Himalayan black bulbul
(Hypsipetes leucocephalus nigerrimus)
Abstract
Melanin-based coloration is found to be often associated with variations in physiological and behavioral traits that are typical targets of sexual selection. However, the function of melanin-based traits to their bearers has been investigated less than that of carotenoid–based ones. It is commonly believed that expression of melanin-based pigmentation is genetically controlled. However, whether it could be modulated by physical condition has remained controversial. In this study, I
demonstrate that the level of melanin-based plumage pigmentation could be negatively correlated with an individual’s oxidative stress level. In this study, I used two groups of black bulbuls (Hypsipetes leucocephalus nigerrimus) obtained from a pet shop in 2010 and 2011 to examine the correlation between the melanin-based plumage coloration and the ratio of lymphocytes and heterocytes, which is an indicator of the oxidative stress experienced by an individual. I found a negative correlation
between oxidative stress and levels of melanin-based pigmentation on the breast and scapular feathers, but the correlation only appeared in the 2011 group individuals of which had higher oxidative stress levels and brighter plumage (i.e. more melanins) than those of the 2010 group. My data suggest that melanin-based plumage could reflect an individual’s physical condition in certain situations. This is consistent with the prediction of the indicator hypothesis. Therefore, melanin-based plumage coloration could be functional in visual communication of Himalayan black bulbuls.
Key words: heterocyte / lymphocyte ratio, Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus), melanin-based trait, oxidative stress
Introduction
According the “indicator hypothesis” , females can acquire information about males’ quality by assessing sexually selected traits that reflect their condition (honest signals), such as coloration or other conspicuous characteristics (Andersson and Simmons 2006). Melanins, mainly responsible for black (eumelanins) or reddish and brown (pheomelanin) colors, are considered one of the main pigment classes associated with variations in the physiological and behavioral traits that are typical targets of sexual selection and animal communication. It has been shown that vertebrates with darker coloration are more aggressive, sexually attractive and resistant to stress than those with lighter ones (Ducrest et al. 2008).
However, compared with the well-studied carotenoid–based coloration, the function of melanin-based traits to their bearers is less understood.
To human eyes, melanin-based ornaments are usually just dull colored (i.e. black or brown), and are less varied within and between species than carotenoid-based ornaments (Badyaev and Hill 2000). However, the extra cones and oil drops that birds have make them have better color
discrimination than humans (Griffith et al. 2006, Jawor and Breitwisch 2003, McGraw 2006). Traditionally, avian melanin-based plumage patterns are characterized by patch size or darkness ranked by human eyes (e.g., Bize et al. 2006), but such methods may highly underestimate variation in darkness and severely hamper our understanding of the functions of melanin-based traits. Fortunately, with the aid of a spectrometer, subtle differences in melanin-based coloration can be quantified, and results indicate that the variation in melanin-based coloration within and between species can be just as much as in
carotenoid-based colorations (e.g., Hill and Brawner 1998).
Animals can synthesize melanins from the aromatic amino acids, phenylalanine and tyrosine. Melanization can be highly heritable (Hill and McGraw 2006, Jawor and Breitwisch 2003). In the past, variations in melanin-based traits were believed mainly to be genetically controlled (Bize et al. 2006). However, more studies suggest that these traits could be condition-dependent: factors such as parasitic infections and diet quality can also influence the level of expression of melanin-based signals (e.g., Fargallo et al. 2007, Jacquin et al. 2011). In particular, oxidative stress could play a critical role in regulating melanization (e.g., Roulin et al. 2008). High oxidative stress, which results from the
imbalance between the rate of production of reactive oxygen species (including free radicals) by cell metabolism and the state of repair and antioxidant machinery, can induce ageing and reduce life span (Finkel and Holbrook 2000). The process of melanization could reduce the concentration of free radicals, making melanin an important antioxidant (Ducrest et al. 2008). However, the correlation between oxidative stress levels and the coloration of melanin-based traits are still controversial in birds (e.g., Ducrest et al. 2008, Galván and Alonso-Alvarez 2009). In the current study, I studied the correlation between the brightness of black plumage and individuals’ oxidative stress in Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus) to test whether melanin-based plumage could be an indicator of the individuals’ physical condition.
The Himalayan black bulbul is widely distributed in Taiwan’s broad-leaf forests at elevations from 100 m to 1500 m. Their plumage coloration is entirely melanin-based; black plumage with grey patches on
the scapular feathers and remige. It makes the Himalayan black bulbul a perfect system to investigate the function of melanin-based signaling. In theory, the process of melanin production should reduce the sensibility of stress-regulation processes and result in lower oxidative stress (Ducrest et al. 2008); therefore, I expected a negative correlation between an
individual’s oxidative stress levels and its black plumage coloration, with brighter individuals (i.e. more melanins) under lower oxidative stress.
The results of this study should enhance knowledge about the role of melanin-based coloration in avian communication.
