Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

The Effect of Copper on the Color of Shrimps: Redder Is Not Always Healthier

  • Ana Martínez ,

    martina@unam.mx

    Affiliation: Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, México DF, México

  • Yanet Romero,

    Affiliation: Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, México DF, México

  • Tania Castillo,

    Affiliation: Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, México DF, México

  • Maite Mascaró,

    Affiliation: Unidad Multidisciplinaria de Docencia e Investigación, Sisal, Universidad Nacional Autónoma de México, Mérida, Yucatán, México

  • Isabel López-Rull,

    Affiliation: Centro Tlaxcala de Biología de la Conducta, Universidad Autónoma de Tlaxcala, Tlaxcala, Tlaxcala, México

  • Nuno Simões,

    Affiliation: Unidad Multidisciplinaria de Docencia e Investigación, Sisal, Universidad Nacional Autónoma de México, Mérida, Yucatán, México

  • Flor Arcega-Cabrera,

    Affiliation: Unidad de Química Sisal, Facultad de Química, Universidad Nacional Autónoma de México, Mérida, Yucatán, México

  • Gabriela Gaxiola,

    Affiliation: Unidad Multidisciplinaria de Docencia e Investigación, Sisal, Universidad Nacional Autónoma de México, Mérida, Yucatán, México

  • Andrés Barbosa

    Affiliation: Departamento de Ecología Evolutiva, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain

The Effect of Copper on the Color of Shrimps: Redder Is Not Always Healthier

  • Ana Martínez, 
  • Yanet Romero, 
  • Tania Castillo, 
  • Maite Mascaró, 
  • Isabel López-Rull, 
  • Nuno Simões, 
  • Flor Arcega-Cabrera, 
  • Gabriela Gaxiola, 
  • Andrés Barbosa
PLOS
x

Abstract

The objective of this research is to test the effects of copper on the color of pacific white shrimp (Litopenaeus vannamei) in vivo. Forty-eight shrimps (L. vannamei) were exposed to a low concentration of copper (1 mg/L; experimental treatment) and forty-eight shrimps were used as controls (no copper added to the water). As a result of this experiment, it was found that shrimps with more copper are significantly redder than those designated as controls (hue (500–700 nm): P = 0.0015; red chroma (625–700 nm): P<0.0001). These results indicate that redder color may result from exposure to copper and challenge the commonly held view that highly pigmented shrimps are healthier than pale shrimps.

Introduction

Yellow, orange and red pigmentation, manifest in animals and plants, is mostly caused by carotenoids [1][4]. These colorful substances are extensively present in nature and are considered useful antioxidants or antiradicals, preventing diseases caused by oxidative stress [5][8]. In particular, the intensity of red pink coloration of crustaceans (as shrimps) is controlled by the concentration of astaxanthin [9], a natural carotenoid that also acts as an effective antioxidant [6][8]. Crustaceans do not synthesize carotenoids de novo. Many crustaceans can synthesize astaxanthin from precursors as ß-carotene ingested from dietary, and as a consequence, they accumulate carotenoids from food [9]. Therefore, high astaxanthin content indicates the availability of a suitable diet during the growth and development of crustaceans, which is why the color of shrimps is regarded as an indication of health, thus affecting its commercial value [10][14]. More intense color suggests better taste and improved food quality [11]. Consequently, an important effort is made in the food industry to intensify the color of products [12][13]. Among crustaceans, an example can be found in Pacific white shrimp (Penaeus vannamei), in which a carotenoid supplementation using Aztec marigold “cempasúchil” (Tagetes erecta) results in redder individuals [13]. Furthermore, there is a variation in price conforming to the redness of these animals [14], so that the more intense the red color, the higher the price. Color of shrimps is so important that several studies have been devoted to the effect of light radiation on the color of organisms [15][17], as well as to the relationship between body color, carotenoid concentration and dietary supplementation [18], [19].

