Considerations for fatty acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism.

Fatty acid accumulation was studied in the parthenogenetic all-female marbled crayfish Procambarus virginalis using six arbitrarily designed experimental feeds and related to individuals with glair glands (sexual maturity) after 100 days of ad libitum feeding at 21 °C, including gravid females from the wild as a reference. Fatty acids 16:0 and 18:1n-9 comprised 40% of the total amount of fatty acids and tended to up-concentrate in bodies. Shorter chain 14:0 depleted from feed to body. Across diets, there was a concomitant decrease in precursor fatty acid and increase in product fatty acid, such as reinforcements in monounsaturated fatty acid (18:1n-9), eicosanoid precursors 20:4n-6 (arachidonic acid, ARA) and 20:5n-3 (eicosapentaenoic acid, EPA) in-vivo, but not 22:6n-3 (docosahexaenoic acid, DHA) except when deficient in CHI or CHI + SPI diets. Saturation kinetics modeling (R2 0.7-0.9, p < 0.05) showed that when the ARA share is ~ 1%, the EPA share is ~ 8%, and the DHA share is ~ 2% in the food lipids, the accumulation of fatty acids in body lipids levels off. The lowest DHA in the CHI (0% glair glands) or CHI + SPI (0-3.9% glair glands) diets, and the lowest ARA in SER (0% glair glands) or SER + SPI (0-3% glair glands) diets, were synchronous with negligible sexual maturity despite a wide range of observed specific growth rates (2.77-3.60% per day), body size (0.44-0.84 g), ≤ 5% crude lipid and 40-46% crude protein feed. The FISH and SHRIMP diets (56% protein, 11-14% lipid) with the highest ARA, EPA, and DHA together seem to be the most conducive diets for sexual maturity (up to 20% of individuals with glair glands). We propose a fatty acid profile mimicking the FISH or SHRIMP diets as a starting point for designing the lipid content required in the marbled crayfish standardized reference diet.

Considerations for protein and amino acids in standardized reference diet for parthenogenetic marbled crayfish Procambarus virginalis model organism.

The concept of a standardized reference diet (SRD) is used in laboratory model organisms to ensure nutritional control between studies and laboratories. Although models using the genetically identical, all female parthenogenetic marbled crayfish (Procambarus virginalis) are growing in popularity, research into nutrition in this species still has many knowledge gaps. To fast track the development of a SRD in terms of protein and amino acids (SRDprotein) for this species, we first analyzed the composition of its body amino acids to determine the ideal protein concept (IPC) of indispensable amino acids in wild-caught P. virginalis (which had an unusually high preponderance of leucine and arginine). Then, we strategically evaluated three common clusters of types of fish feed: (1) ornamental fish feed (SER) fortified with a naturally occurring alga (Spirulina). This type of feed was protein-high in arginine and leucine (SER + SPI) that fulfils the species' IPC for iso-protein (~ 40%), iso-phosphorus (~ 0.8%) and near iso-energetic (~ 475 kcal 100 g-1); (2) freeze-dried live feed consisting of chironomid larvae (CHI) fortified with Spirulina (CHI + SPI) that fulfils the IPC for iso-protein (~ 46%), iso-phosphorus (~ 0.7%) and near iso-energetic (~ 405 kcal 100 g-1); and (3) a commercially standardized 'starter diet' for carnivorous fish larvae (FISH) and post-larval shrimps (SHRIMP) with iso-protein (~ 56%) and iso-phosphorus (~ 1.6%). A total of six diets, embracing a diverse range of proteinaceous feeds, were used in a 100-day ad libitum feeding and growth trial. The FISH group outperformed all the other groups (p < 0.05) and our exploratory multivariate analysis revealed an ideal demand of > 44% protein (tailored to deliver high arginine 3% and leucine 4%, followed by the usual lysine > 3.5% and methionine 1.2%) but also the lowest carbohydrate level (21%). For SRDprotein, our findings show that the FISH diet is ideal and suggest the possibilities of using a CHI + SPI diet for further optimization (more economic use of protein and phosphorus).

Risk Perception: Chemical Stimuli in Predator Detection and Feeding Behaviour of the Invasive Round Goby Neogobius melanostomus.

