The quantification of zebrafish ocular-associated proteins provides hints for sex-biased visual impairments and perception.

Biochemical differences between sexes can also be seen in non-sexual organs and may affect organ functions and susceptibility to diseases. It has been shown that there are sex-biased visual perceptions and impairments. Abundance differences of eye proteins could provide explanations for some of these. Exploration of the ocular proteome was performed to find sex-based protein abundance differences in zebrafish Danio rerio. A label-free protein quantification workflow using high-resolution mass spectrometry was employed to find proteins with significant differences between the sexes. In total, 3740 unique master proteins were identified and quantified, and 49 proteins showed significant abundance differences between the eyes of male and female zebrafish. Those proteins belong to lipoproteins, immune system, blood coagulation, antioxidants, iron and heme-binding proteins, ion channels, pumps and exchangers, neuronal and photoreceptor proteins, and the cytoskeleton. An extensive literature review provided clues for the possible links between the sex-biased level of proteins and visual perception and impairments. In conclusion, sexual dimorphism at the protein level was discovered for the first time in the eye of zebrafish and should be accounted for in ophthalmological studies. Data are available via ProteomeXchange with identifier PXD033338.

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.

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.

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.

Effects of environmental factors on the cellular and molecular parameters of the immune system in decapods.

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.

Hemocyte coagulation and phagocytic behavior in early stages of injury in crayfish (Arthropoda: Decapoda) affect their morphology.

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.

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.

High-Resolution Proteomic Profiling Shows Sexual Dimorphism in Zebrafish Heart-Associated Proteins.

Understanding the molecular basis of sexual dimorphism in the cardiovascular system may contribute to the improvement of the outcome in biological, pharmacological, and toxicological studies as well as on the development of sex-based drugs and therapeutic approaches. Label-free protein quantification using high-resolution mass spectrometry was applied to detect sex-based proteome differences in the heart of zebrafish Danio rerio. Out of almost 3000 unique identified proteins in the heart, 79 showed significant abundance differences between male and female fish. The functional differences were mapped using enrichment analyses. Our results suggest that a large amount of materials needed for reproduction (e.g., sugars, lipids, proteins, etc.) may impose extra pressure on blood, vessels, and heart on their way toward the ovaries. In the present study, the female's heart shows a clear sexual dimorphism by changing abundance levels of numerous proteins, which could be a way to safely overcome material-induced elevated pressures. These proteins belong to the immune system, oxidative stress response, drug metabolization, detoxification, energy, metabolism, and so on. In conclusion, we showed that sex can induce dimorphism at the molecular level in nonsexual organs such as heart and must be considered as an important factor in cardiovascular research. Data are available via ProteomeXchange with identifier PXD023506.

Ultracytochemical visualization of calcium distribution in heart cells and erythrocytes of zebrafish Danio rerio.

Detection of patterns of subcellular calcium distribution in the cardiovascular system can contribute to understanding its role in cardiac and blood function. The present study localized calcium in heart atrium, ventricle, and bulbus arteriosus as well as in erythrocytes of zebrafish Danio rerio using an oxalate-pyroantimonate technique combined with transmission electron microscopy. Intracellular calcium stores were detected in caveolae, mitochondria, and the nuclei of several zebrafish cardiac cell types. Melanin pigmentation containing calcium stores was detected in the pericardial cavity. Melanin might be an extracellular source of calcium for heart beating and/or a lubricant to prevent friction during beating process. Calcium deposits were also detected in the plasma membrane, cytoplasm and nucleus of erythrocytes as well as in blood plasma. Possible exchange of calcium between erythrocytes and blood plasma was observed. Interactions of such calcium stores and possible contribution of extracellular calcium stores such as melanin pigmentation to supply calcium for vital functions of heart cells should be addressed in future studies.

Chronic dietary toxicity of zinc oxide nanoparticles in common carp (Cyprinus carpio L.): Tissue accumulation and physiological responses.

