Geothermal habitats as sites for year-round transmission of Fasciola hepatica
by Robert Stanley Potts, Jr
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Veterinary Molecular Biology
Montana State University
© Copyright by Robert Stanley Potts, Jr (1995)
Abstract:
Transmission of Fasciola hepatica in western Montana usually occurs during the fall months. However,
a reported infection of F. hepatica in a bison herd winter pastured near a thermal site suggested that
geothermally influenced habitats may serve as year-round foci of parasite transmission. To investigate
this hypothesis, snails were collected and climatological conditions were monitored at four geothermal
habitats on a monthly basis from July 1993 to August 1994. Lymnaeid snails were identified and their
seasonal population cycles determined. Climatological data were analyzed to determine suitability of
thermal habitats as year-round sources of F. hepatica miracidia and metacercaria. Laboratory- reared
generations of field-collected lymnaeid snails, originating from thermal and non-thermal habitats, were
exposed to F. hepatica miracidia to determine infection rates. Lymnaeid snails were collected from
three of four thermal habitats and four species were identified. Lymnaeid snails were recovered at least
ten months out of the year at each thermal habitat, with population cycles peaking in March-April and
July-September. Review of climatological profiles indicated that temperature and moisture regimes
were suitable for F. hepatica development and transmission year-round. Snail species of thermal and
non-thermal origins demonstrated significantly different F. hepatica infection rates on a site specific
basis. GEOTHERMAL HABITATS AS SITES FOR YEAR-ROUND TRANSMISSION
OF FASCIOLA HEPATTCA
by
Robert Stanley Potts Jr.
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Veterinary Molecular Biology
MONTANA STATE UNIVERSITY
Bozeman, Montana
May 1995
N S r)?
9S51
11
APPROVAL
o f a thesis submitted by
Robert Stanley Potts Jr.
This thesis has been read by each member o f the thesis committee and has been
found to be satisfactory regarding content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the College o f Graduate Studies.
*V n
Date
3\,W \b
Chairperson, Graduate Committee
Approved for the Major Department
Date
Head, Major Depamnent
Approved for the College o f Graduate Studies
Date
Graduate Dean
-STATEM ENT O F PER M ISSIO N TO USE
In presenting this thesis in partial fulfillment o f the requirements for a master's
degree at Montana State University, I agree that the Library shall make it available to
borrowers under rules o f the Library.
I f I have indicated my intention to copyright this thesis by including a copyright
notice page, copying is allowable only for scholarly purposes, consistent with "fair use"
prescribed in the U.S. Copyright Law. Requests for permission for extended quotation
from or reproduction o f this thesis in whole or in parts may be granted only by the
copyright holder.
Signature
Date
S'C Tuft-e.
iv
, ACKNOWLEDGMENTS
I would like to thank the following individuals for their assistance in the
completion o f my graduate program. My major advisor, Dr. Stuart E. Knapp, for his
guidance, encouragement and commitment to developing my writing skills and use o f the
I
English language. The many individuals within the department and those individuals
within the lab, especially Matthew Rognlie and Kristi Dimke. The members o f my
graduate committee, Dr. David E. Worley and Dr. Al Zale. I greatly appreciate the
assistance o f Dr. Shi-Kuei Wu, who completed the identification o f snail specimens and
Dr. Richard Lund for assistance with statistical analysis. I thank my wife, Camille, who
was my companion in completing the field component o f this project and was a continual
'I
source o f support. Finally, I would like to acknowledge my reliance upon my Savior,
Jesus Christ, without whom, completion o f this project would have been impossible.
V
TABLE O F CONTENTS
Page
LIST OF T A B L E S........................................................................................................... vii
LIST OF F IG U R E S .......................................................................................................
viii
A B STR A C T.................................................................................................................... xi
IN TRO D U C TIO N .........................................................................................................
I
Fasciola hepatica................................................................................................ 2
Life C ycle............................................................................................... 2
Fasciola hepatica in M ontana.............................................................. 3
Lymnaeid Snails...................................... ............. ..............................................4
Life C ycle.............................................................................................. 4
E cology................................................................................................. 5
Lymnaeids in M ontana......................................................................... 5
Thermal H abitats............................................................................................... 6
Lymnaeids and Thermal H abitats....................................................... 6
Rationale for the R esearch.............................................................................. 7
Statement o f Problem ...................................................................................... 8
STUDY S IT E S ................................................................................................................
1.1
Green Springs (H o t)...........................................................................................
New Biltmore H ot Spring..................................................................................
Beaverhead Warm Springs.................................................................
McLeod H ot Spring............................................................................................
11
14
MATERIALS AND M ETH O D S.............................................................................
Snail Collections.............................................................................................
Climatological Characterization.....................................................................
Macroclimatological Characterization ...............................................
Microclimatological Characterization ................................................
Specimen Preservation.....................................................................................
Identification..................................... ................................................................
Snail Culture Technique...................................................................................
Determination o f F. hepatica Experimental Infection R a te s ........................
14
17
20
21
21
22
22
22
23
23
24
vi
Page
R E S U L T S .......................................................................................................................
28
Species o f Snails C ollected..............................................................................
Green Springs.......................................................................................
New Biltmore H ot Spring...................................................................
Beaverhead Warm Springs.................................................................
Lymnaeid Population D ynam ics............................................................
Green Springs......................................................................................
New Biltmore H ot Spring..................................................................
Beaverhead Warm Springs.................................................................
Climatological Characterization o f Thermal H abitats..................................
Green Springs......................................................................................
New Biltmore H ot Spring..................................................................
Beaverhead Warm Springs.................................................................
McLeod H ot Spring............................................................................
Experimental Fasciola hepatica Infection R a te s ...........................................
Lymnaea from Thermal H abitats..................................................
Lymnaea from Non-Thermal H abitats..............................................
Positive Control Snails........................................................................
Statistical Significance.........................................................................
28
28
31
32
34
34
35
37
39
39
42
45
48
51
51
51
52
52
D ISC U SSIO N ...................................................
54
Significance o f R esults.............................................
54
Presence o f Snails at Thermal H abitats............................................ 54
Lymnaeid Species P resen t.................................................................. 55
Seasonal D ynam ics.............................................................................
55
Size Class o f Snails.................................................................................. 56
Climatological Conditions.................................................................. 56
Infection R a te s ..................................................................................... 58
Future W o rk .....................................................................
59
C O N C LU SIO N S...........................................................................................................
60
REFERENCES C IT E D ................................................................................................
61
vii
LIST O F TABLES
Table
1.
Page
List o f lymnaeid snails found in M ontana......................................................... 9
2.
Species o f lymnaeid snails collected in Montana which have previously been
found to harbor natural infections o f F. hepatica ................................ .......... 10
3.
Species o f lymnaeid snails collected in Montana which have been
experimentally infected with F. hepatica ........................................................ 10
4.
Analysis o f water chemistry at the Green Springs, New Biltmore H ot
Spring and McLeod Hot Spring....................................................................... 19
5.
Lymnaeid snails from thermal and non-thermal habitats exposed to
F. hepatica............................................................................................................ 27
6.
List o f snail species collected from each thermal h abitat............................... 29
7.
Experimental infection rate o f lymnaeid snails originated from thermal .
and non-thermal site s........................................................................................
53
Vlll
LIST OF FIGURES
Figure
1.
Page
Location o f the four thermal habitats studied. Distribution o f liver flukes,
lymnaeid snails and thermal habitats in Montana by co u n ty.......................
12
2.
Green Springs, Sanders Co.............................................................................
13
3.
New Biltmore Hot Spring, Beaverhead Co..................................................
15
.4.
Beaverhead Warm Springs, Beaverhead Co..................................................
16
5.
M cLeod H ot Spring, Sweet Grass Co...........................................................
18
6.
Culture for lymnaeid snails............................................................................
26
7.
Percentage o f each snail species collected at the Green Springs from
July 1993 to July 1994 ....................................................................................
30
Number Oflymnaeid snails collected at each sampling site (1-4) from
July 1993 to July 1994 at the Green Springs................................................
30
Percentage o f each snail species collected at the New Biltmore H ot
Spring from July 1993 to July 1994 ...............................................................
31
8.
9.
10.
Number o f Lvmnaea modicella collected at each sampling site (1-4) from
July 1993 to July 1994 at the New Biltmore H ot Spring.............................. 32
11.
Percentage o f each snail species collected at the Beaverhead Warm
Springs from July 1993 to July 1994 ............................................................... 33
12.
Number o f Lvmnaea obrussa collected at each sampling site (1-4) from
July 1993 to July 1994 at the Beaverhead Warm Springs............................. 33
13.
Number o f lymnaeid snails collected each month at the Green Springs
from July 1993 to July 1994 ............................................................................. 34
14.
Maximum, minimum and average shell length o f lymnaeid snails at the
Green Springs from July 1993 to June 1994 ................................................... 35
ix
Figure
Page
15.
