Decline of Diporeia in Lake Michigan: Was Disease Associated With Invasive Species the Primary Factor?

Abstract

Populations of the freshwater amphipod Diporeia spp. have steadily declined in Lake Michigan since the late 1980’s. Prior studies have provided inconclusive data on possible reasons for their decline. However, some authors suggest that food competition and/or diseases associated with aquatic invasive species (AIS), such as zebra mussels ( Dreissena polymorpha ), may have caused the collapse of Diporeia . In this project, the possibility of pathogens as the cause of the collapse of Diporeia has been examined. Linear regression modeling show a significant positive linear association between percent of Diporeia exhibiting a pathogenic infection and year (r=0.7202264, p<=0.0124). Chi-square testing for independence was also used to test if there was an association between year and percent infection (X 2 = 50, df = 10, p<=0.0001), implying significant association between year and infection. Hence, the introduction of zebra mussels and the diseases they carry may have been the root cause for the decline of Diporeia . Future research is needed to examine other invasive species for similar pathogens, including live studies showing direct causality between zebra mussels and the decline in Diporeia .

Full text

International Journal of Biology; Vol. 7, No. 1; 2015
ISSN 1916-9671 E-ISSN 1916-968X
Published by Canadian Center of Science and Education
93
Decline of Diporeia in Lake Michigan: Was Disease Associated With
Invasive Species the Primary Factor?
Courtney S. Cave1 & Kevin B. Strychar1
1Annis Water Resources Institute, Grand Valley State University, Muskegon, Michigan, United States of America
Correspondence: Kevin B. Strychar, Annis Water Resources Institute, Grand Valley State University, Muskegon,
Michigan, 49441, USA. Tel: 1-616-331-8796. E-mail: strychark@gvsu.edu
Received: October 29, 2014 Accepted: November 11, 2014 Online Published: December 4, 2014
doi:10.5539/ijb.v7n1p93 URL: http://dx.doi.org/10.5539/ijb.v7n1p93
Abstract
Populations of the freshwater amphipod Diporeia spp. have steadily declined in Lake Michigan since the late
1980’s. Prior studies have provided inconclusive data on possible reasons for their decline. However, some authors
suggest that food competition and/or diseases associated with aquatic invasive species (AIS), such as zebra
mussels (Dreissena polymorpha), may have caused the collapse of Diporeia. In this project, the possibility of
pathogens as the cause of the collapse of Diporeia has been examined. Linear regression modeling show a
significant positive linear association between percent of Diporeia exhibiting a pathogenic infection and year
(r=0.7202264, p≤0.0124). Chi-square testing for independence was also used to test if there was an association
between year and percent infection (X2 = 50, df = 10, p≤0.0001), implying significant association between year
and infection. Hence, the introduction of zebra mussels and the diseases they carry may have been the root cause
for the decline of Diporeia. Future research is needed to examine other invasive species for similar pathogens,
including live studies showing direct causality between zebra mussels and the decline in Diporeia.
Keywords: Diporeia spp., Lake Michigan, aquatic invasive species, zebra mussels (Dreissena polymorpha), disease
1. Introduction
1.1 Background
Diporeia spp. are freshwater amphipods that used to be the most dominant crustaceans in the benthic layer of the
Laurentian Great Lakes. High in lipid content, Diporeia have previously been considered the primary food
source for many bottom feeders in the Great Lakes including whitefish (Coregonus clupeaformis), bloater
(Coregonus hoyi), and slimy sculpin (Cottus cognatus) (Nalepa et al., 1998). Since the mid-1980’s, however,
populations of Diporeia began to disappear in Lake Michigan, declining over 95% in the last 15 years in some
places (Figure 1). As a consequence, some fish populations also decreased, perhaps resulting from a shift to less
nutritional food sources (Nalepa et al., 1998).
Figure 1. Densities of Lake Michigan Diporeia spp. continually decline from 1994 to 2010. Small red dots show
NOAA (National Oceanic and Atmospheric Administration) sampling locations (from Nalepa et al., 2014)

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94
The disappearance of Diporeia has been postulated to be the result of the invasive Zebra (Dreissena polymorpha)
and Quagga (Dreissena rostriformis) mussels introduced three years prior to the decline of Diporeia. Why
Dresseina spp. had this effect on Diporeia is still not completely understood. One hypothesis is that the mussels
led to decreased food availability due to food competition (Nalepa, 1989). However, there are some
inconsistencies with this hypothesis. Diporeia and Dreissena coexist in Lake Superior, Lake Cayuga and isolated
areas of Lake Michigan. Such observations suggest that the relationship between Diporeia and Dresseina is more
complex than simply competition for food.