The round goby Neogobius melanostomus is a notoriously invasive fish originating from the Ponto-Caspian region that in recent decades has successfully spread across the globe. One of its primary impacts is direct predation; in addition, when entering new ecosystems, the round goby is likely to become a food resource for many higher native predators. However, little is known either about the indirect effects of predators on the round goby as prey or its feeding behaviour and activity. The non-consumptive effect of the presence of higher native predators presumably plays an important role in mitigating the impact of non-native round gobies as mesopredators on benthic invertebrate communities, especially when both higher- and mesopredators occupy the same habitat. We tested the food consumption probability and gut evacuation rates in round gobies in response to chemical signals from a higher predator, the European eel Anguilla anguilla. Gobies were placed individually in experimental arenas equipped with shelters and exposed to water from a tank in which (a) the higher predator had actively preyed on a heterospecific prey, earthworms Lumbricus sp. (the heterospecific treatment; HS); (b) the higher predator had fed on round gobies (the conspecific treatment; CS); or (c) the water was provided as a control treatment (C). To ensure exposure to the chemical stimuli, this study incorporated the application of skin extracts containing damaged-released alarm cues from the CS treatment; distilled water was used for the remaining treatments. No significant differences were observed in either the food consumption probability or gut evacuation rate in the tested treatments. Despite the lack of reaction to the chemical stimuli, round gobies did exhibit high evacuation rates (R = 0.2323 ± 0.011 h-1; mean ± SE) in which complete gut clearance occurred within 16 h regardless of the applied treatment. This rapid food processing suggests high efficiency and great pressure on resources regardless of the presence or not of a higher predator. These findings hint at the boldness of round gobies, which did not exhibit any pronounced threat sensitivity. This would seem to suggest great efficiency in food processing and a potential competitive advantage over local native species when colonising new ecosystems, irrespective of the presence of native predators. Our study did not detect any non-consumptive effect attributable to the higher predator, given that the feeding activity of the invasive round goby was not altered.

Taming the terminological tempest in invasion science.

Standardised terminology in science is important for clarity of interpretation and communication. In invasion science - a dynamic and rapidly evolving discipline - the proliferation of technical terminology has lacked a standardised framework for its development. The result is a convoluted and inconsistent usage of terminology, with various discrepancies in descriptions of damage and interventions. A standardised framework is therefore needed for a clear, universally applicable, and consistent terminology to promote more effective communication across researchers, stakeholders, and policymakers. Inconsistencies in terminology stem from the exponential increase in scientific publications on the patterns and processes of biological invasions authored by experts from various disciplines and countries since the 1990s, as well as publications by legislators and policymakers focusing on practical applications, regulations, and management of resources. Aligning and standardising terminology across stakeholders remains a challenge in invasion science. Here, we review and evaluate the multiple terms used in invasion science (e.g. 'non-native', 'alien', 'invasive' or 'invader', 'exotic', 'non-indigenous', 'naturalised', 'pest') to propose a more simplified and standardised terminology. The streamlined framework we propose and translate into 28 other languages is based on the terms (i) 'non-native', denoting species transported beyond their natural biogeographic range, (ii) 'established non-native', i.e. those non-native species that have established self-sustaining populations in their new location(s) in the wild, and (iii) 'invasive non-native' - populations of established non-native species that have recently spread or are spreading rapidly in their invaded range actively or passively with or without human mediation. We also highlight the importance of conceptualising 'spread' for classifying invasiveness and 'impact' for management. Finally, we propose a protocol for classifying populations based on (i) dispersal mechanism, (ii) species origin, (iii) population status, and (iv) impact. Collectively and without introducing new terminology, the framework that we present aims to facilitate effective communication and collaboration in invasion science and management of non-native species.

Effects of chemical cues and prior experience on predator avoidance in crayfish.

