Dietary effects on carotenoid compositio

JOURNAL OF PLANKTON RESEARCH j VOLUME 29 j SUPPLEMENT j PAGES i73 – i83 j 2007 Dietary effects on carotenoid composition in the marine harpacticoid copepod Nitokra lacustris ADELAIDE C E RHODES* NORTHWEST FISHERIES SCIENCE CENTER, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, WASHINGTON 98112, 2725 MONTLAKE BLVD E, SEATTLE, USA *CORRESPONDING AUTHOR: adelaide.rhodes@noaa.gov Received April 13, 2006; accepted in principle September 12, 2006; accepted for publication October 27, 2006; published online December 5, 2006 Nitokra lacustris, a euryhaline harpacticoid copepod, was reared on two diets, which varied in composition and relative quantities of carotenoid pigments, and analyzed for differences in tissue carotenoid composition Carotenoid compositions were analyzed by means of reverse-phase high performance liquid chromatography (RP-HPLC) Total amounts of unesterified astaxanthin were quantified in the two populations by means of an isocratic HPLC procedure Carotenoid composition and total astaxanthin content for adult copepods differed between the two diets Copepod populations fed the live microalga Tetraselmis suesica averaged 696 mg carotenoids (g dry weight)21 and 185 mg astaxanthin (g dry weight)21 while copepod populations fed a formulated diet with high levels of lycopene, beta-carotene and alpha-carotene averaged 24.3 mg carotenoids (g dry weight)21 and 650 mg astaxanthin (g dry weight)21 Neither diet contained canthaxanthin or astaxanthin, suggesting that the copepods were synthesizing the astaxanthin from precursor pigments The absence of canthaxanthin and echinenone coupled with the presence of other intermediary carotenoid pigments suggests that the carotenoid conversion pathway of N lacustris differs from that described for calanoid copepods and other marine crustaceans N lacustris may utilize the b-carotene ! zeaxanthin ! b-doradexanthin (adonixanthin) ! astaxanthin pathway of conversion rather than the b-carotene ! echninenone ! canthaxanthin ! astaxanthin pathway proposed for other copepods Thus, diets containing higher amounts of b-carotene and zeaxanthin would be more likely to produce high levels of astaxanthin in this species I N T RO D U C T I O N Animals which are not able to biosynthesize the ketocarotenoids (e.g canthaxanthin and astaxanthin) from other carotenoids must acquire them directly from their diets Flamingoes are an example of this phenomenon, because they will lose their pink coloration if they become deficient in the ketocarotenoids obtained from the small crustaceans they consume (Goodwin, 1984) Marine crustaceans, like other animals, must convert dietary sources of carotenoids, usually b-carotene, into the ketocarotenoids astaxanthin and canthaxanthin (Paanakker and Hallegraef, 1978) The ability of marine crustaceans to perform this bioconversion is essential to the marine food web, as many fish species cannot convert dietary carotenoids to ketocarotenoids and must thus accumulate astaxanthin directly from their diets (Katayama et al., 1972; Torrissen and Christiansen, 1995) Nitokra lacustris has a striking reddish-orange coloration which can be observed easily through a microscope, suggesting that it contains the ketocarotenoids astaxanthin and/or canthaxanthin that are found in other crustaceans and copepods This study considered the differences in the overall pigment composition and quantity of astaxanthin present in the harpacticoid copepod N lacustris fed a natural algal diet versus a formulated diet composed of flax seed oil, yeast, vitamin C, vitamin B complex and vegetable juice doi:10.1093/plankt/fbl068, available online at www.plankt.oxfordjournals.org # The Author 2006 Published by Oxford University Press All rights reserved For permissions, please email: journals.permissions@oxfordjournals.org Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 Communicating editor: K.J Flynn JOURNAL OF PLANKTON RESEARCH j VOLUME 29 Neither diet contained astaxanthin nor canthaxanthin, but both contained a variety of precursor carotenoid pigments that could be converted to astaxanthin by crustaceans This study utilized reverse-phase high performance liquid chromatography (RP-HPLC) to conduct the qualitative analyses and an isocratic HPLC procedure to conduct the quantitative analyses of the pigments contained in N lacustris copepods fed two different diets j SUPPLEMENT j PAGES i73 – i83 j 2007 russelli (Bandaranayake and Gentien, 1982) and the calanoid copepods Acartia bifilosa (Lotocka and Styczynska-Jurewicz, 2001) and Pseudocalanus acuspes Giesbrecht (Lotocka et al., 2004) Canthaxanthin and the intermediate 40 -hydroxyechinenone have been