For Reference
NOT TO BE TAKEN FROM THIS ROOM
6bc 11BBB
MM2I$
Digitized by the Internet Archive
in 2020 with funding from
University of Alberta Libraries
https://archive.org/details/Shostak1980
THE UNIVERSITY OF ALBERTA
RELEASE FORM
NAME OF AUTHOR Allen William Shostak
TITLE OF THESIS Survival of first -stage larvae of
Parelaphostrongy lus odocoi lei and
Parelaphostrongy lus tenuis (Nematoda:
Metastrongy loidea) .
DEGREE FOR WHICH THESIS WAS PRESENTED Master of Science
YEAR THIS DEGREE GRANTED 1980
Permission is hereby granted to THE UNIVERSITY OF
ALBERTA LIBRARY to reproduce single copies of this
thesis and to lend or sell such copies for private,
scholarly or scientific research purposes only.
The author reserves other publication rights, and
neither the thesis nor extensive extracts from it may
be printed or otherwise reproduced without the author's
written permission.
THE UNIVERSITY OF ALBERTA
SURVIVAL OF FIRST-STAGE LARVAE OF
PARELAPHOSTRONGYLUS ODOCOILEI AND PARELAPHOSTRONGYLUS TENUIS
( NEMATOD A : MET AS TRONG YLO ID E A)
by
ALLEN WILLIAM SHOSTAK
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES AND RESEARCH
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE
OF MASTER OF SCIENCE
DEPARTMENT OF ZOOLOGY
EDMONTON, ALBERTA
SPRING, 1980
THE UNIVERSITY OF ALBERTA
FACULTY OF GRADUATE STUDIES AND RESEARCH
The undersigned certify that they have read, and
recommend to the Faculty of Graduate Studies and Research,
for acceptance, a thesis entitled "Survival of first-stage
larvae of Pare 1 apho s tr on gv lu s odocoilei and
Pare 1 apho sir onpy i u s tenuis (Nematoda:Metastrongyloidea) . "
submitted by Allen William Sho stale
in partial fulfilment of the requirements for the degree of
Ma s t er of Sc i enc e .
\
ABSTRACT
The survival of first-stage larvae of Parelaphostrongy lus
odocoi lei and Parelaphostrongy lus tenui s was determined in a
variety of temperature and moisture conditions. Following
treatment in some of those conditions, the infectivity of
surviving first -stage larvae to the experimental intermediate host
Triodopsis multilineata was determined.
The survival of larvae of P. odocoilei was strongly influenced
by both moisture and temperature conditions. Hydrated larvae, and
larvae desiccated at low relative humidity, had the lengthiest
survival. Desiccation enhanced survival of larvae at high
temperature, but reduced their survival while frozen. A major
loss of infectivity to the intermediate host was observed for
larvae which survived desiccation.
Repeated temperature changes above freezing did not alter
survival of larvae of P. odocoilei . Repeated freezing or repeated
desiccation resulted in reduced survival of the larvae of
P. odocoilei and P_. tenuis . The reduction in survival was
proportional to the number of treatments administered.
The survival of larvae of P. odocoilei from two sources,
Vancouver Island and Jasper National Park, did not differ following
storage at various temperature and moisture conditions. Differences
were noted between species in both their survival, and in their
infectivity to the intermediate host. Larvae of P_. odocoilei
survived better than P. tenui s following repeated freezing, while
larvae of P. tenuis survived better than P. odocoilei following
IV
repeated desiccation. Following freezing,
a greater proportion
of the surviving first -stage larvae of P. odocoi lei were infective
to the intermediate host than were larvae of P. tenui s , when
compared to the infectivity of the larvae of the two species
which had not been previously frozen.
The results are discussed in light of current concepts
regarding survival of the free-living stages of parasitic nematodes,
and in light of the current distribution of Parelaphostrongy lus spp.
in North America. It is suggested that differences in the tolerance
of first -stage larvae of P. odocoi lei and P. tenui s to temperature
and moisture conditions provide a means by which climatic factors
can differentially influence the survival of the free-living
stage of these two species, and thereby their distributions.
v
I
ACKNOWLEDGEMENTS
I wish to express my deep gratitude to my supervisor.
Dr. W.M. Samuel, for his advice, encouragement, and interest in
this study. I would like to thank the members of my supervisory
and examining committees, Drs. J.C. Holmes, W.M. Samuel, and
F.C. Zwickel, and Drs. B. Heming, J.C. Holmes, W.C. Mackay, and
W0M. Samuel, respectively, for their helpful comments.
Dr. J.M. Mayo, Dr. D.W. Whitfield, and Mr. J, Richards,
Department of Botany, provided helpful comments dealing with the
methods of this study. Ms. M. Barker, Department of Zoology, and
the staff of the University of Alberta Vivarium, particularly
Mr. K. Taylor, provided excellent technical assistance in
maintenance of the experimental animals.
I am also indebted to the members of the parasitology group
at the University of Alberta, for interesting discussions and
probing questions.
Financial support was provided by the Alberta Fish and
Wildlife Division, with special thanks to W. Wishart and D . Neave,
the Natural Sciences and Engineering Council of Canada (Operating
Grant No, A-6603 to W.M. S., and Postgraduate Scholarship to A.W.S.),
the Boreal Institute for Northern Studies, the Alberta Fish and
Game Association, the Saskatchewan Department of Tourism and
Renewable Resources, and the University of Alberta (Teaching
Assistant ship to A.W.S.).
vi
Finally, I would like to thank Shirley Hilger, whose love,
understanding, and encouragement during this study will always
be remembered.
Vll
TABLE OF CONTENTS
ABSTRACT . . . .
ACKNOWLEDGEMENTS
LIST OF TABLES .
LIST OF FIGURES .
CHAPTER
• •000000000000000*0 • O o
0000*00000000000*000*
Page
iv
vi
x
xii
I. INTRODUCTION . . . 1
II. MATERIALS AND METHODS . . 11
A. Definitions . . . . . . . . . 11
B. Source of Experimental Animals . 12
Co Temperature and Humidity Control „ . 13
D. Preparation of Larvae for Experimentation .... 18
E. Experimental Design . 18
F. Data Analysis . . . . . . 40
III. RESULTS . . 42
A. Survival Under Non-varying Conditions . 42
B. Survival Under Varying Temperature Conditions . . 57
C. Survival Under Varying Moisture Conditions . . „ 62
D. Infectivity Trials . . 69
IV. DISCUSSION . „....„ . . 81
A. Survival of P. odocoilei . . 81
B. Infectivity of P. odocoilei . . 96
Co Comparative Studies . 97
D. Epizootiological Considerations . 101
viii
LITERATURE CITED
APPENDIX I . .
APPENDIX II . .
Page
116
128
131
IX
LIST OF TABLES
Table
Description
Page
I
Theoretical and measured values of percentage
relative humidity (% RH) in humidity control
chambers.
17
II
Percentage relative humidity (% RH) over
saturated salt solutions and water.
23
III
Maximum days survival of first -stage larvae of
Parelaphostrongy lus odocoilei under various
temperature and moisture conditions in Experiment
Numbers 1 and 2 (Exp'ts 1,2).
45
IV
Anova table for the survival of first-stage larvae
of P. odocoilei at 25°C, by source of larvae,
position in humidity control chamber, and
treatment (Exp't 3).
51
V
Anova table for the survival of first-stage larvae
of P. odocoilei at -25°C, by source of larvae,
moisture condition of freezing, and length of
freezing (Exp't 4).
52
VI
Survival of first -stage larvae of P. odocoilei,
frozen on fecal pellets at -25°C (Exp't 5).
54
VII
Anova table for the survival of first-stage larvae
of P. odocoilei and Parelaphostrongy lus tenuis
following repeated freezing, by species and number
of freezings (Exp't 9).
61
VIII
Recovery of second- and third-stage larvae from
Triodopsis multilineata exposed to first-stage
larvae of P. odocoilei under various exposure
conditions (Exp't 15).
72
IX
Recovery of second- and third-stage larvae from
T. multilineata exposed to treated first-stage
larvae of P. odocoilei (Exp't 16).
74
X
Recovery of second- and third-stage larvae from
T. multilineata exposed to treated first-stage
larvae of P. odocoilei (Exp't 17).
75
x
'
List of Tables
continued
Table
XI
XII
XIII
XIV
Description Page
Anova table for numbers of larvae recovered
from T. multilineata exposed to treated first-
stage larvae of P. odocoi lei , by moisture
condition, temperature, and length of
treatment (Exp't 17). 76
Recovery of second- and third-stage larvae from
T. multilineata exposed to treated first-stage
larvae of P_, odocoi lei and P. tenui s (Exp't 18). 78
Anova table for numbers of larvae recovered
from T. multilineata exposed to treated first-
stage larvae of P. odocoi lei and P_. tenuis , by
species and temperature of treatment (Exp't 18). 79
Recovery of dor sal -spined larvae from the feces
of white-tailed deer from different regions of
southern Saskatchewan (Shostak, unpub.). 107
xi
LIST OF FIGURES
Figure Page
1. Approximate distributions of Parelaphostrongy lus
tenuis and Parelaphostrongy lus ander soni in
Odocoi leus virginianus in North America. 3
2. Approximate distribution of Parelaphostrongy lus
odocoilei in Odocoi leus hemionus in North
America. 5
3. Generalized life cycle of genus
Parelaphostrongy lus. 8
4. Diagrammatic representation of humidity
control chamber. 15
5. Flow diagram outlining the organization of the
experimental program. 20
6. Schematic representation of the design of
Experiment Number 9 (Exp't 9) showing temperatures
of samples in each of the four groups over time. 30
7. Schematic representation of the basic design of
all varying -moisture experiments (Exp'ts 10-14). 32
8. Schematic representation of the design of
Exp't 16. 38
9. Survival of first-stage larvae of P. odocoilei
at various temperature and moisture conditions
(Exp ' ts 1,2) . 44
10. Survival of first-stage larvae of P, odocoilei
from two sources, at 25°C and various moisture
conditions (Exp't 3). 48
11. Mean percent survival of first -stage larvae of
P. odocoilei from two sources, following
freezing (Exp't 4). 50
12. Survival of first -stage larvae of P. odocoilei
(P.o.) and P. tenuis (P.t.) at 30°C and various
moisture conditions (Exp't 6). 56
xii
List of Figures - continued
Figure Page
13. Survival of first-stage larvae of P. odocoi lei
(P.o.) and P. tenuis (P.t.) following repeated
freezing (Exp't 9). 60
14. Survival of first -stage larvae of P. odocoi lei
following repeated desiccations at 18°C
(Exp'ts 10,11,13,14) . 64
15. Survival of first -stage larvae of P. tenui s
following repeated desiccations at 18°C
(Exp 1 ts 13, 14) . 66
16. Survival of first-stage larvae of P. odocoi lei
(P.o.) and P. tenuis (P.t.) following repeated
desiccations at 18°C (pooled data from
Exp'ts 10,11,13,14) . 68
17. Survival of first-stage larvae of P, odocoilei
following repeated desiccations at 8 and 18°C
(Exp ' ts 10-14) . 71
18. Survival of desiccated free-living larvae of
a variety of plant- and animal -parasitic
nematodes at a variety of relative humidities. 89
19. Potential route of expansion of P. tenuis
range into the foothills of the Rocky Mountains,
as suggested by Bindernagel and Anderson (1972). Ill
xiii
I. INTRODUCTION
Three species of the genus Parelaphostrongy lus (Nematoda:
Metastrongy loidea: Elaphostrongy linae) have been reported in North
America. Parelaphostrongy lus tenuis (Dougherty 1945) , the menin¬
geal worm, is found in its normal host, the white-tailed deer (Odo-
coileus virginianus) , throughout eastern North America (Fig. 1)
(Dougherty 1945; DeGiusti 1955; Anderson 1956; Alibasoglu et al.
1961; Karns 1967; Smith and Archibald 1967; Behrend and Witter 1968;
Prestwood and Smith 1969; Samuel and Trainer 1969; Bindernagel and
Anderson 1972; Carpenter et al . 1972; Pursglove 1977; Thurston and
Strout 1978). Parelaphostrongy lus ander soni Prestwood 1972, a
muscleworm of white-tailed deer, has been reported in that host from
the southeastern United States (Prestwood et al. 1974; Pursglove
1977) and in southeastern British Columbia (M.J. Pybus, pers. comm.)
(Fig. 1). Another muscleworm, Parelaphostrongy lus odocoi lei (Hob-
maier and Hobmaier 1934) , has been reported from Columbian black¬
tailed deer (Odocoi leus hemionus columbianus) and California mule
deer (Odocoileus hemionus calif ornicus) in northcentral California
(Hobmaier and Hobmaier 1934; Brunetti 1969) , in 0. h. columbianus
from Vancouver Island, British Columbia (Platt and Samuel, unpub.),
and from mule deer (Odocoileus hemionus hemionus) in westcentral
Alberta (Platt and Samuel 1978a; Samuel, unpub.) (Fig. 2).
The life cycle of all three species of Parelaphostrongy lus
involves the adult occupying an extraintestinal site in a cervid
definitive host, the first-stage larva (Ll) shed in the feces of the
1
2
Figure 1. Approximate distributions of Parelaphostrongy lus
tenuis and Parelaphostrongy lus ander soni in
Odocoileus virginianus in North America. Star
indicates a report of P. tenuis in Angora goats,
outside of known P. tenuis range in 0. virginianus
(Guthery and Beasom 1979). Deer distribution is
from Stock (1978).
3
P. andersoni
Figure 2. Approximate distribution of Parelaphostrongy lus
odocoi lei in Odocoileus hemionus in North America.
Deer distribution is from Stock (1978).
5
P. odocoilei
definitive host, and the penetration of the foot of a terrestrial
gastropod by the Ll. Development to the infective, third-stage
larva occurs in the foot, and the life cycle is completed when the
gastropod infected with third-stage larvae is accidentally ingested
by another cervid.
The maintenance of Parelaphostrongylus in a area is
dependent upon both suitable intermediate and definitive host
conditions, and sufficient resistance to the external environment
by the free-living stage (the Ll) . Infective conditions require
suitable densities of both hosts, and behavioral characteristics
which facilitate each host acquiring the appropriate stage of the
parasite. The intermediate host must come in contact with the first-
stage larva, and subsequently must be available in sufficient num¬
bers to the definitive host as it grazes. The free-living larval
stage must resist environmental extremes of moisture, temperature,
and solar radiation, either through physiological adaptation or
avoidance .
Many aspects of the relationship between the parasite Par elapho -
strongy lus and its normal intermediate and definitive hosts have been
studied. Ecological aspects of the parasite- intermediate host rela¬
tionship have been studied (Lankester and Anderson 1968; Kearney and
Gilbert 1978; Platt 1978), as has the life cycle in the intermediate
host (Lankester and Anderson 1968; Platt 1978). Work on the defini¬
tive host has provided information not only on the prevalence and
distribution (as previously cited) and pathology (reviewed by Anderson
1971), but also on such aspects as prepatent periods (Anderson 1963;
Nettles and Prestwood 1976; Platt and Samuel 1978b), effect of size,
Figure 3. Generalized life cycle of genus Parelaphostrongy lus .
8
9
and frequency of administration, of infective inocula (Nettles and
Prestwood 1976; Prestwood and Nettles 1977; Platt and Samuel 1978b),
and larval output (Nettles and Prestwood 1976; Platt and Samuel
19 78b) .
The relationship between the first-stage larva and the environ¬
ment has not been as well studied as that between the parasitic
stages and their hosts. For example, although the range of gastro¬
pods and ungulates which can maintain the parasitic stages of
Parelaphostrongy lus has been extensively documented (Lankester and
Anderson 1968; review by Brown et al. 19 78; Platt 19 78; Platt and
Samuel 1978b), the range of environmental conditions that can sup¬
port the free-living stage is almost unknown.
The importance of environmental influences on the free-living
stages of parasitic nematodes has long been recognized in epi-
zootiological studies on parasites of domestic animals (reviewed by
Gordon 1948; Levine 1963; Rogers and Sommerville 1963; Kates 1965;
Gibbs 1973), but Lankester and Anderson (1968) have been the only
investigators to attempt documenting the environmental resistance of
Parelaphostrongy lus free-living larvae. Their study, using an
extremely limited range of conditions, established that the first-
stage larvae of P. tenui s are somewhat resistant to desiccation and
freezing.
