Green-blood pigmentation in lizards

The green pigment in the plasma of the scincid lizard genus Prasinohaema is identified as the bile pigment biliverdin. Concentrations of biliverdin in the plasma of P. flavipes, P. prehensicauda and P. virens are 714 ± 79 μmol/1 (mean ± one standard deviation), 1020 ± 624 μmol/1 and 819 ± 89 μmol/1, respectively. These values represent the highest concentration of plasma biliverdin measured for any organism and are the first report of non-pathological biliverdin accumulation in amniotes. We review the literature for fish species with high concentrations of plasma biliverdin and pathological biliverdin accumulation in humans; we find that Prasinohaema species have plasma biliverdin concentrations approximately 1.5–30 times greater than fish species with green blood plasma and 40 times greater than humans with green jaundice.     

Isolation,identification and characterisation of ballan wrasse Labrus bergylta plasma pigment

This study confirmed that observations of blue‐green colouration in plasma fractions of the ballan wrasse Labrus bergylta were caused by the linear tetra‐pyrrole biliverdin and that the molecule was of the physiologically relevant IXα isomer. Accumulation appears driven by chromogenic association with an unknown protein moiety which precludes enzymatic reduction and would suggest active management. It was demonstrated that the pigment did not fluctuate relative to ontogeny, or indeed binary gender in the species of interest, but mobilisation and depletion in the subset of individuals undergoing sex change at the time of study supports a potential association with gender inversion processes. It is of note that although biliverdin does have some effect on external colouration, the evidence is indicative that crypsis is a supplementary function thus other factors must be considered.     

Bile pigment formation and excretion in the rabbit

Bile and plasma levels of biliverdin and bilirubin, together with the hepatic biliverdin reductase and bilirubin UDP-glucuronosyl transferase activities, were studied in the rabbit. No biliverdin could be detected in the blood plasma. The bilirubin concentration in blood was 7.81 +/- 0.79 mumol/l. Biliverdin was the predominant pigment in bile (63%). Hepatic biliverdin reductase activity was 0.086 +/- 0.016 nmol/mg protein/hr. The synthesis of bilirubin was apparently limited by the enzyme activity. Most of the bilirubin in bile was conjugated (90%) with monoconjugates predominating (75%). Hepatic UDP-glucuronosyl transferase activity was 2.65 +/- 0.18 and 1.14 +/- 0.16 mumol/mg protein/hr with and without activation, respectively.     

Observations on the colours of lumpsuckers, Cyclopterus lumpus L.

Ripe unfertilized eggs derived from fish landed at two Icelandic ports were predominantly red and yellow-red, but a small proportion were red-purple. There was a significant difference between the frequencies of hues of eggs from the two landing places. During the phase of blastula formation, fertilized red-purple eggs showed no discernible change in either hue or chroma. However, as the gastrula formed and covered the yolk, the chroma declined dramatically, although the hue remained unaltered. When the embryo formed, the chroma continued to decrease and the hue also began to change as the red-purple pigment disappeared from the yolk and the orange-yellow oil of the yolk became more visible.
The most common plasma colouration in male fish collected at Hafnarfjordur and Husavik was red, but the observed hues covered 60% of the visible spectrum, ranging from green to purple. No significant differences between Hafnarfjordur and Husavik males in terms of hue or value were discerned, but there was a significant difference in chroma score. Female plasma hues were almost as variable as the males (spanning 45% of the visible spectrum) but centred in the green-yellow zone. Plasma, subcutaneous jelly and skin colours were closely linked.
Fish from the oceanic feeding grounds were all green in skin colour, whatever their age or sex; it is suggested that this colouration has a cryptic function in the near surface waters inhabited by lumpsuckers.     

Blue‐green eggshell coloration reflects yolk antioxidant content in spotless starlings Sturnus unicolor

The hypothesis that eggshell colouration is a sexually selected trait of female birds is based on the fact that biliverdin, the pigment responsible for blue‐green colours of the eggshell, is a potent antioxidant and that only females with high antioxidant capacity can deposit higher concentrations of biliverdin as eggshell pigment. Antioxidants (e.g. carotenoids, vitamins) are also abundant in the egg yolk, which serve as nutrient reserves for the developing embryo, and eggshell colour intensity may reflect maternal investment in yolk antioxidants. Here, we test the relationship between blue‐green eggshell colour intensity and concentration and amount of carotenoids, vitamin A, and vitamin E in the egg yolk of spotless starling Sturnus unicolor, a species for which we have previously shown good evidence of sexual selection driving egg coloration. As could be extrapolated from the hypothesis of sexual selection driving the evolution of blue‐green eggshells, we found that eggshell colour intensity was positively related to the concentration and amount of carotenoids and vitamin E in the yolk. Thus, mothers may use egg colour intensity to signal to fathers the antioxidant status of their offspring. Moreover, we provide evidence suggesting that maternal yolk investment in more coloured eggs can also explain the detected association between feeding decisions of males and egg colour intensity that we have found previously in this species.     

