Same same, but different: exploring the enigmatic role of the pituitary adenylate cyclase-activating polypeptide (PACAP) in invertebrate physiology.

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.

Characterization of corazonin signaling in a molluscan model species, Lymnaea stagnalis.

In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan "GnRH" should be classified as "CRZ".

Issues, challenges, directives, and limitations concerning the improvement of environmental risk assessment of pharmaceutically active compounds.

Nowadays, when tons of different chemicals including pharmaceutically active compounds (PhACs) are known to be released into the environment, applying adequate risk assessment relating to the protection of the ecosystem is very important. A broad body of scientific papers expresses a growing demand for improvement of the method(s) for ecological/environmental risk assessment (ERA). Although certain issues about ERA often emerge in the community, most of them cannot be considered as real problems and its methodology was developed keeping several limitations in mind. Nevertheless, the current approaches can be improved in order to better serve the intended purposes. For example, there is a lack of an integrated, manageable ecotoxicological database. It is not uncommon for basic, but extremely important, influencing factors such as time of exposure, interactions between different compounds, and characteristics of different habitats to be ignored. Discussing under the basic regulatory framework used in the EU, this correspondence paper deals with these and other examples to present the current features of ERA, identify gaps in process and application, and propose possible improvements/directives.

Prevalence of Coxiella burnetii in bovine placentas in Hungary and Slovakia: Detection of a novel sequence type - Short communication.

Cotyledons (n = 167) from 30 Hungarian and 5 Slovakian dairy cattle herds were analysed for Coxiella burnetii by real-time PCR targeting the IS1111 gene. Eighty (88.9%) out of the 90 cotyledons from retained placentas and 31 (40.3%) out of the 77 cotyledons from normally separated placentas tested positive. Seventeen out of the 80 positive samples (21.3%) originating from retained placentas were found to be highly loaded with C. burnetii with a cycle threshold (Ct) value lower than or equal to 27.08, ranging between 11.92 and 27.08. The rest of the positive samples from retained fetal membranes and from normally separated placentas were moderately loaded with C. burnetii DNA. Five out of the ten samples showing the strongest positivity (Ct 11.92-18.28) from retained placentas were genotyped by multispacer sequence typing based on ten loci, which revealed sequence type (ST) 61, a type that had not been detected in Hungary and Slovakia previously. Retained placenta was more likely in cows with C. burnetii PCR-positive cotyledons (odds ratio: 12.61, P = 0.0023). The high C. burnetii DNA load found in retained fetal membranes may be a potential risk factor for human infection and may also be associated with the retention of fetal membranes.

Serological survey of Coxiella burnetii infections in dairy cattle, sheep, goats and zoo animals in Hungary - Short communication.

Q fever is a disease of high zoonotic potential, but interest in its causative agent is rather low although it causes some public health problems in Hungary. The prevalence of Q fever is highly variable by country. The main reservoirs of the disease are the same domestic ruminant species everywhere, but the epidemiological profile depends on the features of the specific reservoir. The aim of this large-scale study was to demonstrate the importance of Q fever in different species as a possible source for human infection in most regions of Hungary. A total of 851 serum samples from 44 dairy farms, 16 sheep flocks, 4 goat farms and 3 zoos located in different parts of Hungary were tested. The presence of antibodies to Coxiella burnetii was surveyed in dairy cattle (n = 547), goats (n = 71), sheep (n = 200) and zoo animals (n = 33). The animal species tested in Hungary showed different seroprevalence values of C. burnetii infection. Seropositivity by the enzyme-linked immunosorbent assay was found in 258 out of 547 (47.2%) cows and in 69 out of 271 (25.5%) small ruminants, among them in 47 out of 200 (23.5%) sheep and in 22 out of 71 (31.0%) goats. Antibodies to C. burnetii were not detected in zoo animals. Seropositivity was demonstrated in 44 out of 44 (100%) dairy cattle farms, with at least one serum sample found to be positive on each farm. The seropositivity rate of small ruminant farms was 55.0% (11 positive out of 20 tested), with 9 out of 16 (56.3%) sheep flocks and 2 out of 4 (50.0%) goat herds showing seropositivity.