Number o f Lymnaea modicella collected each month at the New Biltmore
H ot Spring from July 1993 to June 1 9 9 4 ......................................................... 36
16.
Maximum, minimum and average shell length o f Lvmnaea modicella
collected at the New Biltmore Hot Spring from July 1993 to June 1994 .... 36
17.
Number o f Lvmnaea obrussa collected each month from July 1993 to
June 1994 at the Beaverhead Warm Springs................................................... 37
18.
Maximum, minimum and average shell length o f Lvmnaea obrussa
collected at the Beaverhead Warm Springs from July 1993 to June 1994 ... 38
19.
Air temperature and precipitation values reported for each month at
the Green Springs from July 1993 to June 1994 ............................................ 39
20.
Average water temperature at sampling sites 1-4 from July 1993 to
June 1994 at the Green Springs............................................... ,...................... 40
21.
W ater temperature at sampling sites 1-4 from July 1993 to June 1994 at
the Green Springs...................................... ........................................................ 41
22.
Soil temperature at sampling sites (1-4) from September 1993 to June
1994 at the Green Springs................................................................................ 41
23.
Average air temperature and precipitation each month from July 1993
to June 1994 at the New Biltmore H ot S pring............................................... 42
24.
Average water temperature at sampling sites 1-4 from July 1993 to
June 1994 at the New Biltmore Hot S pring............................. ...... ............... 43
25.
W ater temperature at sampling sites 1-4 from July 1993 to June 1994
at the New Biltmore H ot Spring....................................................................... 44
26.
Soil temperature at sampling sites 1-4 from September 1993 to June 1994
at the New Biltmore H ot Spring....................................................................... 44
27.
Average air temperature and precipitation from July 1993 to June 1994 at
the Beaverhead Warm Springs.......................................................................... 45
28.
Average water temperature at sampling sites 1-4 from July 1993 to
June 1994 at the Beaverhead Warm Springs ........................................ .......... 46
/
X
Figure
29.
30.
31.
32.
33.
34.
35.
Page
W ater temperature at sampling sites 1-4 from July 1993 to June 1994
at the Beaverhead Warm Springs...................................................................
47
Soil temperature at sampling sites 1-4 from October 1993 to June 1994
at the Beaverhead Warm Springs...................................................................
47
Average air temperature and precipitation from July 1993 to June 1994
at the McLeod H ot S pring.............................................................................
48
Average w ater temperature at sampling sites 1-4 from July 1993 to
June 1994 at the McLeod H ot Spring.................................... ......................
49
W ater temperature at sampling sites 1-4 from July 1993 to June 1994
at the McLeod H ot Springs............................................................................
50
Soil temperature at sampling sites 1-4 from July 1993 to June 1994
at the M cLeod H ot Spring.............................................................................
50
Delineation o f thermal and non-thermal habitats at the Beaverhead
Warm Springs.................... ..............................................................................
57
ABSTRACT
Transmission o f Fasciola hepatica in western Montana usually occurs during the fall
months. However, a reported infection o f F. hepatica in a bison herd winter pastured near
a thermal site suggested that geothermally influenced habitats may serve as year-round
foci o f parasite transmission. To investigate this hypothesis, snails were collected and
climatological conditions were monitored at four geothermal habitats on a monthly basis
from July 1993 to August 1994. Lymnaeid snails were identified and their seasonal
population cycles determined. Climatological data were analyzed to determine suitability
o f thermal habitats as year-round sources ofF. hepatica miracidia and metacercaria.
Laboratory- reared generations o f field-collected lymnaeid snails, originating from thermal
and non-thermal habitats, were exposed to F. hepatica miracidia to determine infection
rates. Lymnaeid snails were collected from three o f four thermal habitats and four species
were identified. Lymnaeid snails were recovered at least ten months out o f the year at each
thermal habitat, with population cycles peaking in March-April and July-September.
Review o f climatological profiles indicated that temperature and moisture regimes were
suitable for F. hepatica development and transmission year-round. Snail species o f thermal
and non-thermal origins demonstrated significantly different F. hepatica infection rates on
a site specific basis.
INTRODUCTION
Three factors determine the development o f liver fluke disease caused by members
o f the Fasciolidae. These include characteristics o f the definitive host, the intermediate
host and the parasite (Malone and Zukowski, 1992). The definitive host is usually a wild
or domestic ungulate but may include other herbivorous animals as well as humans. The
snail intermediate hosts for liver flukes belong to the family Lymnaeidae. The principle
species o f liver fluke o f concern to the livestock industry in Montana is Fasciola hepatica
although the N orth American deer fluke, Fascioloides magna. may also be present (Knapp
et a l, 1992). Parasite transmission is highly complex as each o f the components o f the
Fasciola-Lymnaea system may affect the others and is influenced by the environment.
Whereas each component is essential and their biological characteristics affect the
incidence o f disease, environmental conditions are rate limiting with respect to snail and
parasite development in the northwestern United States. The snail intermediate hosts and
parasite are sensitive to temperature and moisture and as a result development and
transmission o f the parasite is usually seasonal in Montana. Additionally, transmission
tends to be focal in nature, often occurring in specific microhabitats. Thermal habitats are
abundant and widely distributed throughout the state and exhibit many characteristics o f
microhabitats that favor the occurrence o f fasciolosis. The relationship o f thermal habitats
to the development o f liver flukes and the possibility o f such areas presenting a year-round
microhabitat for transmission is the topic o f this study.
2
Fasciola hepatica
Fasciola hepatica. the common liver fluke, is believed to have been first described
in 1379 by Jehan de Brie in France, making it one o f the oldest known parasites
(Ollerenshaw, 1959). The life history was worked out in 1882 when Lvmnaea truncatula
was determined to be the intermediate host in Germany (Leuckart, 1882) and England
(Thomas, 1883). In North America, Lymnaea (= Galbal bulimoides was the first
intermediate host identified for Fasciola hepatica (Shaw and Simms, 1929).
Life Cycle
Fasciola hepatica is a digenetic trematode. The intermediate host is a mollusc and
the final host is a vertebrate. The life cycle consists o f an alternation o f developmental and
transport phases, each o f which typically result in a variable level o f mortality
(Ollerenshaw, 1959). Adult flukes occur in the liver and bile duct o f the final host, where
they have been reported to maintain an infection for as long as 11 years (Price, 1956).
Flukes are hermaphroditic and produce thousands o f eggs which are deposited in the bile
duct and pass to the intestine by way o f the common bile duct. Further development to
the hatching stage is dependent upon liberation from the feces as well as temperatures of
at least 10 C and soil moisture conditions at or exceeding soil water holding capacity
(Ollerenshaw, 1959). The time required for the egg to develop to the miracidial stage
varies with environmental conditions between two and six weeks. The ciliated miracidia
have an active period measured in hours during which time contact and penetration o f an
intermediate host must occur. Once penetration has occurred the miracidia change into
sporocysts. Each sporocyst gives rise to many redia which in turn give rise to daughter
3
redia. Ultimately, daughter redia give rise to cercaria which leave the snail through the
mantle. The cercaria are free swimming and eventually encyst on vegetation, losing their
tail in the process and becoming metacercaria. Metacercaria are infective to animals
which ingest them while grazing. Upon ingestion, the metacercaria excyst in the stomach
and bore through the wall o f the gut into the abdominal cavity and eventually reach the
surface o f the liver. The immature flukes then bore into the liver parenchyma, tunneling
through and feeding until they reach the bile ducts. Upon arrival, this new generation o f
adult flukes deposit eggs and the cycle is complete.
Fasciola hepatica in Montana
Until recently, (Knapp et al., 1992) fasciolosis was not considered to be an
important disease o f domestic livestock in Montana as the cold dry climate was considered
to limit the disease to a few o f the western counties. Jacobson and Worley (1969)
reported detecting fluke ova in 2.4% o f 791 samples and noted that infected cattle were
primarily distributed in the western region o f the state. In 1973, 5% o f the livers from
M ontana slaughter cattle were reported to be condemned because o f F. hepatica (Foreyt
and Todd, 1976). Recently, however, a study o f United States Department o f Agriculture
(USDA) inspected slaughter plants in Montana reported that liver flukes were present in
17.24% o f the 6,032 cattle processed during a 12 month period and that the parasite is
widely distributed (Knapp et al., 1992). Subsequently, a survey o f beef cow processing
facilities in the western United States reported the prevalence rate o f F. hepatica to be
19.2 ± 1.2% (Briskey et al., 1994). The parasite has been reported in 26 o f 56 counties in
Montana. Infections appear to be heaviest in the Bitterroot Valley area, where 90% o f the
<
livers from slaughter cattle were reported to be condemned because o f liver fluke (Knapp
et al., 1992). Based on studies using tracer sheep, transmission is thought to occur
primarily in the autumn as metacercaria are not able to overwinter, rendering pasture
essentially parasite free in the spring (Hoover et al., 1984; Knapp and Abrahamsen, 1994).
