| Citation: |
Danqi Cui, Na Wang, Jingru Ge, Jiaying Xu, Weihong Zheng, Jinsong Liu. 2019: The role of temperature as a driver of metabolic flexibility in the Red-billed Leiothrix (Leiothrix lutea). Avian Research, 10(1): 46. DOI: 10.1186/s40657-019-0184-3
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The thermoregulatory ability of animals is strongly influenced by the temperature of their environment. Acclimation to cold requires a range of physiological and morphological adjustments. In this study, we tested the hypothesis that a small passerine, the Red-billed Leiothrix (Leiothrix lutea), can maintain homeothermy in cold conditions by adjusting the physiology and biochemistry of its tissue and organs and return to its former physiological and biochemical state when moved to a warm temperature.
Phenotypic variation in thermogenic activity of the Red-billed Leiothrixs (Leiothrix lutea) was investigated under warm (35 ℃), normal (25 ℃) or cold (15 ℃) ambient temperature conditions. Oxygen consumption was measured using an open-circuit respirometry system. Mitochondrial state-4 respiration and cytochrome-c oxidase (COX) activity in liver, kidney heart and pectoral muscle were measured with a Clark electrode.
Birds acclimated to an ambient temperature of 15 ℃ for 4 weeks significantly increased their basal metabolic rate (BMR) compared to a control group kept at 25 ℃. Birds acclimated to 35 ℃ decreased their BMR, gross energy intake (GEI) and digestible energy intake (DEI). Furthermore, birds acclimated to 15 ℃ increased state-4 respiration in their pectoral muscles and cytochrome-c oxidase (COX) activity in their liver and pectoral muscle, compared to the 25 ℃ control group. Birds acclimated to 35 ℃ also displayed lower state-4 respiration and COX activity in the liver, heart and pectoral muscles, compared to those kept at 25 ℃. There was a positive correlation between BMR and state-4 respiration, and between BMR and COX activity, in all of the above organs except the liver and heart.
Our study illustrates that the morphological, physiological, and enzymatic changes are associated with temperature acclimation in the Red-billed Leiothrix, and supports the notion that the primary means by which small birds meet the energetic challenges of cold conditions is through metabolic adjustments.
As a consequence of the excessive discharge of pollutants from industrial production and other human activities, large amounts of pollutants have made their way into the environment. These accumulate in wetland ecosystems through the effect of runoff and are then transmitted along the food chain through the trophic relations of organisms (Furness and Greenwood, 1993; Kim and Koo, 2007). Recently, colonial waterbirds have attracted much interest because of their capacity to introduce large amounts of wetland-derived nutrients to land (Anderson and Polis, 1999; Garcia et al., 2002; Ellis, 2005). Ardeid birds are the top-level predators in wetland ecosystems, exhibiting behavioral characteristics of colonial breeding and perhaps exerting an enriching effect on pollutants.
Previous studies have found that during the course of colonial breeding, the feces of parent birds and their offspring, food dropped while parent birds feed their offspring, the bodies of dead birds, feathers being shed and the shells of abandoned eggs carry organic matters to the breeding grounds, causing changes in soil properties of the colonial breeding grounds (Hogg and Morton, 1983; Bukacinski et al., 1994; Li et al., 2005). When heavy metal contamination of nesting soils exceeds the limit of tolerance of plants, the absorption and metabolism of these plants will be disrupted and some contaminants will end up inside the plants, affecting their growth, cause serious soil contamination and may even result in the death of plants (Anderson and Polis, 1999). Furthermore, heavy metal contamination of soils may drive plant species turnover in the nesting habitat after a few years, forcing birds to change their breeding location (Vidal et al., 2000; Li et al., 2005). However, not much research has been conducted on the accumulation of heavy metal contamination in soils as a result of colonial breeding in birds (Headley, 1996; Otero, 1998).
In this study, we aim specifically to investigate: 1) what are the concentrations of the heavy metals (V, Cr, Mn, Ni, Cu, Zn, Cd, Se and Pb) as well as of semi-metal element (As) in the feces of Chinese Egret (Egretta eulophotes, Plates Ⅰ and Ⅱ), which has been listed as a vulnerable species with an estimated global population of 2600–3400 birds (IUCN, 2009); 2) whether differences can be found between the topsoil of their nesting and non-nesting areas on islands before and after breeding and 3) if a colonial breeding of E. eulophotes is likely to have an effect on the accumulation of heavy metals in its heronry soil.