The second possibility is that mussels served as a vector for pathogenic organisms infecting Diporeia; this
hypothesis is still being explored (Fanslow, pers. comm., 2013). In addition, anecdotal evidence indicates that
during the early years of decline in Diporeia, crustaceans (i.e. shrimp) in many other locations of the United
States were experiencing severe population declines, purportedly a consequence of disease. Rickettsia-like
infection, i.e. Haplosporidia and Microsporidia, have all been observed in Diporeia tissues (Messick et al.,
2004). The origin of these pathogens is not known. However, it is interesting to note that Rickettsia-like
infections are found in Dreissena located in Greece (Molloy et al., 2001). Similarly, Haplosporidium pathogens
were identified as the primary pathogen causing death in Crassostrea virginica (Eastern oyster) on the east coast
of North America and in fresh water snails (Physella parkeri) in Douglas Lake (Michigan; Barrow, 1961).
Microsporidia is also a common pathogen in freshwater shrimp (Gammarus fasciatus), an amphipod closely
related to Diporeia. The associated pathologies suggest that any one of these diseases could infect and possibly
be the cause of the decline in populations of Diporeia in the Great Lakes.
Taxonomically, Diporeia spp. belongs to the Phylum Arthropod, Subphylum Crustacea, Class Malacostraca,
Order Amphipoda, and Family Pontoporeiidea. In years past, all Diporeia were classified as Pontoporeia hoyi
(anonomous with P. affinis), however, taxonomists today believe there may be as many as eight species in the
Great Lakes (Cavaletto et al., 1996).
1.2 Objectives of the Study
In order to better understand why Diporeia spp. crashed and are still unable to repopulate to their original
concentrations, we designed our project to first (1) update the population density of Diporeia in Lake Superior’s
Batchawana Bay. This location has been identified in prior studies as a “safe haven” with high concentrations of
healthy Diporeia that coexist with a high abundance of zebra mussels. Studies of abundance of Diporeia at this
location have not been done since 2008. Secondly (2), we wanted to examine preserved (~14 years ago) samples
of Diporeia tissue for pathogenic infection prior to the introduction of zebra mussels, and immediately there-after,
from one general locality (i.e. Lake Michigan). In doing so we would be able to examine what pathogens existed
in Diporeia’s tissues before the presence of zebra mussels in Lake Michigan, and what pathogens exist in their
tissues now that the mussels have become established.
2. Materials and Methods
2.1 Field Work
Figure 2. Map is showing sampling sites located in Batchawana Bay, Lake Superior, Canada
In collaboration with the National Oceanic and Atmospheric Association (NOAA) and Great Lakes Environmental
Research lab (GLERL), Diporeia samples were collected from Lake Superior’s Batchawana Bay in Ontario,

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95
Canada (Figure 2). GLERL provided a 7.3 meter research vessel that is equipped with a ponar grab which, when
lowered, collects bottom sediment from the benthos. Using numerous site-specific locations known by GLERL
after many years of collecting Diporeia, the anchored boat at each site allowed technical staff to lower the ponar to
the bottom of the lake. Once bottom sediment was collected and retrieved, it was processed through a series of
water flushes filtered through a ASTM round all-brass 500 µm sieve until only a mixture of large material plus
Diporeia remained. Collecting sediment using the ponar was repeated ten times at each location (ten locations) to
ensure an accurate abundance of Diporeia was collected. After filtering the sediment, Diporeia was manually
removed with tweezers from the remaining material. The collected Diporeia samples were further subdivided into
two aliquots. The first aliquot was placed in liquid nitrogen and later transferred to a -80°C freezer for future
analyses. The second aliquot was placed into a -20°C freezer and retrieved as needed for this project.
2.2 Labwork: Histology
Diporiea collected from this study in addition to samples provided by NOAA, were prepared for histological studies.
Samples of Diporeia provided by NOAA were originally collected from Lake Michigan since the late 1980’s.
All tissues regardless of their date or site of collection were prepared and analyzed using similar methodologies.
All samples were first stained in Rose Bengal dye (Sigma-Aldrich, USA) and then placed in 10% formalin
(Sigma-Aldrich, USA) which maintains and preserves the tissue. Samples are then processed for microscopy.
