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\setlength{\parindent}{0pt}\raggedright \textcolor{GJMediumGrey}{\rule{\textwidth}{2pt}} \vskip16pt \textcolor{GJBlue}{\large\bfseries\abstractname\space} }{% \vskip8pt \textcolor{GJMediumGrey}{\rule{\textwidth}{2pt}} \vskip16pt } \usepackage[absolute,overlay]{textpos} \makeatother \usepackage{lineno} \linenumbers \begin{document} \author[1]{Diogo Barbalho Hungria} \author[2]{Camila Prestes dos Santos Tavares} \author[3]{Ubiratã de Assis Teixeira da Silva} \author[4]{Leandro Ângelo Pereira} \author[5]{Ariana Cella-Ribeiro} \author[6]{Antonio Ostrensky} \renewcommand\Authands{ and } \date{\small \em Received: 8 April 2021 Accepted: 30 April 2021 Published: 15 May 2021} \maketitle \begin{abstract} Soft-shell crab is considered a gastronomic delicacy, reaching high values in the international market. Under normal conditions, the process of hardening of the crab's exoskeleton after moulting takes approximately two days to complete; however, the commercial consistency of soft-shell crab is lost in just 3 hours. The goal of this research was to evaluate the effects of chemical changes of water on the duration of postmoult, specifically at the stage in which they can be marketed as soft-shelled crab. In this research, Callinectes ornatus (n=241) underwent two experiments: One group was maintained in tanks with partial daily water renewal (Experiment 1), and other in tanks without water renewal (Experiment 2). In the experiment 1, the chemical characteristics of the water remained unchanged over time (p > 0.05), and the median time to hardening of the exoskeleton after moulting was 3 hours. \end{abstract} \keywords{acidification; ammonification; portunidae; calcium; moult; soft-shell crab.} \begin{textblock*}{18cm}(1cm,1cm) % {block width} (coords) \textcolor{GJBlue}{\LARGE Global Journals \LaTeX\ JournalKaleidoscope\texttrademark} \end{textblock*} \begin{textblock*}{18cm}(1.4cm,1.5cm) % {block width} (coords) \textcolor{GJBlue}{\footnotesize \\ Artificial Intelligence formulated this projection for compatibility purposes from the original article published at Global Journals. However, this technology is currently in beta. \emph{Therefore, kindly ignore odd layouts, missed formulae, text, tables, or figures.}} \end{textblock*} \begin{textblock*}{10cm}(1.05cm,3cm) {{\textit{CrossRef DOI of original article:}} \underline{10.34257/GJMRGVOL21IS1PG1}} \end{textblock*}\let\tabcellsep& \par Is it Possible to Keep the Exoskeleton of the Crab Callinectes Ornatus Soft for Several Days?\par Slowing the Hardening of the Crab Exoskeleton Diogo Barbalho Hungria ? , Camila Prestes dos Santos Tavares ? , Ubiratã de Assis Teixeira da Silva ? , Leandro Ângelo Pereira ? , Ariana Cella-Ribeiro ¥ \& Antonio Ostrensky § Abstract-Soft-shell crab is considered a gastronomic delicacy, reaching high values in the international market. Under normal conditions, the process of hardening of the crab's exoskeleton after moulting takes approximately two days to complete; however, the commercial consistency of soft-shell crab is lost in just 3 hours. The goal of this research was to evaluate the effects of chemical changes of water on the duration of postmoult, specifically at the stage in which they can be marketed as soft-shelled crab. In this research, Callinectes ornatus (n=241) underwent two experiments: One group was maintained in tanks with partial daily water renewal (Experiment 1), and other in tanks without water renewal (Experiment 2). In the experiment 1, the chemical characteristics of the water remained unchanged over time (p > 0.05), and the median time to hardening of the exoskeleton after moulting was 3 hours. Over the course of experiment 2, there was a reduction (p <0.05) in pH and increases in the ammonia and nitrite concentrations. When moulting occurred in water with a pH below 7.3 and total ammonia concentrations above 6.0 mg/L, the crabs' shells did not harden, and it was possible to keep them soft for up to 5 days.\par Keywords: acidification; ammonification; portunidae; calcium; moult; soft-shell crab. \section[{Introduction}]{Introduction}\par allinectes ornatus Ordway, 1863 (Crustacea, Decapoda, Portunidae) is a swimmer crab found from North Carolina (USA) to the Rio Grande do Sul {\ref (Brazil)}. It occurs in areas with sand, mud or shell bottoms and inhabits estuarine to marine areas at a depth of approximately 75 m \hyperref[b5]{(Carvalho and} {\ref Couto 2011, Melo-Filho 1996)}. Similar to other arthropods, C. ornatus grows through a process of periodic exoskeleton changes; each shedding of the exoskeleton is known as ecdysis or moult {\ref (Drach 1939}\hyperref[b14]{, Freeman and Perry 1985}\hyperref[b33]{, Newcombe, Sandoz et al. 1949}). Immediately after shedding its exoskeleton, the crab presents a soft and flexible integument that has a low level of calcification. In this phase, the animals can be commercialised and consumed whole as "soft-shell crab", a delicacy that is appreciated worldwide and that reaches high market values \hyperref[b21]{(Hungria, Tavares et al. 2017}\hyperref[b47]{, Tavares, Silva et al. 2018}). According to FAO (2020), soft-shell crab aquaculture is considered a millionaire aquaculture practice in the eastern United States.\par Immediately after moult, CaCO 3 deposition begins on the protein matrix of the new exoskeleton. This process involves a complex system of absorption of Ca 2+ , CO 2 , and HCO 3 and the synthesis of CaCO 3 and other elements \hyperref[b19]{(Greenaway 1985}\hyperref[b38]{, Perry, Trigg et al. 2001}\hyperref[b53]{, Wheatly 1999}. The initially fragile exoskeleton undergoes rapid hardening, providing rigidity and mechanical protection for the animal. Under natural conditions, the hardening of the exoskeleton takes about two days to complete \hyperref[b4]{(Cameron and Wood 1985)}.