Abstract
Traits that compete limited internal resources against other somatic needs could lead to trade-offs of resource allocations between them; their expression could therefore be correlate. Because the amount of carotenoids available for use is limited and carotenoids can be used in many physical functions, trade-offs arising among carotenoid-based ornaments and other physical demands. Molting is energetically and nutritionally demanding, but the effects of molting on major somatic needs are vague and contentious. In this study, I aimed to investigate whether carotenoid-based ornaments are quality cues that could reflect individuals’ physical condition following severe physical stress, namely molting induced by artificial plucking in Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus). I conducted a 2×2 grouping design experiment involving plucking and carotenoid supplementation.
My data showed that bill redness was positively correlated with an individual’s immunocompetence and negatively correlated with oxidative stress level it experienced, but such correlations were not found in the carotenoid-supplemented group. My results suggest that the bill is a quality cue in black bulbuls, and additional carotenoids would reduce the negative effect of molting on both decorative traits and physical
condition.
Keywords: carotenoid-based ornaments, Heterocyte/liphocyte ratio, Himalayan black bulbul (Hypsipetes leucocephalus nigerrimus), molting, quality cues, PHA test.
Introduction
Competition for limited internal resources among different functions could lead to life history trait trade-offs, which may occur between physiological traits expressed either during the same stage or different stages of the life cycle (Zera and Harshman 2001). Signal traits, which could reflect individuals’ physical condition or agonistic abilities, are usually costly to their bearers because these traits share resources with other physically demanding ones (e.g., Spencer et al. 2003). Consequently, trade-offs between variable life-history traits and those that could serve as signal traits are to be expected. Among different types of signal traits, carotenoid-based ornaments are the most studied till date.
Besides their ability to producing striking colors (i.e. yellow, orange and red, Fox and Vevers 1960, Latscha 1990), their powerful antioxidant properties also make carotenoids crucial to several life-history traits like breeding (McGraw et al. 2005, Surai 2002), migration (Alan et al. 2013, Metzger and Bairlein 2011) and molting (e.g., McGraw et al. 2006).
Nevertheless, animals can only acquire carotenoids from food, and the ability of an animal to utilize the carotenoids in food depends on its genotypes and physiological condition (Alonso-Alvarez et al. 2004, Olson and Owens 1998). It has been suggested that the quantities of carotenoids available to individuals are limited, although carotenoids may be abundant in the natural environments (Olson and Owens 1998). Due to the limited availability of carotenoids, individuals may have to trade-off carotenoids among different life history traits (Alonso-Alvarez et al.
2004). For instance, females may have to balance the benefits of
carotenoids with those investing in offspring quality, or of impairing their
own antioxidant damage during reproduction (Bertrand et al. 2006, Biard et al. 2005). In migratory birds, early-arriving males, who usually have a longer reproductive season and greater reproductive success (e.g., Klomp 1970, Perrins 1970), may be strong enough to allocate carotenoids into coloration rather than use them as antioxidants during migration (Ninni et al. 2004). In summary, the appropriate allocation of carotenoids among life traits and physiological functions (i.e., decorative plumage and antioxidation) are necessary in order to maximize individual fitness (e.g.
Bertrand et al. 2006, Biard et al. 2005, Faivre et al. 2003, Nordeide et al.
2008).
Molting is energetically and nutritionally demanding (Jenni and Winkler 1994, Klaassen 1995, Kuenzel 2003, Lindstrom et al. 1993) and would decrease the accumulation of circulating plasma carotenoids (Barbosa et al. 2013, Del Val et al. 2014, Del Val et al. 2013). However, to the best of my knowledge, studies that addressing whether molting can affect the expression of carotenoid-based characteristics have been rare.
