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Article

Allantoin and Tissue Specific Redox Regulation in Mud Crab Scylla serrata under Varied Natural Water Physico-Chemical Parameters

by
Samar Gourav Pati
1,2,
Biswaranjan Paital
1,* and
Dipak Kumar Sahoo
3
1
Redox Regulation Laboratory, Department of Zoology, College of Basic Science and Humanities, Odisha University of Agriculture and Technology, Bhubaneswar 751003, India
2
Department of Zoology, School of Life Sciences, Ravenshaw University, Cuttack 753003, India
3
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
*
Author to whom correspondence should be addressed.
Water 2024, 16(3), 480; https://doi.org/10.3390/w16030480
Submission received: 4 January 2024 / Revised: 25 January 2024 / Accepted: 29 January 2024 / Published: 1 February 2024
(This article belongs to the Special Issue Aquatic Ecotoxicology: A Tool for Monitoring the Effects of Anthropogenic Chemical Contamination on Fisheries and Aquaculture)
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Abstract

:
Effects of varied sediment and physico-chemical properties of water on allantoin content in tissues of Scylla serrata and its role in antioxidant homeostasis were investigated. Tissues of crabs were sampled from different coastal natural habitats of S. serrata of India during summer, winter, and rainy seasons and were analyzed to detect the variation in allantoin content and lipid peroxidation (LPx) and oxidative stress (OS) neutralizing antioxidant factors. High allantoin content in hepatopancreas over muscle tissue was observed in all seasons and sampling sites. The correlation coefficient values between allantoin and environmental factors, i.e., temperature, pH, salinity, organic carbon, Mg, and Ca, strongly support the stress-induced allantoin variation level in tissues. The level of allantoin had a negative correlation with levels of ROS, which was probably due to the upregulation of the activity of major antioxidant enzymes and assisting enzymes such as glutathione peroxidase (GPx), glutathione reductase (GR), and biotransforming enzyme glutathione -s- transferases (GST). A significant seasonal variation in the level of allantoin was correlated with the activity of including superoxide dismutase, catalase, GPx, GR, and GST, which was clearly noticed as a function of abiotic factors. Additionally, the level of allantoin did not correlate with small antioxidant molecules, such as ascorbic acid and reduced glutathione. Discriminant function analysis revealed that the level of allantoin and CAT and GR activities were the major contributing factors for the clear discrimination of groups. Therefore, allantoin can be considered as a significant factor for the seasonal modulation of OS physiology in mud crab Scylla serrata.

1. Introduction

Allantoin (5-ureidohydantoin or glyoxyldiureide) is a compound available naturally in many organisms, including microorganisms, plants, and animals [1]. Basically, it is a degraded product of purine base bases [2]. Many animals excrete uric acid, but some oxidize this and excrete allantoin or allantoic acid, a more water-soluble form of nitrogenous waste [3]. Allantoin has been shown to enhance cell proliferation and collagen synthesis in human fibroblasts, which are cells that play a key role in wound healing due to their anti-inflammatory properties [4]. In addition, allantoin is commonly used in cosmetic products due to its moisturizing and skin-soothing properties. It can help to reduce skin irritation and inflammation, as well as improve the overall texture and appearance of the skin [5]. Allantoin influences various biological processes, particularly in wound healing and skin regeneration, making it a valuable compound for medical and cosmetic applications. Out of several plant and animal sources, crustaceans are considered good sources of allantoin, which protects them against stress, facilitates cell growth, regenerates tissues, rebuilds tissue granulation in tissue, and is responsible for slower aging [6,7]. The level of allantoin in most organisms depends on several physiological and environmental factors [8]. Although allantoin is used to study metabolism [9], as a marker for vitamins [10], as a marker of stress [11], atherosclerosis [12], apoptosis studies [13], as a signaling molecule in PI3K/Akt/GSK-3β pathway [14], scaffold formation for medical use [15], clinical markers, especially for chronic kidney disease [16], for protein aggregation [17], the variation of allantoin and its role in modulating oxidative stress physiology as a function of various sedimental and physicochemical factors of water is scantly studied in animals. The nitrogen metabolite ‘allantoin’ has multiple roles in modulating physiological activity in animals under varied abiotic factors. Still, its regulatory effect(s) on (oxidative) stress (OS) responses in organisms in general, and mud crab S. serrata in particular, remains elusive.
Environmental temperature, humidity, and light can modulate the formation of allantoin. For example, high temperatures and low humidity can increase the formation of allantoin in some plants, while low temperatures and high humidity can decrease its formation [18]. Similarly, allantoin production can be seasonally influenced by factors such as alkalinity, salinity, temperature, and pH, influencing the uricase mRNA level responsible for allantoin production in aquatic animals. For instance, a 7.1-fold downregulation of uricase transcript level was recorded in great spider crab Hyas araneus in water with pH 7.54, salinity 33.5 ppt, and temperature 9 °C as compared to a control. The objective of the study was to stop the formation of allantoin in the spider crab [19]. However, little information is available on allantoin-induced changes in invertebrates in general and under environmental fluctuations in particular. In addition to environmental factors, the formation of allantoin can also be influenced by genetic factors, as well as the presence of other compounds in the animal tissue. The altered level of allantoin has several physiological effects on organisms. Studies conducted on chronic renal failure patients have revealed that a 480% increase in allantoin affects enzymatic antioxidants activity, such as a a 96% increase in superoxide dismutase (SOD) and a 32% decrease in glutathione peroxidase (GPx) activity. In contrast to its effects on antioxidant enzymes, the lipid peroxidation (LPx) concentration was found to be 64% elevated [20]. In another study, allantoin pretreatment reduced glutathione (GSH) depletion and restored catalase (CAT) enzyme activity in an ethanol-induced gastric ulcer model [21]. However, free radical scavenging activity was found to be 36% lower in both Helix aspersa and Helix pomatia, when treated with allantoin extracted from animals, than in plants [22]. This indicates that allantoin modulates the oxidative stress physiology of the organism, but its exact role under the varied environmental factors in any natural population is not studied.
To understand the organismal effects of allantoin under the varied physicochemical parameters of water in crustaceans such as mud crab Scylla serrata, it is essential to identify and correlate the capacity of organisms to cope with the projected environmental changes [23,24,25]. As an inhabitant of the intertidal zone, S. serrata (mud crab) is highly influenced by environmental factors, including pH, salinity, and temperature [26]. In this regard, the antioxidant regulatory capacity of allantoin has been noted in microbes and some invertebrates [27,28,29]. Although the combined effects of various aquatic environmental factors in modulating complex cellular processes and oxidative stress responses in S serrata have been studied, the role of allantoin in this crab under such a context is still obscure. In order to fill the knowledge gap between the potentials of allantoin and oxidative stress (physiology) in S. serrata under changing environments, it is highly beneficial to investigate and correlate the allantoin concentration with oxidative stress parameters and environmental factors [30]. Therefore, the hypothesis of this study was “allantoin modulates OS physiology of S. serrata”.

