Download The Effects of Flooding on the Microbial Communities of Sparrow

The University of Maine
DigitalCommons@UMaine
Honors College
Spring 2014
The Effects of Flooding on the Microbial
Communities of Sparrow Eggs in a Temperate
Maine Salt Marsh
Mattie Paradise
University of Maine - Main
Follow this and additional works at: http://digitalcommons.library.umaine.edu/honors
Part of the Environmental Health and Protection Commons, and the Environmental Indicators
and Impact Assessment Commons
Recommended Citation
Paradise, Mattie, "The Effects of Flooding on the Microbial Communities of Sparrow Eggs in a Temperate Maine Salt Marsh" (2014).
Honors College. Paper 151.
http://digitalcommons.library.umaine.edu/honors/151
This Honors Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Honors College by
an authorized administrator of DigitalCommons@UMaine.
THE EFFECTS OF FLOODING ON THE MICROBIAL COMMUNITIES OF
SPARROW EGGS IN A TEMPERATE MAINE SALT MARSH
by
Mattie V. Paradise
A Thesis Submitted in Partial Fulfillment
of the Requirements for a Degree with Honors
(Biology)
The Honors College
University of Maine
May 2014
Advisory Committee:
Dr. Brian Olsen, Assistant Professor, School of Biology and Ecology, Advisor
Dr. Farahad Dastoor, Lecturer, School of Biology and Ecology
Katharine Ruskin, PhD Student, Ecology and Environmental Science
Dr. Mark Haggerty, Preceptor, Honors College
Dr. D. Rabern Simmons, Research Associate, School of Biology and Ecology
Abstract
Microbial infection has been shown to reduce hatching success for the eggs of tropical
birds. In these ecosystems, humidity and temperature encourage bacterial growth and the
transport of microbes through the pores of the egg shell. A single study in a temperate
ecosystem found no noticeable change of microbial communities during the length of the
incubation cycle, and thus no increased risk of microbial infection by the time of
hatching. This study, however, took place in the arid Mediterranean type climate of
California, a locale that likely diminishes the abilities of microbial communities to
colonize and grow on egg surfaces. For this study we explored the ability of microbial
communities to colonize and flourish on eggshells in a temperate salt marsh. These
temperate ecosystems possess much higher humidity and water exposure than
Mediterranean climates, thus possessing conditions that are more conducive to microbial
growth. We analyzed the microbial communities of eggs from Saltmarsh (Ammodramus
caudacutus) and Nelson's (A. nelsoni) sparrows in a tidal salt marsh in southern Maine.
Eggs were swabbed every three days during June and July to test for present microbial
communities. To determine if environmental factors affect the microbial community, we
compared the microbial environment of eggshells that had and had not been flooded by
the tides. We found no significant differences in the microbial communities of these
eggshells.
Acknowledgements
I would like to acknowledge the following people for whom I am grateful for their
assistance and guidance during the research and writing of this thesis.
First and foremost, I would like to thank Brian Olsen and Farahad Dastoor who spent
many hours as my thesis advisors guiding me on the methods and procedures to compose
and defend my research. They gave me assistance during all phases of this project.
I would also like to thank Kate Ruskin who was my field advisor. She made my research
in the salt marsh during the summer of 2013, exciting and fun. It was an experience that I
will never forget.
Without the help of David Simmons, I could not have completed the results portion of my
thesis. He generously donated laboratory space, materials, and technical assistance.
Finally, I would like to thank Elijah Davis, Darlene Turcotte and Andrea Santariello who
edited my thesis during our weekly meetings. Their thoughtful insights and expert skills
helped me to stay on the right track.
iii
Table of Contents
INTRODUCTION .............................................................................................................1
EGGSHELL BACTERIAL COMMUNITIES ..................................................................................2
NELSON’S AND SALTMARSH SPARROWS’ BREEDING HABITAT .............................................3
SALINE ENVIRONMENT MICROBIOLOGY ...............................................................................5
AVIAN MICROBIOLOGY.........................................................................................................6
OBJECTIVE AND HYPOTHESES ...............................................................................................7
METHODS .........................................................................................................................8
STUDY SITE...........................................................................................................................8
EGG MICROBIAL SAMPLING ..................................................................................................9
CHOOSING SAMPLES .............................................................................................................9
BACTERIAL MORPHOLOGY .................................................................................................10
BACTERIAL IDENTIFICATION ...............................................................................................10
STATISTICAL ANALYSIS ......................................................................................................11
RESULTS .........................................................................................................................11
MICROBIAL CONTAMINATION OF SHELL SURFACE ...............................................................11
MICROBIAL SEQUENCE IDENTIFICATION .............................................................................12
DISCUSSION ...................................................................................................................12
TABLES ............................................................................................................................21
TABLE 1 ..............................................................................................................................28
TABLE 2 ..............................................................................................................................29
TABLE 3 ..............................................................................................................................29
TABLE 4 ..............................................................................................................................33
FIGURES ..........................................................................................................................36
FIGURE 1 .............................................................................................................................36
FIGURE 2 .............................................................................................................................37
FIGURE 3 .............................................................................................................................37
FIGURE 4 .............................................................................................................................38
FIGURE 5 .............................................................................................................................39
FIGURE 6 .............................................................................................................................40
FIGURE 7 .............................................................................................................................41
REFERENCES.................................................................................................................21
AUTHOR’S BIOGRAPHY .............................................................................................42
iv
Introduction
Microorganisms are the foundation of the biosphere, both evolutionarily and ecologically,
and their role in the community has some effect on all organisms (Zoetendal, 2006).
Among birds, microbial trans-shell infection has been shown to reduce hatching success
in eggs. This ability to impact egg viability, however, is likely dependent on the
ecological interactions of the microbial community on the eggshell. Little is understood
about how environmental factors impact the membership in egg microbe communities.
This study observed the microbial environment of eggs from a temperate ecosystem
using Saltmarsh (Ammodramus caudacutus) and Nelson's (A. nelsoni) sparrows whose
eggs are subjected to periodic tidal flooding (Gjerdrum et al., 2005), a disturbance event
that may impact both the microbial community of the eggshell and its ability to influence
egg viability. We tested for differences in the microbial community of eggshells that had
and had not been flooded by the tides.
Limited by their size, microbes form symbiotic relations with higher organisms
(Haygood et al., 1999). Symbiotic relationships of microorganisms provide many
essential nutrients to plants and animals, such as methane and nitrogen that would
otherwise not be made available for use (Zoetendal, 2006). These symbiotic interactions
can occur externally or internally and may act as a mutual relationship (where both
partners gain), a commensal relationship (where the microbe gains, but neither helps nor
harms its host), or a pathogenic relationship (where the microbe gains at the expense of
the host). Due to their nature, microbial symbionts can alter host fitness by affecting its
ability to survive, reproduce, compete, grow, or defend itself, both positively and
negatively (Jarosz and Davelos, 1995).
1
Eggshell Bacterial Communities
The avian egg is one example of a host affected by pathogenic microbial
environments. Shortly after laying, eggshells become exposed to environmental
microbes. These microbes can multiply rapidly and penetrate through the shell pores
(Cook et al., 2005). The pores are responsible for all gas exchange across the eggshell.
