Crayfish are emerging as model organisms for various disciplines. Moreover, decapod crustaceans also exhibit pain-like reactions and heightened anxiety when exposed to harmful stimuli, leading to short-term or persistent behavioral shifts. Awareness of decapod crustacean sentience and thus, suffering calls for refinement of current laboratory protocols. This study aims to enhance the standard methodology for injecting substances into crayfish by minimizing stress-inducing manipulation. We examined the impacts of various administration routes on the persistence of injected chemicals in marbled crayfish, its excretion, and animal survival. Fluorescein dye was used as a visual marker. It was administered via three alternative injection routes-intracoelomic (IC), intrapericardial administration through areola (IP-A), and intrapericardial administration through arthrodial membrane (IP-AM). Continuous video observations were made for a 4-h period under UV light, followed by intermittent observations at 12-h intervals over 48 h. The highest mortality (20%) was observed in IP-A administration. The IP-A method also provided the fastest systemic distribution of the dye in the body. Results indicated visibly higher urination frequency in IP-AM compared to IP-A. IC mirrored IP-AM outcomes without any observed mortality. We conclude that IC administration proved superior to intrapericardial methods, offering the least harmful but effective approach for crayfish injections.

School of Natural Sciences University of Hull Hull UK

South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Faculty of Fisheries and Protection of Waters University of South Bohemia in České Budějovice České Budějovice Czech Republic

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“Animal Welfare (Sentience) Bill.” UK Parliament. 2022. 1–5. https://bills.parliament.uk/bills/2867.

Bergman, D. A., A. L. Martin, and P. A. Moore. 2005. “Control of Information Flow Through the Influence of Mechanical and Chemical Signals During Agonistic Encounters by the Crayfish, Orconectes rusticus.” Animal Behaviour 70, no. 3: 485–496.

Bergman, D. A., C. N. Redman, K. C. Fero, J. L. Simon, and P. A. Moore. 2006. “The Impacts of Flow on Chemical Communication Strategies and Fight Dynamics of Crayfish.” Marine and Freshwater Behaviour and Physiology 39, no. 4: 245–258.

Berry, F. C., and T. Breithaupt. 2010. “To Signal or Not to Signal? Chemical Communication by Urine‐Borne Signals Mirrors Sexual Conflict in Crayfish.” BMC Biology 8: 25.

Breithaupt, T. 2011. “Chemical Communication in Crayfish.” In Chemical Communication in Crustaceans, edited by T., Breithaupt and M. Thiel, 257–276. NY: Springer New York.

Breithaupt, T., and P. Eger. 2002. “Urine Makes the Difference.” Journal of Experimental Biology 205, no. 9: 1221–1231.

Breithaupt, T., D. P. Lindstrom, and J. Atema. 1999. “Urine Release in Freely Moving Catheterised Lobsters (Homarus americanus) With Reference to Feeding and Social Activities.” Journal of Experimental Biology 202, no. 7: 837–844.

Chang, Y. H., R. Kumar, T. H. Ng, and H. C. Wang. 2018. “What Vaccination Studies Tell Us About Immunological Memory Within the Innate Immune System of Cultured Shrimp and Crayfish.” Developmental & Comparative Immunology 80: 53–66.

Crump, A., H. Browning, A. Schnell, C. Burn, and J. Birch. 2022. “Sentience in Decapod Crustaceans: A General Framework and Review of the Evidence.” Animal Sentience 7, no. 32: 1. https://www.wellbeingintlstudiesrepository.org/cgi/viewcontent.cgi?article=1691&context=animsent.

Diggles, B. K., R. Arlinghaus, H. I. Browman, et al. 2024. “Reasons to Be Skeptical About Sentience and Pain in Fishes and Aquatic Invertebrates.” Reviews in Fisheries Science & Aquaculture 32, no. 1: 127–150.

Dyuizen, I. V., E. P. Kotsyuba, and N. E. Lamash. 2012. “Changes in the Nitric Oxide System in the Shore Crab Hemigrapsus sanguineus (Crustacea, Decapoda) CNS Induced by a Nociceptive Stimulus.” Journal of Experimental Biology 215, no. 15: 2668–2676.

Edwards, D. H., W. J. Heitler, and F. B. Krasne. 1999. “Fifty Years of a Command Neuron: The Neurobiology of Escape Behavior in the Crayfish.” Trends in Neurosciences 22, no. 4: 153–161.

Fossat, P., J. Bacqué‐Cazenave, P. De Deurwaerdère, D. Cattaert, and J. P. Delbecque. 2015. “Serotonin, but Not Dopamine, Controls the Stress Response and Anxiety‐Like Behavior in the Crayfish Procambarus clarkii.” Journal of Experimental Biology 218, no. 17: 2745–2752.

Fossat, P., J. Bacqué‐Cazenave, P. De Deurwaerdère, J. P. Delbecque, and D. Cattaert. 2014. “Anxiety‐Like Behavior in Crayfish Is Controlled by Serotonin.” Science 344, no. 6189: 1293–1297.

Goessmann, C., C. Hemelrijk, and R. Huber. 2000. “The Formation and Maintenance of Crayfish Hierarchies: Behavioral and Self‐Structuring Properties.” Behavioral Ecology and Sociobiology 48: 418–428.

Gutekunst, J., R. Andriantsoa, C. Falckenhayn, et al. 2018. “Clonal Genome Evolution and Rapid Invasive Spread of the Marbled Crayfish.” Nature Ecology & Evolution 2, no. 3: 567–573.

Hawkins, P., S. Brookes, and J. Bussell, et al. 2019. Avoiding Mortality in Animal Research and Testing. United Kingdom: University of Cambridge: RSPCA Research Animals Department, RSPCA Research Animals Department.