2. Material and Methods

2.1. Sampling of Crabs and Analysis of Physico-Chemical Properties of Water and Sediment

The crabs were sampled from northern, southern, and eastern India along the Bay of Bengal and Arabian Sea. They were sampled from the coastal belts of India, covering west, south, and east regions that encase salt water or seashores. From west India, Gujarat (21°27′52.4″ N 87°02′47.0″ E) was selected as a sampling site, while Tamil Nadu (19°40′43.2″ N 85°28′19.5″ E) was chosen for the south Indian sampling site. Odisha (20°03′35.6″ N 86°20′36.5″ E) was selected for the East Indian site for sampling. Sampling was carried out in rainy (mid-July to early August), winter (mid-November to early December), and summer (from April to early May) seasons from 2018 to 2019. The data set for November to early December of 2018 to mid-July to early August 2019 is presented in this article. Because of the COVID-19 restrictions, sampling for the year 2020 was hindered and incompletely analyzed; data from this period were discarded. Each season in India was verified with district changes in climatic factors, leading to an alteration of the habitat and physiology of the inhabitants [23,26,31]. Therefore, to ascertain the exact impact of the environmental factors on allantoin and its induced change in OS physiology parameters in S. serrata, water physicochemical parameters and water sediment factors were assessed in the sampling site for three consecutive days during sampling. Temperature (by a mini thermometer in °C) was immediately measured. Water samples from the sampling site were quickly brought to the field laboratory in sealed dark bottles. Water physico-chemical parameters such as pH and salinity were recorded using specific electrodes (µp Based Soil and Water Analysis kit, Esico, New Delhi, India). To correlate with the other variables, organic carbon content in sediment samples was measured using the protocols described by Walkley and Black [32]. Mg and Ca content of samples was also measured as per Schofield and Taylor [33].

2.2. Tissue Collection and Processing

Medium size adult male crabs (n = 10) weighting 143 ± 4 g were sacrificed, and hepatopancreas and muscle tissues were dissected and transferred immediately into ice-cold saline solution (0.67%, w/v) and cleaned, soaked in tissue papers, and flash frozen in liquid nitrogen. Then, the frozen tissues were transported and were transferred to a −80 °C refrigerator and stored for further analysis. Then, 10% (w/v) tissue homogenates were centrifuged at 1000× g for 10 min at 4 °C to extract the post-nuclear fraction (PNF). PNF was centrifuged at 10,000× g for 10 min at 4 °C to obtain post-mitochondrial fraction (PMF) as the clear supernatant [34]. Protein concentration was determined in the above fractions using BSA as standard [35].

2.3. Determination of Allantoin

Allantoin was measured in the supernatant (10,000× g, 15 min), which was hydrolyzed in alkaline conditions at 100 °C to form phenyl hydrazone [36]. Then, the concentration was measured at 522 nm using a spectrophotometric assay. The results were calculated from the standard curve of allantoin (100 mg/L) and were expressed as mg of allantoin mg−1 protein.

2.4. Determination of OS Parameter

Thiobarbituric acid reactive substances (TBARS, expressed as nmol mg−1 protein) were determined in the PNF of tissues [34,37].

2.5. Enzymatic Antioxidant Assays

The activity of RRE in PMF was assayed at 25 °C [34]. The activity of SOD (EC1.15.1.1, unit mg−1 protein, [38]), CAT (EC1.11.1.6, nano Kat mg−1 protein, [39,40]), GPx (EC1.11.1.9, [41]), and GR (EC1.6.4.2, [42] nmol of NADPH oxidized min−1 mg−1 protein) was assayed as per Paital and Chainy [34].

2.6. Assay of Total Antioxidant Capacity and Small Antioxidant Molecules

PNF fractions were precipitated in trichloroacetic acid (5% w/v) followed by centrifugation at 10,000× g for 15 min. The level of non-protein sulfhydryl groups and AA (as ng g−1 of wet organ) was determined in the clear supernatant, as described earlier [43,44].

2.7. Statistical Analysis

The results of this study were expressed as mean ± standard deviation (n = 10 in each group). Data were subjected to a test for normal distribution and homogeneity of variance. To compare the means of biochemical estimations, two-way ANOVA followed by Duncan New Multiple range test was employed. Differences between mean values were considered significant at p < 0.05 levels. Effects of the contribution of different variables such as allantoin, LPx, antioxidant enzymes, and small antioxidant molecules on the groups were evaluated employing discriminant function analysis (DFA) according to Paital and Chainy [31,34]. Correlation analysis was performed using Microsoft Excel version 8.1 to check the association among the studied parameters. Mean values of the biochemical parameters between the groups were compared to calculate the absolute change in percentage.

3. Results

3.1. Physico-Chemical Properties of Water and Sediment Factors

A distinct variation of the physicochemical properties of water and sediment is presented in Table 1. In brief, during the summer season, the sediment parameters, such as organic carbon (232 and 44%), Ca (13.88 and 3.96%), and Mg (20 and 13.5%), were found to be significantly (p < 0.05) higher than both the rainy and winter seasons. During the summer, the water temperature of different sampling sites was significantly (82 and 28%) higher (p < 0.05) than in both the winter and rainy seasons. The water pH was also recorded to be 6.38 and 6.1%, significantly (p < 0.05) higher than both the rainy and winter seasons. Finally, the salinity recorded during summer was found to be 23.63 and 78.7%, significantly (p < 0.05) higher than in both the winter and rainy seasons.