The only relevant study showed that molting speed constrains the expression of yellow throat in the rock sparrows (Petronia petronia, (Serra et al. 2007). Meanwhile, the effect of molting on the expression of carotenoids on the bared ornaments like bill, tarsus, or dewlap is also unknown. It is suggested that the expression of carotenoid-based bared parts could reflect an individual’s physical condition faster than that of plumage (e.g. Ardia et al. 2010, Faivre et al. 2003). The effects of molting on major somatic functions are vague and contentious. With
immunocompetence, an indicator of the individual’s immune response, molting has either positive (Sanz et al. 2004) or negative (Martin 2005,
Sanz et al. 2004) correlations, or none at all (Pap et al. 2008). Although it is suggested that oxidative stress is higher during or after molting due to the depletion of plasma carotenoids, the effects of molting on an
individual’s oxidative stress level has not been tested directly (Del Val et al. 2013). To the best of my knowledge, only one study has found that individuals’ oxidative stress levels did not change by natural molting in great tits (Parus major, Vaugoyeau et al. 2015). Knowledge of the effect of carotenoid abundance on molting is lacking; therefore, I would like to know whether the effect of molting can be diminished by carotenoid supplementation, because carotenoids are powerful antioxidants.
In this study, I aim to investigate whether carotenoid-based ornaments are the quality cues that reflect individuals’ physical condition after a severe physical stress. I used Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus), which have carotenoid-based bills and tarsi, as the study species to examine whether the expression of
carotenoid-based characteristics and individuals’ physical condition (both immunocompetence and oxidative stress level) could be affected under the stress of molting. Traditionally, the study of molting effects in birds involves the natural induction of molting, but this may be inappropriate under the current study conditions. Because both natural molting and carotenoid allocation can be influenced by hormones, such as testosterone, thyroxine, or corticosterone (Cherel et al. 1988, Rehder et al. 1986), it would be difficult to control the confounding effects of hormones when conducting experiments based on natural molting. I therefore plucked feathers directly from study animals to initiate molting artificially. A 2×2 grouping design was utilized within the carotenoid supplementary group,
tail and secondary feathers were pulled off on half of the birds, while the other half of them served as a control group with their feathers intact. I predicted that molting would have negative effects on the physical condition and cause trade-off of carotenoids allocation in black bulbuls;
therefore, I should have observed a significant trade-off between
decorative traits and individuals’ physical condition in the molting group.
I also predicted that the effects of molting could be diminished when the carotenoids were in abundant supply.
Materials and methods Captive setting and sampling
Thirty-one Himalayan black bulbuls were purchased from a pet-shop in 2010. They were individually housed in cages with opaque covers
between them, so that the birds could have acoustic contact but not visual perception of others. They were placed in a room at constant temperature (27℃) and humidity (80%). Blood (150 μL) was collected from each bird using a heparinized capillary tube. Blood samples were stored in a −20°C freezer for molecular sex-typing. In addition, a drop of blood,
approximately 5μL, was put on a slide to assay the oxidative stress level.
Molecular sex typing
Genomic DNA was extracted from blood samples with traditional proteinase K digestion followed by LiCl extraction (Gemmell and Akiyama 1996). The polymerase chain reactions (PCRs) program used for molecular sex typing (Fridolfsson and Ellegren 1999) was the same as
that described in Hung et al. (2015 in revision).
Experimental procedures Experimental design
Thirty-one birds were used in this experiment. After sex typing, individuals of each sex were randomly and equally assigned to four treatment groups: carotenoid-supplemented and plucked [Caro(+),
plucked, F = 4, M = 4], carotenoid-supplemented and unplucked [Caro(+), unplucked, F = 2, M = 4], carotenoid-unsupplemented and pluckded [Caro(-), plucked, F = 3, M = 4], or control [Caro(-), unplucked, F = 5, M
= 5]. In the plucked group, individuals’ tails and secondary feathers had been removed. Carotenoid supplementation was initiated on February, 10, 2010, a month prior to the molting experiment. The molting experiment was initiated on the March, 8, 2010, this day also served as the baseline for the immune challenge experiment. During the experimental period, only commercial feed formulated with or without supplementary
carotenoids was fed. Oxidative stress testing was conducted on two dates:
the baseline day, on which tails and secondary feathers were removed and the testing date, on which the tails and secondary feathers of individuals in the experimental group had grown to half of their average length (approximately three weeks later, around 5 to 5.5 cm for tail feathers and 1.5 to 2.0 cm for secondary feathers). The immune challenge was
conducted only on the testing day.
Carotenoid-supplementation experiment
Each individual was fed 40 grams of commercial bird food every day. In the carotenoid-supplemented groups, the additional canthaxanthin (0.9
mg/40 g, Orpharma, Belgium) and lutein (0.32 mg/40g, Orpharma, Belgium) were added to the commercial bird foods.