This study was initiated to expand upon the pioneering work
of Lankester and Anderson (1968) on the environmental resistance of
first-stage larvae of Parelaphostrongy lus. The objectives of this
study were threefold: 1) to determine the range of two major cli-
raatic factors, temperature and moisture, over which the first -stage
larvae of P„ odocoilei could survive; 2) to determine if those
first -stage larvae of P0 odocoilei which survived temperature or
moisture stress retained their infectivity to the intermediate
host, and; 3) to determine whether or not temperature or moisture
stress equally affected the survival and infectivity of two of the
species of Parelaphostrongy lus , P. odocoi lei and P. tenuis ♦
II. MATERIALS AND METHODS
A. Definitions
The following are definitions of terms used throughout the
text which have various meanings in the literature:
1. Surviving larva: one that is living following stor¬
age under specified conditions. The specific criterion used was
that a surviving larva must move on its own, or exhibit active
motion following prodding with a sharp probe. A larva was assumed
dead or moribund if decayed or if not exhibiting active motion
even after prodding. Survival of desiccated larvae was monitored
after they had been given a minimum of three hours in water to
revive. Survival of frozen larvae was monitored no sooner than
one hour after thawing.
2. Infective larva: one possessing the ability to enter
and develop further in the next host of the life cycle.
3. Sample: a group of 100 first-stage larvae (unless
otherwise specified) in a 60 mm Petri dish, used for testing of
survival .
4. Replicate: one in a group of samples prepared at the
same time from a common source of larvae, and used for the same
experimental treatment.
5. Varying: treatment conditions which are changed be¬
tween two levels on a regular basis.
6. Non -varying: treatment conditions maintained at a
constant level throughout an experimental period.
11
7.
Hydrated: a larva in water.
8. Desiccated: a larva in air.
B0 Source of Experimental Animals
First-stage larvae of Parelaphostrongy lus odocoilei were ob¬
tained from experimentally infected mule deer (Odocoi leus hemionus
hemionus) and black-tailed deer (Odocoileus hemionus columbianus)
housed at the University of Alberta Vivarium, Ellerslie, Alberta.
The majority of the larvae used in this study was from mule deer
infected with P. odocoi lei originating from a population of mule
deer in Jasper National Park, Alberta. A smaller number of larvae,
used in only a few experiments, was from a black-tailed deer infec¬
ted with P. odocoilei originating from a population of black¬
tailed deer on Vancouver Island, British Columbia.
First-stage larvae of Parelaphostrongy lus tenui s were obtained,
frozen on feces, from naturally infected white-tailed deer (Odocoi -
leus virginianus) from the Rachelwood Wildlife Research Preserve
in Pennsylvania. A white-tailed deer from Alberta was exposed to
larvae from this source, and P. tenuis was the only helminth recov¬
ered at necropsy (D.R. Anderson, pers. comm.). This deer, hous¬
ed in isolation at the University Vivarium, provided an additional
supply of larvae.
All first-stage larvae were left on fecal pellets until
required. P. odocoilei (Vancouver Island source) larvae were ob¬
tained fresh and were refrigerated (8°C) until required. Larvae of
P. odocoilei (Jasper source) and P. tenuis were available from
■
13
feces both fresh and previously frozen (-25°C).
In experiments involving a comparison of larvae from two
sources, it was ensured that both groups of larvae had similar
prior treatments, to minimize the influence this might have on
experimental outcomes. If fresh larvae were available, they were
used preferentially over previously frozen larvae. The sources
of larvae used in each experiment are itemized in Appendix I.
The snail Triodopsis multi lineata (Say) was used as the
experimental intermediate host. These snails have been maintained
in a laboratory colony at the University of Alberta for several
years. The original stock of the colony was from Nebraska.
C. Temperature and Humidity Control
Temperatures other than room temperature (18°C) were provided
by a variety of incubators, coolers, environmental chambers, and a
freezer. Use of some of these facilities for other purposes placed
minor constraints on the choice of temperatures for experiments.
Relative humidities were maintained at desired levels by the
use of saturated salt solutions (Winston and Bates 1960) in small
chambers (Fig. 4) . The chambers, constructed of plywood, had four
shelves (5 mm mesh, aery lie -coated galvanized metal) capable of
holding a total of 16 samples. Each chamber was enclosed in plastic
to reduce transfer of moisture through the walls. Forty to 50 ml
of appropriate saturated salt solution with precipitate, in a
50 mm diameter glass dish, were placed in the bottom of each cham¬
ber .
I
'
■
0
14
Figure 4. Diagrammatic representation of humidity control
chamber. Measurements are in centimeters.
FRONT SIDE
15
Temperatures were monitored several times throughout the
course of each experiment. The relative humidity in several cham¬
bers was measured in the early stage of the study to confirm the
effectiveness of the apparatus. An electronic probe (Brady-
Array Humidity Module, model PC 2000, from Thunder Scientific in
Albuquerque, N.M.) with a stated accuracy of + 2%, calibrated
shortly prior to measurement, was used to measure relative humid¬
ity (RH) . Chambers were allowed to equilibrate for two days befor
measurements of relative humidity were made. The results are
given in Table I. The discrepancy between expected and measured
relative humidities was small for the intermediate humidity values
but greater for the highest and lowest relative humidities. Since
this method of humidity control relies on a diffusion process, it
was assumed that the discrepancies at the high and low humidities
were due mainly to insufficient equilibration time for the chambers
In all subsequent experimentation the chambers were allowed to
equilibrate for a minimum of one week, and at cooler temperatures
for two weeks, prior to the introduction of samples.
Since saturated salt solutions regulate humidity up or down
towards the theoretical values as long as precipitate remains in
the solution, no further measurements were made. Salt solutions
were frequently checked, more water or salts added if necessary,
and the solutions stirred to prevent the formation of an unsatur¬
ated water layer at their surface.
17
Table I. Theoretical and measured values of percentage relative
humidity (X RH) in humidity control chambers. Measure¬
ments were made only at the temperatures indicated
below. Theoretical values are taken from Table II.
Discrepancy is the deviation of the measured value from
the theoretical value.
Solution
Temperature
(°C)
Theoretical
X RH
Measured
X RH
Discrepancy
(X RH)
h2o
45
100
94.0
- 6.0
KC1
45
81.0
82.5
+ 1.5
35
83.0
82.0
1
o
o
NaCl
35
75.5
77.0
+ 1.5
k2co3
48
40.0
48.0
+ 8»0
35
41.5
42.0
+ 0 o 5
25
43.0
50.0
+ 7.0
LiCl
35
11.5
22.0
+10.5
18
D. Preparation of Larvae for Experimentation
Feces containing larvae were wrapped in a double layer of
cheesecloth and placed overnight in a Baermann apparatus. Approx¬
imately 100 ml of fluid was drawn off and refrigerated (8°C) .
Larvae in this fluid were repeatedly washed in tap water at 8°C to
remove as much fecal debris as possible from the solution. Before
samples were prepared, larvae were allowed to reach room temperature,
and were given a final wash in room-temperature distilled water.
Washing procedure allowed larvae to sediment out by gravity before
drawing off the supernatant by vacuum.
Fluid containing an estimated 100 larvae was pipetted into
60-mm Petri dishes. The water level in all dishes was equilibrated
so that evaporation in all samples that were to be desiccated
would proceed similarly.
Control samples (i.e. those remaining hydrated) were covered
to prevent evaporation. In those to be desiccated, the water was
allowed to evaporate under ambient conditions until only a thin
film remained. At that time, experimental treatment of all
samples, controls included, was started.
E. Experimental Design
The experiments were designed to determine the effects of
several types of environmental factors on the first-stage larvae
of Parelaphostrongy lus . The outline of the experimental program
is given in Figure 5. Two criteria, survival and infectivity, were
Figure 5. Flow diagram outlining the organization of the
experimental program. Experiment numbers correspond
to those used in the text.
EXPERIMENTS INVOLVED
20
VO
r— |
CO
**
< — 1
VO
ID
ro
00
**
i — 1
1 — 1
ro
CTi
• — i
-
-
CN
■v
CN
ro
CO
i — 1
vO
* — i
**
*•
i — 1
i — 1
CN
i — 1
^0
i — 1
in
i — i
i — 1
• — i
O
i — I
21
used to assess the influence of experimental treatments on first-
stage larvae.
The experimental conditions were of two types: one in which
the physical conditions were maintained non-varying, the other in
which they were varied. Two types of non-varying conditions were
used. In one type the larvae were hydrated and stored at
temperatures above freezing, so that they remained in an active
state. In the other type the larvae were desiccated and/or frozen,
so that their response while in a hypobiotic state (sensu Keilin
1959) could be determined.
Varying conditions comprised either temperature fluctuations
with non-varying moisture conditions, or moisture-level
fluctuations with non-varying temperature. Temperature
fluctuations were of two types: one in which the larvae remained
in the active state, the other in which they were repeatedly cycled
between the active state and cryobiotic state (hypobiosis induced
by low temperature [Keilin 1959] ). Moisture fluctuations were
only of the type where larvae were cycled between the active state
and the anhydrobiotic state (hypobiosis induced by water
deficiency [Keilin 1959] ) .
A total of 18 experiments was performed to determine the
survival or infectivity of first-stage larvae of P. odocoilei and
P. tenui s . The following sections describe basic procedures for
each type of experiment along with variations on the basic
procedure that were employed in specific experiments. The
experiments were numbered, and the numbers correspond to those
22
used in Appendix I and in Figure 5.
1. Survival Following Non-varying Treatment
The survival of larvae of P. odocoilei and P. tenuis was
determined at several combinations of temperature and moisture.
Desiccated samples of larvae were stored at a variety of relative
humidities in the humidity control chambers which each contained
an appropriate saturated salt solution. Hydrated samples were
also stored in the chambers, to control for the effects of
possible contaminants in the chambers. Those chambers containing
hydrated samples had relative humidities maintained near 100 percent
with distilled water; this prevented desiccation of the hydrated
samples with their covers removed. Temperature was controlled by
placing the humidity control chambers in either coolers or
incubators set to desired temperatures.
Where larvae from two sources were being compared, each chamber
contained samples from both sources. At various intervals a
number of samples was removed to monitor survival. These samples
were not returned to the experiment following monitoring. If
larvae from two sources were involved, survival of those in the same
chamber was monitored simultaneously.
The relative humidities maintained by saturated salt solutions
vary slightly with temperature. For the solutions used in these
experiments, the relative humidities reported in the literature are
given in Table II for a range of temperatures from 2 to 50°C.
Further reference to the humidities maintained by each of these
Table II. Percentage relative humidity (% RH) over saturated salt solutions and water. Top rows are
23
cc
<±
d
a)
•H
d
O
a
o
u
a)
u
d
CO
d
a
o
■U
■u
o
O
vO
(T
W
<U
4J
d
T3
d
d
d
o
■u
CO
d
•iH
s
a
o
!-i
id
CO
a)
3
r-H
d
>
O
o
n
d
•
•
•
•
*H
r-H
o
o
r-H
<1-
00
(1)
X
o
00
X
1
1
i
1
o
d
o
o
n
o
r-H
d
5^
•
•
•
•
cd
CO
o
00
r“H
n
n-
00
o
n
n
n
O
©
1
•
1
•
•
•
•
1
o
LO
t— 1
1
1
1
o
o
1
o
r-H
00
00
r-H
o
o
o
u~l
•
1
1
1
•
i
•
1
1
1
r- H
1
1
1
n
i
r-H
1
1
1
r—H
CO
„v
o
o
o
o
n
o
n
O
•
1
•
•
•
o
•
•
1
o
r— 1
1
o
o
n
n
CM
CM
1
o
r— 1
<1-
00
00
r-H
LO
n
o
LO
•
1
1
1
t
i
•
1
1
1
co
r—H
1
1
1
n
i
CO
1
1
1
r—H
r-~
00
in
o
n
n
n
n
00
o
•
•
•
1
•
•
•
•
1
o
u
CO
r-H
CO
CO
1
n
n
1
o
o
r-H
r-H
r-
CO
CO
r-H
V
0)
d
o
o
o
o
n
o
o
o
3
m
1
o
4-J
CM
Csl
CO
CO
CO
n
co
n
1
o
d
r-H
r-H
00
00
r-H
d
<u
a
a
in
o
o
o
o
o
o
n
0)
o
1
o
H
CN
n
cO
I^-
n
v£>
1
o
r-H
t— H
00
00
r-H
Ja
o
o
o
o
o
n
n
n
•
. •
•
•
•
•
•
•
1
o
r— 1
CO
n
cO
co
cO
1
o
r-H
r-H
'd
r^.
00
00
r-H
’i'*
in
o
o
n
n
o
n
c
•
1
•
•
•
•
•
•
1
o
i — 1
CO
1
co
cO
00
1
o
r—H
•4*
00
00
r-H
o
o
n
•
1
1
i
•
1
1
1
1
r
1
1
i
n
1
1
1
1
i
r—H
n
o
o
o
o
O
o
o
nj
1
o
cO
o
n
n
00
00
1
o
r—H
r-H
n
CO
00
r—H
CO
r-H
o
r-H
u
u
o
< — 1
o
C\]
d
o
CM
X
X
X
* Determination was made at a temperature slightly different from the column head
24
solutions were expressed as a single percentage relative humidity
(% RH) , with an implied range of + 5% RH. For K^COo, NaCl, and
KCl, the values were 45, 75, and 85% RH, respectively. The values
of H2O and LiCl were 95 and 20% RH, respectively, with allowance
for the difficulty of maintaining very high or very low humidities.
Given the implied ranges of these values, all five solutions
provided a gradient from high to low relative humidity, with no
overlap of relative humidity regardless of temperature.
a. Experiment Number 1
This experiment, in conjunction with the next one, was
designed to determine the survival of P. odocoi lei stored under a
variety of moisture conditions, at temperatures above freezing.
In this experiment, desiccated samples at 20, 45, 75, 85, or
95% RH, and hydrated samples, were stored at 5, 36, or 48°C.
Experimentation at the combination of 20% RH and 5°C was not
performed, due to the inefficiency of humidity control by LiCl
at this low temperature (O'Brien 1948). The survival of larvae was
monitored after up to eight time periods in each condition. In
most cases, four replicates were examined during monitoring.
b. Experiment Number 2
This experiment was similar in design to Experiment Number 1
(Exp't 1), except that intermediate temperatures of storage (14 and
26°C) were used. The conditions tested comprised hydrated samples,
and desiccated samples at 20, 45, 75, or 95% RH.
25
c. Experiment Number 3
To determine if larvae of the same species but with dif¬
ferent sources of origin had similar survival, the following experi¬
ment was performed. Samples of Jasper-source and Vancouver Island-
source larvae of P. odocoilei were placed in a variety of moisture
conditions at 25°C. These were desiccated at 45, 75, or
95% RH, and hydrated. A pair of samples from each source was placed
on each shelf in the humidity control chambers, giving a total of
six samples per moisture treatment per source of larvae. The sur¬
vival of larvae from all six samples of each source was monitored
after 5 days at 95% RH, 7 days at 75% RH, 19 days at 45% RH, and
12 days hydrated. These times were chosen to allow for an inter¬
mediate level of mortality to occur at each moisture condition,
thereby facilitating comparison of survival between sources.
d. Experiment Number 4
The next two experiments were designed to determine the survi¬
val of P. odocoilei while frozen. The purpose of this experiment
was twofold; to determine the effect of desiccation prior to freez¬
ing on survival while frozen, and again to compare the survival of
P. odocoilei larvae from the two sources (Jasper and Vancouver
Island) , this time following freezing.
Six hydrated and six desiccated (at ambient = 35% RH) samples
from each source were frozen at -25°C. Survival was monitored
after 100, 190, and 280 days. Between one and three samples per
source was monitored at each time period.
1
e.
Experiment Number 5
The long-term survival of P. odocoilei while frozen on feces
was estimated in this experiment. Platt (1978) determined larval
output per gram of host feces (LPG) from experimentally infected
mule deer, using the Baermann technique on subsamples of fecal pel¬
let groups. He then placed the remainders of those pellet groups
in a freezer at -25°C. For this experiment, those remainders were
thawed, and LPG were determined by the same method that Platt used.
The duration of freezing was 32 months for the pellet groups
from one deer (n=4) , and 34 months for those from a second deer
(n=4) . Since the Baermann technique tends to result in recovery
of live larvae only, before-and-af ter LPG could be used to estimate
survival of larvae while frozen,
f. Experiment Number 6
This experiment, similar in design to Exp ' t 3, was to determine
if larvae of the two species, P. odocoilei and P. tenui s , had
similar survival following storage under non-varying conditions at
a temperature above freezing. Larvae of each species were placed in
three moisture conditions at 30°C . They comprised hydrated, and
desiccated at 45 or 95% RH. Survival of larvae in each moisture
condition was monitored after up to four time periods. Four repli¬
cates per species and moisture condition were monitored after each
time period.