(Wangensteen, 1972). Microbes appear on the inner membrane after one day of exposure,
invade the albumen after three days and reach the yolk in five days (Cook et al., 2003).
This horizontal transmission of bacteria into the egg is known as trans-shell infection.
Most frequently, gram-negative, motile and non-clustering bacteria penetrate the
eggshell (De Reu et al., 2006). These microorganisms digest the protective shell cuticle
and allow for microbial infection by removing the water resistant properties of the shell,
which causes an increase in the number of open shell pores available for other microbes
to access (Board and Halls 1973, Board et al., 1979). While eggshell characteristics such
as area, shell thickness, and number of pores have no influence on bacterial eggshell
penetration (De Reu et al., 2006), bacteria do penetrate eggs with pore diameters ranging
from 6 to 65 µm (Tyler, 1956).
To protect against trans-shell infection, the mother and egg provide many physical
and chemical barriers against bacteria. The first line of defense against microbial
infection is incubation by the mother, which reduces the presence of bacteria on the
eggshell and changes the microbial community by inhibiting pathogenic shell microbiota
(Cook et al., 2005). Bacteria that can withstand incubation must then pass through the
cuticle layer, a waterproofing agent that helps serve as a barrier for bacterial invasion,
before passing through the eggshell (Board et al., 2003). Although the shell is mainly
seen as a physical barrier, it also possesses many proteins with antimicrobial functions. A
2
study done by Mine et al. (2003) revealed that the shell matrix proteins may inhibit
certain bacteria by interacting with and disrupting their membranes (Pseudomonas
areuginosa, Bacillus cereus, Staphylococcus aureus).
Inside the egg the albumen defends against microbial infection chemically with
many antimicrobial proteins (Rose et al., 2003). Two of the most abundant antimicrobial
proteins found within the shell and albumen are lysozyme and ovotransferrin (WellmanLabadie et al., 2007). Lysozyme specifically targets the cell wall of gram positive
bacteria by hydrolyzing the thick peptidoglycan layer (Hughey et al., 1987). Gram
negative bacteria are not easily degraded by lysozyme because their peptidoglycan wall is
protected by an outer lipopolysaccharide layer. Lipopolysaccharide binds to lysozyme
and reduces the enzymatic activity of the lysozyme to degrade peptidoglycan (Ohno et
al., I989). Ovotransferrin mainly causes iron deprivation and prevents bacterial growth
(Baron et al., 1997), but it also causes cytoplasmic membrane disturbance, which
interferes with normal biological functioning of the bacteria (Aguilera et al., 2003).
While some bacteria are more successful than others at infecting the egg, which microbes
are available for infection are determined by the community of microbiota on the
eggshell itself. The eggshell microbiota, in turn, is determined by the microbial species it
is in contact with: species that reside in the saltmarsh habitat and the mother’s natural
flora.
Nelson’s and Saltmarsh Sparrows’ Breeding Habitat
The Nelson's and saltmarsh sparrows are two of a few passerines (perching birds)
that nest in salt marshes along the northeastern United States and Canadian coast
(Greenlaw and Rising, 1994; DiQuinzio et al., 2001; Hodgman et al., 2002). Since salt
3
marshes are located along the coast and at the mouths of large rivers, their inhabitants are
considered to be especially vulnerable to pollution, habitat change due to development,
and sea-level rise (Adam, 1990). Due to these risks, very few ground-nesting passerines
breed in tidal marshes (Teal, 1986). These marsh habitats are usually dominated by
native vegetation, such as saltmarsh cord-grass (Spartina patens), smooth cordgrass (S.
alterniflora), and black grass (Juncus gerardi) (Woolfenden, 1956, Reinert and Mello,
1995, Brawley et al., 1998, Benoit and Askins, 1999, Shriver et al., 2004). These two
species of birds select nesting sites with deep accumulation of dead plant material
(Shriver, 2002), higher relative elevations (Shriver, 2002, DiQuinzio et al., 2002), and
taller vegetation (DiQuinzio et al., 2002). Both species are promiscuous breeders; males
do not defend territories or provide parental care and females solely incubate the eggs
(Woolfenden, 1956; Post and Greenlaw, 1982; Greenlaw and Rising, 1994).
Although these species build their nests in higher elevations within the marsh,
these elevations are still not sufficient to avoid flooding during the highest tides
(Gjerdrum et al., 2005; Shriver et al., 2007). These unusually high flood tides occur
every 26–28 days during the lunar tide cycle and reduce reproductive success by
destroying active nests (Johnston, 1956; Montevecchi, 1978; Meanley, 1985). In two
separate studies on saltmarsh and Nelson's sparrows, flooding accounted for the majority
of nest failure (Gjerdrum et al., 2005 and Shriver et al., 2007). These flooding events
synchronize female nesting behavior across the marsh because nest losses and subsequent
re-nesting events occur at the same time (Shriver et al., 2007). Compared to Nelson's
sparrows, saltmarsh sparrow females are more tightly synchronized with the tidal cycle
4
and usually initiate nesting within three days after a flood tide, which leads to higher nest
success in saltmarsh versus Nelson’s sparrows (Shriver et al., 2007).
Saline Environment Microbiology
Saline habitats (such as a saltmarsh) are frequently inhabited by an abundance of
microbial communities adapted to these ecosystems (Zahran, 1997). These bacteria play a
major role as important and dominant inhabitants of saline and hypersaline environments.
The bacterial communities in saline environments usually include extreme (halophilic)
and facultative (halotolerant) bacteria. Halotolerant bacteria may also include non-saline
bacteria, which have become adapted to these extreme environments. Gram-negative
bacteria appear to be much more frequent in saline environments (Ventosa et al., 1982
and Del Moral et al., 1987). Gram-positive bacteria are represented in saline habitats, and
members of the genera Bacillus and Micrococcus are dominant among other Grampositive bacteria in saline soils. These spore-forming bacilli are usually moderate or
extreme halo-alkaliphiles. (Zahran et al. 1992).
Bacteria living in an environment with high salinity must cope with a number of
stresses, especially ionic stress (Galinski and Truper 1994) due to osmoregulatory
challenges across the cell membrane (Imhoff and Thiemann 1991; Thiemann and Imhoff,
1991). The adaptation to osmotic stress requires the maintenance of osmotic equilibrium
by establishing a cytoplasmic osmolarity similar to the surrounding medium (Larsen,
1986; Truper and Galinski, 1986). Bacteria living in saline environments have adopted
two strategies for osmoadaptation: the KCl type and the compatible solute type. (Galinski
and Truper, 1994). In the KCl mechanism the bacteria maintain a cytoplasmic KCl
concentration that is similar to that of the surrounding medium to obtain osmotic
5
equilibrium. For the compatible solutes mechanism the bacteria keep their osmolytes
responsible for metabolism and osmotic balance at concentrations above 1M in the
cytoplasm (Galinski and Truper, 1994). It has been hypothesized that osmolytes have two
modes of action under saline conditions: first to increase the intracellular osmotic
strength, and second to stabilize cellular macromolecules. These osmolytes are
sometimes referred to as osmoprotectants. (Csonka and Hanson 1991; Lippert and
Galinski 1992). Bacteria that do not usually grow in saline environments may also exhibit
cell morphological modifications in high salt stress. These modifications include
swelling, elongation and shrinkage, which aids in reducing the cell volume. (Zahran,
1997).