Heavy metals such as copper have an impact on the metabolism of aquatic species [20][26]. Experiments evaluating the toxicity of copper in shrimps indicate that high concentrations of copper particularly affect osmoregulation, molting frequency and survival [20][23]. Recent results from computational chemistry [27] indicate that the presence of metals such as copper, lead, mercury and cadmium, combined with astaxanthin causes the formation of novel complexes that are redder (larger lambda maxima) in appearance. The two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal and form these complexes, as described in experiments by Polyakov et al. [28], who likewise assess the effect of metal on coloration. These results may have important implications, as metal pollutants are commonly present in aquatic ecosystems [24], [29]. Whilst theoretical and experimental results exist, testifying to the modification of astaxanthin in the presence of metal atoms, it is necessary to corroborate that this modification actually occurs among live animals, altering their natural coloration. Despite ample studies, describing the effects of heavy metals on aquatic organisms together with studies that focus on the body color of shrimps, the effect of exposure to heavy metals on the body pigmentation of shrimps remains to be investigated. Therefore the objective of this research is to test in vivo, the effects of copper on the color of the pacific white shrimp (Litopenaeus vannamei).

Materials and Methods

Seawater was extracted from an unprotected marine area, where the laboratory facilities are located (21°09′N 90°01′W). In conformity with Mexican federal and state laws, as this is neither a protected area, nor private land, we did not require any type of permission. Our study did not involve endangered or protected species.

Two independent closed circuit water circulation systems, each with 6 glass aquaria (39×33×19 cm D×W×H; ≈ 25L) and a reservoir; 47 L) were prepared. Seawater was extracted directly from the sea and filtered (sand filter 20 and 30 microns and then subjected to a bag filter 25, 10 and 5 microns). Water was maintained at 25±1°C throughout the entire experiment. 12×12 light/dark period was controlled with white light lamps. Copper was added to one of the independent closed circuits (1 mg/L; experimental treatment) as CuSO4•5H2O (Sigma Aldrich Technical Grade), one day prior to the initiation of the experiment. This concentration of copper far exceeds that found naturally in the ocean (approximately 0.00025 mg/L, [30]), but is much lower than the median lethal concentration (96 h LC50; 37.3 mg/L [29]), thus ensuring shrimp survival and making it possible to analyze the effect of low concentration of copper on the color of shrimps.

Shrimps pertained to the Litopenaeus vannamei species, which is neither endangered, nor under legal protection. They were grown in floc in outdoor tanks. Floc contains microalgae, nematodes and some copepod bloom [31] and is a source of carotenoids [32]. Ninety-six shrimps (2.5–8.5 g wet body weight) were taken from these outdoors tanks and randomly and evenly assigned to one of the independent closed circuit water circulation systems, described previously in this section. Each glass aquarium contained 8 shrimps. During the experiment, shrimps were fed with pellets containing vitamins but not carotenoids. This means that their only source of carotenoids was derived from floc during the growing process.

Forty-eight shrimps (L. vannamei) were exposed to copper at a concentration of 1 mg/L (experimental), whilst forty-eight shrimps were maintained in clean seawater (controls). All shrimps were similar in size (6.95–10.32 cm) and were at the intermolt stage. Two experimental shrimps died during the experiment and were excluded from the analyses. Color was assessed at two stages: after 4 and 9 days under experimental conditions. On day four, 4 shrimps were collected from each aquarium to assess color, so that 4 shrimps remained in each glass aquarium. Those remaining were collected on day 9 (end of the experiment). Shrimps were sacrificed (submerged in boiling distilled water (100°C) for one minute immediately after collection), in order to prevent possible post mortem changes in color.

The most objective and reliable method for assessing color is spectrophotometry, which measures the distribution of wavelengths reflected, via a digital device known as a spectrophotometer. Spectrophotometry can also measure color, other than that which is visible to humans (e.g. ultraviolet and infrared wavelengths), and is therefore useful in the case of animals, as it may be that they perceive colors beyond the spectrum visible to humans [33], [34]. Reflectance spectra were obtained from 300 to 800 nm using a spectrophotometer (Ocean Optics USB2000). The diameter of the illuminated region was 6 mm and measurements were taken at a 45° angle, using an attachment designed for the purpose.