\par During the hardening process, the exoskeleton can be classified into four sequential levels of consistency: soft, leather, paper and hard \hyperref[b13]{(Freeman, Kilgus et al. 1987)}. Only the first two are valued in the international market of soft-shell crabs (Gaude? and Anderson 2011, Oesterling 1995, Perry, Graham et al. 2010). However, the combined duration of the soft and leathery stages is very short in nature, rarely lasting more than 3 hours \hyperref[b4]{(Cameron and Wood 1985)}, which obliges commercial producers to inspect all of the Extending the duration in which the crab shells remain at the consistencies of high market value would significantly reduce production costs \hyperref[b38]{(Perry, Trigg et al. 2001}). Furthermore, it would minimise the damage caused by rapid exoskeleton hardening, providing better quality and uniformity regarding the softness of the product. The goal of this research was to evaluate the effects of chemical changes of water on the duration of postmoult to extend the time during which the animals could be marketed as soft-shell crab. \section[{II.}]{II.} \section[{Material and Methods}]{Material and Methods} \section[{a) Crab collection and maintenance}]{a) Crab collection and maintenance}\par Specimens of C. ornatus were obtained via trawling by professional fishers at the balneary of Shangri-la, municipality of Pontal do Paraná (25°37'S/48°25'O), Paraná, Brazil. Shrimp trawls 12 m in length and with 20 mm mesh were used. In each sampling campaign, on average, three trawls of approximately 50 minutes each were made. Immediately after crab collection from the net, the crabs were separated and transferred to two polyethylene tanks (70 L volume) with lids, each containing 20 L of seawater. The tanks received continuous aeration supplied via an 18W air compressor. Inside each tank were plastic screens with 2 mm mesh positioned to reduce contact and prevent fights between the animals and minimise injuries and deaths. Thereafter, 100\% of the water was renewed every half hour during the campaign.\par Immediately after capture, the animals were transported to the Marine Aquaculture and Restocking Center (CAMAR) of the Integrated Group of Aquaculture and Environmental Studies (GIA), Federal University of Paraná (UFPR), at Pontal do Paraná (25°41'29.94"S, 48°27'57.09"W). The time elapsed between animal capture and arrival at the laboratory was consistently less than 4 hours. Animals that were not used were returned to the sea. In the laboratory, the crabs were maintained in 1,000 L tanks containing 100 L of seawater (30 mg/L) supplied with constant aeration for approximately 6 hours. This period was purposely short since a large proportion of the captured individuals were very close to moult. Dead animals were discarded, and the live animals were classified by sex. Then, the crabs were inspected to determine the phase of the moulting cycle.\par Those individuals at the premoult phase were selected for the experiments based on macroscopic indicators (visualisation of an inner line along the edges of the fifth pair of pleopods) {\ref (Drach 1939}\hyperref[b11]{, Drach and Tchernigovtzeff 1967}\hyperref[b49]{, Wehrtmann and Mena-Castañeda 2003)}. The selected individuals were weighed on an analytical balance (Marte AL 500c, Brazil; accuracy of 0.01 g) and measured (width of the carapace, measured as the distance between the base of the largest lateral spines) with a pachymeter. The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered. As it was an invertebrate research project, it was not necessary to submit it for analysis by the Animal Use Ethics Commission of Federal University of Paraná (UFPR). In any case, all experimental procedures used followed current legislation and the rules of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) for the capture and use of native invertebrates. \section[{b) Pilot experiments}]{b) Pilot experiments}\par Two pilot experiments were carried out. The first experiment tested the influence of fasting on animal survival under laboratory conditions. The animals only began to die after 50 days without access to food. Based on this result and to potential feeding effects on water quality or the process of moult and hardening, the crabs were not fed during the 12 days of each of the main experiments. The second experiment tested the influence of the non-renewal of water on hardening time. The time elapsed between moult and shell hardening was significantly higher under water non-renewal than under the periodic renewal of water. In addition, a higher frequency of moult was observed at night (between 18:00 and 06:00); this information informed the design of the experimental methodology described below. \section[{c) Experimental design}]{c) Experimental design}\par In both experiments, the saltwater had been previously chlorinated and maintained under constant aeration for 24 h. After this period, residual chlorine was neutralised (with 50\% sodium thiosulfate), and the water was stored in the dark in 25,000 L tanks. Before use in the experiments, the water was passed through mechanical filters of 5 and 25 ?m mesh and a UV filter for disinfection. Two experiments were performed and are represented schematically in FIGURE \hyperref[fig_0]{1}. i. Experiment 1: crab maintenance in a collective system with filtration and partial daily water renewal C. ornatus crabs (n=66) were individually placed in perforated pet bottles (600 mL) and distributed in a system consisting of 20 polyethylene tanks (71.0 x 35.5 x 35.0 cm, containing 25 L of seawater each). The tanks were interconnected via a skimmer and a mechanical/biological filter system and were under constant aeration, continuous water recirculation and controlled photoperiod (14:10-light: dark).The animals were separated into two groups: A1, premoultcrabs (n = 46), and AC, control crabs (at the intermoult stage) (n = 20). Each day throughout the experimental period (12 days), 1/3 of the total water volume of the system (333 L) was added, promoting mixing with the water already present, and an approximately equivalent amount was removed, keeping the total water volume in the system constant.