Toxic metal detection on hummingbird feathers captured in the Sabana de Bogotá and characterization of the associated sporulated bacteria

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Toxic Metal Detection on Hummingbird Feathers Captured

in the Sabana de Bogotá and Characterization of the

Associated Sporulated Bacteria

Esteban Gongora, Jenny Dussán

Centro de Investigaciones Microbiológicas, Universidad de los Andes, Bogotá Colombia.

Abstract

Human activities constantly release toxic metals such as lead and chromium to the environment which pollute the air, water and soil. As birds are constantly in contact with these pollutants and their sources, they are an ideal organism to be used as a bioindicator. We evaluated the use of hummingbird feathers captured in three sites of the Sabana de Bogotá, Colombia as bioindicators of toxic metal pollution by a spectrophotometric and a spectroscopic method. We also characterized the bacterial microbiota that is associated with the hummingbirds’ plumage by molecular identification using the 16S rRNA with a special focus on sporulated bacteria. Finally, the interactions that occur naturally between the feathers, their associated bacteria and the pollutants were described. Differences in the Pb and Cr concentrations between the sampling sites were found. The described bacterial communities were mainly composed by sporulated bacilli belonging to the Bacillaceae family. It was concluded that hummingbird feathers can be used as lead and chromium bioindicators and that the associated bacteria may be in fact interacting with these two toxic metals.

Resumen

Las actividades humanas liberan metales tóxicos como el plomo y el cromo constantemente al ambiente lo que lleva a la contaminación del aire, las fuentes de agua y el suelo. Como las aves se encuentran constantemente en contacto con estos contaminantes y sus fuentes, son un organismo ideal para ser usado como bioindicador. En este estudio, evaluamos el uso de las plumas de colibríes capturados en tres sitios de la Sabana de0020Bogotá, Colombia como bioindicadores de contaminación con metales tóxicos por un método espectrofotométrico y uno espectroscópico. También realizamos una caracterización de la microbiota bacteriana asociada al plumaje de los colibríes por una identificación molecular con el ARNr 16S con un enfoque particular en las bacterias esporuladas. Finalmente, se describieron las interacciones que ocurren naturalmente entre las plumas, sus bacterias asociadas y los contaminantes. Se encontraron diferencias entre las concentraciones de Pb y Cr de los sitios de muestreo. Las comunidades bacterianas descritas estaban compuestas principalmente de bacilos esporulados pertenecientes a la familia Bacillaceae. Se concluyó que las plumas de colibríes pueden ser usadas como bioindicadores de plomo y cromo y que las bacterias asociadas podrían efectivamente estar interactuando con estos dos metales tóxicos.

Key words: Trochilidae; Feathers; Bioindicator; Lead; Chromium; Microbiota; Bacillaceae; Sabana de Bogotá

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1. Introduction

Toxic metal pollution is a current issue occurring worldwide. It is mainly caused by the exploitation and usage of several resources. Some of the processes that lead to the release of toxic metals are the release of various compounds that contain impurities, the intentional extraction of heavy metals and waste incineration (He, Yun, Shi & Jiang, 2013). Lead and chromium are two of the most important toxic metal pollutants present in the Sabana de Bogotá, Colombia. Lead is a metal that has no known biological activity or importance, it cannot be biodegraded (Flora, Gupta & Tiwari, 2012) and thus it can be easily bioaccumulated by a large number of organisms. It appears to be toxic on any particular dosage and, due to the fact that it is present in products such as batteries, pipelines, paint, gasoline and is even found frequently on water sources, lead is an ubiquitous toxic metal that may represent an environmental hazard (Flora et al., 2012; Harrison, 2012, Patrick, 2006). Chromium is a metal that has three main stable oxidation states: Cr(0), the inert metallic state; Cr(III), an low toxicity ionic form that is even considered to have some level of biological activity; and Cr(VI), is the most toxic state and its toxicity appears to be dosage-dependent (Lay & Levina, 2012; Song et al., 2012; Zhitkovich, 2005). Environmental Cr contamination is caused mainly by industrial pollution and it is a major by-product of the tannery industry which is vastly present in this area of Colombia (Carrero et al., 2011, Song et al., 2012).

Animals such as birds have various roles inside their respective food chains and are thus prone to suffer from bioaccumulation processes (Malik & Zeb, 2009). Birds can be exposed to toxic metals both by their diets and by environmental pollution they are exposed to on a daily basis (Markowski et al., 2013). Because of this, birds can be used as bioindicators. Bird feathers can be easily sampled and manipulated and are an excellent example of a non-invasive tissue that serves as a bioindicator (Bond, Hobson & Branfireun, 2015; Hahn, Hahn & Stoeppler, 1993; Malik & Zeb, 2009; Markowski et al., 2013). Hummingbirds, belonging to the Trochilidae family, are a very diverse group. They are found on several habitats that are under different levels of human intervention, especially in Colombia as it has the largest number of hummingbird species (Hilty & Brown, 1986; Remsen et al., 2015) and they can be easily manipulated. Because of this, hummingbird feathers are an ideal bioindicator to detect the presence of toxic metals.

