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Colour preference and colour vision of the larvae of the giant freshwater prawn Macrobrachium rosenbergii
Journal of Experimental Marine Biology and Ecology 474 (2016) 67–72
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Colour preference and colour vision of the larvae of the giant freshwater prawn Macrobrachium rosenbergii Gunzo Kawamura a, Teodora Bagarinao b, Annita Seok Kian Yong a,⁎, Ivy Michelle Xavier Jeganathan a, Leong-Seng Lim a a b
Borneo Marine Research Institute, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia Aquaculture Department, Southeast Asian Fisheries Development Center, 5021 Tigbauan, Iloilo, Philippines
a r t i c l e
i n f o
Article history: Received 21 July 2015 Received in revised form 30 September 2015 Accepted 1 October 2015 Available online xxxx Keywords: Colour vision Innate colour preference Macrobrachium rosenbergii Crustacean larvae
a b s t r a c t This paper reports on the innate colour preference and colour vision in the hatchery-reared larvae (10–16 days old, stages IV–VIII) of the giant freshwater prawn Macrobrachium rosenbergii (De Man) based on their response to coloured beads in a grey-walled tank under natural illumination. Plastic beads (4.1 mm in diameter) of different colours (dark blue, light blue, light green, yellow, red, white, black, and grey) in various combinations were suspended in the water 5 cm from the water surface and 12–20 cm from the tank walls where the larvae rested in the absence of aeration. The larvae swam head first straight toward the beads and gathered around them. The number of larvae was highest around the dark blue, light blue, and white beads; lowest around the black, red, and light green beads; and moderate around the yellow bead. Tests with different colours in combination with three shades of grey indicated that the larvae of M. rosenbergii discriminated colours by chromaticity. The preference for blue seemed to be an innate rather than a learned ability since the larvae did not prefer the yellow and red beads that were more similar to the colours of the egg custard and the Artemia nauplii on which they had been reared. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The tropical giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879) (Decapoda: Palaemonidae) is now an important aquaculture commodity and its global production has increased rapidly since the 1990s (FAO, 2014). Efforts are continuing to improve its growth, survival, and production under various conditions. In the hatchery, it is necessary to understand the behaviour of larvae to ensure their welfare (Mench, 1998). Light is a very important factor in larval behaviour particularly for feeding and predator avoidance. The colour of the rearing tank affects M. rosenbergii; juveniles showed a significant preference for black over light blue or white backgrounds (Juarez et al., 1987), and larvae had significantly higher survival in red and green tanks than in white and blue tanks (Yasharian et al., 2005). Most tanks commonly used in commercial M. rosenbergii larviculture in the USA are either blue or black (Yasharian et al., 2005). The colours of the incident light and the background affect spawning and growth of marine penaeid shrimps (Emmerson et al., 1983; Primavera and Caballero, 1992; Wang et al., 2003; You et al., 2006; Luichiari et al., 2012). Adding artificial shelters is a common practise in prawn ponds and orange shelter is recommended for M. rosenbergii (D'Abramo et al., 2006). Meyers and
⁎ Corresponding author. E-mail address: [email protected] (A.S.K. Yong).
http://dx.doi.org/10.1016/j.jembe.2015.10.001 0022-0981/© 2015 Elsevier B.V. All rights reserved.
Hagood (1984) observed that M. rosenbergii larvae accepted light coloured feed flakes better than darker flakes. Light has several biologically relevant characteristics: wavelength (colour), intensity (brightness), and periodicity. Experiments on the effects of light colour and background colour are often based on the assumption that the test animals have colour vision. An animal has colour vision if it can discriminate between two lights of different spectral composition regardless of their relative intensity (Kelber et al., 2003). To a totally colour-blind animal, each colour appears as grey of a certain degree of brightness. Colour vision requires at least two types of photoreceptors with different spectral sensitivities (Tempel and Mimstedt, 1979; Kelber et al., 2003). Colour vision has been claimed or speculated in the Crustacea based on the types of photoreceptors (Leggett, 1979; Stowe, 1980; Marshall et al., 1996; Kashiyama et al., 2009; Rajkumar et al., 2010). Different colours have different contrasts against background colour and influence the efficiency of detecting and catching the prey or feeds by sight. A high contrast leads to higher visibility and better food consumption (Kawamura et al., 2010; El-Sayed and El-Ghobashy, 2011). Preference tests have been widely used to determine the best environmental conditions for captive animals. To make a choice, the test animals must be able to discriminate between pleasant and unpleasant conditions, and choose the ones that best fit their comfort zones (Gonyou, 1994). A colour preference test can show that an animal can distinguish colours, but does not prove that it has colour vision, for
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these are not always the same (von Frisch, 1950). For example, coral larvae without eyes settle on red surfaces over other coloured surfaces, and this was attributed to a photosensitivity to long wavelengths (Mason et al., 2011). Earlier colour preference tests for penaeid and palaemonid shrimps, mentioned above, implied colour vision. However, colour vision in penaeid shrimps is uncertain as most decapod crustaceans have only a single type of photoreceptor (Lindsay et al., 1999; Johnson et al., 2002), and Litopenaeus vannamei has a single visual pigment (Matsuda et al., 2011). Colour vision requires at least two types of spectral receptors (Kelber and Osorio, 2010). Stomatopods are currently the only crustaceans known to possess true, behaviourally demonstrated colour vision (Marshall et al., 1996; Theon et al., 2014). Studies have not been done on the colour vision or visual pigment of M. rosenbergii. The present study examined for the first time the colour preference and possible colour vision of the larvae of M. rosenbergii by means of an experiment that took advantage of their natural swimming behaviour, and particularly the attraction to, and congregation around, large suspended particles in a rearing tank. 2. Materials and methods 2.1. Larval rearing and handling Larvae of M. rosenbergii from wild broodstock were reared in the shrimp hatchery