\par ii. Experiment 2: crab maintenance in a collective system with filtration and without water renewal C. ornatus crabs (n = 176) were individually placed in perforated (600 mL) pet bottles and distributed among 12 polyethylene tanks (71.0 x 35.5 x 35.0 cm, containing approximately 30 L of water each). Each tank contained a protein skimmer and a mechanical/biological filter and was subjected to constant aeration, continuous water recirculation and controlled photoperiod (14:10 -light: dark). The animals were subdivided into 3 groups: B1, premoultcrabs (n = 83); B2, premoultcrabs (n = 40); and BC, control intermoult animals (n = 52). In addition, three tanks containing water only were maintained throughout the experimental period (12 days) for comparison of physical and chemical water variables between these tanks and the 3 treatment groups. There was no water renewal during the experiment. The crabs in the B2 group were housed in the same tanks used to house group B1 and maintained in the same water used for group B1.TABLE 1 provides summary information on the subject animals and design of the two experiments. \section[{d) Experimental procedures}]{d) Experimental procedures}\par During the experiments, the crabs were monitored every three hours on the first four days, every six hours on the following five days, and every 12 hours on the last three days of experimentation, preferably between 18:00 and 06:00 hours. These times were selected based on the results of the pilot experiments. Monitoring consisted of identifying animals undergoing the moulting process, removing any moulted exoskeletons (to prevent the animals from obtaining calcium by feeding on them), evaluating the consistency of the carapace of those animals that had moulted, and removing any dead animals. Evaluating the consistency of the exoskeleton was performed by pressing the carapace with an index finger. Sufficient pressure was applied to deform the carapace but not injure the animal or break the carapace when rigid. Based on the resistance to pressure and texture of the exoskeleton, its consistency (Co) was classified by the evaluator as follows: hard -before ecdysis (1), soft (2), leather (3), soft paper (4), hard (5) or hard paper -after ecdysis (6). To reduce and standardise the error, a single evaluator performed the consistency assessments in both experiments. \section[{e) Water analysis}]{e) Water analysis}\par In both experiments, salinity (refractometer; Instrutemp, Brazil), temperature (digital thermometer), pH (AZ pH/mV/TDS /Temperature Meter 86505, Taiwan), and dissolved oxygen concentration (Oximeter YSI 550A, US states) were monitored daily in all experimental units. Water samples were collected from the units, labelled and immediately frozen (-20°C) for later evaluation of the physical and chemical variables. For group A1, water collection was performed every 24 hours before the new water was added to the system. For groups B1, B2, BC and BA, 50 mL of water was collected every 12 h.\par At the end of the experiments, the frozen water samples were analysed with respect to the following parameters: Na + , K + , Ca 2+ and NO 3 (electrodes of the LAQUA twin series, Horiba Scientific®, Japan) and total ammonia (NH 3 +NH 4 + ) and NO 2 -(SpectraMax® m2 spectrophotometer, US states).Measurements were performed following APHA (2005) and \hyperref[b2]{Büldt and Karst (1999)}. The determinations of Mg 2 + and Clwere performed using colourimetry (Labtest®, Brazil) at a wavelength of 540 nm and 470 nm, respectively (SpectraMax® m2, US states), according to the method described by \hyperref[b6]{Clarke (1950)}.( D D D D ) G f) Statistical analyses\par The survival of the animals during the experiments was analysed through Kaplan-Meier curves. The data were grouped by treatment (groups), and the normality of the distribution of each variable was tested by using Shapiro-Wilk test. Where the normality hypothesis was rejected, non-parametric Mann-Whitney or Kruskal-Wallis tests were used. Multiple linear regression analysis was performed to model the influences of the physical and chemical variables that determine water quality on exoskeleton hardening time.\par The assumption of the independence of the physical and chemical variables was upheld, the hypothesis of autocorrelation and collinearity (using Durbin-Watson and the serial error correlation tests) was rejected, and the normality of the error was confirmed.\par To limit the number of variables and thereby minimize the complexity of the models without a significant loss of the information offered by the total set of original variables, we select only those variables that: 1) were statistically significant (p <0,05) and; 2) contributed more than 5\% to the coefficient of determination (R²) of the model or that caused the R2value to move into a higher category when it was included in the model, following the classification proposed by \hyperref[b31]{Mukaka (2012)}: very weak:: R 2 < 0,19; weak: 0,20> R 2 <0,39; moderate: 0,40> R 2 < 0,69; strong: 0,70> R 2 <0,89; very strong: R 2 > 0,90. \section[{III.}]{III.