The purpose of processing tissue samples is to remove water from the tissue and replace it with a solid medium
that will allow for thin sectioning. Individual Diporeia are removed from the formalin solution and placed into a
histology cassette. Each cassette held ten Diporeia (n=10). The Diporeia in these cassettes are then processed
using a series of increasing graded ethanol (Sigma-Aldrich, USA) solutions to dehydrate the tissue. Once
complete, tissue was then placed in xylene (Sigma-Alrich, USA), which is a clearing agent that removes the
alcohol from the previous step. Each cassette was placed, in order, in an 80%, 90%, 95%, and two changes of
100% ethanol solutions, followed by two washes of 100% xylene. Each cassette was then incubated for 20
minutes in each respective solution (Bergman, per comm., 2013). Following incubation in graded ethonal, the
cassettes were placed in tissue trays sprayed with HistoPrep Mold Releasing Agent (Fisher Scientific, USA).
Each sample of Diporeia was then placed into a metal embedding tray using tweezers and submerged in liquid
paraffin (wax) baths ~30 minutes. Immersion in liquid paraffin for 30 minutes allowed the tissue to become
completely infiltrated with wax. Prior spraying of the trays was important as it helped with the removal of the
solid wax block after cooling. Blocks not prepared in this manner chipped and fell apart and were too difficult to
remove. Once infiltration was complete, each wax block containing a single Diporeia sample were left to cool
for at least 3 hours at room temperature. Due to the small size of Diporeia, the wax outside a 1 cm radius of the
organism was heated to 55°C and removed using a vacuum infiltrator and paraffin dispenser (Lipshaw Inc.).
Preparring the Diporeia sample in this manner assisted in the latter processes of sagitally sectioning the wax
block with a sliding microtome (Bausch & Lomb, Rochester, USA).
In order to collect thin-sections of Diporeia tissues, each wax block needed to be secured in the sliding microtome.
Each collected thin-section (5-8 µm) was transferred to a warm water bath at 36°C to ensure the wax section was
free of “wrinkles”. The sections were then placed on poly-prep-lysine coated glass slides (Sigma-Aldrich, USA)
followed by placement of the slides on a slide warmer (Sigma-Aldrich, USA) at 46°C for 24 hours. This procedure
ensured that the thin sections adhered to the slides. After heating the prepared sections for 24 hours, the slides were
exposed to a graded series of ethanol and xylene solutions in reverse order as described earlier. The graded series of
ethanol and xylene solutions helped to remove the wax from each slide, leaving only the Diporeia tissue. Each slide
was then incubated in a solution consisting of 65% ethanol and 5% hydrochloric acid for 5 minutes. The purpose of
the aforementioned step was to remove any Rose Bengal dye that the tissue may have.
Mayer’s Hematoxylin and Eosin Y stains (Sigma-Aldrich, USA) were then used to stain tissues adhering to the
glass slides, followed by light and fluorescent microscopy to identify and characterize infected Diporeia tissue.
The protocol used was a modified version from Lillie (1965), and is as follows:
• Immerse tissue with Mayer’s Hematoxylin;
• Incubate for 10 minutes;
• Rinse and run room temperature tap water over sections for 10 minutes;
• Immerse with working Eosin Y Stain;
• Allow to incubate for 30 seconds;
• Rinse with tap water until water runs clear off of a slide;
• Clear, and mount tissue

www.ccse
n
2.3 Statist
i
The statis
t
statisticall
y
samples t
o
replicates
However,
regressio
n
independe
analyses
w
to 2005 w
a
3. Results
3.1
F
ield
w
The purp
o
p
opulatio
n
with Dipo
r
sample, th
3.2
Lab w
o
The
p
atho
l
was deter
m
Basophili
c
they abso
r
response
t
Diporeia.
structures
Figure 3.
are obser
v
Examinati
earlier (M
e
the curren
t
infection
v
Diporeia
t
b
odies, ve
r
masses,
w
b
odies or
role in th
e
(r=0.7202
2
associatio
n
10, p≤ 0.0
0
n
et.org
/
ijb
i
cal analysis
t
ical analyses
y
compare pa
t
o
generate a
n
collected ove
r
all Lake Mi
c
n
was used,
u
nce was also
w
ere complete
d
a
s collected b
y
w
or
k
o
se of collect
i
n
density of
D
r
eia tissue for
h
e observed po
p
o
rk: histology
l
ogy of Dipor
e
m
ined to be i
n
c
bo
d
ies (or
m
r
b more dye (
F
t
o pathogens
(
The second p
r
had projectio
n
Analysis usin
g
v
e
d
in
p
roximi
t
were t
y
on of Lake
M
e
ssick, 2004).
