**4.3.2 Immersion technique**

Because feeding shrimps with MEs is time consuming and requires tedious technical work, the possibility of immersing live full-grown crustaceans in an ME solution for inhibiting postharvest melanosis was considered. Martínez-Álvarez et al. (2005) reported the efficacy of immersing shrimp in 0.05% hexylresorcinol (HR) for preventing postharvest melanosis. *In vivo* treatment with antimelanosic agents may be more effective than postmortem treatment because of greater absorption of antimelanosic agents through the gills, increased accumulation in the hemolymph, and enhanced distribution throughout the body. Therefore, the effects of *in vivo* treatment with *F. velutipes* ME, sodium ascorbate (ascorbic acid, AA), sodium sulfate (SS), and HR on postharvest melanosis in crustaceans were compared.

The study conducted by Encarnacion et al. (2011a) showed that immersing live *M. japonicus* shrimps in ME effectively inhibited postharvest melanosis in the shrimps (Fig. 10). Moreover, the development of melanosis in the carapace decreased with increase in the concentration of ME in the immersion solution. These effects may be due to greater absorption and accumulation of ERT in the shrimps with increase in the concentration of ME in the immersion solution. At 0.5 % ME concentration in pure seawater, melanosis was effectively inhibited in shrimp samples after 2 days of ice storage; the effect was the same as that obtained with 500 ppm HR and better than obtained with 500 ppm AA.

The study performed by Encarnacion et al. (2011b) also used this technique for the red queen crab, *Chionoecetes japonicus,* and the result clearly showed that treating live crabs with purified seawater containing 1.0% ME effectively prevented melanosis during 4 days of ice storage; and the effect obtained the same as that with 500 ppm SS or with 500 ppm HR solutions (Fig. 11).

ERT accumulation in shrimp tissue may inhibit PPO activity in the carapace during the postharvest period. Because thiols, such as ERT, are powerful nucleophiles that can chelate Zn2+ and Cu2+ (Hanlon, 1971; Park et al., 2006), the mechanism underlying the inhibitory effect of ERT could be due to its Cu2+ chelating activity. The mechanism of ERT could also be attributed to its Cu2+ chelating activity; thus, melanosis in the shrimps immersed in the ME solution was inhibited.

hemocytes of the control samples, showing that the ME inhibited PPO activation in the hemolymphs of the supplement-fed shrimps (Fig. 9). Because PPO activity mainly depends on the activation of the proPO system, decreasing the expression of proPO genes in

The results of a study performed by Amparyup et al. (2009) indicated the significant role of the proPO activating system as one of the major immune responses in shrimps and also reported that inhibiting proPO activation in shrimps could cause death. However, the study did not show any significant differences in mortality between the supplement-fed shrimps the control samples. A study by Leclerc et al. (2006) reported that microbial infections in *Drosophila* did not require the activation of the proPO system for survival thus, raising questions regarding the precise function of phenoloxidase activation and its level of participation in the immune defense system of invertebrates because they use 2 broad but interacting categories of defense responses against pathogens, namely, the cellular and

Because feeding shrimps with MEs is time consuming and requires tedious technical work, the possibility of immersing live full-grown crustaceans in an ME solution for inhibiting postharvest melanosis was considered. Martínez-Álvarez et al. (2005) reported the efficacy of immersing shrimp in 0.05% hexylresorcinol (HR) for preventing postharvest melanosis. *In vivo* treatment with antimelanosic agents may be more effective than postmortem treatment because of greater absorption of antimelanosic agents through the gills, increased accumulation in the hemolymph, and enhanced distribution throughout the body. Therefore, the effects of *in vivo* treatment with *F. velutipes* ME, sodium ascorbate (ascorbic acid, AA),

sodium sulfate (SS), and HR on postharvest melanosis in crustaceans were compared.

that obtained with 500 ppm HR and better than obtained with 500 ppm AA.