of the Borneo Marine Research Institute, Universiti Malaysia Sabah. Every day the zoeae were with fed newly hatched Artemia nauplii (b 0.5 mm long) in the afternoon and egg custard (egg yolk and albumen, milk powder, mixed vitamin, and cod-liver oil) in the morning. The wet egg custard was passed through a laboratory sieve (Endecotts, UK) with 500 μm mesh for early zoeae and 1 mm mesh for later zoeae. In the absence of food, M. rosenbergii zoeae actively swam backward, tail-oriented, with their dorsal side down. When live Artemia nauplii were added to the rearing tank, the zoeae showed no changes in swimming behaviour. But when egg custard was added to the rearing tank, the zoeae approached the custard particles by swimming forward, head first, with their dorsal side up. The zoeae held on to custard particles 1–5 mm in size. Batches of 10,000 larvae were transferred to a tank (diameter 93 cm, depth 53 cm, water volume 360 l) provided with mild aeration using diffusion stone and placed inside a roofed hatchery for the experiment on colour vision and colour preference. Larvae (12–18 days old, stages IV to VIII) were stocked in an indoor grey-walled conical round tank under natural illumination for a 6-day experiment that took advantage of their natural behaviour in the presence of custard particles. In this experimental tank, the water temperature, dissolved oxygen, pH and salinity were measured twice daily at 08:00 and 16:00 with a pH/ ORD/EC/DO tester (Hanna Instruments, HI 9828, Washington, USA). Water temperature ranged from 25.7–27.4 °C; dissolved oxygen, 6.0–8.2 mg l−1; pH 7.5–8.7; and salinity 8–12. Water quality in the experimental tank stayed within the normal ranges experienced by M. rosenbergii larvae (New, 2002). Animal care and handling was according to the guidelines for the care and use of laboratory animals set by the World Health Organization in Geneva, Switzerland, the Malaysian Animal Handling Code of Conduct, and the National Research Council (2011). 2.2. Colour vision and preference tests Plastic beads (4.1 mm in diameter, density N1.03 g ml−1) of five different colours were displayed to the zoeae in different combinations. Two sizes of beads (4.1 and 2.9 mm in diameter) were commercially available but in a preliminary test, the zoeae responded more strongly to the larger beads (χ2 test, P b 0.005). The reflectance spectra were shown to identify chromatic and achromatic properties of the beads (Fig. 1). The light reflectance of the beads in the wavelength range of 400–700 nm was recorded with a spectroradiometer (HSR-8100, Maki
Fig. 1. Light reflectance spectra for (A) the colour beads and (B) the grey beads used in the colour preference tests on Macrobrachium rosenbergii larvae. The reflectance of the white bead was higher than those of the colour beads at all wavelengths except at the peak of the red bead.
Manufacturing Co., Ltd., Hamamatsu, Japan) under the natural illumination beside the experimental tank placed inside the roofed hatchery. The peak wavelengths of the beads were at 421 and 436 nm for dark blue, 493 nm for light blue, 532 nm for light green, 493 and 532 nm for yellow, and 635 nm for red (Fig. 1A). To determine whether larvae chose a colour bead based on colour per se (chromaticity) or based on the difference in brightness of the beads, grey beads were presented together with different combinations of colour beads. If larvae choose a particular coloured bead among varying shade of grey, then they must be able to perceive the colour of the bead regardless of brightness. Three shades of grey, Grey30, Grey50, and Grey70 were produced by mixing 30%, 50%, and 70% black paint (Kenlux Synthetic Enamel 9103) with white paint (Kenlux Synthetic Enamel 9102). The painted beads were washed in running tap water for 2 days to elute the paint chemicals. The light reflectance spectra for the white, black, and grey beads are shown in Fig. 1B. The tests were done from 11:00 to 16:00 when the natural illuminance ranged from 1062 l× to 22,791 l× (light metre LT300, Extech Instruments, USA) at the water surface of the experimental tank. These light intensities were high enough to allow for colour vision if the larvae had the necessary visual mechanism. The natural light spectrum under direct sun through the hatchery roof is shown in Fig. 2.
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each combination of colour beads was replicated 20 times at 10–15 s intervals or 40 times when the number of larvae attracted to a bead was less than 6. In combinations of a colour bead with grey beads, display was replicated 10 times. The digital photographs taken during the trials were later examined and the number of larvae around each bead was counted, and then summed up for the 10, 20, or 40 trials of a given colour combination. The aggregated larvae oriented with heads toward the beads, at the same depth as the beads, and appeared clear and focused in photographs; those that were not attracted were deeper and farther away and showed poorly in the photographs. The aggregation around each bead did not overlap with those around the adjacent beads, and accurate counting was possible. 2.3. Statistical analysis Fig. 2. Natural light spectrum measured inside the roofed hatchery where the experimental tank was placed. Note that the UVA (b390 nm) irradiance is lower than the measurable level due to the UVA-opacity of the roofing material, polycarbonate.
The plastic beads were tethered by transparent monofilament (0.25 mm thick, 20 cm long) to a transparent plastic ruler at intervals of 6–9 cm depending on the number of beads in the combination to be tested. The display positions along the tank wall and the arrangement of the beads in each combination were interchanged at random. Aeration in the tank was stopped during each test. The larvae distributed at or close to the tank wall and within 25 cm, mostly within a 15 cm depth from the surface (none was at the bottom) during the experiment. Each test consisted of lowering the combination of beads to a depth of 5 cm at a distance of 12–15 cm from the tank wall and allowing the larvae to gather around the beads of choice. The monofilaments were straight and the tethered beads were well separated in the water. The larvae attracted to the beads were visually counted by two observers during each trial. As precise counting of more than 10 larvae was difficult, the larvae were photographed with a digital camera (no flash) within about 5–8 s after the beads were submerged and the number of larvae around the beads had stabilized (Fig. 3). A single photograph was taken for each bead arrangement, care being taken to avoid reflections at the water surface and obtain a clear image. Display of
Fig. 3. Illustration of experimental tank with beads and camera setting.
