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The Fungi in the Feces:
Identifying Pilobolus Species
Casey Gailey1, 2, Beth Barbolla1, 3, and Dr. Dale Beach1
1
Honors Section: Unity of Life (Biology 121)
Biology Major, Rhetoric and Professional Writing Minor
3
Nursing Major
2
Longwood University, Farmville, Virginia
Abstract:
This study investigated a sample of fungi gathered from a farm in Cumberland,
Virginia. The fungi from the sample demonstrated the morphological
characteristics of the genus Pilobolus. Classical spore sizing techniques
provided a morphological basis to identify fungi from the length and width of
individual spores. By comparing the results of this type of technique to the
compiled data from scientists such as Grove and Hu (Foos et al. 2011), the
species the spores originated from could be hypothesized. However, due to
spore-size overlaps and natural distinctive characteristics among individual
strains, this method is inconclusive, leaving an uncertainty of identity as P.
roridus, P. crystallinus, or other species. Thus, DNA methodology was used on
rRNA and β-Tubulin genes in order to provide more substantial evidence for
asserting one species over another. Spore sizing techniques identified the
presence of both P. crystallinus and P. roridus species. These finding were
supported by genetic techniques and classify the original sample as supporting a
heterozygous population of Pilobolus fungi.
INTRODUCTION
Pilobolus fungi are filamentous,
coprophilous, phototropic organisms
characterized by the early development of
trophocysts from hyphae. Pilobolus is
further known for possessing dark
pigmented sporangia which develop atop a
subsporangial swelling and are ejected from
the fungi by ballistic discharge.
Figure 1. Pilobolus Life Cycle as illustrated by
Casey Gailey.
The life cycle of Pilobolus begins
with spores growing in animal dung and
grows taller on one side to aim the
spreading coenocytic hyphae throughout the
sporangium toward light and the fluid inside
feces (A in Figure 1). In two to three days,
of the subsporangial swelling builds up
large yellow-orange nodules called
pressure until it explodes, ejecting the
trophocysts form along the hyphae (B in
sporangium away from the parent fungus
Figure 1). In Figure 1, C through E
(transition from F to A in Figure 1). Due to
illustrate the growth of the fungus, including
the adhesive quality of the sporangial
the development of the stipe, subsporangial
coating, the sporangium clings to whatever
swelling, and sporangium, as well as the
it hits after being ejected, which is typically
invisible maturation of spores within the
vegetation in natural environments, so
sporangium. Once fully mature, Pilobolus
animals will presumably consume the spores
develops the dark sporangial coating which
and later excrete them to begin the Pilobolus
is adhesive (F in Figure 1). The stipe then
life cycle anew.
The genus Pilobolus (Eukaryota,
inoculated onto Simplified Hemin Media in
Fungi, Zygomycota, Mucoromycotina,
petri dishes sealed with parafilm and grown
Mucorales, Pilobolaceae) contains seven to
at 20 to 24°C with a 12 hour light cycle
nine different species and Pilobolus samples
(Foos et. al., 2011). These primary cultures
gathered from one source may potentially
will be further referred to as S-1, S-2, S-3,
contain multiple species (Foos 1997).
S-4, S-5, and S-6.
Therefore, to determine if the population of
Spore Sizing. Three sporangia from each of
Pilobolus that was collected from one pile of
the S-1, S-2, S-3, S-4, and S-5 Pilobolus
dung is homozygous or heterozygous,
cultures were transferred to a glass slide.
morphological characteristics and genomic
The sporangia were then crushed on the
DNA from several cultures were compared.
slide to break the thick sporangial coating
It is expected that this sample will contain
and reveal the individual spores contained
multiple species of Pilobolus.
within for microscopic observation and
MATERIALS AND METHODS
measurement. Approximately ten spores
Culture Method. Dung samples containing
were randomly selected per sporangium for
Pilobolus fungi were attained from a farm in
measurement.
Cumberland, Virginia. The sample was
DNA Extraction. Liquid growth cultures
transferred to a closed container and placed
were developed using agar blocks
in a secure location to allow growth of the
containing the hyphae from S-1, S-2, S-3, S-
fungus on the dung at approximately 20 to
4, and S-6. A MoBio UltraClean Soil Kit
24°C with a 12 hour light cycle. After the
was then used as the basis for genomic
fungi in the sample matured, sporangia
preparation of each as seen through Foos
adhering to the lid of the container were
et.al. (2011).
Table 1. This shows the results from spore sizing: For five of the primary cultures (S-1, S-2, S-3, S-4, and S-5), two
to three sporangium were examined and, from each, approximately ten spores were measured and averaged.