Identification, presence, and possible multifunctional regulatory role of invertebrate gonadotropin-releasing hormone/corazonin molecule in the great pond snail (Lymnaea stagnalis).

In the last years, our interpretation of the origin and function of the gonadotropin-releasing hormone (GnRH) neuropeptide superfamily has changed substantially. A main driver for these conceptual changes came from increased investigations into functions and evolutionary lineage of previously identified molluscan GnRH molecules. Emerging evidence suggests not only reproductive, but also diverse biological effects of these molecules and proposes they should most likely be called corazonin (CRZ). Clearly, a more global understanding requires further exploration of species-specific functions and structure of invGnRH/CRZ peptides. Towards this goal, we have identified the full-length cDNA of invGnRH/CRZ peptide in an invertebrate model species, the great pond snail Lymnaea stagnalis, termed ly-GnRH/CRZ, and characterized the transcript and peptide distribution in the central nervous system (CNS) and peripheral organs. Our results are consistent with previous data that molluscan GnRHs are more related to CRZs and serve diverse functions. Hence, our findings support the notion that peptides originally termed molluscan GnRH are multifunctional modulators and that nomenclature change should be taken into consideration.

Testing the Applicability of MALDI-TOF MS as an Alternative Stock Identification Method in a Cryptic Species Complex.

Knowledge of intraspecific variability of a certain species is essential for their long-term survival and for the development of conservation plans. Nowadays, molecular/genetic methods are the most frequently used for this purpose. Although, the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has become a promising alternative tool to specify intraspecific variability, there is a lack of information about the limitations of this method, and some methodological issues need to be resolved. Towards this goal, we tested the sensitivity of this method on an intraspecific level, using genetically identified individuals of a cryptic fish species complex collected from five distinct populations. Additionally, some methodologic issues, such as the effect of (1) delayed sample preparation, (2) clove oil anaesthetization, and (3) different tissue types (muscle, and brain) were investigated using the MS analysis results. Our results show that the delayed sample preparation has a fundamental effect on the result of MS analysis, while at the same time the clove oil did not affect the results considerably. Both the brain and muscle samples were usable for cryptic species identification, but in our opinion this method has limited applicability for population-level segregation. The application of MALDI-TOF MS to the exploitable toolkit of phylogenetic and taxonomic researches could be used to broaden conclusions.

Relationship of dairy heifer reproduction with survival to first calving, milk yield and culling risk in the first lactation.

OBJECTIVE: The aim of our study was to determine the associations of heifer reproductive performance with survival up to the first calving, first-lactation milk yield, and the probability of being culled within 50 days after first calving. METHODS: Data from 33 large Holstein-Friesian commercial dairy herds were gathered from the official milk recording database in Hungary. The data of heifers first inseminated between January 1, 2011 and December 31, 2014 were analyzed retrospectively, using Cox proportional hazards models, competing risks models, multivariate linear and logistic mixed-effects models. RESULTS: Heifers (n = 35,128) with younger age at conception were more likely to remain in the herd until calving, and each additional month in age at conception increased culling risk by 5.1%. Season of birth was related to first-lactation milk yield (MY1; n = 19,931), with cows born in autumn having the highest milk production (p<0.001). The highest MY1 was achieved by heifers that first calved between 22.00 and 25.99 months of age. Heifers that calved in autumn had the highest MY1, whereas calving in summer was related to the lowest milk production (p<0.001). The risk of culling within 50 days in milk in first lactation (n = 21,225) increased along with first calving age, e.g. heifers that first calved after 30 months of age were 5.52-times more likely to be culled compared to heifers that calved before 22 months of age (p<0.001). Calving difficulty was related to higher culling risk in early lactation (p<0.001). Heifers that required caesarean section were 24.01-times more likely to leave the herd within 50 days after first calving compared to heifers that needed no assistance (p<0.001). CONCLUSION: Reproductive performance of replacement heifers is closely linked to longevity and milk production in dairy herds.

