This page is a work-in-progress regarding carotenoids and their influence on amphibian colouration.
CAROTENOIDS, CAROTENES AND XANTHOPHYLLS
Carotenoids are organic compounds containing 40 carbon atoms, with a tetraterpenoid structure. Carotenoids containing only carbon and hydrogen atoms are referred to as carotenes, while xanthophylls also contain oxygen atoms.
Most carotenoids are brightly coloured, ranging from yellow through orange and red to purple. They are hydrophobic compounds; they are at most sparingly soluble in water and other polar solvents [refs].
CAROTENOIDS IN BIOLOGICAL SYSTEMS
Carotenoids are synthesized by many species of higher and lower plants [refs]. Beta-carotene was recognised as being responsible for the orange colour of carrots as long ago as 1831 (Wackenroder, 1831).
Carrots contain around 100mg/kg (0.01%) total carotenoids (Matejkova & Petrikova, 2010). Other vegetables identified as having more than 50mg/kg (0.005%) total carotenoids include kale (348 mg/kg), parsley(254 mg/kg), spinach(173 mg/kg), lamb's lettuce (160 mg/kg), lettuce (84.8 mg/kg), Brussels sprouts (61.5 mg/kg) red peppers (304 mg/kg) and tomatoes (127 mg/kg) (Muller, 1997).
There is only one animal species known to synthesis carotenoids, this ability being present in certain strains of the aphid Acyrthosiphon pisum. This species is thought to have acquired the ability via pathogenic fungi (Moran & Jarvik, 2010). Excepting this example, all animals must acquire carotenoids from dietary sources.
Carotenoids may be metabolised in animals, or may be deposited in various tissues. The carotenes alpha-carotene, beta-carotene, gamma-carotene, and the xanthophyll beta-cryptoxanthin are converted into vitamin A in many herbivorous or omnivorous animal species [refs]. Beta-carotene can be converted to canthaxanthin in brine shrimp (Artemia) with echinonene being an intermediate (Hsu et al., 1970). Similarly, water fleas (Daphnia) convert beta-carotene and zeaxanthin to canthaxanthin and astaxanthin respectively (Partali et al., 1985).
Deposition in body tissues may influence the colour of those tissues[refs], and the hydrophobic nature of carotenoid compounds means that they are likely to accumulate in the body (Geyer et al., 1991). Thus dietary carotenoids can have a long-term effect on animal colouration.
Carotenoids have been shown to be a major influence on the colouration of many species including flamingoes, fire-bellied toads, and lobsters (Fox & Vevers, 1960; Frost & Robinson, 1984). The colour may not necessarily correspond directly to the colour of the source carotenoids. For example, it is protein-bound astaxanthin which causes the blue colour of live lobsters. When lobster is cooked, astaxanthin is freed from the proteins, resulting in a pink or orange colour (Cianci et al., 2002).
Carotenoids can affect human skin colour- 'carotenosis' can be caused by excess dietary carotenoids, whether from natural sources, or dietary supplements[refs].
CAROTENOIDS IN AMPHIBIAN COLOUR
It has long been known amongst herpetoculturists that colouration in Bombina species is influenced by diet, and that addition of carotenoids to the diet can enhance the red belly colour (e.g. Mattison, 1982). Frost & Robinson (1984) confirmed the presence of carotenoid-containing cells in the red bellies of B. orientalis.
It is perhaps less well known that carotenoids contribute to the colouration of a number of newt species. Sparreboom (1998) suggests that belly colour in Cynops species can be intensified by feeding carotenoid-rich foods, and Matsui et al. (2002) confirm that carotenoids are responsible for the ventral colouration of Cynops pyrrhogaster. Carotenoids have also been identified as responsible for the red spots of the red-spotted newt, Notophthalmus viridescens (Forbes et al., 1973), while Collins et al. (1953) reported the presence of both carotenes and xanthophylls in Triturus carnifex, though they did not suggest any influence on colouration.
My own observations suggest that most species of Triturus sensu lato also exhibit some dietary influence on their ventral colour. For example, captive-bred specimens of Triturus alpestris(=Mesotriton alpestris, Ichthyosaura alpestris) often show a yellow belly colour, whereas that of wild individuals may be deep red. Similarly, the central stripe on the belly of the smaller species (Triturus (=Lissotriton) boscai, helveticus, italicus, montandoni, and vulgaris) has a wide variation in intensity, with many captive-bred individuals being towards the yellow end of the range.