Concerns regarding the potential toxic effects of zinc oxide nanoparticles (ZnO NPs) on aquatic organisms are growing due to the fact that NPs may be released into aquatic ecosystems. This study aimed to investigate the effects of dietary exposure to ZnO NPs on juvenile common carp (Cyprinus carpio). Fish were fed a spiked diets at doses 50 and 500mg of ZnO NPs per kg of feed for 6 weeks followed by a 2-week recovery period. Fish were sampled every 2 weeks for haematology trends, blood biochemistry measures, histology analyses, and determination of the accumulation of zinc in tissues. At the end of the exposure and post-exposure periods, fish were sampled for an assessment of lipid peroxidation levels. Dietborne ZnO NPs had no effects on haematology, blood biochemistry, and lipid peroxidation levels during the exposure period. After the recovery period, aspartate aminotransferase activity significantly (p < 0.05) increased and alanine transferase activity significantly (p < 0.05) decreased in the higher exposure group. The level of lipid peroxidation significantly (p < 0.05) decreased in liver of treated fish after 2 weeks post-exposure period. A histological examination revealed mild histopathological changes in kidneys during exposure. Our results did not show a significant increase of zinc content at the end of experiment in any of tested organs. However, chronic dietary exposure to ZnO NPs might affect kidney and liver function.

Insight into the modulation of intestinal proteome of juvenile common carp (Cyprinus carpio L.) after dietary exposure to ZnO nanoparticles.

ZnO nanoparticles (NPs) are widely used in industrial and consumer products. Therefore understanding their interaction with biological systems is key to their safe application. Proteomics was applied to assess the sub-lethal effects of dietary ZnO NPs on two parts of carp intestine, the intestinal folds and the muscular parts. A commercial carp feed containing 500mgkg-1 of ZnO NPs was fed to fish for six weeks. The abundances of 32 proteins in the treated intestinal folds were significantly changed and in addition, 28 proteins were significantly changed in the muscular parts. Pathways analysis revealed downregulation of pathways attributed to protein synthesis in both parts of the treated intestine. Remodelling of actin cytoskeleton pathways were regulated positively and negatively in intestinal folds and muscular parts, respectively, albeit via different mechanisms. Apoptosis response was indicated in exposed intestinal folds, whereas elevated levels of protein associated with cancerous cell survival were observed in the muscular parts. Results showed that ZnO NPs affected the protein abundances associated with cell motility, immune system response, oxidative stress response, as well as cell metabolism. Data are available via ProteomeXchange with identifier PXD006867.

Quantification of egg proteome changes during fertilization in sterlet Acipenser ruthenus.

Eggs of sterlet are discharged outside into ambient aquatic environment where egg activation and fertilization occur. Effects of different activation media including freshwater and clay suspension on protein abundances of egg were quantified in sterlet Acipenser ruthenus. In-gel digestion and high resolution mass spectrometry were used for label-free protein quantification in the eggs of five females. No significant (p > 0.05) difference was found between protein abundances in eggs activated with different media. However, results showed significant (p < 0.05, fold change ≥2) reduction in the abundances of nine proteins including six glycoproteins, enolase and heat shock protein in activated groups compared to freshly ovulated eggs as control. The fact that abundance of proteasome subunit alpha significantly reduced only in eggs which were activated by clay suspension suggests that activation medium can somehow intervene with protein regulation during fertilization. In conclusion, external fertilization in sturgeon egg is accompanied by huge release of proteins into the external environment that may participate in the construction of a transient microenvironment around egg for attraction and protection of spermatozoa to ensure ensuing fertilization. Data are available via ProteomeXchange with identifier PXD006232.

Subcellular distribution of calcium during spermatogenesis of zebrafish, Danio rerio.

Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio), using a combined oxalate-pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron-dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the process of male gamete development.

Effects of chronic dietary exposure of zinc oxide nanoparticles on the serum protein profile of juvenile common carp (Cyprinus carpio L.).

Zinc oxide (ZnO) nanoparticles (NPs) have been dramatically used in industry, biology, and medicine. Despite their interesting physico-chemical properties for application in various industrial, medical, and consumer products, safe use of ZnO NPs are under challenges due to the inadequate information related to their toxicological endpoints. Proteomics was applied to evaluate the sub-lethal effects of dietary exposure to ZnO NPs on serum proteome profile of juvenile common carp, (Cyprinus carpio). Therefore, ZnO NPs solution (500mgkg-1 of feed) was added to a commercial carp feed for six weeks. We compared the serum proteome profile from 7 controls and 7 treated fish. In addition, zinc accumulation were measured in intestine, liver, gill and brain. In total, we were able to identify 326 proteins from 6845 distinct peptides. As a result of the data analysis, the abundance levels of four proteins were significantly altered (fold change (fc) ≥2 and p<0.05) after dietary exposure to ZnO NPs. The protein levels of the complement component C4-2 (fc 2.5) and the uncharacterised protein encoded by kng1 (fc 5.8) were increased and major histocompatibility class I (fc 4.9) and the uncharacterised protein encoded by lum (fc 3.5) were decreased (fc 2.5). Molecular pathway analysis revealed four canonical pathways including acute-phase response signalling, liver and retinoid X receptors activation, and intrinsic and extrinsic prothrombin activation pathways as significantly regulated in the treated fish. No significant difference was observed for zinc accumulation in exposed fish compared to controls. In summary, despite no apparent accumulation, ZnO NPs exposure to common carp probably disturbs the fish homeostasis by affecting proteins of the haematological and the immune systems.