reported in the cyclopoid copepod Cyclops kolensis Lilljeborg (Czeczuga et al., 2000) However, the intermediates canthaxanthin and echinenone have not been observed in the harpacticoid copepods Tigriopus brevicornis (Davenport et al., 2004) or Canuella perplexa (Buffan-Dubau et al., 1996) The analysis of pigments undertaken in this study allows for further examination of the pigment composition and potential pathways utilized by N lacustris for biosynthesis of astaxanthin from dietary precursors Role of carotenoid pigments in harpacticoid copepods METHOD Copepod production N lacustris (originally collected from a dockside plankton tow at the Gulf Coast Marine Lab in Panacea, FL, USA) has been reared in the laboratory for several years on two diets: (i) Tetraselmis suecica or (ii) a formulated feed containing the carotenoid pigments lycopene, a-carotene, b-carotene, lutein, phytoene and phytofluene The copepod cultures are non-axenic, but are periodically treated with a sodium hypochlorite solution (6.7 mM NaOCl) to remove protozoans and bacteria The formulated diet has been successfully used to rear N lacustris in captivity for extended periods, and no significant differences in population growth rates, size of animals, or fatty acid composition have been documented between the populations fed the two different diets (Rhodes, 2003; Rhodes and Boyd, 2005) The prasinophyte Tetraselmis suecica contains the pigments trans-neoxanthin, violaxanthin, lutein, chlorophyll b, chlorophyll a and a-carotene as identified by Egeland et al (1995) and Bustillos-Guzman et al (2002) The carotenoid content of the formulated feed was assumed to be composed mainly of the pigments found in a tomato-based vegetable juice (V-8 juice, Campbell’s Soup, Ohio) The juice blend of tomatoes, beets, celery, carrots, lettuce, parsley, watercress and spinach results in a formulated feed which contains lycopene (87.8% w/w), a-carotene (2.7%), b-carotene (7.6%), lutein (1.5%) and zeaxanthin (0.4%) (Tonucci et al., 1995; Arab et al., 2002) Copepods were reared in six 10 L trays under identical environmental conditions (salinity 27, 208C, 12-h light:12-h dark (General Electric Cool White No F15T8-CW) Three trays were fed the algae and three i74 Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 Carotenoids are isoprenoid tetraterpenes containing 40 carbon atoms that are synthesized by plants, algae and certain type of bacteria and fungi (Britton, 1995) Copepods and other marine crustaceans may utilize astaxanthin and other carotenoid pigments as protectants against photooxidation and as vitamin A precursors Astaxanthin, the most commonly detected carotenoid in crustacean organs, has been associated with reproductive and visual physiology as well as the stabilization of membranes and proteins (Goodwin, 1984; Linan-Cabello et al., 2002) For marine planktonic copepods that can escape the photic zone, astaxanthin may act as an antioxidant for the protection of unsaturated storage lipids (Juhl et al., 1996; Lotocka et al., 2004) It has been hypothesized that the rapid oxidation of carotenoids decreases the availability of free radicals to react with unsaturated fatty acid molecules and consequently prevent damage to membranes (Woodall et al., 1997; Lotocka et al., 2004) Astaxanthin cannot be synthesized de novo in copepods (Matsuno, 1989; Andersson et al., 2003) Several pathways of astaxanthin synthesis from precursor carotenoids have been suggested for crustaceans (Bandaranayake and Gentien, 1982; Goodwin, 1984; Berticat et al., 2000; Linan-Cabello, 2002) and fish (Katayama et al., 1970; Hata and Hata, 1972; Hsu et al., 1972) Katayama et al (1973) classified aquatic animals into three categories based upon astaxanthin biosynthetic capability: (i) fish that cannot biosynthesize astaxanthin from b-carotene, lutein or zeaxanthin, but which can transfer dietary astaxanthin to body astaxanthin, (ii) fish, such as red carp and goldfish, that can bioconvert astaxanthin from lutein or zeaxanthin, but not from b-carotene, and (iii) crustaceans (e.g prawns) that can bioconvert b-carotene into astaxanthin The metabolic pathway for the conversion of b-carotene to astaxanthin that has been proposed for most crustaceans relies on the intermediates echinenone and canthaxanthin (Goodwin, 1984) Canthaxanthin has been identified in the tropical reef copepod Euchaeta A C E RHODES j DIETARY EFFECTS ON COPEPOD CAROTENOIDS Astaxanthin quantification by isocratic HPLC method The difference in the quantity of astaxanthin between copepod populations fed the two diets was measured using an isocratic HPLC procedure on each of the six populations Duplicate samples of $ 5000 copepods from each of