Lvmnaeid Snails
The Lymnaeidae are amphibious snails that belong to the subclass Pulmonata. The
family contains over one thousand species (Cruz-Reyes, 1982) and is world-wide in
distribution.
Life Cycle
Lymnaeid snails are hermaphroditic, although they may cross fertilize. Eggs are
laid in gelatinous capsules and are attached to surrounding substrate. The capsules
contain a varying number o f eggs ranging from 4 to 180 (Hyman, 1967). The eggs
develop into veliger larva within the egg capsule and emerge as young snails anatomically
complete except for the reproductive system. Length o f development within the egg
capsule is temperature dependent. Snails do not develop at temperatures below 9 C or
above 37 C. At 9 C the snails hatch from their eggs in about 30 days, at 17-19 C in 17-22
days, and at 25 C in only 8-12 days (Roberts, 1950). Snails typically undergo logarithmic
growth with rapid growth in length or diameter at first, then slowing down to a level with
little or no growth. Growth commonly ceases after attainment o f sexual maturity. M ost
pulmonates live about one year and die after one spawning (Hyman, 1967).
5
Ecology
Members o f the family Lymnaeidae can be found in many habitat types, including
lakes, ponds, ditches, rivers, swamps, and irrigated and non-irrigated pastures. Lymnaeids
occur in a wide range o f conditions, from sea level to 10,000 feet elevation, from ice
waters to hot springs, and in lakes from shallow waters to depths o f 250 m (Hyman,
1967). Temperature and moisture have been reported to be the most important factors
regarding the development o f lymnaeid snails (Ollerenshaw, 1959), but other
environmental factors are considered to be important as well. Lymnaeids are generally not
associated with rapidly flowing water, however, a moderate flow rate is beneficial (Boray,
1964). A preference o f soil type has been reported by several investigators. Okland
r
(1935), Peters (1938) and Wetzel (1953) each concluded that lymnaeids prefer clay soil.
Schmid (1934) suggested that an important factor o f soil may be the level o f
impermeability to water. Several authors have suggested the importance o f both soil and
water pH. The range o f soil pH favored is given variously as 6.2 - 7.2 (Mehl, 1932;
Bryant, 1935) and 6 - 9 (Schadin, 1937). Hyman (1967) reported that lymnaeids live in
slightly alkaline waters to a maximum o f pH 8.5. The alkalinity o f the water was due to
the presence o f calcium carbonate, o f which a minimum o f 20 mg/1 is essential for the
well-being o f the snails (Boycott, 1936).
Lymnaeids in Montana
The history o f lymnaeids in Montana dates back to I860 when several species
were collected from the Missouri River and Hell Gate River (Cooper, 1868). Currently, 18
species have been identified (Table I) and specimens have been found in 29 o f 56
6
counties. O f the 18 species o f lymnaeid snails found in the state, 6 have been reported as
natural vectors and 5 have been reported as experimental vectors o f F. hepatica in various
places in the United States (Tables 2 and 3).
Thermal Habitats
There are 96 thermal habitats in the state o f Montana with estimated reservoir
temperatures ranging from 20 C to 136 C (Sonderegger et a l, 1981). The various springs
and wells are located in 29 o f 56 counties. Thermal waters in M ontana are classified
according to water temperature as hot, warm or tepid and they represent the largest and
most varied group o f hot springs in the world (Brues, 1932). Climatic differences relating
to temperature and humidity limit the distribution o f animals, resulting in regionally
distinct fauna (Brues, 1932), but thermal waters present notable exceptions.
In Montana,
thermal habitats provide opportunities for faunal growth year-round that would not
otherwise occur.
Lvmnaeids and Thermal Habitats
Many molluscs, including lymnaeid snails, inhabit hot springs in Europe, Iceland
and America (Brues, 1932). These snails have been found in waters ranging in
temperature up to 45 C. In Montana, Oswald (1979) reported finding lymnaeid snails
from the Ringling thermal well in Meagher county and Dunkel et al. (1995) collected
several species from thermal habitats throughout the state.
7
Rationale for the Research
First, Fasciola hepatica is a parasite o f considerable economic importance in
M ontana and throughout the world (Knapp et al., 1992; Boray, 1994). Liver flukes
negatively affect the condition o f livestock in numerous ways and result in decreased food
production and significant economic losses (Chick, 1979; Dargie, 1987). Second,
agriculture is the top grossing industry in Montana, resulting in 2.2 billion dollars in cash
receipts per year, o f which livestock sales accounted for 52.7% in 1993 (Sands and Lund,
1994). Third, the few studies that have been completed suggested that F. hepatica
transmission in the northern Rocky Mountain region was seasonal, occurring primarily
during the autumn (Hoover et al., 1984; Knapp and Abrahamsen, 1994). However, these
studies did not consider the possible effects o f microclimates, which Smith and Wilson
(1980) and Malone and Zukowski (1992) consider to be important factors in the
transmission o f F. hepatica. Observation o f an outbreak o f fasciolosis in a herd of
American bison (Bison bison) that had been winter pastured near a thermal habitat
(Knapp, personal communication), and a report o f clinical fasciolosis in a goat herd in H ot
Springs Montana in which parasite transmission occurred in November (Leathers et al.,
1982) suggested that transmission could occur year-round in association with thermal
habitats. Fourth, thermal habitats are abundant in Montana, occurring in 35 o f 56
counties, and their spatial distribution is positively correlated (p < 0.01) to the occurrence
o f liver flukes. Finally, environmental conditions related to temperature and moisture are
the most important factors influencing the development o f lymnaeid snails and F. hepatica.
;
8
Statement o f Problem
The purpose o f this study was to explore the effects o f geothermal habitats on
lymnaeid snail populations and liver fluke transmission in Montana. The proposed
research was designed to determine the following:
1. the species o f lymnaeid snails present at several thermal habitats;
2. the seasonal population dynamics o f lymnaeid snails at thermal habitats
(population dynamics refers to the number o f snails present and the age structure o f the
population each month over the course o f the year);
3. the macro- and microclimatological conditions o f several thermal habitats
J
throughout the year and, thereby, indirectly determine their suitability as a year-round
source o f F. hepatica miracidia and metacercaria; and
4. if snails from thermal and non-thermal habitats differ in susceptibility to F.
hepatica.
'i
\
9
Snail Species
Collection Site, Date
Reference
L v m n a e a b u li m o id e s (L e a )
M is s o u r i r iv e r . 1 8 6 0
C ooper, 1868
L d is i d i o s a ( S a v )
M is s o u r i r iv e r , 1 8 6 0
C ooper, 1868
L h u m ilis ( S a y )
M is s o u r i r iv e r , 1 8 6 0
C ooper, 1868
L . o a lu s t r is ( L in n )
H e ll G a te r iv e r , 1 8 6 0
C oop er, 1868
L m o n t a n e n s is
H a v es C reek , 1 9 1 2
B ak er, 1 9 1 3
L. c a p e r a ta
W ir m e c o o k L a k e , 1 9 1 4
B errv, 1 9 1 6
L ob ru ssa (S a y )
E lk C r e e k , 1 9 1 4
B en v, 1916
L p arva
W in n e c o o k L a k e , 1 9 1 4
B errv, 1 9 1 6
L. b in n e y i
M a d is o n R iv e r , 1 9 2 4
T a v lo r , 1 9 5 2
L. h in k le y i ( S a y )
Y e ll o w s t o n e L a k e , 1 9 3 5
T a v lo r , 1 9 5 2
L e lr o d ia n a (B a k e r )
S t. M a r v 's L a k e , 1 9 6 0
R u s s e ll a n d B r u n s o n , 1 9 6 7 b
L s ta e n a lis (S a v )
L a k e M c D o n a ld , 1 9 6 6
R u s s e ll, 1 9 6 7 a
L d a lli
B eaverhead C o, 1989
D u n k e l e t a l„ 1 9 9 5
L e lo d e s
G a lla tin C o .. 1 9 8 9 ,
D u n k e l e t a l., 1 9 9 5
L m o d ic e l la
B e a v e r h e a d C o ., 1 9 8 9
D u n k e l e t a l., 1 9 9 5
L t e c h e lla (H a I d e m a n )
M is s o u r i R iv e r . 1 9 9 0
K n a p p , p e r s . c o m m ., 1 9 9 3
L a u r ic u la r ia
R a ttle s n a k e L a k e , 1 9 9 3
K n a p p , p e r s . c o m m ., 1 9 9 3
L c a t a s c o n iu m
F la th e a d riv e r , 1 9 9 3
K n a p p , p e r s . c o m m ., 1 9 9 3
Table I. List o f lymnaeid snails found in Montana.
