Melanosis in Penaeus monodon: Involvement of the Laccase-like

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Melanosis in Penaeus monodon: involvement of the laccase-like activity of hemocyanin Cédric Le Bris, Benoit Cudennec, Pascal Dhulster, Djamel Drider, Guillaume Duflos, and Thierry Grard J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04997 • Publication Date (Web): 15 Dec 2015 Downloaded from http://pubs.acs.org on December 19, 2015

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Journal of Agricultural and Food Chemistry

Full title: Melanosis in Penaeus monodon: Involvement of the Laccase-like Activity of Hemocyanin

Cédric Le Brisa,b,c,d,e*; Benoit Cudenneca,b; Pascal Dhulstera,b; Djamel Dridera,b; Guillaume Duflosf; Thierry Grarda. a

Univ. Littoral Côte d’Opale, EA 7394 – ICV – Institut Charles Viollette, F-62200 Boulogne sur Mer, France b Univ. Lille, F-59000 Lille, France c Univ. Artois, F-62000 Arras, France d INRA e ISA, F-59000 Lille, France f ANSES, Laboratoire de Sécurité des Aliments – Département des Produits de la Pêche et de l’Aquaculture, Boulevard du Bassin Napoléon, F-62200 Boulogne sur Mer, France

E-mail of each author: Benoit Cudennec: [email protected] Pascal Dhulster: [email protected] Djamel Drider: [email protected] Guillaume Duflos: [email protected] Thierry Grard: [email protected]

*Corresponding author: Cédric Le Bris Tel: +33 3 21 99 45 20; fax: +33 3 21 99 45 24. E-mail address: [email protected]

ACS Paragon Plus Environment


1

Abstract

2

In shrimp, the development of post-mortem melanosis resulting from phenoloxidase activities

3

leads to important economic losses. Phenoloxidase enzymes include catechol oxidases,

4

laccases and tyrosinases, but hemocyanin is also capable of phenoloxidase activities. These

5

activities have been explored in Penaeus monodon, using different substrates. Results

6

highlighted that tyrosinase-specific substrates were little oxidized, whereas hydroquinone

7

(laccase-specific substrate) was more highly oxidized than L-DOPA (non-specific substrate)

8

in the pereopods and pleopods. Global phenoloxidase activity, assayed with L-DOPA, did not

9

appear thermally stable over time and probably resulted from phenoloxidase enzymes.

10

Conversely, the laccase-like activity assayed with hydroquinone was thermally stable over

11

time, reflecting the thermal stability of hemocyanin. Independently of the anatomical

12

compartment, the temperature or the substrate, the highest activities were assayed in the

13

cuticular compartments. This study demonstrates the complexity of phenoloxidase activities

14

in P. monodon, and the importance of considering all the activities, including laccase-like

15

activities such as that of hemocyanin.

16 17

Keywords: Melanosis; Penaeus monodon; Phenoloxidase activities; Laccase; Hemocyanin.

18

Chemical compounds used in this article:

19

Sodium metabisulfite (PubChem CID 656671) ; 3,4-dihydroxy-L-phenylalanine (PubChem CID

20

6047) ; sodium dodecyl sulfate (PubChem CID 3423265) ; Tris hydrochloride (PubChem CID

21

93573) ; Dopamine (PubChem CID 681) ; Catechol (PubChem CID 289) ; L-Tyrosine

22

(PubChem CID 6057); Tyramine (PubChem CID 5610) ; p-phenylenediamine (PubChem CID

23

7814) ; Hydroquinone (PubChem CID 785).

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Introduction

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Today, the global shrimp industry and more generally the crustacean industry, has a high

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market-value. Penaeus vannamei (Boone, 1931) and Penaeus monodon (Fabricius, 1798), the

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two major farmed shrimp species, are mainly exported to the United States, Japan and the

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European Union. One issue with these seafood products is the development of a melanosis

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phenomenon commonly called as “black spot”. This phenomenon, observed both on fresh and

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cooked shrimp, leads to a blackening of the cephalothorax carapace, the abdomen, the pods

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and the whole shrimp from the head to the tail1. While this blackening does not modify the

34

taste of the shrimp and appears harmless to consumers, it causes undesirable appearance and

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radically reduces the consumer acceptability and consequently the market value of the

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product1a, 2. Many studies have focused on the origin of this melanosis and all agree that this

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blackening results from phenoloxidase activities (PAs), independently of the molecules

38

responsible for it. The term “Phenoloxidases” includes three different enzymes: catechol

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oxidases (EC 1.10.3.1), laccases (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1). The PAs

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correspond to the oxidation of monophenols into ortho-diphenols and ortho-diphenols into

41

ortho-quinones as well as to the oxidation of para-diphenols into para-quinones3. These

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different quinones then undergo a series of oxidation and polymerization processes resulting

43

in the formation of melanin derivatives4.

44

In crustaceans, phenoloxidase enzymes are not the only proteins able to exhibit

45

phenoloxidase activity (PA). Indeed, hemocyanin, which is the oxygen carrier for arthropods

46

and some mollusk species, could be activated like the prophenoloxidases and in so doing

47

exhibit PA5. This similarity between phenoloxidases and hemocyanin underlined the

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importance of differentiating the PAs from the phenoloxidase enzymes themselves. In

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crustaceans, hemocyanin is present in the hemolymph plasma in large quantities and



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represents between 90 and 95% of total plasma proteins whereas phenoloxidase enzymes,

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located in hemocytes in their inactivated form (prophenoloxidase), are present in negligible

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quantities6. This imbalance in proportions has logically led to investigation of the role of

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hemocyanin in the melanosis process. The predominant role of hemocyanin in this blackening

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phenomenon has been demonstrated in various crustacean species2,

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been imputed to phenoloxidase enzymes alone.

6-7

whereas it has long

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A growing number of studies8 have investigated the responsibility of both hemocyanin

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and phenoloxidase enzymes in the post-mortem development of melanosis in shrimp. The

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current trend is to find and characterize natural compounds in order to replace the currently

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used sodium metabisulfite which is known to cause allergic reactions in asthmatic people in

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particular9. For example, inhibition of shrimp melanosis using molecules obtained from the

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mushroom Flammulina velutipes10,

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studied. While some of these natural compounds were good inhibitors of PA, few solutions to

63

date appear effective and applicable to the agro-food industry. While a growing number of

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studies consider both phenoloxidase enzymes and hemocyanin as potential sources of PAs,

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there are still few studies on the different PAs: tyrosinase, catechol oxidase and laccase. The

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most commonly used substrate to assay PA is 3,4-dihydroxy-L-phenylalanine (L-DOPA)

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which is a common substrate of the different PAs. This overly general consideration of PA

68

could explain the lack of an effective alternative inhibitor for melanosis in shrimp.