Tissue levels of bilirubin and biliverdin in the sea lamprey, Petromyzon marinus L., before and after biliary atresia

1. Liver, intestine, kidney, muscle and epidermis from larvae, juvenile adults and upstream migrants of the sea lamprey, Petromyzon marinus L., were assayed for the presence of biliverdin and bilirubin. Urine was also examined for these bile pigments in juveniles and upstream migrants. 2. Bilirubin concentration increased dramatically in the liver and caudal intestine following loss of larval bile ducts while biliverdin levels were highest in the liver of upstream migrants and rose sharply in the caudal intestine immediately following the atresia. 3. Small amounts of bile pigment were present in larval kidneys but high concentrations were found in this organ in upstream migrants. The urine of the latter possessed biliverdin. 4. Mucus of the epidermis may be a vehicle for transport and release of bilirubin in upstream migrants. 5. These data indicate that lampreys utilize different avenues for bile pigment storage and elimination over the course of their life cycle.     

Serum pigmentation in Cyclopterus lumpus L

Female lumpsuckers, Cyclopterus lumpus L., have blue-green blood serum and subcutaneous gelatinous tissue; the male's are red/magenta in colour. The female serum contains a chromoprotein with biliverdin as the prosthetic group; the male serum contains the same biliverdin pigment but this is normally masked by a red pigment, probably phycoerythrin. The female serum also contains the red pigment but at a much lower concentration than in the male. These bile pigments are probably formed by the breakdown of haem in the liver. Lumpsucker gall bladders are relatively small and do not enlarge during starvation. Storage of bile pigments in the serum and subcutaneous jelly may have arisen in response to the problems of regular long-term starvation. The pigments now appear to have an additional sexual signalling function.     

Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity, inner filter effect and signal interpretation

Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.     

Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity,inner filter effect and signal interpretation

Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.     

Biliverdin initiates the liver regeneration in the rat--a hypothesis

Biliverdin has been observed to occur in the blood plasma of a 90% hepatectomized rat. It is also shown that the bile pigment induces a rise in mitotic index in the hepatic parenchymal cells of an intact rat at about 30 h and an elevated rate of hepatic DNA synthesis at 26 h after a single intraperitoneal injection. Hemoglobin, bilirubin, hemin, and protoporphyrin exhibited some of the inducing activity at lower degrees. It is hypothesized that biliverdin initiates the liver-cell-multiplication.     

Study of the heme catabolism of fish

• 1.1. The composition of bile pigments in the blood and bile of 39 species were studied.
• 2.2. Conjugated bilirubin (trace to 4.62 mg/100 ml) was detected in the serum of most fish, while biliverdin (trace to 2.0 mg/100ml) was detected only in Anguilla Japonica, Thalassoma lunare and Clinocottus analis.
• 3.3. Analysis showed tht there are two types of bile pigments excretion pattern in these fishes. The first pattern excretes bilirubin (most conjugate) predominantly, the other excretes mostly biliverdin with some bilirubin. However, during starvation, the excretion of conjugate bilirubin gradually shifted to unconjugated biliverdin. The rate of shifting varies with species.
• 4.4. Introduction of bilirubin into Anguilla japonica produced an initial excretion of mono-conjugates, followed by di-conjugates. Introduction of biliverdin caused an increased in the excretion of unconjugated biliverdin, but no significant increase of bilirubin in the bile was detected.
• 5.5. A binary excretion pathway of bile pigments in fish is proposed. The evolutionary characteristics of heme catabolism in terrestrial animals with respect to this pathway is discussed.

     

The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea)

Summary Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of³H-biliverdin were detected in hepatic bile following the intravenous administration of³H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.     

Biglutaminyl-biliverdin IX alpha as a heme degradation product in the dengue fever insect-vector Aedes aegypti

Hemoglobin digestion in the midgut of hematophagous animals results in the release of its prosthetic group, heme, which is a pro-oxidant molecule. Heme enzymatic degradation is a protective mechanism that has been described in several organisms, including plants, bacteria, and mammals. This reaction is catalyzed by heme oxygenase and results in formation of carbon monoxide, ferrous ion, and biliverdin IXalpha. During digestion, a large amount of a green pigment is produced and secreted into the intestinal lumen of Aedes aegypti adult females. In the case of another blood-sucking insect, the kissing-bug Rhodnius prolixus, we have recently shown that heme degradation involves a complex pathway that generates dicysteinyl-biliverdin IX gamma. The light absorption spectrum of the Aedes purified pigment was similar to that of biliverdin, but its mobility on a reverse-phase chromatography column suggested a compound less hydrophobic than biliverdin IXalpha. Structural characterization by ESI-MS revealed that the mosquito pigment is the alpha isomer of biliverdin bound to two glutamine residues by an amide bond. This biglutaminyl-biliverdin is formed by oxidative cleavage of the heme porphyrin ring followed by two subsequent additions of glutamine residues to the biliverdin IXalpha. The role of this pathway in the adaptation of this insect vector to a blood-feeding habit is discussed.     