In this investigation, color was assessed in the head region immediately after sacrifice, using the spectrophotometer. Using the reflectance spectra, we calculated three colorimetric variables [35]: hue (the wavelength at maximum reflectance); red chroma saturation (the proportion of total reflectance in the red region; i.e. the proportion of light reflected at the wavelengths of red chroma); and light (the total amount of light reflected). Shrimp color was measured in the head area just below the eye three times over, and the arithmetic mean of these measurements was used for subsequent analysis. Red shrimp coloration was tested using a General Linear Mixed Model (GLMM), where hue, red chroma and total light were the dependent variables; tank was included as a random factor, whereas time (4 or 9 days of exposure) and treatment were applied as fixed factors, and weight and body size as covariate. The copper concentration in shrimps was measured using the atomic absorption technique. Shrimp heads were separated from their bodies. Heads were freeze dried in a Labconco FreeZone 2.5 freeze dryer. Dried shrimp heads were weighed and placed in Teflon vessels containing 9 mL of HNO3 (JT Baker ACS) for total digestion, in a microwave digestion system. Total copper was determined by absorption spectrometry, applying the acetylene flame technique [36].

Results and Discussion

Results related to copper concentration indicated that it was higher among experimental shrimps than among controls (ANOVA F1,89 = 16.735, P = 0.00009; control 172.32±13.41 mg/Kg and experimental 250.31±13.55 mg/Kg). Figure 1 presents the reflectance spectra of head coloration, in control (black line) and experimental (grey line) shrimps. Reflectance with wavelengths that fall between 625 and 700 nm (red chroma) was greater in the case of experimental shrimps (grey line). This means that individuals exposed to copper (B) were redder than individuals without added copper (A).

thumbnail
Figure 1. Color of shrimps.

Reflectance spectra for head coloration in control (black line) and experimental (grey line) shrimps. Visual aspects of selected shrimps not exposed (A) and exposed (B) to 1 mg/L of Cu during 9 days are shown in the image, in order to highlight the effect.

http://dx.doi.org/10.1371/journal.pone.0107673.g001

Our results indicate that shrimps are significantly redder in hue and chroma, when copper is present (see Figures 2 and 3). Among all variables tested, treatment emerged as the only significant factor, indicating that the presence of copper affected the coloration of shrimps (hue (500–700 nm): control = 589.44±7.7230; experimental = 625.63±7.8891; GLMM treatment F = 10.75, DF = 1,82, P = 0.0015. Red chroma (625–700 nm): control = 0.198±0.005; experimental = 0.2352±0.005; GLMM treatment F = 28.27, DF = 1,82, P<0.0001. Amount of light did not differ between control and experimental shrimps (P = 0.8335). Neither did time period (assessed on day 4 or 9), tank number or body size affect shrimp coloration (all p>0.66)). These results indicate a clear change in color of shrimps, due to the presence of copper.

thumbnail
Figure 2. Red chroma.

Differences in red chroma in control and copper exposed shrimps.

http://dx.doi.org/10.1371/journal.pone.0107673.g002

thumbnail
Figure 3. Hue.

Differences in hue (between 500 and 700 nm) in control and copper exposed shrimps.