Multisensory stimuli provide organisms with information to assess the threat present in the surroundings. Olfactory cues show dominance over other sensory modalities in the aquatic environment. The impact of chemical predator cues combined with experiences gained (learning) in species without previous contact is not fully understood. We investigated the foraging and shelter-seeking behaviour of naïve and experienced marbled crayfish Procambarus virginalis juveniles in response to the chemical signals of pumpkinseed Lepomis gibbosus alone and in combination with alarm chemicals produced by preyed-upon conspecifics. Naïve and experienced (previously exposed to pumpkinseed predation) juveniles were stocked in an arena with shelter and feed and exposed (1) to water from a tank containing a predator actively feeding on conspecifics, (2) water from a tank with predator only and (3) water only as control. Crayfish exposed to the combined stimuli avoided the inlet zone and gravitated to shelter zone of the arena to a greater extent than did those exposed to predator-only cues and the control. Regardless of the treatment, experienced crayfish showed significantly reduced interest in feeding. Our findings imply that crayfish response to threat-associated odours with the greatest potency when visual or tactile cues are present, while previous encounters with predators may make them more cautious.

Quantification of proteomic profile changes in the hemolymph of crayfish during in vitro coagulation.

Hemolymph is the circulatory fluid that fills the body cavity of crustaceans, analogous to blood in vertebrates. Hemolymph coagulation, similar to blood clotting in vertebrates, plays a crucial role in wound healing and innate immune responses. Despite extensive studies on the clotting process in crustaceans, no comparative quantitative analysis of the protein composition of non-clotted and clotted hemolymph in any decapod has been reported. In this study, we used label-free protein quantification with high-resolution mass spectrometry to identify the proteomic profile of hemolymph in crayfish and quantify significant changes in protein abundances between non-clotted and clotted hemolymph. Our analysis identified a total of two-hundred and nineteen proteins in both hemolymph groups. Furthermore, we discussed the potential functions of the top most high and low-abundant proteins in hemolymph proteomic profile. The quantity of most of the proteins was not significantly changed during coagulation between non-clotted and clotted hemolymph, which may indicate that clotting proteins are likely pre-synthesized, allowing for a swift coagulation response to injury. Four proteins still showed abundance differences (p < 0.05, fold change>2), including C-type lectin domain-containing proteins, Laminin A chain, Tropomyosin, and Reverse transcriptase domain-containing proteins. While the first three proteins were down-regulated, the last one was up-regulated. The down-regulation of structural and cytoskeletal proteins may affect the process of hemocyte degranulation needed for coagulation, while the up-regulation of an immune-related protein might be attributed to the phagocytosis ability of viable hemocytes during coagulation.

Effect of psychoactive substances on cardiac and locomotory activity of juvenile marbled crayfish Procambarus virginalis.

Pharmaceutically active compounds are common and increasing in the aquatic environment. Evidence suggests they have adverse effects on non-target organisms, and they are classified as emerging pollutants for a variety of aquatic organisms. To determine the effects of environmentally relevant levels of psychoactive compounds on non-target organisms, we analyzed cardiac and locomotory activity in early developmental stages of marbled crayfish Procambarus virginalis. Responses to sertraline, methamphetamine, and a mixture of citalopram, oxazepam, sertraline, tramadol, venlafaxine, and methamphetamine at a concentration of 1 µg L-1 of each compound were assessed. On day four of exposure, cardiac activity was recorded for 5 min, and on day eight, locomotory activity was recorded for 15 min. There was a significant increase (p < 0.01) in heart rate in methamphetamine-exposed and Mix-exposed juveniles compared to the unexposed control and there was significant difference (p < 0.01) in proportion of time (activity %) was observed with sertraline-exposed, whereas velocity, and distance moved did not significantly differ (p > 0.05) in exposed and control animals. These findings revealed that low concentrations of chemicals and their mixtures can modify the physiological state of aquatic animals without outward manifestations (activity, distance moved, and velocity). Aquatic animals can be impacted earlier than is visible, but effects can potentially lead to substantial changes in populations and in ecosystem processes. Additional research to investigate chemical combinations, exposure systems, and organism physiological and molecular responses may provide evidence of broad impact of environmental pharmaceuticals.

Short-term effects of an environmentally relevant concentration of organic UV filters on signal crayfish Pacifastacus leniusculus.