COLOUR FEEDING WITH CAROTENOIDS
Carotenoids are routinely used to intensify colour in captive animals [refs]. Canthaxanthin (a xanthophyll) has traditionally been used to intensify colour in farmed salmon, but following the identification of crystalline deposits in the retinas of human subjects exposed to large quantities (e.g. Espaillat et al., 1999; McGuinness & Beaumont, 1985), its use is now restricted in many countries (e.g. EC, 2003). Astaxanthin (another xanthophyll) is now promoted as a replacement in aquaculture (DSM Nutritional Products, 2006), and is more effectively absorbed than canthaxanthin (Choubert & Storebakken, 1996).
EU legislation (EC, 2003) specifies that salmon feed can contain no more than 25mg/kg (0.0025%) canthaxanthin, while concentrations of up to 100mg/kg (0.01%) astaxanthin are permitted (EC, 2004).
Canthaxanthin and astaxanthin are available as animal feed supplements under the tradenames 'Carophyll Red' and 'Carophyll Pink' respectively (both TM Hofmann-La Roche). These are finely granular preparations of the carotenoid embedded in a gelatine and carbohydrate matrix, and coated with starch. They are specified at no less than 10% active content. These products are intended for addition to commercial foods at the manufacturing stage (DSM Nutritional Products, 1999, 2006).
The microalgae Haematococcus pluvialis is high in carotenoids, containing up to 30000mg/kg (3%) astaxanthin in dry matter (Lorenz & Cysewski, 2000). Powdered Haematococcus products are currently (2016) commercially available as human dietary supplements containing from 1.2% to 10% astaxanthin.
The algae 'Spirulina' (Arthrospira platensis, A. maxima) is another source of carotenoids for aquaculture. Miki et al. (1986) found up to 6480mg/kg (0.6%) total carotenoids in spray-dried A. maxima, while a commercial producer of A. platensis specifies >5000mg/kg in its dried algae preparation ('Spirulina Pacifica', Cyanotech Corporation, 2010), with beta-carotene composing 2250mg/kg (0.225%), and zeaxanthin 3000mg/kg (0.3%). 'Spirulina' is commonly used by hobbyist aquarists, often as a component of commercial feeds (e.g. Sera, undated).
Paprika (dried, powdered fruits of Capsicum annuum) also contains high carotenoid concentrations. Minguez-Mosquera et al. (2000) found 3451.5-5046.0mg/kg (0.3-0.5%) total carotenoids in commercial paprika.
Marigold (Calendula officinalis) flowers contain high carotenoid concentrations. Pintea et al. (2003) found 1110-2760mg/kg (0.1-0.3%) total carotenoids in fresh flowers, with carotenes (beta-carotene, gamma-carotene, and lycopene) dominating in orange varieties, and xanthophylls (flavoxanthin, lutein, rubixanthin) dominating in yellow. Dried marigold petals are commercially available for use in herbal medicine.
Brine shrimp (Artemia spp.) are also known to contain moderately high concentrations of carotenoids. Kumar & Marian (2006) found 600mg/kg (0.06%) total carotenoids in dried cysts of A. parthenogenitica. Krinsky (1965) found only canthaxanthin and echinenone in A. salina, with 95% being canthaxanthin. Dried decapsulated cysts are commonly available to hobbyist aquarists, primarily for feeding (usually after rehydration) to fish fry.
Freshwater shrimp contain similar concentrations of carotenoids. Gaillard et al (2004) found 203mg/kg (0.02%) total carotenoids in fresh Gammarus pulex and 252mg/kg (0.03%) in G. roeseli, with astaxanthin being the major component. As water content of Gammarus is approximately 75% (Lockwood & Inman, 1973), this would represent around 900mg/kg (0.09%) of dry matter. Gaillard et al also suggest that Gammarus transform dietary carotenoids, particularly beta-carotene, into astaxanthin, and state that this is "a process that has been shown in most crustaceans".
Water fleas (Daphnia spp.) also contain moderately high carotenoid concentrations. De Meester & Beenaerts (1993) found 249.9-457.2mg/kg (0.02-0.05%) of dry mass in Daphnia magna.
Water lice (Asellus spp.) contain moderate to low carotenoid concentrations. Czeczuga et al. (2005) found 13.9mg/kg (0.001%) of dry mass in A. aquaticus, with astaxathin (37.5%) and canthaxanthin (21.4%) being the major components. Protein-bound carotenoids were also found in this species.
Yellow corn meal (polenta) contains low to moderate carotenoid concentrations. Perry et al. found 6.94mg/kg (0.0007%) total carotenoids (5.31mg/kg (0.0005%) zeaxanthin) in commercial cornmeal. They suggested that this was a major source of zeaxanthin in human diets.
AMPHIBIAN COLOUR FEEDING WITH CAROTENOIDS
Some keepers have successfully intensified belly colour by dusting food items with carotenoid supplements; e.g. S. Hartley (personal communication) for Bombina. Others have used foods supposed to be naturally high in carotenoids; e.g. Sparreboom (1998) for Cynops.