the six populations were pooled, filtered and lyophilized at 2708C for 36 h (Model No 6201 – 3218; The Virtis Company, Inc., Gardiner, NY) Lyophilized samples were extracted in 10 mL of 100% HPLC-grade acetone in a glass tissue homogenizer tube kept on ice, flushed with nitrogen and set aside for 0.5 h in the dark at 2208C Samples were centrifuged for at 200 g (Marathon Micro A; Fisherbrand) All supernatant was collected and the pellet was extracted two more times Collected supernatant was evaporated under a nitrogen flush in the dark, weighed and reconstituted in the injection solvent [60% tetrahydrofuran (THF), 40% methanol] Samples were injected with a Waters U6K loop injector (Waters, Massachusetts) into a 3.5 mm Waters Symmetry C18 (4.6  150 mm) column with a flow rate of 1.4 mL/min controlled by a Waters Model 510 pump The isocratic mobile phase was made up of acetonitrile-methanol-chloroform (100:20:5, v/v/v) and the ultraviolet visible detector (Spectroflow 757; Kratos Instruments, New York) was set at 474 nm Astaxanthin peaks were identified by comparison to an astaxanthin standard (Sigma A9335) The standard was prepared under yellow fluorescent lighting and injected on the same day as the samples All samples were run on the same day Astaxanthin was quantified using an external standard curve plotting astaxanthin peak area versus mg mL21 of astaxanthin injected into the HPLC Peak areas for each the cis- and trans-astaxanthin isomers were compared to a standard curve developed using an Carotenoid separation by RP-HPLC To analyze the complete carotenoid composition of the copepods with RP-HPLC, a representative sample of approximately five thousand N lacustris adults each from a culture fed live Tetraselmis and a culture fed the formulated feed were collected and filtered in the same manner as described previously Pigments were collected from freshly harvested samples by placing the filters containing the animals in mL THF and sonicating for After centrifugation at 200 g for at 48C, the supernatant was drawn off One mL of supernatant was evaporated under nitrogen flush in the dark and reconstituted in a 200 mL mixture of 1:4 ethyl acetate:ethanol for RP-HPLC injection Peaks identities from the RP-HPLC analysis were analyzed with the assistance of Craft Technologies (Wilson, NC, USA) In brief, this technique involved RP-HPLC on a mm C30 Column (YMC carotenoid column 25 cm  4.6 mm, mm diameter; YMC, Wilmington, NC, USA) using HPLC-grade solvents and a linear tertiary gradient system (100% isopropyl alcohol:1 N Methanol with 0.15 mM ammonium acetate:100 % THF) at a flow rate of mL/min Pigments were quantified at 450 nm and spectrally characterized on a Waters 991 M photodiode array detector system (Waters; Milford, MA) which recorded spectra at nm intervals (Millenium Software) at a rate of 300 spectra per minute Data were downloaded and analyzed using Microsoft Excel 97 software (Microsoft; Redmond, WA) Peaks were identified at Craft Technologies by comparison to the spectra and retention times under the same conditions of a standard mix containing lutein, i75 Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 astaxanthin standard prepared from crystallized astaxanthin (Sigma A 9335) The standard curve was prepared by serial dilution of the stock solution [500 mg astaxanthin (mL solvent)21] to final concentrations of 100, 50, 25, 10 and mg astaxanthin (mL solvent)21 All sample concentrations fell within this standard curve (R ¼ 99%) Areas under the peaks that correlated to cis-astaxanthin and trans-astaxanthin were compared to the standard curve to determine the concentration of astaxanthin in mg (mL solvent)21 Results were converted into mg astaxanthin (g dry weight)21 in order to facilitate comparison Astaxanthin values from the duplicate samples were averaged for each of the three replicates in each treatment Concentrations of cis-astaxanthin, trans-astaxanthin and total astaxanthin were compared within and between the three populations assigned to each treatment by analysis of variance (ANOVA) were fed the formulated feed ad libitum The algae continued to grow throughout the experiment, and the formulated feed remained suspended with the assistance of aeration The cultures were reared for 10 days and terminated by harvesting all adult copepods through a 105 mm mesh and starving for 24 h in fresh seawater to allow them to void enteric contents Duplicate samples for each replicate were rinsed with sterile deionized water to remove excess salt before filtration onto a glass fiber filter (Fisherbrand GF/C, Fisher Scientific, Pittsburgh, PA) As the copepods were killed by the filtration process, all procedures from this