Complex molecular changes induced by chronic progestogens exposure in roach, Rutilus rutilus.

In our previous study, we measured 0.23-13.67ng/L progestogens (progesterone, drospirenone, levonorgestrel) in natural waters in the catchment area of the largest shallow lake of Central Europe, Lake Balaton. Progestogen contaminations act as potent steroids with mixed progestagenic, androgenic and mild estrogenic effects that is why our aim was to investigate the morphological and molecular effects of mixture of progesterone, drospirenone, and levonorgestrel in environmentally relevant (10ng/L) and higher (50 and 500ng/L) exposure concentrations in common roach, Rutilus rutilus. Steroids (e.g. progestogens) and the protein deglycase DJ-1 chaperon molecule aim the same target molecules in cells, therefore, we hypothesized that a relationship may exist between progestogens and DJ-1. Furthermore, our other aim was to follow the changes of signal molecules of different biological function due to progestogen treatment in serum and brain. Adult roaches were exposed to 10, 50 and 500ng/L of mixture of progestogen for 42 days and their somatic indices (brain-somatic, liver-somatic, gonadosomatic and kidney-somatic) were measured. Vitellogenin (VTG) expression (estrogen effect) or inhibition (androgen effect) in fish is a widely used biomarker so we measured its changes in liver by ELISA. To determine the quantity and to map the spatial distribution of DJ-1 chaperon protein the brain and liver tissues were analyzed by ELISA and immunohistochemistry. Furthermore, we also studied molecular alterations: a) in the serum by measuring cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride concentrations and b) in brain homogenate using a cell stress array kit (26 protein). The somatic index of liver and kidney significantly in all the treated groups, whereas the gonadosomatic index of 500ng/L treated group showed significant decrease compared to control animals. VTG level increased significantly in 500ng/L progestogen treated group. Since the concentration of DJ-1 significantly increased in brain and liver in all progestogen treatment groups, the DJ-1 protein could be able to a more sensitive marker than VTG. Serum LDL and cholesterol levels of exposed fish were significantly decreased. DJ-1 was mediated through the stimulation of the expression of LDL-receptor which facilitates reuptake subsequently. In summary, our observations unfolded new data about molecular alterations induced by the combined action of environmental progestogens. In addition, the DJ-1 chaperon protein as a possible biomarker helped to trace the abiotic chemical environmental contaminations, like progestogens in the freshwater ecosystems.

Proteomic insight into the primycin fermentation process of Saccharomonospora azurea.

Saccharomonospora azurea SZMC 14600 is a member of the family Pseudonocardiaceae exclusively used for industrial scale production of primycin a large 36-membered non-polyene macrolide lactone antibiotic belonging to the polyketide class of natural products. Even though maximum antibiotic yield has been achieved by empirically optimized two-step fermentation process, little is known about the molecular components and mechanisms underlying the efficient antibiotic production. In order to identify differentially expressed proteins (DEPs) between the pre- and main-fermentation stages of primycin, comparative 2D-PAGE experiments were performed. In total, 98 DEP spots were reproducibly detected, out of which four spots were excised from gels, and identified through MALDI-TOF/TOF mass spectrometry. Peptide mass fingerprint analysis revealed peptide matches to HicB antitoxin for the HicAB toxin-antitoxin system (EHK86651), to a nucleoside diphosphate kinase regulator ((Ndk; EHK81899) and two other proteins with unknown function (EHK88946 and EHK86777).

Persistent suppression of type 1 diabetes by a multicomponent vaccine containing a cholera toxin B subunit-autoantigen fusion protein and complete Freund's adjuvant.