Samples of feces and soil were collected from the heron colony of the endangered Chinese Egret on Caiyu Island in Zhangpu County, Fujian Province, China (23°46'N, 117°39'E). The distance of the uninhabited Caiyu Island from the mainland is about 8 km. This uninhabited island is an ideal place for studying the impact of the colonial breeding of birds on the concentration of heavy metals in its soils since the island is relatively far off the coast and the concentration of elements in its soil is not subject to the impact of agriculture and other human activities. The nearby mainland areas of this island are characterized by several artificial wetland habitats, including seawater culture ponds of shrimp and fish, rice fields and various sloughs and ditches. The breeding waterbird species at Caiyu Island are Egretta eulophotes and E. sacra of the Ardeidae family, Larus crassirostris of the Laridae family and Sterna anaethetus and S. sumatrana of the Sternidae family. E. eulophotes nests on trees in the center of the island and the other four species nest on the rocky areas around the edge of the island, thus making the E. eulophotes to form a separate heron colony.
Sampling was conducted on Caiyu Island in mid-April, 2008 before the Ardeidae birds started breeding and in mid-August of the same year after the parent birds and their offspring had left their nests. Feces samples of E. eulophotes were collected from the trunks of 10 nesting trees on the island during the 2008 breeding season.
For soil sampling, we designated 10 randomly selected breeding nest trees at the E. eulophotes nesting habitat as 10 sample locations. These 10 trees largely covered the entire nesting habitat. Nine soil sampling points were established under each tree according to a checkerboard distribution method for soil extraction. The checkerboard was 100 cm long and 100 cm wide. At each soil sampling point, a small stainless steel spade was used to extract approximately 0.25 kg of topsoil (about 0 to 5 cm deep). Each soil sample was sealed in a numbered bag and taken back to the laboratory. We also selected 10 non-nesting sample locations, corresponding to nesting sample locations in terms of properties (elevation, soil color, soil grain size and vegetation cover) and conducted sampling using the same method.
The instruments used included a Muffle furnace (Tianjin Northern China Lab Instruments Company), a high-pressure digestion vessel, an oven, PE DRC-e Inductively Coupled Plasma Mass Spectroscopy (ICP-MS), a constant-temperature drying oven and ultra-pure water equipment (Millipore, France). The reagents used were a mixed standard solution, nitric acid (GR), ultra-pure water (Millipore), ultra-pure water and acetone.
Soil samples were spread on clean sheets of kraft paper for drying and removal of gravel, root residues and other impurities. After being compacted with a mallet, the samples were extracted using a quartering method (Li et al., 1998). Samples were then ground in a mortar before being screened with a 20-mesh nylon sieve. Afterwards, the soil samples were spread and grouped into a few cells. Then, the samples, totaling approximately 200 g, were taken from multiple points using a medicine spoon. The samples were ground again and screened with a 100-mesh nylon sieve before being sealed in a bag. The E. eulophotes feces was dried in the constant-temperature drying oven and screened with a 100-mesh nylon sieve.
In the digestion of soil samples, 0.1 g soil samples were placed inside the high-pressure digestion vessel, where 5 mL HNO3 and 1 mL HClO4 were added. The vessel was tightly recapped and placed in the oven, where it was heated at 100℃ for an hour and 120℃ for four hours. Afterwards, the tightly-sealed digestion vessel was reopened and the samples were removed and placed into clean 50 mL PET bottles. The digestion vessel and its cover were cleansed in ultra-pure water three to four times and the cleansing solution was put into the volumetric flask to a level of 50 mL. A sample was also prepared for purposes of contrast.
In the digestion of feces samples, 0.2 g samples were placed into a crucible and 2 mL of HNO3 was added to the crucible, which was then placed into the Muffle furnace for heating at 120℃ for four hours. Afterwards, the samples were removed and another 2 mL HNO3 was added. The samples were then placed in a clean 50 mL volumetric flask where they were rinsed with ultra-pure water; in the end, the samples were filtered and the flask filled to a level of 50 mL.
Before the analysis of the elements, standard curves were formulated. Mixed standard solutions were progressively digested with ultra-pure water to 10, 20 and 40 ppb to obtain a series of mixed standard solutions. A series of blank and standard solutions was collected under optimized instrumental conditions, with the instrument automatically drawing standard curves. The correlation coefficient of the standard curves of the elements, i.e., r, was greater than 0.9995 in all cases.
The concentration of heavy metals was measured using inductively coupled plasma-mass spectrometry (ICP-MS). We established the following ICP-MS analytical procedure: given the property of self-absorption of the PFA micro-flow centrifugal atomizer, the digestion solution of samples was atomized and then the plasma was entered. The analytical procedure was then activated to measure the signal strength of V, Cr, Mn, Ni, Cu, Zn, Cd, As, Se and Pb and their corresponding concentrations were measured using the standard curves.