Additionally, bird feathers contain a wide variety of bacterial communities that include the feather degrading bacteria (FDB) that possess enzymes that can degrade β-keratin, the main component of feathers and can thus change the feather’s structure (Czirják et al., 2013; Jacob, Colmas, Parthuisot & Heeb, 2014). Some of these FDB such as Bacillus licheniformis (Ramnani, Singh & Gupta, 2005) can also form spores as resistance structures. Sporulated environmental bacteria such as B. licheniformis and Lysinibacillus sphaericus are especially important for their biotechnological potential. B. licheniformis is important for its enzymatic production (Ramnani

et al., 2005) and L. sphaericus has a role in various environmental processes which include their use in bioremediation of toxic metals and hydrocarbon compounds such as crude oil (Carrero et al., 2011; Córdoba, Vargas, Dussan, 2008; Manchola & Dussán, 2014; Shaw & Dussan, 2015; Velásquez & Dussán, 2009) and environmental control (Lozano & Dussán, 2013; Peña-Montenegro, Lozano & Dussán, 2015), among others.

In this study, we detected the presence of two toxic metals (Pb and Cr) on hummingbird feathers and evaluated their use as potential bioindicators of environmental toxic metal pollution.

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We also focused on the characterization of the associated bacterial communities present in such feathers with a particular interest on the sporulated bacteria.

2. Methods

2.1. Sampling Sites

Different hummingbird species were captured using mist nets from three sites on different municipalities of the Sabana de Bogotá: Reserva Biológica del Encenillo (which will be referred as Encenillo), a biological reserve in Guasca (4o 47’ N – 73o 54’ W); Hacienda San Carlos (which will be referred as Zipacón), a cattle farm with various forest patches in Zipacón (4o 46’ N – 74o 25’ W); and a small forest patch inside Universidad de los Andes (which will be referred as Uniandes) located downtown in Bogotá (4o 35’ N – 73o 3’ W).

2.2. Capture and Sample Collection

Birds were photographed and identified using field guides (Hilty & Brown, 1986; Asociación Bogotana de Ornitología, 2000).Before the birds were taken from the nets for manipulation and sampling, we sterilized our hands with antibacterial hand sanitizer to avoid bacterial contamination and to facilitate bird manipulation (Bisson, Marra, Burtt, Sikaroodi & Gillevet, 2007). Birds were swabbed throughout their body with a sterile cotton swab that was previously submerged in a sterile LB broth. The swabs were kept on a Falcon tube with LB broth at ambient temperature until they were sent to the Centro de Investigaciones Microbiológicas (CIMIC) laboratory where they were stored at 4oC until they were processed. Additionally, a minimum of 5 feathers were taken from the breast region of each bird and were stored on sealed plastic bags for further analysis. Feathers that were loosened due to the manipulation of the animals were also collected.

2.3. Metal Detection

We developed two detection methods using a single feather in order to detect and quantify the Pb and Cr levels on bird feathers.

2.3.1. Spectrophotometric Method

In order to quantify the total metal concentration in the feathers, the organic material in the sample must digested first so that all metal particles are available for detection. This was accomplished by placing a single feather on a test tube with cap and adding 1-2 mL of HNO3

65% so that the feather was completely covered with the acid. The solution was then processed for 30 minutes at 111oC with the Spectroquant® Crack set 10 (Merck) according to the manufacturer’s instructions. The samples were then analysed with the Spectroquant®

NOVA 60A photometer using the Spectroquant® Lead and Chromate test (Merck) based on the manufacturer’s instructions. This procedure was performed by triplicate for each bird.

2.3.2. Energy Dispersive X-ray (EDX) Analysis

Pb and Cr, among other elements were also detected by X-ray Energy Disperse Spectroscopy (EDS) using a JEOL JSM-6490LV scanning electron microscope (SEM) coupled with an OXFORD Inca Energy 250 EDS System LKIE250. Feathers were first mounted on clean copper coins with double-sided adhesive carbon tape and were then coated with gold-plate on the

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Desk®IV cold sputtering platform (Denton Vacuum). Feathers were then examined and analysed on the SEM at 20 kV in the Characterization Laboratory (MEB) at Universidad de los Andes.