Avian Microbiology
Although few studies have observed natural flora of wild avian species, a study
conducted in 1988 tested 364 passerines and woodpeckers for the presence of common
bacteria and found the following community members: Escherichia coli (1%),
Pseudomonas
spp.
(22%),
Salmonella
spp.(0%),
Staphylococcus
spp.
(15%),
Streptococcus spp. (18%), and Yersinia spp. (1%) (Brittingham et al., 1988). More
recently studies have been conducted on feather degrading bacteria in response to the
pollution of poultry feathers. Most of the keratin (which constitutes ninety percent of a
feather’s mass: Onifade et al., 1998) degrading microorganisms reported belong to the
genera Bacillus (Brandelli, 2008; Lin et al., 1999; Kim et al., 2001; Bressolier et al.,
1999). Keratin is difficult to degrade because the polypeptide is densely packed and
strongly stabilized by several hydrogen bonds and hydrophobic interactions, in addition
to several disulfide bonds (Brandelli, 2008). In order to breakdown keratin the bacteria
6
must be able to produce keratinase. Most of the keratin degrading microorganisms
reported belong to the genera Bacillus (Brandelli, 2008; Lin et al., 1999; Kim et al.,
2001; Bressolier et al., 1999). A study done on swamp sparrows, that compared the
abundance of feather degrading bacilli between the sparrows in a tidal marsh to those in
inland freshwater marshes, found that the coastal swamp sparrow had a much higher
abundance of feather degrading bacilli than the inland swamp sparrow (Peele et al.,
2009).
Objective and Hypotheses
Microbial communities are known to change along a number of environmental
gradients including temperature (Ratkowsky et at., 1982), salinity (Herlemann et al.,
2011), and moisture (Yeager, 1981). Given the ability of abiotic conditions to alter
microbial communities, we expect that eggs in certain environments are more prone to
trans-shell infection than others. In tropical ecosystems, for instance, humidity and
temperature encourage bacterial growth and the transport of microbes through the pores
of the eggshell (Cook et al., 2003). Whereas a similar study conducted in a dryer, cooler
environment found no noticeable effect of microbial infection on egg viability (Wang et
al., 2011).
This purpose of this study was to observe the microbial inhabitants of eggshells
from a temperate ecosystem using Saltmarsh (A. caudacutus) and Nelson's (A. nelsoni)
sparrows who construct their nests in a tidal salt marsh in southern Maine, an
environment that possesses characteristics that promote microbial growth. To determine
if the flooding, caused by the high monthly tide, would affect the microbial egg
7
community, we compared the microbial environment of eggs that had and had not been
flooded by the tides.
We hypothesized that salt water would have one of four effects on the microbial
communities of eggshells: (1) flooding would increase the abundance of microbes on the
eggshells because the presence of water promotes microbial growth (Board and Halls,
1973), and the decrease in temperature caused by flooding would decrease antimicrobial
enzymatic activity (Board and Tranter, 1986); (2) Flooding would have no effect on
microbial communities on the eggshell because salt marshes are saline habitats, which are
frequently inhabited by bacterial communities that are adapted to these halophilic
ecosystems (Zahran, 1997); (3) Flooding would decrease the abundance of microbes on
the eggshells because the microbes will exhibit hyperosmotic shock, which causes
shrinkage of the cytoplasmic volume or plasmolysing of the cell. This process occurs
when the environment of the microbe increases in osmolarity compared to the interior
osmolarity of the cytoplasm, (Csonka, 1989); (4) Flooding would cause a change in
microbial diversity because some microbes would be more tolerant to halophilic
environments than others. Methods
Study Site
This study was conducted within two marshes in Scarborough, Maine
(Scarborough and Jones Creek Marshes Figure 1) during two months of the saltmarsh and
Nelson’s sparrow breeding season (June and July) in 2013. In the marsh, each nest found
was marked with a numbered flag, positioned one meter away. Temperature was
8
measured at each nest using Maxim iButtons; (hereafter as “iButtons”) to determine nest
flooding.
Egg Microbial Sampling
We sampled bacteria on each egg by swabbing half of the egg with a sterile swab,
moistened with sterile phosphate buffered saline solution (PBS). The side of the egg
swabbed was marked with a permanent marker and the unmarked side was swabbed
during a later visit. During each nest visit we swabbed half of two different eggs. Nests
were visited every two to three days during incubation, which allowed us to sample the
entire clutch throughout the 12-day incubation phase, assuming a mean clutch size of four
eggs. After taking the sample, the swab was placed in a sterile tube containing 0.5 ml of
sterile glycerol PBS/(0.05% Tween 80/15% glycerol) which we placed in a cooler on ice
(Wang et al., 2011). Samples were frozen for later use once we were out of the field.
Choosing Samples
To determine if a nest was flooded, we examined iButton data for each nest
(Figures 2 and 3). A rapid drop in temperature, corresponding to the timing of a high tide,
was defined as a flooding event (Gjerdrum et al., 2008). The swabs from nests collected
before flooding were compared to the swabs collected after flooding. We also chose to
compare nests that did flood, with the nests that did not flood (hatch and non-hatch in
both conditions). The non-flooded swabs were chosen on corresponding dates to the nonflooded swabs with the flooding event (+/- 7 days). This served as a time control to
minimize the bias of a changing microbial community.
9
Bacterial Morphology
Samples were spread onto nutrient agar and incubated for 72 hours at 35 ºC (to
mimic the average incubation temperature of incubating sparrows). Colony forming units
(CFUs) and percent coverage were noted for each plate. We first identified all bacteria
present using morphological characteristics. Each colony was placed into a type using
the following characteristics: whole shape of colony, size of colony, edge/margin of
colony, chromogenesis (color) of colony, opacity of colony, elevation of colony, surface
of colony, and texture of colony.
Bacterial Identification
The most abundant types of bacteria from the samples were re-plated onto new
nutrient agar plates to form pure colonies. These pure colonies were removed from the
plate with 200 µl of PBS and transferred to 1.5 ml eppendorf tubes for DNA extraction.