10
Snail Species
Collection Site
Reference
Lvmnaea bulimoides
Eastern Washington
Lang, 1977
L. caperata
Sanders Co., Montana
Knapp, pers. comm., 1993
L. modicella
Eastern Washington
Lang, 1977
L. obrussa
Beaverhead Co., Montana
Knapp, pers. comm., 1993
L. palustris
Eastern Washington
Lang, 1977
L. staenalis
Eastern Washington
Lang, 1977
Table 2. Species o f lymnaeid snails collected in Montana which have previously been
found to harbor natural infections o f F. hepatica.
Snail Species
Reference
Lvmnaea modicella
Krull, 1933
L. montanensis
Rowan et al., 1966
L. palustris
Lang, 1977
L. stagnalis
Lang, 1977
L. bulimoides
Foreyt and Todd, 1978
T able 3. Species o f lymnaeid snails collected in Montana which have been experimentally
infected with F. hepatica.
11
STUDY SITES
The four thermal habitats represented three geographically distinct regions o f
the state and were selected based on existing knowledge o f the distribution o f liver flukes,
lymnaeid snails (Knapp et a l, 1992) and thermal habitats (Sonderegger and Bergantino,
1981) (Fig. I). Each thermal habitat had previously been visited as part o f a state-wide
survey for lymnaeid snails (Dunkel et al., 1995).
Green Springs (Hot)
Green Springs (Fig. 2) effluent emanates from a Precambrian Piegan rock
formation and Alluvium sediments (Sonderegger et al., 1981) in Sanders Co., Latitude
47D 26M 35S, Longitude 114 D 40M 7S. An analysis o f water chemistry is presented in
Table 4. The thermal habitat was a marshy depression in the middle o f a pasture which
was continuously filled by the spring. The area covered about 0.2 ha and was 0.6 meters
deep near the center. Cattle were present much o f the year and created a mud flat region
around the perimeter o f the spring. The elevation was 850 meters. Regional land cover
was designated as strongly sloping range (8-15%) and the soil had a water holding
capacity o f 10.7 cm and an average pH o f 7.0 (Caprio et al., 1990). The climax vegetation
was Subalpine Fir climax forest, with a typical overstory o f 50% Subalpine Pine, 35%
Douglas Fir, 10% Ponderosa Pine and 5% Engelmann Spruce (Caprio et al., 1990).
F i g u r e I . Location of the 4 thermal habitats studied. Distribution of liver
flukes (shaded counties), lymnaeid snails (•) and thermal habitats (*) in
Montana by county. (I), Green Springs; (2), New Biltmore Hot Spring; (3),
Beaverhead Warm Springs; (4), McLeod Hot Spring.
Fig. 2. Green Springs, Sanders Co.
14
N ew Biltmore H ot Spring
1
N ew Biltmore H ot Spring (Fig. 3) effluent emanates from the Madison aquifer in
Beaverhead Co., Latitude 45D 27M 58S, Longitude 112D 29M IS. The estimated
reservoir temperature was 71 C and an analysis o f water chemistry is presented in Table 4
(Sonderegger et al., 1981). The spring continuously flowed from a pipe into a ditch that
was approximately 1.5 meters across and had high vertical sides. The water column was
less than 0.3 meters deep. The elevation was 1,460 meters. Regional land cover was
designated as gently sloping range (2-4%) and the soil had a water holding capacity o f
12.4 cm and an average pH o f 7.9 (Caprio et al., 1990). The climax vegetation was Saline
Lowland Range, characterized by various grass communities (Caprio et al., 1990).
Beaverhead Warm Springs
Beaverhead Warm Springs (Fig. 4) effluent emanates from tertiary sediments over
the Madison aquifer (Sonderegger et al., 1981) in Beaverhead Co., Latitude 45D 22M
47S, Longitude 112D 26M 33 S. The spring percolated through the sediments o f a stream
bed. The channel o f the stream was 3.0 - 4.5 meters wide and the banks ranged from
vertical to gently sloping. The average depth o f the water column was 0.76 meters at the
center o f the channel. The study site was at an elevation o f 1,480 meters. Regional land
cover was designated as gently sloping range (2-4%) and the soil had a water holding
capacity o f 13.0 and an average pH o f 7.9 (Caprio et al., 1990). The climiax vegetation
was Silty Range, characterized by various grass communities (Caprio et al., 1990).
15
Fig. 3. New Biltmore Hot Spring, Beaverhead Co.
16
Fig. 4. Beaverhead Warm Springs, Beaverhead Co
17
McLeod H ot Spring
M cLeod H ot Spring (Fig. 5) effluent emanates from a Kibbey rock in Sweet Grass
Co., Latitude 45D 40M 8S, Longitude IlOD SM 41S. The estimated reservoir
temperature was 50 C and an analysis o f water chemistry is presented in Table 4
(Sonderegger et al., 1981). The spring continuously flowed through a trench and into a
holding tank. The sampling sites were located along a marshy region created by water
that spilled over the edges o f the trench. The elevation was 1460 meters. Regional land
cover was designated as strongly sloping range (8-15%) The soil had a water holding
capacity o f 12.2 cm and an average pH o f 7.1 (Caprio et al., 1990). The climax vegetation
was Hardwood climax forest with a typical overstory comprised o f 80% Cottonwoods
(Caprio et al., 1990).
18
Fig. 5. McLeod Hot Spring, Sweet Grass Co.
Table 4. Analysis o f water chemistry at the Green Springs, New Biltmore Hot Spring and McLeod Hot
Spring (Sonderegger et al., 1981). Measurements given in mg/L. (ND), not detected/detection limit
unknown. Data not available for the Beaverhead Warm Springs.
Ca
HCO3
Green Springs
ND
101.0
New Biltmore Hot Springs
290.0
McLeod Hot Spring
473.0
O
P
Site
Cl
SO4
K
Fe
NO3
12.0
5.0
18.0
61.0
0.1
ND
230.0
<1.0
46.0
1100.0
ND
0.10
ND
122.0
0.0
3.0
1350.0
ND
0.40
0.0
20
MATERIALS AND METHODS
Snail collections and observations o f climatological conditions were made from
July 1993 through July 1994. Sampling was done at four sampling sites along a 75 meter
transect at each o f the thermal habitats. The transects originated at the source o f thermal
activity and proceeded along the descending thermal gradient. The sampling sites (1-4)
were spaced at 25 meter intervals, covering an area not exceeding Im2, and were situated
at the interface o f the water and bank so as to allow for sampling o f aquatic, transitional
and terrestrial environments. Sampling was done according to a repeated measures
design, although other methods were considered. This method was selected because the
origin o f thermal activity was a point source, creating a temperature gradient along the
length o f the habitat. Therefore, regions differing in distance from the source represented
unique microhabitats, each o f which were likely to be more or less suitable for lymnaeid
snails. The repeated measures design works well in this situation because it is non-random
and the same microhabitats are sampled during each effort. This is important because it
allows the population dynamics for each microhabitat to be determined. Also, the
microhabitat which is best suited for lymnaeid snails could be determined. I f a random
design were used, inferences about population dynamics and habitat suitability would be
limited to the system as a whole.
21
Snail Collections
A 10 minute visual search o f the soil and vegetation was made within each o f the
Im2 study sites along the transect. Forceps were used to recover snails from the terrestrial
and transitional sections o f the study site and an aquatic net was used to sample the
-
-
aquatic section. Snails were transported to the laboratory in sealed plastic containers filled
with water collected on site. Containers were marked to indicate the thermal habitat and
sampling site (1-4) where the snails originated. Visual searches were used as opposed to
the water extraction o f snails from soil cores (Ross and O'Hagan, 1968; Malone et al.,
1984) because the objective was to identify active lymnaeid populations. A distinction
was made between active and non-active or aestivating snails because o f the underlying
objective to understand F. hepatica transmission. According to Lynch (1965) lymnaeid
snails burrow prior to aestivation 85% o f the time. Therefore, while active snails would
be susceptible to acquiring new F. hepatica infections, aestivating snails generally would
not.
Climatological Characterization
Climatological conditions o f the macro- and microhabitats associated with the
thermal habitats were monitored each month over the course o f the year. The region
immediately surrounding a thermal habitat was considered to be representative o f the
macrohabitat and the sampling sites (1-4) were each considered to be microhabitats.
Climatological conditions, such as precipitation, have been reported to be useful as
indicators o f the probability o f F. hepatica transmission (Ross, 1970; Malone et al., 1984),
however. Smith and Wilson (1980) stated that such factors are useful only to the extent to
)
22
which they affect microclimates. The purpose o f this experiment was to compare the
suitability o f the macro- and microhabitats for lymnaeid snails and F. hepatiCa over the
course o f a year. Temperature and moisture were the principle climatological conditions
measured because they are the rate limiting factors for growth and development o f
lymnaeid snails and F. hepatica (Ollerenshaw, 1959; Boray, 1969; Malone et al., 1984).