69

Furthermore, Sodium Dodecyl Sulfate (SDS) is frequently used as a phenoloxidase activity

70

activator without verification of its actual activation effect.

11

or from the green tea Camellia sinensis12 has been

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Therefore, in this study, in an attempt to better understand the post-mortem melanosis

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phenomenon in shrimp, the different PAs have been explored. Thus, global PAs of the

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different compartments were compared using different substrates, more or less specific to one

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or more phenoloxidase subclasses. In order to understand whether different molecules are


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involved in post-mortem PAs in shrimp, the thermal stability of these enzymatic activities was

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studied in the pods and tail of shrimp over a storage period of more than 4 months. Finally,

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the PAs were studied in 4 anatomical shrimp compartments after seven days of “natural”

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development of melanosis at 7.3 °C and 22.9 °C. These post-mortem PA assays were

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performed both with and without SDS, in order to compare occasional and potential

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enzymatic activities. This study was conducted with the general objective of globally

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reconsidering post-mortem PAs in P. monodon.

82 83

Material and Methods

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Experimental animals

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All shrimps used for this study were obtained from UNIMA Frais (Isques, Pas-de-Calais,

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France) which is a shrimp’s cooker. The average length of the shrimps (from the tip of the tail

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to the tip of the rostrum) used for the different experiments was 17.19 ± 0.99 cm. The P.

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monodon shrimp were bred in a farm in Madagascar and then sent frozen at -20 °C to France.

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Thus, the studied shrimp were thawed only at the time of their use in experiments. The full

90

traceability from the hatchery until their arrival in frozen form at the laboratory ensured that

91

the studied animals came from a limited number of different clutches. In other words, the

92

genetic diversity of the batches of shrimp used in this study was relatively low, enabling

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comparisons across individuals and ensuring that if significant differences were observed,

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they were not due to shrimp affiliations with different populations or sub-populations with

95

heterogeneous genetic heritage. Furthermore, the shrimp used in this study did not receive any

96

treatments with sulfite (or other PAs inhibitors) before their use.

97

The experiments of this study focused on different shrimp compartments. The term

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“Cephalothorax” refers to the samples from the carapace (=exoskeleton) of the cephalothorax,

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the term “Abdomen” refers to the samples from the carapace of the abdomen, the term “Pods”



100

refers to the samples from the pereopods and the pleopods and the term “Tail” refers to the

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samples from the uropods and the telson.

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Chemicals

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Except for the BioRad Protein Assay Dye Reagent Concentrate, which was obtained from

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BioRad (France), all the chemicals used were purchased from Sigma-Aldrich (France).

105

Preparation of enzyme extracts

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For each series of experiments, the targeted compartments were isolated from the rest of

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the animal and finely cut. Three volumes (w/v) of extracting buffer (sodium phosphate buffer,

108

0.1 M, pH 6.4) were added and the sample was then homogenized at a speed of 19000 rpm

109

using an Ultra Turrax homogenizer (T25 basic, Janke and Kunkel, IKA-Labortechnik,

110

Germany) twice for one minute. All these operations were performed on ice. The homogenate

111

was then centrifuged (21460 × g, 1h, 4 °C) and the resulting supernatant was used as the

112

crude enzyme preparation and stored at -80 °C if it was not immediately used.

113

Protein determination and phenoloxidase activity assays

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For each compartment, the total protein content of crude enzyme preparation was

115

determined by the Bradford method13 using BioRad Protein Assay Dye Reagent Concentrate

116

and bovine serum albumin as the protein standard. Results were expressed in mg.mL-1.

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PAs were assayed spectrophotometrically in 96-well microplates (Nunc, France) by

118

recording the formation of quinones according to the protocol adapted from Le Bris et al.14.

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Briefly, 50µL of Tris hydrochloride (Tris-HCl) buffer (0.10 M, pH 8.4) were added to 50µL

120

of crude enzyme preparation (“Cephalothorax”, “Abdomen”, “Pods” or “Tail”). The mixture

121

was then incubated for 10 min at 25 °C before adding 100µL of substrate (dissolve in pure

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water) to each well. PAs were monitored for 30 min following the increase of absorbance at

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the wavelength imposed by the substrate using a microplate reader (BioTek µQuant


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microplate spectrophotometer, INC., Vermont, USA). The spontaneous oxidation of substrate

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was simultaneously measured and the values obtained were subtracted from the test values in

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order to consider enzymatic oxidation alone. When SDS was used as a PA activator it was

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directly dissolved in Tris-HCl buffer. All the PA assays were performed in triplicate.

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Phenoloxidase-specific activities were expressed in arbitrary unit (U) per milligram of total

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protein and were calculated thusly: Phenoloxidase specific activity (U. ) =

ΔAλ. min × dilution factor Protein concentration

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Where ∆Aλ.min-1 is the value of the increment of absorbance per minute at the wavelength

131

imposed by the substrate, the dilution factor is the crude enzyme preparation’s factor of

132

dilution, and the protein concentration is the preparation’s total protein concentration.

133

Effects of pH and final SDS concentration

134

The protocol for measuring the PA was optimized by testing different concentrations of

135

SDS, which is known as the most commonly used phenoloxidase activator. Tris-HCl buffer

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adjusted to pH values of 7.4, 8.4 and 9.4 was tested. These enzymatic assays were performed

137

on “Pods” and “Tail” obtained from three shrimp. For each compartment, the PA levels in the

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presence of 0.1% SDS or 1% SDS were compared in order to evaluate the impact of the

139

concentration on the activator effect.

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Phenoloxidase activity substrates

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The PAs were explored in “Tail” and “Pods” using more or less specific substrates at various

142

concentrations. This experiment was done pooling three shrimp tails and three shrimp pods.

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L-DOPA (40 mM, i.e. in excess) was used as a reference (non-specific and most

144

conventionally used substrate). Two other non-specific substrates were used: dopamine (from

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1 to 30 mM) and catechol (from 1 to 40 mM). PA assays were also performed using two

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tyrosinase-specific substrates: L-tyrosine (from 1 to 30 mM) and tyramine (from 1 to 40 mM);



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and two laccase-specific substrates: p-phenylenediamine (PPD; from 1 to 40 mM) and

148

hydroquinone (from 1 to 40 mM). The resulting absorbance of the oxidation of these

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substrates was monitored at wavelengths equal to 490nm and 405nm for L-DOPA, dopamine

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and L-tyrosine and for catechol, tyramine, PPD and hydroquinone respectively.