point forward were conducted under yellow fluorescent light (Philips F40GO) to prevent pigment destruction JOURNAL OF PLANKTON RESEARCH j VOLUME 29 SUPPLEMENT j PAGES i73 – i83 j 2007 reflected their diets (Table I) Both populations of copepods contained unesterified astaxanthin, antheraxanthin, b-carotene, a ketocarotenoid peak characteristic of b-doradexanthin and chlorophylls a and b Copepods fed the live alga contained the unique carotenoid pigments violaxanthin, lutein, zeaxanthin, and a-carotene (Fig 1) The copepods fed the live microalgae also exhibited significant amounts of three unknown carotenoids ( peaks 1, and 7) as well as astaxanthin esters (four peaks labeled 13) Only copepods fed the formulated diet contained the pigments 1,1-carotene, lycopene and cis-lycopene as well as two unique unidentified carotenoids ( peaks and 6) (Fig 2) A single peak was identified as unesterified astaxanthin in both sets of animals based on its retention time and absorption spectra (Figs and 2) Two distinct spectra corresponding to cis- and trans-astaxanthin coeluted in this single peak during the RP-HPLC method ( peaks and 9) The isocratic HPLC method was able to separate these two peaks and it was possible to determine their relative concentrations Other peaks were found with similar absorption spectra, which correspond to astaxanthin esters and isomers (Yuan and Chen, 1998) Canthaxanthin or echinenone were not detected, based on retention time, absorption spectra, and comparison to the controls Based on retention time and the absorption spectra, peak was putatively identified as the intermediate compound b-doradexanthin (adonixanthin) found when zeaxanthin is converted to astaxanthin in the crayfish Astacus leptodactylus (Berticat et al., 2000) (Fig 3) R E S U LT S Qualitative carotenoid analysis by RP-HPLC The separation peaks at 470 nm for copepods fed the live alga Tetraselmis (Fig 1) differed from the separation peaks at 470 nm for copepods fed the formulated feed (Fig 2) Both copepod populations contained astaxanthin as well as several carotenoid and chlorophyll pigments (Table I, Figs and 2) Copepod populations fed the two diets had pigment compositions that Fig Retention time and absorbance at 470 nm for copepods fed the live alga T suecica All peak identifications are provided in Table I Peaks identified as carotenoids are numbered Peaks that represent the same compounds are given the same number on all figures Unlabeled peaks are chlorophyll-related peaks i76 Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 lycopene, b-cryptoxanthin, a-carotene and b-carotene, b-carotene isomers (Hoffman-LaRoche Co and Sigma Chemical Co.), as well as a control mixture prepared from homogenized sea cucumber The sea cucumber control had identifiable peaks for b-carotene, canthaxanthin, astaxanthin, zeaxanthin, phoenicoxanthin (adonirubin) and echinenone Peak identifications were confirmed by comparison to published absorption spectra from various sources (Nelis and De Leenheer, 1988; Canjura, 1990; Britton, 1995; Matsuno and Tsushima, 1995; Yuan and Chen, 1997; Hyvarinen and Hynninen, 1999; Berticat et al., 2000; Ston and Kosakowska, 2002) Relative amounts of carotenoids were quantified by dividing peak area at 450 nm by the relative response factors for each type of carotenoid (Britton, 1995) The response factors were provided by Craft Technology for their system and controls at 450 nm j A C E RHODES j DIETARY EFFECTS ON COPEPOD CAROTENOIDS Quantification of astaxanthin in two copepod populations During preparation of the formulated feed, 236 mL of V-8 juice, which contains 17 mg lycopene per serving (Arab et al., 2002; Campbell’s Soup, Ohio) were diluted into 1500 mL total volume The copepods were fed one mL (L culture media)21 of this dilute solution once, corresponding to 11 mg lycopene (L culture media)21 available to the copepods About 2000 adult copepods were harvested from each liter of media; hence, about 5.5 ng lycopene was available to each copepod over the entire 10 days Based on the relative amount of lycopene to astaxanthin determined from the RP-HPLC analysis (Table I), the copepods contain about 2.7 ng lycopene individual21 after 10 days in cultures Lycopene was not found in the copepods fed the live algal diet ANOVA analyses of the isocratic HPLC results indicated that the tanks fed the formulated feed had significantly higher amounts of trans- and cis-astaxanthin, as well as a higher proportion of transastaxanthin when compared to total astaxanthin content; 47% (+4% S.D., n ¼ 3) versus 26% (+2% S.D., n ¼ 3), p , 0.05 (Fig 4) The dry weights and amount of lipid per adult copepod did not differ significantly between treatments