2. Survival Following Varying Temperature Treatment
To determine the effect of repeated temperature changes, such
as those that might occur on a daily basis in natural conditions,
on larvae of P„ odocoi lei and P. tenui s , hydrated larvae were rep¬
eatedly moved between two temperature levels a number of times.
a. Experiment Number 7
This experiment examined the effects of repeated changes
in temperature, during which the larvae remained in the active
state. Three hydrated samples of 200 larvae of P. odocoi lei
were repeatedly moved between 8 and 37°C, for a total of 16 complete
cycles of temperature over a four day period. To control for the
effects of high temperature per se on survival, a control group of
three hydrated samples (200 larvae each) was placed non -varying at
37°C for a similar timespan (89 hours) to that spent by larvae in
the experimental samples at the high temperature of the cycle (86
hours at 37°C) . If survival in the experimental groups was lower
than that observed in the control group, then the excess mortality
would be a result of the change of temperature, and not the
lethal action of the high temperature alone.
b. Experiment Number 8
The next two experiments involved repeatedly changing the tem¬
perature of samples between above -freezing and below-freezing levels,
so larvae were repeatedly cycled between the active state and the
cryobiotic state.
This experiment was of a preliminary nature, to see if repeated
freezing would reduce survival of the larvae. Two hydrated
samples were repeatedly frozen at -25°C and thawed to +14°C, for a
total of 13 cycles over a four -day period. Following the final
28
thawing, survival of larvae was monitored,
c. Experiment Number 9
This experiment was designed to determine whether or not
survival following repeated freezing differed between P. odocoi lei
and P_. tenuis. Twelve hydrated samples of each species were
subdivided into four groups; two controls and two experimental s
(Fig. 6). The non-frozen control remained at +14°C. the frozen
control was placed at -25°C for the duration of the experiment,
11 complete days, when the samples were thawed and survival
monitored. The two experimental groups were repeatedly forzen and
thawed, 10 and 20 times, respectively. Survival was monitored after
the final thawing.
3. Survival Following Varying Moisture Treatment
As with temperature, moisture conditions experienced by
larvae may change on a daily or other basis. Five experiments
were performed to determine the effects of repeated desiccation on
larvae of P. odocoi lei or P_. tenui s . A generalization of the
experimental design is presented schematically in Figure 7. All
experiments involved varying the moisture conditions between
hydration and desiccation, so that larvae were repeatedly moved
between the active, hydrated state, and the anhydrobiotic state.
Several hydrated samples were divided into control and
experimental groups. There were two control groups in each
experiment. The samples in the hydrated control were covered
29
Figure 6. Schematic representation of the design of Experiment
Number 9 (Exp't 9) showing temperatures of samples in
each of the four groups over time. "S" indicates
the time when survival of larvae was monitored.
30
CO
+
CO-L
CO
CO
10
CN
+
«n
<N
I
m
CN
0o d IAI 3 X
CO
to
o
CC
I-
z
o
o
LU
N
o
CC
LL
I
LU
N
o
CC
LL
< CO
I- LU
Z _l
UJ O
5 >
- O
CC
LU O
Ql t-
X
LU
CO
LU
_J
o
>
o
o
CN
DAY OF EXPERIMENT
31
Figure 7. Schematic representation of the basic design of all
varying-moisture experiments (Exp'ts 10-14).
Moisture states of samples in each group over time
are shown (H= hydrated; D= desiccated). "S" indicates
time when survival of larvae was monitored.
CONTROL
32
3 1 VIS 3uniSIOIfl
(/>
o
-1
LU
<
o
1-
h-
LU
<
z
h-
o
LU
<
o
5
cc
—
Q
CO
QC
>
LU
LU
X
o
0.
X
LU
I-
<
o
o
c n
LU
Q
CO
If)
DAY OF EXPERIMENT
33
to prevent evaporation of the water (Fig. 7) . The
samples in the desiccated control, were allowed to desiccate
and remain that way for the duration of the experiment. These
samples were rehydrated prior to survival monitoring (Fig. 7) . The
two control groups served to account for the effects of continuous
hydration or desiccation on the larvae. The samples in the experi¬
mental groups were repeatedly desiccated, with a small amount of
distilled water added at various intervals to rehydrate the larvae.
The example of experimental design (Fig. 7) has three experimental
groups, receiving 3, 5, or 10 desiccations.
All experiments were conducted at ambient relative humidity
(30-40% RH) . Evaporation of the water in the samples occurred with¬
in 24 hours at room temperature (18°C) or 48 hours when refriger¬
ated (8°C) . All groups, control and experimental, were prepared at
the same time, and survival monitored on the same days. The number
of replicates in each group varied from one to four.
a. Experiment Number 10
Larvae of P. odocoi lei were divided into two control groups and
one experimental group, which received five desiccations. The
experiment was run over eight days, at room temperature.
b. Experiment Number 11
Larvae of P. odocoi lei were divided into two control and three
experimental groups. The experimental groups received 3, 5, or 10
desiccations. The experiment was carried out at room temperature
over a nine day period.
34
c. Experiment Number 12
This experiment was identical in design to Exp ’ t 11, but was
carried out at a lower temperature (8°C) , and over a longer time
period (22 days) .
d. Experiment Number 13
Larvae of P„ odocoi lei and P. tenui s were each divided into
two control and three experimental groups. Five, six, or nine des¬
iccations were administered to the experimental groups over
15 days, at room temperature.
e. Experiment Number 14
This experiment was similar to Exp ' t 13, except that the
experimental groups received three, six, or nine desiccations, over
12 days.
4. Infectivity Trials
Infectivity of first-stage larvae was determined by exposing a
known number of surviving larvae to the snail Tr iodopsi s multilineata.
After a period of one month, larvae were recovered from the snails
by artificial pepsin digestion (0o6 g pepsin powder, 0o7 ml HCl per
100 ml distilled water; incubated at 37°C) of the snail tissues.
Larvae were exposed to snails in 10 -cm diameter glass dishes
with two discs of filter paper lining the bottom,, A suspension of
larvae in water, with numbers of live larvae estimated by standard
survival monitoring on a subsample of the suspension, was pipetted
onto the filter paper. A group of snails was then placed on the
filter paper and the dish covered. The snails were allowed to crawl
35
on the filter paper for several hours. At intervals of about one-
half hour, any snails on the sides or cover of the dish were placed
back on the filter paper.
In Exp ' t 15, numbers of larvae and snails varied between expo¬
sure dishes, since this experiment was designed to determine the
influence of different density conditions of exposure on final lar¬
val recoveries. In Exp'ts 16-18 the same number of larvae and
snails was used in all dishes; the variable here was the prior
treatment of the larvae. Prior to being exposed to snails, larvae
were allowed to revive, if necessary, from the treatments they were
administered. For example, if the experimental treatment had invol¬
ved desiccation of the larvae, they would be placed in water for
several hours to revive,
a. Experiment Number 15
To determine the effect of exposure conditions on subsequent
recovery of second- and third-stage larvae, P. odocoilei was exposed
to snails under four different densities of snails and first -stage
larvae: 1) many larvae/many snails, 2) many larvae/few snails,
3) few larvae/many snails, and 4) few larvae/few snails. "Many"
and "few" larvae were 4710 and 1570 total, or 60 and 20 larvae per
cm^ on the filter paper, respectively. For snails, "many" and "few"
2
were 15 and 5 per dish, or 0.192 and 0.064 snails per cm on the
filter paper, respectively. Larvae and snails were chosen at random
from common sources prior to allocation. Exposure was for nine
hours, and digestion of the snails was after four weeks.
36
b. Experiment Number 16
This was a preliminary experiment to determine the influence
of temperature and moisture stress on infectivity of first-stage
larvae. A large quantity of P. odocoilei was washed repeatedly in
water at 8°C, and subdivided into four groups. The summarization
of experimental design (Fig. 8) illustrates the sequence of events
for these four groups regarding: changes in moisture state of the
larvae between hydrated and desiccated, changes in temperatures,
and times of exposure of the surviving larvae to snails.
One group of larvae was designated as initial control, and was
exposed to snails at the start of the experiment to determine the
initial infectivity of the larvae.
Two of the remaining groups were designated as experimental
groups. One was allowed to desiccate and was then placed at 95% RH
and 26°C for six days. This was the high -humidity experimental
group. The second group, a low-humidity experimental, was allowed
to desiccate, and was then placed at 45% RH and 26°C for 14 days.
Following desiccation, the larvae were rehydrated and placed over¬
night at 8°C to revive. They were then exposed to snails. A shor¬
ter treatment time was chosen at the high humidity based on other
experimental results which indicated that few or no larvae would
survive 95% RH and 26°C for 14 days.
The fourth group of larvae was designated as final control,
and was stored in water at 8°C for the duration of the experiment.
It was designed to account for any effects that passage of time
since the start of the experiment would have on infectivity of the
Figure 8. Schematic representation of the design of Exp ' t 16.
Moisture states of samples in each group over time
are shown (H= hydrated; D= desiccated) . Temperature
of samples (degrees C) is shown above each graph.
"E" indicates time when larvae were exposed to snails.
CONTROL .. I- 8~l
38
1 1 1
3ivis 3amsioi/v
DAY OF EXPERIMENT
39
original stock of larvae. Any reduction in infectivity of the
experimental groups below that of the final control would be due
to experimental treatment, and not loss of infectivity over time.
The exposure of larvae from each of the four groups was as
follows. Two units of 10 000 surviving larvae were selected from
each group, and each unit was exposed to 10 snails for 6.5 hours.
Snails were digested four weeks after exposure to the first-stage
larvae .
c. Experiment Number 17
This experiment was designed to determine the influence of
moisture conditions, temperature, and length of treatment on the
infectivity of first-stage larvae of P. odocoi lei . A single stock
of larvae was subdivided into 12 groups, each to receive a differ¬
ent treatment. Four groups of larvae received one of three mois¬
ture treatments: hydrated, desiccated at 45% RH, or desiccated at
75% RH. Within each moisture condition two groups were treated at
20°C, two at 26°C. At each of these six temperature-moisture com¬
binations, one group was treated for 2 days, the other for 10 days.
The choice of conditions was such that even under the harshest of
them (95% RH at 26°C for 10 days) there would be sufficient numbers
of surviving larvae to expose to snails.
Following treatment, 10 000 surviving larvae from each group
were exposed to 10 snails for six hours. Snails were digested five
weeks post -exposure. For logistic reasons, all treatments were not
started simultaneously, but were staggered so that exposure took
place in three~group units for each of four successive weeks.
40
d. Experiment Number 18
The objectives of this experiment were twofold: first, to
examine the effects of freezing on infectivity of first-stage
larvae of P_. odocoilei and P. tenuis; second, to determine whether
infectivity of the two species differed following freezing.
Two sources of larvae of each species were used: one fresh on
feces; the other collected from the same animals one month previ¬
ously, and stored on the feces since then at -25°C. All fecal
samples, fresh or frozen, were brought to room temperature on the
same day, and larvae recovered from them in a Baermann apparatus.
The recovered larvae were washed five times over the next week; their
temperature during this period was kept at 8°C.
The exposure of larvae from each of the four groups was as
follows. Two units of 5000 surviving larvae were selected from
each group, and each unit exposed to five snails for 4 hours. At
28 days post -exposure, the snails exposed to one unit of larvae
from each of the four groups were digested. The remaining snails
were digested 38 days post -exposure.
F. Data Analysis
Data were analyzed statistically using procedures outlined
by Snedecor and Cochran (1967) and Sokal and Rohlf (1969). Regres¬
sion analyses were performed on an Amdahl 470 computer using APL,
with programs obtained from the public library of the University of
Alberta Computing Center.
Pairs of means were compared using t-tests. Where critical
41
values of t had to be calculated, these are given along with
their probability values. Groups of means were compared by
analysis of variance (anova) . Heteroscedasticity was reduced to
within acceptable limits by appropriate transformations. Angular
transformation was used in the case of percentage data, while
logarithmic transformation was used for count data. Reduction of
heteroscedasticity was confirmed using either the F -max test, or
Bartlett's test. Departures from normality were tested for using
the Kolgomorov -Smirnov test.
Where overall statistical analysis of a given set of data
could not be done due to violation of necessary assumptions, butwhere
analyses on subsets of the data could be validly performed, the
technique of combining probability values was used to test the
hypothesis (Sokal and Rohlf 1969) . The proviso of this test was
that the separate tests, from which probability values were obtained,
all tested the same scientific, though not necessarily statistical,
hypothesi s .
Means, standard errors, and confidence limits on all percent¬
age data were calculated following angular transformation, but are
reported here following conversion back to the percentage scale.
The same information on count data was calculated, and is reported,
in the original scale.
Any departure from the procedures outlined here will be noted
when they occur.
III. RESULTS
A. Survival Under Non-varying Conditions
The survival of first -stage larvae of Parelaphostrongy lus
odocoi lei under non-varying conditions at temperatures above
freezing (Exp'ts 1,2) is shown in Figure 9 (a more complete
presentation of the data, including confidence limits for the mean
percentages, is in Appendix II). Generally, survival declined in
a linear manner.
The maximum length of survival could only be accurately
determined by direct inspection of the data in a few cases, such
as for the samples stored at 36 or 48°C. In other cases, either
the time period between observations was too great to determine
when the last larvae died (e.g. 75% RH at 14°C) , or insufficient
numbers of samples were available to monitor survival for the
entire lifespan of all the larvae (e.g. 45% RH at 5°C) . In the
latter two cases maximum length of survival was estimated by
regression analysis. Linear regression was used to estimate the
2
x- intercept (maximum days survival) , unless a significant x
component was detected, in which case polynomial regression was
used. The maximum survival times of the larvae in all conditions,
as determined by the appropriate one of the above methods, are
given in Table III.
At all moisture conditions survival was inversely related to
temperature of storage. Survival of desiccated larvae was
inversely related to the relative humidity of storage, regardless
42
Figure 9. Survival of first -stage larvae of P. odocoi lei at
various temperature and moisture conditions
(Exp ' t s 1,2).
44
957.RH
85% RH
757.RH
45% RH
20% RH
IN
WATER
MONTHS
DAYS
Table III. Maximum days survival of first -stage larvae of Parelaphostrongy lus odocoilei under
45
CN
to
U~|
m Lo cn
• • •
-<1- i — - — •
o
m
O
o
o
•
•
•
•
•
•
CN
CN
CN
oo
r— l
cn
00 OO r— I
MO MO LT)
LO
i r^
CN
r**
i
cn
rH
CNJ
cn
co
00
o
M0
ON
O'
00
o
r~H
r— 1
cn
Mt
30
33
33
33
33
o3
03
03
03
B-S
S'?
B'-S
B-S
B-S
in
m
in
in
O
ON
00
CM
"d
<u
4-1
ctf
o
o
•H
c n
0)
Q
Hydrated 413 269
46
of temperature. The survival of larvae desiccated at the lower
relative humidities was similar to that of hydrated larvae when at
low temperatures, but was lengthier than hydrated larvae when at
high temperatures.
Thus Po odocoilei can survive at high temperatures for up to
one week, and at low temperatures from six months to over one year,
depending on moisture conditions.
The survival of P. odocoilei originating from Vancouver
Island did not differ from survival of P. odocoilei originating
from Jasper, either at above-freezing temperatures (Exp't 3; Fig. 10)
or below -freezing temperatures (Exp't 4; Fig. 11). When survival
of the larvae was correlated with source of larvae, shelf position
in the humidity control chamber, and moisture treatment above
freezing (Exp't 3) by anova, there was no significant effect of shelf
position or larval source (Table IV). However, the different
moisture conditions did have a significant effect on survival of
the larvae. When mean survival of P. odocoilei larvae from the
two sources was correlated with source of larvae, moisture condition
prior to freezing, and length of freezing (Exp't 4) by anova, again
there was no significant difference of survival between sources of
larvae. However, there was a significant decrease in survival
over time, dependent upon the moisture state of the larvae prior
to freezing, as indicated by the significant interaction between
moisture and time (Table V) .
First-stage larvae of P. odocoilei can withstand freezing
on feces for much longer than the 32 and 34 month periods used
47
Figure 10. Survival of first-stage larvae of P.
two sources, at 25°C and various moi
(Exp't 3). Bars represent mean + SE
a. Hydrated for 12 days.
b. Desiccated for 19 days at 45% RH0
c. Desiccated for 7 days at 75% RH.
d. Desiccated for 5 days at 95% RH.
odocoilei from
ture conditions
48
<u
>
w. 3^
0) o c
® a o *
w c oj
to (D —
r
o
o
1
o
Q
O
<
“IVAIAanS 1 N 3 0 d 3 d
TREATMENT
Figure 11. Mean percent survival of first -stage larvae of
P. odocoilei from two sources, following freezing
(Exp't 4). Open circles and dashed lines represent
Jasper source larvae; closed circles and solid lines
represent Vancouver Island source. Group "H" samples
were frozen while hydrated; group "D" were frozen
while desiccated.
