Dietary effects on carotenoid compositio

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 ca

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 , 2725 MONTLAKE BLVD E , SEATTLE ,

WASHINGTON 98112, USA

*C ORRESPONDING 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

Communicating editor: K.J Flynn

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

formulated diet with high levels of lycopene, beta-carotene and alpha-carotene averaged 24.3 mg

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

lacus-tris differs from that described for calanoid copepods and other marine crustaceans N lacuslacus-tris

astax-anthin 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

keto-carotenoids (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 color-ation which can be observed easily through a micro-scope, 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

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

Role of carotenoid pigments in

harpacticoid copepods

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

protec-tants against photooxidation and as vitamin A

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

unsatu-rated 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

con-sequently prevent damage to membranes (Woodall

et al., 1997; Lotocka et al., 2004)

Astaxanthin cannot be synthesized de novo in

cope-pods (Matsuno, 1989; Andersson et al., 2003) Several

pathways of astaxanthin synthesis from precursor

(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

biosyn-thetic capability: (i) fish that cannot biosynthesize

astax-anthin from b-carotene, lutein or zeaxastax-anthin, but which

can transfer dietary astaxanthin to body astaxanthin,

(ii) fish, such as red carp and goldfish, that can

biocon-vert 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

russelli (Bandaranayake and Gentien, 1982) and the

Styczynska-Jurewicz, 2001) and Pseudocalanus acuspes Giesbrecht (Lotocka et al., 2004) Canthaxanthin and

reported in the cyclopoid copepod Cyclops kolensis Lilljeborg (Czeczuga et al., 2000) However, the inter-mediates canthaxanthin and echinenone have not been observed in the harpacticoid copepods Tigriopus

(Buffan-Dubau et al., 1996) The analysis of pigments undertaken in this study allows for further examination

of the pigment composition and potential pathways uti-lized by N lacustris for biosynthesis of astaxanthin from dietary precursors

M E T H O D

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 formu-lated feed containing the carotenoid pigments lycopene, a-carotene, b-carotene, lutein, phytoene and phyto-fluene 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 sig-nificant differences in population growth rates, size of animals, or fatty acid composition have been documen-ted 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, chloro-phyll b, chlorochloro-phyll 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 identi-cal 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

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

fil-tration process, all procedures from this point forward

were conducted under yellow fluorescent light (Philips

F40GO) to prevent pigment destruction

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

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 5 min 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%

tetra-hydrofuran (THF), 40% methanol]

Samples were injected with a Waters U6K loop

injec-tor (Waters, Massachusetts) into a 3.5 mm Waters

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

stan-dard curve plotting astaxanthin peak area versus

areas for each the cis- and trans-astaxanthin isomers

were compared to a standard curve developed using an

astaxanthin (Sigma A 9335) The standard curve was prepared by serial dilution of the stock solution [500 mg

All sample concentrations fell within this standard curve

cis-astaxanthin and trans-astaxanthin were compared to the standard curve to determine the concentration of

facilitate comparison Astaxanthin values from the duplicate samples were averaged for each of the three

cis-astaxanthin, trans-astaxanthin and total astaxanthin were compared within and between the three popu-lations assigned to each treatment by analysis of variance (ANOVA)

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 col-lected from freshly harvested samples by placing the filters containing the animals in 5 mL THF and sonicat-ing for 5 min After centrifugation at 200 g for 5 min at 48C, the supernatant was drawn off One mL of super-natant 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 5 mm C30 Column (YMC carotenoid

Wilmington, NC, USA) using HPLC-grade solvents and a linear tertiary gradient system (100% isopropyl

acetate:100 % THF) at a flow rate of 1 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 5 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 com-parison to the spectra and retention times under the same conditions of a standard mix containing lutein,

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,

(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

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

astax-anthin as well as several carotenoid and chlorophyll

pigments (Table I, Figs 1 and 2) Copepod populations

fed the two diets had pigment compositions that

reflected their diets (Table I) Both populations of copepods contained unesterified astaxanthin, antherax-anthin, b-carotene, a ketocarotenoid peak characteristic

Copepods fed the live alga contained the unique caro-tenoid pigments violaxanthin, lutein, zeaxanthin, and a-carotene (Fig 1) The copepods fed the live

unknown carotenoids ( peaks 1, 4 and 7) as well as astaxanthin esters (four peaks labeled 13) Only cope-pods fed the formulated diet contained the pigments 1,1-carotene, lycopene and cis-lycopene as well as two unique unidentified carotenoids ( peaks 5 and 6) (Fig 2)

A single peak was identified as unesterified astax-anthin in both sets of animals based on its retention time and absorption spectra (Figs 1 and 2) Two distinct spectra corresponding to cis- and trans-astaxanthin coeluted in this single peak during the RP-HPLC method ( peaks 8 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 2 was putatively identified as the intermediate compound b-doradexanthin (adonixanthin) found when zeaxanthin

is converted to astaxanthin in the crayfish Astacus lepto-dactylus (Berticat et al., 2000) (Fig 3)

Fig 1 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.