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Thermal stability of phenoloxidase activities

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The aim of this experiment was to study the thermal stability of PAs over time. Crude

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enzyme preparations were obtained from a pool of pods from three shrimp and from a pool of

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tails from three shrimp respectively. Once the crude enzyme preparations were made, the PAs

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were assayed a first time before freezing and the remainder of each crude enzyme preparation

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was aliquoted and stored, for half at -20 °C and half at -80 °C. In order to evaluate the

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freezing effect, a second PAs assay was conducted 2 hours after freezing had begun (0.1 day).

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PA assays were performed after 0.1, 5, 18, 32, 49, 61, 77, 95, 105 and 125 freezing days.

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These assays were both performed on L-DOPA (40 mM) and hydroquinone (40 mM) to

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assay the total PA and the laccase-like activity respectively. No SDS was used for these

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assays because the aim was to evaluate the levels of real activity at a given time and not the

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potential activity.

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Phenoloxidase activities following “natural” development of melanosis

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SDS activator effect and PA profiles in shrimp

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For this experiment, 10 shrimp were placed for 7 days in the dark, exposed to air: 5 at a

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temperature of 7.3 ± 1.1 °C and the 5 others at a temperature of 22.9 ± 1.4 °C. At the end of

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these 7 days, the animals were dissected and for each shrimp, “Tail”, “Pods”, “Abdomen” and

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“Cephalothorax” preparations were made. The crude enzyme preparations were made

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individually for each shrimp and each compartment and stored at -80 °C until use. For each

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sample, the total PA was assayed using L-DOPA (40 mM) as substrate and the laccase-like


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activity was assayed using hydroquinone (40 mM) as substrate. For each sample and each

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substrate, phenoloxidase assays were performed with 0.1% SDS and without SDS in order to

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evaluate the activator effect of SDS on each shrimp compartment.

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Effect of the thermal factor on the PA profiles in shrimp

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The melanosis phenomenon does not occur when the animals are frozen. Thus, it has been

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decided to study the melanosis phenomenon at ambient temperature, on one hand inside the

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laboratory (22.9 ± 1.4°C) and, on the other hand in a cold chamber (7.3 °C ± 1.1 °C), which

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are both places where the temperature could be consistently followed, and would not

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experience important disparities. The PA profiles in shrimp maintained at a temperature of 7.3

180

°C were compared with those obtained for animals maintained for 7 days at a temperature of

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22.9 °C to estimate the potential effect of temperature on the development of the melanosis

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phenomenon.

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Statistical analysis

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All statistical analyses were conducted using SigmaPlot software (11.0, Systat Software

185

Inc, San Jose, CA). The data are submitted as mean values and their standard deviation of

186

mean. One-way ANOVA and Tukey’s test were used to identify significant differences

187

between groups. Results were considered significant at p < 0.05.

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Results

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Effects of pH and final SDS concentration

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The results showed in the first instance that the levels of phenoloxidase-specific activities

191

in “Tail” were significantly higher than those measured in “Pods” (Fig. 1). The highest

192

specific activity in these samples was equal to 0.223 (± 0.009) U.mg-1 and corresponded to the

193

value measured in presence of 0.1% SDS in “Tail” for a pH of 8.4.



194

The results also showed that for pH of 7.4 and 8.4, the modification of the SDS

195

concentration did not lead to any significant difference in the specific activity levels, neither

196

for “Pods” nor for “Tail”. Conversely, for a pH value of 9.4, the phenoloxidase-specific

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activity values were higher with 1% SDS than those obtained with 0.1% SDS for both

198

compartments.

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Phenoloxidase activity substrates

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In this section, L-DOPA was used as a reference substrate. Two other non-specific

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substrates, dopamine and catechol, were tested, both of which underwent significant

202

enzymatic oxidation (Fig. 2). Nevertheless, for both compartments, the PA levels assayed

203

were significantly lower with catechol or dopamine than with L-DOPA (40 mM), regardless

204

of the substrate concentration.

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Two tyrosinase-specific substrates were also tested: L-tyrosine and tyramine, which are

206

both monophenols. Using L-tyrosine as substrate, no significant PA was detected either in

207

“Tail” or in “Pods” regardless of the tested concentration. Using tyramine as substrate, no

208

significant oxidation was detected for “Pods”, but although activity levels for “Tail” were

209

very low, significant oxidation could be observed for tyramine concentrations up to 10 mM

210

(Fig. 2).

211

Finally, two laccase-specific substrates were tested: PPD (non-phenolic) and

212

hydroquinone (p-diphenol). With the former, PA levels were low but significant for

213

concentrations up to 10 mM for “Tail”. For “Pods”, except for the 40 mM concentration, the

214

activity levels were not significant. Using hydroquinone as substrate, a significant dose effect

215

was observed for both “Pods” and “Tail”. For “Pods”, the PA levels assayed were

216

significantly higher with hydroquinone than for other substrates, specific or non-specific. For

217

“Tail”, the activity levels were also significantly higher than those obtained with other

218

substrates excepted than for 40 mM L-DOPA (Fig. 2).


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Thermal stability of phenoloxidase activities

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The stability of PA of both “Tail” and “Pods” was explored over 125 days using L-DOPA

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and hydroquinone to evaluate global PA and laccase-like activity respectively (Fig. 3).

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Results regarding oxidation of the different substrates were partially confirmed during storage

223

experiments at various temperatures. In fact, overall, the PA levels were higher using

224

hydroquinone (40 mM) as substrate than using L-DOPA (40 mM), even for “Tail”. The mean

225

total protein concentration in the samples over the 125 days was 10.95 ± 0.51 mg.mL-1 and

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10.30 ± 0.70 mg.mL-1 for “Pods” and “Tails” respectively.

227

Thermal stability of PAs in “Pods”

228

Apart from the differences in PA level mentioned above, the thermal stability of the

229

enzymatic activity was variable from one substrate to another. Two hours after freezing (0.1

230

day), the activity levels were significantly higher than at T0, which seems to indicate that

231

freezing had an activating effect on the activity, especially when using L-DOPA as substrate

232

(Fig. 3. A). With this substrate, the average level of PA in samples stored at -80 °C remained

233

relatively high over time whereas it significantly decreased with the “freezing activation

234

effect” for samples stored at -20 °C, and it remained low until the end of the experiment.

235

Nevertheless, except for assays done after 5 and 49 days, the level of PA measured in the

236

samples stored at -80 °C was always significantly higher than those measured in samples

237

stored at -20 °C. Conversely, for the activity assays on hydroquinone, no significant

238

difference between the activity level of samples stored at -20 °C and those stored at -80 °C

239

was observed for any of the sampling times (Fig. 3. B). In fact, except after 32 days of

240

freezing, the activity levels were not significantly different between both storage conditions.