and had an average value of 5.5 mg dry weight individual21 (+0.7 S.D., n ¼ 6) and 0.56 mg lipid individual21 (+0.2 S.D., n ¼ 6) The total amount of astaxanthin in copepods fed live microalgae Table I: Carotenoid composition of two populations of copepods fed different diets Peak number Retention time (min) Identification absorption maxima Copepods fed Tetraselmis (% of identified carotenoids) Copepods fed formula (% of identified carotenoids) and 10 11 12 13 14 15 16 17 18 19 20 4.49 5.19 5.5 5.91, 10.88 5.91 7.09 8.43 8.8, 8.92 9.62 10.11 12.62 17.51, 21.92, 31.47, 36.95 23.63 26.39 29.12 29.88 38.82 40.15 41.88 Unknown ketocarotenoid 450 (470) b-doradexanthin (adonixanthin) 450 475 Violaxanthin 430 455 485 Lutein-like 425 450 475 cis-ketocarotenoid 330 440 465 Unknown ketocarotenoid 445 465 Unknown carotenoid 450 475 cis- and trans-Astaxanthin (coeluted) 475 Antheraxanthin 415 445 475 Lutein 420 445 475 Zeaxanthin 420 450 470 Astaxanthin ester 475 1,1-carotene 415 440 470 b,1-carotene (a carotene) 445 475 b,b-carotene (b carotene) 430 455 480 9-cis b-carotene 425 445 475 cis-Lycopene (365) 445 470 500 Astacene 485 Lycopene 450 475 505 3.02 14.42 1.11 5.45 — — 1.82 26.58 1.26 8.36 0.70 15.68 — 5.12 2.42 2.25 — 11.81 — — 67.44 — — 10.30 4.32 — 2.68 6.35 — — — 0.27 — 4.17 1.12 1.09 0.24 2.02 i77 Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 Fig Retention time and absorbance at 470 nm for copepods fed the formulated feed All peak identifications are provided in Table I Peaks identified as carotenoids are numbered Peaks that represent the same compounds are given the same number on all figures Unlabeled peaks are chlorophyll-related peaks Inset provided to scale peak JOURNAL OF PLANKTON RESEARCH j VOLUME 29 j SUPPLEMENT j PAGES i73 – i83 j 2007 T suecica was 1.0 ng astaxanthin individual21 (+0.2 S.D., n ¼ 3) or 185 mg astaxanthin (g dry weight)21 (+32 S.D., n ¼ 3) According to the results of the one-way ANOVA, the copepods fed the formulated feed had a statistically significantly higher astaxanthin content, $3.6 ng astaxanthin individual21 (+1.7 S.D., n ¼ 3) or 650 mg astaxanthin (g dry weight)21 (+303 S.D., n ¼ 3) (Fig 4) Buffan-Dubau et al., 1996; Juhl et al., 1996; Czeczuga et al., 2000; Andersson et al., 2003; Davenport et al., 2004) As described in previous studies, the carotenoid composition varied with diet (Buffan-Dubau et al., 1996; Juhl et al., 1996; Kleppel, 1998; McLeroyEtheridge and McManus, 1999; Andersson et al., 2003; Davenport et al., 2004; Van Nieuwerburgh et al., 2005) The amounts of cis-, trans- and total astaxanthin were significantly greater in the copepods fed the formulated feed The amount of trans-astaxanthin per total astaxanthin was also significantly higher for the copepods fed the formulated feed N lacustris astaxanthin values for all populations in the experiment ranged from 0.8 to 5.9 ng astaxanthin individual21 and 146 to 1066 mg astaxanthin (g dry DISCUSSION N lacustris contained many of the same pigments as other copepods (Hairston, 1976; Hairston, 1979; Bandaranayake and Gentien, 1982; Goodwin, 1984; Fig Trans-, cis- and total astaxanthin mg (g dry weight)21 for N lacustris fed two different diets, live Tetraselmis (clear bars) and a formulated feed (shaded bar) Values represent an average of three replicates in each treatment, each replicate was tested in duplicate Significantly, different means (one-way ANOVA) are indicated by separate letters for each measurement Bars denote S.D i78 Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 Fig Absorption spectra of putative b-doradexanthin (solid line, peak in Figs and 2) and trans-astaxanthin (broken line, peak in Figs and 2) A C E RHODES j DIETARY EFFECTS ON COPEPOD CAROTENOIDS phaeophytins in Calanus pacificus that were reported to be derived from the digestive breakdown of chlorophyll from algae in the diet McLeroy-Etheridge and McManus (1999) also reported the rapid breakdown of chlorophyll into phaeopigments by copepods when food was limiting This phenomenon has been found in harpacticoid copepods as well, Canuella perplexa which subsists on diatoms, cyanobacteria and/or green microalgae converts ingested chlorophyll a into phaeophytin and phaeophorbide-like compounds (Buffan-Dubau et al., 1996) The formulated feed had an effect on the copepod carotenoid composition, most notably in the amount of lycopene The formulated feed contains mostly lycopene (87% w/w), which equates to an availability of $5.5 ng lycopene per individual copepod for the duration of the experiment Copepods fed the live algae did not contain lycopene; whereas the copepods fed the formulated feed contain about 2.7 ng lycopene individual21, suggesting that the