The DNA was extracted using a Qiagen® Blood & Tissue Kit. The 16s rDNA gene was
amplified from these samples using PCR (Table 1a. and 1b.). We analyzed our results
from PCR using gel-electrophoresis and identified our 16s rDNA band at 1.5-1.6 kB
(Figure 7). We removed our samples from the gel using a QIAquick® Gel Extraction Kit
and sent our samples to the UMaine sequencing lab for sequencing and editing. These
sequences were identified to the genus level using the National Center for Biotechnology
Information
(NCBI)
Basic
Local
Alignment
Search
Tool
(BLAST)
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). BLAST is an algorithm for comparing primary
biological sequence information, in this case nucleotides of sequenced DNA (Altschul et
al., 1990). This search allows for a sequence of DNA to be compared with a database of
sequences, which will identify library sequences that resemble the unknown sequence. 10
Statistical Analysis
For this experiment, eggshells were classified into one of three treatments:
flooded and failed, not flooded and hatched, and not flooded and failed (we did not
collect any eggshell samples that came from nests that flooded and hatched). We
compared the microbial communities of flooded eggs with two types of non-flooded eggs
to eliminate hatching success as a variable that affected microbial communities because
there were eggs that did not flood and hatched and eggs that did not flood and failed. We
used unpaired, two-tailed t-tests to compare the difference (before and after flooding
event) in microbial community for each of these treatments. Microbial communities of
eggshells were compared by: CFUs per sample, percent microbial coverage per sample,
and microbial diversity per sample. We compared the diversity of each sample using the
Shannon’s Index. This diversity index is a mathematical measure of species diversity in
a community (Shannon, 1948). We used this test to measure species richness and species
abundance for each sample.
Results
Microbial contamination of shell surface
We examined the eggshell surface after incubation for presence of
microorganisms on 38 eggs from 19 clutches from 2 separate sites. We compared 11
clutches that flooded and failed with 4 clutches that did not flood but failed (due to
predation) and 4 clutches that did not flood and hatched. Microbes were evident on
100% of the eggs from each type of nest. The difference in percent microbial coverage
of the plates was not significant between the two marsh sites (N=19, t =1.2, df = 9.3,
p=0.26). The percent coverage of microbes present on plates did not differ between those
11
that flooded and did not hatch and those that did not flood and hatched (Figure 4a). There
was also no difference between those that flooded and those that did not flood and failed
(Figure 4b). The difference in colony forming units (CFUs) between flooded and not
flooded and failed eggshells was not significant (N=15, t=0.05, df = 13, p=0.96). The
difference in CFUs between flooded and not flooded and hatched was also not significant
(N=15, t=0.18, df=13, p=0.86) (Figure 5).
Using morphological characteristics, we identified 22 different types of bacteria
growing on nutrient agar from the 38 samples. Comparing the two sites we found no
difference in species composition between Jones marsh and Scarborough marsh (N=19,
t=0.7, df=14.3, p=0.49). There was also no difference found in community composition
between eggs that flooded and eggs that did not flood and hatched (Figure 6a), nor was
there a difference found between the eggs that flooded and did not hatch and the eggs that
did not flood and failed (figure 6b).
Microbial Sequence Identification
Of the 22 types of bacteria found on our plate samples (Table 2), 14 types were
sequenced and identified (Tables 3 & 4). Of the 14 samples, 2 were identified as
Paenibacillus spp., 5 identified as Staphylococcus spp., and 7 identified as Bacillus spp.
Discussion
A crucial precondition for microbial trans-shell infection is the presence of
bacteria or other pathogens on the shell surface (Board and Tranter, 1986). The purpose
of this experiment was to observe and compare the microbial communities of two types
of eggshells (flooded and non-flooded). We predicted that if a nest flooded with salt
12
water, we would observe at least one type of change in the microbial community of the
eggshells (increase in microbial abundance, decrease in microbial abundance, or change
in microbial diversity). Our results did not support this hypothesis, and we did not
observe any significant change in the microbial communities of eggshells from before
and after flooding. Without additional, more in-depth research, no link can be made
between salt water flooding and a change in the microbial community of Nelson’s and
saltmarsh sparrow eggs.
Our first major finding was that neither the percent microbial coverage of the
bacterial plate nor the number of colony forming units (CFUs) per eggshell significantly
increased from before and after flooding. We originally predicted that flooding would
increase the bacterial coverage of eggshells because water has been shown to increase
microbial growth on eggshells (Cook et al., 2004). This result could have occurred due
to continued incubation by the female after flooding.
Unlike other trans-shell infection studies (Cook et al., 2003, 2004, 2005, and
Wang et al., 2011), our research did not use incubation as a variable and we did not
remove any eggs from their natural environment. Using iButton data we confirmed that
even though the nests flooded, the mothers continued to incubate their nests once the tide
receded. Incubation decreases water presence on the egg and has been shown to
minimize the growth of pathogenic and cuticle digesting microorganisms: fungi and gram
negative bacteria (Cook et al., 2005). The heat from incubation also increases the
internal temperature of the albumen to levels at which antimicrobial enzymes function
optimally (Board and Tranter 1986). In the 2005 study done by Cook et al., they showed
that the incubation by thrasher (another passerine bird) females reduced bacteria on the
13
eggshells and moved the community towards a more benign composition. Besides
reducing the presence of water on eggshells, incubation is also believed to prevent
microbial presence on eggshells because the female parent’s brood patch inoculates the
shells with antibiotic agents such as preen gland oils or other epidermal fatty acids (Jacob
1978; Menon & Menon 2000; Shawkey et al., 2003), or with protective microbial species
(Baggot & Graeme-Cook 2002). Although the eggs that flooded were exposed to
elements that are known to increase microbial growth, the mother continued to incubate,
which therefore may have prevented a significant change in the microbial communities
between flooded and non-flooded eggshells.
Our second major finding was that the diversity of species on the eggshells did
not vary between flooded and non-flooded treatments. We hypothesized that the salt
water would cause a change in the diversity because halophilic conditions cause
plamolysis of bacterial cells walls that are not salt tolerant (Zahran, 1996). Although our
results showed no significant difference between the microbial diversity of eggshells that
flooded and eggshells that did not flood, this may indicate that the species of bacteria
inhabiting the eggs were mostly salt tolerant.
In these marshes salt water floods every 26 to 28 days (Gjerdrum et al., 2005 and
Shriver et al., 2007), therefore the species of bacteria inhabiting must be able to tolerate
increases in salinity to survive (Zahran, 1997). Since these sparrows inhabit the
saltmarsh during their breeding season, their bodies and nests are constantly in contact
with bacteria that reside within this halophilic environment. If the bacteria living on the
eggshell consist mainly of the halotolerant bacteria that live within the saltmarsh, it
would be expected that the saltwater flooding would not affect their viability.
14
Of the 22 DNA samples extracted from pure colony samples, only 14 amplified
after PCR. These samples may not have amplified during PCR for a number of reasons.
One reason may be that there was an error during the DNA extraction or PCR that
prevented the samples from amplifying. Another reason could be that the samples were
not all bacterial species. During PCR we used 16s rRNA primers which only work with
bacterial species to replicate the 16s rRNA gene. If any of the samples were non-bacterial
samples they would not have amplified because only bacteria contain the16s rRNA gene.
Since previous research has shown that fungal species are able to infect the eggshell,
future research should test for fungal species that are present on the eggshell.
