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.

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.

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.

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.

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.

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.

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.

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.

Functional characterization and related evolutionary implications of invertebrate gonadotropin-releasing hormone/corazonin in a well-established model species.

In vertebrates, gonadotropin-releasing hormone (GnRH) peptide is the central mediator of reproduction. Homologous peptides have previously also been identified in molluscan species. However, emerging evidence suggests that these molecules might serve diverse regulatory functions and proposes to consider them as corazonin (CRZ). We previously isolated the full-length cDNA of the invGnRH/CRZ peptide (termed ly-GnRH/CRZ) in the well-established invertebrate model species, the great pond snail Lymnaea stagnalis; however, its predicted functions remain to be verified. In this study, we first confirmed the presence of the deduced active peptide from the central nervous system of L. stagnalis. Further, we performed in vivo and in vitro studies to explore the functions of ly-GnRH/CRZ. Injection of sexually mature specimens with synthetic active peptide had an inhibitory effect on locomotion and an acceleratory effect on egg-laying, but had no effect on feeding. The previously predicted modulatory effect of ly-GnRH/CRZ was supported by its identified co-localization with serotonin on the surface of the heart atria. Lastly, we demonstrated not only the presence of ly-GnRH/CRZ in the penial complex but also that ly-GnRH/CRZ-containing neurons project to the efferent penis nerve, suggesting ly-GnRH/CRZ may directly modulate the motor output of this peripheral tissue. Overall, our findings strongly support that ly-GnRH/CRZ is a multifunctional neuropeptide. These results contribute to the understanding of the GnRH superfamily and, more broadly, disciplines such as comparative endocrinology and neurobiology.

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.

Effects of chronic sublethal progestogen exposure on development, reproduction, and detoxification system of water flea, Daphnia magna.

The presence of sex steroid hormones in aquatic ecosystems is of rapidly growing concern worldwide since they can affect the different non-target species including cladocerans. Although data are available on the effects of estrogens on the well-established ecotoxicological model organism Daphnia magna, the molecular or behavioural alterations induced by environmentally relevant concentrations (from a few ng L-1 to a few hundred ng L-1 in average) of progestogens have not been investigated on this species. In the present study, we exposed neonates of D. magna to relevant equi-concentrations (1, 10, 100, 500 ng L-1) of mixtures of four progestogens (progesterone, drospirenone, gestodene, levonorgestrel) in short-term (6 days) and long-term (21 days) experiments. Significant alterations were observed at the molecular, cellular, and individual levels. During the short-term exposure, all of the mixtures increased the gene expression of glutathione S-transferase (GST) detoxification enzyme, moreover, the activity of GST was also significantly increased at the concentrations of 10, 100, and 500 ng L-1. In long-term exposure, the number of days until production of the first eggs was reduced at the 10 ng L-1 concentration compared to control, furthermore, the maximum egg number per individual increased at the concentrations of 1 and 10 ng L-1. Based on the authors' best knowledge, this is the first study to investigate the effects of progestogens in mixtures and at environmentally relevant concentrations on D. magna. Our findings contribute to the understanding of the possible physiological effects of human progestogens. Future research should be aimed at understanding the potential mechanisms (e.g., perception) underlying the changes induced by progestogens.

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.

Slowly seeing the light: an integrative review on ecological light pollution as a potential threat for mollusks.

Seasonal changes in the natural light condition play a pivotal role in the regulation of many biological processes in organisms. Disruption of this natural condition via the growing loss of darkness as a result of anthropogenic light pollution has been linked to species-wide shifts in behavioral and physiological traits. This review starts with a brief overview of the definition of light pollution and the most recent insights into the perception of light. We then go on to review the evidence for some adverse effects of ecological light pollution on different groups of animals and will focus on mollusks. Taken together, the available evidence suggests a critical role for light pollution as a recent, growing threat to the regulation of various biological processes in these animals, with the potential to disrupt ecosystem stability. The latter indicates that ecological light pollution is an environmental threat that needs to be taken seriously and requires further research attention.

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 Impact of Paratuberculosis on Milk Production, Fertility, and Culling in Large Commercial Hungarian Dairy Herds.

Paratuberculosis (PTBC) is a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), which is common in dairy herds worldwide, although the scale of its impact on herd productivity is unclear. The aim of our study was to determine the differences between MAP ELISA positive vs. negative cows in terms of milk production and quality, reproductive parameters, and culling. The data of five large dairy herds that participated in the voluntary PTBC testing program in Hungary were analyzed. Cows were tested by ELISA (IDEXX Paratuberculosis Screening Ab Test, IDEXX Laboratories, Inc., Westbrook, ME, USA) using milk samples collected during official performance testing. The outcome of the initial screening test involving all milking cows in the herds was used for the classification of the cows. The 305-day milk production, reproduction and culling data of 4,341 dairy cows, and their monthly performance testing results (n = 87,818) were analyzed. Multivariate linear and logistic models, and right censored tobit model were used for the statistical analysis. Test-day and 305-day milk production of ELISA positive cows decreased by 4.6 kg [95% CI: 3.5-5.6 kg, P < 0.0001 (-13.2%)] and 1,030 kg [95% CI: 708-1,352 kg, P < 0.0001 (-9.4%)], compared to their ELISA negative herdmates, respectively. Milk ELISA positive cows had 35.8% higher [95% CI: 17.9-56.4%, P < 0.0001] somatic cell count, on average. Test positive cows conceived 23.2 days later [95% CI: 9.2-37.3 days, P = 0.0012 (+16.5%)] and their calving interval was 33.8 days longer [95% CI: 13.2-54.4 days, P = 0.0013, (+9.7%)], compared to the negative cows, on average. Milk ELISA positive cows were less likely to conceive to first insemination (odds ratio: 0.49, 95% CI: 0.31-0.75, P = 0.0013), and required 0.42 more inseminations to conceive [95% CI: 0.07-0.77, P = 0.0192 (+13.7%)], on average. Milk ELISA positive cows were culled 160.5 days earlier after testing compared to their ELISA negative herdmates (95% CI: 117.5-203.5 days, P < 0.0001). Our results suggest that MAP ELISA positive cows experience decreased milk production, milk quality, fertility, and longevity, which supports the need to control the prevalence of PTBC in dairy herds.

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.

A critical evaluation of some of the recent so-called 'evidence' for the involvement of vertebrate-type sex steroids in the reproduction of mollusks.

Many studies on the control of reproduction in mollusks have focused on hormones (and proteins associated with the production and signaling of those hormones) which were originally discovered in humans, in the belief that if they are also present in mollusks, they must have the same role. However, although human sex steroids can be found in mollusks, they are so readily absorbed that their presence is not necessarily evidence of endogenous synthesis. A homolog of the vertebrate nuclear estrogen receptor has been found in mollusks, but it does not bind to estrogens or indeed to any steroid at all. Antibodies against human aromatase show positive immunostaining in mollusks, yet the aromatase gene has not been found in the genome of any invertebrates (let alone mollusks). This review will deal with these and other examples of contradictory evidence for a role of human hormones in invertebrate reproduction.

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.