copepods were most likely bioaccumulating the lycopene It is not possible to determine from this experiment alone how much the carotenoid content of the copepods depends on bioconversion versus bioaccumulation However, due the absence of any astaxanthin in the diets, it is possible to state that N lacustris bioconverts astaxanthin from precursors in the diet Role of ketocarotenoids in copepod metabolism Some evidence for a conversion pathway involving the putatively identified b-doradexanthin (adonixanthin) is found in the extremely large peak ( peak 2) found preceding free astaxanthin (Figs and 2) These peaks have absorption spectra very close to spectra observed in the freshwater crayfish for b-doradexanthin (Katayama et al., 1970; Berticat et al., 2000) (Fig 3) b-doradexanthin has also been found to be more polar than astaxanthin in other HPLC analyses (Linan-Cabello et al., 2002), which matches the observation from this study that the putative b-doradexanthin elutes before astaxanthin Furthermore, no absorption spectra found in the survey corresponded with the intermediates canthaxanthin and echinenone, suggesting that the copepods may be using an alternative bioconversion pathway It has been suggested that it is the structure of astaxanthin and canthaxanthin which makes them more effective as antioxidants (Tera˜o, 1989); hence, other carotenoids with similar structures should share some of the same properties It is usually a minor carotenoid in relation to astaxanthin and canthaxanthin (Linan-Cabello et al., 2002) If peak is indeed Transfer of pigments from food to copepods All Tetraselmis sp pigments identified by Bustillos-Guzman et al (2002) and Egeland et al (1995) were found in the copepods fed Tetraselmis in this experiment except trans-neoxanthin Many of the same chlorophyll degradation products found in the copepod tissues and fecal pellets of the Pseudodiaptomous euryhalinus in Bustillo-Guzman et al.’s (2002) study were also found in the copepod tissues of N lacustris: chlorophyllide a, phaeophytin b, phaeophytin a and pyrophaeophytin a Juhl et al (1996) found phaeophorbides and i79 Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 weight)21 This falls within the middle of the range of values reported for other copepods In a survey of the ontogenetic stages of Acartia bifilosa and Pseudocalanus acuspes, Lotocka et al (2004) found that nauplii had the highest concentration of unesterified astaxanthin, with mean respective concentrations of 427 mg (g dry weight)21 and 321 mg (g dry weight)21 The amount of astaxanthin reported for Calanus pacificus by Juhl et al (1996) (11 to 92 ng astaxanthin female21) equals $ 65 to 541 mg astaxanthin (g dry weight)21 since C pacificus adult females dry weight averages 170 mg (Frost, 1972), which is 30 times heavier than N lacustris Hairston (1980) found mg pigment (g dry weight)21 for Diaptomus nevadensis, or about 250 ng individual21, which is an order of magnitude larger than what is found in N lacustris However, D nevadensis inhabits high alpine lakes that are exposed to more ultraviolet radiation resulting in higher astaxanthin content (Hairston, 1976) Tigriopus brevicornis, a bright orange harpacticoid copepod found in rocky tide pools, contained 10.35 mg astaxanthin (g dry weight)21 when raised in the laboratory with a substratum of Enteremorpha sp and a diet of Tetraselmis sp, exceeding the natural levels found in wild-harvested T brevicornis [4.86 mg (g dry weight)21] (Davenport et al., 2004) Interestingly, no astaxanthin was detected in the laboratory reared T brevicornis when raised in the dark on a diet of baker’s yeast (Davenport et al., 2004) Conversely, Canuella perplexa, a harpacticoid copepod which lives in the sediments of salt marshes, was reported to contain very low amounts of astaxanthin, about 0.0044 to 0.012 ng astaxanthin individual21 when harvested from the wild (Buffan-Dubau et al., 1996), which is two orders of magnitudes less than what is found in laboratory-reared N lacustris N lacustris populations fed the two different diets fall in the middle range of astaxanthin content for marine calanoid and harpacticoid copepods JOURNAL OF PLANKTON RESEARCH j VOLUME 29 SUPPLEMENT j PAGES i73 – i83 j 2007 Fig Suggested pathways for b-carotene conversion to astaxanthin (Thommen and Wackernagel, 1964; Goodwin, 1984; Katayama et al., 1970; Bandaranayake and Gentien, 1982; Berticat et al., 2000, Linan-Cabello, 2002) Black arrows indicate the pathway proposed for most crustaceans White arrows indicate variations from this main pathway in other crustaceans The arrow with a dotted line is a hypothesized pathway for lutein to astaxanthin The arrows that are shaded gray