2.4. Bacterial Community Characterization

In order to better simulate the micro-environment were the swabbed bacteria were obtained from and to reduce the amount of feathers needed, liquid and solid feather media were developed by modifying a feather meal agar (Tork, Aly & Nawar, 2010). The composition of the liquid feather medium was: NaCl (0.5 g/L), K2HPO4 (1.4 g/L), KH2PO4 (0.7 g/L), MgSO4 (0.1 g/L) and 1

ground feather obtained from a particular hummingbird as a carbon and nitrogen source. The solid feather medium had the same composition as the liquid medium and, additionally, agar (15 g/L) was added. As each medium is prepared using feathers from a different bird and so it is specific for each bacterial community.

Two different recovery methods were used: in the first method, which was used to recover the total bacteria found in the samples, an aliquot of 1 mL of the stored LB broth that contained the cotton swabs was added to its respective liquid feather medium and incubated at 30oC and 150 RPM for 3-4 days (until the medium became turbid). For the second method, which was used to recover the sporulated bacteria from the samples, before adding the aliquot to the liquid medium, we performed a heat shock by adding 1 mL of the LB broth to a sterile Eppendorf tube which was heated for 20 minutes at 80oC and was then quickly placed at 4oC until the samples were cooled. The heat socked samples were then added to the liquid feather medium and incubated under the same conditions as in the first method. After this initial incubation period, the media were subcultured to their respective solid liquid medium by replica plating by triplicate. The solid media were incubated at 30oC for 4-5 days. Finally, the bacterial colonies obtained from the solid feather medium were streaked on standard plate count (SPC) agar in order to obtain pure isolates.

The microscopic morphology of the pure isolates was observed by Gram staining which also allowed us to confirm that a single morphology was present in each isolate. These isolates were then identified by sequencing the V3V5 region of the 16S rRNA using the forward primer

27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and the reverse primer 1492R

(5’-GGTTACCTTCTTACGACTT-3’) (Villegas-Torres, Bedoya-Reina, Salazar, Vives-Florez, Dussan, 2011). The cycling parameters and the PCR amplification conditions are the same as the ones followed by Villegas-Torres et al. (2011). The obtained sequences were identified using the BLAST algorithm (Zhang, Schwartz Wagner & Miller, 2000).

2.5. Feather-Bacteria-Metal Interaction

In order to identify if the metals present on the collected feathers could interact with the bacteria there present, feathers from the different hummingbirds were fixed overnight in an Eppendorf tube with 2.5 % glutaraldehyde at 4oC in Milloning’s phosphate buffer, pH 7.2, and were then dehydrated consecutively with 70, 95 and 100 % ethanol. Fixed feathers were mounted on copper coins, plated with gold-plate observed on the SEM to look for bacteria present on the feathers (see Section 2.3.2. for details on the mounting and plating processes). When the bacteria were found, an EDX analysis was performed to detect the presence of Pb and Cr on the surface of both the bacteria and the feathers.

Additionally, a pure culture of the L. sphaericus strain OT4b.31 obtained from the CIMIC bacteria collection was incubated for 5 hours in an LB broth that was supplemented with a Pb at a concentration of 5mM. Feathers were briefly submerged on this medium, dried for 1 h

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at 30oC and then fixed, mounted and observed in the SEM as described above. This procedure was performed so that we could have a reference sample for bacteria-feather-metal interaction observation on the SEM and EDX analysis.

2.6. Statistical Analysis

All statistical analyses were performed using R (R Core Team, 2015). A significance value of P<0.05 was established for all tests. A Shapiro-Wilk test was performed to confirm whether the data had a normal distribution. A Kruskal-Wallis rank sum test was used to determine statistical differences between the metal concentrations for each site as the data did not present a normal distribution. Tukey’s Honest Significant Difference method was used to determine an estimate of which of the groups was different from the others.

3. Results

A total of 22 hummingbirds were collected in the three sampling sites: 9 were caught at Encenillo, 9 were caught at Zipacón and the remaining 4 were caught at Uniandes. These 22 specimens comprised 12 different bird species which are presented in Table 1.

Table 1. Hummingbirds captured in the three sampling sites. Encenillo: Reserva Biológica del Encenillo; Zipacón: Hacienda San Carlos; Uniandes: Universidad de los Andes.