The number of larvae was counted for each bead in each display and the counts were summed for each combination (rows, Table 1). The data was analysed by chi-square χ2 test to assess the goodness of fit between a set of observed values and those expected theoretically, i.e., on the hypothesis that the probability of choice for each colour is 1/k, where k is the number of beads in a combination. The significance level was set at α = 0.05. Across the different test combinations, the variation in the numbers of zoeae attracted to each colour bead was computed as the mean ± standard deviation (columns, Table 1). 3. Results 3.1. Response of larvae to colour beads M. rosenbergii larvae stages IV to VIII responded to any single suspended plastic bead, of any colour, by swimming head first straight toward it in the same typical swimming posture as toward particles of egg custard. Most larvae approached from above, some horizontally, and a few from below. Larvae gathered around a bead for some time (2–10 s) before dispersing. When combinations of several colour beads were displayed, the larvae swam straight to the beads of specific colours from the tank walls and all directions from distances as far as 20 cm. Larvae gathered around the beads orienting with the head; some of them touched a bead several times and then hopped away. As larvae gathered around a bead, the latter became less visible from a distance and attracted no more larvae. After about 10 s the gathered larvae gradually dispersed. The larvae exhibited the same response during each of the 10–40 trials at 10–15 s intervals for each colour combination. Table 1 summarizes the number of larvae gathered around the coloured and grey beads displayed in different combinations. Larvae aggregated around the dark blue, light blue, and white beads, indicating a biased attraction to blue and white. In the combinations of dark blue, light blue, and white beads (tests 5, 6, and 7), the largest aggregation formed around the light blue bead (totalling 676 larvae to light blue, 534 larvae to dark blue and 601 larvae to white), but the numbers varied with the arrangement of the three beads (χ2 = 16.72, P b 0.005). The reflectance spectra show that the light blue bead was much brighter than the dark blue bead (Fig. 1A), yet there was no significant difference in the aggregation of larvae around dark blue and light blue beads (test 3). The fewest larvae gathered around the black bead (tests 4 and 15), and some more responded to light green (tests 1 and 2) and red (tests 4 and 13). Aggregations were moderate for yellow beads in combination with other colours (tests 1 and 2), but not significantly different from those around the three shades of grey (test 12). The preference for the different colours is also evident in the mean numbers of larvae that gathered around the beads (bottom row in Table 1). Colour preference did not vary with age from 12 days to 18 days during the 7-day experiment, nor with the 20 × variation in
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Table 1 Number of Macrobrachium rosenbergii larvae attracted to colour and grey beads displayed in different combinations. Colour of test beads Test
Combination [no. of trials]
1 F, D A, C and B [n = 40] 2 D, A, C and B [n = 40] 3 A and B [n = 20] 4 F, E and G [n = 20] 5 A, B and F [n = 20] 6 B, A and F [n = 20] 7 A, F and B [n = 20] 8 H, I and J [n = 20] 9 A, H, I and J [n = 10] 10 B, H, I and J [n = 10] 11 C, H, I and J [n = 10] 12 D, H, I and J [n = 10] 13 E, H, I and J [n = 10] 14 F, H, I and J [n = 10] 15 G, H, I and J [n = 10] Mean larvae per trial ± SD†
A Dark blue
B Light blue
C D Light green Yellow
504 569 163
565 624 168
8 8
179 210 145
258 216 202
E Red
25 38
150 45
11.6 ± 3.1 12.8 ± 2.5 0.7 ± 1.1
G Black
H Grey30
I Grey50
J Grey70
302 182 206 213
1
106 31 23 57 50 40 10 46 220 39 7 34 1.3 ± 1.3 0.8 ± 0.8 12.2 ± 4.2 0.3 ± 0.5 4.2 ± 1.1
χ2 for grey beads
χ2 951.516⁎ 1068.167⁎ 0.076 534.573⁎ 19.428⁎
457
15
201
F White
0.241 14.275⁎ 99 30 22 53 44 22 24 22 3.3 ± 1.1
126 38 32 48 48 55 37 23 4.5 ± 1.2
3.560 282.747⁎ 1.152 ⁎ 192.901 2.363 1.670 0.772 1.297 0.727 39.180⁎ 14.195⁎ 328.325⁎ 3.980 17.128⁎ 3.368
⁎ Significant at α = 0.05. † Standard deviation.
natural illuminance, nor with the ambient pH 7.5–8.7 or other water quality variables. 3.2. Response of larvae to the brightness of beads Table 1 also shows that in the combinations of a coloured bead with grey beads, the aggregations of larvae were significantly higher around the dark blue, light blue, and white beads (tests 9, 10, 14). The number of larvae attracted to light green and yellow beads was not significantly different from those around the grey beads (tests 11 and 12; 0.75 N P N 0.50). Significantly fewer larvae came to the red bead and the black bead than to the three grey beads (tests 13 and 15). In the combination with three grey beads, the number of larvae attracted was not significantly different (test 8; 0.25 N P N 0.05). The three grey beads attracted similar number of larvae (P N 0.25) but only in the combination of the red and the three grey beads (test 13), G50 attracted significantly fewer larvae than G30 and G70 (P b 0.005). 4. Discussion In the present study, the existence of colour vision in M. rosenbergii larvae is argued based on colour preference behaviour. Most knowledge of the visual system necessarily comes from physiological and opsin transcript studies, but only behavioural (psychophysical) studies can tell what the animal's visual system is truly capable of achieving (Douglas and Hawryshyn, 1990). The visual response of M. rosenbergii larvae to the plastic beads was qualitatively different from their thigmotactic response to the tank walls. The visual attraction was strongest for the light blue, white and dark blue beads, and weakest for the black, light green, and red beads. Choice of the coloured beads was not based on brightness—light blue bead and dark blue bead were equally preferred, whereas the brighter yellow and light green beads were not preferred over the darker grey beads. If larvae had not discriminated between colours, they would have been unable to distinguish the blue beads from the three shades of grey beads. In fact, the larvae discriminated blue beads from grey beads based mainly on colour (chromaticity) and thus have true colour vision. The strong response to blue, moderate response to yellow, and poor response to red are consistent with the fact that the main rhabdome in decapod shrimps contains a visual pigment with λmax between 460 nm and 550 nm (Wald and Seldin, 1968; Cronin and Hariyama, 2002). The M. rosenbergii larvae may be like grass shrimp Palaemonetes pugio larvae in which the visual system is already functional at hatching (Douglass