indicate an alternative conversion pathway proposed for crustaceans that may not rely on echinenone and canthaxanthin as intermediates Geryon quinquedens (Kuo et al., 1976) and the crayfish Astacus leptodactylus Eschscholtz (Berticat et al., 2000) One occurrence of b-doradexanthin (adonixanthin) has been reported in a species of copepod surveyed from the Great Barrier Reef (Bandaranayake and Genien, 1982) However, this copepod also contained the intermediate canthaxanthin, which makes it difficult to determine which, if any, pathway is being used to produce astaxanthin The bioconversion of astaxanthin from b-carotene and zeaxanthin cannot be specifically elucidated from the information presented in this paper However, some evidence for a conversion pathway involving the ketocarotenoid b-doradexanthin (adonixanthin) is found when the absorption spectra for the ketocarotenoid peak ( peak 2) preceding the free astaxanthin peaks ( peaks and in Figs and 2) are analyzed This peak has absorption spectra different from the astaxanthin spectra observed in the RP-HPLC phase of the experiment (Fig 3), and which is almost identical to spectra observed in the freshwater crayfish for b-doradexanthin (Katayama et al., 1970; Berticat et al., 2000) b-doradexanthin has also been found to be more polar than astaxanthin in other HPLC analyses, which matches the observation from this study that the putative b-doradexanthin elutes before astaxanthin Furthermore, no absorption spectra found in the survey corresponded with the intermediates canthaxanthin and echinenone utilized by other crustaceans (e.g shrimp, lobster and crabs) to convert b-carotene to Astaxanthin bioconversion pathways The exact pathway of bioconversion has not been determined for most aquatic animals Thommen and Wackernagel (1964) first suggested the pathway b-carotene ! echninenone ! canthaxanthin ! astaxanthin (Fig 5) This pathway has been found to be the most probable one for conversion of dietary carotenoids to canthaxanthin in the brine shrimp Artemia salina (Czygan, 1968; Hsu et al., 1970) However, Artemia are not able to continue the conversion process to astaxanthin (Hsu et al., 1970) Two alternative pathways which not rely on canthaxanthin and echinenone for the conversion of the carotenoids b-carotene and lutein to astaxanthin use b-doradexanthin (adonixanthin) as an intermediate The complete conversion of lutein to astaxanthin has not been definitively proven for any animal, because the bioconversion of a-doradexanthin into its isomer b-doradexanthin has not been demonstrated (Ohkubo et al., 1999) However, it has been demonstrated that zeaxanthin is metabolised into astaxanthin via b-doradexanthin (adonixanthin) in goldfish (Hata and Hata, 1972; Ohkubo et al., 1999), the deep sea red crab i80 Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 b-doradexanthin (adonixanthin), it may be serving as an intermediate between zeaxanthin and astaxanthin b-doradexanthin has a substituent group at the C-4, which b-carotene and zeaxanthin not (Katayama et al., 1970) The position of this group may have an effect on the antioxidant capacity of carotenoids (Woodall et al., 1997) For example, astaxanthin and canthaxanthin have been found to have greater free radical quenching ability than zeaxanthin or b-carotene in vitro (Tera˜o, 1989) The antioxidant properties of b-doradexanthin (adonixanthin) have not been tested in relation to the other ketocarotenoids The large proportion of the peak ketocarotenoid putatively identified as b-doradexanthin in the copepods fed the formulated feed suggests that astaxanthin may not be the only ketocarotenoid that serves as an antioxidant for N lacustris The harpacticoid copepod N lacustris is an epibenthic detritovore found in salt marshes, and will migrate up into the water column periodically The role of the pigmentation in N lacustris may be more than solely photoprotective, as has been described in high radiation environments for tide pool dwelling harpacticoid copepods (Davenport et al., 2004) For example, Lotocka et al (2004) hypothesized that carotenoid molecules such as astaxanthin serve as a physiological oxygen replacement mechanism for upwardly migrating calanoid copepods which are rapidly combusting lipid materials as they swim j A C E RHODES j DIETARY EFFECTS ON COPEPOD CAROTENOIDS astaxanthin are preferentially absorbed (Østerlie et al., 2000) In contrast, lower digestibility of cis-astaxanthin relative to trans-astaxanthin has been observed in Atlantic halibut (Hippoglossus hippoglossus) and Atlantic salmon (Bjerkeng and Berge, 2000) This difference in digestibility suggests that fish species may have different mechanisms than mammals