Personal care products, including organic UV filters, are considered emerging contaminants, with their toxic effects being a concern in recent decades. UV filters continually enter surface waters via wastewater and human activity. Despite the presence of organic UV filters in the freshwater environment, little is known of their impact on aquatic biota. In this study, we evaluated the cardiac and locomotor responses of signal crayfish Pacifastacus leniusculus exposed to environmentally relevant concentrations of either 2-Phenylbenzimidazole-5-sulfonic acid (PBSA, 3 µg/L) or 5-Benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid (BP4, 2.5 µg/L). Specimens exposed to the tested compounds for 30 min exhibited significantly greater changes in distance moved and time active than did unexposed controls. Significant differences of mean heart rate change compared to control were detected in both PBSA and BP4 experimental groups. Such behavior and physiological alterations demonstrate ecological effects of personal care products with the tested sunscreen compounds even with a short exposure. Evidence of the consequences of organic UV filters on aquatic organisms is scarce and is an important topic for future research.

Granules of immune cells are the source of organelles in the regenerated nerves of crayfish antennae.

Regeneration refers to the regrowing and replacing of injured or lost body parts. Crayfish antennae are nervous organs that are crucial for perceiving environmental signals. Immune cells (hemocytes) are responsible for neurogenesis in crayfish. Here, we used transmission electron microscopy to investigate at ultrastructural levels the potential roles of immune cells in nerve regeneration in crayfish antennae after amputation. The results showed that, while all three types of hemocytes were observed during nerve regeneration, granules of semi-granulocytes and granulocytes are the main sources of new organelles such as mitochondria, the Golgi apparatus and nerve fibres in the regenerated nerves of crayfish antennae. We describe the transformation of immune cell granules into different organelles in the regenerating nerve at ultrastructural levels. Also, we observed that the regeneration process speeds up after crayfish moulting. In conclusion, the granules are compacted packages of versatile materials carried by immune cells and can be converted into different organelles during nerve regeneration in crayfish antennae.

Strong temporal variation of consumer δ13C value in an oligotrophic reservoir is related to water level fluctuation.

Using stable carbon and nitrogen isotope analysis (δ13C and δ15N) to assess trophic interactions in freshwater ecosystems is a well established method, providing insight into ecosystem functioning. However, the spatial and temporal variability of isotope values, driven by environmental fluctuation is poorly understood and can complicate interpretations. We investigated how the temporal variation of stable isotopes in consumers (fish, crayfish and macrozoobenthos) of a canyon-shaped oligotrophic reservoir is associated with environmental factors such as water temperature, transparency, flooded area, and water quality measures. Consumers and their putative food sources were sampled and analyzed for carbon and nitrogen stable isotopes annually, and environmental parameters were measured monthly from 2014 to 2016. Results revealed significant differences in δ13C and δ15N values in each consumer among studied years. Over the years, fish and crayfish expressed differences in δ13C between 3 and 5‰, whereas in zoobenthos differences were 12‰. Variability in δ15N was similar across all consumers (2-4‰). Moreover, results suggest that the flooded area of the reservoir was a major driver of δ13C stable isotope values variation in consumers, while variation in δ15N was not linked to any of the studied environmental factors. Bayesian mixing models further showed significant changes in the origin of detritivorous zoobenthos carbon sources (reversal shift from terrestrial detritus to algae origin) between years with low water level to years with the standard water level. Other species showed only slight differences in food source utilization among years. Our study highlights the importance of environmental factors as sources of variation in consumer's stable isotope values which should be considered especially when studied ecosystem strongly fluctuate in some environmental factor.

Comparative ultrastructure of the antennae and sensory hairs in six species of crayfish.

Background: Antennae in crayfish are essential for gaining information about the local topography and localising food, chemicals, conspecifics or predator. There are still gaps in the research on the morphology of antennae in decapods compared to other arthropods. Methodology: Biometrical and ultrastructural methods were applied using light and cryo-scanning electron microscopies to study the morphology of antennae in six different crayfish species, including marbled crayfish Procambarus virginalis, Mexican dwarf crayfish Cambarellus patzcuarensis, red swamp crayfish Procambarus clarkii, signal crayfish Pacifastacus leniusculus, common yabby Cherax destructor, and spiny-cheek crayfish Faxonius limosus to find their potential morphological differences. Results: Significant differences in the antenna length, length and width of each segment to carapace length ratios, and the number of segments were found in the six crayfish species. The ultrastructure revealed differences in the distribution of sensory hairs on the antenna and the morphology of the antennal surface. Conclusions: The different morphology of antennae might reflect adaptation to the conditions of their specific habitats. In addition, results showed that a combination of differences in the morphological features and biometrical measurements of antennae could be used for the distinguishment of different studied crayfish species.