and Forward, 1989), ready for the ecological tasks during the pelagic phase and then modified at metamorphosis to meet the new visual demands of the demersal phase. Metamorphic change in visual pigment does not occur in decapod crustaceans (Cronin et al., 1995), so colour vision will persist throughout the ontogeny of M. rosenbergii. The distinct preference of M. rosenbergii larvae for blue and white beads could not be explained by associative learning, but rather seems to be an innate ability. The larvae in this study had been feeding on orange Artemia nauplii and yellow egg custard. If colour preference was learned, then the larvae should have preferred orange and/or yellow. Instead the larvae showed moderate response to the yellow bead and poor response to the red bead. The larvae in this study retained the innate colour preference for blue even after feeding on the yellow egg custard. One adaptive significance of innate colour preference is that it helps naïve animals to find food (Giurfa et al., 1995; Weiss, 1997). Innate colour preferences have been reported also among fishes (Kawamura et al., 2010) and birds (Honkavaara et al., 2004; Schmidt and Schaefer, 2004; Bascuñán et al., 2009). The M. rosenbergii larvae were observed immediately headed straight toward the custard particles and the tasteless and odourless plastic beads from distances of 10–20 cm; this was clearly a visual response. But the larvae IV to VIII probably already have multiple sensory modalities. Moller (1978) observed the feeding behaviour of M. rosenbergii larvae and postlarvae in Petri dishes under a stereomicroscope and concluded that the larvae detect and discriminate food from non-food particles by chemosensory mechanisms rather than vision, just like juvenile banana shrimp Penaeus merguiensis De Man (Hindley, 1975). The ingestion rate of Artemia nauplii by M. rosenbergii larvae is a function of nauplii density (Barros and Valenti, 2003). You et al. (2006) found that food intake of juvenile white leg prawn L. vannamei is significantly lower in an illuminated tank than in a dark tank, and concluded that light is not necessary for the shrimp to locate food. The feeding modality usually changes with growth and metamorphosis (Kawamura and Hara, 1980). Juvenile and adult M. rosenbergii are active at night and probably rely on chemoreception and mechanoreception to detect food (Nakamura, 1975; Scudder et al., 1981). The larvae of M. rosenbergii live as part of the zooplankton community in spectrally poor turbid environments such as estuaries and coastal lagoons (New and Singholka, 1985; Nandlal and Pickering, 2005). The planktonic larvae must feed, avoid predators, and travel to a location where they can successfully metamorphose. Their diets consist mainly of minute crustaceans and worms (New, 2002; Nandlal and Pickering, 2005). Zoeae must eat continuously to survive and vision is very important in daytime feeding (New, 2002). Turbidity can affect prey detection
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by increasing or diminishing the contrast between prey and background due to the scattering of light. The positive effect of turbidity is pronounced for planktivores and fish larvae (Utne-Palm, 2002). Contrast is detected in the form of brightness or colour. Colour vision would help larvae detect food organisms in turbid habitats and allow them to choose food items by colour. M. rosenbergii probably evolved colour vision at larval stages to enhance contrast and, thus, improve the visibility of objects against the background. The reaction distance of M. rosenbergii larvae to the beads was about 10–20 cm, much greater than prey detection distances (via the vibration sense) of other zooplankton: less than 1 mm in copepods Oithona plumifera and O. similis; and less than 3 mm in chaetognaths (arrow worms) (Svensen and Kiørboe, 2000; Jian and Paffenhöfer, 2008; Horridge and Boulton, 1967; Feigenbaum and Reeve, 1977). The significance of the strong wavelength-specific response to blue beads in M. rosenbergii larvae is unknown. The bias for blue and white may be adaptive for visual function under dim light, as has been shown for the foraging nocturnal hawk moth Manduca sexta (Goyret et al., 2008). Floral surveys confirmed that the flowers visited by M. sexta were white but never blue, thus the innate preference for blue is not adaptive in M. sexta and remains a mystery (Goyret et al., 2008). The diurnal hawk moth M. stellatarum also has a bias for blue (Kelber, 1997). 5. Conclusions Behavioural experiments indicate that hatchery-reared M. rosenbergii larvae (10–18 days old, stages IV–VIII) aggregate preferentially on blue and white objects (including artificial food); this colour preference is based on chromaticity rather than brightness, and strongly suggest that the larvae are capable of colour vision. Competing interest The authors declare no competing or financial interests. Author contribution G.K. designed, conducted the experiment and wrote the manuscript, T.B and Y.A. contributed to revision of the manuscript, I.M.X and L.L.S conducted the experiment. References Barros, H.P., Valenti, W.C., 2003. Ingestion rates of Artemia nauplii for different larval stages of Macrobrachium rosenbergii. Aquaculture 217, 223–233. Bascuñán, A.L., Tourville, E.A., Toomey, M.B., McGraw, K.J., 2009. Food color preferences of molting house finches (Carpodacus mexicanus) in relation to sex and plumage coloration. Ethology 115, 1066–1073. Cronin, T.W., Hariyama, T., 2002. Spectral sensitivity in crustacean eyes. In: Wiese, K. (Ed.), The Crustacean Nervous System. Springer, Heidelberg, pp. 499–511. Cronin, T.W., Marshall, N.J., Caldwell, R.L., Pales, D., 1995. Compound eyes and ocular pigments of crustacean larvae (Stomatopoda and Decapoda, Brachyura). Mar. Freshw. Behav. Physiol. 26, 219–231. D'Abramo, L.R., Tidwell, J.H., Fondren, M., Ohs, C.L., 2006. Pond production of the freshwater prawn in temperate climates. Southern Regional Aquaculture Center Publication No. 484 (8 pp.). Douglas, R.H., Hawryshyn, C.W., 1990. Behavioral studies of fish vision; an analysis of visual capabilities. In: Douglas, R.H., Djamgpoz, M.B.A. (Eds.), The visual system of fish. Chapman and Hall, London, pp. 373–418. Douglass, L., Forward Jr., R.B., 1989. The ontogeny of facultative superposition optics in a shrimp eye: hatching through metamorphosis. Cell Tissue Res. 258, 289–300. El-Sayed, A.F.M., El-Ghobashy, A.E., 2011. Effects of tank colour and feed colour on growth and feed utilization of thin lip mullet (Liza ramada) larvae. Aquac. Res. 42, 1163–1169. Emmerson, W.D., Hayes, D.P., Ngonyame, M., 1983. Growth and maturation of Penaeus indicus under blue and green light. S. Afr. J. Zool. 18, 71–75. FAO, 2014. Cultured Aquatic Species Information Programme—Macrobrachium rosenbergii (De Man, 1879) Retrieved from http://www.fao.org/fishery/culturedspecies/ Macrobrachium_rosenbergii/en#tcNA002B.