3.2. Allantoin

Hepatopancreas tissue had 322.68, 301.52, and 315.35% higher allantoin levels than muscle tissue during the rainy, winter, and summer seasons, respectively (Figure 1). Allantoin content in hepatopancreas tissue collected from Gujarat was 22.22 and 8.79% significantly (p < 0.05) higher than crab samples collected from Tamil Nadu and Odisha, respectively. However, during the summer season, hepatopancreas tissue collected from Gujarat was found to be 15.21 and 3.11% significantly (p < 0.05) higher than samples collected from Tamil Nadu and Odisha, respectively (Figure 1a). Allantoin content of muscle tissue collected from Gujarat was found to be 17.85 and 29.48% significantly (p < 0.05) higher than crab samples collected from Tamil Nadu and Odisha, respectively. However, during the summer, muscle tissues collected from Tamil Nadu and Odisha were 23 and 35.29%, respectively, lower than samples collected from Gujarat (Figure 1b).

3.3. Oxidative Stress, ROS, and Antioxidant Parameters

The TBARS level in muscle tissues was 95% lower than in hepatopancreas tissues, with the latter showing higher levels. During the summer season, both muscle (37 and 27%) and hepatopancreas (46 and 18%) tissues showed significantly (p < 0.05) higher TBARS than the winter and rainy seasons, respectively. Major enzymatic antioxidants, including CAT, GPx, GR, and GST, also increased during the summer when compared to the rest seasons. During the summer, the CAT activity was found to be 10 and 18% significantly (p < 0.05) higher in hepatopancreas and muscle tissues, respectively, than in rainy season samples. The GPx activity of hepatopancreas and muscle tissues were shown to be 63 and 80% higher enzyme activity, respectively, during the summer season with respect to the rainy season. Similarly, in that season, the hepatopancreas (61 and 27%) and muscle (18 and 72%) tissues showed a significant (p < 0.05) elevation of GR activity in comparison to rainy and winter season tissue samples, respectively. The GST activity with respect to the summer season was found to be significantly (p < 0.05) higher in hepatopancreas (27 and 52%) and muscle (38 and 54%) tissues than in the winter and rainy seasons, respectively. However, the SOD activity recorded during the summer season was found to be 22.36 and 21.64% significantly (p < 0.05) lower in muscle and hepatopancreas tissues, respectively, compared to the rainy and winter season. In contrast to most major antioxidant enzymes, the small antioxidant molecules, including AA and GSH, exhibited an alleviated level during the summer season in both tissues. An elevation of the total antioxidant capacity in hepatopancreas tissue of crabs was found to be 14 and 43% (p < 0.05) higher during the summer season as compared to the winter and rainy seasons, respectively (Table 2).

3.4. Correlation of Allantoin and Environmental Factors

The physicochemical properties of water and sediment factors, including pH, temperature, salinity, organic carbon, Ca, and Mg, have been correlated with measured allantoin of different seasons and sites. The allantoin concentration of hepatopancreas tissues collected from Gujarat, Odisha, and Tamil Nadu shows a strong positive correlation with pH, i.e., 0.96, 0.97, and 0.97, respectively (Table 3, Figure S1A). However, on average, allantoin and temperature Pearson correlation coefficient falls to 0.69 in muscle and 0.77 in hepatopancreas (Table 3, Figure S1A,B). Similarly, the correlation between allantoin and salinity has also been found to be 0.78 in hepatopancreas and 0.76 in muscle, irrespective of collection sites (Table 3, Figure S1A,B). In addition to that, organic carbon and Ca have a similar pattern of correlation coefficient with allantoin of the crab tissue sample. The average correlation coefficient of organic carbon is 0.85 for hepatopancreas and 0.82 for muscle tissue. Micronutrients like Ca and Mg show a strong positive correlation with allantoin in the hepatopancreas and muscle tissues of crabs. The correlation coefficient between levels of allantoin and Ca was 0.74 in hepatopancreas and 0.76 in muscle tissue in all seasons. Similarly, for Mg, the average Pearson correlation coefficient reached 0.89 in the hepatopancreas and 0.88 in the muscle of the crab sample, irrespective of collection sites (Table 3, Figure S2A,B) Thus, both physicochemical properties of water and sedimental parameters have a strong positive correlation with allantoin of carb tissue samples, especially for hepatopancreas tissues collected from different sampling sites in India.

