three ionic conductances. Here we demonstrate that intrinsically produced NO affected two of the same conductances in another buccal neuron, B19, where it had the opposite, namely inhibitory, effect on neuronal activity. Using single-cell RT-PCR, we show that B19 neurons express NO synthase (NOS) mRNA. The inhibition of intrinsic NO production with NOS inhibitors caused membrane potential depolarization, transient spiking and an increase in input resistance. Inhibition of the main intracellular receptor of NO, soluble guanylyl cyclase, had similar effects on the parameters mentioned above. An investigation of the effects of NO on ion channels revealed that intrinsic NO mediated neuronal hyperpolarization by activating voltage-gated calcium channels that in turn caused the tonic opening of apamin-sensitive calcium-activated potassium channels. The analysis of action potentials in B5 and B19 neurons suggested that the opposite effects on neuronal excitability elicited by intrinsic NO were probably determined by differences in the ionic conductances that shape their action potentials. In summary, we describe a mechanism by which B19 neurons utilise intrinsically produced NO in a cell-type-specific fashion to decrease their neuronal activity, highlighting an important physiological role of NO within NO-producing neurons.

Contextual modulation of a multifunctional central pattern generator.

The multifunctional buccal central pattern generator in snails, which controls different oral behaviors, has been well characterized. In this work we propose a role for the group of about 40 electrotonically coupled buccal A cluster cells as a context-dependant switch for the buccal central pattern generator, modulating motor patterns that elicit different oral behaviors. We characterize these cells based on location and morphology, and provide evidence for their selective activation under two different stimuli - Listerine perfusion and intestinal nerve stimulation - triggering buccal motor patterns putatively underlying egestion and substrate cleaning. A new role for these electrotonically coupled buccal A cluster neurons is shown. They serve as a context-dependant switch that alters buccal motor patterns depending on input stimuli, thereby eliciting the appropriate behavioral response.

Indirect effects of fish on macrophytes in Bays Mountain Lake: evidence for a littoral trophic cascade.

We began this experiment to test specific hypotheses regarding direct and indirect effects of fish predation on the littoral macroinvertebrate community of Bays Mountain Lake, Tennessee. We used 24 m2 enclosures in which we manipulated the presence and absence of large redear sunfish (Lepomis microlophus>150 mm SL), and small sunfish (L. macrochirus and L. microlophus <50 mm SL) over a 16-mo period. Here we report on effects of fish predation on gastropod grazers that appear to cascade to periphyton and macrophytes.Both large redear sunfish and small sunfish maintained low snail biomass, but snails in fish-free controls increased significantly during the first 2-mo of the experiment. By late summer of the first year of the experiment, the difference in biomass between enclosures with and without fish had increased dramatically (>10×). Midway through the second summer of the experiment, we noted apparent differences in the abundance of periphyton between enclosures containing fish and those that did not. We also noted differences in the macrophyte distribution among enclosures. To document these responses, we estimated periphyton cover, biovolume and cell size frequencies as well as macrophyte distributions among enclosures at the end of the experiment. When fish were absent, periphyton percent cover was significantly reduced compared to when fish were present. Periphyton cell-size distributions in enclosures without fish were skewed toward small cells (only 12% were greater than 200 m3), which is consistent with intense snail grazing. The macrophyte Najas flexilis had more than 60 x higher biomass in the fish-free enclosures than in enclosures containing fish; Potamogeton diversifolius was found only in fish-free enclosures. These results suggest a chain of strong interactions (i.e. from fish to snails to periphyton to macrophytes) that may be important in lake littoral systems. This contrasts sharply with earlier predictions based on cascading trophic interactions that propose that fish predation on snails would enhance macrophyte biomass.