Data presented here demonstrate multifunctional vaccination strategies that harness vaccinia virus mediated delivery of a gene encoding an immunoenhanced diabetes autoantigen in combination with complete Freund's adjuvant (CFA) that can maintain safe and durable immunologic homeostasis in NOD mice. Systemic coinoculation of prediabetic mice with recombinant vaccinia virus rVV-CTB::GAD and undiluted or 10-fold diluted CFA demonstrated a significant decrease in hyperglycemia and pancreatic islet inflammation in comparison with control animals during 17-61 and 17-105 weeks of age, respectively. Synergy in these beneficial effects was observed during 43-61 and 61-105 wks of age, respectively. Inflammatory cytokine and chemokine levels in GAD-stimulated splenocytes isolated from vaccinated mice were generally lower than those detected in unvaccinated mice. The overall health and humoral immune responses of the vaccinated animals remained normal throughout the duration of the experiments.

Copper-transporting ATPases throughout the animal evolution - From clinics to basal neuron-less animals.

Copper-transporting ATPases are a group of heavy metal-transporting proteins and which can be found in all living organisms. In animals, they are generally referred to as ATP7 proteins and are involved in many different physiological processes including the maintaining of copper homeostasis and the supply of copper to cuproenzymes. A single ATP7 gene is present in non-chordate animals while it is divided into ATP7A and ATP7B in chordates. In humans, dysfunction of ATP7 proteins can lead to severe genetic disorders, such as, Menkes disease and Wilson's disease, which are characterized by abnormal copper transport and accumulation, causing significant health complications. Therefore, there is a substantial amount of research on ATP7 genes and ATP7 proteins in humans and mice to understand pathophysiological conditions and find potential therapeutic interventions. Copper-transporting ATPases have also been investigated in some non-mammalian vertebrates, protostomes, single-cellular eukaryotes, prokaryotes, and archaea to gain useful evolutionary insights. However, ATP7 function in many animals has been somewhat neglected, particularly in non-bilaterians. Previous reviews on this topic only broadly summarized the available information on the function and evolution of ATP7 genes and ATP7 proteins and included only the classic vertebrate and invertebrate models. Given this, and the fact that a considerable amount of new information on this topic has been published in recent years, the present study was undertaken to provide an up-to-date, comprehensive summary of ATP7s/ATP7s and give new insights into their evolutionary relationships. Additionally, this work provides a framework for studying these genes and proteins in non-bilaterians. As early branching animals, they are important to understand the evolution of function of these proteins and their important role in copper homeostasis and neurotransmission.

A novel and unique refraction-based optical recording system for pharmacological investigations on isolated muscle preparations.

In the field of neuroscience and ecotoxicology, there is a great need for investigating the effect(s) of a variety of different chemicals (e.g., pharmacologically active compounds, pesticides, neurotransmitters, modulators) at different biological levels. Different contractile tissue preparations have provided excellent model systems for in vitro pharmacological experiments for a long time. However, such investigations usually apply mechanical force transducer-based approaches. Thus, a rapid, easy, cheap, digital, and reproducible in vitro pharmacological method based on an effective, 'non-invasive' (compared to the force-transducer approaches), refraction-based optical recording approach and isolated heart preparations was developed.•A versatile and unique refraction-based optical recording system with a Java application was developed.•The recording system was tested and validated on isolated heart preparations obtained from the widely used invertebrate model organism, the great pond snail (Lymnaea stagnalis).•The recording system illustrates the progression of technology from the mechanical force transducer system and can represent a suitable tool in ecotoxicology or neuroscience.

Temperature related toxicity features of acute acetamiprid and thiacloprid exposure in Daphnia magna and implications on reproductive performance.