MEAN
PERCENT SURVIVAL
50
0
MONTHS
51
4-1
o
g
u
u
3
o
G
03
u
G
So
3
cO
4-1
o
CO
o
a
in
CM
•
fa
G
00
fa
fa
o
03 03 o
c c
o
v
o
o
•
H
v
cO
in
•
o>
ON
03 03 03 03
g c g c
o
o
•
r-H
V
ON
o
o
o
•
r-H
V
o
o
•
r-H
V
•H
4J
G
-
i— l
fa
•H
X
o
fa
o
* — '
o
"G
4-1
O
C
a)
•
£
fa
4-1
cO
4-1
<U
O
g
4-1
G
CO
TO
>
£
g
G
cO
I— 1
r\
g
G
0)
£30
fa
cO
£
4-1
CO
03
X
i
o
4J
03
t— H
$-1
o
•H
g
fa
4-1
G
fa
o
O
o
tfa
So
cO
4-J
>
*H
*i-l
T5
>
•H 1 fa
G
£ S>
3
G ^
03
rC C
•H
g
C M
jG
•H O
4-1
G
C G
g
O
o
•H
fa
4J fa
•i— l
G
03 "G
r— )
O G
fa
fa £
cO
G
4-J
- O
G fa
cO
G co
>
> C
O
G G
g
G G
<5
r— 1 4-J
G
r-H
^3
G
H
a)
m
o
CO
00
o
00
ON
o
u
•
•
•
•
•
•
v
C
ctf
co
03
CM
o
fa
r-H
LO
co
G
G
g
D
cr
C/0
CO
r-H
00
CO
fa
CO
CO
CM
r-H
co
CO
OO
r-H
03
00
o
oo
oo
fa
<U
•
•
•
•
•
•
•
•
Jh
co
cc
fa
r-H
co
o
LO
r-H
ctf
CO
CO
00
fa
o
CO
cr
<Tn
D
OO
fa
r-H
cr
o
CO
CM
fa
a
cm co
CM CO v£)
40 <f
CM
fa
c
a)
£
+j
G
a)
03
i— •
EH
G
fa
o
O
C
•H
X
fa
fa
G
fa
4-J
G
G
£
a
G
c
o
O
G
fa
G
O
o
G
•G
E
G
G
•H
•H
G
fa
fa
G
G
fa
4-1
G
•f-i
G
G
fa
•H
G
4-1
03
G
H
G
03
fa
c
O
G
fa
o
G
fa
fa
Eh
X
fa
G
co
4-J
G
>
4-J
a
G
G
X
X
C
G
X
G
o
G
G
o
"3
fa
G
G
•H
£
"O
G
G
•H
G
G
O
fa
O
4-J
4-J
G
O
G
4J
O
O
fa
G
•iH
G
O
G
G
•H
l
G
G
fa
G
CO
G
l
G
G
CO
T3
G
O
O
O
G
fa
O
O
O
G
O
G
G
C
CO
cu
H
03
C/3
CO
O
CO
O
G
•H
G
a
G
O
fa
G
G
CO
g
fa
CO
fa
vO
Total 47 21815.
Table V. Anova table for the survival of first -stage larvae of P. odocoi lei at -25°C, by
52
CO
4-J
cO
Q
to
X
to
00
e
•H
N
0)
(0
U
<44
<4-1
o
to
4-1
O0
d
<0
"d
d
<0
00
d
to
N
CO
<0
54
<4-4
<4-1
o
c
o
to
TO
d
o
o
<0
54
3
co
to
o
e
<0
CO
>
54
cO
<4-4
O
CO
o
!-4
3
O
co
d
co
o
54
cO
>o
~0
<0
E
5-4
O
<4-4
CO
C
<0
54
4-J
<0
}-l
CO
£
i — 1
o
<n
<n
co
o
o
CO
co
o
to
d
•
•
d
d
•
o
o
o
V
V
V
00
o
cO
o
o
co
o
r-H 1
<40
o
o
ON
EtJ
•
•
•
•
•
•
CM
r-'
r— 1
r-H
CO
On
O’
V
V
CM
r^
r— 1
<0
LO
r-4
r—l
oo
CM
00
54
•
•
•
•
•
•
•
c
CO
<n
U")
o
o
CM
CM
cO
3
o
CO
00
(0
cr
t-H
r— 4
a
CO
IT)
CO
<4-4
CO
O'*
LO
CM
r-H
<n
r^-
p— H
o
54
•
•
•
•
•
•
•
•
cO
<4")
O
o
r-H
<40
CM
s
3
o
Or
'sO
<40
3
o'
r— 1
CM
t-H
<40
co
co
<40
<40
to
r—l
r-H
CM
r-H
CM
CM
CM
t-H
Q
t— H
54
O
54
-d
54
44
to
oo
•w'
d
CO
d
to
d
o
o
•H
X
•H
44
4-J
<0
rd
O
<0
C
O
54
44
03
54
O
co
3
to
00
54
3
•r4
54
44
44
d
(0
44
4-J
(0
co
00
<0
44
CO
CO
4-J
to
d
to
d
•pH
•H
d
O
<0
to
o
U
to
a
to
X
a
cO
co
54
>
4J
<0
54
X
X
CO
(0
X
o
54
<0
54
"d
<4-1
<0
CO
3
to
"d
CO
<0
3
54
<0
O
<4-1
o
4-J
4-J
54
CJ
o
44
O
o
<4-1
5-4
CO
00
O
54
54
co
1
54
CO
to
3
•r-4
d
1
3
3
to
T3
3
o
O
0
<0
4-1
O
O
O
d
O
r-H
54
d
CO
a
to
CO
CO
CO
a
o
CO
ctf
3
•rH
54
o
44
O
ctf
to
<0
o
CO
a
to
CO
H
53
in Exp 1 1 5. Survival after that time period was still close to
75 percent (Table VI). Median, rather than mean, survival was
used due to the asymmetric distribution of survival values from
the four samples from each deer. The survival estimate of 75
percent for survival on feces is only slightly less than the
86 percent which is predicted from the data of Exp 1 1 4
(Y= 98.8 - 0.01319X) , for survival of Jasper -source P. odocoi lei
which were frozen while hydrated.
The survival of first -stage larvae of the two species,
P. odocoi lei and Parelaphostr ongy lus tenuis, under non -varying
conditions at a temperature above freezing (Exp't 6) is shown in
Figure 12. Survival could not be analyzed by anova as was done for
the results of the similarly designed Exp't 3 (using P. odocoi lei
from two different sources) due to overall heteroscedasticity which
could not be reduced to an acceptable level. However, t- tests on
the difference in survival between species could be performed for
the individual treatment groups. There was a significant difference
between species in two cases: Class "B" (t=4.920, df=6, p<0.01),
and "D" (t=3.506, df=6, p<0.05). Probability values were
suggestively low in several other classes (Class "A", t=2.057,
df =2 , p<0 . 20; "E", t=2.287, df=6, p<0.10; and "H", t=2.263, df=6,
p<0 . 10) .
The probability values from the t- tests on the six treatment
groups that were not significantly different were combined, the
null hypothesis being that survival of P. odocoilei = P. tenuis
2
survival. The resulting statistic, -223 InP (distributed as X )
I
'
54
40
4-1
cm
x
w
a)
E
G
CD
a
o -G
o -u
in
CM 4M
' O
G
>
•H
>
G
3
CD
4-1
G
a;
o
G
a)
P-i
oo
00
OS
MO
00
•vf-
CO
00
r-
SO
r-
13
00 00 r-M CO
r — vxo r^-
■u
CD
cO
a)
(—i
CD
CM
■u
E
3
G
t-M
CD
r4
-Q
3
3
cm
CD
r-H
E
cO
o
a
G
3
4-4
4-4
3
c
G
o
G
G
co
0)
C
N
o
o
•H
4J
4M
G
C
*\
•tM
•rH
E
3
g
r-H
a )
*H
4-J
o
3
o
T3
o
G)
o
O
PM
•
hG
PM
O
PM
h4
CO
G
•H
pH
Os
as
CM
o
OO
40
o
m
as
sO
sO
as
CM
in
00
CO
<t
as
i— H
as
o
CO
CO
CO
t^.
40
r-»
<!■
c
3
N
O
CD
s*
Y*
G
-G
in
40
o
o
CN
40
m
in
40
LO
4M
4-J
•
•
•
•
•
•
•
•
•
•
3
E
•H
H
c
CM
CM
CM
CM
CN
o
E
CO
CO
CO
CO
OO
CO
OO
OO
OO
OO
o
Q-*
H-J
a3
*H
4-J
CM
CO
40
o
r-H
40
as
r-H
CO
r->
as
CM
rH
r-H
in
o
OJ
o
CN
o
00
as
4M
E
G
O
G
M
G
3
00
G
1
>
G
G
3
T)
G
CM
3
rH
1
4-1
3
a
/•— N
3
G
3
G
00
>
r-H
G
G
G
rH
3
4-4
G
o
X
CO
f-M
a
i
sO
sD
sO
40
40
LO
in
1
-G
r-
r-
r-»
r^.
1 —
4-4
i (
CD
4-4
i
i
i
i
i
1
i
i
co
G
3
c
M
M
M
H
H
M
M
M
G
G
3
o
M
l-M
H
M
H
H
M
MM
*G
•H
3
s
1
1
1
1
X!
X
X
X
4M
4M
•
4M
1
<t
sO
SO
a>
i
1
1
1
CM
X
r-M
r-H
CM
CO
vt
as
4M
t3
3
3
G
rM
r-H
rH
r-H
o
G
O
4-1
a
G
G
G
rH
00
Q
G
r— M
>
G
4-4
•H
•H
3
>
4M
r-H
i — 1
CO
G
•r4
rH
3
c
3
r-H
•
o
CO
M
CM
G
O
o
E
s
X
•H
•
c
G
G
M
G
3
3
>
3
3
3
Q
Q
3
O
r-H
G
3
3
-Q
3
1 - 1
r-H
G
O
3
3
EH
CO
s
£
JL*
4"*
median
55
Figure 12. Survival of first-stage larvae of P. odocoilei (P.o.)
and P. tenuis (P.t.) at 30°C and various moisture
conditions (Exp't 6). Bars represent mean + SE.
a. Hydrated for 20 days.
b. Desiccated for 3 days at 45% RH.
c. Desiccated for 9 days at 45% RH.
d. Desiccated for 20 days at 45% RH.
e. Desiccated for 2 days at 95% RH.
f. Desiccated for 4 days at 95% RH.
g. Desiccated for 6 days at 95% RH.
h. Desiccated for 8 days at 95% RH.
56
IVAIAdns !N30d3d
TREATMENT
57
was 20.12, df-12, p<0.05. Of the two species in this experiment,
P. tenuis generally had higher survival at low relative humidity
(Fig. 12b-d), while P. odocoi lei generally had higher survival at
high relative humidity (Fig. 12e-h) .
Three experiments in this section (Exp'ts 3,4,6) compared the
survival of larvae of different types following storage under non¬
varying conditions. In two of them (Exp'ts 3,4) the different
types of larvae used were of the same species, but from different
geographical regions, and the null hypothesis that their survival
under non-varying conditions is equal was accepted. In the other
(Exp't 6) the different types of larvae were of different species,
and the null hypothesis that their survival is equal was rejected.
B. Survival Under Varying Temperature Conditions
The survival of first-stage larvae of P_. odocoi lei was not
affected by the repeated cycling of temperature between 8 and
37°C (Exp't 7). Mean percent survival with 95% confidence limits
was 90 (88- 93) after 16 complete cycles between the two
temperatures, compared with 87 (74-97) in the control. The control
had spent almost as much total time at 37°C as the experimental
group but was not cycled. The difference in survival between
control and experimental groups was not significant (t=l.ll, df=4,
p<0 .40) .
The cycling of larvae of P. odocoi lei between above- and
below- freezing temperatures did reduce their survival. In the
preliminary experiment (Exp't 8), mean percent survival was reduced
58
to 82 percent (95% confidence limits: 45- 93% survival) after
13 complete freeze-thaw cycles.
The reduction in survival following repeated cycles of
freezing was confirmed for ]?. odocoi lei , and also observed for
tenui s , in Exp ' t 9 (Fig. 13). Survival of both species
decreased in relation to the number of cycles. There was no
significant difference in survival between the non-frozen controls
of the two species (t=1.027, df=4, p<0.40). The survival of the
three groups which underwent freezing (the control a single time,
and the two experimental groups 10 or 20 times) was correlated
with species and number of freezings by anova (Table VII) . The
effects of both species and number of freezings were significant,
with no interaction. P. tenuis not only had lower survival than
P. odocoi lei in the two experimental groups (10 freezings, t=2.979,
df=4, p<0.05; 20 freezings, t=5.560, df=4, p^O.Ol) , but also in
the frozen control, which had just a single freezing (t=3.463,
df =4, p<0 . 05) .
In summary, temperature variation not involving a change of
state of the hydrated first-stage larvae of P. odocoi lei did
not affect their survival. However, when the temperature
variations involved a change of state for the larvae (from active
to cryobiotic) , survival of both P. odocoi lei and P. tenuis was
reduced in relation to the number of times the change occurred.
P. tenui s was slightly more susceptible than P. odocoi lei to
repeated freezing.
59
Figure 13. Survival of first-stage larvae of P. odocoilei (P.o.)
and P. tenuis (P.t.) following repeated freezing
(Exp't 9). Bars represent mean + SE.
60
“IVAI Ad ns 1 N 3 0 d 3 d
NO. OF FREEZINGS
61
•
M
M
>
0)
r-H
fa
a
H
r-H
r-H
o
o
o
o
Cfl
P-I
•
o
c
o
o
V
V
CM
o
r'-
ON
CO
•
•
•
fa
CD
r-H
vt
fa
CO
< - i
03
ON
CM
CO
U
•
•
•
•
a
o 3
r— 4
CO
VsO
03
0
CM
CO
03
cr
CM
S
CO
fa
w
ON
CO
o
r-H
>4-1
0)
•
•
•
•
o
34
r—l
CD
Csl
00
03
CM
fa
t— H
fa
fa
e
D
CM
ON
CM
3
cr
CM
CO
CO
CO
r-H
r-H
fa
r—l
CM
CsJ
CM
Q
r— 4
r-H
e
o
•r4
4J
C
O
O
03
<D
•H
34
X)
44
03
G
03
4-1
3
•H
0
&
U
1-4
03
w
X
>
44
u
O
W
03
CO
>4-1
03
03
34
T3
0)
o
<4-4
•r4
03
34
*rH
<4-1
O
fa)
o
CJ
0)
fa
03
g
1
03
o
(fa
3
4-1
(fa
34
r-H
S-4
c
CO
s
03
CO
O
03
D
•r4
34
34
44
o
03
•r4
34
o
CO
s
fa
fa
H
62
C. Survival Under Varying Moisture Conditions
The results obtained in the four repeated-desiccation
experiments which involved P, odoco ilei at 18°C (Exp'ts 10,11,13,14)
were in close agreement (Fig. 14). In only one desiccation class
("3-5") was there a significant difference in survival between
experiments (F=54.8, df=3,ll, p<0.01). The results from the two
experiments involving repeated desiccation of P. tenuis at 18°C
(Exp'ts 13,14) were also in close agreement (Fig- 15), with
only one desiccation class ("9-10") having a difference in
survival between experiments (t=3.613, df=6, p<0.05). Because of
the general similarity of results from the different experiments,
they were pooled to allow comparison between species (Fig. 16) .
For each species, survival of the two control groups
(hydrated and desiccated) was high, although survival in the desic¬
cated controls, which had undergone one desiccation, was about
3 percent lower than that of the corresponding hydrated controls,
The survival in all experimental groups was lower than in the
corresponding controls, and decreased in relation to the number
of desiccations. P. tenui s had significantly higher survival than
P. odocoi lei in the hydrated control (t=2.697, df=19, p<0.05) and
in the three experimental groups (Class "3-5", t=2.891, df=20,
t [q -g c r i t . = 2.702; "6-8", t=7.614, df=14, p<0.01; "9-10", t=2.996,
df =18 , p<0.0l) .
A lowered temperature (Exp't 12) reduced, but did not
eliminate, the effect of repeated desiccation on survival of
63
Figure 14. Survival of first-stage larvae of P. odocoilei
following repeated desiccations at 18°C (Exp'ts 10,
11,13,14). Bars represent mean + SE.
64
r
o
o
T
o
lVAIAdDS ± N 3 0 d 3 d
NO. O F DESICCATIONS
65
Figure 15. Survival of first-stage larvae of P. tenuis
repeated desiccations at 18°C (Exp'ts 13,14)
following
. Bars
represent mean + SE.
66
c
£ o
— ja
i e
a 3
X Z
LU
HVAIAanS I N30 y 3d
NO. OF DESICCATIONS
67
Figure 16. Survival of first-stage larvae of P. odocoilei (P.o.)
and P. tenuis (P.t.) following repeated desiccations
at 18°C (pooled data from Exp'ts 10,11,13,14). Bars
represent mean + SE.