3.5. Correlation between Allantoin and Oxidative Stress Physiology Parameters

Among different sites, the most significant correlation coefficient of LPx and allantoin is Odisha and Gujarat, and the Pearson correlation coefficient reached 0.98 and 0.9, respectively, in hepatopancreas tissue. In comparison to hepatopancreas, the muscle tissue allantoin concentration shows a weak positive correlation (r < 0.65) with measured LPx at respective sampling sites (Table 4, Figure S3A,B). Like oxidative stress parameters, the enzymatic antioxidants, including CAT, GPx, GR, and GST, have correlated with measured allantoin (Table 4, Figures S3–S5). The measured allantoin at different sampling sites and SOD shows a very weak negative correlation, i.e., 0.59, 0.66, and 0.48 at Gujarat, Odisha, and Tamil Nadu, respectively, in hepatopancreas tissue, but in muscle tissue, this correlation becomes insignificant (Table 4, Figure S3A,B). Interestingly, a strong positive correlation between allantoin and CAT is apparent in both hepatopancreas and muscle tissue. The r values for allantoin and CAT are 0.69, 0.97, and 0.9 in hepatopancreas and 0.77, 0.66, and 0.78 in muscle tissue collected from Gujarat, Odisha, and Tamil Nadu, respectively (Table 4, Figure S3A,B).
A major enzyme of the glutathione system GPx also possesses a strong positive correlation, i.e., 0.97, 0.76, and 0.72 in hepatopancreas tissue and 0.88, 0.9, and 0.9 in muscle tissue collected from Gujarat, Odisha, and Tamil Nadu, respectively (Table 4, Figure S4A,B). The GR activity also shows similar r values with allantoin measured at respective collection sites. The glutathione-s-transferase, a xenobiotic neutralizer, also exhibits a strong positive correlation with measured allantoin. Here, again, the hepatopancreas tissue leads with a stronger positive correlation, i.e., 0.99, 0.83, and 0.95, than muscle tissue r values such as 0.86, 0.79, and 0.88 of Gujarat, Odisha, and Tamil Nadu, respectively (Table 4, Figure S4A,B). In contrast to these major AD enzymes, the small AD molecules are mostly insignificant; however, allantoin in muscle tissue from Gujarat and hepatopancreas tissue from Odisha show a strong positive correlation with GSH, i.e., 0.92 and 0.78, respectively. Similarly, AA in muscle tissue collected from Odisha only offers a significant correlation (0.63) with measured allantoin (Figure S5A,B). Thus, major AD enzymes have more substantial correlation coefficients with measured allantoin than small AD molecules.
Overall, the total antioxidant capacity showed a different correlation pattern than the mentioned AD enzymes and molecules. Here, allantoin in muscle and hepatopancreas tissues show a similar positive correlation with DPPH inhibition percentage. The r values for hepatopancreas tissue are 0.76, 0.75, and 0.7; similarly, for muscle tissue, 0.71, 0.07, and 0.55 of Gujarat, Odisha, and Tamil Nadu, respectively (Table 4, Figure S3A,B). Therefore, antioxidants have a strong positive correlation with the allantoin concentration in tissues collected from different sampling sites.

3.6. Multivariate Statistical Analysis

The contribution of different parameters on mud crab physiology under seasonal changes was examined through DFA. The mud crab’s hepatopancreas and muscle tissue were tested for allantoin and antioxidant parameters in the summer, winter, and rainy seasons. Each tissue was split into three groups, and both tissues were divided into six groups (Figure 2a–c). When hepatopancreas tissue was individually selected for the DFA study, the % of the variance for the first function was 78.5, and 21.5% for the second. Similarly, for muscle tissue, the percentage of the variance for the first function is 88.6, and 11.4% for the second (Figure 2a,b). Interestingly, when both tissues were considered together for DFA, it showed 97.3% and 2% variance in the first and second functions, respectively (Figure 2 and Table 5). Thus, whether tissues are taken together or separately, the percentage of the variance with respect to antioxidant parameters for both tissues is in the same pattern. It seems that when all the factors are taken together, the association among the parameters in the hepatopancreas or muscle is distinctly visible, as the parameters were clumped together (Figure 2c).