241

The data also showed that these activity levels were relatively stable over time before

242

significantly increasing from the 95th day and the 77th day for samples stored at -20°C and -

243

80°C respectively.



244

Thermal stability of PAs in “Tail”

245

Results obtained with “Tail” were consistent with those obtained with “Pods”. The

246

“freezing effect” previously described, mainly observed using L-DOPA as substrate, was also

247

observed for “Tail” stored at -80 °C but not for samples stored at -20 °C (Fig. 3. C). In fact,

248

whereas for samples stored at -80 °C the PA level significantly increased between 0 and 0.1

249

days, it slightly and significantly decreased for samples stored at -20 °C until the 18th day.

250

Then, the activity levels remained significantly lower than those of samples stored at -80 °C,

251

which did not significantly differ from the level measured after 0.1 days freezing (except after

252

105 days of storage). As for “Pods”, no significant difference in PA levels between samples

253

stored at -20 °C and those stored at -80 °C was observed using hydroquinone as substrate

254

regardless of the number of freezing days (Fig. 3. D). Levels of PA measured on samples

255

stored at -20°C and at -80°C significantly increased after 77 days of storage.

256

Phenoloxidase activities following “natural” development of melanosis

257

For this experiment, shrimps were placed for 7 days in the dark, exposed to natural

258

atmosphere at a temperature of 7.3 ± 1.1 °C or 22.9 ± 1.4 °C. After 7 days, “Tail”, “Pods”,

259

“Abdomen” and “Cephalothorax” were prepared for each shrimp and PAs were assayed on

260

each compartment with or without 0.1% SDS (Fig. 4.). The total protein concentration in the

261

samples stored at 7.3 °C, after 7 days, was 7.84 ± 1.40 mg.mL-1, 20.22 ± 2.36 mg.mL-1, 10.98

262

± 1.28 mg.mL-1 and 3.57 ± 0.43 mg.mL-1 respectively for “Tail”, “Pods”, Abdomen” and

263

“Cephalothorax”. For the samples stored at 22.9 °C, the total protein concentration was equal

264

to 10.02 ± 2.72 mg.mL-1, 26.39 ± 5.90 mg.mL-1, 5.076 ± 2.81 mg.mL-1 and 1.48 ± 0.41

265

mg.mL-1 respectively for “Tail”, “Pods”, Abdomen” and “Cephalothorax”.

266

Activator effect of SDS

267

Using L-DOPA as substrate (Fig. 4. A, B), SDS had contrasting effects depending on the

268

storage temperature and to the relevant compartment. For shrimp maintained at 7.3 °C the


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level of specific PA in “Pods” was quite similar with or without 0.1% SDS (0.03 and 0.04

270

U.mg-1, respectively), whereas it was almost three-fold higher in “Tail” with the addition of

271

SDS (0.05 and 0.14 U.mg-1, respectively). For animals maintained at 22.9 °C, the level of

272

activity in “Cephalothorax” was quite similar with or without 0.1% SDS (0.42 and 0.43 U.mg-

273

1

274

respectively). Using L-DOPA as substrate, the addition of 0.1% SDS thus had an activation

275

effect or no effect on PA. Using hydroquinone as substrate, the effects were even more

276

contrasted (Fig. 4. C, D). In fact, for shrimp stored at 7.3 °C and for those stored at 22.9 °C,

277

the PA levels in “Tail”, “Pods” or “Abdomen” were quite similar, with or without 0.1% SDS.

278

However, the level of activity in “Cephalothorax” was almost 1.5 times higher for animals

279

stored at 22.9 °C (1.33 and 1.90 U.mg-1, respectively), whereas for those maintained at 7.3 °C

280

the level of activity was almost 1.5 times lower when 0.1 % SDS was added (1.13 and 0.74

281

U.mg-1 with or without 0.1 % SDS respectively).

, respectively), whereas it was twice as high in “Abdomen” (0.10 and 0.23 U.mg-1,

282

Because of these nuanced or opposing effects of SDS on PAs depending of the storage

283

temperature, compartment and substrate, only the real activity (without SDS) will be

284

discussed in the following sections.

285

PA profiles in shrimp

286

Independently of the storage temperature, the PA profiles obtained with L-DOPA (Fig.

287

4. A) and hydroquinone (Fig. 4. B) were quite similar. “Cephalothorax” was systematically

288

the compartment with the upmost specific activity. The PA levels assayed on each substrate

289

on other compartments were lower and not significantly different from each other.

290

Effect of thermal factor on profile of PAs within shrimp

291

There were no significant differences between the activity level of shrimp stored at 7.3

292

°C and those stored at 22.9 °C for all compartments using hydroquinone as substrate (Fig. 4.

293

C). Similarly, the differences were not significant for “Pods”, “Tail” or “Abdomen” using L-



294

DOPA as substrate (Fig. 4. A). The only case in which the thermal factor had a significant

295

effect was for “Cephalothorax” using L-DOPA. Indeed, for this compartment and this

296

substrate, rise of storage temperature from 7.3 to 22.9 °C increased PA.

297

Discussion

298

The PAs have already been investigated in different species of the Penaeus and

299

Parapenaeus genus, including P. monodon15, Penaeus japonicus1b, 6, 16, P. vannamei2, 15a, 17,17b

300

or Parapenaeus longirostris7, 18. The results in P. monodon were in accordance with these

301

studies and highlight thereof the great complexity of PAs.

302

Concerning PA assays, the experiments were initially carried out on “Pods” and “Tail”

303

because compared to the carapace, it was more convenient to obtain a significant amount of

304

proteins and also because industry professionals detect the first signs of melanosis onset in

305

these compartments. The pH used for the experiments was equal to 8.4 for “Tail” and “Pods”

306

and was fully in agreement with results obtained for the plasma of P. vannamei19 emphasizing

307

increasing PAs associated with rising substrate auto-oxidation for pH values ranging from 5.8

308

to 7.8. Auto-oxidation, measured simultaneously, was always subtracted to ensure that higher

309

activities did not reflect a higher auto-oxidation.