Site Species

Encenillo Eriocnemis vestitus

Encenillo Chaetocercus mulsant

Encenillo Metallura tyrianthia

Encenillo Ramphomicron microrhynchum

Encenillo Eriocnemis cupreoventris

Zipacón Ocreatus underwoodii

Zipacón Adelomyia melanogenys

Zipacón Coeligena bonapartei

Zipacón Thalurania colombica

Zipacón Adelomyia melanogenys

Zipacón Ocreatus underwoodii

Zipacón Coeligena prunellei

Uniandes Metallura tyrianthia

Uniandes Lesbia nuna

Uniandes Metallura tyrianthia

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3.1 Metal Detection

We found that Pb could be detected in all the feathers that were sampled using both detection methods. Cr could be detected on the majority of the analysed feathers by the spectrophotometric method but, in some of them, the concentration obtained happened to be under the detection limit of the Chromate test. For the EDX analysis, Cr was only detected in a smaller percentage of the feathers compared to the spectrophotometric analysis.

3.1.1. Spectrophotometric Method

The metal concentrations obtained from each feather were grouped according to the sampling site from which it was collected in order to compare the Pb and Cr levels for each site. The Pb and Cr concentrations for the three sites are presented on Figure 1. The average Pb concentration was 3.25 ± 3.27 ppm for Encenillo, 4.01 ± 0.93 ppm for Zipacón and 4.69 ± 1.68 ppm for Uniandes. The Kruskal-Wallis test evidenced that there is a statistical difference between the three sites (P = 0.008) but Tukey’s test could not be performed due to the non-parametric nature of the data. The average Cr concentration for Encenillo was 0.89 ± 1.33 ppm, for Zipacón it was 0.52 ± 0.23 ppm and 3.00 ± 2.02 ppm for Uniandes. Statistical differences were found between sites with the Kruskal-Wallis test (P = 1.66 x 10-5). Despite that the data were not normally distributed, Tukey’s test could be performed and it was determined that there was a statistical difference between the Uniandes and Zipacón sites (P = 2.0 x 10-6) and the Uniandes and Encenillo sites (P = 1.14 x 10-3).

Figure 1. Lead and chromium concentration (in ppm) by site. The number of samples for each site is presented above the each boxplot. Letters above the chromium boxplot indicate statistical differences between groups.

3.1.2. EDX Analysis

The metal detection performed with the SEM allowed us to observe the microscopic particulate material that was trapped inside and in between the analysed feathers (Figure 2a). We could then determine if in any point we selected of said particles contained Pb, Cr or any other element with the EDS, as can be observed in Figure 2. The EDX analysis presented its results in terms of a

Encenillo Zipacón Uniandes

0 2 4 6 8 10 12 14

Lead Concentration on Feathers per Site

Sampling Site P b C onc e nt ra ti on (p pm )

N=8 N=24 N=12

Encenillo Zipacón Uniandes

0 2 4 6 8 10

Chromium Concentration on Feathers per Site

Sampling Site Cr C onc e nt ra ti on (p pm )

a a b

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percentage of the sampled area that contained any particular element. Due to the fact that we made punctual analyses of microscopic material, we could not determine or estimate the weight of those particles in order to obtain a concentration based on the percentage results. Because of this, we used the EDX analysis, on one hand, as a validation method to confirm presence of the two metals we analysed by spectrophotometry and, on the other, to determine if there are any other pollutants that may be present in the feathers (Figure 2b).

Figure 2. EDX analysis of particulate material on the surface of a hummingbird feather. a SEM imaging at 20 kV of a feather. Particulate material can be observed on various points of the feather. The image was used to select the point where the EDS spectrum is measured. b EDS spectrum of the selected point. Peaks represent elements that are present in the sampled point.

The elemental composition of the particles present in the feathers was obtained based on the results of the EDX analysis. The percentage of feathers that contained any given element the EDS detected is presented in Figure 3. Lead was found in 100% of the examined feathers while

a

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chromium was found in only 36.3%. Other elements that can be considered toxic pollutants such as arsenic or mercury were found in 4.5% of the sampled feathers.

Figure 3. Percentage of the total number of feathers (N=22) that contained a given element. Elemental compositions were obtained by EDS.

3.2 Bacterial Community Characterization

Over 50 morphotypes were obtained from the two recovery methodologies utilized of which 37 were chosen to be identified by the sequencing of the V3V5 region of the 16S rRNA. The molecular characterization of each of the sequenced morphotypes is presented in Table 2. Amongst those bacteria were chosen for sequencing and identification, 54.3% of the isolates belonged to the family Bacillaceae and 8.6% belonged to the Pseudomonadaceae family. The rest of the morphotypes could not be identified because of two reasons: either no sequences were obtained for the isolates labeled as “Unidentified” on Table 2 as they did not amplify during the PCR or sequencing processes (28.6% of the 37 morphotypes), or the microscopic characteristics and the recovery method did not match the molecular identification for the bacteria labeled as “Misidentified” on Table 2 (8.6%).