4. Discussion

In this study, the allantoin was measured in two different tissues, i.e., muscle and hepatopancreas of S. serrata as well as water physicochemical properties and sediment factors from sampling sites, which were analyzed in different seasons. Tissues such as hepatopancreas (metabolically active) and muscle (used for locomotion) were sampled to study the effects of allantoin on stress physiology. Levels of antioxidant regulatory factors such as the activity of SOD, CAT, GPx, glutathione reductase (GR) and glutathione-S-transferase, the levels of ascorbic acid (AA) and the reduced glutathione (GSH), and total antioxidant capacity in the form of DPPH inhibition capacity were quantified in the present study to draw a correlation between allantoin and the above factors [23,24,25,26,30,45]. Thus, the present study provides possible insights into the modulation of allantoin and its subsequent effects on the studied oxidative stress physiology parameters under the varied physicochemical parameters of the habitats. The quantification of allantoin concentration, activity of different enzymatic antioxidants, small antioxidants, and lipid peroxidation can be used to uncover its physiological effects on the organism [46,47,48,49]. Furthermore, it might be easy to use statistical tools to define the connection among potential factors that play crucial roles in modulating crab physiology in mangrove areas or in situ studies.
Being an intermediate product of the purinolytic sequence, the presence of allantoin in lower mammals is familiar but is rarely traced in marine invertebrates as they further metabolize it to ammonia and CO2 [50]. This is the first study on mud crabs, as well as on marine crustaceans that detects the level of allantoin and investigates its seasonal variation depending on fluctuating environmental factors. The significant impact of seasons on allantoin formation describes the role of various abiotic factors and physiological responses of tissues of mud crabs. In particular, the concentration of allantoin in hepatopancreas tissue is more relevant than in the muscle tissue of crabs, as the hepatopancreas is the central hub of metabolism. The observed allantoin level in mud crabs and its strong correlation coefficient with the physicochemical properties of water during the summer indicate that pH, temperature, and salinity collectively affect the formation of allantoin to regulate several physiological, immunological, and stress-related activities. Additionally, in previous studies, it has been shown that during drought, solar radiation, and high salinity, the accumulation of allantoin occurs in both animal and plant species. During physiological wear and tear, the allantoin modulates wound healing, cell proliferation, and inflammatory responses, which generally rise during summer as they are easily exposed to predators and dry mud [22]. According to studies, sediment carbon content is inversely associated with urea; however, our results show a strong positive correlation with allantoin. The correlation values of Ca with allantoin are consistent with previous studies, but the reason behind such correlation is still unclear [51]. In contrast, the correlation coefficient with Mg is opposite to the earlier findings that suggest Mg inhibits uricase, which is the primary enzyme for the conversion of uric acid to allantoin; this requires further investigation [52]. Apart from the measured environmental factor, exercise-induced oxygen consumption is another potential explanation for the observed content of allantoin during dry seasons. Overall, environmental factors have a significant impact on allantoin formation and their subsequent role in modulating various physiological processes.
Allantoin has multiple roles in physiology and inflammatory responses, immunomodulation, and biomarkers of OS [47,53], so it is necessary to analyze the role of antioxidant homeostasis of the mud crab in association with allantoin. The observed TBAR level in both hepatopancreas and muscle tissues clearly indicates thermal stress during summer [31]. In addition, the observed correlation between allantoin and LPx defines the subsequent ROS-neutralizing effects of allantoin [54,55,56]. Thus, the correlation study could indirectly identify allantoin as a potent antioxidant, but this requires further study to prove it. The observed correlation values of allantoin with SOD enzyme activity suggest that the scarcity of ROS during thermal stress is due to the participation of uric acid as an antioxidant, which is evident from the increased allantoin content in tissues [57]. However, the observed CAT activity in both hepatopancreas and muscle tissues indicates the subsequent neutralization of H2O2, which is the by-product of uric acid oxidation [58]. Furthermore, the correlation coefficient values between allantoin and CAT activity support the conclusion that uric acid, rather than SOD, plays a significant role in neutralizing ROS in the present study [55]. Apart from this, allantoin also directly upregulates the expression of SIRT1 and NRF2, which elevates CAT and SOD activity [45,57]. Thus, the above observation can be interpreted as the allantoin concentration being substantially related to the peroxide formation as well as a modulator of CAT and SOD activity.
According to previous studies, the elimination of peroxides from different sources like urate, amino acid oxidase, etc., is driven by glutathione systems that include GPx, GR, and GSH. The observed activity of GPx, GR, and allantoin concentration in the present study indicates peroxide formation and their neutralization. This is strongly supported by the correlation coefficient values observed between allantoin and GPx in both muscle and hepatopancreas tissues. Similarly, the r values of allantoin and GR enzymes of both tissues strongly define the presence of peroxide and its subsequent neutralization. However, the r values of GSH with allantoin do not support the same. The xenobiotics undergo detoxification through GST activity, where GSH also contributes a significant role during dry seasons, but a direct link between allantoin concentration and GST activity is still missing. However, our present correlation study strongly defines the effect of allantoin on GST activity through observed “r” values in each tissue from different sampling sites. In addition to enzymatic antioxidants, the small Ads, such as AA and GSH, of both tissues were observed to be fluctuating from the pattern of most enzymatic antioxidants, preferably due to the active participation of allantoin and uric acid-like molecules. The lack of a significant correlation between small antioxidants and allantoin further emphasizes the unsupportive role of small AD. In contrast to small AD, the observed total antioxidant capacity of both tissues was found to be significantly correlated to allantoin content, suggesting its significant role in antioxidant homeostasis.
Allantoin, considered as a potential modulator of oxidative stress physiology in a range of organisms [56], varies seasonally in the crab tissues in the present study. Figure 1 clearly indicates its augmented values in the summer season as compared to the other two seasons. During the summer period, all the studied environmental parameters, such as pH, temperature, salinity, organic carbon level, Ca, and Mg, were high, and the level of these parameters, along with the titer of allantoin, was low in the rainy season as compared to the other two seasons. Following the trends, all the above-studied parameters were almost observed to be at a moderate level. Such data are in agreement with the earlier observation [24,25,30]. As such, the studied parameters, especially temperature and salinity, were able to raise allantoin levels in these ectothermic animals in their natural population during the summer season. As noted earlier, allantoin is metabolized to ammonia and CO2 in invertebrates [50]. Therefore, high salt concentrations in the hot summer season, compared to winter and rainy seasons, could be associated with the low catabolism rate of allantoin. Further detailed study is suggested on this aspect. The crab S. serrata slowly adapts uricotelic excretory metabolism over ammonotelic metabolism as salinity increases in their environment [34,59]. The reverse mechanism, i.e., adaption of ammonotelic nature at low salinity observed in the rainy season, is also noted in this animal, indicating the involvement of salinity in allantoin production. This could be an adapted mechanism the crab follows to produce less ammonia at high salinity. A controlled laboratory experiment to map the allantoin level in these crabs at a constant temperature with altered salinity levels is suggested to prove this mechanism. The results confirm the rise in oxidative stress markers, such as LPx level, in the crab during summer [24,25,30]. Therefore, allantoin can also be considered as one of the markers of oxidative stress in invertebrates, as already observed in vertebrates, including humans [54,55,57].
Finally, a two-way ANOVA and discriminant function analysis (DFA) are used to investigate the potential role of parameters at the group or individual level. Collectively, the allantoin content and its relation between environmental factors, oxidative stress parameters, and antioxidants reveal the importance of allantoin as a participant in various physiological roles, significantly modulating antioxidant homeostasis in mud crabs. The standardized canonical coefficient values recorded individually for muscle tissue indicate that allantoin and GPx are the most reliable factors for discriminating the three groups. Similarly, for hepatopancreas tissue the canonical coefficient value suggests the importance of GR and allantoin over other factors for the discrimination of groups. These values differ when considering both tissues together, which show CAT, GR, and allantoin as the most reliable factors of discrimination among groups (Table 6). The non-overlapping of factors is predominant when muscle and hepatopancreas tissues are taken separately. However, considering both tissues together shows a clear overlap among 4, 5, and 6, while 1 and 2 are only slightly overlapped, suggesting hepatopancreas as a significant tissue for antioxidant homeostasis over the muscle tissue (Figure 2c). The study was conducted in nature without any control over the intrinsic or extrinsic factors of crabs; therefore, multiple such studies are suggested to overcome this limitation.
Alternatively, under the altered pH, temperature, salinity, level of organic carbon, Ca, and Mg in the habitat in the summer period, allantoin values in animals rise. In these animals, under these altered conditions of physicochemical parameters of the environment, the magnitude of oxidative damage markers such as LPx was increased, the activity of SOD was decreased, and the activity of the other antioxidant regulatory enzymes such as catalase and GPx was increased. Consequently, the level of small antioxidant-regulating enzymes and the biotransformation enzyme level was also high during the summer. It indicates the active role of the studied antioxidants in alleviating the (oxidative) stress in these animals in summer compared to the other two seasons [60]. Therefore, the increase in the level of (oxidative) stress was accompanied by a rise in antioxidant levels (to compensate for the diminished level of SOD activity) to ameliorate the latter in animals [55]. The elevated level of DPPH scavenging activity in the crabs at such elevated salinity and temperature is not surprising. However, the fact that it is positively correlated with allantoin levels suggests that the former plays a role in reducing stress in crabs. The antioxidant role of allantoin measured via DPPH scavenging activity in plants has clearly been noted [61]. The chemical nature of a compound must be identical in animals and plants. So, allantoin must have ROS scavenging activity in the crabs. The role of allantoin in modulating stress primarily via NRF2 pathways is known to control several integral processes of aging, neural function, stress relieving, etc., observed in animal models [7,14,29]. Furthermore, this phenomenon has been observed in plant models [62]. However, such studies are necessary to be conducted in animals in general and specifically in ectotherms. In the present study, the adaptation of crabs to ameliorate stress via allantoin-dependent pathways is clearly noted. The rise in the allantoin level may be considered favorable to indicate its role in distressing the animals under high temperatures and salinity.