This study investigated the potential for elevated temperature to alter the toxicity of acetamiprid (ACE) and thiacloprid (Thia) in the ecotoxicity model Daphnia magna. The modulation of CYP450 monooxygenases (ECOD), ABC transporter activity (MXR) and incident cellular reactive oxygen species (ROS) overproduction was screened in premature daphnids following acute (48 h) exposure to sublethal concentrations of ACE and Thia (0.1-, 1.0 µM) at standard 21 °C and elevated 26 °C temperatures. Delayed outcomes of acute exposures were further evaluated based on the reproduction performance of daphnids monitored over 14 days of recovery. Exposures to ACE and Thia at 21o C elicited moderate induction of ECOD activity, pronounced inhibition of MXR activity and severe ROS overproduction in daphnids. In the high thermal regime, treatments resulted in significantly lower induction of ECOD activity and inhibition of MXR activity, suggesting a suppressed metabolism of neonicotinoids and less impaired membrane transport activity in daphnids. Elevated temperature on its own, caused a three-fold rise in ROS levels in control daphnids, while ROS overproduction upon neonicotinoid exposure was less accentuated. Acute exposures to ACE and Thia caused significant decreases also in the reproduction of daphnids, indicating delayed outcomes even at environmentally relevant concentrations. Both the cellular alterations in exposed daphnids and decreases in their reproductive output post exposures evidenced closely similar toxicity patterns and potentials for the two neonicotinoids. While elevated temperature elicited only a shift in baseline cellular alterations evoked by neonicotinoids, it significantly worsened the reproductive performance of daphnids following neonicotinoid exposures.

Automated pose estimation reveals walking characteristics associated with lameness in broilers.

Walking ability of broilers can be improved by selective breeding, but large-scale phenotypic records are required. Currently, gait of individual broilers is scored by trained experts, however, precision phenotyping tools could offer a more objective and high-throughput alternative. We studied whether specific walking characteristics determined through pose estimation are linked to gait in broilers. We filmed male broilers from behind, walking through a 3 m × 0.4 m (length × width) corridor one by one, at 3 time points during their lifetime (at 14, 21, and 33 d of age). We used a deep learning model, developed in DeepLabCut, to detect and track 8 keypoints (head, neck, left and right knees, hocks, and feet) of broilers in the recorded videos. Using the keypoints of the legs, 6 pose features were quantified during the double support phase of walking, and 1 pose feature was quantified during steps, at maximum leg lift. Gait was scored on a scale from 0 to 5 by 4 experts, using the videos recorded on d 33, and the broilers were further classified as having either good gait (mean gait score ≤2) or suboptimal gait (mean gait score >2). The relationship of pose features on d 33 with gait was analyzed using the data of 84 broilers (good gait: 57.1%, suboptimal gait: 42.9%). Birds with suboptimal gait had sharper hock joint lateral angles and lower hock-feet distance ratios during double support on d 33, on average. During steps, relative step height was lower in birds with suboptimal gait. Step height and hock-feet distance ratio showed the largest mean deviations in broilers with suboptimal gait compared to those with good gait. We demonstrate that pose estimation can be used to assess walking characteristics during a large part of the productive life of broilers, and to phenotype and monitor broiler gait. These insights can be used to understand differences in the walking patterns of lame broilers, and to build more sophisticated gait prediction models.

Bioconversion of Different Waste Streams of Animal and Vegetal Origin and Manure by Black Soldier Fly Larvae Hermetia illucens L. (Diptera: Stratiomyidae).

Black soldier fly larvae (BSFL) are considered a commercially viable solution for global organic waste problems. The objective of this study was to assess the feasibility of rearing BSFL on a wide range of low-value waste streams and its potential to transform them into high-quality animal feed and fertilizer. Six waste streams of different origins were selected and each tested in triplicate. Several parameters were analysed: growth performance, waste reduction index (WRI), conversion efficiency (ECI) and larval composition. Frass composition was also analysed. Larvae reared on fast food waste (FFW) had the highest ECI and WRI and the lowest values when reared on pig manure slurry mixed with silage grass (PMLSG) and slaughter waste (SW). The highest protein content was found for larvae reared on mushroom stems (MS) although this substrate had the lowest protein content. Moreover, the frass nutritional profile was proportionally related to the substrate's nutritional profile: the protein-rich substrate (SW) resulted in protein-rich frass and the low-protein substrate (MS) resulted in protein-poor frass. The same was true for the lipid content. In conclusion, this study showed that BSFL can be successfully reared on a wide range of waste streams that can affect the larval and frass chemical compositions.