Materials and methods
Study species and captive setting
In total, 18 and 48 Himalayan black bulbuls were bought from a pet shop in Taipei in 2010 and 2011 respectively. Each bird was housed
individually in the laboratory. I collected 150 μL blood from each
individual for molecular sex typing. A drop of blood, approximately 5 μL, of each bird was used as the blood smear for the oxidative stress test.
Molecular sex typing
Genomic DNA was extracted from blood samples with traditional proteinase K digestion followed by LiCl extraction (Gemmell and Akiyama 1996). The detailed programing of polymerase chain reactions (PCRs) for molecular sex typing (Fridolfsson and Ellegren 1999) was the same as in Hung et al. (2015 in revision). I identified 8 females and 10 males in 2010 and 18 females and 30 males in 2011.
Coloration measurements
For each individual, the reflectance of eight regions of melanin-based plumage- including the forehead, nape, back, breast, belly, tail, remige and scapular feathers- was measured using an USB2000 spectrometer (Ocean Optics) with a HL2000 deuterium-halogen light source (Ocean Optics).The measuring procedures were as in Hung et al. (2015 in revision). The brightness of each region was defined as the average of total reflectance within the range of 300-700nm. The data used in this study were extracted and reanalyzed from the data in Hung et al. (2015 in revision). (McGraw et al. 2005) demonstrated that the levels of both eumelain and phaeomelanin concentration in feathers were significantly and positively correlated with brightness. According to their results, a higher brightness indicates more accumulation of melanin in the feathers Oxidative stress test
I calculate the ratio of heterocytes (H) to lymphocytes (L) in total 100 leukocytes as a measure of oxidative stress for each bulbul individual following the methods used by Vleck et al. (2000). The blood smear was first dyed with a Wright-Giemsa stain for 3 min and then with 5% PBS for 70 sec. After drying, the numbers of lymphocytes and heterocytes were counted under a microscope at a magnification of 100X with oil immersion and the H/L ratio calculated. The H/L ratio has been proved to be a good indicator of oxidative stress: higher levels of corticosterones can cause higher oxidative stress and also increase the number of heterocytes in the blood (Davis et al. 2008, Gross and Siegel 1983).
Because leukocyte numbers change more slowly in response to stress than corticosterone does (Maxwell 1993), H/L ratios provide a more useful and accurate measure of long-term stress than a single measure of
plasma corticosterone (McFarlane and Curtis 1989, Vleck et al. 2000).
Statistical analysis
Multiple regression was used to test whether the brightness of each body part correlated with oxidative stress (H/L ratio) while taking into account the birds’ sex and the interaction between sex and oxidative stress. In the tests, the H/L ratio was log transformed to fit the normal distribution.
Two-way ANOVA was conducted to test whether each individual’s oxidative stress and the brightness of each its body parts differed significantly between years. In these tests, sex, year and the interaction between sex and year were taken into account.
Results
The average H/L ratio in 2010 was 0.35 ± 0.05 and ranged from 0.1 to 0.4 while the average H/L ratio in 2011 was 1.05 ± 0.13 and ranged from 0.2 to 4.6. Individuals in the 2010 group were under significant lower oxidative stress (higher H/L ratio) than those in the 2011 group (Fig. 4.1, two-way ANOVA, F= 15.36, p=0.0002). The females’ H/L ratio was 0.76
± 0.15 and the males’ was 0.92 ± 0.14; there was no significant difference of H/L ratios between the two sexes (Fig. 4.1, two-way ANOVA, F= 0.73, p=0.72).
The brightness of melanin-based colors differed significantly between Himalayan black bulbuls in the 2010 and 2011 groups (data extracted and reanalyzed from (Hung et al. 2015), in revision). Members of the 2011 group were brighter (i.e. more melanins) than those of the 2010 group, mainly in belly (two-way ANOVA, Table 4.1; Ls mean± SE, 2010 group
4.45 ± 0.31%, 2011 group 5.34 ± 0.19%, post-hoc (student’s t) test, CL:
-1.61; -0.1) and breast (Table 4.1; 2010 group 3.22 ± 0.21%, 2011 group 3.82 ± 0.13%, post-hoc (student’s t) test, CL: -1.10; -0.11). The same tendencies were observed at the remige and scapulars, although there were significant interactions with two factors- sex and year (two-way ANONA Table 4.1).
The results of multiple regression indicated brighter breast and scapular feathers might be related to lower oxidative stress: there was a significant negative correlation between the brightness of breast and scapulars and oxidative stress (multiple regressions, Table 4.2; linear correlation coefficients in breast, rfemale= -0.23, rmale= -0.47; correlations in scapulars, rfemale= -0.54, rmale= -0.23), but not between oxidative stress and the brightness of the other six body parts. However, this relationship only applied in 2011 and not in 2010: the H/L ratio was not significantly correlated with the brightness of any melanin-based parts (multiple regressions, Table 4.2).