Macroclimatological Characterization. Characterization o f the macroclimate was
achieved by monitoring air temperature and precipitation. Air temperature was measured
at 1.2 meters above the ground, to the nearest 0.1 C, with a Tegam 87IA digital
thermometer using a 20 cm air/gas thermocouple probe. Average monthly precipitation
was retrieved from the Montana Agricultural Potential System Atlas, Version 5.0,
Geographic Information System (Caprio et al., 1990).
Microclimatological Characterization. Characterization o f microclimates included
monitoring water temperature and electrical conductivity and soil temperature. W ater
temperature was measured to the nearest 0.1 C and electrical conductivity to the nearest
0 .1 mS with an Extech (Model 650) digital conductivity meter/thermometer.
Measurements were made by suspending the probe within the aquatic section o f the
sampling site. Soil temperature was measured, to the nearest 0 .1 C, with a Tegam 871A
digital thermometer using a 10 cm surface thermocouple probe. Measurements were
made by fully inserting the probe within the terrestrial section o f the sampling site.
Specimen Preservation
Snails were relaxed prior to preservation using menthol crystals. Specimens were
placed in a container o f water and powdered menthol was sprinkled over the water surface
23
(50 mg/ml). The container was sealed and maintained at 10 C for 10-24 hours.
Specimens were relaxed until their bodies were extended from the shell and insensitive to
touch. Snails were placed in hot water (60 C) for 2 seconds and preserved in 70%
ethanol. Specimens were stored as wet collections (70% ethanol) in 25, ml screw top
vials. Each collection was assigned an accession number which was indicated on both the
collecting data label and the identification label, which were placed in the vials. A
duplicate set o f each accession was made and submitted to the mollusc collection at the
Museum o f Natural History at the University o f Colorado, Boulder.
Identification
Snails were identified by Dr. Shi-Kuei Wu at the University o f Colorado, Boulder.
Shell characters were relied upon for most identifications. Dissection o f specimens and
microscopic examination o f soft body parts were performed when necessary to place snails
into the proper species group.
Snail Culture Technique
Field collected snails were cultured to produce generations free o f F. hepatica for
experimental use. Cultures were prepared as described by Taylor and Mozley (1948) with
a few modifications (Fig. 6). Twenty-five centimeter unglazed clay pots were soaked in
distilled water for 2 days and mud collected from the sampling sites was sterilized by
autoclave to remove any potential for parasite transmission. Sterile aquaria gravel was
placed in the bottom o f the clay pot and mud was formed into a slope (m = 0 .1) extending
from near the lip o f the pot on one side to the bottom o f the pot on the other. Distilled
w ater was added to the pots to form a pool at the base o f the slope creating an
24
approximate 2:1 ratio o f exposed soil to submerged soil. Pots were placed in plastic spill
trays filled with distilled water. The cultures were misted with distilled w ater daily to
/
compensate for evaporative losses. Cultures were placed in a controlled environment and
allowed to equilibrate for 2 days, at which time 5-10 snails were introduced. Temperature
was maintained at 22-27 C, and lighting, which was controlled by a timer, was set to 12
hour light:dark cycles. Cultures were placed in racks that had wide spectrum (40 W) plant
and aquarium fluorescent lighting mounted at a height o f 45 cm overhead. Snails were fed
-red leaflettuce to supplement the algal growth which occurred in the cultures. Lettuce
was prepared by soaking in distilled water for 24 hours and then rinsing in distilled water.
Chalk was added ad libitum as a source o f calcium.
Determination o f F. hepatica
Experimental Infection Rates
Generations o f snails reared in laboratory cultures were exposed to F. hepatica
miracidia to determine infection rates o f the various species . Exposures were designed to
compare infection characteristics among species o f lymnaeids and also within species o f
lymnaeids which originated from thermal and non-thermal habitats (Table 5). Snails
ranging in size from 2-6 mm were individually exposed to 3-8 miracidia (Montana bovine
origin) for 6 hours and then maintained in cultures. Snails were crushed and examined
microscopically for redia, sporocysts and cercaria at 50 days post-exposure to determine
infection rates. Lvmnaea columella was used as a positive control during experiments as it
has been consistently demonstrated to be susceptible to infection at a rate o f 60- 80% in
the laboratory (Knapp, pers. comm., 1993). Differences in infection rates o f specimens
25
from thermal and non-thermal habitats were tested for statistical significance; the
/
comparison o f infection rates was based on the difference o f the sample proportions o f the
two populations and used the z statistic (Moore and McCabe, 1989). Lymnaea obrussa
and L. modicella were used in this experiment because o f their availability from thermal
and non-thermal origins. Laboratory reared snails were used to ensure that the snails
being exposed were free o f R hepatica. Field collected specimens could not be used
because it is impossible to detect the parasite without crushing the snail. First generation
snails were used whenever possible to ensure that they were not appreciably different than
the parental generation. It is generally accepted that lymnaeid snails evolve from their
wild type when maintained in culture. Boray (1966) reported that lymnaeids acclimate to
laboratory conditions within 2-3 generations. Therefore, while first generation snails
would still be likely to respond to parasite exposure in a similar manner as the thermal and
non-thermal field collected snails, second generation and beyond might not.
26
Fig.
6 . C u l t u r e f o r I y m n a e id s n a ils .
27
Table 5. Lymnaeid snails from thermal and non-thermal habitats exposed to F. hepatica.
(a), based on identification o f snails used to start cultures, (b), average water temperature
o f the sampling site from which snails originated.
T em p eratu reb
Snaila
O rigin
L obrussa
Beaverhead Warm Springs
18.6 C ± 3.3
L. obrussa
Beaverhead Warm Springs
14.4 C ± 5 .2
L modicella
New Biltmore Hot Spring
23.5 C ± 4 .0
L modicella
Beaverhead River
12.9 C ± 8 .6
I
28
RESULTS
Species o f Snails Collected
In this study 3,500 snails representing 2 subclasses and 5 genera were collected,
including 4 species o f lymnaeids (Table 6). Lymnaeid snails were collected from the
Green Springs, New Biltmore Hot Spring and Beaverhead Warm Springs, but were not
found at the McLeod H ot Spring. Snails demonstrated some variability in habitat type
selection, however, the majority o f snails was recovered from the transitional segment o f
the study sites on gently sloping mud flats at the interface o f the water and bank.
Green Springs
Lymnaeid snails comprised 93% o f the 498 snails collected from July 1993 to July
1994 (Fig 7). Lymnaea caperata and L. montanensis were found at sampling sites I, 2 and
4; the majority was collected at site I (Fig. 8). The snails were always found in the
transitional section o f the sampling sites, usually in the tracks o f livestock that grazed near
the springs perimeter. Oxvloma spp. was the only other snail collected at the Green
Springs and it always appeared on the terrestrial segment o f the sampling sites.
,1
29
Table 6. List o f snail species collected from each thermal habitat.
Subclass
Genus/Species
Collection Site
Pulmonata
Lvmnaea canerata
Green Springs
L obrussa
Beaverhead Warm Springs
L. modicella
New Biltmore Hot Spring
L. montanensis
Green Springs
Phvsa skinneri
New Biltmore H ot Spring,
Beaverhead Warm Springs,
McLeod Hot Spring
Gvraulus parvus
New Biltmore Hot Spring
Beaverhead Warm Springs
McLeod Hot Spring
Oxvloma spp.
Green Springs
New Biltmore Hot Spring
Beaverhead Warm Springs
Prosobranchia
Juea spp.
Beaverhead Warm Springs
30
Fig 7. Percentage o f each snail species collected at the Green Springs from July 1993 to
June 1994.
400
300
200
100
Il
S itel
Site 2
Site 3
Site 4
Fig 8. Number o f lymnaeid snails collected at each sampling site (1-4) from July 1993 to
July 1994 at the Green Springs.
31
New Biltmore Hot Spring
Lymnaea modicella. the only lymnaeid snail found at the New Biltmore Hot
Spring, comprised 30% o f the 1,090 snails collected from July 1993 to July 1994 (Fig 9).
Lvmnaea modicella were found only at sampling sites 3 and 4 (Fig. 10). The snails were
always found in the transitional segment o f site 3, but were found in both the transitional
and aquatic segments o f sampling site 4. Phvsa skinneri. Gvraulus parvus and Oxyloma
spp. snails were also collected. Phvsa skinneri was found in all 4 sampling sites and was
always in the aquatic segment o f the sites, usually on the surface o f rocks or woody debris.
Gvraulus parvus and Oxvloma spp. were collected at site 4 and were always found in the
aquatic and terrestrial segments o f the site, respectively.
Fig 9. Percentage of each snail species collected at the New Biltmore Hot Spring from
July 1993 to July 1994.
32
Fig 10. Number o f Lvmnaea modicella collected at each sampling site (1-4) from July
1993 to July 1994 at the New Biltmore Hot Spring.