Presence, variation, and potential ecological impact of microplastics in the largest shallow lake of Central Europe.

The presence of microplastics (MPs) in the global ecosystem has generated a rapidly growing concern worldwide. Although their presence in the marine environment has been well-studied, much less data are available on their abundance in freshwaters. MPs alone and in combination with different chemicals has been shown to cause acute and chronic effects on algae and aquatic invertebrate and vertebrate species at different biological levels. However, the combined ecotoxicological effects of MPs with different chemicals on aquatic organisms are still understudied in many species and the reported data are often controversial. In the present study, we investigated, for the first time, the presence of MPs in Lake Balaton, which is the largest shallow lake of Central Europe and an important summer holiday destination. Moreover, we exposed neonates of the well-established ecotoxicological model organism Daphnia magna to different MPs (polystyrene [3 µm] or polyethylene [≤ 100 µm]) alone and in combination with three progestogen compounds (progesterone, drospirenone, levonorgestrel) at an environmentally relevant concentration (10 ng L-1) for 21 days. The presence of 7 polymer types of MPs in the size range of 50-100 µm was detected in Lake Balaton. Similarly to the global trends, polypropylene and polyethylene MPs were the most common types of polymer. The calculated polymer-independent average particle number was 5.5 particles m-3 (size range: 50 µm - 100 µm) which represents the values detected in other European lakes. Our ecotoxicological experiments confirmed that MPs and progestogens can affect D. magna at the behavioral (body size and reproduction) and biochemical (detoxification-related enzyme activity) levels. The joint effects were negligible. The presence of MPs may lead to reduced fitness in the aquatic biota in freshwaters such as Lake Balaton, however, the potential threat of MPs as vectors for progestogens may be limited.

Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17ß-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

Progestogen-induced alterations and their ecological relevance in different embryonic and adult behaviours of an invertebrate model species, the great pond snail (Lymnaea stagnalis).

The presence of oral contraceptives (basically applying estrogens and/or progestogens) poses a challenge to animals living in aquatic ecosystems and reflects a rapidly growing concern worldwide. However, there is still a lack in knowledge about the behavioural effects induced by progestogens on the non-target species including molluscs. In the present study, environmental progestogen concentrations were summarised. Knowing this data, we exposed a well-established invertebrate model species, the great pond snail (Lymnaea stagnalis) to relevant equi-concentrations (1, 10, 100, and 500 ng L-1) of mixtures of four progestogens (progesterone, drospirenone, gestodene, levonorgestrel) for 21 days. Significant alterations were observed in the embryonic development time, heart rate, feeding, and gliding activities of the embryos as well as in the feeding and locomotion activity of the adult specimens. All of the mixtures accelerated the embryonic development time and the gliding activity. Furthermore, the 10, 100, and 500 ng L-1 mixtures increased the heart rate and feeding activity of the embryos. The 10, 100, and 500 ng L-1 mixtures affected the feeding activity as well as the 1, 10, and 100 ng L-1 mixtures influenced the locomotion of the adult specimens. The differences of these adult behaviours showed a biphasic response to the progestogen exposure; however, they changed approximately in the opposite way. In case of feeding activity, this dose-response phenomenon can be identified as a hormesis response. Based on the authors' best knowledge, this is the first study to investigate the non-reproductive effects of progestogens occurring also in the environment on molluscan species. Our findings contribute to the global understanding of the effects of human progestogens, as these potential disruptors can influence the behavioural activities of non-target aquatic species. Future research should aim to understand the potential mechanisms (e.g., receptors, signal pathways) of progestogens induced behavioural alterations.

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.