Average Spore Sizes
S-1
S-2
S-3
S-4
S-5
S-6
Sporangium 1
6.7 x 5.2 µm
6.3 x 4.0 µm
5.1 x 4.4 µm
6.3 x 4.2 µm
5.6 x 4.1 µm
-
Sporangium 2
9.1 x 6.3 µm
5.8 x 4.5 µm
5.0 x 5.2 µm
5.9 x 3.8 µm
6.0 x 4.2 µm
-
Sporangium 3
-
-
-
6.3 x 4.0 µm
8.1 x 4.1 µm
-
PCR Amplifications. Using the DNA
used primers pTub-vF1 and pTub-vR1. Each
extracted from the S-1, S-2, S-3, S-4, and
reaction then underwent a Thermal Profile,
S-6 liquid growth cultures, two PCR
including an initial denaturation step of
reactions were set up for each sample. The
95°C for 4 minutes, with thirty incubation
first reaction was designed to target
cycles of 30 seconds at 95°C, 90 seconds at
ribosomal RNA using the primers ITS4 and
50°C, and 90 seconds at 72°C, concluded
ITS5. To do so, 25 µl of 2X MasterMix
with an additional seven minutes at 72°C to
solution (containing the 10X buffer, 2.5 mM
polish off the amplicons.
deoxynucleotide triphosphate or dNTP,
Agarose gel electrophoresis was then
TAQ polymerase and 25 mM MgCl2), 1 µl
used to measure the basepair counts of each
of each rRNA 20 µM primer, 2 µl of the
template, which determines whether they
template, and 21 µl of H2O were combined
correctly isolated rRNA and β-Tubulin since
to make a 50 µl solution. The second
the rRNA should consist of approximately
reaction was designed to target β-tubulin
700 bases and β-Tubulin approximately 900
and incorporated the same mixture except
bases.
The β-Tubulin gene, unlike rRNA,
Table 2. These are the results from Agarose Gel
Electrophoresis.
may have multiple possible sequences, so
Basepair Counts
rRNA
β-Tubulin
S-1
750
1000
S-2
700
900
S-3
700
800
S-4
700
800
S-5
-
-
S-6
750
900
the isolated β-Tubulin was cloned using the
TA-Cloning Kit as seen in Foos et al.
(2011). From the cloned cells, the colonies
containing the β-Tubulin gene were
segregated through a blue-white screening.
Then, using the manufacturer’s protocol for
Sequence Analysis. Contigs were developed
the High-Speed Plasmid Mini Kit, the
plasmid DNA was isolated from β-Tubulin
through the PRABI-Doua CAP3 Sequence
Assembly Program and submitted to the
colonies for sequencing.
National Center for Biotechnology
RESULTS
Information’s BLAST database. Shown in
Spore Sizes. As seen in Table 1, all
Figures 4 to 8, the database identified both
examined cultures demonstrated similar
Pilobolus crystallinus and Pilobolus roridus
spore sizes, typically averaging a length
as the top results for S-1, S-2, S-3, and S-6.
between 5 and 6.7 µm and a width between
These results furthermore had E Values of
3.8 and 5.2 µm.
0.0, demonstrating how significant the
Gel Electrophoresis. After PCR
matches are to the template. The database
amplification, agarose gel electrophoresis
also identified P. crystallinus and P. roridus
determined the basepair counts for rRNA
and β-Tubulin in each culture of Pilobolus,
as the top results for S-4, however none of
the results demonstrated an E Value of 0.0.
as shown in Table 2.
Figure 2. Phylogenetic tree based on five of the primary culture’s (S-1, S-2, S-3, S-4, and S-6) rRNA sequences, as
well as the following controls gathered from the NCBI’s taxonomy database: Pilaira anomala (strain ATCC 36774),
Pilobolus crystallinus (strains ATCC 11505, ATCC 36186, and ATCC 46942), and Pilobolus roridus (strains IUE
920 and IUE 918).
The β-Tubulin sequences were also
Microeléctronique Montpellier (LIRMM), a
submitted to the BLAST database. Shown in
phylogenetic tree was created for the rRNA
Figures 9 to 14, the results for all of the β-
sequences (Figure 2). Controls of
Tubulin templates, S-1, S-2A, S-2B, S-3, S-
previously identified Pilobolaceae fungi,
4A, and S-4B, listed 30 to 50 results with a
including a Pilaira, three P. crystallinus,
0.0 E Values. All of these matches were the
and two P. roridus strains, were also
β-Tubulin genes of various fungi.
included in the tree to obtain genomic based
Phylogenetic Tree. Using the One Click
evidence for species identification of the S-
Phylogeny Analysis system of the
1, S-2, S-3, S-4, and S-6 fungi.
Montpellier Laboratory of Informatics,
A phylogenetic tree was created for
Robotics and Microelectronics, or the
the β-Tubulin templates as well (Figure 3).
French Laboratoire Informatique Robotique
The tree includes β-Tubulin genes from
Figure 3. Phylogenetic tree based on four of the primary culture’s (S-1, S-2, S-3, and S-4) β-Tubulin gene
sequences, as well as the following controls gathered from the NCBI’s database: Actinomucor elegans strain
FSU276, Utharomyces epallocaulus strain FSU854, Mucor mucedo strain FSU621, Pilaira anomala strain FSU268,
Rhizopus homothallicus strain FSU2530, and Blakeslea trispora strain FSU391.