http://dx.doi.org/10.1371/journal.pone.0107673.g003

At the biochemical level, the precise mechanism that causes more intense red color (hue and red chroma) among shrimps, in the presence of metals is still unknown. One explanation is that shrimps in the presence of copper consume more astaxanthin to avoid the oxidative stress caused by the presence of a heavy metal. However, it is not possible that exposure to a contaminated environment increases intake rate of astaxanthin, because shrimps under experimental conditions are fitted with pellets that do not contain astaxanthin. In fact, experiments indicate that it takes two weeks to observe color changes, directly resulting from dietary manipulation [10], [18][19]. These results ruled out this possibility in our experiment, as we detected color changes four days after treatment. One possible explanation is that copper alters the form of astaxanthin. Results from computational chemistry indicate that copper bonded to astaxanthin produces a complex that is redder in color. However, complexities emerge when intra-cell chemical mechanisms that involve organic pigments, are used to explain changes in color due to the presence of metal. Carotenoids are hydrophobic in nature and the concentration of transition metals in the cellular environment is very low [37]. It was reported that transition metal ions such as copper, iron and zinc mainly react with proteins. Considering this information, it is difficult to infer that these metal ions bind to the carotenoids, as these are not available in the cellular environment because they manifest limited solubility in water. All these assumptions need to be clarified. The mechanism causing more intense red color (hue and red chroma) in shrimps in the presence of metals is still undefined. However, it is clear that under experimental conditions, shrimps exposed to copper are significantly redder in color, than shrimps in the absence of copper. Other effects of copper on our shrimps were not analyzed; however shrimps presented normal aspect and behavior.

In summary, the presence of copper makes the body color of shrimps (L. vannamei) redder, but more research is needed in order to ascertain; the extent to which our results are replicated in other species and whether the effect is similar in the presence of other heavy metals, whilst also determining the intake mechanism of heavy metals at a biochemical level.

Supporting Information

Table S1.

Hue, Red Chroma, Lightness, Size, Body Weight and Cu concentration of each studied shrimp.

doi:10.1371/journal.pone.0107673.s001

(PDF)

Table S2.

Absorption spectra for each studied shrimp.

doi:10.1371/journal.pone.0107673.s002

(PDF)

Acknowledgments

This study was made possible due to resources provided by the Instituto de Investigaciones en Materiales IIM and Unidad Multidisciplinaria de Docencia e Investigación (UMDI-Sisal) of Universidad Nacional Autónoma de México. We thank Caroline Karslake (Masters, Social Anthropology, Cambridge University, England) for reviewing the grammar and style of the text in English. The authors would like to acknowledge Manuel Valenzuela, Miguel Arévalo, Adriana Paredes and Gabriela Palomino for shrimp production and Gemma L. Martínez Moreno, Oralia L Jiménez and María Teresa Vázquez for their technical support.

Author Contributions

Conceived and designed the experiments: AM YR TC MM NS FAC GG AB. Performed the experiments: AM YR TC MM FAC AB. Analyzed the data: AM YR TC MM ILR AB. Contributed reagents/materials/analysis tools: GG FAC. Contributed to the writing of the manuscript: AM MM ILR AB. Carried out the statistical analysis of the data: AM YR TC MM AB ILR. Contributed to the design of the experiment and revising the manuscript: AM YR TC MM NS FAC GG ILR AB.