Biliverdin IX delta and neobiliverdin IX delta, isolated from the ovaries of the marine snail, Turbo cornutus

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.     

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.     

Mechanism of action of heme oxygenase. A study of heme degradation to bile pigment by 18O labeling

The formation of bile pigment from heme by a reconstituted heme oxygenase system containing purified bovine spleen heme oxygenase, NADPH-cytochrome P-450 reductase, and biliverdin reductase was studied under an atmosphere containing 18,18O2. The product, bilirubin, was isolated and subjected to mass spectrometry, which revealed incorporation of 18O consistent with a two-molecule mechanism, whereby the product bile pigment contains oxygen atoms derived from two different oxygen molecules.     

The breeding colouration of male three-spined sticklebacks (Gasterosteus aculeatus) as an indicator of energy investment in vigour

Summary A necessary condition of most models of intersexual selection requires that secondary sexual traits are costly so that cheating is prevented. If the conspicuous breeding colouration of male three-spined sticklebacks (Gasterosteus aculeatus L.) is such a handicap, it must involve costs. I examined the energetic costs of the breeding colouration by varying the energy contents of the daily food supply among five groups of sticklebacks over a 10 week period. The nutritional carotenoid level, i.e. the colour pigment used in the breeding colouration, was constant for all fish. Both the increase of their condition factor and the condition level they finally achieved correlated positively with the food ration of the groups. Individuals whose condition increased during the experiment developed a more intensive red colouration. However, a direct correlation between food quantity and the red breeding colouration reached at the end of the experiment did not exist. Nevertheless, given the limitation of pigment availability, there was still variation in the breeding colouration and the costs for the metabolism of the colouration were sufficient to render it an honest signal: a female stickleback can assess a male's condition and condition change over the past few weeks by the intensity of the colour of his blue eyes (which is not based on carotenoids and whose pigments were therefore not controlled in the food) and his red jaw, respectively. How much an individual male fish invests in increase of length and increase of condition (which correlate negatively with each other) seems to be, at least partly, his own strategic decision, which could have important consequences in the competition for female mates. It is eventually this decision that a male stickleback seems to signal with his red jaw.     

Serum biliverdin as source of colouration upon sexual maturation in male blue‐throated wrasse Notolabrus tetricus

The blue colour observed in the serum of female blue‐throated wrasse Notolabrus tetricus was related to the presence of biliverdin. The biliverdin is probably stored in the female's serum until deposited in the skin upon transition to the male reproductive status, resulting in the brilliant blue colouration typical of males of this protogynous labrid species.     

An experimental test of the effect of food and antioxidants on blue‐green eggshell coloration in mountain bluebirds Sialia currucoides

Evolutionary biologists have long debated the functional relevance of the ornamental traits that occur in many animal taxa, and yet, female ornaments have received relatively little attention compared to those produced by males. A greater understanding of these traits, particularly those that are unique to females, may shed light on the potential for sexual selection to shape female phenotypes. Recently, blue‐green eggshell colour, derived from the antioxidant pigment biliverdin, has been proposed as a candidate trait that advertises female quality to males in species of birds with biparental care. However, studies have been equivocal in their support for blue‐green eggshell colour being an informative signal, and correlations between eggshell colour and other female characteristics have been inconsistent. We conducted a supplementation experiment to test if improving the access of female birds to food resources and micronutrients, thereby improving their condition prior to egg laying, would intensify the blue‐green colouration of the eggs they laid. We provided mountain bluebirds Sialia currucoides with food and carotenoids during nest building and egg laying in two breeding seasons, and assessed both within‐ and among‐clutch variation in colour. Supplementation did not affect patterns of within‐clutch variation, but did result in differences in colour among clutches. Specifically, we found that food, but not carotenoids, resulted in higher colour saturation, and decreased the brightness of blue‐green eggshells. Although this trend was observed in both years, the effect was statistically significant only in one year. Our results suggest that food supplementation influences eggshell colour, but that conditions, such as weather and natural food availability, which can vary annually, may also determine how female birds allocate pigment to eggshells.