S-1, S-3, and two from S-2 and S-4, as well
particular, S-1 exhibited spore size
as control β-Tubulin gene sequences from
characteristics of P. crystallinus, S-2 and S-
various other fungi of the Mucorales order.
3 exhibited spore sizes that resemble both P.
DISCUSSION
crystallinus and P. roridus, and S-4 and S-5
P. crystallinus is 8-10 µm long and 5-6 µm
exhibited spore sizes that more closely
wide according to Grove, and 7-10 µm long
resemble P. roridus.
and 4.5-6 µm wide according to Hu. P.
The BLAST results for rRNA,
roridus is 6-8 µm long and 3-4 µm wide
Figures 4 to 8, also support this: Each of the
according to Grove, and 4.5-7.5 µm long
five primary cultures sequenced, S-1, S-2, S-
and 3-4.5 µm wide according to Hu. Based
3, S-4, and S-6, the top four results were of
on this, the primary cultures that underwent
P. roridus or P. crystallinus. In all but S-4,
spore sizing, S-1, S-2, S-3, S-4, and S-5,
these results have E Values of 0.0,
demonstrate both of these species. In
emphasizing the significance of the species
as the potential identity of the cultures.
However, these techniques lack
absolute distinction between these two
species which are so characteristically
likely because the database doesn’t have
Pilobolus β-Tubulin sequences in the system
yet.
The phylogenetic tree for β-Tubulin,
analogous, so the phylogeny analysis
Figure 3, also confirms this as it illustrates
provides more substantial evidence for
the cultures as having close genetic
species identification. Figure 4 illustrates
relationships with the β-Tubulin controls.
that S-1, S-2, S-3 and S-6 are all the same
Oddly, the phylogenetic tree shows two
species and strain. The figure also shows
primary branches, and splits the samples
that these four are more closely related to P.
from this experiment between the two:
crystallinus. On the other hand, S-4 is
Samples from S-1 and S-3 reside on one
farther away from the other four cultures,
branch, while the S-2A, S-2B, S-4A, and S-
which represents that it is genetically
4B samples reside on the second branch
dissimilar from them. Based on branch
with all of the controls. Considering that Gel
length and structure, S-4 is more closely
Electrophoresis supports each template of
related to the P. roridus controls.
rRNA and of β-Tubulin as being the target
The BLAST results for β-Tubulin,
sequences, because the basepair counts of
Figures 9 to 14, support that the β-Tubulin
each are relatively close to the expected
templates are of the correct gene, because
values, S-1 and S-3 should be β-Tubulin
the top results are all of β-Tubulin for
genes. As such, the tree may demonstrate
various fungi. And, while the database
that these two samples merely used a
doesn’t show Pilobolus as a result, it does
different variant of the gene. After all,
show fungi of the Mucorales order, most
Pilobolus is a diploid organism, so there are
National Park." Mycological
two copies of each gene.
Research 101.12 (1997): 1535-1536.
Thus, between morphological and
genetic methods, this experiment surmises
that the sample gathered from Cumberland,
Virginia, is a heterozygous population
containing both Pilobolus crystallinus and
Pilobolus roridus species.
AKNOWEDGEMENTS
We thank Longwood University and Dr.
Dale Beach, Assistant Professor of Biology,
for supporting this project by supplying the
laboratory, materials and guidance
necessary. We also thank Shane Crean,
Alexus Edwards, Bailey Graebner, Molly
Kabis, Brianna Kelly, Christina Mertz,
Samantha Pritchett, and Kelly Ward for
providing the data from their Pilobolus
cultures.
CITATIONS
Foos, K. Michael. "Pilobolus and lungworm
disease affecting elk in Yellowstone
---, et al. "Phylogeny of Pilobolaceae."
Mycologia 103.1 (2011): 36-44.
SUPPLEMENTARY DATA
Figure 4. (Above) These are the top four BLAST results for S-1 rRNA template.
Figure 5. (Above) These are the top four BLAST results for S-2 rRNA template.
Figure 6. (Above) These are the top four BLAST results for S-3 rRNA template.
Figure 7. (Above) These are the top four BLAST results for S-4 rRNA template.
Figure 8. (Above) These are the top four BLAST results for S-6 rRNA template.
Figure 9. (Above) These are the top five of thirty-six S-1 β-Tubulin BLAST results with a 0.0 E Value.
Figure 109. (Above) These are the top five of thirty S-2A β-Tubulin BLAST results with a 0.0 E Value.
Figure 101. (Above) These are the top five of forty-two S-2B β-Tubulin BLAST results with a 0.0 E Value.
Figure 112. (Above) These are the top five of forty-two S-3 β-Tubulin BLAST results with a 0.0 E Value.
Figure 123. (Above) These are the top five of thirty-seven S-4A β-Tubulin BLAST results with a 0.0 E Value.
Figure 134. (Above) These are the top five of fifty S-4B β-Tubulin BLAST results with a 0.0 E Value.