Physiological conditions
Immune challenge: Phytohaemagglutinin assay (PHA assay)
The PHA assay was conducted on May, 26,2010. PHA solution (80μl of 2.5ng/μL, Sigma L-1668, Sigma Chemical Co., St. Louis, Mo, USA) was injected into individuals, according to the method of Smits et al. (1999).
A pressure-sensitive caliper (TecLock Inc., Japan) was used to measure the thickness of the right wing web before injection to the nearest 0.01 mm. Twenty-four hours after the injection, the level of swelling was measured on the wing web of each individual to determine the degree of immune response (Smits et al. 1999). Individuals with larger swollen wing webs were considered to have stronger immune responses.
Oxidative stress test: Heterocyte to lymphocyte ratio (H/L ratio) I calculated the ratio of lymphocytes (L) to heterocytes (H) in a total of 100 leukocytes as a measure of oxidative stress for each bulbul individual, according to the method of Vleck et al. (2000). The detailed procedures were the same as that described by Hung et al. (2015 in revision). A higher H/L ratio indicates that the individual were under a higher level of oxidative stress (Gross and Siegel 1983). The assay of oxidative stress was conducted on the baseline and testing dates.
Molting speed measurements
To test whether carotenoid supplementation affected molting speed, I measured the regrowth lengths of tail and secondary feathers on the testing date in the plucked groups, then divided by 21 days to determine each individual’s molting speed.
Statistical analysis
Multiple regression analyses were conducted to test whether bill coloration was correlated with the individuals’ physical condition, factors, such as sex, plucking treatment and carotenoid supplement treatment, were included in the analysis. In the test of whether molting would affect an individual’s condition, due to the interaction effects of plucking and carotenoid supplement treatments that my analysis
uncovered, factors including sex, plucking treatment and their interaction were used in Caro(+) and Caro(-) groups respectively.
Results
My data showed that the carotenoid supplementation could affect an individual’s physical condition in Himalayan black bulbuls. Coloration comparisons between the Caro(+) and Caro(-) groups before
feather-plucking showed that bill coloration in the Caro(+) group was significant redder than that of the Caro(-) group (Hue mean±se, Caro(+) 591.16 ± 1.56 nm, Caro(-) 587.02 ± 1.11, two-way ANOVA, t=2.58, p=0.01). My data also showed that the negative correlations between plucking, bill coloration and the individual’s physical condition were significant in the group without carotenoid supplementation. In the Caro(-) group, individuals in the plucked group had lower wing web swelling after PHA injection (Table 3.1 a, plucked (mean±SE) 1.24 ± 0.21%, unplucked 1.96 ± 0.21%, multiple regression, t = -2.42, p = 0.039) and higher H/L ratios (Table 3.1 b, plucked 0.55 ± 0.16 %, unplucked 0.08 ±
0.12 %, multiple regression, t = 2.26, p = 0.049); they also had paler bills (Table 3.1c, plucked 581.3±1.7nm, unplucked 587.2 ± 1.7, t = -2.45, p = 0.03). However, in the Caro(+) group, there were no inter-individual differences within plucked or unplucked groups in any physical indicators (Table 3.1). These data suggest that adding carotenoids buffers the
negative effect of molting in Himalayan black bulbuls. I also found that, regardless of plucking, carotenoid supplementation was associated with an increase in the intensity of the red bill coloration (Ls mean ± SE, Caro(-) 584.3 ± 1.9, Caro(+) 601.1 ± 1.9, two-factor ANOVA, Fplucking treatment =1.46, p = 0.24, Fcarotenoid treatment = 39.31, p < 0.0001,
Fplucking*carotenoid= 0.70, p = 0.41); again, these data suggest that the carotenoid supplementation enhances the red bill coloration. However, I found no effect of carotenoid supplement on molting speed in either secondary or tail feathers (the secondary: Molting SpeedCaro(+)= 0.53 ± 0.11 cm/day, Molting SpeedCaro(-) 0.52 ± 0.11 cm/day, t = -1.07, p = 0.30;
Tail: Molting SpeedCaro(+) = 0.20 ± 0.08 cm/day, Molting SpeedCaro(-) = 0.26 ± 0.01 com/day, t = -0.51, p = 0.62 ).