3.3 Feather-Bacteria- Metal Interaction

Figure 4 shows some of the bacteria that were found on the hummingbird feathers along with their respective EDS spectra. These images suggest that the bacteria can interact with the metals, both of which are naturally present on the sampled feathers. Figure 5 shows the L. sphaericus

OT4b.31 cells that were artificially placed on the feathers. This feather-bacteria-metal interaction presents a similar pattern as the ones observed in Figure 4.

C O S Cl Pb Al Si Cr Na Ca K Fe Mg Pt Zn Ti Zr Hg Br Y As Ge Mo Rb Bi

Element

P

e

rc

e

nt

a

ge

(%)

0

20

40

60

80

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Table 2. Molecular identification of the bacterial isolates obtained from the three sites and by both recovery methods. Encenillo: Reserva Biológica del Encenillo; Zipacón: Hacienda San Carlos; Uniandes: Universidad de los Andes; Unidentified: Isolates for which no sequences were obtained; Misidentified: Isolates for which the molecular identification did not match the microscopic morphology and/or the recovery method.

Isolate

ID Site

Recovery

Method Identification

1 Zipacón Direct Unidentified

3 Zipacón Heat Shock Lysinibacillus sp.

4 Zipacón Direct Pseudomonas sp.

7 Zipacón Heat Shock Bacillus sp.

9 Zipacón Heat Shock Paenibacillus sp.

13 Zipacón Heat Shock Misidentified

14 Zipacón Heat Shock Unidentified

16 Zipacón Direct Paenibacillus sp.

18 Zipacón Direct Pseudomonas sp.

20 Zipacón Heat Shock Cohnella sp.

21 Zipacón Direct Bacillus sp.

23 Zipacón Heat Shock Bacillus anthracis

25 Uniandes Heat Shock Misidentified

26 Uniandes Direct Bacillus sp.

27 Encenillo Heat Shock Unidentified

28 Uniandes Direct Unidentified

29 Uniandes Heat Shock Misidentified

30 Uniandes Direct Bacillus cereus

31 Uniandes Direct Pseudomonas sp.

32 Uniandes Direct Unidentified

33 Uniandes Direct Bacillus sp.

34 Uniandes Heat Shock Cohnella sp.

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Figure 4. EDX analysis of native bacteria present on the surface of a hummingbird feather. a-b SEM imaging at 20 kV of bacteria on feathers. Black arrows indicate where the analysed bacteria are located. Black squares represent points selected to perform the EDX analysis. c-d EDS spectra of the selected points. Peaks represent elements that are present in the sampled point.

a

c

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Figure 5. EDX analysis of L. sphaericus OT4b.31 bacteria present on the surface of a hummingbird feather. a SEM imaging at 20 kV of bacteria on the feather. The black arrow indicates where the analysed bacterium is located. The square represents the selected point to perform the EDX analysis. c EDS spectrum of the selected point. Peaks represent elements that are present in the sampled point.

4. Discussion

Our results show that both spectrophotometry and EDS are valid methods to detect the presence of lead and chromium in the hummingbird feathers. It must be noted that and advantage of these methods is that they are based on the use of a single feather and so a specific and possibly more strict feather weight is not required for the analyses as happens in previous studies (Bond et al., 2015; Hahn et al., 1993; Pérez-López et al., 2005; Malik & Zeb, 2009). This is of great

a

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importance as it decreases the number of feathers that must be removed from each individual in order to detect the presence of metals and other elements which is advantageous as it reduces the sampling times and efforts. Additionally, it is a less invasive strategy that reduces the stress of the captured birds as only feathers from the breast region are used and so feathers from other regions such as the primary and secondary flight feathers are not removed.

The comparison of the metal concentrations between sites revealed that there are significant differences between the three sampling sites. It must be noted that, Tukey’s test was performed for the chromium concentrations even though the data did not have a normal distribution. Because of this, the results for this test should not be considered as strong statistical evidence of differences between groups but rather as an insight to the origin of said differences. The differences found can be explained by the nature of the three sampling sites and the activities performed in them. The Reserva Biológica del Encenillo is a biological reserve, currently being reforested, located on an area that used to be a mining site which was exploited to obtain raw material used to produce bricks. The mining activity may have released toxic metals to the environment and this might explain the presence of lead and cadmium on the bird feathers. Hacienda San Carlos is a cattle farm that is located less than an hour away by car from a mining site for a cement company and less than thirty minutes away from a main highway. These two activities can explain the pollution levels we observed. Finally, Universidad de los Andes is located in downtown Bogotá where various construction sites are often found. The forest patch studied is surrounded on its eastern and southern sides by streets that are frequently used by motor vehicles. Volatile particles originated from the construction sites and the combustion of gasoline nearby can explain why the lead and chromium levels found at Universidad de los Andes where the highest in this study.