Statistical tests were performed using SPSS software, version 13.0. One-way ANOVA was used to detect any differences in the mean concentrations of heavy metals and other elements in the soil. Following the ANOVAs, Student-Newman-Keuls (SNK) tests were used for pairwise multiple comparisons. Significance was accepted at the 0.05 probability level.
We did not detect any Se or Cd in the nesting or non-nesting soils before or after breeding. As shown in Table 1, a comparison of the data from the non-nesting soils before and after breeding reveals no significant difference in heavy metal concentrations in the non-nesting soils before and after breeding. Another comparison from the nesting soil before and after breeding shows that all elements in the soil had a higher concentration after breeding than they did before breeding and that there was a highly significant difference (p < 0.01) in the concentrations of Zn and Pb before and after breeding.
| Sample | V | Cr | Mn | Ni |
| Before-non | 0.678±0.191 | 32.334±37.333 | 92.484±24.407 | 3.498±3.712 |
| After-non | 0.641±0.089a | 27.176±28.830 | 82.929±17.043 | 7.097±12.395a |
| Before-nest | 0.781±0.244 | 39.273±37.642 | 87.116±19.096 | 20.591±45.894 |
| After-nest | 0.868±0.190b | 121.653±303.890 | 102.349±40.525 | 68.105±112.834b |
| Sample | Cu | Zn | As | Pb |
| Before-non | 0.647±0.576 | 29.888±7.392a | 0.365±0.350 | 12.012±1.563a |
| After-non | 0.708±0.481a | 33.351±2.455ab | 0.245±0.245 | 12.739±1.637a |
| Before-nest | 1.874±0.930 | 39.248±2.240c | 0.498±0.267 | 12.871±1.759ab |
| After-nest | 3.136±3.002b | 45.742±2.351d | 0.756±0.383 | 15.309±1.500c |
| Note: "Before-non" stands for non-nest soil before breeding, "after-non" for non-nest soil after breeding, "before-nest" for nest soil before breeding, and "after-nest" for nest soil after breeding. The values with different letters in rows for means (± SD), represent significant differences (p < 0.05). | ||||
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A further comparison of the data from the nesting and non-nesting soils during the same period indicated that the nesting soil contained higher concentrations of heavy metals (except Mn) than non-nesting soils, both before and after breeding. Before breeding, the concentration of Zn was significantly greater in the nesting soil (p < 0.01) than in the non-nesting soil, while after breeding, the concentrations of V, Cu, Zn, As and Pb were considerably greater in the nesting soil (p < 0.01) than in the non-nesting soil. The concentration of Ni was also significantly greater in the nesting soil (p < 0.05) than in the non-nesting soil.
No Se or Cd was detected in E. eulophotes feces in this study. The concentrations of V, Cr, Mn, Ni, Cu, Zn, As and Pb in E. eulophotes feces are shown in Fig. 1. The highest concentration of Zn reached 424.292 ± 268.727 μg·g−1, while the concentration of Mn was 218.590 ± 112.298 μg·g−1, that of Pb 26.333 ± 9.370 μg g−1, of Cr 20.000 ± 37.223 μg·g−1, of Cu 7.038 ± 2.569 μg·g−1, of Ni 6.968 ± 5.245 μg·g−1, of V 0.037 ± 0.0562 μg·g−1 and the concentration of As was 0.014 ± 0.0439 μg·g−1.
Headley (1996) found that the feces of seabirds in the Arctic contained high levels of heavy metals and therefore drew the conclusion that the feces of seabirds was the primary reason for heavy metal contamination in breeding grounds. The concentrations of Zn, Mn and Cu in the feces of E. eulophotes in our study (424.292 ± 268.727, 218.590 ± 112.298 and 7.038 ± 2.569 μg·g−1, respectively) were higher than those in feces of Larus hyperboreus as measured by Headley (76.0, 39.3 and 6.3 μg·g−1). The concentrations of Zn, Mn and Pb (26.333 ± 9.370 μg·g−1) in our study were also higher than those in the feces of Rissa tridactyla as measured by Headley (the concentration of Zn, Mn and Pb stood at 176.0, 24.1, and 21.6 μg·g−1, respectively). However, the concentration of Ni (6.968 ± 5.245 μg·g−1) in our study was lower than that of L. hyperboreus (9.9 μg·g−1), while the concentrations of Cu and Ni in this study were also lower than those of Rissa tridactyla (51.2 and 8.4 μg·g−1). Compared with the heavy metal concentrations in Larus cachinnans on Cies Island (Otero, 1998), the concentrations of Zn and Cr (20.000 ± 37.223 μg·g−1) in the feces of E. eulophotes were higher than those in the feces of L. cachinnans (305.1 ± 158.5 and 9.8 ± 5.1 μg·g−1), whereas the concentrations of Cu and Pb in the feces of E. eulophotes were lower than those in the feces of L. cachinnans (60.1 ± 33.3 and 39.9 ± 5.8 μg·g−1, respectively). Some studies suggest that heavy metal concentrations in birds are related to their feeding habits, offspring rearing behavior and physiological characteristics and that heavy metals in feces are useless metals which have accumulated in their internal organs and are eventually expelled by various physiological means (Norheim, 1987; Rainbow, 1990; Elliot et al., 1992). The feces of E. eulophotes in this study contained higher levels of Zn, Cr and Mn, lower levels of Ni and average levels of Cu and Pb compared with previous reports (on the heavy metal concentrations in the feces of seabirds in the Arctic and Larus cachinnans on Cies Island). Whether these differences were caused by food, metabolism or other factors requires further study.