Although this research focused primarily on the pathogenicity of eggshell
microbiota, the species of bacteria found on eggshells may alternatively be commensal or
mutualistic. These species may inhabit the eggshell without trans-shell infecting
(commensal) or they may prevent certain species from inhabiting the eggshell that are
known to be pathogenic (mutualistic). The species identified from the samples were all
gram positive. This could potentially mean that only gram-positive species grow on
eggshells in a saltmarsh, or it could mean that gram-positive species prevent gramnegative species from colonizing on eggshells. In order to test their role in this
environment it would be necessary to grow these species on eggshells and observe how
they colonize and interact with other species of bacteria. If it were discovered that gramnegative species do not inhabit the eggshells of saltmarsh and Nelson’s sparrows it would
reinforce our hypothesis from this study: trans-shell infection does not reduce the
hatching success of sparrow eggs in a Maine temperate saltmarsh.
15
The most frequent type of bacteria identified from the samples were species of the
genus Bacillus. One of these species was Bacillus weihenstephanensis. B.
weihenstephanensis is a soil-dwelling, gram positive rod-shaped bacterium that can be
found in various climatic zones (Lechner et al., 1998 and von Stetten et al., 1999). B.
weihenstephanensis is a bacterial strain belonging to the Bacillus cereus group. In 1998,
B. weihenstephanensis was suggested as a new species on the basis of sequence
differences in ribosomal RNA genes and cold-shock protein genes (Lechner et al., 1998).
This species is known to be psychrotolerant, meaning that it optimally grows at 7 °C or
below (Lechner et al., 1998), a characteristic that is likely to make trans-shell infection
unlikely given an incubating female and nest temperatures above 30 °C. Although this
bacteria only varies by a couple nucleotides in the 16s rDNA from other species within
the B. cereus group, it can be rapidly identified using recombinant DNA or cold shockprotein A (cspA) targeted PCR (Lechner et al. 1998).
Another type of species within the Bacillus genus found in the samples was
Bacillus megaterium. B. megaterium is a gram positive, spore-forming bacterium that is
easily recognized by its large size of vegetative cells and spores. This species of
bacterium can be found in a wide range of ecological habitats. It is generally considered
to be a soil organism, but can be found in diverse environments such as rice paddies,
dried food, seawater, sediments, fish and normal flora of humans (Vary, 1994). This
bacterium produces important enzymes such as penicillin amidase and steroid hydrolases.
It is the major aerobic producer of vitamin B12, and is one of the organisms involved in
fish spoilage (Vary, 1994). Although this species is classified within the B. subtilis group,
16
it is more closely related to the B. cereus group by 16s rRNA sequence analysis (Ash et
al., 1991 and Priest, 1993).
The last type of species within the Bacillus genus found in the samples was
Bacillus toyonensis. B. toyonensis is a gram positive, spore forming bacteria that was
initially classified as B. cereus, until genomic analysis confirmed that it was a different
strain within the Bacillus cereus group. B. toyonensis is found in a wide array of
environments and has recently been used as a probiotic for swine, poultry, cattle, rabbits
and aquaculture (Jiménez, 2013).
We can hypothesize that these types of Bacilli came into contact with the eggshell
from one of three places: the mother’s feathers during incubation, the sea water from
flooding, or the dead vegetative material used for building the nest. A way to test for this
would be to sample the water in the marsh and to swab the mother and the nest to test for
the presence of these bacteria. It would also be reasonable to suggest that these bacteria
may produce keratinase because bacteria of the genus Bacillus are the most common
bacteria that degrade feathers (Brandelli, 2008; Lin et al., 1999; Kim et al., 2001;
Bressolier et al., 1999). A way to test for keratinase activity and production would be to
test these different species of Bacilli on feather agar (Cai et al., 2008). While the 16s
rRNA sequencing can match sequence data closely with matching species, 16s rRNA is
known to lack resolution between highly related organisms (Fox et al., 1992). Since all of
these bacteria are closely related to the B. cereus group another form of identification
should be done to confirm their identity.
Another genus of bacteria found within the samples was Paenibacillus.
Paenibacillus are gram positive straight rods with a central endospore. Paenibacillus are
17
found in a large number of environments such as the gut of earthworms (Horn et al,
2005), in industrial wastewater (Meehan et al. 2001), and feathers of crested and least
auklets (Paul et al., 2013). Although most of the keratin degrading microorganisms
belong to the genera Bacillus (Brandelli, 2008), Paenibacillus is a keratinase producer
and has feather degrading ability, which was observed in a study on chicken feather
degradation (Paul et al., 2013). We can hypothesize that this bacteria transferred from the
mother's feathers onto the egg because this bacteria has been found on feathers of other
avian species. If this species is found on the feathers of female sparrows, it may be one
type of bacteria responsible for feather degradation. This theory can be tested by
swabbing the mother’s feathers to test for the presence of these bacteria. Keratinase
activity and production could again be tested using feather agar.
The last types of bacteria found within our samples were from the genera
Staphylococcus. Staphylococci are ubiquitous in the environment and are found on the
skin of most warm-blooded animals (Mitschlerlich and Marth, 1984). One species found
within our samples was Staphylococcus pasteuri. S. pasteuri is a gram positive bacterium
that optimally grows at 15 to 45 °C and can grow in salinities up to 15% (Chesneau et al.,
1993). This species of staphylococci is distinguished from all the others by the production
of yellow pigment and the resistance to lysostaphin (Chesneau et al., 1993). We can
hypothesize that Staphylococci bacteria came into contact with the eggshell from the
mother’s skin while incubating the egg. A way to confirm this would be to swab the
mother’s skin to test for the presence of staphylococci bacteria.
Although this study did not focus on the mechanics of trans-shell infection, future
studies would need to be done to observe how flooding affects the pores of sparrow
18
eggshells. The tide only covers the eggshell for a few hours, but salt crystals may form in
the pores, after the water evaporates, preventing gas exchange from occurring. If
saltwater flooding did cause salt crystal formation on the eggshell, it would affect gas
exchange across the eggshell and may be a cause of decreased hatching success due to
flooding (Wangensteen, 1972). In order to test for salt crystal formation in the pores,
eggshells could be observed under a scanning electron microscope. While observing the
eggshell, it would also be important to look at the porosity of the eggshell because the
type of pore may control how readily bacterial penetration takes place (Board & Fuller,
1994).
Originally we planned to use community sequencing for microbial analysis, a
method that would identify all types of microbes present in a sample, but this did not
happen due to unexpected drawbacks and time constraints. Our initial trial with
community sequencing did not work because the samples were too dilute. Although
another method of sample extraction was attempted for community sequencing, our
second trial did not return any positive results either. As an alternative we used a nutrient
agar, a non-selective media, to grow our samples. While this agar allows for most types
of bacteria to grow, this agar cannot grow fastidious species (species that require extra
nutrients in order to grow) or anaerobic species (bacteria that cannot grow in the presence
of oxygen) (Difco & BBL manual, 2003). There may have also been bacteria in the
samples that did not grow in the laboratory because the bacterial plate did not replicate
essential aspects of their environment (nutrients, pH, osmotic conditions, temperature,
microbial symbionts and many more) necessary to grow (Stewart et al., 2012). Since
nutrient agar does not allow for all types of bacteria to grow, we may not have observed a
19
change in diversity or abundance because the media did not permit growth for the types
of bacteria that were or were not affected by salt water. Growing our samples on a
selective media would have been advantageous compared to nutrient agar because we
could have chosen a media for types of bacteria that are known to influence trans-shell
infection such as MacConkey agar, which selects for gram negative bacteria (Cook et al.,
2004).