Previous studies have shown the effectiveness of using feathers, a non-invasive tissue, as bioindicators of metal pollution. The average Pb and Cr levels obtained using feathers for some studies are presented on Table 3. It can be seen that the concentrations found in the present study are similar to those found for other geographical regions. However, the other studies were performed using different bird species and taxonomical groups and the characteristics of the studied regions may not be the same as the ones present in the Sabana de Bogotá. Because of this, we cannot directly compare our results with the others here presented. In order to give a better insight to the pollution levels present in the studied area, further samplings must be held in new sites and with a larger sample size.

Table 3. Lead and chromium concentrations obtained using feathers as bioindicators for various studies.

Pb (ppm) Cr (ppm) Study Site Study

4.06 ± 1.78 1.26 ± 1.59 Sabana de Bogotá, Colombia This study

37.5 ± 10.7 5.38 ± 1.0 Punjab province, Pakistan Malik & Zeb, 2009 2.72 ± 0.70 0.60 ± 0.08 Bali, Indonesia Burger & Gochfeld, 1997

1.04 7.02 Papua New Guinea Burger & Gochfeld, 1995

4.83 ± 1.08 N/A Antwerp, Belgium Dauwe et al., 2000

~16 N/A Central Poland Markowski et al., 2013

The two feather media that were developed for this study, along with the initial enrichment in LB broth, allowed us to recover a large amount of bacterial morphotypes that were associated to the hummingbird’s plumage. However, the molecular identification was not very

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effective as over 37% of the isolates chosen for sequencing could not be identified. Other studies have had similar difficulties when using molecular identification for their bacterial morphotypes recovered from feathers (Shawkey, Mills, Dale & Hill, 2005; Shawkey, Pillai & Hill, 2003). For those morphotypes that could be correctly identified, the majority belonged to the Bacillaceae family and included the genera Lysinibacillus, Bacillus, Paenibacillus and Cohnella. These results are consistent with what was expected as we mainly focused on the recovery of sporulated bacteria that mainly belong to this family. Additionally, both the Bacillaceae and Pseudomonadaceae families include environmental bacteria which were previously found in other studies that describe the feather microbiome and feather associated bacteria (Table 4).

Table 4. Bacterial taxa associated with feathers of different birds described in various studies.

Bacterial Taxa Study

Lysinibacillus, Bacillus, Paenibacillus, Cohnella, Pseudomonas This study

Bacillus, Microbacterium, Pantoea, Serratia Shawkey et al., 2005

Bacillus, Pseudomonas, Staphylococcus Shawkey et al., 2003

Bacillus, Streptomyces, Fervidobacterium, Vibrio Lucas et al., 2003

Bacillus, Pseudomonas, Xanthomonas, Rhizobium Bisson et al., 2007

Bacillus, Staphylococcus, Entreococcus, Kocuria, Micrococcus, Steptomyces, Nesterenkonia, Pseudomonas, Stenotrophomonas, Vibrio, Chryseobacterium,

Flavobacterium

Gunderson, 2008

Finally, we could observe that the bacteria that naturally inhabit feathers appear to be in contact with metals and other elements found on the feathers. As some of these bacteria are FDB, this suggests that they may in fact have some type of interaction with the present lead and chromium. It is then possible then that these bacteria may be able to tolerate toxic metals (Lozano & Dussán, 2013), reduce them (Córdoba et al., 2008), and they could even absorb/adsorb them by different mechanisms such as efflux pumps (Shaw & Dussan, 2015) or using the S-layer protein (Carrero et al., 2011; Velásquez & Dussan, 2009). Since the FDB use the keratin present in the feathers as a carbon and nitrogen source, it has been shown that the presence of toxic metals reduces the ability of said bacteria to degrade keratin (Kainoor & Naik, 2010; Vigneshwaran, Shanmugam & Kumar, 2010). Future studies should focus on evaluating the metal tolerance and accumulation ability of the isolated strains and the change in their keratinase activity under a toxic metal pressure. This will allow us to have a better understanding of the feather-bacteria-metal interactions that naturally occur in the bird’s plumage.

5. Conclusions

This study showed that hummingbird feathers that inhabit the Sabana de Bogotá can be used as bioindicators of environmental pollution by toxic metals such as lead and chromium with a single feather based method. However, our sampling efforts were low and this affected our statistical analyses. Larger sample sizes and an increased number of sampling sites should be performed in future studies. We also performed a characterization of the feather’s bacterial microbiome which was mainly composed of members of the Bacillaceae family and a few members of the Pseudomonadaceae family. This is consistent with what was found in previous studies but it must be noted that our study was biased towards the sporulated bacteria and this might be the reason why the majority of our isolates were sporulated bacilli. In conclusion,

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through this study, we were able to give an insight to the system and the interactions that occur between birds, their associated bacteria and the environment they inhabit.