t
state of Dipo
r
v
ersus 2010 sa
t
hat had path
o
r
sus those tha
t
w
ith the remai
n
masses. A lin
e
e
density decl
i
2
64, p≤0.012
4
n
between yea
r
0
01, implying
and figures i
n
t
hogens of Di
p
n
y historical
t
r
time and one
c
higan sampl
e
u
sing year as
used to deter
m
d
using the R-
y
Messick et a
l
i
ng Diporeia
D
iporeia since
future researc
h
p
ulation was
5
e
ia was assess
e
n
fected if it h
a
m
ass) are stru
c
F
igure 3A). T
(
Martinez, 20
0
r
evalent struc
t
n
s off of the c
e
g
fluorescent
m
t
y to one anot
h
y
pically obser
v
M
ichigan Dipo
r
Examination
o
r
eia tissue and
mples, which
o
genic infecti
o
t
had (2) budd
n
ing being a
s
e
ar regression
i
ne of Dipore
i
4
). A Chi-squ
a
r
and percent
p
significant as
s
Internation
a
n
this study o
n
p
oreia tissues
c
t
rends. Statist
i
with too few
(
e
s were teste
d
the predicto
r
m
ine if there
i
Statistic (R C
o
l
. (2004).
from Batch
w
presence/abs
e
h
projects. Ba
s
5
85 Diporeia
m
e
d using both
l
a
d a basophili
c
c
tures within t
h
hese masses
a
0
7). Basophili
c
t
ure found in
D
e
llular body su
g
m
icroscopy. (
A
h
er. (B) Buddi
n
v
ed scattered t
h
r
eia has been
d
o
f these histori
disease. For
D
showed ~29.2
o
ns are furth
e
ing structures
.
s
sociated with
model was u
s
i
a
p
opulations
a
re test for in
d
p
athogens obs
s
ociation bet
w
a
l Journal of Bi
o
96
n
ly pertain to
c
ollected fro
m
i
cally compa
r
(
or none), intr
o
d
using linea
r
r
and percent
i
s association
o
re Team, 201
w
ana Bay (La
k
e
nce monitori
n
s
ed on the ave
m
-2
.
l
ight and fluo
r
c
body (Figur
h
e tissue that
a
re involve
d
i
n
c
bodies are
o
D
iporeia tissu
e
g
gesting poss
i
A
) Basophilic
b
n
g structure (
b
h
roughout the
d
one previou
s
cally relevant
D
iporeia colle
c
% pathogenic
e
r categorized
.
In 2005, ~63
budding stru
s
ed as a predi
(Figure 4).
T
d
ependence, (
F
erved in Dipo
r
w
een year and
p
o
logy
Lake Michig
a
m
Lake Superi
o
r
ing these tw
o
o
duces signifi
c
r
regression
a
of pathogen
s
between yea
r
3). The patho
g
k
e Superior)
w
n
g began in
1
e
rage number
o
r
escent micros
c
e 3A) and/or
a
are abnormal
n
the innate i
m
o
ften found in
e
s were buddi
i
ble fungal inf
e
b
odies (black
a
b
lack arrow) o
b
body tissues
o
s
ly on sample
s
samples was t
o
c
ted in 2005, it
infection.
into those t
h
.6% of Dipor
e
u
ctures. In 20
1
ctor to deter
m
T
he linear regr
F
igure 5), was
r
eia tissues.
Va
p
athogen inci
d
a
n Diporeia s
a
o
r as there we
r
o
localities,
o
c
ant error.
a
nd chi-squar
e
s
present. A
c
r
and presenc
e
g
en data for
D
w
as to updat
e
1
977, and to
p
o
f Diporeia co
l
c
opy in all tis
s
a budding str
u
and are dark
e
m
mune syste
m
the legs and
i
ng structures
(
e
ction.
a
rrow) observ
e
b
served at 40
0
o
f Diporeia sp
p
s
that were co
l
o
provide mor
e
was found th
a
h
at had (1) ba
e
ia exhibited
b
1
0, ~71.4% e
x
m
ine if year o
f
ession was si
g
also used to
t
Va
lues obtaine
d
d
ence.