Comparison of Behavior and Space Use of the European Bullhead Cottus gobio and the Round Goby Neogobius melanostomus in a Simulated Natural Habitat.

The round goby is an invasive fish in Europe and North America that threatens native species by predation and competition. Its habitat preferences are similar to those of the European bullhead, which it displaces from shelters and out-competes for available resources. We assessed the microhabitat preferences, shelter use, and activity of the round goby and European bullhead in single-species experiments in habitat simulator systems to investigate their behavior in a novel environment. Fish were video-recorded for 28 h in the presence of shelter and feed with water velocity ranging from 0.00 to 0.96 m s-1. The two species showed similar behavior under given conditions. A primary difference was in stress-induced behavior in the initial phases of observation. The round goby spent more time in movement when outside the shelter and a longer time in the escape zone in the exploration period during light. Our results confirmed a significant preference of round goby for low velocity areas and a preference for higher velocities in the European bullhead. Both species were able to cope with velocities > 0.7 m s-1. Therefore, the reported invasion success of round goby is probably not driven by space use or activity patterns, but rather by higher adaptability.

The Invasive Round Goby Neogobius melanostomus as a Potential Threat to Native Crayfish Populations.

Despite the spread of round goby Neogobius melanostomus into freshwater streams, there is a lack of information with respect to its effect on macroinvertebrate communities, especially crustaceans. We studied foraging efficiency of N. melanostomus on Procambarus virginalis and Asellus aquaticus, using a functional response (FR) approach. Stocking density of the prey species was manipulated to determine its effect on consumer utilization, with prey offered separately or combined at 1:1, 3:1, and 1:3 at each tested density. For both prey species, N. melanostomus exhibited type II FR, occasionally with a high proportion of non-consumptive mortality. Procambarus virginalis suffered a significantly higher attack rate compared to A. aquaticus. Neogobius melanostomus killed significantly more of the most prevalent prey, regardless of species. In trials with prey species of equal proportions, a difference in the number of each species killed was observed only at the highest density, at which P. virginalis was preferred. Neogobius melanostomus may be an important driver of population dynamics of prey species in the wild. The non-selective prey consumption makes N. melanostomus a potential threat to macrozoobenthic communities of river tributaries.

Survival, Growth, and Reproduction: Comparison of Marbled Crayfish with Four Prominent Crayfish Invaders.

Biological invasions are increasingly recognized ecological and economic threats to biodiversity and are projected to increase in the future. Introduced freshwater crayfish in particular are protruding invaders, exerting tremendous impacts on native biodiversity and ecosystem functioning, as exemplified by the North American spiny-cheek, signal and red swamp crayfish as well as the Australian common yabby. The marbled crayfish is among the most outstanding freshwater crayfish invaders due to its parthenogenetic reproduction combined with early maturation and high fecundity. As their introduced ranges expand, their sympatric populations become more frequent. The question of which species and under what circumstances will dominate in their introduced communities is of great interest to biodiversity conservation as it can offer valuable insights for understanding and prioritization of management efforts. In order to examine which of the aforementioned species may be more successful as an invader, we conducted a set of independent trials evaluating survival, growth, claw injury, and reproduction using single-species stocks (intraspecific interactions) and mixed stocks (interspecific interactions) of marbled crayfish vs. other crayfish invaders since the onset of exogenous feeding. In both single and mixed stocks, red swamp crayfish and yabby grew faster than marbled crayfish, while marbled crayfish were superior to both spiny-cheek and signal crayfish in terms of growth. With the exception of signal crayfish, the faster-growing species consistently reached a higher survival rate. The faster-growing species tended to negatively impair smaller counterparts by greater claw injury, delayed maturation, and reduced fecundity. Only marbled crayfish laid eggs as early as 14 weeks in this study, which is earlier than previously reported in the literature. Thus, the success of marbled crayfish among invasive crayfish is significantly driven by relatively fast growth as well as an early and frequent reproduction. These results shed light on how interactions between invasive populations can unfold when their expansion ranges overlap in the wild, thereby contributing to the knowledge base on the complex population dynamics between existing and emerging invasive species.