68
“IVAIAHflS 1 N 3 0 a 3 d
NO. OF DESICCATIONS
69
P. odocoilei larvae (Fig. 17) . At both temperatures survival
decreased in relation to the number of desiccations. At five or
fewer desiccations, survival at 8°C was only a few percentage
points higher than at 18°C, but after 6 to 10 desiccations the
margin increased to nearly 40 percent.
The results from this section are similar to the observations
on larval survival following repeated freezing. Moisture
fluctuations which elicited a change of state of the larvae (in this
case between active and anhydrobiotic) reduced their survival in
relation to the number of fluctuations. In contrast to their
survival following repeated freezing, larvae of P. odocoilei were
slightly more susceptible than P. tenuis to the effects of
repeated desiccation on survival.
D. Infectivity Trials
The conditions under which experimental exposure of
P. odocoilei to Triodopsis multi lineata occurs appeared to markedly
affect the number of larvae which successfully entered the snails
and developed (Table VIII). Using product -moment correlations, the
total number of larvae recovered from all snails in a given
exposure (Exp't 15) was significantly correlated with the overall
density of the exposure conditions (r=0.996, p<0.01), but not with
either snail density (r=0.702, ns) or larval density (r=0.602, ns),
the two components of overall density. Further evidence for the
importance of density of both snails and larvae during exposure
comes from the lack of correlation between numbers of first -stage
Figure 17. Survival of first-stage larvae of P. odocoi lei
following repeated desiccations at 8 and 18°C
(Exp'ts 10-14). Bars represent mean + SE.
71
“IVAIAanS 1 N 3 0 d 3 d
NO. OF DESICCATIONS
Table VIII. Recovery of second- and third-stage larvae from Triodopsis multilineata exposed to
72
CO
CL
X
w
co
C
o
•H
3
O
o
a)
3
3
CO
o
Cl
X
<11
co
3
O
•H
3
3
>
3
<3
dJ
3
3
•H
CD
rH
•H
o
o
o
"d
o
3d
44
O
3
3
>
3
3
DO
3
4—1
CO
I
4-1
CO
3
•3
4-4
3
3
>
3
3
4-1
O
>>
3
3
>
O
O
3
Pi
3
3
3
CO
o
CL
X
3
44
O
co
3
3
3
3
4J
O
H
*
>>
44
•H
CO
3
3
-d
3
3
3
co
o
a
x
w
co
3
•r4
44
44
O
u
3
rO
e
3
s
•H
3
3
CO
3
3
&
3
3
1
3
3
3
3,
3
3
>
54
3
3
00
3
44
CO
co
vD
CO
CO
o
•
•
•
r-H
CO
CM
00
00
00
CM
CO
CO
CH
00
CM
00
CO
Mt
00
•
CO
CNJ
LO
CNJ
•H
fi
co
o
i
S4
•H
44
3
44
CO
a
, — s
*p4
3
CM
CM
d>
•H
3
s
E
3
44
3
a
3
O
>
o
O
u
44
3
CM 14
cO
3
O
3
3
3
rH
II CL
II
CL
44
'w/
3
3
40
00
o
o
O
O
s
3
co
1 — 1
( - 1
3
44
•H
co
r^
CO
S
co
T3
r-H
r-H
co
<-4
•H
3
3
co
CM CM
-d- CM
as
E
44
CD E
\ — 1
3
O
-3
O 3
•
CO
•
o
3
3
*r4
O 3
3
3
d)
3
II
CL
rO
II CL
' _ '
E
54
' — /
3
3
CO
S
CL
CO
r-H
CM
CO
r-H
Mt"
O
r-H
co
rH
CO
c
Number of snails per cmz x number of larvae per cm'
73
larvae available per snail and the recovery of second- and
third-stage larvae from those snails (r=0.333, ns). In both the
few snails/few larvae, and many snails/many larvae conditions of
Exp 1 1 15 there were 314 larvae per snail in the exposure dish, but
in the denser conditions of the latter group, five times as many
second- and third-stage larvae were recovered per snail.
In the first experiment to examine the effect of desiccation
and high temperature on the infectivity of first-stage larvae of
P. odocoilei (Exp't 16), there was much lower recovery of larvae
from the snails in the two experimental groups than from
either of the control groups (Table IX) . There was no loss of
infectivity of the larvae as a result of the two-week timespan
over which the experiment was run, since recovery of larvae from
the final control was not significantly different from the initial
control (t=2.753, df=2, p<0.20).
The conditions under which first-stage larvae of P. odocoilei
were stored prior to exposure to snails (Exp't 17) had great
influence on their infectivity (Table X) . Desiccation of the
first-stage larvae, treatment at higher temperatures or for longer
periods of time, all resulted in some loss of their infectivity.
Those larvae desiccated at lower (45%) relative humidity retained
their infectivity more than those stored at higher (95%) relative
humidity, even though both desiccated groups had lower infectivity
than the corresponding groups of hydrated larvae. The correlation
of total numbers of larvae recovered with treatment conditions
of the first -stage larvae by anova (Table XI) showed that the
74
■u
03
?H
•rH
<4H
03
3
jj
03
a)
■u
o
4-1
o3
cu
03
O
3
0)
44
03
3
d
•H
r-H
•rH
44
r— 4
3
E
Hi
E
o
<4-4
a)
03
>
•
J-l
y - N
03
43
r-H
i — 1
<3
4-1
00
—
03
Cu
44
X
03
w
1
S_x
03
U
■H
•rH
3
43
r-H
4-1
•H
o
03
3
C
O
o3
03
I
O
03
•
S3
Hi
O
CJ
4H
3
O
03
3
4-4
3
O
>
Jh
X
3
U
r-H
3
>
3
O
00
a
3
3
4-1
Pi
03
X
M
a)
r-H
rO
n3
H
03
3
Jh
3
>
O
s — s
LO
r-H
o
CM
3
w
•
•
3
GO
r-H
CM
r-H
03
U
+ 1
Cl
+ 1
+ 1
+ 1
+
3
d
UO
CO
r^.
t-Q
0 3
<01
43
co
E
3
O
r-
CO
CM
d
E
CM
CM
X
' _ /
43
4-1
03
oo
X
rH
<t
43
<3"
d
03
r-H
t-H
3
03
H
3
U
d
03
4J
S3
03
O
Jh
•H
3
00
00
43
43
4-1
a
CM
OH
•rH
E
a
03
3
o
d
H
' _ '
o
o
4-1
d
3
r- N
E
33
33
44
Pi
Pi
03
3
s-s
^2
Sh
<0
40
H
3
03
5h
'w'
•--4
d
4-1
03
03
03
3
3
•rH
03
03
4-1
44
o
3
3
a3
OS
S
4-1
4-J
3
3
03
03
O
3
!h
5h
•rH
•H
o3
o3
03
03
X
X
3
3
t-T-»
r-H
33
P
Q
t-H
4-1
r-H
03
0!
d
44
3
d
d
S
3
o
•rH
E
•rH
J4
•rH
4-1
3
Sh
o3
CU
3
d
X
CU
00
i — 1
3
X
•rH
o
3
03
u
r-H
X
3
4-1
o
4-1
X
03
d
Jh
•rH
44
o
4—1
03
•H
CU
a
d
•rH
03
d
o
g
•rH
o
r-H
o
3
g
JH
cd
43
3
o
•H
1 — 1
1
43
4J
cd
43
1
a
oo
£
c
•H
•rH
O
M
Ph
33
H
75
76
I
-U 4J
3
•rH
3
a
05
CO
05
co
CO
co
m-i
4-1
03
ft
•
o
3
•
o
P
3
3
T3
a)
a)
34
M
V
44
4-1
3
a)
44
3
O
44
ft
o
4J
CM
O-N
M2
rH
r-
O
4J
20
cO
O
CO
o
O
K
ft
•
•
•
•
•
•
T3
3
00
20
o
CM
00
rH
(1)
to
r—l
CM
CO
V
o
"3
a
3
X
03
a)
03
a)
4-1
34
3
3
3
4-1
3
03
3
•H
34
34
CO
rH
CO
CO
rH
IT)
rH
a)
3
03
20
o
LO
O
20
o
*H
&
o3
3
O
co
CO
r—l
CO
o
o
4-J
a
3
cr
•
•
•
•
•
•
•
1— — 1
3
a
3
| |
a
CO
rH
o
rH
o
o
o
o
•
3
H
o
•f-i
•
S
4-1
,, — N
o
•H
r— 4
co
3
T3
+
44
3
20
rH
CO
02
20
rH
o
M-4
3
X
O
34
CM
o
40
o
CM
o
O
3
rH
CO
CO
CM
I-'-
o
o
T3
o
o
E
3
•
.
•
•
•
•
•
0)
r—l
3
cr
CM
o
rH
o
o
o
o
3-i
0)
00
CO
CO
3
34
o
>
3
r— 4
O
4-1
O
CO
kO
3
•H
42
34
O
a
03
3
3
ft
CM
rH
rH
Osl
CM
rH
CM
3
x
a
Q
>
42
34
34
O
03
r\
44
r— H
•H
CO
3
3
44
rH
3
O
*H
34
o
44
CO
o
42
34
o
3
44
ai
ft
34
00
42
O
3
3
3
E
3
O
3
3
•
34
ft
3
ft
03
3
4-1
W
X
34
44
03
_ _ '
O
O
Q
3
3
44
co
34
3
3
3
3
3
43
O
3
a)
3
•
O
44
44
•t— 1
44
t — 1
>
s
■r4
3
oo
44
3
ft
34
r-
44
34
43
3
3
3
o 3
3
rH
3
o
3
44
3
3
3
4-1
rH
O
3
ft
00
ft
3
ft
4-J
•H
34
a
3
3
a
03
3
—
44
3
3
3
X
44
3
>
00
pH
03
44
H
ft
3
H
O
3
•H
3
3
3
H
3
44
w
34
34
H
X
X
3
X
<2
CO
3
CO
3
3
3
>
44 3
44
34
3
3
44
3
3
O 34
3
3
34
34
3
3)
3
•
44
3 3
34
4=
ft
3
3
3
3
3
M
O
44 44
3
44
34
44
44
3
O
44
X
44 CO
ft
GO
O
co
CO
ft
l
CO
3
ft -H
a
3
1
•H
•H
a
33
•H
0)
O
O
3
3
44
o
o
3
3
O
i — 1
34
3 g
H
ft
CO
a
S
H
O
a
as
3
%H
34
o
03
O
3
•r4
3
H
CO
2
ft
CO
Total 11 4.806
effects of moisture condition and duration of treatment were
significant, while the 6°C difference between the two temperature
treatments did not have a significant effect. In all groups of
larvae recovered from snails, the majority had reached the third
st age .
Several differences between the infectivity of P. odocoi lei
and P. tenuis (Exp't 18) were observed (Table XII). The mean
number of P. odocoilei recovered from T. multilineata that were
exposed to previously -frozen first-stage larvae was not signif¬
icantly different from those exposed to fresh ones (t= 0, df=2, ns)
However, significantly fewer (about one -twentieth the number)
P. tenuis were recovered from snails exposed to previously -frozen
first-stage larvae than from those exposed to fresh ones (t= 6.212,
df=2, p<0.05). Total numbers of larvae recovered from
T. multilineata was correlated with species and prior treatment
of first-stage larvae by anova (Table XIII). The significant
interaction indicates that significantly fewer larvae of P. tenuis
retained their infectivity following freezing, compared to first-
stage larvae of P. odocoilei . Under the exposure conditions
used in this experiment, the first-stage larvae of P. tenuis were
generally much more infective to T. multi lineat a than those of
P. odocoi lei exposed in a similar manner.
In all four groups in Exp't 18 the proportion of third-stage
larvae recovered from snails was greater in the second replicate,
which was digested 10 days after the first. Twenty-eight days
after exposure to T. multilineata, the proportion of third-stage
Table XIX. Recovery of second- and third-stage larvae from T. multilineata exposed to treated
78
4J CO
CO >,
54 G
•H T 3
4-1
00
CM
-G
g
4-1
CO
CU
00
•H
TO
CO
G
£
CO
•
*H
co
0
to
C
03
Q)
T3
4-J
00
•
on
P4
54
t3
cu
G
4-1
03
4-1
G
•r4
a)
r\
r-4
CM
•H
o
(U
a
4J
o
G
"G
O
O
■H
r-4
•
a
Pu
cu
54
4-1
O
• •>
G
a)
54
03
G
>
CO
5-i
O
oJ
a
H
X
cu
(U
i
00
4-1
G
CO
4-1
O
co
C4
cO
4-J
o
4-4
O
T3
CU
54
<U
>
O
o
0)
54
54
G
"§
3
52
Pj|
O
O
o
T3
O
Ph I
cO
4-1
O
H
0)
00
cO
4-1
co
I
"G
5j
•H
-C
H
a)
oo
cO
cO
4-1
o
H
CD
oo
cO
4-1
CO
I
TO
54
•H
-C
H
<U
00
cO
4-1
CU
CO
On
ON
00
00
ON
on
00
On
43
on
on
oo
on
cO
on
ON
40
00
no
Mt"
00
CvJ
40
40
CM
4-J
rH
40
00
LO
CO
00
t— 1
00
1
TO
*w'
c
CM
CM
ON
o
o
<u
CO
CM
on
l o
r-H
04
rH
43
04
O
on
vt
40
CO
o
'w'
o
o
o
CM
o
N — /
o
LO
O'.
on
co
O
O
i
T3
o
o
LO
i- H
O
rH
CM
G
v _ '
'w'
O
a
vD
o
CO
1 - 1
cu
4-1
03
O
•r4
Cu
CU
Pd
CM
G
CU
E
4J
g
cu
u
H
G
rG
G
co
N
G
O
54
54
p4
p4
Mean
79
4-1
CO
Pi
•H
<4-4
"O
44
3
3
4-1
3
3
s
3
44
Pi
3
4J
3
3
o
44
4-1
>44
"3
O
a)
m
3
o
34
a
3
X
44
3
3
U
3
3
4-1
a
3
e
3
3
c
44
•rH
t—4
T3
•H
3
4-1
3
T— 4
3
3
a
3
•H
•
o
H
3
a
E
3
•
O
N
Pi
>1
X
>44
43
v— '
TJ
r\
o
a)
co
r-H
Pi
•H
00
<D
0
o
>
c
r-H
o
a)
o
4-J
x
3
43
Pi
•
P4
T3
3
3
3
od
e
>
3
u
Pi
3
O
03
>44
r-H
•H
3
3
3
>44
1-4
3
o
■H
Pi
O
44
3
3
Pi
O
3
<D
03
3
43
O
3
E
g
3
•
3
CP
3
44
!4
M4
3
O
O
Q
>44
3
0)
3
•
r— 1
>
N
43
00
03
3
r— H
44
1 - 1
44
03
Q)
—
>
00
CL,
O
ctf
X
g
4-J
w
<
CO
\ _ /
x
3
i — I
"3
H
PL,
r-l LO
o o
• •
o o
V \x
in
o
•
o
v
CM
00
<n
CM
<1*
•
•
•
in
in
m
o
r-H
r-H
3
CM
!4
vD
CN
CM
3
3
00
00
3
3
co
r^
n-
3
c r
•
•
•
S
C/3
in
o
o
144
3
3
r^
CM
O
Pi
vr>
CM
CM
3
CO
00
a
3
co
n-
3
O'1
•
•
•
CO
CO
m
o
o
Pp
Q
t-i <t
a)
3
Pi
O
3
•H
44
4-J
3
3
a
Pi
O
ctf
3
•H
u
CP
44
a)
E
3
4-J
3
•H
3
r-1
EH
Pi
Pi
M
3
CO
3
X
>
44
44
U
3
3
3
CD
3
>44
3
3
Pi
TD
3
O
144
•rl
3
u
•H
>44
O
CP
o
o
3
w
3
6
1
3
a
CL,
3
4-J
CP
Pi
,—1
Pi
3
CO
H
CO
CO
O
3
3
•H
u
Pi
44
O
CTJ
•H
Pi
O
CO
s
Ph
W
Eh
2028 0.0507
80
larvae of P_. tenui s recovered did not differ between the fresh and
2
previously-frozen groups (X = 0*613, df=l, p<0.50). However, after
38 days there was a significantly lower proportion of third-stage
to second-stage larvae recovered in the frozen group compared to
the fresh group (X^= 21.23, df=l, p<0.0l).
IV. DISCUSSION
A. Survival of P. odocoi lei
First-stage larvae of Parelaphostrongy lus odocoilei appear
extremely resistant to both high and low temperatures: the maximum
temperature they can tolerate is at least 48°C; at temperatures
just above freezing they can survive in the neighborhood of one
year, depending on moisture conditions; while frozen they can
survive several years. Their infectivity to snails does not appear
excessively reduced by any particular temperature condition.