310

The importance of the chemical reagents used has already been highlighted for EDTA

311

used in anticoagulant solutions19, whereas it is a known inhibitor of PA20. In order to simulate

312

conditions as close as possible to those of the agri-food sector, the choice was made to use

313

neither anticoagulant solutions nor protease inhibitors. The results revealed the relative

314

importance of activators. For both compartments, there were no significant differences

315

between activity levels with 0.1% and 1% SDS at pH of 7.4 or 8.4. With a pH of 9.4, PAs

316

were significantly higher using 1% SDS than using 0.1% SDS. Regardless of its

317

concentration, the use of any activators is also considered controversial in the literature. Thus,

318

the use of trypsin as PA activator in the plasma of P. vannamei led to levels more than 5 times


Page 14 of 27

Page 15 of 27


319

higher than physiological ones19. The use of activators may be useful for biochemical

320

characterization experiments but not for physiological ones. Even for biochemical

321

characterization, the results showed that the activator effect could vary according to the

322

substrate used and the sample storage conditions. In fact, after 7 days of natural atmospheric

323

exposure at 22.9 °C, 0.1% SDS did not have any significant activator effect on the PA of

324

“Cephalothorax” assayed on L-DOPA, whereas the activity when assayed on hydroquinone

325

was really activated. Conversely, for samples stored at 7.3 °C, 0.1% SDS activated the PA

326

assayed on this compartment using L-DOPA but inhibited the activity assayed using

327

hydroquinone. The lack of an activator effect for SDS has already been shown with the

328

cephalothorax of P. japonicus1b, P. vannamei21 and P. longirostris18. However, an inhibitory

329

effect of SDS has never been reported, maybe because its effect has only rarely, or never,

330

been tested using hydroquinone. These results demonstrated that data obtained using

331

activators must be interpreted with caution.

332

Among the molecules with potential PAs, there are the phenoloxidases themselves (i.e.

333

tyrosinases, laccases and catechol oxidases) and hemocyanin5d,

6, 22

334

molecules capable of PAs may be the cause of the differential effects of SDS on observed

335

PAs. Tyrosinase, catechol oxidase and hemocyanin have a very similar type 3 active site23 but

336

their activation could differ due to the absence/presence of an N-terminal protein domain

337

covering the active site5b, 24. Thus, the activation of PA using SDS could lead to an active site

338

more or less accessible to the different substrates23. Laccase, whose catalytic site contains

339

both type 3 and type 2 copper ions25, may have differential sensitivity to activation by SDS.

340

Despite these differences, SDS is often used as an activator without knowing the source of the

341

studied PA.

. These two kinds of

342

Regardless of the proteins responsible for PAs, the activation differences observed

343

may also be attributed to different activities. Tyrosinases oxidize both monophenols and o-



344

diphenols, catechol oxidases only oxidize o-diphenols and laccases oxidize o-diphenols, p-

345

diphénols and some non-phenolic substrates3. PAs gather the oxidation of these different

346

compounds. In Parapanaeus and Penaeus genera, different kinds of PAs have been

347

evidenced. Tyrosinase activity was identified in the hemolymph of P. vannamei using

348

tyramine and tyrosine as substrates 2 and the authors emphasized a 30 minute lag-period for

349

tyrosine oxidation. Garcia-Carrenno et al. 2 also measured high oxidation of tyramine without

350

any lag-time. This lag-phase is a significant feature of tyrosinases reflecting slow activation

351

from the inactive met-state to the active oxy-state26. The results obtained with P. monodon

352

differed from those obtained with P. vannamei because no oxidation was observed using

353

tyrosine as substrate and only very low oxidation levels were measured using tyramine. Given

354

that lag phase usually ranges from 1 to 10 minutes27 it is unlikely that measurements showing

355

no or low monophenolase activity in P. monodon reflected a lag phase longer than the assay

356

duration. The results led to the conclusion that in “Tail” and “Pods” there was no tyrosinase

357

or residual activity contradicting the idea that tyrosine is the main substract of phenoloxidases

358

in crustaceans28. There is no study supporting the presence of catechol oxidase in shrimp

359

although its international nomenclature number (EC 1.10.3.1) is often used to define

360

phenoloxidases. The presence of catechol oxidase is more difficult to determine because no

361

specific substrate exists3. Conversely, p-diphenols such as hydroquinone are laccase-specific

362

substrates. Despite this, laccase activity is rarely investigated, whereas in “Tails” of P.

363

monodon, hydroquinone was the most highly oxidized substrate. Laccase-like enzyme was

364

also found in the cephalothorax of P. longirostris18 and the authors highlighted that a non-

365

specific inhibitor, such as 4-hexylresorcinol, failed to inhibit activity of the laccase-like

366

enzyme. Therefore, the inefficiency of some melanosis inhibitors could be due to their failure

367

to affect the neglected laccase-like activities.


Page 16 of 27

Page 17 of 27


368

In the hemolymph of crustaceans, the hemocyanin concentration is about 1000 times

369

higher than phenoloxidase enzymes27a. Hemocyanin, whose main role in live crustaceans is to

370

carry oxygen, could also have PAs5a. It is therefore important to distinguish PAs from the

371

phenoloxidase enzymes. A means of considering the molecules responsible for the PAs, and

372

hence the melanosis, is to study their thermal stability. Phenoloxidase enzymes are instable

373

proteins which aggregate and are easily denatured at low temperatures whereas hemocyanin

374

remains stable when stored at -20 °C and could be activated even after several months2, 29.

375

During the experiments, the effects of the storage temperature mainly showed that activity

376

assayed using L-DOPA was activated by freezing itself, especially in “Pods”. For these

377

samples, PA was significantly increased by freezing (2 h at -20 °C or -80 °C) and a similar

378

type of activation was observed for “Tail” stored at -80 °C. This kind of activation has already

379

been observed in the plasma of P. vannamei put on ice or stored at -20 °C19 and reflected the

380

fact that PAs could also be activated by mechanical and abiotic stressors30. In P. monodon,

381

results also emphasized a difference in thermal stability between PAs assayed using L-DOPA.

382

For both “Tail” and “Pods”, the PA levels were significantly higher for samples stored at -80

383

°C than for those stored at -20 °C. This difference could be explained by the fact that at -80

384

°C, the molecules responsible for the PA were protected from denaturation, whereas at -20 °C

385

these proteins were in part denatured. In Nephrops norvegicus, cellular PA in hemolymph was

386

reduced by 98% after freeze-thawing at -25 °C, whereas hemocyanin retained its integrity22

387

confirming the difference in thermal stability between these proteins. The differences between

388

PA levels observed using L-DOPA during the experiments reflected thermal instability and

389

led to the conclusion previously stated by Huang et al.19 that samples need to be used in

390

assays immediately after collection or preserved at -80 °C without thawing multiple times.

391

More generally, this thermal instability means that the enzymatic activity thus assayed

392

appeared to have been more the result of phenoloxidase enzymes than of hemocyanin.