} \section[{Results}]{Results} \section[{a) Ecdysis}]{a) Ecdysis}\par Significant effects of sex on survival rate, ecdysis, or exoskeleton postmoult hardening time were not observed. Therefore, the data from males and females were pooled. In addition, water temperature (27.0 ± 1.1°C), salinity (31.0 ± 2.1 mg/L) and dissolved oxygen concentration (5.0 ± 0.52 mg/L) remained largely stable and did not significantly influence any of the dependent variables. The moulting rate of the animals in premoult at the beginning of the experiments ranged from 40 to 95\%. Most moulting events occurred during the night, and 50\% of the animals moulted between 52 and 80 hours after the beginning of the experiments. There was a significant effect of moulting on final mortality rate and on survival time after ecdysis (TABLE \hyperref[tab_1]{2}). \section[{b) Physical and chemical water parameters}]{b) Physical and chemical water parameters}\par In Experiment 1, the total ammonia (NH 3 +NH??) concentrations remained below the limit of analytical detection, and the median pH was 8.5, with variation between 8.1 and 8.5. The remaining physical and chemical parameters were relatively stable throughout the experimental period (TABLE \hyperref[tab_2]{3}). In Experiment 2, only potassium and sodium concentrations presented differences between the groups B1 and B2. There was a reduction in pH and increases in total ammonia and nitrite concentrations in the experimental treatments (premoult crabs, B1 and B2) in relation to the control (BA, tanks containing water only \section[{B1: crabs in premoult. B2: tanks containing water previously used for group B1 and premoult crabs. BA (Control): tanks containing water only. BC (Control): tanks with crabs in intermoult. Different letters indicate significant differences (p < 0.05) between groups according to the Kruskal-Wallis test. TA: Total ammonia (NH 3 +NH4 + ). c) Influence of the physical and chemical water parameters on the survival and moulting of C. ornatus}]{B1: crabs in premoult. B2: tanks containing water previously used for group B1 and premoult crabs. BA (Control): tanks containing water only. BC (Control): tanks with crabs in intermoult. Different letters indicate significant differences (p < 0.05) between groups according to the Kruskal-Wallis test. TA: Total ammonia (NH 3 +NH4 + ). c) Influence of the physical and chemical water parameters on the survival and moulting of C. ornatus}\par As expected, crab survival time was influenced by moulting regardless of the experiment. The crabs of group B1 that underwent moult in the first 36 h showed rapid exoskeleton hardening and low mortality rates. Therefore, the B1 data were divided into two categories: M1, animals that moulted within the first 36 h, and M2, those that moulted after 36 h. The survival of M1 animals was strongly influenced by pH and total ammonia and nitrite concentrations, whereas the survival of the M2 animals was moderately influenced by the same variables. TABLE \hyperref[tab_4]{5} shows the multiple linear regression results. Crab survival rate was significantly influenced by pH, nitrite and total ammonia in all of the experiments. The remaining parameters had no significant influence (p < 0.05) on the crab survival time. The results of the multiple linear regression analysis of the effects of the physical and chemical parameters on the time until either the shells fully hardened (reached Co6) or death are presented in TABLE \hyperref[tab_5]{6}. In Experiment 1, only pH had an influence (weak) on the results. In experiment 2, pH, ammonia and nitrite had moderate influences on the results. The duration at which the shell was at consistency 2 (i.e., the consistency with the highest market value) was significantly higher in the M2 animals than in the M1 animals. Furthermore, none of the M2 individuals that moulted after 36 h achieved Co6 (hard) shells, whereas in the M1 group, more than half of the individuals had shells that reached this consistency. In addition, 68\% of individuals with shells that hardened remain alive. Among those that did not achieve shell hardening, the survival rate was only 15\% (TABLE \hyperref[tab_6]{7}). G Table {\ref 8} shows the median, minimum and maximum values of the physical and chemical parameters of water quality that most influenced crab survival time and exoskeleton consistency: pH, ammonia and nitrite. The duration at each level of consistency was highly related to the pH and concentration of total ammonia at the time of moulting. When the levels of the physical and chemical parameters favoured hardening, the time until the crabs reached Co4 (soft paper) was short (between two and four hours). In contrast, when the pH was below 7.3 and the total ammonia concentration remained above 6 mg/L, the median time to Co4 was 60 hours. G Table {\ref 8}: Water quality parameter measurements and crab survival data registered in the experimental units in which the crabs (Callinectes ornatus) moulted. \section[{Experiment 1 (collective treatment with filtration and partial daily water renovation). A1: crabs in premoult. Experiment 2 (collective treatment with filtration but no water renewal). B1: crabs in premoult; B2: tanks containing water previously used for the B1 group, with crabs also in premoult; M1: crabs that moulted within 36 hours; M2: crabs that moulted after 36 hours. TA: Total ammonia (NH 3 +NH4 + ).}]{Experiment 1 (collective treatment with filtration and partial daily water renovation). A1: crabs in premoult. Experiment 2 (collective treatment with filtration but no water renewal). B1: crabs in premoult; B2: tanks containing water previously used for the B1 group, with crabs also in premoult; M1: crabs that moulted within 36 hours; M2: crabs that moulted after 36 hours. TA: Total ammonia (NH 3 +NH4 + ).