5. Conclusions

Endogenous allantoin formation under fluctuating abiotic factors significantly effects mud crab physiology, especially in antioxidant homeostasis. The rising temperature and other factors, including pH, salinity, organic carbon, Mg, and Ca, elevate allantoin in hepatopancreas tissue as a response to reduce stress by acting directly as an antioxidant or indirectly by up-regulating the primary AD enzyme level in mud crabs. Additionally, being a byproduct of uric acid oxidation, it can be indirectly considered as an indicator of rising stress during the summer season (Figure 3). Thus, allantoin is a potent biomolecule in maintaining the mud crab antioxidant homeostasis in response to seasonal changes and can be considered as one of the biomarkers in invertebrates.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w16030480/s1, Figure S1: Correlation graph of physico-chemical properties of water and allantoin. title; Figure S2: Correlation graph of sediment factors and allantoin, Figure S3: Correlation graph of OS and AD enzymes with allantoin, Figure S4: Correlation graph of AD enzymes with allantoin, Figure S5: Correlation graph of small AD molecules and total antioxidant capacity with allantoin.

Author Contributions

B.P.: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing—original draft, Writing—review and editing. S.G.P.—Data curation, Formal analysis, Investigation, Writing—original draft, Writing—review and editing. D.K.S.—Writing—original draft, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

The work was generously supported by the funding to B.P. from the Science and Engineering Research Board, Department of Science and Technology, Govt. of India New Delhi, India (No. ECR/2016/001984) and Department of Science and Technology, Government of Odisha (Grant letter number 1188/ST, Bhubaneswar, dated 01.03.17, ST-(Bio)-02/2017).

Institutional Review Board Statement

The study does not require any approval from the institutional review board/ethics committee. Since Scylla serrata belongs to an edible group of invertebrate plentifully available in India, the ethical permission was not required for any experiment to enhance their production or culture.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

Encouragement rendered by Pravat Kumar Raul, Honourable Vice Chancellor, OUAT is duly acknowledged.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

AA—ascorbic acid, CAT—catalase, DPPH-2,2-diphenyl-1-picryl hydrazyl, GPx—Glutathione peroxidase, GR—Glutathione reductase, GSH—Reduced glutathione, GST—Glutathione-S-transferase, H2O2—hydrogen peroxide, HP—hepatopancreas, LPx—lipid peroxidation, OS—oxidative stress, PMF—post mitochondrial fraction, PNF—post nuclear fraction, ROS—reactive oxygen species, SOD—superoxide dismutase