References

  1. 1. Goodwin TW (1984) The biochemistry of carotenoids. Chapman and Hall, New York.
  2. 2. Olson VA, Owens IPF (1998) Costly sexual signals: are carotenoids rare, risky or required?. Trends in Ecology and Evolution 13: 510–14. doi: 10.1016/s0169-5347(98)01484-0
  3. 3. Hill GE (1991) Plumage coloration is a sexually selected indicator of male quality. Nature 350: 337–339. doi: 10.1038/350337a0
  4. 4. Hill GE, McGraw KJ (2006) Bird Coloration. Mechanisms and Measurements Harvard University Press: Cambridge, MA, Vol. 1.
  5. 5. Burton GW, Ingold KU (1984) Betacarotene an unusual type of lipid antioxidant. Science 224: 569–573. doi: 10.1126/science.6710156
  6. 6. Krinsky NI (2001) Carotenoids as antioxidants. Nutrition 17: 815–817. doi: 10.1016/s0899-9007(01)00651-7
  7. 7. Martínez A, Rodríguez-Gironés MA, Barbosa A, Costas M (2008) Donor acceptor map for carotenoids, melatonin and vitamins. Journal of Physical Chemistry A 112: 9037–9042. doi: 10.1021/jp803218e
  8. 8. Galano A, Vargas R, Martínez A (2010) Carotenoids can act as antioxidants by oxidizing the superoxide radical anion. Physical Chemistry Chemical Physics 12: 193–200. doi: 10.1039/b917636e
  9. 9. Maoka T (2011) Carotenoids in Marine Animals. Marine Drugs 9, 278–293.
  10. 10. Parisenti J, Beirão LH, Maraschin M, Mouriño JL, Do Nascimento Vieira F, et al. (2011) Pigmentation and carotenoid content of shrimp fed with Haematococcus pluvialis and soy lecithin. Aquaculture Nutrition 17: e530–e535. doi: 10.1111/j.1365-2095.2010.00794.x
  11. 11. Stahl W (2012) Carotenoids in Nutrition and Health - Developments and Future Trends. Molecular Nutrition and Food Research 56: 1–352. doi: 10.1002/mnfr.201100232
  12. 12. Soto-Salanova MF (2003) Natural pigments: practical experiences. In: Garnsworthy PC and Wiseman J (eds.) Recent Advances in Animal Nutrition. Nottingham University Press, Nottingham, UK. 67–75.
  13. 13. Vernon-Carter EJ, Ponce-Palafox JT, Pedroza-Islas R (1996) Pigmentation of Pacific White shrimp (Penaeus vannamei) using Aztec marigold (Tagetes erecta) extracts as the carotenoids source. Archivos Latinoamericanos de Nutrición 46: 243–246.
  14. 14. Lucien-Brun H, Vodal F (2006) Quality issues in marketing White shrimp. AQUA Culture Asia Pacific Magazine May/June: 32–3.
  15. 15. Wang F, Dong S, Huang G, Wu L, Tian X, et al. (2003) The effect of light color on the growth of Chinese shrimp Fenneropenaeus chinensis. Aquaculture 228: 351–360. doi: 10.1016/s0044-8486(03)00312-0
  16. 16. Guo B, Mu Y, Wang F, Dong S (2012) Effect of periodict light color change on the molting frequency and growth of Litopenaeus vannamei. Aquaculture 362–363: 67–71. doi: 10.1016/j.aquaculture.2012.07.034
  17. 17. You K, Yang H, Liu Y, Liu S, Zhou Y, et al. (2006) Effects of different light sources and illumination methods on growth and body color of shrimps Litopenaeus vannamei. Aquaculture 252: 557–565. doi: 10.1016/j.aquaculture.2005.06.041
  18. 18. Ponce-Palafox JT, Arredondo-Figueroa JL, Vernon-Carter EJ (2006) Carotenoids from plants used in diets for the culture of the pacific white shrimp (Litopenaeus vannamei). Revista Mexicana de Ingeniería Química 5: 157–165.
  19. 19. Cruz-Suárez LE, León A, Peña-Rodríguez A, Rodríguez-Peña G, Moll B, et al. (2010) Shrimp/Ulva co-culture: A sustainable alternative to diminish the need for artificial feed and improve shrimp quality. Aquaculture 301: 64–68. doi: 10.1016/j.aquaculture.2010.01.021
  20. 20. Bambang Y, Thuet P, Charmantier-Daures M, Trilles JP, Charmantier G (1995) Effect of copper on survival and osmoregulation of various developmental stages of the shrimp Penaeus japonicus Bate (Crustacea, Decapoda). Aquatic Toxicology 33: 125–139. doi: 10.1016/0166-445x(95)00011-r