Discussion
My study shows that melanin-based characteristics could be duller in individuals that suffered higher oxidative stress. It consists with the work of Ducrest et al. (2008). They reviewed six studies and discovered that there is significantly negative correlation between the expression of melanin-based characteristics and the level of oxidative stress. It implies that expression of melanin-based traits is condition-dependent and could be the quality cue, which can reflect individual’s physical condition (Hill
and McGraw 2006), in the focal species.
However, the significant overall negative relationship between coloration and oxidative stress was not found within any given year. In contrast, individuals in 2011 suffered higher oxidative stress but had brighter plumage (i.e. more melanins). Several recent studies also report similar, positive correlations between oxidative stress and melanin-based coloration (Galván and Alonso-Alvarez 2008, Galván and
Alonso-Alvarez 2009, Hõrak et al. 2010), suggesting that an alternative hypothesis about a different interaction between melanin coloration and oxidative stress should be considered in the role of melanin-based signaling.
Glutathione (GSH), another key intracellular antioxidant, has been suggested to inhibit eumelanogenesis and eumelanin-based black
ornaments, and might be crucial to the expression of melanin-based traits (Benedetto et al. 1982). GSH is a tripeptide thiol found in virtually all animal cells, and often considered as a vital antioxidant. It functions in the reduction of the disulfide linkages of proteins, in the synthesis of the deoxyribonucleotide precursors of DNA and in the protection of cells against free radicals (Meister 1983). GSH also serves as an agent regulating the process of melanogenesis. Low GSH levels have been associated with the deposition of melanin in bird feathers, whereas high GSH levels inhibit melanogenesis (Galván and Alonso-Alvarez 2008, Galván and Alonso-Alvarez 2009, Hõrak et al. 2010). Hence, GSH might play a role in trade-offs between antioxidants and melanogenesis. I suspect this might be the case in the black bulbuls: individuals in 2011 might had lower GSH than those in 2010, therefore had brighter plumage
but higher oxidative stress.
The different correlation between melanin-based ornaments and oxidative stress in two years- I only discovered a significant relationship in the 2011 group, not in the 2010 group- might also provide support for the GSH hypothesis. A study in rats suggested that corticosterone could decrease glutathione levels (Patel et al. 2002). In my study, individuals in 2011 suffered almost three times the oxidative stress of those in 2010, there it might be expected a relatively lower level of GSH in 2011 than in 2010. With a lower level of GSH, individuals could enhance the darkness or the size of patch of melanin-based ornaments (Galván and
Alonso-Alvarez 2008, Galván and Alonso-Alvarez 2009, Hõrak et al.
2010). Therefore the positive correlation between oxidative stress and coloration of melanin-based traits would be expected.
The status of oxidative stress I test here should not reflect the physical condition in real time or during molting, but is rather the long-term condition of an individual. Such long-term oxidative stress could be affected by many factors: genetic, environmental or different life-history stage (Monaghan et al. 2009). Therefore, black bulbuls might be able to use melanin-based coloration to evaluate individual’s long-term physical condition. Consequently, the traits might be important in sexual selection or individual assessment in animal aggressive behavior in the Himalayan black bulbul.
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Table 4.1. Two-way ANOVA tests of brightness in eight body parts
1Factor Year: including 2010 and 2011
2Df=1
*p < 0.05
Table 4.2 Multiple regressions of brightness in different body parts in Belly Intercept 6.09 <.0001 24.15 <.0001
Sex(F) -1.37 0.194 1.53 0.134 Log (H/L ratio) 0.78 0.447 -1.35 0.183 Sex(F)*Log (H/L ratio) -0.85 0.409 0.35 0.728 Breast Intercept 9.00 <.0001 24.53 <.0001
Sex(F) 0.65 0.525 0.93 0.358 Scapulars Intercept 8.07 <.0001 40.47 <.0001
Sex(F) -2.22 0.043* 0.03 0.976
Log (H/L ratio) -1.01 0.329 -2.37 0.022 * Sex(F)*Log (H/L ratio) -0.76 0.461 -0.93 0.358 Remige Intercept 5.70 <.0001 49.39 <.0001
Sex(F) -2.41 0.032* 0.59 0.555 1 Log transformation of heterocytes / lymphocyte ratio due to the
non-normal distribution of original H/L data.
*p < 0.05
Figure 4.1. The Log (H/L ratio) of two sexes in different years. The circles indicate the average Log (H/L) ratios for 2011 and the triangles indicate those for 2010. The lines indicate the Standard Error. The solid symbols represent the females and the hollow ones represent the males.
Two-way ANOVA, factor year includes 2010 and 2011. Df =1, *p <
0.05
Log (H/L ratio)
Sex F=0.13, p=0.715 Year F=15.36, p=0.0002*
Sex*Year F=0.004, p=0.835