The study conducted by Encarnacion et al. (2011a) showed that immersing live *M. japonicus* shrimps in ME effectively inhibited postharvest melanosis in the shrimps (Fig. 10). Moreover, the development of melanosis in the carapace decreased with increase in the concentration of ME in the immersion solution. These effects may be due to greater absorption and accumulation of ERT in the shrimps with increase in the concentration of ME in the immersion solution. At 0.5 % ME concentration in pure seawater, melanosis was effectively inhibited in shrimp samples after 2 days of ice storage; the effect was the same as

The study performed by Encarnacion et al. (2011b) also used this technique for the red queen crab, *Chionoecetes japonicus,* and the result clearly showed that treating live crabs with purified seawater containing 1.0% ME effectively prevented melanosis during 4 days of ice storage; and the effect obtained the same as that with 500 ppm SS or with 500 ppm HR

ERT accumulation in shrimp tissue may inhibit PPO activity in the carapace during the postharvest period. Because thiols, such as ERT, are powerful nucleophiles that can chelate Zn2+ and Cu2+ (Hanlon, 1971; Park et al., 2006), the mechanism underlying the inhibitory effect of ERT could be due to its Cu2+ chelating activity. The mechanism of ERT could also be attributed to its Cu2+ chelating activity; thus, melanosis in the shrimps immersed in the

hemocytes consequently reduces the activity of PPO.

hormonal responses.

solutions (Fig. 11).

ME solution was inhibited.

**4.3.2 Immersion technique** 

Fig. 10. Development of melanosis and changes in the mean gray value of the carapace of *Marsupenaeus japonicus* shrimps before and after ice storage: (a) dose-dependent effect of ME, (b) comparison with other antimelanosic compounds. Results are presented in terms of mean ± standard deviation (*n* = 3). The superscript letters above each data point represent statistically significant differences (*p* < 0.05) (Encarnacion et al., 2011a).

Fig. 11. (a) Digital photographs of the development of melanosis in *Chionoecetes japonicus* crabs pretreated with purified seawater (C), 1.0% mushroom extract (ME), 500 ppm sodium sulphite (SS), and 500 ppm 4-hexyl-1,3-benzenediol (HR) solutions during ice storage. (b) Relative changes in the mean gray value of the carapace of *C. japonicus* during ice storage, analyzed using the ImageJ software. Results are presented in terms of mean (SD) (*n* = 3). The superscript letters above each data point represent statistically significant differences (*p* < 0.05) (Encarnacion et al., 2011b).

In addition to inhibiting PPO activity, ME also suppressed proPO gene expression in the hemolymph of *M. japonicus* (Fig. 12) and *C. japonicus* (Fig. 13). The same result was obtained in the feeding trial. Because immersing shrimp in ME significantly reduced PPO activity in

Fig. 11. (a) Digital photographs of the development of melanosis in *Chionoecetes japonicus* crabs pretreated with purified seawater (C), 1.0% mushroom extract (ME), 500 ppm sodium sulphite (SS), and 500 ppm 4-hexyl-1,3-benzenediol (HR) solutions during ice storage. (b) Relative changes in the mean gray value of the carapace of *C. japonicus* during ice storage, analyzed using the ImageJ software. Results are presented in terms of mean (SD) (*n* = 3). The superscript letters above each data point represent statistically significant differences (*p* <

In addition to inhibiting PPO activity, ME also suppressed proPO gene expression in the hemolymph of *M. japonicus* (Fig. 12) and *C. japonicus* (Fig. 13). The same result was obtained in the feeding trial. Because immersing shrimp in ME significantly reduced PPO activity in

0.05) (Encarnacion et al., 2011b).

Fig. 12. (a) Enzymatic oxidation of L-DOPA in the hemolymph of kuruma shrimps (*M. japonicus*) immersed in control or treatment solutions. In each graph, the average absorbances of samples are shown relative to the average absorbance of the control (defined as 100%). Results are presented in terms of mean ± SD (*n* = 5). (b) Gene expression analysis of prophenoloxidase (proPO) transcripts in the hemocytes of *M. japonicus* shrimp immersed in control or treatment solutions. The top and bottom panels show representative gels and quantitative analyses of the band intensities obtained using the ImageJ software, respectively. The expression level of the elongation factor 1α (EF1-α) gene was used as the reference for calculating the relative expression level for each band. B denotes a blank sample (negative control). Results are presented in terms of mean ± SD (*n* = 3). The superscript letters above each bar represent statistically significant differences (*p* < 0.05) (Encarnacion et al., 2011a).