I found that regardless of carotenoid supplementation, individuals with redder bill had more severe swelling in the PHA test (Fig. 3.1, multiple regression, estimatecarotenoid treatmet -7.45 ± 1.31, t = -5.66, p <
0.0001; estimateswelling 5.61 ± 2.45, t = 2.28, p = 0.03) and lower H/L ratio (Fig. 3.1, estimatecarotenoid treatmet -7.87 ± 1.27, t = -6.21, p < 0.0001;
estimateLog (H/L ratio) -6.73 ± 2.81, t = -2.39, p = 0.02), indicating that individuals with redder bills had better immunocompetence and were
under lower oxidative stress, relative to those with less intensely colored bills.
Discussion
My data clearly demonstrate that the bill coloration can serve as a quality cue in the Himalayan black bulbuls: the redness of the bill is positively correlated with an individual’s immunocompetence and negatively correlated with the oxidative stress level. I also propose that the addition of carotenoids can reduce the effect of molting on both decorative traits and individuals’ physical condition in the focal species.
According to the communication theory, informative cues may be perceived and function in animal communication (Dale 2006, Stevens 2013), particularly when such cues could reflect individuals’ qualities (Senar 2006). Hence, bill coloration may play essential roles in the communication of Himalayan black bulbuls. Previous data showed that the bill coloration alone may not be a sexually selected cue in the focal species (Chapter Five). A recently published paper suggested that the carotenoid-based bill should be more likely to function as a social signal, as opposed to asexually selected trait (Dey et al. 2015). Unlike feathers, in which carotenoids are deposited during molting, the outer layers of the bill are continuously replaced; therefore bill coloration could reflect a more recent physical condition of its bearer. It is more useful for animals that constantly live in groups to use fast-reacting signals in social
interactions, such as the assessment of rivals or the formation of social rankings, than in choosing a mate. Himalayan black bulbuls are socially
monogamous during the breeding season but live in groups during the nonbreeding season. Therefore, it is possible that bill coloration may play an important role in their social interactions, because the presence of social signals could be very beneficial for them to diminish the frequent conflicts that occur in their collective society.
I have clearly demonstrated that molting would have negative effects on individuals’ physical conditions and carotenoid-based ornaments. My data suggest that there may be trade-offs of carotenoids occurring between molting and these traits. The trade-offs between the immune response and molting have been found in several avian species (e.g.
Kuenzel 2003, Sanz et al. 2004). These trade-offs may occur for various of reasons: first, as noted above, both molting and immune activities impart high resource demands. Martin (2005) found that both activities would increase metabolic rates in house sparrows (Passer domasticus).
Other lines of evidence indicate that trade-offs between molting and immune activity may also be driven by the availability of protein (e.g.
Lochmiller and Deerenberg 2000) or particular hormones, such as prolactin (Kuenzel 2003). My data provide direct evidence that molting would increase individuals’ oxidative stress. To the best of my knowledge, this is the first study to provide such evidence. My results also provide evidence that molting decreases the expression of carotenoid-based bared parts. The reasons for the negative effects of molting on these traits have not been tested; however, I suspect that it may be due to competition to the limited resource - carotenoids.
In this study, I did not find the molting effect when the carotenoid supplement was added. A study of molting in gentoo penguins
(Pygoscelis papua) showed that plasma carotenoid could be depleted during molting within a week (Barbosa et al. 2013), suggesting that carotenoids could be used rapidly during molting. Therefore, a sufficient supply of carotenoids could decrease the effect of molting on the
carotenoid content in our species. Similar results were also found in the study of European starlings (Sturnus vulgaris, Casagrande et al. (2015);
they discovered that birds receiving extra carotenoids kept singing even during the sickness phase induced by inflammation. Aside from
carotenoids, such trade-offs could also be influenced by nutritional quality; reduced nutrient availability can substantially magnify an apparent trade-off, while increased nutrient availability can diminish or obviate it (Zera and Harshman 2001).
In conclusion, my data provide evidence that carotenoid-based bill coloration is informative and may play roles in animal communication in this species. These results also provide direct evidence for a mechanism by which molting would influence several crucial physical traits.
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