Beaverhead Warm Springs
Lvmnaea obrussa. the only lymnaeid found at the Beaverhead Warm Springs,
comprised 67% o f the 1,600 snails collected from July 1993 to July 1994 (Fig 11).
Lymnaea obrussa was collected from all 4 sampling sites, but most were found in the
muddy portion o f site 3 (Fig 12). Phvsa skinneri and Juga spp. were collected from all 4
sampling sites and Gyraulus parvus and Oxvloma spp. were collected from sites I, 2 and
4. Phvsa skinneri. Juga spp and Gyraulus parvus were always found in the water and
Oxvloma spp. was always found in the terrestrial section o f the sampling sites.
33
Oxyloma spp (1 62%)
Physa skinneri (27 48%)
Juga spp. (2.81%)Gyraulus panAJS (0 87%)
Lymnaea obrussa (67 21%)
Fig 11. Percentage of each snail species collected at the Beaverhead Warm Springs from
July 1993 to July 1994.
Fig 12. Number o f Lvmnaea obrussa collected at each sampling site (1-4) from July 1993
to June 1994 at the Beaverhead Warm Springs.
34
Lvmnaeid Population Dynamics
Green Springs
Lymnaeid snails were found during 10 months o f the year. The number o f snails
collected per month ranged from a low o f 2 in June to a high o f 116 in September. The
population cycled seasonally with peaks in September and April (Fig 13). Two
generations o f snails were produced during the year, as indicated by the presence o f
juvenile snails in September and June (Fig 14).
'5 »-
Jl 93
Sept 93
Nov 93
Jan 94
Mar 94
May 94
Month/Year
Fig 13. Number o f Iymnaeid snails collected each month at the Green Springs from July
1993 to June 1994.
- U O l C D O t O - b .
I
I
I
_ _ L i _ _ _ I. . . . .
I
....
U
I
I
I
--1
I
I
I .
I
I-
I '
i ,
I
j
I
I
I
i
i
I
;
I
Jl 93
Z +Z
I
Sept 93
........
.......
J .....
I
I
I
,1, ■
■i
D M
Snail Shell Length (mm)
35
I
Nov 9:
Jan &
Month/Year
....
I
Ht
........
! . V
I
I
I
I
I
I
_ _ _ _ _ I_ _
I
I
I
I
I
Mar 94
May 94
Fig 14. Maximum, minimum and average shell length o f lymnaeid snails at the Green
Springs from July 1993 to June 1994.
New Biltmore H ot Spring
Lvmnaea modicella was found throughout the year. The number o f snails
collected per month ranged from a low o f 2 in February to a high o f 65 in March. In
general, the population cycled seasonally with peaks in March and October followed by a
gradual decline in the size o f the population, however, the number o f snails collected in
April did not follow this trend (Fig 15). Two generations o f snails were produced during
the year, as indicated by the presence o f juvenile snails in October and April (Fig 16).
36
Sepl 93
Nov 93
Jan 94
Mar 94
May 94
Month/Year
Fig 15. Number o f Lymnaea modicella collected each month at the New Biltmore Hot
Spring from July 1993 to June 1994.
£ 4
Sept 93
Nov 93
Jan 94
Mar 94
May 94
MonttVYear
Fig 16. Maximum, minimum and average shell length o f Lvmnaea modicella collected at
the New Biltmore Hot Spring from July 1993 to June 1994.
37
Beaverhead Warm Springs
Lymnaea obrussa was found during 10 months o f the year. The number o f snails
collected per month ranged from a low o f 3 in January to a high o f 473 in July. The
population size cycled 3 times during the year, reaching peaks in April, July and October
(Fig 17). At least 2 generations o f snails were produced during the year, as indicated by
the presence o f juvenile snails in January, March, April and May and July (Fig 18).
-o 100 -
w 60
Sept 93
Nov 93
Jan 94
Month/Year
Mar 94
May 94
Fig 17. Number o f Lvmnaea obrussa collected each month from July 1993 to June 1994 at
the Beaverhead Warm Springs.
38
i
i
i
i
I
I
I
I
i
i
i _ :
,...
I
I
- ,- i
8
I
I
___ .1___ I...... L__ I___ i...... I
9 --1
|
10
r
"|...«—
T ''
-.--...I . *
...
.... - j — r
::± :
5
= I ----- i .....I..... T......T......T"' " T ' : B...
i
i
:
: 1 i ,. I
i
I
I
i _ :
i
j 1
• *" j
:
;
i X :
I
...i T i
t i- i-X h- i |...v-..—...^..........k....---1--------- Q I {
i—i—il—i—<i—
4
d r
—...... ....... ....... j— *......-i—I
I 3
-T
BI*............
I-j—I
2
d rz
?;
I
I
0
.......I.......I.......I...... i
i
i
i
i
I
i
:
I
:
I
Jl 93
:
I
:
i
:
;
j
I
May 94
Month/Year
Fig 18. Maximum, minimum and average shell length o f Lymnaea obrussa collected at the
Beaverhead Warm Springs from July 1993 to June 1994.
39
Characterization o f Thermal Habitats
Green Springs
Macroclimatological Conditions. Air temperature cycled seasonally reaching a
maximum o f 16.7 C in July and a minimum o f -5.0 C in January. The mean annual
precipitation was 43.2 cm (Caprio et al., 1990). Precipitation reached a maximum o f 5.8
cm in June and a minimum o f 2.5 cm in April and October. Air temperature and
precipitation values for each month from July 1993 to June 1994 are presented in Figure
19.
I
0
1QI- o
£
CL
Jl 93
Sept 93
Nov 93
Jan 94
Mar 94
May 94
MonthZYear
Fig 19. Air temperature and precipitation values reported for each month at the Green
Springs from July 1993 to June 1994. ( • ) , air temperature. (■), precipitation.
40
Microclimatological Conditions. The average water temperature o f study sites I -4
ranged from 2 7.1 C (±4.2) to 15.4 C (±8.3) (Fig 20). The water temperature cycled
seasonally reaching a maximum o f 31.8 C at site I in August and a minimum o f 5.2 C at
site 4 in November (Fig 21). The average electrical conductivity o f the water ranged from
0.18 mS (±0.1) at sites 1-3 to 0.19 mS (±0.1) at site 4. Soil temperature cycled
seasonally, similar to the water temperature, reaching a maximum o f 16.6 C at site I in
May and a minimum o f -3.4 C at site 2 in February (Fig 22).
S itel
Site 2
Site 3
Site 4
Fig 20. Average water temperature (± standard deviation) at sampling sites I -4 from July
1993 to June 1994 at the Green Springs.
41
Fig 21. W ater temperature at sampling sites 1-4 from July 1993 to June 1994 at the Green
Springs. ( • ) , site I; (■ ), site 2; 0 ) , site 3; (▼), site 4.
Sept 93
Nov 93
Jan 94
Mar 94
May 94
Fig 22. Soil temperature at sampling sites (1-4) from September 1993 to June 1994 at the
Green Springs. ( • ) , site I. (*), site 2; (■), site 3; (▼), site 4.
42
New Biltmore Hot Spring
Macroclimatological Condition. Air temperature cycled seasonally reaching a
maximum o f 16.7 C in July and a minimum o f -5.0 C in January. The mean annual
precipitation was 22.9 cm (Caprio et al., 1990). Precipitation reached a maximum o f 4.8
cm in June and a minimum o f 0.5 cm in January, February and December. Air temperature
and precipitation values for each month are presented in Figure 23.
20
-10
Jl 93
Sept 93
Nov 93
Jan 94
Month/Year
Mar 94
May 94
Fig 23. Average air temperature and precipitation each month from July 1993 to June
1994 at the New Biltmore Hot Spring. ( • ) , air temperature; (■), precipitation.
43
Microclimatological Conditions. The average water temperature o f study sites I -4
ranged from 34.6 C (±4.8) to 20.6 C (±4.6) (Fig 24). The water temperature cycled
seasonally reaching a maximum o f 44.9 C at site I in July and a minimum o f 15.7 C at site
4 in December (Fig 25). The average electrical conductivity o f the water ranged from
1.32 to 1.45 mS (±0.1). Soil temperature cycled seasonally, similar to water temperature,
reaching a maximum of 33.8 C at site I in May and a minimum o f -0.7 C at site 4 in
January (Fig 26).
Fig 24. Average water temperature (± standard deviation) at sampling sites I-4 from July
1993 to June 1994 at the New Biltmore Hot Spring.
44
45
I....I.... I
Sept 93
Nov 93
Jan 94
Month/Year
Mar 94
May 94
Fig 25. W ater temperature at sampling sites 1-4 from July 1993 to June 1994 at the New
Biltmore Hot Spring. ( • ) , site I; (*), site 2; (■), site 3; (▼), site 4.
Fig 26. Soil temperature at sampling sites 1-4 from September 1993 to June 1994 at the
New Biltmore Hot Spring. ( • ) , site I; ( a), site 2; (■), site 3; (▼), site 4.