In this study, the concentration of elements in non-nesting soil did not show any significant differences before and after breeding, indicating that during the breeding season of Egretta eulophotes on the island, no external factors (such as absorption of plants, rainfall and soil runoff) had caused changes in heavy metal concentrations in the soils of the island. On the other hand, heavy metal concentrations in the nesting soil after breeding were larger than those in the same soil before breeding, with the concentrations of Zn and Pb showing a highly significant difference (p < 0.01). As such, one year colonial breeding of E. eulophotes may cause an increase of heavy mental concentrations in the nesting soil.
Furthermore, the concentrations of heavy metals (except Mn) were greater in the nesting soil than in the non-nesting soil, both before and after breeding. After breeding, the concentrations of V, Cu, Zn, As and Pb were greater in the nesting soil (p < 0.01) than in the non-nesting soil, with the concentration of Ni (p < 0.05) significantly greater in the nesting soil than in the non-nesting soil. This indicates that during the colonial breeding of E. eulophotes, heavy metals resulting from microbiological degradation of the feces of parent birds and their offspring, dropped food, dead birds, shed feathers and abandoned egg shells increased the heavy metal concentrations in the nesting soil. This is consistent with the findings of previous studies (Headley, 1996; Otero, 1998; Garcia et al., 2002). Before breeding, only the concentration of Zn was significantly greater in the nesting soil (p < 0.01) than in the non-nesting soil. This is consistent with the high concentration of Zn in the feces of E. eulophotes, indicating that the high concentration of Zn in the feces had resulted in an increased difference in the concentration of Zn in nesting and non-nesting soils after years of colonial breeding and Zn accumulation.
Taken together, it is clear that colonial breeding activities of E. eulophotes play an important role in the transfer of heavy metals between wetland and island ecosystems. However, the heavy metal concentrations on the island as measured in this study do not exceed the level specified in the Grade 1 standard in China's Environmental Soil Quality Standards (GB15618-95) (Xia et al., 1995). This perhaps can be explained by the small size of the E. eulophotes population on Caiyu Island (approximately 200 birds per year) and by the short history of documented E. eulophotes breeding on the island (no documentation until 2003). As such, it is necessary to conduct long-term monitoring to identify changes in the size of the colonial breeding of E. eulophotes and in heavy metal concentrations in the nesting soil in order to provide a scientific basis for the formulation of management strategies of the nesting soil of E. eulophotes.
This research was supported by the National Natural Science Foundation of China (Grant Nos. 40876077, 30970380) and the Fujian Natural Science Foundation of China (2008S0007, 2009J01195).
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Figures(6) / Tables(1)
| Sample | V | Cr | Mn | Ni |
| Before-non | 0.678±0.191 | 32.334±37.333 | 92.484±24.407 | 3.498±3.712 |
| After-non | 0.641±0.089a | 27.176±28.830 | 82.929±17.043 | 7.097±12.395a |
| Before-nest | 0.781±0.244 | 39.273±37.642 | 87.116±19.096 | 20.591±45.894 |
| After-nest | 0.868±0.190b | 121.653±303.890 | 102.349±40.525 | 68.105±112.834b |
| Sample | Cu | Zn | As | Pb |
| Before-non | 0.647±0.576 | 29.888±7.392a | 0.365±0.350 | 12.012±1.563a |
| After-non | 0.708±0.481a | 33.351±2.455ab | 0.245±0.245 | 12.739±1.637a |
| Before-nest | 1.874±0.930 | 39.248±2.240c | 0.498±0.267 | 12.871±1.759ab |
| After-nest | 3.136±3.002b | 45.742±2.351d | 0.756±0.383 | 15.309±1.500c |
| Note: "Before-non" stands for non-nest soil before breeding, "after-non" for non-nest soil after breeding, "before-nest" for nest soil before breeding, and "after-nest" for nest soil after breeding. The values with different letters in rows for means (± SD), represent significant differences (p < 0.05). | ||||
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