While resistant to a wide range of temperatures, moisture
conditions are critical to the first-stage larvae. When hydrated
or desiccated their survival was lengthy. However, while survival
was not necessarily reduced by desiccation, infectivity of the
surviving larvae always was. Thus, first-stage larvae of
P. odocoilei appear susceptible to desiccation, in terms of its
apparent potential for reducing their transmission.
Survival of larvae was modified by several factors. Both
repeated freezing and repeated desiccation reduced survival,
although repeated temperature changes above freezing did not.
Desiccation of the larvae, while increasing their resistance to
high temperatures, reduced their resistance to freezing.
These features of larval survival of P. odocoi lei are inter¬
esting from two points of view. First, they indicate that the abil
ity of the first-stage larvae to resist extremes of environmental
moisture and temperature conditions (at least in terms of survival)
is exceptional, although the mechanisms by which they do so appear
82
unusual for a nematode (see later). Second, they provide a
groundwork for discussing the epizootiology of P. odocoi lei . While
much of their life in the intermediate and definitive hosts has been
documented, prior to this study little information other than
on morphology or prevalence was available for the free-living,
first -stage larvae of any species of Parelaphostrongylus. However,
before examining the epizootiological implications of this study,
the relationship of P. odocoilei survival characteristics to those
of the free-living stages of other nematodes will be examined.
Survival of the free-living stages of parasitic nematodes
depends not only upon whether they are in water or desiccated, but
also if desiccated upon the relative humidity of the environment
(Rose 1957; Prasad 1959; Hansson 1974; Nath 1978). It is therefore
important, in comparing survival of different groups of nematodes,
that moisture as well as temperature conditions for which survival
was determined be precisely specified. Unfortunately, most of the
earlier studies reported the moisture conditions to which larvae
were subjected with such ill-defined terms as "humid" and "moist",
in addition to the more accurate descriptors, "wet", and "dry" or
"desiccated" .
The following paragraphs discuss the survival of first -stage
larvae of IP. odocoilei in relation to that of the free-living
stages of other parasitic nematodes. The purpose will be to point
out both that great variation in temperature and moisture tolerance
exists between species, and that the tolerance of P. odocoilei to
the range of conditions tested in this study is similar or superior
83
to that reported for other nematode species. The greatest use
will be made of literature in which experimental conditions are
accurately described. A large number of studies, though vague in
description of some aspects of experimental conditions, contain
valid qualitative information, and will be referred to where
appropriate .
The high-temperature tolerance of P_. odocoi 1 ei larvae is
similar to that of other metastrongyles . The larvae survived a
temperature of 48°C for one day in water; one week when desiccated
at 20 or 45% relative humidity (% RH) . The larvae of Cy stocaulus
ocr eatus and Protostrongylus sp. tolerated temperatures of 45-
50°C for one-half hour (Morev 1966). The upper lethal temperature
for Elaphostrongy lus cervi in water was near 50°C (Mitskevich 1964) ,
but a temperature of 40°C could be tolerated for one week (Lorentzen
and Halvorsen 1976) under unspecified moisture conditions. Some
larvae of Protostrongylus sti lesi could survive at least one day
on dried fecal pellets at 72°C (Forrester and Senger 1963); this
was the only metastrongy le studied at such a high temperature.
These studies indicate that species in this group have similar
resistance to high temperatures, being able to survive exposure to
temperatures of 45- 50°C at least for a short while.
Lower survival of hydrated than desiccated larvae at high
temperatures has been reported for many species. In studies with
larvae on feces (Rose 1957; Forrester and Senger 1963) this might
be interpreted as due to the presence of contaminants such as
bacteria, rotifers and fungi on the decaying, moist pellets.
84
Susceptibility of first-stage metastrongy le larvae to such
conditions has been reported (Hobmaier and Hobmaier 1930; O'Roke
1936; Pillraore 1956). In this study however, isolated, clean,
desiccated larvae of P. odocoi lei exhibited superior survival over
similarly treated but hydrated larvae at high temperature. Third-
stage larvae of trichostrongy le nematodes also reacted similarly
(Poole 1956, on Nematodirus filicollis; Andersen and Levine 1968,
on Trichostrongy 1 us colubriformis; Todd et al. 1976, on Haemonchus
contortus) . This suggests that desiccation of larvae promotes
their high-temperature survival, not by reducing decay of their
surroundings, but by action on the larvae themselves.
While resistance of first -stage larvae to high temperature is
similar among the metastrongyles , their long-term survival at
cooler, more favorable temperatures differs markedly. In water,
Protostrongylus kochi lived only 4 months at 2- 4°C (Davtian 1949,
cited by Morev 1966) as did P. stilesi at an unspecified temperature
(Pillmore 1956) , yet larvae of Protostrongylus ruf escens lived in
water over a year at an unspecified temperature (Hobmaier and
Hobmaier 1930) . P. odocoilei , also able to survive a year in water
at 5°C, lived even longer (18 months) when desiccated at 45% RH.
Protostrongylus (=Synthetocaulus) hobmaier i was able to survive
19 months at 10- 20°C, while desiccated at 35- 50% RH (Matekin
et al. 1954, cited by Forrester and Senger 1963). Under similar
conditions, survival of P. odocoilei was only slightly shorter
(14 months) .
85
In studies on other strongylids (Belle 1959; Gupta 1961;
Herlich 1966; Andersen and Levine 1968; Todd et al. 1976) a range
in maximum lifespan (from 4 to over 18 months) similar to that
existing among the metastrongy les has been reported. In contrast
to the response of P_. odocoi lei , studies on trichostrongy les have
shown a lower survival at cool temperatures when desiccated than
when hydrated (Andersen and Levine 1968; Todd et al. 1976).
Thus while desiccation appears to promote high -temperature
survival of strongylid larvae, regardless of species, the
benefits at cooler temperatures vary between species. Too few
studies have been done to permit generalization of particular
types of survival responses as characteristic of any of the
major taxa within the Strongylida.
There is as much variation in the resistance of various
species of strongylid larvae to freezing as was demonstrated in
their ability to survive at above -freezing temperatures. Studies
on strongyles (Belle 1959; Balasingam 1964) indicated a range
in survival from two hours to over 30 days when frozen at -20°C;
on trichostrongy les (Andersen and Levine 1968; Todd et al. 1976),
from a few days to two months at -28°C; and on metastrongy les
(Pillmore 1956; Rose 1957; Lankester and Anderson 1968; Hansson
1974), from nearly two weeks to 10 months at -20°C. The ability
of larvae of P. odocoilei to survive freezing for several years
thus appears exceptional for this group of nematodes.
86
Two of the factors that can influence the survival of larvae
while frozen are: whether the larvae are on or off feces, and
whether they are hydrated or desiccated prior to freezing. For
example, Skr ;abingylus nasicola survived freezing at -20°C when on
pellets, but not when off (Hansson 1974); H. contortus survived
freezing better when off fecal pellets (Todd et al. 1976).
Survival estimates for P. odocoi lei frozen on feces or in water
were similar.
In summary, larvae of IP. odocoi 1 ei generally have higher
survival, under comparable conditions, than most other Strongylida
studied. However, the extremes of high and low temperature and
desiccation that have been examined in some other studies were
not part of this study, and the response of P. odocoi lei to those
conditions was not determined. The range of conditions chosen for
this study was similar to what might be expected in nature; the
data obtained would aid in understanding the epizootiology of
Parelaphostrongy lus . The use of temperature and moisture extremes
would not serve that purpose, but rather would be useful for
studying the mechanisms of resistance to high temperature,
freezing, and desiccation; such was not the purpose of this study.
However, while the elucidation of survival mechanisms was not the
purpose of this study, some observations on P. odocoilei warrant
special attention in that regard.
The consistent observation in studies on other nematodes has
been that while desiccated, survival was lengthiest at the highest
relative humidities, and shortest at the lowest. To illustrate
.1
87
this point, survival data for several species whose desiccation
tolerance was studied over a range of relative humidities is shown
in Figure 18. Because absolute lengths of survival varied
between species, survival at the different relative humidities in
each study was converted to a proportion of the maximum survival
noted for each species. Thus the differences between species in
their sensitivities to the level of relative humidity are emphasized,
while differences in the length of survival at a given relative
humidity are not.
Strongy loides papillosus (Fig. 18a) was most sensitive to the
level of relative humidity, surviving only if it was above 90% RH.
Ditylenchus myceliophagus , S_. nasicola , and Buno stomum tr igono -
cephalum (Fig. 18b-d) were somewhat less sensitive, surviving
desiccation at relative humidities as low as 20 percent. Heterodera
rostochiensi s and Heterodera schachtii (Fig. 18e) , although
demonstrating rapid decrease in survival at relative humidities
below 100 percent, still had some survival at almost 0% RH. The
survival of Ditylenchus dipsaci , Muellerius capillaris, and
Trichostrongylus retortaeformis (Fig. 18f-h) did not fall as
rapidly with decreasing relative humidity as the previous species,
and in the case of T. retortaeformis and M. capillaris was quite
substantial, even at the lowest relative humidities.
These nine species, while showing a continuum from extreme
to mild sensitivity to reduced relative humidity, all responded
in the same qualitative manner. The survival of P. odocoi lei , when
displayed in the same manner (Fig 18i) had the opposite qualitative
Figure 18, Survival of desiccated free-living larvae of a variety
of plant- and animal -parasitic nematodes at a variety
of relative humidities.
a. Infective larvae of Strongy loides papillosus
(after Nath 19 78).
b. Fourth-stage larvae of Ditylenchus myceliophagus
(after Perry 1977).
c. First-stage larvae of Skr j abingylus nasicola
(after Hansson 19 74).
d. Infective larvae of Bunostomum trigonocephalum
(after Belle 1959) .
e. Second-stage larvae of Heterodera schacht ii and
Heterodera rostochiensi s (after Ellenby 1968b) .
f. Fourth-stage larvae of Ditylenchus dipsaci (after
Perry 19 77) .
g. First-stage larvae of Muel lerius capillar is (after
Rose 1957) .
h. Infective larvae of Tr icho strongy 1 us
retortaef ormis (after Prasad 1959) .
i. First-stage larvae of P. odocoilei.
SURVIVAL
89
RELATIVE HUMIDITY (%)
I
90
response to the level of relative humidity; greater survival with
decreasing relative humidity.
This suggests three possibilities. First, the observed
survival pattern of P_. odocoi lei may merely be an extension of the
continuum of increasing resistance to low relative humidity, with
the same mechanism for survival while anhydrobiotic (see p. 21)
operating as in the nine other species. A second possibility is
that a different mechanism for survival operates in larvae of
P. odocoilei than in the other nine species studied. The third
possibility is that the results obtained in this or the other
studies were artif actual , arising only from the techniques employed.
The last possibility will nowbe addressed in detail.
The experimental protocol in this study comprised five parts.
First, larval survival in this study was examined using larvae that
had been removed from feces, cleaned, and placed in clear water
prior to desiccation. Of the eight other studies used to compile
Figure 18, all but Hansson (1974) used larvae prepared similarly.
Second, in order to desiccate the larvae, evaporation of the water
in which they were contained was done in a Petri dish under
ambient room conditions. All the other studies but those of
Belle (1959), Prasad (1959), and Nath (1978) also used a smooth
substrate for evaporation of the water. Rose (1957) and Nath (1978)
allowed the samples to desiccate under ambient conditions, as in
the present study. Third, humidity control in this study was by
saturated salt solutions. Only Rose (1957), Ellenby (1968b), and
Perry (1977a) used other methods. Fourth, survival in this and all
91
other studies was monitored after rehydrating larvae, without a
period of "pre-hydration" (see later). Finally, survival of larvae
in this study, as in all the others, was monitored using motion
of the larvae as a criterion. In summary, the methods of
this study were similar to those of the other eight studies, and
likely did not contribute to the different qualitative results
observed.
From this study there are two other pieces of supporting
evidence that the unusual desiccation survival of P. odocoilei is
a real phenomenon. First, the congener Parelaphostrongy lus tenuis
had a similar response; survival after nine days at low relative
humidity (Fig. 12c) was greater than after a shorter time at a
higher relative humidity (Fig. 12g) . Second, the infectivity of
larvae surviving storage at high relative humidity was lower than
infectivity after the same time spent at lower relative humidity
(Exp ' t 17) .
If the evidence that survival of first -stage larvae of
P. odocoilei (and P. tenui s) is indeed better when desiccated at
lower than high humidity is accepted, the question is raised: what
mechanisms could be responsible?
Although the ability of many organisms to survive lengthy
periods of desiccation has been recognized for over two centuries
(Keilin 1959) , the study of anhydrobiosis is unfortunately still
largely in the descriptive stage (as in this study) . Several
factors are known to enhance desiccation resistance of nematodes,
although the mechanisms by which they do so are still poorly
i
92
understood.
Evaporative water loss, for example, must be slow to result
in maximum survival while desiccated (Ellenby 1968a_,b; Crowe and
Madin 1975). This can be accomplished by several means: coiling
of the body, aggregations into groups ("eel-worm wool"), retention
of cuticles from previous molts, or water loss occurring at high
relative humidities (Ellenby 1968a, 1969; Bird and Buttrose 1974;
Crowe and Madin 1975; Rtfssner and Perry 1975; Perry 1977a).
Similarly, slow water uptake during rehydration also promotes
revival of larvae. With Aphy lenchus avenae stored at 0% RH, a
period of "pre-hydration" at 95% RH before the addition of water
resulted in 95% revival of the larvae and adults; without the
"pre-hydration" treatment, revival following the addition of
water was only 60 percent (Crowe and Madin 1975).
These studies however, have not provided answers as to how
larvae survive desiccation, apart from suggestions that "prepara¬
tory" biochemical events must occur (Crowe and Madin 1975), or
that slow dehydration might permit an orderly packing of tissues
during shrinking (Bird and Buttrose 1974). No one has addressed
the question of why survival at different relative humidities
differs, let alone why some species should favor low relative
humidities and others high.
Pigori and Weglarska (1955) and Bhatt and Rohde (1964) have
suggested that metabolism in anhydrobiotic organisms, which is
almost non-existent at low relative humidities, increases sharply
at relative humidities of 90 percent and above. Burns (1964)
93
suggested that a water content just sufficient to support oxygen
consumption, but not other biochemical reactions (20- 60%) may be
lethal. This may be of little consequence to the nematode, either
hydrated or desiccated. Studies on water content during dehydration
(Ellenby 1968.a; Rdssner and Perry 1975; Perry 1977b) have resulted
in estimates of the initial water content of several species at
about 75 percent, exceeding the lethal value of 60 percent
estimated by Burns (1964). As well, upon drying the water content
is reduced to below the 20 percent lethal value suggested by Burns
in a very short time. For example, even when dried slowly at
ambient relative humidity of 95-99 percent, water content of
Roty lenchus r ob u s t u s dropped to below 5 percent after only 5 min¬
utes (Rdssner and Perry 1975). Only a brief time was spent in the
critical zone, even when dried slowly. Thus, factors other than
a lethal water content must be operating during mortality of larvae
experiencing desiccation. No solution appears at hand to explain
the effect of relative humidity on survival. Subsequent discussion
of the phenomenon will deal only with its epizootiological
consequences .
The previous sections have dealt with survival of P. odocoilei
under a variety of constant conditions. The information gained from
controlled studies of that nature can be applied to field
situations only if it is assumed that the influence of different
conditions in combination is additive; that under changing
conditions the changes themselves do not have any influence
beyond what the summation of their individual components would
SUZi
■
94
have. The results from several experiments in this study
indicate that this assumption is valid only under certain circum¬
stances.
The factor which appears to determine whether added influence
on larval survival beyond the summation of the components occurs,
is whether a change of the physical state of the larvae is
involved. In the case where repeated temperature changes occurred,
but larvae remained in the active state (Exp't 7), no added
mortality in the temperature-cycled group of larvae beyond that
in the control group was detected. However, when repeated
temperature changes resulted in the larvae repeatedly moving
between the active and cryobiotic (see p. 21) states (Exp'ts 8,9),
additional mortality of larvae was observed, above the level
expected when they were constantly in either of the two states.
This phenomenon was also observed when moisture changes involved
larvae repeatedly moving between the active and anhydrobiotic
(see p. 21) states (Exp'ts 10-14).
Changing states acted on the larvae by killing or weakening
a certain proportion of the individuals each time a change of
state occurred. There was slightly reduced
survival observed after even one freezing (or desiccation) , and
the increased mortality after each succeeding freezing (or
desiccation) cycle. The processes involved in freezing larvae
appeared to be less demanding than those involved in desiccating
larvae. Survival of larvae of P. odocoilei after 20 freezings
was similar to the survival resulting from only 10 desiccations.