45
Beaverhead Warm Springs
Macroclimatological Conditions. Air temperature cycled seasonally reaching a
maximum o f 18.3 C in July and a minimum o f -6.7 C in January. The mean annual
precipitation was 22.9 cm (Caprio et al., 1990). Precipitation reached a maximum o f 4.8
cm in June and a minimum o f 0.5 cm in January, February and December. Air temperature
and precipitation values for each month are presented in Figure 27.
Jl 93
Sept 93
Nov 93
Jan 94
Month/Year
Mar 94
May 94
Fig 27. Average air temperature and precipitation from July 1993 to June 1994 at the
Beaverhead Warm Springs. ( • ) , air temperature. (■), precipitation.
46
Microclimatological Conditions. The average water temperature o f study sites I -4
ranged from 21.7 (±6.0) to 18.3 C (±3.3) (Fig 28). Water temperature reached a
maximum o f 28.3 C at site I in September and a minimum o f 10.2 C in June. The effect
o f seasonal influences on water temperature cycling was evident from September to
February, however, local irrigation practices also had an effect, by increasing water
volume, from May - August (Fig 29). The average electrical conductivity o f the water
ranged from 0.46 mS (±0.03) at site I to 0.48 mS (±0.04) at site 2. Soil temperature
cycled seasonally, similar to water temperature, reaching a maximum o f 18.7 C at site I in
April and a minimum o f -5.0 C at site 4 in November (Fig 30).
Fig 28. Average water temperature (± standard deviation) at sampling sites 1-4 from July
1993 to June 1994 at the Beaverhead Warm Springs.
47
Sept 93
Nov 93
Jan 94
MonthZYear
Mar 94
May 94
Fig 29. W ater temperature at sampling sites 1-4 from July 1993 to June 1994 at the
Beaverhead Warm Springs. ( • ) , site I; (*), site 2; (■), site 3; (»), site 4.
Fig 30. Soil temperature at sampling sites 1-4 from October 1993 to June 1994 at the
Beaverhead Warm Springs. ( • ) , site I; (*), site 2; (■), site 3; (▼), site 4.
48
McLeod Hot Spring
Macroclimatoloeical Conditions. Air temperature cycled seasonally reaching a
maximum o f 18.3 C in July and a minimum o f -7.8 C in January. The mean annual
precipitation was 38.1 cm (Caprio et al., 1990). Precipitation reached a maximum o f 6.8
cm in May and June and a minimum o f 1.0 cm in February. Air temperature and
precipitation values for each month from July 1993 to June 1994 are presented in Figure
31.
.
_
I
V j
\!
_ _ u
.Z
, i . . . . :*••*
...... ...... ...... ...... - - - - L . / j ,
E f
*. . . .
I
I
G 10
I..
P-I
i
3 0
... j
I
/
i
I
Jl 93
E *
:
.I . . . . .
it
I'
I
I
I
I
Sept 93
"
Nov 93
Jan 94
MonttVYear
I
I
Mar 94
I
May 94
Fig 31. Average air temperature and precipitation from July 1993 to June 1994 at the
McLeod Hot Spring. ( • ) , air temperature; (■), precipitation.
49
Microclimatological Conditions. The average water temperature o f study sites 1-4
ranged from 4 8 .1 C (±1.2) to 25.9 (±8.5) (Fig 32). The water temperature remained
nearly constant at sites I and 2 and did not cycle seasonally; the water temperature at sites
3 and 4 cycled seasonally reaching a maximum o f 45.9 C at site 3 in June and a minimum
o f 14.1 C in March (Fig 33). The average electrical conductivity o f the water was 1.28
mS (±0.09). Soil temperature cycled seasonally, similar to water temperature, reaching a
maximum o f 38.2 C at site I in April and a minimum o f 1.6 C at site 4 in December (Fig
34).
Sitel
Site 2
Site 3
Site 4
Fig 32. Average water temperature (± standard deviation) at sampling sites 1-4 from July
1993 to June 1994 at the McLeod Hot Spring.
50
Sept 93
Nov 93
Jan 94
Month/Year
Mar 94
May 94
Fig 33. W ater temperature at sampling sites 1-4 from July 1993 to June 1994 at the
McLeod Hot Spring. ( • ) , site I; (*), site 2; (■), site 3; (*), site 4.
! / I...\j....T
I O |
Jl 93
Sept 93
Nov 93
Jan 94
MonttVYear
Mar 94
May 94
Fig 34. Soil temperature at sampling sites 1-4 from July 1993 to June 1994 at the McLeod
Hot Spring. ( • ) , site I; (*), site 2; (■), site 3; (▼), site 4.
SI
Experimental Fasciola hepatica Infection Rates
Lymnaea from Thermal Habitats
A total o f 233 L obrussa were exposed during 4 replicates. Only 148 snails were
examined 50 days post-exposure because an average mortality rate o f 36.5% occurred
while snails were in culture. There was no evidence o f infection in any o f the snails
examined (Table I).
A total o f 221 L. modicella were exposed during 3 replicates. Only 95 snails were
examined .50 days post-exposure because an average mortality rate o f 61.3% occurred
while snails were in culture. Lymnaea modicella had an average infection rate o f 3.2%
(Table 7). Redia and cercaria were observed in 2 o f 52 snails (3.8%) in replicate I and in
I o f 21 snails (4.8%) in replicate 2, but there was no evidence o f infection in replicate 3.
Lvmnaeids from Non-Thermal Habitats
(
A total o f 168 L. obrussa were exposed during 2 replicates. Only 65 snails were
examined at 50 days post-exposure because an average mortality rate o f 61.3% occurred
while snails were in culture. Lymnaea obrussa had an average infection rate o f 10.7%
(Table 7). There was no evidence o f infection in snails in replicate I, but immature redia
were observed in 7 o f 53 snails (13.2%) in replicate 2.
A total o f 103 L modicella were exposed during 2 replicates. Only 61 snails were
examined at 50 days post-exposure because an average mortality rate o f 41.7% occurred
while snails were in culture. Lvmnaea modicella had an average infection r a t e f 4.9%
(Table 7). Redia were observed in 3 o f 45 snails (6.7%) in replicate I, but there was no
evidence o f infection in snails in replicate 2.
52
Positive Control Snails
A total o f 356 L. columella from laboratory stock was used as a positive control in
the experiment. Only 173 snails were examined 50 days post-exposure because an
average mortality rate o f 51.4 % occurred while snails were in culture. Lvmhaea
columella had an average infection rate o f 43.9% (Table 7). During the 6 replicates o f the
experiment the infection rate ranged from 15% to 96%. Redia and cercaria were observed
during each o f the replicates.
Statistical Significance
Data were analyzed to determine if there was a statistically significant difference
between the infection rates o f L obrussa or L modicella from thermal and non-thermal
habitats. The null hypothesis (H0) stated that there was no difference between the
infection rates in snails from thermal and non-thermal habitats. The alternative hypothesis
(Ha) stated that a difference existed between the infection rates o f snails from thermal and
non-thermal habitats.
The null hypothesis was rejected (p<0,05) when analyzing the infection rates o f L.
obrussa from thermal and non-thermal habitats. The average infection rate o f L. obrussa
from non-thermal habitats was greater than that o f specimens from thermal habitats and
the difference was statistically significant.
The null hypothesis was not rejected (p>0.05) when analyzing the infection rates o f
L. modicella from thermal and non-thermal habitats. The average infection rate o f L.
modicella from thermal habitats was greater than specimens from non-thermal habitats, but
the difference was not statistically significant.
Table 7. Experimental infection rate o f lymnaeid snails originated from thermal and non-thermal sites, (a), based on
identification o f snails used to start cultures, (b), average temperature o f the sampling site where snails used to start the
cultures were collected, (c), average o f replicates. (N), number o f snail examined.
Snail* (N)
O rigin
L. obrussa fl48)
Beaverhead Warm Springs
18.6 ± 3 .3
0.0
L. obrussa (65)
Beaverhead Warm Springs
14.4 ± 5 .2
10.8
L. modicella (95)
New Biltmore Hot Spring
23.5 ± 4 .0
3.2
L. modicella (61)
New Biltmore Hot Spring
12.9 ± 8 .6
4.9
L. columella (173)
Laboratory Stock
Not Applicable
43.9
W ater T em peratureb (C)
Infection R atec (% )
54
DISCUSSION
Significance o f Results
Presence o f Snails at Thermal Habitats
Although snails had been collected from many habitats in M ontana as part o f a
state wide survey (Dunkel et al, 1995), this is the first study to focus on the presence o f
lymnaeid snails at a specific habitat type. This is an important distinction because, while
lymnaeid snails are present in many habitat types, F. hepatica transmission is focal in
nature (Malone and Zukowski, 1992). This aspect o f transmission can be attributed to the
effects o f climatic variation on lymnaeid snail intermediate host populations. In fact,
variation in habitat suitability, resulting from year to year weather changes, can lead to
100-fold differences in the infection rate o f definitive host populations (Malone and
Zukowski, 1992). Conversely, a habitat that is both suitable and stable with respect to
climatological conditions would be likely to consistently serve as a focus o f transmission
as they would positively affect the development o f lymnaeid snails and F. hepatica. For
example, incubation o f L. truncatula eggs at constant temperatures resulted in a hatching
success o f at least 55%, depending on the temperature, while hatching success was only
28.7% when the temperature was cycled (Khallaayoune et al., 1990) The presence o f
lymnaeid snails at three o f four thermal habitats studied is significant for two reasons.