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Flake diets and larval crustacean culture. Prog. Fish Cult. 46, 225–229. Moller, T.H., 1978. Feeding behaviour of larvae and postlarvae of Macrobrachium rosenbergii. J. Exp. Mar. Biol. Ecol. 35, 251–258. Nakamura, R., 1975. A preliminary report on the circadian rhythmicity in the spontaneous locomotor activity of Macrobrachium rosenbergii and its possible application to prawn culture. In: Avault, J.W., Miller, R. (Eds.), Proceedings of the Sixth Annual Meeting of the World Mariculture Society, Louisiana State University Press, Baton Rouge, USA, pp. 37–41. Nandlal, S., Pickering, T., 2005. Freshwater prawn Macrobrachium rosenbergii farming in Pacific Island countries. Volume 1. Hatchery Operation. Secretariat of the Pacific Community, Noumea, New Caledonia (31 pp.). National Research Council, 2011. In: Institute of Laboratory Animal Resources (Ed.), Guide For The Care And Use Of Laboratory Animals, 8th ed. National Academies Press, Washington, D.C. (220 pp.). New, M.B., 2002. 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Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
Colour preference and colour vision of the larvae of the giant freshwater prawn Macrobrachium rosenbergii Gunzo Kawamura a, Teodora Bagarinao b, Annita Seok Kian Yong a,⁎, Ivy Michelle Xavier Jeganathan a, Leong-Seng Lim a a b
Borneo Marine Research Institute, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia Aquaculture Department, Southeast Asian Fisheries Development Center, 5021 Tigbauan, Iloilo, Philippines
a r t i c l e
i n f o
Article history: Received 21 July 2015 Received in revised form 30 September 2015 Accepted 1 October 2015 Available online xxxx Keywords: Colour vision Innate colour preference Macrobrachium rosenbergii Crustacean larvae
a b s t r a c t This paper reports on the innate colour preference and colour vision in the hatchery-reared larvae (10–16 days old, stages IV–VIII) of the giant freshwater prawn Macrobrachium rosenbergii (De Man) based on their response to coloured beads in a grey-walled tank under natural illumination. Plastic beads (4.1 mm in diameter) of different colours (dark blue, light blue, light green, yellow, red, white, black, and grey) in various combinations were suspended in the water 5 cm from the water surface and 12–20 cm from the tank walls where the larvae rested in the absence of aeration. The larvae swam head first straight toward the beads and gathered around them. The number of larvae was highest around the dark blue, light blue, and white beads; lowest around the black, red, and light green beads; and moderate around the yellow bead. Tests with different colours in combination with three shades of grey indicated that the larvae of M. rosenbergii discriminated colours by chromaticity. The preference for blue seemed to be an innate rather than a learned ability since the larvae did not prefer the yellow and red beads that were more similar to the colours of the egg custard and the Artemia nauplii on which they had been reared. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The tropical giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879) (Decapoda: Palaemonidae) is now an important aquaculture commodity and its global production has increased rapidly since the 1990s (FAO, 2014). Efforts are continuing to improve its growth, survival, and production under various conditions. In the hatchery, it is necessary to understand the behaviour of larvae to ensure their welfare (Mench, 1998). Light is a very important factor in larval behaviour particularly for feeding and predator avoidance. The colour of the rearing tank affects M. rosenbergii; juveniles showed a significant preference for black over light blue or white backgrounds (Juarez et al., 1987), and larvae had significantly higher survival in red and green tanks than in white and blue tanks (Yasharian et al., 2005). Most tanks commonly used in commercial M. rosenbergii larviculture in the USA are either blue or black (Yasharian et al., 2005). The colours of the incident light and the background affect spawning and growth of marine penaeid shrimps (Emmerson et al., 1983; Primavera and Caballero, 1992; Wang et al., 2003; You et al., 2006; Luichiari et al., 2012). Adding artificial shelters is a common practise in prawn ponds and orange shelter is recommended for M. rosenbergii (D'Abramo et al., 2006). Meyers and
⁎ Corresponding author. E-mail address: [email protected] (A.S.K. Yong).
http://dx.doi.org/10.1016/j.jembe.2015.10.001 0022-0981/© 2015 Elsevier B.V. All rights reserved.
Hagood (1984) observed that M. rosenbergii larvae accepted light coloured feed flakes better than darker flakes. Light has several biologically relevant characteristics: wavelength (colour), intensity (brightness), and periodicity. Experiments on the effects of light colour and background colour are often based on the assumption that the test animals have colour vision. An animal has colour vision if it can discriminate between two lights of different spectral composition regardless of their relative intensity (Kelber et al., 2003). To a totally colour-blind animal, each colour appears as grey of a certain degree of brightness. Colour vision requires at least two types of photoreceptors with different spectral sensitivities (Tempel and Mimstedt, 1979; Kelber et al., 2003). Colour vision has been claimed or speculated in the Crustacea based on the types of photoreceptors (Leggett, 1979; Stowe, 1980; Marshall et al., 1996; Kashiyama et al., 2009; Rajkumar et al., 2010). Different colours have different contrasts against background colour and influence the efficiency of detecting and catching the prey or feeds by sight. A high contrast leads to higher visibility and better food consumption (Kawamura et al., 2010; El-Sayed and El-Ghobashy, 2011). Preference tests have been widely used to determine the best environmental conditions for captive animals. To make a choice, the test animals must be able to discriminate between pleasant and unpleasant conditions, and choose the ones that best fit their comfort zones (Gonyou, 1994). A colour preference test can show that an animal can distinguish colours, but does not prove that it has colour vision, for