6. References

Asociación Bogotana de Ornitología (2000). Aves de la Sabana de Bogotá: guía de campo. Asociación Bogotana de Ornitología, Corporación Autónoma Regional de Cundinamarca. Bogotá.

Bisson, I. A., Marra, P. P., Burtt Jr, E. H., Sikaroodi, M., & Gillevet, P. M. (2007). A molecular comparison of plumage and soil bacteria across biogeographic, ecological, and taxonomic scales. Microbial Ecology, 54(1), 65-81.

Bond, A. L., Hobson, K. A., & Branfireun, B. A. (2015). Rapidly increasing methyl mercury in endangered ivory gull (Pagophila eburnea) feathers over a 130 year record. Proceedings of the Royal Society of London B: Biological Sciences, 282(1805), 20150032.

Burger, J., & Gochfeld, M. (1995). Biomonitoring of heavy metals in the Pacific Basin using avian feathers. Environmental Toxicology and Chemistry, 14(7), 1233-1239.

Burger, J., & Gochfeld, M. (1997). Heavy metal and selenium concentrations in feathers of egrets from Bali and Sulawesi, Indonesia. Archives of environmental contamination and toxicology, 32(2), 217-221.

Carrero, D. M., Morales, J. M., Garcia, A. C., Florez, N., Delgado, P. A., Dussan, J., Córdoba, A. & Barrios, A. F. G. (2011). Comparative analysis for three different immobilisation strategies in the hexavalent chromium biosorption process using Bacillus sphaericus S‐ layer. The Canadian Journal of Chemical Engineering, 89(5), 1281-1287.

Córdoba, A., Vargas, P., & Dussan, J. (2008). Chromate reduction by Arthrobacter CR47 in biofilm packed bed reactors. Journal of hazardous materials, 151(1), 274-279.

Czirják, G. Á., Pap, P. L., Vágási, C. I., Giraudeau, M., Mureşan, C., Mirleau, P., & Heeb, P. (2013). Preen gland removal increases plumage bacterial load but not that of feather-degrading bacteria. Naturwissenschaften, 100(2), 145-151.

Dauwe, T., Bervoets, L., Blust, R., Pinxten, R., & Eens, M. (2000). Can excrement and feathers of nestling songbirds be used as biomonitors for heavy metal pollution?. Archives of Environmental Contamination and Toxicology, 39(4), 541-546.

Flora, G., Gupta, D., & Tiwari, A. (2012). Toxicity of lead: A review with recent updates.

Interdisciplinary toxicology, 5(2), 47-58.

Gunderson, A. R. (2008). Feather-degrading bacteria: a new frontier in avian and host-parasite research?. The Auk, 125(4), 972-979.

Hahn, E., Hahn, K., & Stoeppler, M. (1993). Bird feathers as bioindicators in areas of the German environmental specimen bank-bioaccumulation of mercury in food chains and exogenous deposition of atmospheric pollution with lead and cadmium. Science of the Total Environment, 139, 259-270.

Harrison, R. (2012). Lead pollution: causes and control. Springer Science & Business Media. He, B., Yun, Z., Shi, J., & Jiang, G. (2013). Research progress of heavy metal pollution in China:

sources, analytical methods, status, and toxicity. Chinese Science Bulletin, 58(2), 134-140.

Hilty, S. L., & Brown, B. (1986). A guide to the birds of Colombia. Princeton: Princeton University Press.

(15)

Jacob, S., Colmas, L., Parthuisot, N., & Heeb, P. (2014). Do feather-degrading bacteria actually degrade feather colour? No significant effects of plumage microbiome modifications on feather colouration in wild great tits. Naturwissenschaften, 101(11), 929-938.

Kainoor, P. S., & Naik, G. R. (2010). Production and characterization of feather degrading keratinase from Bacillus sp. JB 99. Indian journal of Biotechnology, 9(4), 384-390. Malik, R. N., & Zeb, N. (2009). Assessment of environmental contamination using feathers of

Bubulcus ibis L., as a biomonitor of heavy metal pollution, Pakistan. Ecotoxicology,

18(5), 522-536.

Manchola, L., & Dussán, J. (2014). Lysinibacillus sphaericus and Geobacillus sp Biodegradation of Petroleum Hydrocarbons and Biosurfactant Production. Remediation Journal, 25(1), 85-100.