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

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

found in the copepods fed the live algal diet

Quantification of astaxanthin in two copepod populations

indicated that the tanks fed the formulated feed

cis-astaxanthin, as well as a higher proportion of

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 signifi-cantly between treatments and had an average value of

amount of astaxanthin in copepods fed live microalgae

Fig 2 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 2.

Table I: Carotenoid composition of two populations of copepods fed different diets

Peak number Retention time

(min)

Identification absorption maxima absorption maxima Copepods fed Tetraselmis Tetraselmis

(% of identified carotenoids)

Copepods fed formula (% of identified carotenoids)

T suecica was 1.0 ng astaxanthin individual21 (+0.2

(+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

(+1.7 S.D.,

S.D., n ¼ 3) (Fig 4)

D I S C U S S I O N

N lacustris contained many of the same pigments as

Bandaranayake and Gentien, 1982; Goodwin, 1984;

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; McLeroy-Etheridge 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 astax-anthin 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

Fig 3 Absorption spectra of putative b-doradexanthin (solid line, peak 2 in Figs 1 and 2) and trans-astaxanthin (broken line, peak 9 in

Figs 1 and 2).

Fig 4 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 1 S.D.

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

astaxanthin reported for Calanus pacificus by Juhl et al

adult females dry weight averages 170 mg (Frost, 1972),

which is 30 times heavier than N lacustris Hairston

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

(Hairston, 1976)

Tigriopus brevicornis, a bright orange harpacticoid

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

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

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

harpacti-coid copepods

Transfer of pigments from food

to copepods

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

phaeophytins in Calanus pacificus that were reported to

be derived from the digestive breakdown of chlorophyll

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 micro-algae 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

lycopene per individual copepod for the duration of the experiment Copepods fed the live algae did not contain lycopene; whereas the copepods fed the

suggesting that the copepods were most likely bioaccu-mulating 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 pre-ceding free astaxanthin (Figs 1 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 alterna-tive bioconversion pathway

It has been suggested that it is the structure of astax-anthin and canthaxastax-anthin 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

(Linan-Cabello et al., 2002) If peak 2 is indeed

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 do 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 2 ketocarotenoid

putatively identified as b-doradexanthin in the

cope-pods 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

upwardly migrating calanoid copepods which are

rapidly combusting lipid materials as they swim

Astaxanthin bioconversion pathways

The exact pathway of bioconversion has not been

deter-mined for most aquatic animals Thommen and

astaxanthin (Fig 5) This pathway has been found to be

the most probable one for conversion of dietary

caro-tenoids 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

astax-anthin (Hsu et al., 1970)

Two alternative pathways which do 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

b-doradexanthin (adonixanthin) in goldfish (Hata and

Hata, 1972; Ohkubo et al., 1999), the deep sea red crab

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 inter-mediate 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 keto-carotenoid b-doradexanthin (adonixanthin) is found when the absorption spectra for the ketocarotenoid peak ( peak 2) preceding the free astaxanthin peaks ( peaks 8 and 9 in Figs 1 and 2) are analyzed This peak has absorption spectra different from the astaxanthin spectra observed in the RP-HPLC phase of the exper-iment (Fig 3), and which is almost identical to spectra observed in the freshwater crayfish for b-doradexanthin

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 puta-tive b-doradexanthin elutes before astaxanthin

Furthermore, no absorption spectra found in the survey corresponded with the intermediates canthax-anthin and echinenone utilized by other crustaceans (e.g shrimp, lobster and crabs) to convert b-carotene to

Fig 5 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.