In the future, steps should be taken to improve this research. First it would be
crucial to increase the test sample size. Although we sampled over 400 eggs in the
summer, only a small portion of these eggs were analyzed due to time and money
restrictions, therefore the sample size used for this research was not large enough to
possess the statistical power necessary to detect small differences in the microbial
community. Secondly, it would be necessary to improve the method of bacterial
community analysis. This could be achieved using a selective media, which would only
allow species to grow that are known to cause trans-shell infection, or it could be
achieved using community sequencing, which would analyze the entire community for
each sample. Lastly, in order to confirm that trans-shell infection is or is not occurring, it
would be necessary to open some eggs and test the different layers for the presence of
microbes. Without direct evidence for trans-shell infection there is no way to support the
supposition that difference in microbial communities, if detected, are important for egg
viability in saltmarsh and Nelson’s sparrows’ eggs.
20
References
Adam, P. 1990. Saltmarsh ecology. Cambridge University Press, Cambridge, UK.
Aguilera, O., L. M. Quiros, and J. F. Fierro. (2003). Transferrins selectively cause ion
efflux through bacterial and artificial membranes. FEBS Lett. 548:5–10.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local
alignment search tool. Journal of molecular biology, 215(3), 403-410.
Ash, C., Farrow, J. A. E., Wallbanks, S., & Collins, M. D. (1991). Phylogenetic
heterogeneity of the genus Bacillus revealed by comparative analysis of
small-­‐subunit-­‐ribosomal RNA sequences. Letters in Applied Microbiology, 13(4), 202206.
Baron, F., M. Gautier, and G. Brule. (1997). Factors involved in the inhibition of growth
of Salmonella Enteritidis in liquid egg white. J. Food Prot. 60:1318–1323.
Benoit, L. K., & Askins, R. A. (1999). Impact of the spread of Phragmites on the
distribution of birds in Connecticut tidal marshes. Wetlands, 19(1), 194-208.
Board, R. G., & Fuller, R. (Eds.). (1994). Microbiology of the avian egg. London:
Chapman & Hall.
Board, R.G., Halls, N.A., (1973). The cuticle: a barrier to liquid and particle penetration
of the shell of hen's egg. British Poultry Science 14, 67–97.
Board, R. G. & Tranter, H. S. (1986). The microbiology of eggs. In Egg science &
technology, 3rd edn (ed. W. J. Stadelman & O. J. Cotterill), pp. 75–96. Westport, CT:
AVI Publishing Co.
Brandelli, A. (2008). Bacterial keratinases: useful enzymes for bioprocessing
agroindustrial wastes and beyond. Food and Bioprocess Technology, 1(2), 105-116.
Brawley, A. H., Warren, R. S., & Askins, R. A. (1998). Bird use of restoration and
reference marshes within the Barn Island Wildlife Management Area, Stonington,
Connecticut, USA. Environmental Management, 22(4), 625-633.
Bressolier, P., Letourneau, F., Urdaci, M., & Verneuil, B. (1999). Purification and
characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus.
Applied and Environmental Microbiology, 65, 2570–2576.
Brittingham, M. C., Temple, S. A., & Duncan, R. M. (1988). A survey of the prevalence
of selected bacteria in wild birds. Journal of Wildlife Diseases,24(2), 299-307.
21
Cai, C. G., Lou, B. G., & Zheng, X. D. (2008). Keratinase production and keratin
degradation by a mutant strain of Bacillus subtilis. Journal of Zhejiang University
Science B, 9(1), 60-67.
Chesneau, O., Morvan, A., Grimont, F., Labischinski, H., & El Solh, N. (1993).
Staphylococcus pasteuri sp. nov., isolated from human, animal, and food specimens.
International journal of systematic bacteriology, 43(2), 237-244.
Cook, M. I., Beissinger, S. R., Toranzos, G. A., & Arendt, W. J. (2005). Incubation
reduces microbial growth on eggshells and the opportunity for trans-­‐shell infection.
Ecology Letters, 8(5), 532-537.
Cook, M. I., Beissinger, S. R., Toranzos, G. A., Rodriguez, R. A., & Arendt, W. J.
(2003). Trans–shell infection by pathogenic micro–organisms reduces the shelf life of
non–incubated bird's eggs: a constraint on the onset of incubation?.Proceedings of the
Royal Society of London. Series B: Biological Sciences,270(1530), 2233-2240.
Csonka, L. N. (1989). Physiological and genetic responses of bacteria to osmotic stress.
Microbiological Reviews, 53(1), 121-147.
Csonka, L. N., & Hanson, A. D. (1991). Prokaryotic osmoregulation: genetics and
physiology. Annual Reviews in Microbiology, 45(1), 569-606.
Del Moral, A., Quesada, E., & Ramos-Cormenzana, A. (1987, February). Distribution
and types of bacteria isolated from an inland saltern. In Annales de l'Institut
Pasteur/Microbiologie (Vol. 138, No. 1, pp. 59-66). Elsevier Masson.
De Reu, K., Grijspeerdt, K., Messens, W., Heyndrickx, M., Uyttendaele, M., Debevere,
J., & Herman, L. (2006). Eggshell factors influencing eggshell penetration and whole egg
contamination by different bacteria, including Salmonella enteritidis. International
journal of food microbiology, 112(3), 253-260.
Difco & BBL manual: manual of microbiological culture media. Becton Dickinson and
Company, 2003.
DiQuinzio, D. A., P. W C. Paton, and W. R. Eddleman. (2001). Site fidelity, philopatry,
and survival of promiscuous Saltmarsh Sharp-tailed Sparrows in Rhode Island. Auk
118:888–899.
DiQuinzio, D. A., Paton, P. W., & Eddleman, W. R. (2002). Nesting ecology of
Saltmarsh Sharp-tailed Sparrows in a tidally restricted salt marsh. Wetlands,22(1), 179185.
Fox, G. E., Wisotzkey, J. D., & Jurtshuk, P. (1992). How close is close: 16S rRNA
sequence identity may not be sufficient to guarantee species identity.International
Journal of Systematic Bacteriology, 42(1), 166-170.
22
Galinski, E. A., & Trüper, H. G. (1994). Microbial behaviour in salt-stressed ecosystems.
FEMS Microbiology Reviews, 15(2), 95-108.
Gjerdrum, C., Elphick, C. S., & Rubega, M. (2005). Nest site selection and nesting
success in saltmarsh breeding sparrows: the importance of nest habitat, timing, and study
site differences. The Condor, 107(4), 849-862.
Gjerdrum, C., Sullivan-Wiley, K., King, E., Rubega, M. A., & Elphick, C. S. (2008). Egg
and chick fates during tidal flooding of Saltmarsh Sharp-tailed Sparrow nests. The
Condor, 110(3), 579-584.
Greenlaw, J. S. and J. D. Rising. (1994). Sharp-tailed Sparrow (Ammodramus
caudacutus). In The Birds of North America, no. 112 (A. Poole and F. Gill, Eds.).