Markowski, M., Kaliński, A., Skwarska, J., Wawrzyniak, J., Bańbura, M., Markowski, J., Zieliński, P. & Bańbura, J. (2013). Avian feathers as bioindicators of the exposure to heavy metal contamination of food. Bulletin of environmental contamination and toxicology, 91(3), 302-305.

Lay, P. A., & Levina, A. (2012). Chromium: biological relevance. Encyclopedia of Inorganic and Bioinorganic Chemistry.

Pérez-López, M., Cid-Galán, F., Hernández-Moreno, D., Oropesa-Jiménez, A. L., López-Beceiro, A., Fidalgo-Álvarez, L., & Soler-Rodríguez, F. (2005). Contenido de metales pesados en hígado y plumas de aves marinas afectadas por el accidente del “Prestige” en la costa de Galicia. Revista de toxicología, 22(3), 191-199.

Lozano, L. C., & Dussán, J. (2013). Metal tolerance and larvicidal activity of Lysinibacillus sphaericus. World Journal of Microbiology and Biotechnology, 29(8), 1383-1389.

Lucas, F. S., Broennimann, O., Febbraro, I., & Heeb, P. (2003). High diversity among feather-degrading bacteria from a dry meadow soil. Microbial Ecology, 45(3), 282-290.

Ramnani, P., Singh, R., & Gupta, R. (2005). Keratinolytic potential of Bacillus licheniformis

RG1: structural and biochemical mechanism of feather degradation. Canadian journal of microbiology, 51(3), 191-196.

Remsen, J. V., Jr., J. I. Areta, C. D. Cadena, A. Jaramillo, M. Nores, J. F. Pacheco, J. Pérez-Emán, M. B. Robbins, F. G. Stiles, D. F. Stotz, and K. J. Zimmer. Version 18 February 2015. A classification of the bird species of South America. American Ornithologists' Union. http://www.museum.lsu.edu/~Remsen/SACCBaseline.html

Patrick, L. (2006). Lead toxicity, a review of the literature. Part 1: Exposure, evaluation, and treatment. Alternative medicine review: a journal of clinical therapeutic, 11(1), 2-22. Peña-Montenegro, T. D., Lozano, L., & Dussán, J. (2015). Genome sequence and description of

the mosquitocidal and heavy metal tolerant strain Lysinibacillus sphaericus CBAM5.

Standards in genomic sciences, 10(1), 2.

R Core Team (2015). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Retrieved from http://www.R-project.org

Shaw, D. R., & Dussan, J. (2015). Mathematical Modelling of Toxic Metal Uptake and Efflux Pump in Metal-Resistant Bacterium Bacillus cereus Isolated From Heavy Crude Oil.

Water, Air, & Soil Pollution, 226(4), 1-14.

Shawkey, M. D., Mills, K. L., Dale, C., & Hill, G. E. (2005). Microbial Diversity of Wild Bird Feathers Revealed through Culture-Based and Culture-Independent Techniques.

(16)

Shawkey, M. D., Pillai, S. R., & Hill, G. E. (2003). Chemical warfare? effects of uropygial oil on feather‐degrading bacteria. Journal of Avian Biology, 34(4), 345-349.

Song, Y., Zhang, J., Yu, S., Wang, T., Cui, X., Du, X., & Jia, G. (2012). Effects of chronic chromium (VI) exposure on blood element homeostasis: an epidemiological study.

Metallomics, 4(5), 463-472.

Tork, S., Aly, M. M., & Nawar, L. (2010). Biochemical and Molecular Characterization of a New Local Keratinase Producing Pseudomomanas sp., MS21. Asian J Biotechnol, 2(1), 1-13.

Velásquez, L., & Dussan, J. (2009). Biosorption and bioaccumulation of heavy metals on dead and living biomass of Bacillus sphaericus. Journal of Hazardous Materials, 167(1), 713-716.

Vigneshwaran, C., Shanmugam, S., & Kumar, T. S. (2010). Screening and Characterization of Keratinase from Bacillus licheniformis Isolated From Namakkalpoultry Farm.

Researcher, 2(4), 89-96.

Villegas-Torres, M. F., Bedoya-Reina, O. C., Salazar, C., Vives-Florez, M. J., & Dussan, J. (2011). Horizontal arsC gene transfer among microorganisms isolated from arsenic polluted soil. International Biodeterioration & Biodegradation, 65(1), 147-152.

Zhang, Z., Schwartz, S., Wagner, L., & Miller, W. (2000). A greedy algorithm for aligning DNA sequences. Journal of Computational biology, 7(1-2), 203-214.

Zhitkovich, A. (2005). Importance of chromium-DNA adducts in mutagenicity and toxicity of chromium (VI). Chemical research in toxicology, 18(1), 3-11.