for astaxanthin transport into the enterocytes and incorporation into lipoproteins (Bjerkeng and Berge, 2000) Alterations in the base carotenoid composition of the lower trophic food levels could therefore affect the digestibility and nutritional value of copepods to natural predators Nitokra lacustris is a good model organism for further research on carotenoid bioconversion in marine harpacticoid copepods, as it will readily consume inert and live feeds Future work will hopefully allow for manipulation of dietary carotenoid concentrations to determine which precursors are directly correlated to astaxanthin production The relative importance of environmental conditions such as light cycles versus nutritional conditions can also be tested using harpacticoid copepods, as suggested by the work of Davenport et al (Davenport et al., 2004), which showed that diet as well as rearing conditions directly affected astaxanthin content in Tigriopus brevicornis Understanding how copepod carotenoid compositions are affected by their diets will help elucidate the vital role that copepods play in the transfer of astaxanthin and other ketocarotenoids to upper trophic levels in the marine environment AC K N OW L E D G E M E N T S I thank Dr Leon Boyd and Ruth Watkins at NC State University for their guidance and assistance in developing this project I would also like to thank the personnel at Craft Technologies, Inc., especially John Estes, for technical assistance in the preparation of the samples and use of their HPLC system I would also like to acknowledge Dr Peter Ferket, Dr Sam Mozley and Dr Donna Wolcott of North Carolina State University, Raleigh, NC, USA and Ron Johnson of the National Oceanic and Atmospheric Administration Northwest Fisheries Science Center, Seattle, WA, USA for their helpful comments on the manuscript REFERENCES Andersson, M., Van Nieuwerburgh, L and Snoeijs, P (2003) Pigment transfer from phytoplankton to zooplankton with emphasis on astaxanthin production in the Baltic Sea food web Mar Ecol Prog Ser 254, 213–224 i81 Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 astaxanthin (Goodwin, 1984; Linan-Cabello, 2002) The astaxanthin conversion from dietary carotenoids may be following an alternative pathway to the one proposed for other copepods (Lotocka and Styczynska-Jurewicz, 2001; Lotocka et al., 2004) All experiments were conducted on adult copepods, so it is not possible to determine whether all life stages are deficient in these carotenoids It is possible that the carotenoid composition of different life stages might contain alternative pigments, as is found in some calanoid species which contain canthaxanthin primarily in the early life stages and not in the adult stages (Lotocka et al., 2004) Due to the sensitivity of the RP-HPLC procedure, it is not likely that these carotenoids were present and not detected, unless they have an almost instantaneous lifespan during the conversion process The amount of astaxanthin conversion by the copepods depended on the diet Even though the copepods fed the formulated feed had a lower compositional ratio of astaxanthin, copepods fed the formulated feed had higher amounts of free astaxanthin than copepods fed the live alga Tetraselmis due to the high overall quantities of carotenoid A spectral absorbance peak characteristic of b-doradexanthin (adonixanthin) found in both treatments suggests that N lacustris converts b-carotene to astaxanthin using an alternative pathway that has been found in crayfish and deep sea crabs; however, further clarification on the identify of this ketocarotenoid peak needs to be done using a method such as mass spectrometry Copepods fed the 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Downloaded from http://plankt.oxfordjournals.org/ by guest on January 11, 2014 Yuan, J P and Chen, F (1998) Chromatographic separation and purification of trans-astaxanthin from the extracts of Haematococcus pluvialis J Agr Food Chem., 46, 3371– 3375 Tera˜o, J (1989) Antioxidant activity of b-carotene-related carotenoids in solution Lipids, 24, 659–661  ... research on carotenoid bioconversion in marine harpacticoid copepods, as it will readily consume inert and live feeds Future work will hopefully allow for manipulation of dietary carotenoid concentrations... for their system and controls at 450 nm j A C E RHODES j DIETARY EFFECTS ON COPEPOD CAROTENOIDS Quantification of astaxanthin in two copepod populations During preparation of the formulated feed,... Table I: Carotenoid composition of two populations of copepods fed different diets Peak number Retention time (min) Identification absorption maxima Copepods fed Tetraselmis (% of identified carotenoids)