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these are not always the same (von Frisch, 1950). For example, coral larvae without eyes settle on red surfaces over other coloured surfaces, and this was attributed to a photosensitivity to long wavelengths (Mason et al., 2011). Earlier colour preference tests for penaeid and palaemonid shrimps, mentioned above, implied colour vision. However, colour vision in penaeid shrimps is uncertain as most decapod crustaceans have only a single type of photoreceptor (Lindsay et al., 1999; Johnson et al., 2002), and Litopenaeus vannamei has a single visual pigment (Matsuda et al., 2011). Colour vision requires at least two types of spectral receptors (Kelber and Osorio, 2010). Stomatopods are currently the only crustaceans known to possess true, behaviourally demonstrated colour vision (Marshall et al., 1996; Theon et al., 2014). Studies have not been done on the colour vision or visual pigment of M. rosenbergii. The present study examined for the first time the colour preference and possible colour vision of the larvae of M. rosenbergii by means of an experiment that took advantage of their natural swimming behaviour, and particularly the attraction to, and congregation around, large suspended particles in a rearing tank. 2. Materials and methods 2.1. Larval rearing and handling Larvae of M. rosenbergii from wild broodstock were reared in the shrimp hatchery of the Borneo Marine Research Institute, Universiti Malaysia Sabah. Every day the zoeae were with fed newly hatched Artemia nauplii (b 0.5 mm long) in the afternoon and egg custard (egg yolk and albumen, milk powder, mixed vitamin, and cod-liver oil) in the morning. The wet egg custard was passed through a laboratory sieve (Endecotts, UK) with 500 μm mesh for early zoeae and 1 mm mesh for later zoeae. In the absence of food, M. rosenbergii zoeae actively swam backward, tail-oriented, with their dorsal side down. When live Artemia nauplii were added to the rearing tank, the zoeae showed no changes in swimming behaviour. But when egg custard was added to the rearing tank, the zoeae approached the custard particles by swimming forward, head first, with their dorsal side up. The zoeae held on to custard particles 1–5 mm in size. Batches of 10,000 larvae were transferred to a tank (diameter 93 cm, depth 53 cm, water volume 360 l) provided with mild aeration using diffusion stone and placed inside a roofed hatchery for the experiment on colour vision and colour preference. Larvae (12–18 days old, stages IV to VIII) were stocked in an indoor grey-walled conical round tank under natural illumination for a 6-day experiment that took advantage of their natural behaviour in the presence of custard particles. In this experimental tank, the water temperature, dissolved oxygen, pH and salinity were measured twice daily at 08:00 and 16:00 with a pH/ ORD/EC/DO tester (Hanna Instruments, HI 9828, Washington, USA). Water temperature ranged from 25.7–27.4 °C; dissolved oxygen, 6.0–8.2 mg l−1; pH 7.5–8.7; and salinity 8–12. Water quality in the experimental tank stayed within the normal ranges experienced by M. rosenbergii larvae (New, 2002). Animal care and handling was according to the guidelines for the care and use of laboratory animals set by the World Health Organization in Geneva, Switzerland, the Malaysian Animal Handling Code of Conduct, and the National Research Council (2011). 2.2. Colour vision and preference tests Plastic beads (4.1 mm in diameter, density N1.03 g ml−1) of five different colours were displayed to the zoeae in different combinations. Two sizes of beads (4.1 and 2.9 mm in diameter) were commercially available but in a preliminary test, the zoeae responded more strongly to the larger beads (χ2 test, P b 0.005). The reflectance spectra were shown to identify chromatic and achromatic properties of the beads (Fig. 1). The light reflectance of the beads in the wavelength range of 400–700 nm was recorded with a spectroradiometer (HSR-8100, Maki
Fig. 1. Light reflectance spectra for (A) the colour beads and (B) the grey beads used in the colour preference tests on Macrobrachium rosenbergii larvae. The reflectance of the white bead was higher than those of the colour beads at all wavelengths except at the peak of the red bead.
Manufacturing Co., Ltd., Hamamatsu, Japan) under the natural illumination beside the experimental tank placed inside the roofed hatchery. The peak wavelengths of the beads were at 421 and 436 nm for dark blue, 493 nm for light blue, 532 nm for light green, 493 and 532 nm for yellow, and 635 nm for red (Fig. 1A). To determine whether larvae chose a colour bead based on colour per se (chromaticity) or based on the difference in brightness of the beads, grey beads were presented together with different combinations of colour beads. If larvae choose a particular coloured bead among varying shade of grey, then they must be able to perceive the colour of the bead regardless of brightness. Three shades of grey, Grey30, Grey50, and Grey70 were produced by mixing 30%, 50%, and 70% black paint (Kenlux Synthetic Enamel 9103) with white paint (Kenlux Synthetic Enamel 9102). The painted beads were washed in running tap water for 2 days to elute the paint chemicals. The light reflectance spectra for the white, black, and grey beads are shown in Fig. 1B. The tests were done from 11:00 to 16:00 when the natural illuminance ranged from 1062 l× to 22,791 l× (light metre LT300, Extech Instruments, USA) at the water surface of the experimental tank. These light intensities were high enough to allow for colour vision if the larvae had the necessary visual mechanism. The natural light spectrum under direct sun through the hatchery roof is shown in Fig. 2.
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each combination of colour beads was replicated 20 times at 10–15 s intervals or 40 times when the number of larvae attracted to a bead was less than 6. In combinations of a colour bead with grey beads, display was replicated 10 times. The digital photographs taken during the trials were later examined and the number of larvae around each bead was counted, and then summed up for the 10, 20, or 40 trials of a given colour combination. The aggregated larvae oriented with heads toward the beads, at the same depth as the beads, and appeared clear and focused in photographs; those that were not attracted were deeper and farther away and showed poorly in the photographs. The aggregation around each bead did not overlap with those around the adjacent beads, and accurate counting was possible. 2.3. Statistical analysis Fig. 2. Natural light spectrum measured inside the roofed hatchery where the experimental tank was placed. Note that the UVA (b390 nm) irradiance is lower than the measurable level due to the UVA-opacity of the roofing material, polycarbonate.
The plastic beads were tethered by transparent monofilament (0.25 mm thick, 20 cm long) to a transparent plastic ruler at intervals of 6–9 cm depending on the number of beads in the combination to be tested. The display positions along the tank wall and the arrangement of the beads in each combination were interchanged at random. Aeration in the tank was stopped during each test. The larvae distributed at or close to the tank wall and within 25 cm, mostly within a 15 cm depth from the surface (none was at the bottom) during the experiment. Each test consisted of lowering the combination of beads to a depth of 5 cm at a distance of 12–15 cm from the tank wall and allowing the larvae to gather around the beads of choice. The monofilaments were straight and the tethered beads were well separated in the water. The larvae attracted to the beads were visually counted by two observers during each trial. As precise counting of more than 10 larvae was difficult, the larvae were photographed with a digital camera (no flash) within about 5–8 s after the beads were submerged and the number of larvae around the beads had stabilized (Fig. 3). A single photograph was taken for each bead arrangement, care being taken to avoid reflections at the water surface and obtain a clear image. Display of
Fig. 3. Illustration of experimental tank with beads and camera setting.