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Water 16 00480 g001
Figure 1. Spatiotemporal variation of tissue specific allantoin content in Scylla serrata. Allantoin content of (a) hepatopancreas and (b) muscle of S. serrata. Data are presented as the mean ± S.D. (n = 10). Two-way ANOVA was followed to compare the means. Different letters above the bars denote the statistical differences between mean values at p < 0.05.
Figure 1. Spatiotemporal variation of tissue specific allantoin content in Scylla serrata. Allantoin content of (a) hepatopancreas and (b) muscle of S. serrata. Data are presented as the mean ± S.D. (n = 10). Two-way ANOVA was followed to compare the means. Different letters above the bars denote the statistical differences between mean values at p < 0.05.
Water 16 00480 g001
Water 16 00480 g002
Figure 2. DFA for spatio-temporally varied allantoin, OS, and AD parameters levels in S. serrata tissues. Distinct group discrimination was observed in three groups in individual tissue without overlapping with each other (a,b) and six groups by taking two tissues together (c).
Figure 2. DFA for spatio-temporally varied allantoin, OS, and AD parameters levels in S. serrata tissues. Distinct group discrimination was observed in three groups in individual tissue without overlapping with each other (a,b) and six groups by taking two tissues together (c).
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Water 16 00480 g003
Figure 3. Allantoin modulated changes of oxidative stress physiology in mud crab S. serrata under the changing physicochemical properties of water and soil. The function of allantoin may be considered as positive because of the rise in its level to ameliorate stress. The stress amelioration role of allantoin in invertebrate is for the first time resented. The arrows indicate the direction of the event.
Figure 3. Allantoin modulated changes of oxidative stress physiology in mud crab S. serrata under the changing physicochemical properties of water and soil. The function of allantoin may be considered as positive because of the rise in its level to ameliorate stress. The stress amelioration role of allantoin in invertebrate is for the first time resented. The arrows indicate the direction of the event.
Water 16 00480 g003
Table 1. Spatio-temporal pattern of physico-chemical and sediment factors.
Table 1. Spatio-temporal pattern of physico-chemical and sediment factors.
Water Quality of S. serrata Sampling SitesSoil Quality of S. serrata Sampling Sites
LocationSeasonpHTempSalinityOrganic Carbon %Ca Mg (mg kg−1)
(°C)(ppt)(mg kg−1)
OdishaR7.94 ± 0.1 a21.3 ± 0.85 a17.48 ± 0.71 a0.84 ± 0.004 a445.28 ± 11 a88.3 ± 3.5 a
W7.85 ± 0.06 b17.2 ± 0.77 b22.66 ± 0.88 b1.80 ± 0.05 b497.3 ± 11 b96.8 ± 2.1 b
S8.22 ± 0.03 c28.1 ± 0.94 c25.58 ± 0.97 c2.98 ± 0.03 c503.62 ± 13 c104.1 ± 2.2 c
GujaratR7.5 ± 0.06 a23.6 ± 0.7 a12.32 ± 0.65 a0.6 ± 0.002 a512.5 ± 15 a94 ± 1.2 a
W7.53 ± 0.05 b12.5 ± 0.8 b22.78 ± 0.2 b1.92 ± 0.006 b546 ± 16 b97.5 ± 2.3 b
S7.92 ± 0.1 c27.3 ± 0.67 c23.8 ± 0.9 c2.46 ± 0.08 c568.17 ± 14 c103.5 ± 3.2 c
Tamil NaduR7.13 ± 0.04 a22.22 ± 0.1 a17.68 ± 0.6 a0.96 ± 0.004 a447.5 ± 16 a97.6 ± 2.2 a
W7.22 ± 0.3 b17.34 ± 0.91 b23.22 ± 0.9 b1.80 ± 0.001 b495.8 ± 13 b102.9 ± 3.1 b
S7.84 ± 0.2 c30.51 ± 0.93 c35.49 ± 1.8 c2.54 ± 0.04 c528.15 ± 13 c128.5 ± 1.5 c
Note(s): Patterns of water parameters such as pH, temperature (Temp), and salinity along with soil parameters such as organic carbon, Ca, and Mg (mg kg−1) of the different collection sites in different time points such as during rainy (R), summer (S), winter (W) are presented in the table. Data are represented as mean ± SD. Data from three consecutive days were considered for analysis. Mean values designated with different superscripts such as a, b, and c are regarded as statistically significant from one another at p < 0.05 level (two-way ANOVA, post hoc test). ppt: parts per thousand.
Table 2. Enzymatic and non-enzymatic antioxidant modulation as a function of season in abdominal muscle and hepatopancreas (HP) of mud crab Scylla serrata.
Table 2. Enzymatic and non-enzymatic antioxidant modulation as a function of season in abdominal muscle and hepatopancreas (HP) of mud crab Scylla serrata.
LocationsTSnLPxSODCATGPxGRGSTAAGSHDPPH
GJHPR3.43 ± 0.035 d6.23 ± 0.247 b7654.63 ± 25.7 b2.4 ± 0.106 c117.49 ± 3.14 c1.85 ± 0.026 d122.1 ± 4.09 b0.121 ± 0.0038 a12.36 ± 0.48 b
W3.28 ± 0.052 b7.85 ± 0.381 c7059.57 ± 17.8 a2.7 ± 0.063 d135.28 ± 1.79 d1.87 ± 0.031 e103.9 ± 3.8 a0.259 ± 0.0062 d14.84 ± 0.48 c
S4.35 ± 0.066 e5.21 ± 0.25 a8123.94 ± 24.8 c3.6 ± 0.072 e180.132 ± 1.96 f2.66 ± 0.039 f111.86 ± 3.7 a0.196 ± 0.0043 c16.66 ± 0.56 e
MuscleR0.162 ± 0.007 c1.18 ± 0.060 a567.97 ± 5 e0.47 ± 0.0091 b23.944 ± 0.26 b0.155 ± 0.001 b69.66 ± 0.69 c0.405 ± 0.0038 c5.71 ± 0.22 b
W0.131 ± 0.003 a1.75 ± 0.091 c500.74 ± 5.2 d0.84 ± 0.0076 d42.7 ± 0.21 c0.169 ± 0.003 c57.056 ± 4.44 a0.406 ± 0.0029 c6.54 ± 0.42 c
S0.18 ± 0.004 d1.05 ± 0.138 a640.97 ± 6.3 f1.24 ± 0.0375 e63.484 ± 1.07 d0.191 ± 0.003 e71.52 ± 4.4 c0.334 ± 0.0072 b7.42 ± 0.25 d
TNHPR3.55 ± 0.024 d8.65 ± 0.219 d8271.15 ± 20.6 d1.3 ± 0.0353 a69.636 ± 0.89 a1.86 ± 0.060 d125.26 ± 4.4 b0.143 ± 0.0019 a11.24 ± 0.63 a
W3.22 ± 0.02 b10.65 ± 0.191 f8097.94 ± 35.1 c1.6 ± 0.0403 b83.33 ± 1.48 b2.23 ± 0.034 ef114.15 ± 2.7 ab0.232 ± 0.0036 d16.41 ± 0.47 e
S4.15 ± 0.04 de7.81 ± 0.251 cd8994.07 ± 32 e1.7 ± 0.0626 b88.742 ± 2.12 b3.18 ± 0.056 g123.57 ± 3.96 a0.167 ± 0.0063 b17.85 ± 0.45 f