Vo l . 7 , N o. 1;
a
mples. We di
d
r
e too few arc
h
o
ne with plen
t
e
analyses.
L
c
hi-square te
s
e
of pathogen
s
D
iporeia tissue
e
the status o
p
rovide resear
c
l
lected in the
p
s
ue samples.
T
u
cture (Figure
e
r in color be
c
m
of an amphi
p
along the spi
n
(
Figure 3B).
T
e
d at 400x. B
o
0
x. These stru
c
p
.
l
lected in 200
0
e
recent insig
h
a
t ~18.9% exh
i
sophilic mass
b
asophilic bod
i
x
hibited baso
p
f
infection pla
y
g
nificantly po
s
t
est if there w
a
d
were X
2
= 50
,
2015
d
not
h
ived
t
y of
inear
t for
s
. All
prior
f the
c
hers
p
onar
T
issue
3B).
c
ause
p
od’s
n
e of
T
hese
dies
tures
0
and
t
into
b
i
t
ed
e
s or
i
es or
p
hilic
y
ed a
s
itive
a
s an
,
df =

www.ccse
n
Figure 4.
L
D
Figure 5.
C
4. Discus
s
The Dipo
r
introducti
o
p
er squar
e
that local
i
p
opulatio
n
m
-2
in 20
0
shows the
also abun
d
resistance
Superior
h
sub-type
o
type of to
l
consider i
s
Lakes, fo
r
Michigan
whether
m
Diporeia
a
n
et.org
/
ijb
L
inear regress
i
D
iporeia tissu
e
C
hi-square an
a
collect
e
s
ion
r
eia populatio
n
o
n of zebra
mu
e
meter. The in
i
ty of Diporei
a
n
crashed is n
o
0
8, and as we
population o
f
d
ant in this re
g
to diseases i
t
h
ave evolved/
m
o
f the original
p
l
erance to the
s
food availab
i
r
instance, L
a
was possibly
d
m
ore food is
a
a
nd zebra mus
s
i
on analysis us
e
collected fr
o
a
lysis for inde
p
e
d from Lake
M
n
in Batchaw
a
mu
ssels. In our
crease in dens
a
initially fel
l
o
t known. Ho
w
observed, 58
5
f
Diporeia not
o
g
ion, we can
o
t
shares with
t
m
utated from
b
p
opulation. It
specific dise
a
i
lity as it is al
s
a
ke Michigan
.
d
ue to food c
o
a
vailable in
L
s
els.
Internation
a
ing R-statistic
o
m Lake Mich
i
p
endence usi
n
M
ichigan (ana
l
a
na Bay, Lak
e
abundance a
n
ity of Diporei
a
l
in 1977 fro
m
w
ever, in 2001
5
Diporeia m
-
o
nly stabilizi
n
o
nly speculate
t
he mussels.
A
b
reeding with
is also
p
lausi
b
a
ses that are
c
s
o possible th
a
.
N
alepa et
a
o
mpetition, re
s
L
ake Superior
a
l Journal of Bi
o
97
s. Year was us
e
i
gan (analyses
n
g R-statistics
f
l
yses resulted
e
Superior (C
a
n
alyses, we o
b
a
follows an i
m
m
675 Dipore
i
the populatio
2
in 2013. Th
i
n
g, but it conti
n
that this parti
c
A
lternatively,
i
other healthi
e
b
le that the po
p
c
ausing popul
a
a
t more food e
x
a
l. (2006) sug
s
ulting from t
h
and as a co
n
o
logy
ed as a predic
t
resulted in r
=
f
or percent pa
t
in X
2
= 50, df
a
nada), does
n
b
served an inc
m
portan
t
tren
d
i
a m
-2
to 481
o
n increased t
o
i
s is particula
r
n
ues to exhibi
t
c
ular Diporei
a
i
t is possible
t
e
r populations
p
ulation in La
k
a
tion crashes
e
x
ists in Lake
S
gested that t
h
h
e introduc
t
io
n
n
sequence, th
e
t
or for percent
=
0.7202264,
p
t
hogens found
= 10, p≤0.00
0
n
ot appear to
rease in the n
d
where the na
t
Diporeia m
-
2
o
529 Diporei
a
r
ly important
t
growth. Sinc
e
a
population
m
t
hat the Dipo
r
and are now,
k
e Superior ha
v
e
lsewhere. An
o
S
uperior com
p
h
e decline of
n
of zebra mu
s
e
re is less co
Vo l . 7 , N o. 1;
pathogens fo
u
p
≤0.0124)
in Diporeia ti
0
1)
be affected b
y
umber of Dip
t
ural populati
o
2
in 1978; wh
y
a
m
-2
, 570 Dip
as the densit
y
e
zebra musse
l
m
ay have deve
l
r
eia living in
perhaps, a g
e
ve developed
o
ther possibil
i
p
ared to other
G
Diporeia in
s
sels. We spe
c
o
mpetition bet
w
2015
n
d in
s
sue,
y
the
o
reia
o
n for
y
the
o
reia
data
l
s are
l
oped
Lake
e
netic
s
ome
i
ty to
G
reat
Lake
c
ulate
w
een

www.ccsenet.org/ijb International Journal of Biology Vol. 7, No. 1; 2015
98
Microscopy analyses of Lake Michigan Diporeia samples from 2005 and 2010 compared with prior tissue
analyses show an overall increase in the prevalence of pathogens found in Diporeia since 1986. This is apparent
in both the linear regression model (Figure 4) and the chi-square analysis (Figure 5). The overall increasing trend
is consistent with the hypothesis that the invasion of zebra mussels in the Great Lakes has caused Diporeia’s