Hawkins, P., D. B. Morton, and O. Burman, et al. 2011. “A Guide to Defining and Implementing Protocols for the Welfare Assessment of Laboratory Animals: Eleventh Report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement.” Laboratory Animals 45, no. 1: 1–13.

Holdich, D. M. 2002. Biology of Freshwater Crayfish, Vol. 72. Oxford: Blackwell Science.

Hossain, M. S., J. Kubec, W. Guo, et al. 2021. “A Combination of Six Psychoactive Pharmaceuticals at Environmental Concentrations Alter the Locomotory Behavior of Clonal Marbled Crayfish.” Science of the Total Environment 751: 141383.

Hossain, M. S., J. Patoka, A. Kouba, and M. Buřič. 2018. “Clonal Crayfish as Biological Model: A Review on Marbled Crayfish.” Biologia 73, no. 9: 841–855.

Imeh‐Nathaniel, A., V. Orfanakos, L. Wormack, R. Huber, and T. I. Nathaniel. 2019. “The Crayfish Model (Orconectes rusticus), Epigenetics and Drug Addiction Research.” Pharmacology Biochemistry and Behavior 183: 38–45.

Kamada, S., and T. Nagayama. 2021. “Anxiety Induces Long‐Term Memory Forgetting in the Crayfish.” Journal of Comparative Physiology A 207, no. 4: 459–467.

Kouba, A., M. Buřič, and P. Kozák. 2010. “Bioaccumulation and Effects of Heavy Metals in Crayfish: A Review.” Water, Air, & Soil Pollution 211, no. 1–4: 5–16.

Kouba, A., B. Lipták, J. Kubec, et al. 2021. “Survival, Growth, and Reproduction: Comparison of Marbled Crayfish With Four Prominent Crayfish Invaders.” Biology 10, no. 5: 422.

Liu, H., R. Chen, Q. Zhang, H. Peng, and K. Wang. 2011. “Differential Gene Expression Profile From Haematopoietic Tissue Stem Cells of Red Claw Crayfish, Cherax quadricarinatus, in Response to WSSV Infection.” Developmental & Comparative Immunology 35, no. 7: 716–724.

Longshaw, M. 2011. “Diseases of Crayfish: A Review.” Journal of Invertebrate Pathology 106, no. 1: 54–70.

Nguyen, K. Y., K. Sakuna, R. Kinobe, and L. Owens. 2014. “Ivermectin Blocks the Nuclear Location Signal of Parvoviruses in Crayfish, Cherax quadricarinatus.” Aquaculture 420–421: 288–294.

Palillo, M., J. Palillo, N. Williams, et al. 2022. “Anesthesia With Tricaine Methanesulfonate (MS222) and Propofol and Its Use for Computed Tomography of Red Swamp Crayfish (Procambarus clarkii).” Journal of the American Association for Laboratory Animal Science 61, no. 3: 275–282.

Rotllant, G., P. Llonch, J. A. García del Arco, Ò. Chic, P. Flecknell, and L. U. Sneddon. 2023. “Methods to Induce Analgesia and Anesthesia in Crustaceans: A Supportive Decision Tool.” Biology 12, no. 3: 387.

Safari, O., D. Shahsavani, M. Paolucci, and M. M. S. Atash. 2014. “Single or Combined Effects of Fructo‐ and Mannan Oligosaccharide Supplements on the Growth Performance, Nutrient Digestibility, Immune Responses and Stress Resistance of Juvenile Narrow Clawed Crayfish, Astacus leptodactylus leptodactylus Eschscholtz, 182.” Aquaculture 432: 192–203.

Sang, H. M., R. Fotedar, and K. Filer. 2011. “Effects of Dietary Mannan Oligosaccharide on the Survival, Growth, Immunity and Digestive Enzyme Activity of Freshwater Crayfish, Cherax Destructor Clark (1936): Effects of Dietary Mannan Oligosaccharide.” Aquaculture Nutrition 17, no. 2: e629–e635.

Skelton, J., K. M. Geyer, J. T. Lennon, R. P. Creed, and B. L. Brown. 2017. “Multi‐Scale Ecological Filters Shape the Crayfish Microbiome.” Symbiosis 72, no. 3: 159–170.

Sneddon, L. U., R. W. Elwood, S. A. Adamo, and M. C. Leach. 2014. “Defining and Assessing Animal Pain.” Animal Behaviour 97: 201–212.

Turner, P. V., T. Brabb, C. Pekow, and M. A. Vasbinder. 2011. “Administration of Substances to Laboratory Animals: Routes of Administration and Factors to Consider.” Journal of the American Association for Laboratory Animal Science: JAALAS 50, no. 5: 600–613.

Vogt, G. 2008. “The Marbled Crayfish: a New Model Organism for Research on Development, Epigenetics and Evolutionary Biology.” Journal of Zoology 276, no. 1: 1–13.

Vogt, G. 2011. “Marmorkrebs: Natural Crayfish Clone as Emerging Model for Various Biological Disciplines.” Journal of Biosciences 36, no. 2: 377–382.

Wofford, S. J., P. M. Laplante, and P. A. Moore. 2017. “Information Depends on Context: Behavioural Response to Chemical Signals Depends on Sex and Size in Crayfish Contests.” Behaviour 154, no. 3: 287–312.

Xu, N., W. Sun, L. Gong, et al. 2022. “Comparative Pharmacokinetics of Doxycycline in Crayfish (Procambarus clarkii) Following Oral, Intramuscular, and Intrasinus Administrations.” Aquaculture 551: 737941.