I have identified a number of approaches for colour feeding:
'Direct feeding' involves feeding an animal directly with a carotenoid-rich preparation. Although this is clearly the most efficient (and most easily controlled) method for carotenoid feeding, it is not always applicable. As post-metamorphic amphibians prefer live prey, it is unlikely to be useful for adult animals, though some species (particularly aquatic newts) may be persuaded to accept artificial food preparations.
This may be appropriate for feeding anuran larvae- Cothran et al (2015) added carotenoids to a preparation of fishmeal and flour used to feed Hyla and Lithobates tadpoles. As tadpoles are essentially filter feeders (McDiarmid & Altig, 1999), it may also be possible to add carotenoids in suspension, or in solution, to their environment.
'Dusting' involves roughly coating the surface of prey items with a carotenoid-rich powder. Although this method is very convenient (it takes a matter of seconds to shake prey items with powder), dosage control is difficult, aquatic feeding is impossible, and the powder may affect palatability.
This method will result in a prey item with a carotenoid concentration very much lower than that of the supplement, and so this method is most applicable where an extremely carotenoid-rich supplement is available.
'Gut loading' involves feeding prey items with a carotenoid-rich foodstuff shortly before feeding to the target animal, the prey item serving mainly as a palatable wrapper for the carotenoids. If the digestive transit time of the prey item is known, this should be a very predictable method.
This method will produce a prey item with a carotenoid concentration similar to that of the foodstuff, depending on the relative volume of its digestive tract.
'Accumulating' involves feeding prey items with a relatively carotene-rich foodstuff over a long period of time, so carotenoids will accumulate in the prey item. The prey item may itself exhibit deeper colour as it accumulates carotenoids.
This will give a prey item which is more carotene-rich than its foodstuff, and so this method is more suitable for use with supplements relatively low in carotenoids.
'Transforming' involves feeding prey items with a foodstuff containing precursors for the desired carotenoids. As the prey items must metabolise the carotenoids, this is essentially an extension of 'accumulating', and could involve relatively low concentrations of precursors in the foodstuffs.
Waterfleas are organisms supposed to be rich in carotenoids, and suggested as suitable for intensifying amphibian colour (e.g. Sparreboom, 1998). As no animals can synthesize carotenoids, any carotenoids present in Daphnia must originate from their diet. Daphnia feed by filtering suspended particles from their environment, this being supplied in culture by a suspension of yeast, algae, or bacteria [refs]. The minimum food size available to Daphnia is of the order of 0.5um (Gophen & Geller, 1984). The maximum available food size increases rectilinearly with body length of the organism, an individual of 2mm in length being able to ingest particles up to about 40um in diameter (Reynolds, 2006).
The majority of Carophyll Red and Pink granules are more than 150um in size (85% and 70% of Carophyll Red and Pink respectively is specified to be retained by a No. 100 sieve (mesh 150um) (DSM Nutritional Products, 1999, 2006). This makes these products unsuitable for use as a carotenoid source for Daphnia. 'Spirulina Pacifica' is specified as having a maximum particle size of 125um, making it more suitable.
Lesser Waxworm (Achroia grisella)
Lesser waxworms are the larva of a Pyralid moth, and are simple to culture over long periods of time. They can be reared successfully using an artifical diet, to which it would be trivial to add carotenoid supplements. Greater wax moth (Galleria mellonella) can be reared under similar conditions, and could be substituted if a larger prey item is desirable.
FURTHER WORK: PREPARATION OF COLOUR FOODS
Live Daphnia (tentatively identified as D. magna) are available from local tropical fish suppliers. A carotenoid-enriched culture could be prepared by feeding with a suspension of bakers' yeast (Saccharomyces cerevisiae), gram flour (chickpea, Cicer arietinum), and 'Spirulina Pacifica'. A control culture could also be prepared, fed only with yeast and gram flour in suspension. Aquatic newts could be fed with control or enriched Daphnia, and colour changes monitored.
Carophyll Red can be obtained from retail aviary suppliers. This could be added to a lesser waxworm (Achroia grisella) culture at the rate of 0.1%. At 10% canthaxanthin, this would correspond to 100mg/kg in feed, similar to that used for intensifying colour in salmon. Aquatic or terrestrial newts could be fed with control or enriched waxworms, and colour changes monitored.
Carophyll Red could also be used to dust any foodstuff used for terrestrial newts.
FURTHER WORK: COLOUR MONITORING
Colour could be quantified either by eye, or by imaging methods.
Quantification by eye would be most effective by reference to standard colours, e.g. Pantone charts. Imaging methods could include digital photography, or use of a digital flatbed scanner. Either method would require calibration of each image by including standard colours (e.g. red, green and blue chips) in the image captured.
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