} \section[{Exp.: Experiment 1 (collective treatment with filtration and partial daily water renovation), Experiment 2 (collective treatment with filtration but no water renewal). A1 and B1}]{Exp.: Experiment 1 (collective treatment with filtration and partial daily water renovation), Experiment 2 (collective treatment with filtration but no water renewal). A1 and B1}\par When water renewal was not performed (Experiment 2), the pH and total ammonia and nitrite concentrations varied significantly (FIGURE \hyperref[fig_5]{2}). As a result, there was an increase in the carapace hardening time and a decrease in the number of individuals reaching Co6. The crabs of Experiment 1 (A1) and the animals that moulted within the first 36 hours of Experiment 2 (M1) spent significantly less time at Co2 and Co 3 than did those that moulted after the first 36 hours (M2) in group B1 and those in group B2. \section[{Discussion}]{Discussion}\par An issue repeatedly debated among those who investigate the shedding and hardening process in crustaceans is the importance of calcium, the main constituent element of the exoskeleton \hyperref[b19]{(Greenaway 1985)}, in this process \hyperref[b3]{(Cameron 1985} + in the exoskeleton and thereby significantly increase the time that these animals remain soft after moult.\par The organisms of Experiment 1 (subjected to daily water renewal) that underwent ecdysis hardened rapidly, achieving paper consistency (Co 4) a median of 4 hours after moult. This finding is consistent with studies conducted with C. sapidus \hyperref[b3]{(Cameron and} {\ref Wood 1985, Freeman, Kilgus et al. 1987}). Similar results were observed among the crabs in Experiment 2 that moulted in water with a pH above 7.6 and a total ammonia concentration below 4.8 mg/L, with Co 4 achieved after a median time of 2 to 3 hours. However, among the animals that began moulting in water with a pH below 7.3 and a total ammonia concentration above 6 mg/L, up to 129 hours (median of more than 60 hours) elapsed before either reaching Co4 or death.\par To understand this result, it is necessary to understand the chemical processes involved in the calcification of the crab exoskeleton. In a closed system G with water recirculation, it is expected that over time there will be a reduction in the concentration of free Ca 2 + , due mainly to the immobilisation of Ca 2 + in the form of CaCO 3 during exoskeleton hardening \hyperref[b38]{(Perry, Trigg et al. 2001}). This immobilisationcan be represented by the following equation:Ca 2+ + HCO 3 -? CaCO 3 + H + (1)\par With the increased demands for Ca 2 + and HCO 3 -, crabs begin to consume both metabolic and external CO 2 . CO 2 reaches its highest internal concentrations at moulting time \hyperref[b26]{(Mangum, deFur et al. 1985)}, increasing the availability of internal HCO 3 - \hyperref[b4]{(Cameron and Wood 1985)}. As soon as moulting occurs, the enzyme carbonic anhydrase (CA), present mainly in the epithelium and the gills, is activated \hyperref[b26]{(Mangum, deFur et al. 1985)}, accelerating the reaction:CO 2 + H 2 O?H 2 CO 3 ?H + + HCO 3 - (2)\par As explained by \hyperref[b8]{Detours, Armand et al. (1968)} and Zeebe and Wolf-Gladrow (2001), the formed carbonic acid tends to be buffered by the carbonatebicarbonate system. This process results in an increase in the fraction of CO 3 -and acidification of the medium \hyperref[b17]{(Greenaway 1974}\hyperref[b26]{, Mangum, deFur et al. 1985}\hyperref[b52]{, Wheatly 1997)}. However, over time, the natural acid neutralisation capacity of the system becomes compromised, and the medium tends to acidify as a result, increasingly compromising the crab's capacity to deposit CaCO 3 in its exoskeleton. According to \hyperref[b4]{Cameron and Wood (1985)}, the calcification process can be compromised if the pH outside the body is less than 0.3 to 0.5 above the internal pH.\par However, in addition to consuming HCO 3postmoult, the crab excretes H+ or an equivalent ion such as NH \hyperref[b39]{(Rijn, Tal et al. 2005)}. Under these conditions, the metabolism of excretion also contributes to the acidification of the medium, further reducing the capacity for calcium mobilisation by the crab, as observed in experiment 2. There is evidence that water acidification is more critical for the hardening process of marine crustaceans than for that of freshwater crustaceans. Unlike freshwater crustaceans, marine crustaceans have almost no internal reserves of calcium (gastroliths) and depend exclusively on the environment to supply the demand for Ca 2+ \hyperref[b19]{(Greenaway 1985}\hyperref[b36]{, Passano 1960}\hyperref[b52]{, Wheatly 1997)}.\par In a similar manner, acidification might affect the deposition of magnesium in the crustacean exoskeleton. Although magnesium concentrations in water are relatively lower than those of calcium, magnesium also plays an important role in the hardening of the exoskeleton, and it is also obtained through water \hyperref[b4]{(Cameron and Wood 1985}\hyperref[b7]{, Clarke and Wheeler 1922}\hyperref[b50]{, Welinder 1974})in a process that might be affected by pH \hyperref[b46]{(Tao, Zhou et al. 2009}).\par In addition to Ca 2+ and Mg 2+ concentrations, the concentrations of Na + and K + were monitored in this study. These two ions directly participate in important enzymatic activities that occur postmoult \hyperref[b48]{(Towle and Mangum 1985)}. Studies have shown that if the relative proportions of these two ions are altered, ammonia toxicity can occur due to the retention of ammonia by the organism and potentially compromise the anima's survival \hyperref[b35]{(Pan, Luan et al. 2006}, Romano and Zeng 2011, Zanotto and Wheatly 1993). However, were observed no significant effects of these ions in our experiments. It is possible that" the factors described above were much more important in influencing exoskeleton hardening and the probability of survival in C. ornatus.