393

Conversely, PA assayed using hydroquinone appeared thermally stable and the

394

difference between the PAs of samples stored at -20 °C and that of samples stored at -80 °C

395

was rarely significant. This stability measured using hydroquinone, observed on both “Tail”

396

and “Pods”, showed that molecules with laccase-like activities could not be phenoloxidase

397

enzymes, and were more likely hemocyanin. Furthermore, the PA levels assayed using

398

hydroquinone were higher than those measured using L-DOPA, so the results matched those

399

obtained for P. japonicus which showed that hemocyanin was more responsible for the post-

400

mortem melanosis than phenoloxidase enzymes6. To confirm these results, purification and

401

identification experiments on the molecules responsible for PAs are currently underway.

402

Increasing activity was observed using hydroquinone for both “Pods” and “Tail” from

403

the 77th and the 95th storage day respectively. These increases, independent of the storage

404

temperature, could result from the auto-activation of hemocyanin. A similar auto-activation of

405

PA has never been mentioned, but few studies have monitored PA for 4 months and even

406

fewer have focused on laccase-like activities. This activation might be explained by a

407

theoretical release of endogenous factors following a period of post-mortem freezing. As

408

freezing does not completely stop endogenous enzyme activities31, these ones could lead to

409

activation of hemocyanin activities since no inhibitors (protease inhibitors in particular) were

410

added to the samples.

411

In crustaceans, hemocyanin and phenoloxidase enzymes are both present in the

412

hemolymph plasma6 as well as in the cuticle6, 27a, 32. Among the 4 compartments assayed for

413

PAs, after 7 days of natural development of melanosis, the two cuticular compartments (i.e.

414

“Abdomen” and “Cephalothorax”) were those with the highest PA levels for both substrates.

415

For these cuticular compartments, the total amount of proteins extracted was relatively low,

416

but even when PAs were expressed in volumetric activities (not relative to the total protein

417

concentration), these compartments had the highest levels. The shrimp’s appearance, after 7


Page 18 of 27

Page 19 of 27


418

days of natural melanosis development, confirmed the activity levels measured. In fact, the

419

carapaces of both the abdomen and cephalothorax clearly changed color, becoming brown or

420

reddish. Similar differences in PA reaction products and thus in coloration of the carapace

421

have already been demonstrated in P. japonicus16. The melanosis phenomenon was also

422

visible in the remaining compartments, especially at the junction between the exoskeleton

423

segments where an abundance of phenoloxidase enzymes/hemocyanin had already been

424

shown in P. japonicus1b. The effect of storage temperature on PAs, assayed after 7 days, was

425

not evident. Except for the “Cephalothorax” activity, assayed using L-DOPA, no significant

426

differences were observed between animals maintained at 7.3 °C and those maintained at 22.9

427

°C. The PA of “Cephalothorax”, assayed using L-DOPA, was significantly higher for shrimp

428

stored at 22.9 °C than for those stored at 7.3 °C. This rise could be attributed to the

429

temperature, closer to the optimal temperature for PA, as demonstrated in P. japonicus whose

430

optimal temperature for cephalothorax PA was around 30 °C1b. Nevertheless, this does

431

explains neither the lack of impact in the other compartments nor of activity assayed with

432

hydroquinone.

433

As mentioned in previous study, if hemocyanin, and more specifically its laccase-like

434

activity, also has a structural role in shrimp18, it could explain the large amounts of this

435

substance found in the cuticle and so the usual and rapid development of post-mortem

436

melanosis.

437

To conclude, PAs include tyrosinase-, catechol oxidase- and laccase-like activities but

438

these last ones are often neglected. Indeed, laccase-like activities are often poorly investigated

439

while their levels may be significantly higher than those measured using non-specific

440

substrates. The apparent thermal stability of PAs may vary significantly depending on the

441

substrate used. Thus, activity assayed with L-DOPA (thermally unstable over time) results

442

more probably from phenoloxidase enzymes than from hemocyanin, whereas the activity



443

assayed with hydroquinone (thermally stable over time) results from hemocyanin. Purification

444

and identification experiments on the molecules responsible for these PAs will bring more

445

complete answers. Nevertheless, this complexity of PAs and especially of laccase-like

446

activities must be considered in order to find an effective natural melanosis inhibitor that is

447

also an alternative to sulfites.

448

Abbreviations used

449

PAs, phenoloxidase activities; PA, phenoloxidase activity; L-DOPA, 3,4-dihydroxy-L-

450

phenylalanine; SDS, sodium dodecyl sulfate; Tris-HCl, tris hydrochloride; PPD, p-

451

phenylenediamine; EDTA, Ethylenediaminetetraacetic acid.

452

Acknowledgements

453

Cédric Le Bris is grateful to the University of Littoral Côte d’Opale (ULCO) and Innovation

454

Platform Nouvelles Vagues (PFINV) for its postdoctoral fellowship. The authors are also

455

grateful to UNIMA Frais for proving the biological material for the experiments and

456

particularly to Didier Le Tetour for his involvement in the scientific project.

457

References

458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476

1. (a) Montero, P.; Lopez-Caballero, M.; Perez-Mateos, M., The Effect of Inhibitors and High Pressure Treatment to Prevent Melanosis and Microbial Growth on Chilled Prawns (Penaeus Japonicus). Journal of Food Science 2001, 66 (8), 1201-1206; (b) Benjakul, S.; Visessanguan, W.; Tanaka, M., Properties of Phenoloxidase Isolated from the Cephalothorax of Kuruma Prawn (Penaeus Japonicus). Journal of food biochemistry 2005, 29 (5), 470-485. 2. García-Carreño, F. L.; Cota, K.; Navarrete del Toro, M. a. A., Phenoloxidase Activity of Hemocyanin in Whiteleg Shrimp Penaeus Vannamei: Conversion, Characterization of Catalytic Properties, and Role in Postmortem Melanosis. Journal of Agricultural and Food Chemistry 2008, 56 (15), 6454-6459. 3. Walker, J. R.; Ferrar, P. H., Diphenol Oxidases, Enzyme-catalysed Browning and Plant Disease Resistance. Biotechnology and genetic engineering reviews 1998, 15 (1), 457-498. 4. Roulin, A.; Almasi, B.; Meichtry-Stier, K.; Jenni, L., Eumelanin- and Pheomelanin-based Colour Advertise Resistance to Oxidative Stress in Opposite Ways. Journal of evolutionary biology 2011, 24 (10), 2241-2247. 5. (a) Decker, H.; Jaenicke, E., Recent Findings on Phenoloxidase Activity and Antimicrobial Activity of Hemocyanins. Developmental & Comparative Immunology 2004, 28 (7), 673-687; (b) Decker, H.; Schweikardt, T.; Nillius, D.; Salzbrunn, U.; Jaenicke, E.; Tuczek, F., Similar Enzyme Activation and Catalysis in Hemocyanins and Tyrosinases. Gene 2007, 398 (1), 183-191; (c) Ballarin, L.; Franchi, N.; Schiavon, F.; Tosatto, S. C.; Mičetić, I.; Kawamura, K., Looking for Putative