  21. 21. Cheng JC, Lin CH (2001) Toxicity of copper sulfate for survival, growth, molting and feeding of juveniles of the tiger shrimp, Penaeus monodon. Aquaculture 192: 55–65. doi: 10.1016/s0044-8486(00)00442-7
  22. 22. Brooks SJ, Lloyd Mills C (2003) The effect of copper on osmoregulation in the freshwater amphipod Gammarus pulex. Comparative Biochemistry and Physiology Part A 135: 527–537. doi: 10.1016/s1095-6433(03)00111-9
  23. 23. Jung K, Zauke GP (2008) Bioaccumulation of trace metals in the brown shrimp Crangon crangon (Linnaeus, 1758) from the German Wadden Sea. Aquatic Toxicology 88: 243–249. doi: 10.1016/j.aquatox.2008.05.007
  24. 24. Frías-Espericueta M, Abad-Rosales S, Nevárez-Velázquez AC, Osuna-López I, Páez-Osuna F, et al. (2008) Histologial effects of a combination of heavy metals on Pacific white shrimp Litopenaeus vannamei juveniles. Aquatic Toxicology 89: 152–157. doi: 10.1016/j.aquatox.2008.06.010
  25. 25. Arce Funck J, Danger M, Gismondi E, Cossu-Leguille C, Guérold F, et al. (2013) Behavioural and physiological responses of Gammarus fossarum (Crustacea Amphipoda) exposed to silver. Aquatic Toxicology 142–143: 73–84. doi: 10.1016/j.aquatox.2013.07.012
  26. 26. De Jonge M, Tipping E, Lofts S, Bervoets L, Blust R (2013) The use of invertebrate body burdens to predict ecological effects of metal mixtures in mining-impact waters. Aquatic Toxicology 142–143: 294–302. doi: 10.1016/j.aquatox.2013.08.018
  27. 27. Hernández-Marin A, Barbosa A, Martínez A (2012) The Metal Cation Chelating Capacity of Astaxanthin. Does This Have Any Influence on Antiradical Activity? Molecules 17: 1039–1054. doi: 10.3390/molecules17011039
  28. 28. Polyakov NE, Focsan AL, Bowman MK, Kispert LD (2010) Free radical formation in novel carotenoids metal ion complexes of astaxanthin. Journal of Physical Chemistry B 114: 16968–16977. doi: 10.1021/jp109039v
  29. 29. Frías-Espericueta MG, Izaguirre-Fierro G, Valenzuela-Quiñonez F, Osuna-López JI, Voltolina D, et al. (2007) Metal content of the Gulf of California blue shrimp Litopenaeus stylirostris (Stimpson). Bulletin of Environmental Contamination and Toxicology 79: 214–17. doi: 10.1007/s00128-007-9165-z
  30. 30. Chester R (2000) Marine Geochemistry, Blackwell Sciences Ltd, London, UK. 698.
  31. 31. Emerenciano M, Cuzon G, Paredes A, Gaxiola G (2013) Evaluation of biofloc technology in pink shrimp Farfantepenaeus duorarum culture: growth performance, water quality, microorganisms profile and proximate analysis of biofloc. Aquaculture International 21: 1381–1394 DOI 10.1007/s10499-013-9640-y.
  32. 32. Ju ZY, Forster I, Conquest L, Dominy W (2008) Enhanced growth effects on shrimp (Litopenaeus vannamei) from inclusion of whole shrimp floc or floc fractions to a formulated diet. Aquaculture Nutrition 14: 533–543. doi: 10.1111/j.1365-2095.2007.00559.x
  33. 33. Zuk M, Decruyenaere J (1994) Measuring individual variation in colour: a comparison of two techniques. Biological Journal of the Linnean Society 53: 165–173. doi: 10.1111/j.1095-8312.1994.tb01007.x
  34. 34. Laczi M, Török J, Rosivall B, Hegyi G (2011) Integration of spectral reflectance across the plumage: implications for mating patters. Plos ONE 6: e23201 doi:10.1371/journal.pone.0023201.
  35. 35. Montgomerie R (2006) Analyzing colors. In: Hill, G.E. & McGraw, K.J. (eds) Bird coloration. Mechanisms and Measurements. Cambridge, MA: Harvard University Press. Cambridge, MA, USA. 90–147.
  36. 36. Sadiq M, Zaidi TH, Sheikheldin S (1995) Concentration of metals of health significance in commonly consumed shrimps in the eastern province of Saudi Arabi. Journal of Environmental Science and Health. Part A: Environmental Science and Engineering and Toxicology A (30)-1: 15–30. doi: 10.1080/10934529509376181
  37. 37. Kraatz HB, Metzler-Nolte N, eds (2006) Concepts and models in bioinorganic chemistry. Wiley-VCH, Weinheim.