\par There was also a direct relationship between the time to exoskeleton hardening and the mortality rate. However, the mortality rate was only 25\% among those crabs that moulted after approximately 60 h. Those that did not moult died or remained alive until the end of the experiment. In addition, in all of the groups except those receiving periodic water renewal, there was an increase in mortality in the postmoult phase. In this case, the analyses again indicated the influences of pH and total ammonia.\par It is known that crabs (notably C. sapidus, the most studied species of the genus Callinectes) can tolerate a pH range of 6.5 to 8.5 \hyperref[b20]{(Hochheimer 1988}). Nevertheless, in artificial environments, it is recommended that pH be maintained between 7.0 and 8.0 \hyperref[b34]{(Oesterling 1995)}. It is also known that there are behavioural and tolerance differences between young and adult animals in relation to pH \hyperref[b24]{(Laughlin, Cripe et al. 1978)}. In Experiment 2 of the present study, pH values of 5.5 and 5.3 were recorded in groups B1 and B2, respectively. In addition to having a direct effect on the organisms, a reduction of pH causes an increase in the nitrous acid fraction (HNO 2 ) present in water; The toxicity of ammonia, in turn, is directly proportional to pH andNH3 concentrations. Romano and Zeng (2007) estimated an LC 50 for juveniles of Scylla serrata of 6.81 mg/L NH 3 -N. \hyperref[b22]{Koo, Kim et al. (2005)} reported that at least 50\% of juveniles of Orithyiasinica survived for 30 days at approximately 2.33 mg/L NH 3 -N. \hyperref[b23]{Lakshmi (1984)} reported a mortality rate of 20\% in C. sapidus in premoult at 1.41 mg/L NH 3 , which increased to 100\% at 2.31 mg/L NH 3 . In our experiments, a pH reduction was observed over time, which indicated that the NH 3 concentrations remained sufficiently low as to rule out any toxic effects of ammonia on C. ornatus. \section[{G}]{G}\par Regarding nitrite, there is no consensus regarding the concentrations at which this compound is toxic to crabs. \hyperref[b23]{Lakshmi (1984)} and \hyperref[b0]{Ary and Poirrier (1989)} reported that the survival of C. sapidus was only affected at NO 2 -concentrations above 10 mg/L. According tothose authors, crab mortality reached 100\% only after 96 h of exposure to concentrations between 50 and 150 mg/L in water with a pH close to 8.In contrast, \hyperref[b27]{Manthe, Malone et al. (1984)} found that the moulting efficiency of C. sapidus was affected by nitrite concentrations close to 2 mg/L. In the present study, the nitrite concentration reached 7.6 mg/L.Thus, it is possible that the observed mortality might have been influenced by both pH and nitrite levels during the experiments and that they had a cumulative effect. Moreover, a long hardening time, which exposed the animals to unfavourable physiological conditions, appears to have significantly increased the risk of death.\par Moulting in C. ornatus exhibited strong relationships with the characteristics of the crab's aquatic medium. The crabs drastically altered the physical and chemical characteristics of the water, mainly through processes related to acidification and ammonification. These alterations, in turn, directly interfered with exoskeleton hardening, causing the exoskeletons of the animals to remain at soft or paper consistency for periods of up to 5 days. Commercially, the establishment of such periods would allow crabs to be marketed as soft-shell crabs within a time window more than 20 times longer than that typically observed. If the results observed here can be replicated at the commercial scale, large reductions in workload and operational costs could be obtained, increasing the efficiency and viability of large-scale crab production.\begin{figure}[htbp] \noindent\textbf{1}\includegraphics[]{image-2.png} \caption{\label{fig_0}Figure 1 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{2}\includegraphics[]{image-3.png} \caption{\label{fig_5}Figure 2 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{1} \par \begin{longtable}{P{0.013385826771653543\textwidth}P{0.020078740157480315\textwidth}P{0.23202099737532808\textwidth}P{0.05800524934383202\textwidth}P{0.03011811023622047\textwidth}P{0.17066929133858266\textwidth}P{0.1784776902887139\textwidth}P{0.0646981627296588\textwidth}P{0.08254593175853019\textwidth}} Experiment\tabcellsep Group\tabcellsep Experimental Unit\tabcellsep Stage\tabcellsep N\tabcellsep Width (mm) (±sd)\tabcellsep Weight (g) (±sd)\tabcellsep Water Volume (L)\tabcellsep Water Sampling Frequency\\ 1\tabcellsep A1 AC\tabcellsep Tank Tank\tabcellsep Premoult Intermoult\tabcellsep 46 20\tabcellsep 45.4 (±6.16) 61.55 (±10.53)\tabcellsep 15.04 (±6.11) 35.57 (±16.9)\tabcellsep 0.6 0.6\tabcellsep 24 h 24 h\\ \tabcellsep \tabcellsep Tank (Replicate 1)\tabcellsep \tabcellsep 27\tabcellsep 50.7 (±7.78)\tabcellsep 16.64 (±7.03)\tabcellsep 32.5\tabcellsep 12 h\\ \tabcellsep B1\tabcellsep Tank (Replicate 2)\tabcellsep Premoult\tabcellsep 27\tabcellsep 3.10 (±7.36)\tabcellsep 14.36 (±7.47)\tabcellsep 32.5\tabcellsep 12 h\\ \tabcellsep \tabcellsep Tank (Replicate 3)\tabcellsep \tabcellsep 29\tabcellsep 47.5 (±5.12)\tabcellsep 15.11 (±4.92)\tabcellsep 32.5\tabcellsep 12 h\\ \tabcellsep \tabcellsep Tank (Replicate 1)\tabcellsep \tabcellsep 14\tabcellsep 48.1 (±8.25)\tabcellsep 16.46 (±8.08)\tabcellsep 30\tabcellsep 12 h\\ 2\tabcellsep B2\tabcellsep Tank (Replicate 2) Tank (Replicate 3)\tabcellsep Premoult\tabcellsep 13 13\tabcellsep 46.4 (±7.02) 47.0 (±6.64)\tabcellsep 15.40 (±6.7) 14.75 (±6.3)\tabcellsep 30 30\tabcellsep 12 h 12 h\\ \tabcellsep \tabcellsep Tank (Replicate 1)\tabcellsep \tabcellsep 17\tabcellsep 56.2 (±2.9)\tabcellsep 26.51 (±5.07)\tabcellsep 32.5\tabcellsep 12 h\\ \tabcellsep BC\tabcellsep Tank (Replicate 2)\tabcellsep Intermoult\tabcellsep 17\tabcellsep 59.7 (±3.8)\tabcellsep 28.31 (±6.1)\tabcellsep 32.5\tabcellsep 12 h\\ \tabcellsep \tabcellsep Tank (Replicate 3)\tabcellsep \tabcellsep 18\tabcellsep 56.65 (±4.93)\tabcellsep 26.54 (±7.95)\tabcellsep 32.5\tabcellsep 12 h\\ \tabcellsep BA\tabcellsep Tank (3 Replicates)\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep 32.5\tabcellsep 12 h\end{longtable} \par \caption{\label{tab_0}Table 1 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{2} \par \begin{longtable}{P{0.2910958904109589\textwidth}P{0.016634050880626222\textwidth}P{0.03160469667318982\textwidth}P{0.016634050880626222\textwidth}P{0.043248532289628175\textwidth}P{0.10313111545988257\textwidth}P{0.049902152641878667\textwidth}P{0.016634050880626222\textwidth}P{0.07818003913894324\textwidth}P{0.0149706457925636\textwidth}P{0.016634050880626222\textwidth}P{0.0598825831702544\textwidth}P{0.0016634050880626223\textwidth}P{0.074853228962818\textwidth}P{0.0149706457925636\textwidth}P{0.019960861056751465\textwidth}} \multicolumn{4}{l}{Exp. Group Stage n 1}\tabcellsep Weight Gain (\%)\tabcellsep \multicolumn{3}{l}{Ecdysis Performed Period n 2 \%}\tabcellsep \multicolumn{3}{l}{Time to Ecdysis (h) 25\% 50\% 75\%}\tabcellsep \multicolumn{2}{l}{Mortality Rate (\%)}\tabcellsep \multicolumn{3}{l}{Survival Time (h) 25\% 50\% 75\%}\\ 1\tabcellsep A1\tabcellsep Pre\tabcellsep 46\tabcellsep 56\tabcellsep day night\tabcellsep 14 a 24 b\tabcellsep 83\tabcellsep 44.5\tabcellsep 80\tabcellsep 124\tabcellsep \multicolumn{2}{l}{24 a}\tabcellsep 230\tabcellsep -\tabcellsep -\\ \tabcellsep AC\tabcellsep Inter\tabcellsep 20\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep 0\tabcellsep b\tabcellsep -\tabcellsep -\tabcellsep -\\ \tabcellsep B1\tabcellsep Pre\tabcellsep 83\tabcellsep 69\tabcellsep day night\tabcellsep 19 a 59 b\tabcellsep 94\tabcellsep 36\tabcellsep 52\tabcellsep 78\tabcellsep \multicolumn{2}{l}{78 c}\tabcellsep 107\tabcellsep 168\tabcellsep 212\\ 2\tabcellsep B2\tabcellsep Pre\tabcellsep 40\tabcellsep 46\tabcellsep day night\tabcellsep 3 a 13 b\tabcellsep 40\tabcellsep 102\tabcellsep -\tabcellsep -\tabcellsep \multicolumn{2}{l}{78 c}\tabcellsep 174\tabcellsep 228\tabcellsep 413\\ \tabcellsep BC\tabcellsep Inter\tabcellsep 53\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep NA\tabcellsep \multicolumn{2}{l}{26 a}\tabcellsep 255\tabcellsep -\tabcellsep -\\ \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep B1:\\ \multicolumn{16}{l}{premoult organisms; B2: tanks containing water previously used for group B1, with organisms in premoult; BC (Control): tanks with}\\ \multicolumn{3}{l}{organisms in intermoult.}\tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \end{longtable} \par {\small\itshape [Note: Exp.: experiment number; Pre: premoult; Inter: Intermoult; n 1 : number of individuals; Weight Gain: increase in postmoult weight (\%); Period: the period in which moult occurred; n 2 : number and percentage of crabs that performed ecdysis; Time to Ecdysis: time (h) at which 25, 50 and 75\% of the animals had moulted; Mortality rate (\%);Survival Time: time (h) at which 25, 50 and 75\% of the animals survived after ecdysis; NA: Not Applicable. Different letters indicate significant differences (p < 0.05) between the groups according to the Kruskal-Wallis test. Experiment 1 (collective treatment with filtration and partial daily water renovation). A1: premoult organisms; AC (Control): organisms in intermoult. Experiment 2 (collective treatment with filtration but no water renewal).]} \caption{\label{tab_1}Table 2 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{3} \par \begin{longtable}{P{0.85\textwidth}} D D D D ) G\\ (\end{longtable} \par \caption{\label{tab_2}Table 3 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{4} \par \begin{longtable}{P{0.09622641509433963\textwidth}P{0.19245283018867926\textwidth}P{0.195368782161235\textwidth}P{0.19974271012006858\textwidth}P{0.16620926243567752\textwidth}} \tabcellsep \tabcellsep Groups\tabcellsep \tabcellsep \\ \tabcellsep B1\tabcellsep B2\tabcellsep BA\tabcellsep BC\\ Parameter\tabcellsep Median\tabcellsep Median\tabcellsep Median\tabcellsep Median\\ \tabcellsep 25-75\%\tabcellsep 25-75\%\tabcellsep 25-75\%\tabcellsep 25-75\%\\ pH\tabcellsep 6.7 b (6.3-7)\tabcellsep 6.5 b (6.2-7)\tabcellsep 8.4 a (8.3-8.4)\tabcellsep 7.8 ab (6.8-8.1)\\ K? (mg/L)\tabcellsep 420.00 a (370-540)\tabcellsep 280.00 b (230-330)\tabcellsep 330.00 ab (260-370)\tabcellsep 390.00 ab (290-420)\\ Ca 2 ? (mg/L)\tabcellsep 430.00 a (400-480)\tabcellsep 350.00 a (320-420)\tabcellsep 430.00 a (360-490)\tabcellsep 450.00 a (390-480)\\ Mg 2 ? (mg/L)\tabcellsep 545.6 b (472.3-585.8)\tabcellsep 551.2 b (531.8-622.5)\tabcellsep 586.3 ab (573.3-597.4)\tabcellsep 592.0 a (555.2-607.9)\\ Na? (mg/L)\tabcellsep 12,000 a (10,000-14,000)\tabcellsep 7,500 b (6,500-9,000)\tabcellsep 11,500 ab (10,000-13,000)\tabcellsep 12,000 ab (10,000-14,000)\\ Cl -(mg/L)\tabcellsep 18279 a (16,507-20,245)\tabcellsep 11995 a (9,158-15,782)\tabcellsep 15830 a (14,705-17,473)\tabcellsep \end{longtable} \par \caption{\label{tab_3}Table 4 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{5} \par \begin{longtable}{P{0.026288659793814433\textwidth}P{0.04162371134020618\textwidth}P{0.38775773195876284\textwidth}P{0.03286082474226804\textwidth}P{0.17744845360824743\textwidth}P{0.08543814432989691\textwidth}P{0.09858247422680412\textwidth}} Experiment\tabcellsep Group\tabcellsep \multicolumn{2}{l}{Correlated Parameters Cases n}\tabcellsep p\tabcellsep Adjusted R 2\tabcellsep Correlation\\ \tabcellsep \tabcellsep pH (max.)\tabcellsep \tabcellsep 0.000\tabcellsep \tabcellsep \\ 1\tabcellsep A1\tabcellsep TA (min.) NO2? (min.)\tabcellsep 384\tabcellsep 0.000 0.000\tabcellsep 0,372\tabcellsep Weak\\ \tabcellsep AC\tabcellsep NA\tabcellsep \tabcellsep NA\tabcellsep NA\tabcellsep NA\\ \tabcellsep \tabcellsep pH (max.)