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

con-ducted on adult copepods, so it is not possible to

deter-mine whether all life stages are deficient in these

carotenoids It is possible that the carotenoid

compo-sition 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

life-span during the conversion process

The amount of astaxanthin conversion by the

cope-pods depended on the diet Even though the copecope-pods

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

treat-ments 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 live alga Tetraselmis had

proportionately twice as much unesterified astaxanthin

(27%) in comparison to the putatively identified

b-doradexanthin (14%), as well as various astaxanthin

esters In contrast, the copepods fed the formulated feed

contained a very large proportion of the putatively

identified b-doradexanthin (67%) in comparison to

unesterified astaxanthin (3%) This may indicate that

the enzymatic conversion from this intermediate to

astaxanthin may be limited, or it may not be necessary

to convert the ketocarotenoid potentially identified as

b-doradexanthin to astaxanthin if it fulfills the same

antioxidant role as astaxanthin for the copepods

Because the amount and proportion of astaxanthin

depended on the diet, diets containing higher amounts

of b-carotene and zeaxanthin would be more likely

to produce high levels of astaxanthin in this species

The copepod populations fed the formulated diet still

relative proportions due to the extremely high content

b-doradexanthin The fact that N lacustris diets affect

final carotenoid composition has implications for the

upper trophic levels In mammals, cis-isomers of

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 harpac-ticoid 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 pro-duction The relative importance of environmental con-ditions such as light cycles versus nutritional concon-ditions 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 caro-tenoid 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 O W 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 develop-ing 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

R E F E R E N C E S 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.

Arab, L., Steck-Scott, S and Fleishauer, A T (2002) Lycopene and

the lung Exp Biol Med 227, 894 – 899.

Bandaranayake, W M and Gentien, P (1982) Carotenoids of Temora

turbinata, Centropages furcatus, Undinula vulgaris and Euchaeta russelli.

Comp Biochem Phys B, 72, 409 – 414.

Berticat, O., Negre-Sadargues, G and Castillo, R (2000) The

metab-olism of astaxanthin during the embryonic development of the

cray-fish Astacus leptodactylus Eschscholtz (Crustacea, Astacidea) Comp.

Biochem Phys B, 127, 309 – 318.

Bjerkeng, B and Berge, G M (2000) Apparent digestibility

coeffi-cients and accumulation of astaxanthin E/Z isomers in Atlantic

salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus

L.) Comp Biochem Physiol B, 127, 423 – 432.

Britton, G (1995) UV/V is spectroscopy in carotenoids In Britton,

G., Liaaen-Jensen, S and Pfander, H (eds.), Carotenoids Vol 1B.

Chapter 2 Birkhau¨ser Verlag, Basel, 13 –62.

Buffan-Dubau, E., de Wit, R and Castel, J (1996) Feeding selectivity

of the harpacticoid Canuella perplexa in benthic muddy environments

demonstrated by HPLC analysis of chlorin and carotenoid

pig-ments Mar Ecol Prog Ser., 137, 71 – 82.

Bustillos-Guzman, J., Lopez-Cortes, D., Mathus, M E et al (2002)

Dynamics of pigment degradation by the copepodite stage of

Pseudodiaptomus euryhalinus feeding on Tetraselmis suecica Mar Biol.

140, 143 – 149.

Canjura, F L (1990) Separation, identification and degradation kinetics of

chlorophylls and chlorophyllides in spinach MS Thesis North Carolina

State University, Raleigh, NC, USA.

Czeczuga, B., Kozlowska, M and Czeczuga-Semeniuk, E (2000)

Adaptive role of carotenoids and carotenoproteins in Cyclops kolensis

Lilljeborg (Crustacea: Copepoda) specimens to extremely

eutrophi-cal conditions Folia Biol Krakow, 48, 77– 84.

Czygan, F C (1968) On metabolism of carotenoids in crustacean

Artemia salina Z Naturforsch Pt B, 23, 1367– 1368.

Davenport, J., Healy, A., Casey, N et al (2004) Diet-dependent UVAR

and UVBR resistance in the high shore harpacticoid copepod

Tigriopus brevicornis Mar Ecol Prog Ser., 276, 299 – 303.

Egeland, E S., Eikrem, W., Throndsen, J et al (1995) Carotenoids

from further prasinophytes Biochem Syst Ecol., 23, 747 – 755.

Frost, B W (1972) Effects of size and concentration of food particles

on the feeding behaviour of the marine planktonic copepod Calanus

pacificus Limnol Oceanogr., 17, 805 – 815.

Goodwin, T W (1984) The Biochemistry of the Carotenoids, 2nd edn.

Vol 2 Chapman and Hall, London, 64 – 96.

Hairston, N G (1976) Photoprotection by carotenoid pigments in

the copepod Diaptomus nevadensis Proc Natl Acad Sci USA, 73,

971 – 974.

Hairston, N G (1979) The effect of temperature on carotenoid

photo-protection in the copepod Diaptomus nevadensis Comp Biochem Phys A,

62, 445 – 448.