!l
95
Susceptibility of nematode larvae to repeated desiccations
has been reported frequently (Poole 1954, in Todd et al. 1970;
Keilin 1959; Schmidt et al. 1974; Evans and Perry 1976; Todd et
al. 1977). This susceptibility has been attributed to an increase
of solutes with each desiccation, resulting from impurities in the
water added for each rehydration (Todd et al. 1970). The
susceptibility to repeated desiccation varies between species.
Third-stage larvae of H. contortus were able to survive 70 days
of daily desiccation in triple -distilled water (Todd et al. 1970).
They survived at least 64 days while constantly desiccated under
similar ambient conditions (Todd et al. 1976), indicating no added
mortality due to the repeated desiccations. On the other hand,
larval T. colubr if ormi s could survive only 30 days of repeated
daily desiccation in triple-distilled water (Schmidt et al. 1974),
but survived constant desiccation under similar ambient conditions
for at least 128 days (Andersen and Levine 1968) . Resistance of
Cooper ia punctata to repeated desiccation was found to be
intermediate between that of H. contortus and T. colubriformis
(Todd et al. 1977). The survival of P. odocoi lei , determined in
this study using distilled water as the medium, was similar to
that of T. colubriformis in distilled water (Schmidt et al. 1974).
The effects of repeated freezing have been examined only
infrequently. Species of Nematodirus have been shown to resist at
least a dozen cycles of freezing and thawing with little reduction
in survival (Turner 1953; Poole 1956) . Survival of P. odocoilei
was slightly poorer after a similar number of freezing cycles.
In summary, it appears that survival data derived under
y
96
constant temperature and moisture conditions can be interpreted
in relation to the varying conditions of nature only with caution.
If ambient conditions are such that freezing or desiccation of
larvae occurs on a repeated basis, then their survival will
probably be less than that predicted using data derived under
constant experimental conditions.
B. Infectivity of P. odocoi lei
Forrester and Senger (1963), studying the survival of larvae of
P. stilesi under temperature and moisture stress, concluded
that, "... it seems unlikely that temperature and humidity can
influence the survival of first stage protostrongy lid larvae on
fecal material to a significant degree." They felt that due to
the remarkable survival capabilities exhibited by the larvae of
P_. stilesi , they would survive environmental stresses and
probably still be available in sufficient quantities to allow
infection of the intermediate host. They acknowledged that they
had not examined the viability (= infectivity) of the surviving
larvae, but that the surviving larvae could have been affected
physiologically, and may have been unable to complete their life
cycle. Their statement now appears prophetic in light of recent
studies dealing with the infectivity of surviving larvae.
The infectivity of nematode larvae which survived a variety
of different storage conditions has been shown to be reduced
gradually over time. First-stage larvae of Angiostrongy lus
(= Parastrongylus) cost aricen sis had a period of infectivity that
was
shorter than their period of survival under both hydrated and
9 7
desiccated storage conditions (Arroyo and Morera 1978; Bullick and
Ubelaker 1978). Third-stage larvae of T. colubriformis gradually
lost infectivity after 12 months storage on moist filter paper at
4°C (Herlich 1966). First-stage larvae of P. tenuis also lost infec¬
tivity after desiccation or freezing (Lankester and Anderson 1968;
this study) .
The results of this study indicate that P. odocoi lei also loses
infectivity before death occurs. Those conditions which resulted in
the poorest survival also resulted in the lowest infectivity among
the survivors. Two conditions which resulted in lengthy survival of
the larvae, storage in water at temperatures just above freezing, and
freezing, resulted in little loss of infectivity among the surviving
larvae. On the other hand, desiccation of larvae resulted in drastic
reduction of the survivor's infectivity. Reduction in infectivity,
just as in survival, was a function of the degree of desiccation. At
45% RH, survival was better and the loss of infectivity less than for
larvae at 75% RH (Table XI) . It is critical to note that while
first -stage larvae of P_. odocoi lei survived as long when desiccated
at low relative humidities as when hydrated, the infectivity of
larvae surviving desiccation was never found to be more than
15 percent that of larvae which had not been desiccated.
Co Comparative Studies
The experiments just discussed pertain to larvae of P_. odocoi lei
originating from mule deer of Jasper National Park. The remaining
discussion along those lines will deal with the survival of
Jasper-source larvae of P. odocoi lei in relation to P. odocoi lei
■
98
larvae of Vancouver Island source, and to larvae of P. tenuis
originating from Pennsylvania.
No difference in survival between the Vancouver Island and
Jasper source larvae was detected, though a broad range of
conditions was tested: hydration and desiccation; above -freezing
and below-freezing temperatures. Climatic conditions in the
coastal areas of Vancouver Island are certainly more moderate
than those of Jasper. Based on mean values for the period 1941-
1970, daily minimum temperatures reported from Vancouver Island
meteorological stations were generally only a few degrees below
freezing in January, while those for Jasper averaged -17°C
(Anonymous 1973). Greater total annual precipitation and annual
days with measurable rain were much greater on the Island (Anonymous
1973). Snowfall was much less frequent on the coastal areas of
Vancouver Island than in Jasper (Anonymous 1973). This would
result in more direct exposure to ambient conditions for larvae
shed on Vancouver Island sites, while those larvae shed in
Jasper would have a higher probability of being covered with
snow, thereby receiving a degree of protection from ambient
conditions. The sites for transmission of P. odocoi lei on
Vancouver Island are not known, but the results of this study suggest
that not only would transmission be possible in the coastal areas,
but that cooler temperatures in the upland regions would not
likely be limiting.
Differences in survival between P. odocoi lei and its congener
P. tenuis were observed. The differences, slight when under
99
constant conditions, were more prominent under fluctuating
temperature and moisture conditions. These differences comprised
a lower survival of ]?. tenuis than P. odocoi lei following repeated
freezing, but superior resistance of P. tenui s over P. odocoilei
to repeated desiccation. Many other species also exhibited
differences from close relatives in their survival under various
storage conditions. Several examples follow.
Studies on congeneric species pairs of plant -parasitic
nematodes (Ellenby 1968b; Perry 19 77a) have demonstrated that one
member of the pair had superior desiccation resistance to the
other. Ellenby (1968b) suggested that in the case of Heterodera
schachtii and Heterodera rostochiensis that the difference in
resistance was related to differences in the characteristics of
the host plants on which they evolved.
Balasingam (1964), studying Uncinaria (= Dochmoides )
stenocephala, Arthrocephalus (= Placoconus) lotori s , and
Ancy lostoma caninum , demonstrated that the first species, which
has a northerly distribution, had greater freezing resistance
than larvae of A. caninum, a parasite southern in distribution.
A. lotoris, which has a distribution somewhat intermediate to
the other two species, had characteristics which were intermediate.
Differences in survival capabilities also exist between
species of Trichostrongy lus (Gupta 1961; Herlich 1966; Andersen
and Levine 1968; Rojo-Vazquez 1976). It is of interest that
although larvae of T. colubrif ormis exhibited greater resistance
to continuous desiccation than did H. contor tus (Todd et al. 1976;
■
100
Hsu and Levine 1977), H. contortus was the more resistant of the
two to repeated desiccation (Todd et al. 1970).
Todd et al . (1970) suggested that the resistance to
repeated desiccation exhibited by H. contortus and T. colubr if ormi s
may be of adaptive value in places such as Urbana, Illinois,
where daily dew formation and evaporation occur during the summer
months. The difference in resistance between the two was
suggested as a contributing cause of the difference in distribution
of the two species (Schmidt et al. 1974).
While the differences in survival between P. odocoi lei and
P_. tenui s were slight, the difference in the infectivity of the
surviving larvae was not. Following freezing, the infectivity of
P. tenui s was reduced to only 5 percent that of larvae which had
not been frozen, while larvae of P. odocoilei had only slight loss
of infectivity. Following desiccation though, the infectivity of
P. odocoilei was reduced to only about 15 percent of its normal
levels, as discussed previously. Infectivity of P. tenui s larvae
following desiccation was not determined in this study, but
similar information can be derived from the study of Lankester and
Anderson (1968). They reported a recovery of P. tenuis larvae from
snails (Mesodon thyroidus) exposed to first-stage larvae in dried
and remoistened soil that was 50% that of the recovery from snails
exposed to larvae in moist soil that had not been dried. The
recovery from dried and remoistened feces was about 25% that from
fresh feces containing P. tenuis larvae. The 25 and 50% infectivity
retention figures for P. tenuis following desiccation are not
strictly comparable with the infectivity figures derived in this
study. They do not take into account the number of live larvae
available to infect the snails, as this study does; therefore
if mortality of larvae of ]?. tenui s in soil or feces occurred,
those estimates are conservative. Compared to the maximum
infectivity retention for P. odocoi lei following desiccation, those
figures lend credence to the argument that P. tenuis can better
resist desiccation.
D. Epizootiological Considerations
It has been alluded to in the previous discussion that slight
differences in survival of the free-living stages of different
species of nematodes can serve to segregate them geographically.
However, conditions in the environment which we might perceive as
being potentially limiting to free-living larvae may or may not
have any bearing on the conditions actually experienced by the
nematode larva in the soil. Temperature and moisture conditions
occurring in the vegetation mat or in the soil may be markedly
different from the conditions measured less than two meters above
ground in a standard weather shelter (Levine and Todd 1975).
Crofton (1963) reported that relative humidities in the
vegetation mat were still above 90 percent even after three weeks
of drought. Collis -George (1959) outlined several factors regardin
moisture conditions within the soil which would be of importance
to soil-inhabiting nematodes. Even at water levels low enough to
elicit wilting in plants, the soil atmosphere is still above
98.5% RH. The relative humidity in soil near the surface could,
at midday with a drying wind, be as low as 50 percent. To keep
102
from desiccating the nematode must either expend energy sufficient
to retain water, which becomes more expensive as the soil dries,
or migrate deeper into the soil; failing these two routes it must
become anhydrobiotic (Col li s -George 1959) .
Changes in temperature result in temporary modifications of
soil relative humidity: increases in temperature result in a
drop in relative humidity; decreases in temperature result in an
increase through temporary saturation of the air. Despite
occasional drops in relative humidity, soil nematodes spend the
majority of the time in a water film sufficient to keep them
hydrated (Collis -George 1959), in a soil atmosphere of high relative
humidity. Movement of nematodes through the soil is restricted
to within relatively narrow ranges of water suction, which is in
part related to soil structure. In shrinking -soi 1 systems, pore
spaces might be only one-tenth the size they would be in a rigid-
soil system with the same water suction (Collis-George 1959). Once
the pores empty of water, movement of nematodes becomes difficult,
for although a film of water remains on the walls of the pores,
even with relatively abundant water it may only be a fraction of
a micron thick.
It is unfortunate that measurements of soil water suction are
made only infrequently, for they would provide much information of
use in understanding local microclimates; the climate the parasite
experiences. As a generalization, the soil offers a degree of
protection for the larvae, compared with conditions above its
surface.
103
The majority of first-stage larvae of P.. tenui s is located
near the periphery of the fecal pellet (Lankester and Anderson
1968) , and although the mucus coat on a fecal pellet may reduce
the rate of drying by up to 30 percent in the case of sheep
pellets (Crofton 1963) , it is conceivable that larvae on the exposed
portions of the pellets would desiccate rather quickly, suffering
any associated loss of infectivity. It is not known whether
first -stage larvae of Parelaphostrongylus spp, migrate readily
off the fecal pellets soon after they are shed, thus reaching
the protection of the soil or vegetation; another metastrongy le ,
M. capi 1 lar i s , does not (Rose 1957), even though it survives better
in soil (Nickel 1960). Pellets containing first-stage larvae
could be protected if dropped in areas of lush vegetation as
opposed to open areas, and frequent rains would aid survival by
washing larvae into the protective environment of the soil.
The extent to which transmission of Parelaphostrongylus spp.
would be impaired or aided by larvae leaving the fecal pellets
and entering the soil has not been carefully examined, but
Lankester and Anderson (1968) observed that M. thyroidus acquired
substantial numbers of P. tenuis larvae after crawling on infected
pellets or infected soil. However, previous drying of the soil did
not reduce the numbers of larvae acquired by M. thyroidus as much
as previous drying of fecal material containing first-stage larvae.
This indicates that first-stage larvae of P. tenuis may have been
more protected from desiccation by the soil than by fecal material.
While the results of the present study indicate that transmission
104
°f odocoi lei to Triodopsis multilineata is enhanced by crowded
conditions of both snails and first-stage larvae, the movement of
larvae off the fecal material into the surrounding soil may not
have any deleterious consequences for transmission as long as
the larvae are not dispersed too far.
The first-stage larva is the age class in the life cycle
of Parelaphostrongy lus most susceptible to climatic factors.
Regardless of the availability of suitable intermediate and
definitive hosts, if this stage succumbs to environmental pressure,
transmission will not occur. Environmental pressure on the first-
stage larvae is very likely involved in the current known
distribution of Parelaphostrongy lus spp.
There is an apparent segregation of P. tenuis and
Parelaphostrongy lus ander soni in the southeastern United States.
White-tailed deer were examined for both parasite species in 24
counties in the southeast (Prestwood et al. 1974). P. ander soni
was present alone in 12 counties, P. tenuis in 10, but in only two
counties were the two species found together. A particularly
striking example of this segregation occurred in South Carolina,
where 11/30 white-tailed deer from five counties were infected
with P. ander soni . None had P. tenui s even though a large
proportion of the deer to the north and south harbored P. tenuis
(Prestwood et al. 1974). As well, in a previous study (Prestwood
and Smith 1969) none of 87 white-tailed deer from nine other
counties in South Carolina was infected with P. tenuis .
:!
Arguments were put forth by Platt (1978) that the elapho-
strongylines are relatively non-specific at the intermediate host
level. While such appears to be the case for P. tenuis
(Lankester and Anderson 1968) and IP. odocoi lei (Platt 1978),
little is known of the intermediate-host specificity of
ander soni . Barring any extreme idiosyncracies in intermediate
host suitability for P. andersoni , it is unlikely that
intermediate hosts serve as the means for segregating these two
parasite species in this region. The white-tailed deer is the
host for both parasite species, and concomitant infections of
the two parasites have been reported (Prestwood et al. 1974), so
it is unlikely the definitive host plays a role in the segregation
This leaves, of course, the first-stage larvae of the two
species as the point where a differential influence on the
transmission of the two species is occurring. It was observed
that P. tenui s occurred primarily in the oak -hickory -pine
subclimax and the climax deciduous forest habitats, but tended to
be absent from the southern floodplain and southern mixedwood
vegetation habitats (Prestwood and Smith 1969; Prestwood et al.
1974). In contrast, P. ander soni tended to be found in the
southern floodplain and southern mixed vegetation habitats as well
as in the oak -hickory-pine subclimax (Prestwood et al. 1974).
The distributional pattern related to vegetation types in this cas
is strongly suggestive of microclimatological conditions having
a differential influence on the survival or subsequent infectivity
of the larvae of these
two species.
.
106
A second oddity in the distribution of Parelaphostrongy lus
spp . is the apparent lack of any representatives in the North
American grassland biome, as defined by Carpenter (1940). The
reported distribution of P. tenui s extends westward only to the
boundaries of the grassland. The only place where the confirmed
distribution of P. tenui s has not reached the grassland is in
Illinois, but Schaeffer and Levine (unpub., cited by Levine 1968,
p. 287) found dor sal -spined larvae, similar to those of P. tenuis ,
in white-tailed deer feces from Illinois.
Unfortunately, there are few reports of searches for
elaphostrongy line nematodes from the grassland; parasite surveys
of deer from that region (Boddicker and Hugghins 1969; Worley and
Eustace 1972) have not included examination for them. Samuel and
Holmes (1974) reported finding dor sal -spined larvae in deer feces
only from the forested regions of Alberta. Pellet groups from
parkland (n= 43) and grassland (n= 13) were negative, while feces
from adjacent forested regions were infected.
Bindernagel and Anderson (1972) found elaphostrongy line -like
larvae in the feces of white-tailed deer from eastcentral
Saskatchewan. Positive samples were found in areas of forest or
parkland, not in grassland areas. Prevalence of dorsal-spined
larvae decreased from a high in the mixedwood forest to complete
absence in the grassland (Table XIII) during a followup study
(1976-1978) in Saskatchewan (Shostak, unpub.). These pieces of
evidence, admittedly scanty, suggest that deer in the grassland do not
harbor elaphostrongy line nematodes.
Table XIV. Recovery of dor sal -spined larvae from the feces of
10 7
white-tailed deer from different regions of
southern Saskatchewan (Shostak, unpub.).