Academy of Natural Sciences, Philadelphia, and American Ornithologists' Union,
Washington, D.C.
Haygood, M. G., Schmidt, E. W., Davidson, S. K., & Faulkner, D. J. (1999). Microbial
symbionts of marine invertebrates: opportunities for microbial biotechnology. Journal of
molecular microbiology and biotechnology, 1(1), 33-43.
Herlemann DPR, Labrenz M, Jürgens K, Bertilsson S, Waniek JJ, Andersson AF. (2011).
Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic
Sea. ISME J 5: 1571–1579.
Hodgman, T. P., W. G. Shriver, and P. D. Vickery. (2002). Redefining range overlap
between the sharp-tailed sparrows of coastal New England. Wilson Bulletin 114:38–43.
Horn, M. A., Ihssen, J., Matthies, C., Schramm, A., Acker, G., & Drake, H. L. (2005).
Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov.,
Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain MH72, N2O-producing
bacteria isolated from the gut of the earthworm Aporrectodea caliginosa. International
journal of systematic and evolutionary microbiology,55(3), 1255-1265.
Hughey, V. L., and E. A. Johnson. (1987). Antimicrobial activity of lysozyme against
bacteria involved in food spoilage and foodborne disease. Appl. Environ. Microbiol.
53:2165–2170.
Imhoff, J. F., & Thiemann, B. (1991). Influence of salt concentration and temperature on
the fatty acid compositions of Ectothiorhodospira and other halophilic phototrophic
purple bacteria. Archives of microbiology, 156(5), 370-375.
Jarosz, A. M., & Davelos, A. L. (1995). Tansley Review No. 81. Effects of disease in
wild plant populations and the evolution of pathogen aggressiveness.New Phytologist,
371-387.
23
Jiménez, G., Urdiain, M., Cifuentes, A., López-López, A., Blanch, A. R., Tamames, J., ...
& Rosselló-Móra, R. (2013). Description of Bacillus toyonensis sp. nov., a novel species
of the Bacillus cereus group, and pairwise genome comparisons of the species of the
group by means of ANI calculations. Systematic and applied microbiology, 36(6), 383391.
Johnston, R. F. (1956). Population structure in salt marsh Song Sparrows. Part I.
Environment and annual cycle. Condor 58:24–44.
Kim, J. M., Lim, W. J., & Suh, H. J. (2001). Feather-degrading Bacillus species from
poultry waste. Process Biochemistry, 37, 287–291.
Larsen, H. (1986). Halophilic and halotolerant microorganisms-­‐an overview and
historical perspective. FEMS Microbiology Letters, 39(1-­‐2), 3-7.
S. Lechner, R. Mayr, K.P. Francis, B.M. Prüss, T. Kaplan, E. Wiessner-Gunkel, G.S.A.B.
Stewart, S. Scherer. (1998). Bacillus weihenstephanensis sp nov. is a new psychrotolerant
species of the Bacillus cereus group Int. J. Syst. Bacteriol., 48, 1373–1382
Lin, X., Inglis, G. D., Yanke, L. J., & Cheng, K. J. (1999). Selection and characterization
of feather degrading bacteria from canola meal compost. Journal of Industrial
Microbiology and Biotechnology, 23, 149–153.
Lippert, K., & Galinski, E. A. (1992). Enzyme stabilization be ectoine-type compatible
solutes: protection against heating, freezing and drying. Applied microbiology and
biotechnology, 37(1), 61-65.
Meanley, B. (1985). The Marsh Hen—A Natural History of the Clapper Rail of the
Atlantic Coast Salt Marsh. Tidewater, Centreville, Maryland.
Meehan, C., Bjourson, A. J., & McMullan, G. (2001). Paenibacillus azoreducens sp. nov.,
a synthetic azo dye decolorizing bacterium from industrial wastewater. International
journal of systematic and evolutionary microbiology, 51(5), 1681-1685.
Mine, Y., Oberle, C., & Kassaify, Z. (2003). Eggshell matrix proteins as defense
mechanism of avian eggs. Journal of agricultural and food chemistry,51(1), 249-253.
Mitscherlich, E., & Marth, E. H. (1984). Microbial survival in the environment. Bacteria
and rickettsiae important in human and animal health. Springer-Verlag.
Montevecchi, W. A. (1978). Nest site selection and its survival value among Laughing
Gulls. Behavioral Ecology and Sociobiology 4:143–161.
24
Ohno, N., & Morrison, D. C. (1989). Lipopolysaccharide interaction with lysozyme.
Binding of lipopolysaccharide to lysozyme and inhibition of lysozyme enzymatic
activity. Journal of Biological Chemistry, 264(8), 4434-4441.
Onifade, A. A., Al-Sane, N. A., Al-Musallam, A. A., & Al-Zarban, S. (1998). A review:
potentials for biotechnological applications of keratin-degrading microorganisms and
their enzymes for nutritional improvement of feathers and other keratins as livestock feed
resources. Bioresource technology, 66(1), 1-11.
Paul, T., Halder, S. K., Das, A., Bera, S., Maity, C., Mandal, A., ... & Mondal, K. C.
(2013). Exploitation of chicken feather waste as a plant growth promoting agent using
keratinase producing novel isolate Paenibacillus woosongensis TKB2. Biocatalysis and
Agricultural Biotechnology, 2(1), 50-57.
Peele, A. M., Burtt Jr, E. H., Schroeder, M. R., & Greenberg, R. S. (2009). Dark color of
the Coastal Plain Swamp Sparrow (Melospiza georgiana nigrescens) may be an
evolutionary response to occurrence and abundance of salt-tolerant feather-degrading
bacilli in its plumage. The Auk, 126(3), 531-535.
Post, W. and J. S. Greenlaw. (1982). Comparative costs of promiscuity and monogamy:
A test of reproductive effort theory. Behavioral Ecology and Sociobiology 10:101–107.
Priest, F. G. (1993). Systematics and ecology of Bacillus subtilis. Bacillus subtilis and
other Gram-positive bacteria, 3-16.
Ratkowsky, D. A., Olley, J., McMeekin, T. A., & Ball, A. (1982). Relationship between
temperature and growth rate of bacterial cultures. Journal of Bacteriology, 149(1), 1-5.
Reinert, S. E., & Mello, M. J. (1995). Avian community structure and habitat use in a
southern New England estuary. Wetlands, 15(1), 9-19.
Rose, M. L., & Hincke, M. T. (2009). Protein constituents of the eggshell: eggshellspecific matrix proteins. Cellular and molecular life sciences, 66(16), 2707-2719.
Shannon, C. E. (1948) A mathematical theory of communication. The Bell System
Technical Journal, 27, 379–423 and 623–656.
Shriver, G. W. (2002). Conservation ecology of salt marsh birds in New England
(Doctoral dissertation, State University of New York. College of Environmental Science
and Forestry, Syracuse, NY).
Shriver, W. G., Hodgman, T. P., Gibbs, J. P., & Vickery, P. D. (2004). Landscape
context influences salt marsh bird diversity and area requirements in New England.