Ultrastructural Localization of Intracellular Calcium During Spermatogenesis of Sterlet (Acipenser ruthenus).

Calcium regulates many intracellular events such as growth and differentiation during different stages of gamete development. The aim of this study was to localize and quantify the intracellular distribution of calcium during different developmental stages of spermatogenesis in sterlet, Acipenser ruthenus, using a combined oxalate-pyroantimonate technique. The distribution of calcium was described in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. In the spermatogonium and spermatocyte, calcium deposits were mainly localized in the nucleus and cytoplasm. The spermatid had calcium in the nucleus, developing acrosomal vesicle, and cytoplasm. Intracellular calcium transformed from scattered deposits in spermatogonia and spermatocyte stages into an unbound form in spermatid and the spermatozoon. The proportion of area covered by calcium increased significantly (p<0.05) from early to late stages of spermatogenesis. The largest proportion of area covered by calcium was observed in the nucleus of the spermatozoon. In conclusion, although most of the intracellular calcium is deposited in limited areas of the spermatogonium and spermatocyte, it is present an unbound form in the larger area of spermatids and spermatozoa which probably reflects changes in its physiological function and homeostasis during the process of male gamete production in spermatogenesis.

Fine structure of the spermatozoon in three species of Cambaridae (Arthropoda: Crustacea: Decapoda) Cambarus robustus, Orconectes propinquus and Orconectes rusticus: a comparative biometrical study.

The ultrastructure of spermatozoa in three species of cambarid crayfish, Cambarus robustus, Orconectes propinquus, and Orconectes rusticus, were studied and compared with eight previously studied species from different crayfish families using morphological features and biometrical data. The ultrastructure of spermatozoa show a generally conserved pattern including an acrosome and nucleus in the anterior and posterior parts of the cell, respectively, radial arms that wrap around the nucleus, and the whole cell is enclosed by an extracellular capsule. The most outstanding morphological feature in spermatozoa of three studied cambarid crayfish is the crest-like protrusions in the anterior part of the acrosome that can be used as one of the features for distinguishing the members of this family. Results of biometrical data reveal that acrosome size in the representatives of Parastacidae are the smallest, while representatives of Astacidae show the biggest acrosome. The acrosome size in species belonging to Cambaridae occupy an intermediate position between the two other families of freshwater crayfish. In conclusion, a combination of morphological features and biometrical data of spermatozoa can help distinguishing different species of the freshwater crayfish.

Protein modification in the post-mating spermatophore of the signal crayfish Pacifastacus leniusculus: insight into the tyrosine phosphorylation in a non-motile spermatozoon.

After mating, spermatophores of signal crayfish are stored on the body of the female for a period before fertilization. This study compared the post-mating protein profile and pattern of protein tyrosine phosphorylation of the signal crayfish spermatophore to that of the freshly ejaculated spermatophore and found substantial differences. Two major bands of tyrosine-phosphorylated proteins of molecular weights 10 and 50kDa were observed in the freshly ejaculated spermatophore of the signal crayfish. While the tyrosine-phosphorylated protein band with molecular weight 10kDa was formed by protein(s) of similar pH, the band with molecular weight of 50kDa consisted of proteins of varying pH. In the post-mating spermatophore, the band with molecular weight of 50kDa was not detected, and an increase in the level of protein tyrosine phosphorylation was observed in the 10kDa band. The microtubular radial arms of the spermatozoon showed a positive reaction to an anti-tyrosine antibody conjugated with gold particles in both the freshly ejaculated and post-mating spermatophores. In conclusion, the male gamete of the signal crayfish undergoes molecular modification during post-mating storage on the body of the female including changes in the level of protein expression and protein tyrosine phosphorylation. Structural similarity of the radial arms in the crayfish immotile spermatozoon with flagellum, which is the main site of protein tyrosine phosphorylation in the mammalian motile spermatozoa, raises questions regarding evolution and function of such organelles across the animal kingdom that must be addressed in the future studies.