Hairston, N G (1980) On the diel variation of copepod pigmentation.

Limnol Oceanogr., 25, 742 – 747.

Hata, M and Hata, M (1972) Carotenoid pigment in gold fish IV.

Carotenoid metabolism B Jpn Soc Sci Fish., 38, 331 – 338.

Hsu, W J., Chichester, C O and Davies, B H (1970) The

metab-olism of b -carotene and other carotenoids in the brine shrimp,

Artemia salina L (Crustacea: Branchiopoda) Comp Biochem Phys.,

32, 69 – 79.

Hsu, W J., Rodriguez, D B and Chichester, C O (1972) Biosynthesis

of astaxanthin—VI Conversion of [c-14] lutein and [c-14]

b-carotene in goldfish Int J Biochem., 3, 333 – 338.

Hyvarinen, K and Hynninen, P H (1999) Liquid chromatographic separation and mass spectrometric identification of chlorophyll b allomers J Chromatogr A, 837, 107 – 116.

Juhl, A R., Ohman, M D and Goericke, R (1996) Astaxanthin in Calanus pacificus: assessment of pigment-based measures of omnivory.

Limnol Oceanogr., 41, 1198– 1207.

Katayama, T., Yokoyama, H and Chichester, C O (1970) The bio-synthesis of astaxanthin I The structure of a-doradexanthin and b-doradexanthin Int J Biochem., 1, 438 – 444.

Katayama, T., Shintani, K., Shimaya, M et al (1972) Biosynthesis of astaxanthin-IX Transformation of labeled astaxanthin from diet of sea bream, Chrysophrys major Temminck and Schlegel, to their body astaxanthin B Jpn Soc.

Sci Fish., 38, 1399 – 1403.

Katayama, T., Kunisaki, Y., Shimaya, M et al (1973) The biosynthesis

of astaxanthin XIV The conversion of labelled b-carotene-15.

150-3H2 into astaxanthin in the crab, Portunus trituberculatus Comp.

Biochem Phys B, 46, 269 – 272.

Kleppel, G S (1998) Plant and animal pigments as trophodynamic indicators In Soule, D F and Kleppel, G S (eds), Marine Organisms

as Indicators Springer-Verlag, New York, 73 – 90.

Kuo, H C., Lee, T C., Lee, C O et al (1976) The carotenoids in the deep sea red crab Comp Biochem Phys B, 54, 387 – 390.

Linan-Cabello, M A., Paniagua-Michel, J and Hopkins, P M (2002) Bioactive roles of carotenoids and retinoids in crustaceans Aquacult.

Nutr., 8, 299 – 309.

Lotocka, M and Styczynska-Jurewicz, E (2001) Astaxanthin, canthax-anthin and astaxcanthax-anthin esters in the copepod Acartia bifilosa (Copepoda, Calanoida) during ontogenetic development.

Oceanologia, 43, 487 – 497.

Lotocka, M., Styczynska-Jurewicz, E and Bledzki, L A (2004) Changes in carotenoid composition in different developmental stages of copepods: Pseudocalanus acuspes Giesbrecht and Acartia spp.

J Plankton Res., 26, 159 – 166.

Matsuno, T (1989) Animal carotenoid In Krinsky, N I., Mathews-Roth, M M and Taylor, F (eds), Carotenoids Chemistry and Biology Plenum Press, New York, 59 – 74.

Matsuno, T and Tsushima, M (1995) Comparative biochemical studies of carotenoids in sea cucumbers Comp Biochem Phys B,

111, 597 – 605.

McLeroy-Etheridge, S L and McManus, G B (1999) Food type and concentration affect chlorophyll and carotenoid destruction during copepod feeding Limnol Oceanogr., 44, 2005– 2011.

Miki, W (1991) Biological functions and activities of animal caroten-oids Pure Appl Chem., 63, 141 – 146.

Nelis, H J C F and De Leenheer, A P (1988) Reversed-phase liquid chromatography of astacene J Chromatogr., 452, 535 – 542.

Ohkubo, M., Tsushima, M., Maoka, T et al (1999) Carotenoids and their metabolism in the goldfish Carassius auratus (Hibuna) Comp.

Biochem Phys B, 124, 333 – 340.

Østerlie, M., Bjerkeng, B and Liaaen-Jensen, S (2000) Plasma appearance and distribution of astaxanthin E/Z and R/S isomers in plasma lipoproteins of men after single dose administration of astax-anthin Nutr Biochem., 11, 482 – 490.