Fig. 13. (a) Enzymatic oxidation of L-DOPA in the hemolymph of red queen crabs (*Chionoecetes japonicus*) immersed in control or treatment solutions. In each graph, the average absorbances of samples are shown relative to the average absorbance of the control (defined as 100%). Results are presented in terms of mean ± SD (*n* = 5). (b) Gene expression analysis of prophenoloxidase (proPO) in the hemocytes of *C. japonicus* immersed in purified seawater (Control), 1.0% mushroom extract (ME), 500 ppm sodium sulphite (SS), or 500 ppm 4-hexyl-1,3-benzenediol (HR) solutions, performed using reverse transcription–polymerase chain reaction. (b) Quantitative analysis of the band intensities obtained using the ImageJ software. The expression level of the β-actin gene was used as the reference for carculating the relative expression level for each band. Results are presented in terms of mean (SD). The superscript letters above each bar represent statistically significant differences (p < 0.05) (Encarnacion et al., 2011b).

Fig. 13. (a) Enzymatic oxidation of L-DOPA in the hemolymph of red queen crabs (*Chionoecetes japonicus*) immersed in control or treatment solutions. In each graph, the average absorbances of samples are shown relative to the average absorbance of the control (defined as 100%). Results are presented in terms of mean ± SD (*n* = 5). (b) Gene expression analysis of prophenoloxidase (proPO) in the hemocytes of *C. japonicus* immersed in purified seawater (Control), 1.0% mushroom extract (ME), 500 ppm sodium sulphite (SS), or 500 ppm 4-hexyl-1,3-benzenediol (HR) solutions, performed using reverse transcription–polymerase chain reaction. (b) Quantitative analysis of the band intensities obtained using the ImageJ software. The expression level of the β-actin gene was used as the reference for carculating the relative expression level for each band. Results are presented in terms of mean (SD). The superscript letters above each bar represent statistically significant differences (p < 0.05)

(Encarnacion et al., 2011b).

the hemolymph, it is possible that the ME containing ERT inhibited PPO activation. The observation that proPO gene expression in the hemocytes of ME-treated shrimps was lower than that in control shrimps supports this hypothesis. Thus, decreasing the expression of proPO reduces the amount that can be proteolytically activated into PPO (Adachi et al., 2003).

The efficacy of ME in inhibiting postharvest melanosis is comparable to the efficacy of existing antimelanosic compounds, such as HR, AA and SS in this regard. HR forms an inactive complex with PPO that is incapable of catalyzing melanosis development (Guererro-Beltran et al., 2005). Therefore, PPO activity significantly decreases in the presence of HR (Encarnacion et al., 2010; 2011). HR possesses antimicrobial activity in addition to its antimelanosic activity. The antimicrobial activity of HR (Martínez-Álvarez et al., 2005) may be related to the decrease in proPO gene expression because invading microorganisms trigger a proteolytic cascade culminating in the release of active PPO (Leclerc et al., 2006). Because AA did not significantly affect proPO gene expression, its mechanism of inhibiting postharvest melanosis is most likely to be due to its reducing power. The slight melanosis that occurred in AA-immersed shrimps after 2 days of ice storage may be due to the oxidation of AA, which prevented it from reducing *o*-quinones, thereby allowing melanin development (Guererro-Beltran et al., 2005). Sulfites are known to inhibit both enzymatic and nonenzymatic browning reactions. Bisulphite is reported to inhibit melanosis by 2 mechanisms: 1) by reacting with intermediate quinones in the melanosis reaction that forms sulfoquinones; and 2) by irreversibly reacting with PPO, causing complete inactivation (Ferrer et al., 1989). Therefore, PPO activity also significantly decreases in the presence of SS. SS and HR posses antimicrobial activities in addition to their antimelanosic activities, SS and HR are also antimicrobial agents. The antimicrobial activity of SS (McFeeters et al., 2004; Martínez-Álvarez et al., 2005) may also be related to the decrease in proPO gene expression.