Subcellular localization of calcium deposits in the noble crayfish Astacus astacus spermatophore: Implications for post-mating spermatophore hardening and spermatozoon maturation.

The freshly ejaculated spermatophore of crayfish undergoes a hardening process during post-mating storage on the body surface of female. The ultrastructural distribution of calcium deposits were studied and compared in freshly ejaculated and post-mating noble crayfish spermatophores, using the oxalate-pyroantimonate technique, to determine possible roles of calcium in post-mating spermatophore hardening and spermatozoon maturation. Small particles of sparsely distributed calcium deposits were visible in the wall of freshly ejaculated spermatophore. Also, large amount of calcium deposits were visible in the membranes of the freshly ejaculated spermatozoon. Five minutes post-ejaculation, granules in the spermatophore wall appeared as porous formations with numerous electron lucent spaces. Calcium deposits were visible within the spaces and scattered in the spermatophore wall matrix, where smaller calcium deposits combined to form globular calcium deposits. Large numbers of the globular calcium deposits were visible in the wall of the post-mating spermatophore. Smaller calcium deposits were detected in the central area of post-mating spermatophore, which contains the sperm mass, and in the extracellular matrix and capsule. While the density of calcium deposits decreased in the post-mating spermatozoon membranes, numerous small calcium deposits appeared in the subacrosomal zone and nucleus. Substantial changes in calcium deposit distribution in the crayfish spermatophore during post-mating storage on the body of female may be involved in the processes of the spermatophore hardening and spermatozoon maturation.

Subcellular localization of calcium deposits during zebrafish (Danio rerio) oogenesis.

Calcium plays prominent roles in regulating a broad range of physiological events in reproduction. The aim of this study was to describe the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish using a combined oxalate-pyroantimonate technique. The oocyte development of zebrafish was categorized into four stages: primary growth, cortical-alveolus, vitellogenic, and maturation, based on morphological criteria. Calcium deposits in the primary growth stage were detected in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells and deposited in the cortical alveoli. In the vitellogenic stage, some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. In the maturation stage, calcium deposits in all oocyte compartments decreased, with the exception of those in mitochondria. The proportion of area covered by calcium deposits in the mitochondria and cortical alveoli of oocytes at different stages of development was significantly different (p<0.05). The extent of calcium deposits in the cortical alveoli of mature oocytes was substantially lower than in earlier stages. Basic information about calcium distribution during zebrafish oogenesis may contribute to better understanding of its role in oogenesis.

Label-free protein quantification in freshly ejaculated versus post-mating spermatophores of the noble crayfish Astacus astacus.

Crayfish spermatophores are deposited on the body surface of the female during mating and remain there for a period of time before fertilization ensues. Post-mating changes in protein expression level in the noble crayfish Astacus astacus spermatophore were quantified. In-gel digestion and high resolution mass spectrometry were used for label-free protein quantification. One hundred twelve proteins were identified in the spermatophore of noble crayfish. After 7 days of storage on the body of the female, 6 proteins were identified in the post-mating spermatophore that showed significant up-regulation and 4 significant down-regulations (p < 0.05, fold change ≥ 2). The highest rate of up-regulation was observed in sodium/hydrogen exchanger, which may indicate the importance of intracellular pH adjustment for final maturation of the crayfish spermatozoon. The highest rate of down-regulation was observed in histone H2A. This may increase chromatin flexibility and facilitate its transfer into the oocyte during fertilization. The vitellogenin protein was identified in the crayfish spermatophore and its level changed during storage on the body surface of female. Extensive proteomic modification of male gametes during storage on the body surface of the female suggests post-mating final maturation of the crayfish spermatozoon. BIOLOGICAL SIGNIFICANCE: Freshwater crayfish comprise a large and diverse group of ecologically and commercially important animals. Molecular studies of gametes in the crayfish can provide insight into the complex process of reproduction in this diverse group of animals. The results of such studies can be used for development of new techniques for artificial reproduction of these economically important species.