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Phenoloxidases of Compound Ascidians: Haemocyanin-like Proteins in Polyandrocarpa Misakiensis and Botryllus schlosseri. Developmental & Comparative Immunology 2012, 38 (2), 232-242; (d) Idakieva, K.; Raynova, Y.; Meersman, F.; Gielens, C., Phenoloxidase Activity and Thermostability of Cancer Pagurus and Limulus Polyphemus Hemocyanin. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2013, 164 (3), 201-209. 6. Adachi, K.; Hirata, T.; Nagai, K.; Sakaguchi, M., Hemocyanin a most Likely Inducer of Black Spots in Kuruma Prawn Penaeus Japonicus During Storage. Journal of Food Science 2001, 66 (8), 1130-1136. 7. Martínez-Alvarez, O.; Gómez-Guillén, C.; Montero, P., Presence of Hemocyanin with Diphenoloxidase Activity in Deepwater Pink Shrimp (Parapenaeus Longirostris) Post Mortem. Food chemistry 2008, 107 (4), 1450-1460. 8. (a) Siddiqui, N. I.; Akosung, R. F.; Gielens, C., Location of Intrinsic and Inducible Phenoloxidase Activity in Molluscan Hemocyanin. Biochemical and biophysical research communications 2006, 348 (3), 1138-1144; (b) Yan, F.; Zhang, Y.-l.; Luo, H.-q.; Hu, Z.; Huang, T.w.; Ye, X.-q., The Phenoloxidase Activity of Hemocyanin from White Leg Shrimp Litopenaeus Vannamei. Fisheries Science 2008, 1, 001; (c) Xu, J.; Wu, S.; Zhang, X., Novel Function of QM Protein of Shrimp (Penaeus Japonicus) in Regulation of Phenol Oxidase Activity by Interaction with Hemocyanin. Cellular Physiology and Biochemistry 2008, 21 (5-6), 473-480. 9. Chapman, J. A.; Bernstein, I.; Lee, R. E.; Oppenheimer, J.; Nicklas, R. A.; Portnoy, J. M.; Sicherer, S. H.; Schuller, D. E.; Spector, S. L.; Khan, D., Food Allergy: a Practice Parameter. Annals of allergy, asthma & immunology 2006, 96 (3), S1-S68. 10. Jang, M. S.; Sanada, A.; Ushio, H.; Tanaka, M.; Ohshima, T., Inhibitory Effect of Enokitake Extract on Melanosis of Shrimp. Fisheries Science 2003, 69 (2), 379-384. 11. Encarnacion, A. B.; Fagutao, F.; Hirono, I.; Ushio, H.; Ohshima, T., Effects of Ergothioneine from Mushrooms (Flammulina Velutipes) on Melanosis and Lipid Oxidation of Kuruma Shrimp (Marsupenaeus Japonicus). Journal of Agricultural and Food Chemistry 2010, 58 (4), 2577-2585. 12. Nirmal, N. P.; Benjakul, S., Retardation of Quality Changes of Pacific White Shrimp by Green Tea Extract Treatment and Modified Atmosphere Packaging During Refrigerated Storage. International journal of food microbiology 2011, 149 (3), 247-253. 13. Bradford, M. M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-dye Binding. Analytical biochemistry 1976, 72 (1), 248254. 14. Le Bris, C.; Paillard, C.; Stiger-Pouvreau, V.; Guérard, F., Laccase-like Activity in the Hemolymph of Venerupis Philippinarum: Characterization and Kinetic Properties. Fish & shellfish immunology 2013, 35 (6), 1804-1812. 15. (a) Encarnacion, A. B.; Fagutao, F.; Jintasataporn, O.; Worawattanamateekul, W.; Hirono, I.; Ohshima, T., Application of Ergothioneine-rich Extract from an Edible Mushroom Flammulina Velutipes for Melanosis Prevention in Shrimp, Penaeus Monodon and Litopenaeus Vannamei. Food Research International 2012, 45 (1), 232-237; (b) Radha, S.; Mullainadhan, P.; Arumugam, M., Detection of two Distinct Types of Hemolymphatic Prophenoloxidase and their Differential Responses in the Black Tiger Shrimp, Penaeus Monodon, upon Infection by White Spot Syndrome Virus. Aquaculture 2013, 376–379 (0), 76-84. 16. Adachi, K.; Hirata, T.; Nishioka, T.; Sakaguchi, M., Hemocyte Components in Crustaceans Convert Hemocyanin into a Phenoloxidase-like Enzyme. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2003, 134 (1), 135-141. 17. (a) Luna-González, A.; Almaraz-Salas, J. C.; Fierro-Coronado, J. A.; del Carmen FloresMiranda, M.; González-Ocampo, H. A.; Peraza-Gómez, V., The Prebiotic Inulin Increases the Phenoloxidase Activity and Reduces the Prevalence of WSSV in Whiteleg Shrimp (Litopenaeus Vannamei) Cultured under Laboratory Conditions. Aquaculture 2012, 362, 28-32; (b) Nirmal, N. P.; Benjakul, S., Melanosis and Quality Changes of Pacific White Shrimp (Litopenaeus Vannamei) Treated with Catechin During Iced Storage. Journal of Agricultural and Food Chemistry 2009, 57 (9), 3578-3586. 18. Martínez-Alvarez, O.; Montero, P.; Gómez-Guillén, C., Evidence of an Active Laccase-like Enzyme in Deepwater Pink Shrimp (Parapenaeus Longirostris). Food chemistry 2008, 108 (2), 624632.