First, it suggests that thermal habitats are generally suitable as lymnaeid snail habitats.
This is important considering the abundance o f thermal habitats in the state. Second,
thermal habitats are extremely stable with respect to physical and chemical properties,
indicating that F. hepatica transmission would likely be consistent from year to year.
Lymnaeid Species Present
The four species o f lymnaeid snails that were recovered are o f interest as each has
been identified as a natural or experimental vector o f F. hepatica and/or Fascioloides
maena. Grriffiths (1959) and Foreyt and Todd (1978) reported L. caperata to be a natural
vector o f F. magna. Lang (1977) reported L. modicella to be a natural vector o f both F.
hepatica and F. magna. Rowan et al. (1966) reported L. montanensis as an experimental
vector o f F. hepatica. L. obrussa was experimentally infected with F. hepatica in the
present study and Knapp (pers. comm., 1995) found naturally infected specimens.
Seasonal Dynamics
Previous studies have indicated that lymnaeid snail activity is restricted to a few
months o f the year in Montana because o f the harsh climate (Hoover et al., 1984; Seesee
et al., 1988). The results o f this study, however, indicate that lymnaeid snails are active
year-round at thermal habitats. Lymnaea caperata and L. montanensis. and L. obrussa
were collected during 10 months o f the year at the Green Springs and Beaverhead Warm
Springs, respectively, and L. modicella was collected throughout the year at the New
Biltmore H ot Spring. It is likely that snails were actually present year-round at the Green
Springs and Beaverhead Warm Springs, but they were not found because o f the limited
size o f the study sites and the restriction o f the collections to 10 minute visual searches o f
the surface; additionally, other genera o f snails were observed to remain active at the
thermal habitats throughout the year. Fluke ova have been observed to be shed
throughout the year in this region (Knapp, personal communication, 1993). Therefore, the
56
presence o f active lymnaeid snail populations year-round at thermal habitats is important
as it allows for infection o f snails and intra-molluscan parasite development throughout the
year in a region where it would otherwise be unlikely.
Size Class o f Snails
Based on the shell lengths o f the snails collected, it is clear that multiple
generations o f lymnaeids were produced at each o f the thermal habitats during the year.
It has been demonstrated that F. hepatica infection rates o f several lymnaeid species vary
according to the age o f the snail (Boray, 1966; Smith, 1981). Therefore, the fact that
multiple generations were produced, and snails o f various ages were present throughout
the year, suggests that the acquisition o f F. hepatica infection would be probable
throughout the year. This is significant as it would result in the continual propagation o f
the life cycle.
Climatological Conditions'Surface soil temperature and moisture have been reported to be the most critical
factors in the development o f the life cycle o f F. hepatica. including the fluke eggs, intramolluscan larval stages, cercaria and metacercaria. Development o f fluke eggs, intramolluscan stages and the release o f cercaria occur at a minimum temperature o f 9-10 C
(Ollerenshaw and Rowlands, 1959). Metacercaria were viable and infective at -5 C for
28-56 days and for at least 92 days at -2 C (Boray, 1969) and for 11 months on wet grass
at 0 C (Shaw, 1932). Soil temperatures measured at a depth o f 10 cm remained above 10
C throughout the year at the New Biltmore H ot Spring and M cLeod H ot Spring and
estimated surface soil temperatures, based on techniques described by Smith et al., (1964),
57
r e m a i n e d a b o v e 1 0 C y e a r - r o u n d at a ll 4 o f t h e t h e r m a l h a b i t a t s .
W a te r te m p e r a tu r e a ls o
r e m a in e d a b o v e 1 0 C a t I o r m o r e s a m p lin g s it e s at e a c h o f t h e th e r m a l h a b it a t s y e a r -
rou n d
6 .7
C
C o n v e r s e ly , th e a v e r a g e s o il te m p e r a tu r e o f th e s u r r o u n d in g m a c r o h a b ita ts w a s
T h e s e d a ta s u g g e s t th a t th e r m a l h a b ita ts p r o v id e s u it a b le e n v ir o n m e n t s f o r p a r a s it e
d e v e lo p m e n t a n d t r a n s m is s io n to d e f in itiv e h o s t s y e a r -r o u n d , w h e r e a s th e s u r r o u n d in g
m a c r o h a b ita ts d o n o t.
F o r e x a m p le , ic e fo r m e d im m e d ia te ly u p s tr e a m o f th e s o u r c e o t
th e r m a l a c tiv ity at th e B e a v e r h e a d W a rm S p r in g s , b u t th e w a te r w a s ic e fr e e y e a r -r o u n d
d o w n s tr e a m o f th e s o u r c e (F ig 3 5 ).
Fig. 35.
D e lin e a t io n o f th e r m a l a n d n o n - th e r m a l h a b ita ts a t t h e B e a v e r h e a d W a r m S p r in g s .
58
Infection Rates
The difficulty associated with the experimental exposures was maintaining snails
in culture to 50 days post-exposure. Mortality occurred within the first several days after
snails were placed in culture, but the cause o f death could not be determined. It is likely,
however, that mortality resulted as a consequence o f exposure to F. hepatica miracidia as
unexposed snails maintained under identical conditions have been observed to live in
cultures for six months or more. The possible effects o f F. hepatica on snail mortality
could not be avoided, as this was the purpose o f the experiment, but efforts were made to
ensure that the environment was suitable with respect to moisture and temperature.
O f particular interest, this constitutes the first report o f L. obrussa as a vector o f F.
hepatica. Lvmnaea modicella was confirmed as a vector. Also, L. obrussa from thermal
and non-thermal habitats expressed differences in infection rates that were statistically
significant (p<0.05), but L. modicella from thermal and non-thermal habitats did not.
v ■
It should be noted that while L. obrussa from thermal and non-thermal origins expressed
statistically significant differences with respect to infection rate, the differences were not
necessarily biologically significant. At 50 days post exposure, there was no evidence o f '
cercaria in those snails that were infected. This would indicate that L. obrussa is not a
good intermediate host and, therefore, the differences would not be important to parasite
transmission. Hence, the differences would not be biologically significant. However, it
may be that cercaria would have developed if the infection were carried beyond 50 days.
Regardless o f the biological significance in the case o f L. obrussa. the differences in
infection rates are important for another reason. As the snails from thermal and non-
t
59
thermal origins were subjected to identical external conditions, it is likely that the
differences in infection rates are indicative o f strain differences. This would be important
in terms o f understanding F. hepatica transmission patterns. The wide variability in the
occurrence o f R hepatica transmission at different locations within enzootic regions has
largely been attributed to climatic effects on habitat suitability for lymnaeid snails (Malone
and Zukowski, 1992), however, it is likply that strain variations would play a role as well.
A final point o f interest was the observation o f Chaetogaster spp. upon crushing
the snails for microscopic examination at day 50 post-exposure. Chaetogaster limnaei, an
annelid that inhabits the mantle cavity o f snails, has been reported to feed on parasitic
cercaria (Khalil, 1961). Although the specimens were not observed to destroy R hepatica
larval stages, the possibility exists, which would have the effect o f reducing the observed
infection rate.
Future Work
In connection with the work completed, the infection rate o f L caperata should be
determined. Additionally, other thermal habitats in the state should be sampled to
determine the lymnaeid species present and their suitability as year-round habitats.
The natural progression o f the investigation beyond the issues addressed in the
current research should include the following:
1. An analysis o f the mechanisms o f intermediate host susceptibility and
resistance.
2. The development o f a standard technique for identifying lymnaeid snails.
60
CONCLUSIONS
1. Thermal habitats provide suitable conditions for lymnaeid snails, including L.
caperata. L. modicella. L. mohtanensis and L obrussa. and it is likely that populations
remain active throughout the year.
2. Thermal habitats maintain conditions suitable to the development o f the intramolluscan and extra-mammalian stages o f R hepatica year-round in contrast to the
surrounding macrohabitats in Montana.
3. Transmission o f R hepatica to the definitive host may occur year-round in
association with thermal habitats, whereas the prevailing transmission pattern in the
northwestern United States is seasonal and occurs primarily in the fall.
4. Statistically significant differences in R hepatica infection rates o f lymnaeid
snails from thermal and non-thermal origins are site specific and do not necessarily equate
to biologically significant differences.
61
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