The effect of chronic exposure to chloridazon and its degradation product chloridazon-desphenyl on signal crayfish Pacifastacus leniusculus.

The effects of chloridazon (Ch) and its metabolite chloridazon-desphenyl (Ch-D) at the environmentally relevant concentrations of 0.45 µg/L and 2.7 µg/L on signal crayfish Pacifastacus leniusculus were assessed in a 30-day exposure followed by a 15-day depuration period. Locomotion, biochemical haemolymph profile, oxidative and antioxidant parameters, and histopathology were evaluated. Crayfish exposed to Ch at 0.45 µg/L and 2.7 µg/L showed significantly (p < 0.01) higher CAT activity and GSH level in hepatopancreas and gill compared to controls. The concentration of Ch at 2.7 µg/L was associated with significantly (p < 0.01) higher levels of GLU, LACT, ALT, AST in haemolymph compared to controls. Chloridazon-desphenyl exposure at both tested concentrations caused significantly higher (p < 0.01) GLU, LACT, ALT, AST, NH3, and Ca in haemolymph; lipid peroxidation (TBARS) levels in hepatopancreas; and CAT activity and GSH level in hepatopancreas and gill. Alterations of structure including focal dilatation of tubules, increased number of fibrillar cells, and haemocyte infiltration in the interstitium were observed with 2.7 µg/L Ch and with both Ch-D exposures. Locomotion patterns did not vary significantly among groups. A 15-day recovery period was insufficient to restore normal physiological parameters in exposed groups. Chloridazon and its metabolite Ch-D exerts harmful effects on crayfish.

Genetic diversity, phylogenetic position and morphometric analysis of Astacus colchicus (Decapoda, Astacidae): a new insight into Eastern European crayfish fauna.

The phylogeny of European crayfish fauna, especially with respect to Eastern European species, is still far from being completely resolved. To fill this gap, we analyzed most of the European crayfish species focusing on the phylogenetic position of the endemic crayfish Astacus colchicus, inhabiting Georgia. Three mitochondrial and one nuclear marker were used to study evolutionary relationships among European crayfish species, resulting in the unique phylogenetic position of A. colchicus indicating independent species status to A. astacus. Phylogenetic analyses revealed a deep molecular divergence of A. colchicus in comparison to A. astacus (6.5-10.9% in mtDNA and 1.1% in nDNA) as well as to Pontastacus leptodactylus and P. pachypus (5.5-10.0% in mtDNA and 1.4-2.4% in nDNA). Absent ventral process on second male pleopod and abdominal somites II and III with pleura rounded lacking prominent spines clearly indicate taxonomic assignment to the genus Astacus; however, the species is distributed almost in the middle of Ponto-Caspian area typical by occurrence of the genus Pontastacus. Several morphological indices linked to head length, carapace, and total body length and width were found to demonstrate apparent differences between A. colchicus and A. astacus. Although this study provides a novel insight into European crayfish phylogeography, we also point out the gaps in comprehensive study of the P. leptodactylus species complex, which could reveal details about the potential species status of particular species and subspecies within this genus.

A combination of six psychoactive pharmaceuticals at environmental concentrations alter the locomotory behavior of clonal marbled crayfish.