\tabcellsep \tabcellsep 0.000\tabcellsep \tabcellsep \\ \tabcellsep M1\tabcellsep TA (min.)\tabcellsep 233\tabcellsep 0.000\tabcellsep 0,724\tabcellsep Strong\\ \tabcellsep B1\tabcellsep NO?? (max) pH (max.)\tabcellsep \tabcellsep 0.000 0.000\tabcellsep \tabcellsep \\ \tabcellsep M2\tabcellsep TA (min.)\tabcellsep 318\tabcellsep 0.000\tabcellsep 0,681\tabcellsep Moderate\\ 3\tabcellsep \tabcellsep NO?? (med.) pH (max.)\tabcellsep \tabcellsep 0.000 0.000\tabcellsep \tabcellsep \\ \tabcellsep B2\tabcellsep TA (min.)\tabcellsep 495\tabcellsep 0.000\tabcellsep 0,430\tabcellsep Moderate\\ \tabcellsep \tabcellsep NO? (med.)\tabcellsep \tabcellsep 0.000\tabcellsep \tabcellsep \\ \tabcellsep \tabcellsep pH (max.)\tabcellsep \tabcellsep 0.000\tabcellsep \tabcellsep \\ \tabcellsep BC\tabcellsep TA (max.)\tabcellsep 452\tabcellsep 0.000\tabcellsep 0,743\tabcellsep Strong\\ \tabcellsep \tabcellsep NO?? (max.)\tabcellsep \tabcellsep 0.000\tabcellsep \tabcellsep \end{longtable} \par {\small\itshape [Note: Experiment 1 (collective treatment with filtration and partial daily water renovation). A1: premoult crabs; AC (Control): intermoult crabs. Experiment 2: Collective treatment with filtration but no water renewal. B1: premoult crabs; B2: tanks containing water previously used for the B1 group, with premoult crabs; BC (Control): tanks with crabs in intermoult; M1: crabs of B1 Group that performed ecdysis within the first 36 hours; M2: crabs that moulted after 36 hours. NA: Not applicable. TA: Total ammonia (NH 3 +NH4 + ).]} \caption{\label{tab_4}Table 5 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{6} \par \begin{longtable}{P{0.04415584415584416\textwidth}P{0.02207792207792208\textwidth}P{0.033116883116883114\textwidth}P{0.2906926406926407\textwidth}P{0.07359307359307359\textwidth}P{0.12142857142857141\textwidth}P{0.16190476190476188\textwidth}P{0.10303030303030303\textwidth}} Experiment\tabcellsep \tabcellsep Group\tabcellsep Correlated parameters\tabcellsep Cases (n)\tabcellsep p\tabcellsep \multicolumn{2}{l}{Adjusted R 2 Correlation}\\ 1\tabcellsep A1\tabcellsep \tabcellsep pH (max.)\tabcellsep 68\tabcellsep 0.000\tabcellsep 0,196\tabcellsep Weak\\ \tabcellsep \tabcellsep M1\tabcellsep pH (max.)\tabcellsep 116\tabcellsep 0.000\tabcellsep 0,559\tabcellsep Moderate\\ 2\tabcellsep B1\tabcellsep M2\tabcellsep pH (max.) AT (min.)\tabcellsep 136\tabcellsep 0.000 0.000\tabcellsep 0,410\tabcellsep Moderate\\ \tabcellsep B2\tabcellsep \tabcellsep AT (min.) NO?? (min.)\tabcellsep 203\tabcellsep 0.000 0.001\tabcellsep 0,657\tabcellsep Moderate\end{longtable} \par \caption{\label{tab_5}Table 6 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{7} \par \begin{longtable}{P{0.12263681592039802\textwidth}P{0.09303482587064676\textwidth}P{0.021144278606965175\textwidth}P{0.10572139303482588\textwidth}P{0.11417910447761194\textwidth}P{0.19452736318407962\textwidth}P{0.19875621890547263\textwidth}} \multicolumn{2}{l}{Group Co 2/3 (h)(min-max)}\tabcellsep n\tabcellsep \multicolumn{2}{l}{Hardening(\%)}\tabcellsep Ecdysis (n (\%))\tabcellsep Dead (n (\%))\\ M1\tabcellsep 3 a (1-18)\tabcellsep 32\tabcellsep Yes No\tabcellsep (54\%) a (37\%) a\tabcellsep 19 (23\%) 13 (16\%)\tabcellsep 8 (42\%) a 11 (85\%) b\\ M2\tabcellsep 61 b (3-129)\tabcellsep 46\tabcellsep Yes No\tabcellsep -(100\%) b\tabcellsep 0 46 (55\%)\tabcellsep -43 (93\%) b\end{longtable} \par {\small\itshape [Note: Individuals who moulted before (M1) or after (M2) 36 hours.]} \caption{\label{tab_6}Table 7 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{} \par \begin{longtable}{P{0.14604247104247103\textwidth}P{0.2133204633204633\textwidth}P{0.21660231660231657\textwidth}P{0.2133204633204633\textwidth}P{0.06071428571428571\textwidth}} : water not used previously; B2: tanks containing water used previously for the group B1;\tabcellsep M1: crabs that moulted within the first 36 hours; M2: crabs that moulted after 36 hours. Co: consistency. TA: total ammonia. Time:\tabcellsep Length of stay at a given consistency. Different letters indicate significant differences (p < 0.05) according to the Kruskal-Wallis\tabcellsep test. Lowercase letters indicate differences in carapace consistency within the same group. Uppercase letters indicate differences\tabcellsep in carapace consistency among groups.\end{longtable} \par \caption{\label{tab_7}}\end{figure} \begin{figure}[htbp] \noindent\textbf{} \par \begin{longtable}{} \end{longtable} \par \caption{\label{tab_8}}\end{figure} \footnote{© 2021 Global Journals} \backmatter \subsection[{Acknowledgements}]{Acknowledgements}\par This research was supported by a Research Productivity Fellowship from the CNPq (process 302609/2013-0) granted to Dr Antonio Ostrensky and by CNPq financing of the projects associated with this manuscript (processes 381091/2014-7, 473959-2013 and 403705/2013-4) and the project (processes 468251/2014-6) coordinated by Dr Leandro Ângelo Pereira, granting a DTI scholarship to Diogo Barbalho Hungria (Process N.381091/2014-7), granting a doctoral's degree to Camila Tavares (process N.141022/2017-5) and Dr Ariana Cella-Ribeiro receives a scholarship from CAPES, Pró-Amazônia Program (process 1644571). \subsection[{Conflict of Interest}]{Conflict of Interest}\par We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. \subsection[{Author Contribution}]{Author Contribution}\par We recognize that all authors contributed significantly and agree with the content of the manuscript and all individuals listed as authors qualify as authors and have approved the submitted version. \subsection[{Data Availability Statement}]{Data Availability Statement}\par The data that support the findings of this study are available from the corresponding author upon reasonable request. \begin{bibitemlist}{1} \bibitem[ Progress. 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