population crash (Nalepa et al., 2006). Zebra and quagga mussels invaded the Great lakes in the late 1980’s
(Nalepa et al., 1998); Diporeia populations have crashed in most areas since that time. Our data also shows an
overall increase in pathogens found in Diporeia tissue since the introduction of the zebra mussel. Although it is
possible that the increase in tissue pathogens observed in Diporeia might be caused by stress associated with
increased feeding pressures, it seems more likely that the diseases (not present before the arrival of zebra
mussels) are a direct consequence of a foreign invader(s), such as zebra mussels. Hence, an increase in infected
Diporeia tissue might suggest that competition for food may have been a secondary effect caused by the primary
effect, namely disease. The correlation between population decline in Diporeia, increased pathogenic infection
and disease, and purported increased population of zebra mussels (Nalepa et al. 2006) supports this hypothesis.
Although most areas in the Great Lakes have experienced a decline in Diporeia populations since the
introduction of zebra mussels, there are some locations that have not been affected by declining populations.
Isolated areas of Lake Michigan and Lake Huron still support minimal Diporeia populations (Nalepa et al.,
1998). Lake Superior’s population of Diporeia has remained largely unchanged as supported by our sampling of
Batchawana Bay. Diporeia’s stability in Lake Superior may be attributed to multiple factors as discussed above.
One more recent anecdote for their survival is that the greater depths of Lake Superior may have provided a
‘safe-haven’ compared to other shallower areas that Diporeia typically inhabited.
Lastly, it is also possible that the budding structure found in the tissue’s of Diporeia may not be pathogenic
and/or may be present as a commensal (Messick et al., 2004). As a consequence, more studies are needed to
confirm any speculation. Because the identity of these budding structures is unknown, it should not be assumed
that they are necessarily harmful to Diporeia. Future research needs to identify what these budding structures are
and whether they are “infecting” Diporeia tissue and having a negative consequence.
5. Conclusions
Analyses in this study have shown a significant increase in pathogenic infection and immune-type response since
the invasion of the zebra mussels in 1986. The positive correlations suggest that zebra mussels may have acted as
a vector for pathogen(s) that infected Diporeia. Some inconsistencies exist with this hypothesis, however. For
instance, healthy Diporeia populations have remained steady since the invasion of zebra mussels in certain areas
in Canada. Future research should involve identifying these pathogens (e.g. genomics) and how the infections
are affecting Diporeia physiology. In addition, both zebra and quagga mussel tissues should be analyzed for
similar pathogens that are identified in Diporeia tissue.
Acknowledgments
This work could not have been done without support from the R.B. Annis Water Resources Institute Foundation
and the D.J Angus-Scientech Undergraduate Student Internships for summers 2013 and 2014 provided by The
Annis Water Resources Institute, Grand Valley State University. Additional support was provided by NOAA
through David Fanslow whose technical help, in-depth knowledge, and patience made this work possible. We also
thank Patrick McEnaney who helped collect samples and Gavin Christie (Division Manager) at the Great Lakes
Laboratory for Fisheries and Aquatic Sciences for organizing permits allowing us to collect samples in Canada.
We appreciate help from Dr. Sango Otieno at Grand Valley State University, statistical consulting center, who
reviewed and analyzed this data, without whom this work would not have seen completion.
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