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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Crustacean hemocytes are important mediators of immune functions such as coagulation and phagocytosis. We employed an in situ approach to investigate the ultrastructural behavior of hemocytes during coagulation and phagocytosis in the early stages after injury caused by leg amputation, using transmission electron microscopy technique in marbled crayfish Procambarus virginalis. Hemocytes underwent drastic morphological changes during coagulation. The morphology of the cytoplasmic granules changed from electron-dense to electron-lucent forms in an expanding manner. The transformed granules containing amorphous electron-lucent material were observed to merge and discharge their contents into extracellular space for coagulation. We also observed that the contents of the nucleus participate in the process of coagulation. In addition, leg amputation induced extensive muscle degeneration and necrotic tissues were avidly taken up by the phagocytic hemocytes containing distinct phagosomes. Interestingly, we observed for the first time how the digested contents of phagocytized necrotic tissues are incorporated into granules and other cellular components that change the cell morphology by increasing the granularity of the hemocytes. Nevertheless, the degranulation of hemocytes during coagulation can also reduce their granularity. Given that morphological traits are important criteria for hemocyte classification, these morphological changes that occur during coagulation and phagocytosis must be taken into account., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Crustaceans and in particular decapods (i.e. shrimp, crabs and lobsters) are a diverse, commercially and ecologically important group of organisms. They are exposed to a range of environmental factors whose abiotic and biotic components are prone to fluctuate beyond their optimum ranges and, in doing so, affect crustaceans' immune system and health. Changes in key environmental factors such as temperature, pH, salinity, dissolved oxygen, ammonia concentrations and pathogens can provoke stress and immune responses due to alterations in immune parameters. The mechanisms through which stressors mediate effects on immune parameters are not fully understood in decapods. Improved knowledge of the environmental factors - above all, their abiotic components - that influence the immune parameters of decapods could help mitigate or constrain their harmful effects that adversely affect the production of decapod crustaceans. The first part of this overview examines current knowledge and information gaps regarding the basic components and functions of the innate immune system of decapods. In the second part, we discuss various mechanisms provoked by environmental factors and categorize cellular and molecular immune responses to each environmental factor with special reference to decapods., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)