MuscleR0.179 ± 0.004 d1.71 ± 0.088 c234.76 ± 9.4 a0.45 ± 0.0065 b23.07 ± 0.16 b0.156 ± 0.0043 b56.49 ± 5.14 a0.327 ± 0.0026 b4.78 ± 0.61 a
W0.132 ± 0.003 a2.66 ± 0.188 d250.96 ± 8.2 b0.81 ± 0.03 d41.89 ± 0.83 c0.187 ± 0.0035 d56.08 ± 4.71 a0.288 ± 0.0026 a6.28 ± 0.64 c
S0.229 ± 0.009 f1.49 ± 0.074 b364.6 ± 11.2 c1.80 ± 0.026 f90.56 ± 1.18 f0.287 ± 0.0044 f65.2 ± 4.45 b0.32 ± 0.0022 b7.51 ± 0.52 d
ODHPR3.33 ± 0.034 c9.74 ± 0.308 f8412.42 ± 23.6 d2.24 ± 0.040 c114.52 ± 1.32 c1.245 ± 0.035 a123.8 ± 7.19 b0.181 ± 0.0025 b12.73 ± 0.35 b
W3.05 ± 0.052 a13.19 ± 0.329 g7889.27 ± 23.2 b3.20 ± 0.244 e168.32 ± 9.42 e1.415 ± 0.0253 b131.4 ± 3.56 c0.192 ± 0.0024 c14.49 ± 0.6 d
S4.61 ± 0.042 d9.043 ± 0.269 e9827.03 ± 26.7 f4.2 ± 0.209 f220.15 ± 6.57 g1.781 ± 0.045 c137.4 ± 4.46 c0.171 ± 0.0021 b17.70 ± 0.65 f
MuscleR0.163 ± 0.0022 c2.94035 ± 0.22 e803.04 ± 6.07 h0.4 ± 0.0045 a20.62 ± 0.32 a0.14 ± 0.0026 a62.7 ± 4.1 b0.404 ± 0.0022 c5.87 ± 0.23 b
W0.145 ± 0.0026 b3.483 ± 0.035 f707.09 ± 10.98 g0.7 ± 0.021 c39.08 ± 0.88 c0.18 ± 0.002 cd59.9 ± 3.4 a0.584 ± 0.0029 f7.61 ± 0.29 d
S0.187 ± 0.0035 e2.7823 ± 0.182 d889.53 ± 8.47 h1.2 ± 0.041 e64.19 ± 1.3 e0.21 ± 0.0039 e74 ± 3.28 d0.421 ± 0.0023 d6.82 ± 0.27 cd
Note(s): Each value represents the mean ± SD of 10 individuals. Superscripts (a, b, c, d, e, f, g, and h) indicate statistical significance within the groups at p < 0.05. Groups with different superscripts are statistically different from each other. T—tissue, Sn—season, GJ—Gujarat, TN—Tamil Nadu, OD—Odisha, S—summer, W—winter, R—rainy, SOD—superoxide dismutase, CAT—catalase, GPx—glutathione peroxidase, GR—glutathione reductase, GST—glutathione s transferase, AA—ascorbic acid, GSH—the reduced glutathione and DPPH—total antioxidant capacity.
Table 3. Correlation between the studied parameters and sediment factors.
Table 3. Correlation between the studied parameters and sediment factors.
ParametersGujaratOdishaTamil Nadu
HPMuscleHPMuscleHPMuscle
pH0.960.950.970.840.970.88
Temp0.530.590.950.780.810.72
Salinity0.690.610.690.810.970.88
OC0.810.750.830.880.920.84
Ca0.870.810.470.660.880.81
Mg0.950.920.760.860.970.88
Note(s): Correlation Coefficient (r) was taken as significant at 5% confidence level with degrees of freedom as 14. OC-organic carbon, Ca-calcium, Mg- magnesium, temp-temperature. Sampling was performed in Gujarat, Odisha, and Tamil Nadu.
Table 4. Correlation analyses of the redox regulatory parameters with allantoin in crab tissues.
Table 4. Correlation analyses of the redox regulatory parameters with allantoin in crab tissues.
ParametersGujaratOdishaTamil Nadu
HPMuscleHPMuscleHPMuscle
LPx0.900.540.980.650.800.60
SOD−0.59−0.46−0.66−0.14−0.48−0.26
CAT0.690.770.970.660.900.78
GPx0.970.880.760.900.720.90
GR0.950.880.750.910.620.90
GST0.990.860.830.790.950.88
GSH0.20−0.92−0.78−0.23−0.03−0.15
AA0.180.280.310.630.050.26
DPPH0.760.710.750.070.700.55
Note(s): A positive (+) or negative (−) ccorrelation coefficient (r) was considered as significant at a 5% confidence level. Digits presented in bold numbers are significantly correlated with degrees of freedom as 14. LPx—lipid peroxidation, SOD—superoxide dismutase, CAT—catalase, GSH—reduced glutathione, GST—glutathione-s-transferase, AA—ascorbic acid, GR—glutathione reductase, and DPPH—total antioxidant capacity.
Table 5. DFA for allantoin and OS physiology parameters in Scylla serrata in different seasons.
Table 5. DFA for allantoin and OS physiology parameters in Scylla serrata in different seasons.
FunctionEigenvalue% of VarianceCumulative %Canonical Correlation
1576.148 97.397.30.999
211.694 299.20.96
33.448 0.699.80.88
40.866 0.11000.681
50.269 01000.46
Note(s): Function 1 and 2 shows show a high level of Eigenvalues as well as canonical correlation.
Table 6. Standardized Canonical Discriminant Function Coefficients values of all parameters.
Table 6. Standardized Canonical Discriminant Function Coefficients values of all parameters.
ParametersFunction
12345
AA−0.1180.3180.120.174−0.816
GSH−0.1240.065−0.1880.0810.428
CAT1.281−1.3910.172−0.060.74
SOD0.9850.103−0.3240.5060.044
GST0.3650.507−0.5030.201−0.155
DPPH0.133−0.060.4560.6410.609
GR1.0131.4941.065−1.6560.787
GPx−0.425−1.416−1.4091.964−1.213
LPx0.1241.615−0.124−0.295−0.055
Allantoin0.75−0.3540.7910.108−0.048
Note(s): Antioxidant parameters such as CAT, SOD, GR and allantoin show high canonical discriminant coefficient values.
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Pati, S.G.; Paital, B.; Sahoo, D.K. Allantoin and Tissue Specific Redox Regulation in Mud Crab Scylla serrata under Varied Natural Water Physico-Chemical Parameters. Water 2024, 16, 480. https://doi.org/10.3390/w16030480

AMA Style

Pati SG, Paital B, Sahoo DK. Allantoin and Tissue Specific Redox Regulation in Mud Crab Scylla serrata under Varied Natural Water Physico-Chemical Parameters. Water. 2024; 16(3):480. https://doi.org/10.3390/w16030480

Chicago/Turabian Style

Pati, Samar Gourav, Biswaranjan Paital, and Dipak Kumar Sahoo. 2024. "Allantoin and Tissue Specific Redox Regulation in Mud Crab Scylla serrata under Varied Natural Water Physico-Chemical Parameters" Water 16, no. 3: 480. https://doi.org/10.3390/w16030480

APA Style

Pati, S. G., Paital, B., & Sahoo, D. K. (2024). Allantoin and Tissue Specific Redox Regulation in Mud Crab Scylla serrata under Varied Natural Water Physico-Chemical Parameters. Water, 16(3), 480. https://doi.org/10.3390/w16030480

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