Biological Conservation, 119(4), 545-553.
25
Shriver, W. G., Vickery, P. D., Hodgman, T. P., & Gibbs, J. P. (2007). Flood tides affect
breeding ecology of two sympatric sharp-tailed sparrows. The Auk,124(2), 552-560.
Stewart, E. J. (2012). Growing unculturable bacteria. Journal of bacteriology,194(16),
4151-4160.
Teal, J. M. (1986). The ecology of regularly flooded salt marshes of New England: A
community profile. U.S. Department of Interior, Fish and Wildlife Service, Biological
Report 85 (7.4).
Thiemann, B., & Imhoff, J. F. (1991). The effect of salt on the lipid composition of
Ectothiorhodospira. Archives of microbiology, 156(5), 376-384.
Trüper, H. G., & Galinski, E. A. (1986). Concentrated brines as habitats for
microorganisms. Experientia, 42(11-12), 1182-1187.
Tyler, C., (1956). Studies on egg shells. VII: some aspects of structure as shown by
plastic models. Journal of Science Food and Agriculture 7, 483–493.
Vary, P. S. (1994). Prime time for Bacillus megaterium. Microbiology (Reading,
England), 140, 1001-1013.
Ventosa, A., Quesada, E., Rodriguez-Valera, F., Ruiz-Berraquero, F., & RamosCormenzana, A. (1982). Numerical taxonomy of moderately halophilic Gram-negative
rods. Journal of General Microbiology, 128(9), 1959-1968.
Von Stetten, F., Mayr, R., & Scherer, S. (1999). Climatic influence on mesophilic
Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical,
temperate and alpine soil. Environmental microbiology,1(6), 503-515.
Wang, J. M., Firestone, M. K., & Beissinger, S. R. (2011). Microbial and environmental
effects on avian egg viability: Do tropical mechanisms act in a temperate environment?.
Ecology, 92(5), 1137-1145.
Wangensteen, O. D. (1972). Gas exchange by a bird's embryo. Respiration physiology,
14(1), 64-74.
Webb, D.R. (1987). Thermal tolerance of avian embryos: a review. Condor, 89, 874–898.
Wellman-Labadie, O., J. Picman, and M. T. Hincke. (2007). Avian antimicrobial
proteins: structure, distribution and activity. Worlds Poult. 63:421–438.
Woolfenden, G. E. (1956). Comparative breeding behavior of Ammospiza caudacuta and
A. maritima. Publications of the Museum of Natural History, University of Kansas
10:45–75.
26
Yeager, J. G., & Ward, R. L. (1981). Effects of moisture content on long-term survival
and regrowth of bacteria in wastewater sludge. Applied and environmental microbiology,
41(5), 1117-1122.
Zahran, H. H. (1997). REVIEW ARTICLE Diversity, adaptation and activity of the
bacterial flora in saline environments, 211–223.
27
TABLES
Table 1
a. PCR Cocktail mix for 25 µml reactions
CONCENTRATIONS
12.5 µl
1.25 µl
1.25 µl
5 µl
5 µl
REAGENTS
Qiagen® Master PCR MIX
Integrated DNA Technologie® 16s rRNA Forward Primer
(5’ AGA GTT TGA TCC TGG CTC AG)
Integrated DNA Technologie® 16s rRNA Reverse Primer
(5’ AGA GCT ACC TTG TTA CGA CTT)
Nuclease Free Water
Extracted Bacterial DNA
b. PCR Protocol for 16s rDNA amplification
PCR STEPS
Initial activation step
TIME and CYCLES
3 min
TEMPERATURE
95°C
Denaturation
30 s
95°C
Annealing
30 s
50°C
Extension
1 min/kb (90s for 1.5 kB)
72°C
Number of cycles
35 cycles
End of PCR cycling
Indefinite
3-step cycling
4°C
28
Table 2
Bacteria types and CFU abundances calculated by morphological characteristics.
29
Table 3
Sequences of 16s rDNA
30
Table 3 (Continued)
31
Table 3 (Continued)
32
Table 4
Sequences identified using Nucleotide BLAST
Sequence Description
Sequence Sample Picture
Alignment
A
Paenibacillus spp.
99.00%
B
Bacillus weihenstephanensis
99.00%
C
Staphylococcus pasteuri
99.00%
D
Staphylococcus spp.
99.00%
E
Bacillus megaterium
99.00%
33
Table 4 (Continued)
F
Bacillus spp.
99.00%
G
Staphylococcus pasteuri
99.00%
H
Staphylococcus spp.
99.00%
I
Staphylococcus spp.
99.00%
J
Bacillus spp.
99.00%
K
Bacillus toyonensis
99.00%
34
Table 4 (Continued)
L
Bacillus megaterium
99.00%
M
Bacillus weihenstephanensis
99.00%
N
Paenibacillus taichungensis
99.00%
35
FIGURES
Figure 1
Scarborough marsh sites. For this study egg shell swabs were collected from nests located at the Jones
Creek (JO) and Scarborough (SC) sites.
36
Figure 2
Sample iButton Graph: Not Flood/Hatch
Figure 3
Sample iButton Graph: Flood/Not Hatch
37
Figure 4
a.
b.
Compares the difference in percent
microbial coverage of plates from
samples that flooded and did not hatch
with samples that did not food and did
not hatch (N = 15, t = -0.3, df = 3.2, p =
0.79).
38
Compares the difference in percent
microbial coverage of plates from
samples that flooded and did not hatch
with samples that did not flood and
hatched (N = 15, t = 0.7, df = 5.2, p =
0.48)
Figure 5
Compares the colony forming units (CFUs) of eggshells before and after the flooding event for each of the
three treatments. Flood/Fail compared with No Flood/Fail (N=15, t=0.05, df = 13, p=0.96). Flood/Fail
compared with No Flood/Hacth (N=15, t=0.18, df=13, p=0.86).
39
Figure 6
a.
b.
Compares the difference in microbial
diversity of samples that did not flood
and hatched with samples that flooded
and did not hatch (N=15, t =-0.04,
df=4.2, p=0.97).
40
Compares the difference in microbial
diversity of samples that flooded and
did not hatch with samples that not
flood and did not hatch (N =15, t=-1.1,
df =3.9, p=0.49).
Figure 7
Gel from PCR amplification of 16s rRNA gene.
41
Author’s Biography
Mattie V. Paradise was born in York, Maine on May 10, 1992. She was raised in Wells,
Maine and graduated from Wells High School as Salutatorian in 2010. Majoring in
Biology, with a focus in pre-dental studies, she is a dean's list student at the University of
Maine and will graduate in May, 2014. Mattie played trombone in the marching and pep
bands and is currently the Class Liaison for the Class of 2014. She was an active member
in the University of Maine Student Government and served as a student Senator for all
four years of college.
After graduation, Mattie will attend dental school at Dalhousie University in Halifax,
Nova Scotia where she will receive her Doctor of Dental Surgery (D.D.S.) degree. She
plans on having a traveling dental clinic, which will provide dental care for under-served
communities in Maine.
42