Pharmaceutically active compounds (PhACs) are ubiquitous in the aquatic environment worldwide and considered emerging contaminants. Their effects on growth, behavior, and physiological processes of aquatic organisms have been identified even at very low concentrations. Ecotoxicological investigations have primarily focused on single compound exposure, generally at a range of concentrations. In the natural environment, pollutants seldom occur in isolation, but little is known about the effects and risks of combinations of chemicals. This study aimed to investigate the effects of concurrent exposure to six psychoactive PhACs on locomotory behavior and life history traits of clonal marbled crayfish Procambarus virginalis. Crayfish were exposed to ~1 µg L-1 of the antidepressants sertraline, citalopram, and venlafaxine; the anxiolytic oxazepam; the opioid tramadol; and the widely abused psychostimulant methamphetamine. In the absence of shelter, exposed crayfish moved significantly shorter distances and at lower velocity and showed significantly less activity than controls. With available shelter, exposed crayfish moved significantly more distance, showed higher activity, and spent a significantly more time outside the shelter than controls. Molting, mortality, and spawning frequency did not vary significantly between the groups. Hemolymph glucose level did not vary among groups and was not correlated with observed behaviors. Results suggest that environmental concentrations of the tested compounds in combination can alter the behavior of non-target aquatic organisms as individual exposure of these compounds, which may lead to disruption of ecosystem processes due to their reduced caution in polluted conditions. Further research is needed using varied chemical mixtures, exposure systems, and habitats, considering molecular and physiological processes connected to behavior alterations.

The effects of the herbicides terbuthylazine and metazachlor at environmental concentration on the burrowing behaviour of red swamp crayfish.

Despite their low concentrations in many aquatic environments, evidence exists to suggest that herbicides do affect non-target organisms. Given that burrowing is a primary life-history trait in crayfish, herbicides could potentially have serious negative effects on these ecologically important freshwater macroinvertebrates. In this study, we exposed the red swamp crayfish Procambarus clarkii to terbuthylazine (a triazine) and metazachlor (a chloroacetanilide) at an environmental concentration of 2.0 µg/L for 28 days, and then observed their burrowing behaviour for two days. The metazachlor-exposed males excavated a greater number of burrows than the other tested groups, with comparable depths and volumes relative to individual specimen weight. The relative depth and volume of female burrows were identical in all groups. The natural habit of female crayfish of constructing deeper burrows than males was marginally significant in the control and META groups but was not significant for relative volume. The hypothesized adverse effects of chronic exposure to real environmental concentrations of herbicides were not documented in terms of either relative depth or volume. However, the increased number of burrows in metazachlor-exposed animals may mean that this invasive species will cause greater damage to embankments and river banks. The mechanisms behind these effects require closer study.

Effects of chloridazon on early life stages of marbled crayfish.

The effects of chloridazon exposure at concentrations of 2.7 µg/L (maximal real environmental concentration in the Czech Republic), 27 µg/L, 135 µg/L and 270 µg/L on early life stages of marbled crayfish (Procambarus virginalis) were evaluated. Significantly higher glutathione S-transferase activity and reduced glutathione level was observed at all tested concentrations of chloridazon compared with the control. Chloridazon in concentrations 27, 135 and 270 µg/L caused delay ontogenetic development and slower growth. Histopathological changes in hepathopancreas were found in two highest tested concentrations (135 µg/L and 270 µg/L). Crayfish behaviour was not altered in control vs. exposed animals, while the activity parameters tend to decline with increasing chloridazon concentrations.

Cardiac and Locomotor Responses to Acute Stress in Signal Crayfish Pacifastacus leniusculus Exposed to Methamphetamine at an Environmentally Relevant Concentration.

Methamphetamine (METH), a central nervous system stimulant used as a recreational drug, is frequently found in surface waters at potentially harmful concentrations. To determine effects of long-term exposure to environmentally relevant levels on nontarget organisms, we analysed cardiac and locomotor responses of signal crayfish Pacifastacus leniusculus to acute stress during a 21-day exposure to METH at 1 µg L-1 followed by 14 days depuration. Heart rate and locomotion were recorded over a period of 30 min before and 30 min after exposure to haemolymph of an injured conspecific four times during METH exposure and four times during the depuration phase. Methamphetamine-exposed crayfish showed a weaker cardiac response to stress than was observed in controls during both exposure and depuration phases. Similarly, methamphetamine-exposed crayfish, during METH exposure, showed lower locomotor reaction poststressor application in contrast to controls. Results indicate biological alterations in crayfish exposed to METH at low concentration level, potentially resulting in a shift in interactions among organisms in natural environment.