The number of larvae was counted for each bead in each display and the counts were summed for each combination (rows, Table 1). The data was analysed by chi-square χ2 test to assess the goodness of fit between a set of observed values and those expected theoretically, i.e., on the hypothesis that the probability of choice for each colour is 1/k, where k is the number of beads in a combination. The significance level was set at α = 0.05. Across the different test combinations, the variation in the numbers of zoeae attracted to each colour bead was computed as the mean ± standard deviation (columns, Table 1). 3. Results 3.1. Response of larvae to colour beads M. rosenbergii larvae stages IV to VIII responded to any single suspended plastic bead, of any colour, by swimming head first straight toward it in the same typical swimming posture as toward particles of egg custard. Most larvae approached from above, some horizontally, and a few from below. Larvae gathered around a bead for some time (2–10 s) before dispersing. When combinations of several colour beads were displayed, the larvae swam straight to the beads of specific colours from the tank walls and all directions from distances as far as 20 cm. Larvae gathered around the beads orienting with the head; some of them touched a bead several times and then hopped away. As larvae gathered around a bead, the latter became less visible from a distance and attracted no more larvae. After about 10 s the gathered larvae gradually dispersed. The larvae exhibited the same response during each of the 10–40 trials at 10–15 s intervals for each colour combination. Table 1 summarizes the number of larvae gathered around the coloured and grey beads displayed in different combinations. Larvae aggregated around the dark blue, light blue, and white beads, indicating a biased attraction to blue and white. In the combinations of dark blue, light blue, and white beads (tests 5, 6, and 7), the largest aggregation formed around the light blue bead (totalling 676 larvae to light blue, 534 larvae to dark blue and 601 larvae to white), but the numbers varied with the arrangement of the three beads (χ2 = 16.72, P b 0.005). The reflectance spectra show that the light blue bead was much brighter than the dark blue bead (Fig. 1A), yet there was no significant difference in the aggregation of larvae around dark blue and light blue beads (test 3). The fewest larvae gathered around the black bead (tests 4 and 15), and some more responded to light green (tests 1 and 2) and red (tests 4 and 13). Aggregations were moderate for yellow beads in combination with other colours (tests 1 and 2), but not significantly different from those around the three shades of grey (test 12). The preference for the different colours is also evident in the mean numbers of larvae that gathered around the beads (bottom row in Table 1). Colour preference did not vary with age from 12 days to 18 days during the 7-day experiment, nor with the 20 × variation in
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Table 1 Number of Macrobrachium rosenbergii larvae attracted to colour and grey beads displayed in different combinations. Colour of test beads Test
Combination [no. of trials]
1 F, D A, C and B [n = 40] 2 D, A, C and B [n = 40] 3 A and B [n = 20] 4 F, E and G [n = 20] 5 A, B and F [n = 20] 6 B, A and F [n = 20] 7 A, F and B [n = 20] 8 H, I and J [n = 20] 9 A, H, I and J [n = 10] 10 B, H, I and J [n = 10] 11 C, H, I and J [n = 10] 12 D, H, I and J [n = 10] 13 E, H, I and J [n = 10] 14 F, H, I and J [n = 10] 15 G, H, I and J [n = 10] Mean larvae per trial ± SD†
A Dark blue
B Light blue
C D Light green Yellow
504 569 163
565 624 168
8 8
179 210 145
258 216 202
E Red
25 38
150 45
11.6 ± 3.1 12.8 ± 2.5 0.7 ± 1.1
G Black
H Grey30
I Grey50
J Grey70
302 182 206 213
1
106 31 23 57 50 40 10 46 220 39 7 34 1.3 ± 1.3 0.8 ± 0.8 12.2 ± 4.2 0.3 ± 0.5 4.2 ± 1.1
χ2 for grey beads
χ2 951.516⁎ 1068.167⁎ 0.076 534.573⁎ 19.428⁎
457
15
201
F White
0.241 14.275⁎ 99 30 22 53 44 22 24 22 3.3 ± 1.1
126 38 32 48 48 55 37 23 4.5 ± 1.2
3.560 282.747⁎ 1.152 ⁎ 192.901 2.363 1.670 0.772 1.297 0.727 39.180⁎ 14.195⁎ 328.325⁎ 3.980 17.128⁎ 3.368
⁎ Significant at α = 0.05. † Standard deviation.
natural illuminance, nor with the ambient pH 7.5–8.7 or other water quality variables. 3.2. Response of larvae to the brightness of beads Table 1 also shows that in the combinations of a coloured bead with grey beads, the aggregations of larvae were significantly higher around the dark blue, light blue, and white beads (tests 9, 10, 14). The number of larvae attracted to light green and yellow beads was not significantly different from those around the grey beads (tests 11 and 12; 0.75 N P N 0.50). Significantly fewer larvae came to the red bead and the black bead than to the three grey beads (tests 13 and 15). In the combination with three grey beads, the number of larvae attracted was not significantly different (test 8; 0.25 N P N 0.05). The three grey beads attracted similar number of larvae (P N 0.25) but only in the combination of the red and the three grey beads (test 13), G50 attracted significantly fewer larvae than G30 and G70 (P b 0.005). 4. Discussion In the present study, the existence of colour vision in M. rosenbergii larvae is argued based on colour preference behaviour. Most knowledge of the visual system necessarily comes from physiological and opsin transcript studies, but only behavioural (psychophysical) studies can tell what the animal's visual system is truly capable of achieving (Douglas and Hawryshyn, 1990). The visual response of M. rosenbergii larvae to the plastic beads was qualitatively different from their thigmotactic response to the tank walls. The visual attraction was strongest for the light blue, white and dark blue beads, and weakest for the black, light green, and red beads. Choice of the coloured beads was not based on brightness—light blue bead and dark blue bead were equally preferred, whereas the brighter yellow and light green beads were not preferred over the darker grey beads. If larvae had not discriminated between colours, they would have been unable to distinguish the blue beads from the three shades of grey beads. In fact, the larvae discriminated blue beads from grey beads based mainly on colour (chromaticity) and thus have true colour vision. The strong response to blue, moderate response to yellow, and poor response to red are consistent with the fact that the main rhabdome in decapod shrimps contains a visual pigment with λmax between 460 nm and 550 nm (Wald and Seldin, 1968; Cronin and Hariyama, 2002). The M. rosenbergii larvae may be like grass shrimp Palaemonetes pugio larvae in which the visual system is already functional at hatching (Douglass