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19. Huang, J.; Yang, Y.; Wang, A., Reconsideration of Phenoloxidase Activity Determination in White Shrimp Litopenaeus Vannamei. Fish & shellfish immunology 2010, 28 (1), 240-244. 20. Sung, H.-H.; Chang, H.-J.; Her, C.-H.; Chang, J.-C.; Song, Y.-L., Phenoloxidase Activity of Hemocytes Derived from Penaeus Monodon and Macrobrachium Rosenbergii. Journal of invertebrate pathology 1998, 71 (1), 26-33. 21. Nirmal, N. P.; Benjakul, S., Inhibitory Effect of Mimosine on Polyphenoloxidase from Cephalothoraxes of Pacific White Shrimp (Litopenaeus Vannamei). Journal of Agricultural and Food Chemistry 2011, 59 (18), 10256-10260. 22. Coates, C. J.; Nairn, J., Hemocyanin-derived Phenoloxidase Activity: a Contributing Factor to Hyperpigmentation in Nephrops Norvegicus. Food chemistry 2013, 140 (1), 361-369. 23. Decker, H.; Schweikardt, T.; Tuczek, F., The First Crystal Structure of Tyrosinase: all Questions Answered? Angewandte Chemie International Edition 2006, 45 (28), 4546-4550. 24. Nagai, T.; Kawabata, S.-i., A Link Between Blood Coagulation and Prophenol Oxidase Activation in Arthropod Host Defense. Journal of Biological Chemistry 2000, 275 (38), 29264-29267. 25. Skálová, T.; Dohnálek, J.; Østergaard, L. H.; Østergaard, P. R.; Kolenko, P.; Dušková, J.; Štěpánková, A.; Hašek, J., The Structure of the Small Laccase from Streptomyces Coelicolor Reveals a Link Between Laccases and Nitrite Reductases. Journal of molecular biology 2009, 385 (4), 11651178. 26. Naish-Byfield, S.; Riley, P. A., Tyrosinase Autoactivation and the Problem of the Lag Period. Pigment cell research 1998, 11 (3), 127-133. 27. (a) Adachi, K.; Endo, H.; Watanabe, T.; Nishioka, T.; Hirata, T., Hemocyanin in the Exoskeleton of Crustaceans: Enzymatic Properties and Immunolocalization. Pigment cell research 2005, 18 (2), 136-143; (b) Jaenicke, E.; Decker, H., Tyrosinases from Crustaceans form Hexamers. Biochem. J 2003, 371, 515-523. 28. Rolle, R.; Guizani, N.; Chen, J.; Marshall, M.; Yang, J.; Wei, C., Purification and Characterization of Phenoloxidase Isoforms from Taiwanese Black Tiger Shrimp (Penaeus Monodon). Journal of food biochemistry 1991, 15 (1), 17-32. 29. Adachi, K.; Hirata, T.; Nagai, K.; Fujisawa, S.; Kinoshita, M.; Sakaguchi, M., Purification and Characterization of Prophenoloxidase from Kuruma Prawn Penaeus Japonicus. Fisheries Science 1999, 65 (6), 919-925. 30. Galko, M. J.; Krasnow, M. A., Cellular and Genetic Analysis of Wound Healing in Drosophila Larvae. PLoS biology 2004, 2 (8), e239. 31. Sikorski, Z. E.; Kotakowski, E., Endogenous Enzyme Activity and Seafood Quality: Influence of Chilling, Freezing, and Other Environmental Factors. 2000; p 451-488. 32. Ali, M.; Marshall, M.; Wei, C.; Gleeson, R., Monophenol Oxidase Activity from the Cuticle of Florida Spiny Lobster (Panulirus Argus). Journal of Agricultural and Food Chemistry 1994, 42 (1), 53-58.

569


Page 22 of 27

Page 23 of 27


0.08

A a ab ab ab

-1

Phenoloxidase specific activity (U.mg )

0.06

bc 0.04

c

0.02 0.3

B Col 1 vs pattes SDS 0.1 Col 1 vs Pattes SDS 1

a a

a a

0.2

a

b

0.1

0.0 7.0

7.5

8.0

8.5

9.0

9.5

10.0

pH

Fig. 1. Phenoloxidase specific activity assayed in “Pods” (A) and “Tail” (B) for a buffer pH value of 7.4, 8.4 and 9.4 with 0.1% SDS (○) or 1% SDS (▼). Values are the means of triplicate assays ± S.D. Means without a common letter are significantly different (p < 0.05).



Page 24 of 27

-1 Phenoloxidase specific activity (U.mg )

0.25

0.20

0.15

0.10

0.05

0.00 1 10 20 30 40

1 5 10 20 30

40

Cat.

Dop.

L-Dop.

non-specific (mM)

1 5 10 20 30

L-Tyr.

1 10 20 30 40

1 10 20 30 40

1 10 20 30 40

Tyr.

PPD

Hyd.

tyrosinase-specific (mM)

laccase-specific (mM)

Fig 2. PA assays on “Pods” (Yellow) and “Tail” (Blue) using non-specific substrates (catechol (Cat.), dopamine (Dop.), L-DOPA (L-Dop.)), tyrosinase-specific (L-tyrosine (L-Tyr.), tyramine (Tyr.) and laccase-specific (PPD, hydroquinone (Hyd.)) at different concentrations. Phenoloxidase specific activities are expressed in U.mg-1 of total proteins and values are the means of triplicate assays ± S.D.


Page 25 of 27


0.16

0.6

A - Pods (L-DOPA)

* * *

phenoloxidase specific activity (U.mg-1)

*

B - Pods (Hydroquinone)

*

*

* *

* 0.08

0.3

*

0.00

0.0

0.16

0.4

C - Tail (L-DOPA)

D - Tail (hydroquinone)

* *

* *

0.08

*

*

* *

*

* *

0.00

0.2

0.0

0

0.1

5

18 32 49 61 77 95 105 125

storage time (days)

0

0.1

5

18 32 49 61 77 95 105 125

storage time (days)

Fig. 3. Specific PA as a function of storage time for “Pods” (A and B) and “Tail” (C and D) using 40 mM L-DOPA (A and C) or 40 mM hydroquinone (B and D) as substrate. Samples were stored at -20 °C (White) or -80 °C (Grey). Phenoloxidase-specific activities are expressed in U.mg-1 of total proteins and values are the means of triplicate assays ± S.D. “*” means a significant difference (p < 0.05) between the 2 storage conditions.



phenoloxidase specific activity (U.mg-1)

0.6

3

A - L-DOPA

a

Page 26 of 27

C - Hydroquinone

2

0.4

a

0.2

bc bc bc

c

c

1

b

b

b b

c

a

b b

b

0

0.0 0.6

a

B - L-DOPA, 0.1% SDS

3

D - Hydroquinone, 0.1% SDS a

0.4

2

b bc

0.2

1

bc bc c

bc bc

c

0

s pod

b

bc c

0.0

tail

bc

ax en om hor abd alot h p ce

t a il

s pod

bc c

ax en om hor abd alot h p ce

Fig. 4. Specific PAs according to the studied anatomical compartment for shrimp stored for 7 days at 7.3 ± 1.1 °C (Yellow) or 22.9 ± 1.4 °C (Blue). PA levels were assayed using L-DOPA as substrate and without SDS (A) or with 0.1% SDS (B) or using hydroquinone as substrate without SDS (C) or with 0.1% SDS (D). Means values without a common letter (in each section) were significantly different (p < 0.05).


Page 27 of 27


TOC Graphic 87x47mm (300 x 300 DPI)


Melanosis in Penaeus monodon: Involvement of the Laccase-like

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