and Forward, 1989), ready for the ecological tasks during the pelagic phase and then modified at metamorphosis to meet the new visual demands of the demersal phase. Metamorphic change in visual pigment does not occur in decapod crustaceans (Cronin et al., 1995), so colour vision will persist throughout the ontogeny of M. rosenbergii. The distinct preference of M. rosenbergii larvae for blue and white beads could not be explained by associative learning, but rather seems to be an innate ability. The larvae in this study had been feeding on orange Artemia nauplii and yellow egg custard. If colour preference was learned, then the larvae should have preferred orange and/or yellow. Instead the larvae showed moderate response to the yellow bead and poor response to the red bead. The larvae in this study retained the innate colour preference for blue even after feeding on the yellow egg custard. One adaptive significance of innate colour preference is that it helps naïve animals to find food (Giurfa et al., 1995; Weiss, 1997). Innate colour preferences have been reported also among fishes (Kawamura et al., 2010) and birds (Honkavaara et al., 2004; Schmidt and Schaefer, 2004; Bascuñán et al., 2009). The M. rosenbergii larvae were observed immediately headed straight toward the custard particles and the tasteless and odourless plastic beads from distances of 10–20 cm; this was clearly a visual response. But the larvae IV to VIII probably already have multiple sensory modalities. Moller (1978) observed the feeding behaviour of M. rosenbergii larvae and postlarvae in Petri dishes under a stereomicroscope and concluded that the larvae detect and discriminate food from non-food particles by chemosensory mechanisms rather than vision, just like juvenile banana shrimp Penaeus merguiensis De Man (Hindley, 1975). The ingestion rate of Artemia nauplii by M. rosenbergii larvae is a function of nauplii density (Barros and Valenti, 2003). You et al. (2006) found that food intake of juvenile white leg prawn L. vannamei is significantly lower in an illuminated tank than in a dark tank, and concluded that light is not necessary for the shrimp to locate food. The feeding modality usually changes with growth and metamorphosis (Kawamura and Hara, 1980). Juvenile and adult M. rosenbergii are active at night and probably rely on chemoreception and mechanoreception to detect food (Nakamura, 1975; Scudder et al., 1981). The larvae of M. rosenbergii live as part of the zooplankton community in spectrally poor turbid environments such as estuaries and coastal lagoons (New and Singholka, 1985; Nandlal and Pickering, 2005). The planktonic larvae must feed, avoid predators, and travel to a location where they can successfully metamorphose. Their diets consist mainly of minute crustaceans and worms (New, 2002; Nandlal and Pickering, 2005). Zoeae must eat continuously to survive and vision is very important in daytime feeding (New, 2002). Turbidity can affect prey detection
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by increasing or diminishing the contrast between prey and background due to the scattering of light. The positive effect of turbidity is pronounced for planktivores and fish larvae (Utne-Palm, 2002). Contrast is detected in the form of brightness or colour. Colour vision would help larvae detect food organisms in turbid habitats and allow them to choose food items by colour. M. rosenbergii probably evolved colour vision at larval stages to enhance contrast and, thus, improve the visibility of objects against the background. The reaction distance of M. rosenbergii larvae to the beads was about 10–20 cm, much greater than prey detection distances (via the vibration sense) of other zooplankton: less than 1 mm in copepods Oithona plumifera and O. similis; and less than 3 mm in chaetognaths (arrow worms) (Svensen and Kiørboe, 2000; Jian and Paffenhöfer, 2008; Horridge and Boulton, 1967; Feigenbaum and Reeve, 1977). The significance of the strong wavelength-specific response to blue beads in M. rosenbergii larvae is unknown. The bias for blue and white may be adaptive for visual function under dim light, as has been shown for the foraging nocturnal hawk moth Manduca sexta (Goyret et al., 2008). Floral surveys confirmed that the flowers visited by M. sexta were white but never blue, thus the innate preference for blue is not adaptive in M. sexta and remains a mystery (Goyret et al., 2008). The diurnal hawk moth M. stellatarum also has a bias for blue (Kelber, 1997). 5. Conclusions Behavioural experiments indicate that hatchery-reared M. rosenbergii larvae (10–18 days old, stages IV–VIII) aggregate preferentially on blue and white objects (including artificial food); this colour preference is based on chromaticity rather than brightness, and strongly suggest that the larvae are capable of colour vision. Competing interest The authors declare no competing or financial interests. Author contribution G.K. designed, conducted the experiment and wrote the manuscript, T.B and Y.A. contributed to revision of the manuscript, I.M.X and L.L.S conducted the experiment. References Barros, H.P., Valenti, W.C., 2003. Ingestion rates of Artemia nauplii for different larval stages of Macrobrachium rosenbergii. Aquaculture 217, 223–233. Bascuñán, A.L., Tourville, E.A., Toomey, M.B., McGraw, K.J., 2009. Food color preferences of molting house finches (Carpodacus mexicanus) in relation to sex and plumage coloration. Ethology 115, 1066–1073. Cronin, T.W., Hariyama, T., 2002. Spectral sensitivity in crustacean eyes. In: Wiese, K. (Ed.), The Crustacean Nervous System. Springer, Heidelberg, pp. 499–511. Cronin, T.W., Marshall, N.J., Caldwell, R.L., Pales, D., 1995. Compound eyes and ocular pigments of crustacean larvae (Stomatopoda and Decapoda, Brachyura). Mar. Freshw. Behav. Physiol. 26, 219–231. D'Abramo, L.R., Tidwell, J.H., Fondren, M., Ohs, C.L., 2006. Pond production of the freshwater prawn in temperate climates. Southern Regional Aquaculture Center Publication No. 484 (8 pp.). Douglas, R.H., Hawryshyn, C.W., 1990. Behavioral studies of fish vision; an analysis of visual capabilities. In: Douglas, R.H., Djamgpoz, M.B.A. (Eds.), The visual system of fish. Chapman and Hall, London, pp. 373–418. Douglass, L., Forward Jr., R.B., 1989. The ontogeny of facultative superposition optics in a shrimp eye: hatching through metamorphosis. Cell Tissue Res. 258, 289–300. El-Sayed, A.F.M., El-Ghobashy, A.E., 2011. Effects of tank colour and feed colour on growth and feed utilization of thin lip mullet (Liza ramada) larvae. Aquac. Res. 42, 1163–1169. Emmerson, W.D., Hayes, D.P., Ngonyame, M., 1983. Growth and maturation of Penaeus indicus under blue and green light. S. Afr. J. Zool. 18, 71–75. FAO, 2014. Cultured Aquatic Species Information Programme—Macrobrachium rosenbergii (De Man, 1879) Retrieved from http://www.fao.org/fishery/culturedspecies/ Macrobrachium_rosenbergii/en#tcNA002B.
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