Region
Number of
s amp 1 e s
examined
Number of
s amp 1 e s
positive
Percent
of samples
positive
Prairie
35
0
0
Prairie/Parkland transition
61
2
3
Parkland
57
8
14
Parkland/Mixedwood transition
25
4
16
Mixedwood
386
123
32
Total
564
137
27
108
The grassland has populations of white-tailed and mule deer,
providing a definitive host for elaphostrongy lines . Gastropods
that are known intermediate hosts for Par elaphostrongy lus (see
Lankester and Anderson 1968; Platt 1978) range throughout the
grassland (Carpenter 1940; Burch 1962). The availability of
suitable hosts for the parasitic phases of the life cycle would
not appear to be limiting for transmission of Par elaphostrongy lus
in the grassland. It is possible that transmission of elapho-
strongylines in the grassland is being at least partially impaired
at the point involving the first -stage larvae, as was suggested
already as partial explanation for the disjoint distribution of
P_. tenuis and P. ander soni in the southeastern United States.
The seasonal precipitation pattern in the grassland has been
implicated in limiting the activity of small animals and plants
(Carpenter 1940) . Conditions of desiccation (on the soil surface
at least) are easily conceivable given the characteristic drought
at the end of the hot season. Prior desiccation has been shown
to reduce the infectivity of P. odocoi lei (this study) and
P. tenuis (Lankester and Anderson 1968) . Exposure to ultraviolet
light, either from sunlight or atificial sources, has also been
found to result in mortality of tr ichostrongylid (Senger 1964;
Conder 1978) and metastrongylid (Rose 1957) nematode larvae. In
contrast, Mitskevich (1964) reported that first-stage larvae of
E. cervi were resistant to direct sunlight. Desiccation and
exposure to high levels of solar radiation, working in conjunction,
may result in sufficient larval mortality or loss of infectivity
.
109
to seriously impair transmission of Parelaphostrongy lus in the
grassland. Reduced gastropod activity under dry conditions might
also act to reduce the efficiency of parasite transmission. It
seems probable that, even if conditions in the majority of the
grassland are severe enough to impair elaphostrongy line trans¬
mission, these parasites may be present there in a discontinuous
distribution, associated with protected habitats such as river
valleys.
It was mentioned previously that the westward distribution
of P. tenuis in North America appears limited by the grassland
biome. In 1972, Bindernagel and Anderson hypothesized that
P. tenuis could reach the foothills of the Rocky Mountains in
western Alberta by spreading westwards from Manitoba along the
aspen parkland in central Saskatchewan and Alberta (Fig. 19) .
Thus, P. tenuis could circumvent the grassland barrier. The
arrival of P. tenuis in Alberta could have disastrous consequences
for a variety of wild ruminants there. Bindernagel and Anderson's
hypothesis was met with an alternative hypothesis (Samuel and
Holmes 1974) which stated that some ecological feature, possibly
associated with drier western conditions, limited P. tenuis
populations in the west. They cited the apparent absence of
P. tenuis from the grassland, and from the sandy-soiled pine
forests of the southeastern United States, as supporting evidence.
The results from the present study may be used to test the
hypothesis of Samuel and Holmes (1974) as it might apply to the
first-stage larvae of P. tenuis . Their hypothesis can be restated
no
Figure 19. Potential route of expansion of P. tenuis range into
the foothills of the Rocky Mountains, as suggested
by Bindernagel and Anderson (1972). Vegetation
regions are from Rowe (1972).
Ill
112
as follows: "The first-stage larvae of P. tenui s have insufficient
tolerance of climatic conditions in the Canadian west
for the parasite to become established there".
A conclusive test of this hypothesis would involve the
seeding of an area in the west with white-tailed deer feces
containing larvae of P. tenuis , and the subsequent documentation
of any spread of the parasite into non-infected deer in the area.
Unfortunately, such an exercise would have grave consequences if
the hypothesis were disproved. The hypothesis can be tested in a
more indirect manner.
The life cycle of P. tenuis is almost identical to the
life cycle of P. odocoi lei , apart from the definitive hosts. Both
parasites even share several species of intermediate hosts (see
Lankester and Anderson 1968; Platt 1978). Since P. odocoilei is
native to western Alberta (Fig. 19) it is obviously adapted to
the climatic conditions found there. If the larvae of P. tenuis
have environmental tolerances equal to or superior to those
possessed by the native P. odocoilei , the hypothesis that they
cannot tolerate western climatic conditions should be rejected.
This method uses the environmental tolerances of P. odocoilei as
a biological measurement of the stress imposed by western Alberta
microclimates .
This study examined the effect of two climatic factors,
temperature and moisture, on the two parasite species. In terms
of moisture stress, the difference between survival of the two
species under a variety of constant conditions (Fig. 12) was
p
113
never more than a few percentage points. In the case of repeated
desiccation (Fig. 16) , P. tenuis survival was always slightly
greater. The similar or superior survival of P. tenuis compared
to P. odocoi lei , coupled with greater retention of infectivity
by P. t enui s larvae following desiccation (see previous discussion),
leads to rejection of the hypothesis that moisture conditions limit
distribution of P. tenuis in the west.
In terms of temperature stress, P. odocoilei had consistently
greater survival than P. tenuis following repeated freezing
(Fig. 13), although the difference in survival between the two
species was slight. Length of survival while continuously frozen
was considerably longer for P. odocoilei (at least 34 months) than
P. tenuis (10 months; Lankester and Anderson 1968). The infectiv¬
ity of P. tenui s was also considerably reduced by freezing
(Table XII) . Not only did just 5 percent of the P. tenuis larvae
retain their infectivity, compared with over 85 percent for
P. odocoilei , but those larvae of P. tenuis which had been frozen
appeared delayed in their development from second-stage larvae to
third-stage larvae. The data suggest that a susceptibility to
freezing might be a means by which P. tenui s is prevented from
expanding its range westwards.
To support the hypothesis that susceptibility to freezing
may bar P. tenuis from the west, it must be assumed that winter is
an important time for transmission of the parasite. It is
obvious that infection of the intermediate host cannot occur in
winter, but must await spring. An advantage accruing
114
Parelaphostrongy lus in having larvae highly resistant to freezing
would be that a winter's accumulation of first-stage larvae would
survive until spring, and following snow-melt would flood the
environment with massive numbers of larvae, increasing the chances
for larval contact with molluscs. The results from this study
(Table VIII) suggest that larval acquisition by molluscs might be
facilitated under conditions of high larval density.
The necessity for infection of the intermediate host to
occur in the spring has not been established. However, the
strategy of releasing greatest numbers of first-stage larvae to
the environment during the winter months appears to have been
adopted by at least two species of metastrongylid nematodes . An
increase in larval shedding by infected definitive hosts has
been reported during the late winter and early spring months for
Protostrongy lus spp. (Forrester and Senger 1964; Uhazy et al. 1973)
and P. odocoilei (Platt 1978; Samuel, unpub.). Platt (1978)
discussed this phenomenon in relation to the epizoot iology of
P. odocoilei in Jasper National Park. He suggested that dispersal
of mule deer during the summer months would tend to drastically
reduce contact between larvae and molluscs, and infected molluscs
and deer; additionally, larvae shed during the summer months
would run serious risk of desiccation due to only sporadic rain¬
fall at that time. The winter's accumulation of larvae, on the
other hand, would be swept to the protection of the soil,
surviving there to infect molluscs throughout the summer months.
Lengthy survival of larvae in the soil would make it unnecessary
115
that infection of the intermediate host occur only in the spring.
Deer, returning to the wintering grounds in the autumn, would
thus face densities of infected molluscs sufficient to result in
acquisition of P_. odocoi lei by many of the returning deer. There
is evidence suggesting that fawns in Jasper do not acquire
P_. odocoilei until they return to the wintering grounds (Samuel,
unpub . ) .
Seasonal changes in larval output might be unimportant for
the transmission of parasites possessing that characteristic.
It may represent only a host -regulated phenomenon. The scheme
suggested by Platt (1978) for the transmission of P. odocoilei
might be applicable to P. tenuis only if the latter species has
a seasonal fluctuation in larval output. Such information is
lacking for IP. tenuis. Until more information is available on
the extent to which winter is important in the transmission of
P. tenuis, the potential of its reduced freezing resistance to
serve as a means for preventing its spread westwards can only be
speculated on.
The present study can go no further than to suggest that at
least two mechanisms exist by which climatic factors might
influence the transmission of P_. odocoilei and P. tenui s in a
differential manner. One, a greater susceptibility to repeated
desiccation, may serve to prevent the establishment of
P. odocoilei in areas which P. tenuis could occupy. The other,
a greater susceptibility of P. tenuis to freezing (both continuous
and repeated), might also serve to segregate the two species
under certain circumstances.
LITERATURE CITED
Alibasoglu, M. , D.C0 Kradel and H.W. Dunne. 1961. Cerebral
nematodiasis in Pennsylvania deer (Odocoileus virgineanus) .
Cornell Vet. 51:431-441.
Andersen, F.L. and N.D. Levine. 1968. Effect of desiccation on
survival of the free-living stages of Trichostrongy lus
colubriformis. J. Parasitol. 54: 11 7 - 128
Anderson, R.C. 1956. Elaphostrongy lus odocoilei Hobmaier and
Hobmaier, 1934 in the cranial case of Odocoileus virginianus
borealis Miller. Can. J. Zool. 34:167-173.
_ . 1963. The incidence, development, and experimental
transmission of Pneumostrongylus tenui s Dougherty
(Metastrongy loidea : Protostrongy lidae) of the meninges of
the white-tailed deer (Odocoileus virginianus borealis) in
Ontario. Can. J. Zool. 41:775-792.
_ . 1971. Metastrongy loid lungworms. In Davis, J.W. and
R.C. Anderson (Eds.). Parasitic Diseases of Wild Mammals.
The Iowa State University Press, Ames, Iowa. pp . 81-126.
Anonymous. 1973. Environment Canada (Atmospheric Environment):
Canadian Normals. Vol. 1, Temperature (1941-1970); Vol. 2,
Precipitation (1941-1970). Downsview, Ontario.
Arroyo, R. and P. Morera. 1978. Viability of the first stage
larvae of Angiostrongylus costaricensis in rat feces.
J. Parasitol. 64:146.
116
Balasingam, E. 1964. Comparative studies on the effects of
temperature on free-living stages of Placoconus lotoris,
Dochmoides stenocephala , and Ancy lostoma caninum.
Can. J. Zool . 42:907-918.
Behrend, D.F. and J.F. Witter. 1968. Pneumostrongy lus tenuis in
white-tailed deer in Maine. J. Wildl. Manage. 32:963-966.
Belle, EoA. 1959. The effect of microenvironment on the free-
living stages of Bunostomum trigonocephalum.
Can. J. Zool. 37:289-298.
Bhatt, B.D. and R.A. Rohde. 1970. The influence of environmental
factors on the respiration of plant -parasitic nematodes.
J. Nematol. 2:277-285.
Bindernagel, J.A. and R.C. Anderson. 1972. Distribution of the
meningeal worm in white-tailed deer in Canada. J. Wildl.
Manage. 36:1349-1353.
Bird, A.F. and M.S. Buttrose. 1974. Ultrastructural changes in
the nematode Anguina tritici associated with anhydrobiosis .
Jo Ultrastruct. Res. 48:177-189.
Boddicker, M.L. and E.J. Hugghins. 1969. Helminths of big game
mammals in South Dakota. J. Parasitol. 55:1067-1074.
Brown, T„T0,Jr., H.E. Jordan and C0N. Demorest. 1978.
Cerebrospinal parelaphostrongylosis in llamas.
J. Wildl. Dis. 14:441-444.
Brunetti, O.A„ 1969. Redescription of Parelaphostrongy lus (Boev
and Schuls, 1950) in California deer, with studies on its
life history and pathology. Calif. Fish and Game 55:307-316.
118
Bullick, G.R. and J0E. Ubelaker. 1978. Ecological factors
involved in survival of Parastrongy lus cost aricen sis free-
living larvae. Abstract No. 247, Annual Meeting of American
Society of Parasitologists. Chicago Illinois, 5-10 November
19 78.
Burch, J.B. 1962. How to Know the Eastern Land Snails. Wm. C.
Brown Publishers, Dubuque, Iowa. 214 p.
Burns, M.E. 1964. Cryobiology as viewed by the microbiologist.
Cryobiology 1:18-39.
Carpenter, J.R. 1940. The grassland biome.
Ecol. Monogr. 10:617-684.
Carpenter, J„W. , H. E. Jordan and J.A. Morrison. 1972. Meningeal
worm (Parelaphostrongy lus tenui s) infection in white-tailed
deer in Oklahoma. J. Wildl. Dis. 8:381-383.
Collis-George , N. 1959. The physical environment of soil
animals. Ecology 40:550-557.
Conder, G.A. 1978. The effect of UV radiation on survival of the
free-living stages of Haemonchus contortus. Proc. helminthol.
Soc. Wash. 45:230-232.
Crofton, H.D. 1963. Nematode parasite population in sheep and on
pasture. Commonwealth Agric. Bur. Technical Comm. No. 35.
104 p.
Crowe, J„H. and K.A.C. Madin. 1975. Anhydrobiosi s in nematodes:
evaporative water loss and survival. J. exp. Zool. 193:323-
333.
119
Davtian, E.A. 1949. [Larval development of lung nematodes of
sheep and goats in Armenia]. Zool. Sbornik AN Arm. SSR
Vol . 6. (cited by Morev, Yu. B. 1966) (In Russian).
DeGiusti , DCL. 1955. The occurence of a protostrongy lid
nematode in the meninges of Michigan deer. J. Parasitol.
(Suppl.) 41:42.
Dougherty, E.C. 1945. The nematode lungworms (Suborder Strongylina)
of North American deer of the genus Odocoileus.
Parasitology 36:199-208.
Ellenby, C. 1968a. Desiccation survival of the infective larva
of Haemonchus contortus. J. Exp. Biol. 49:469-475.
_ . 1968b. The survival of desiccated larvae of Heterodera
rostochiensis and H. schachtii . Nematologica 14:544-548.
_ . 1969. Dormancy and survival in nematodes. Soc. Exp.
Biol. Symposium No. 23. pp. 83-97.
Evans, A.A.F. and R.N. Perry. 1976. Survival strategies in
nematodes. In Croll, N.A. (Ed.). The Organisation of
Nematodes. Academic Press, London, pp. 383-424.
Forrester, D.J. and C.M. Senger. 1963. Effect of temperature and
humidity on survival of first stage Protostrongy 1 us sti lesi
larvae. Exp. Parasitol. 13:83-89.
_ and _ . 1964. A survey of lungworm infection in
bighorn sheep of Montana. J. Wildl. Manage. 28:481-491.
Gibbs, H.C. 1973. Transmission of parasites with reference to the
strongyles of domestic sheep and cattle. Can. J. Zool.
51:281-289.
1
120
Gordon, H. McL. 1948. The epidemiology of parasitic diseases,
with special reference to studies with nematode parasites of
sheep. Aust. vet. J. 24:17-45.
Gupta, SoP0 1961. The effects of temperature on the survival and
development of the free-living stages of Trichostrongylus
retortaeformis Zeder (Nematoda) . Can. J. Zool. 39:47-53.
Hansson, I. 1974. Seasonal and environmental conditions affecting
the invasion of mustelids by larvae of the nematode
Skr j abingy lus nasicola. Oikos 25:61-70.
Herlich, H. 1966. Effects of cold storage on the infectivity of
third-stage larvae of the intestinal worm, Trichostrongylus
colubriformis (Giles, 1892) Ransom, 1911. Proc. helminthol.
Soc. Wash. 33:101-102.
Hobmaier, A. and M. Hobmaier. 1930. Life history of
Protostrongy lus ( Synthetocaulus) ruf escens . Proc. Soc. exp.
Biol. Med. 28:156-158.
and _ . 1934. Elaphstrongylus odocoi lei n. sp., a
new lungworm in black tail deer (Odocoileus columbianus) .
Description and life history. Proc. Soc. exp. Biol. Med.
31:509-514.
Hsu, C. -K. and N.D. Levine. 1977. Degree-day concept in
development of infective larvae of Haemonchus contor tus and
Trichostrongylus colubrif ormi s under constant and cyclic
conditions.
Am. J. vet. Res. 38:1115-1119.
121
Karns, P0D0 1967. Pneumostrongy lus tenuis in deer in Minnesota
and implications for moose. J. Wildl. Manage. 31:299-303.
Kates, K0Co 1965. Ecological aspects of helminth transmission
in domesticated animals. Am. Zoologist 5:95-130.
Kearney, S0R. and F.F. Gilbert. 1978. Terrestrial gastropods
from the Hims'worth Game Preserve, Ontario, and their
significance in Pare laphostrongy lus tenuis transmission.
Can. J. Zool. 56:688-694.
Keilin, D. 1959. The problem of anabiosis or latent life:
history and current concept. Proc. R. Soc. Lond. Ser. B
150:149 -191.
Lankester, M.W. and R.C. Anderson. 1968. Gastropods as
intermediate hosts of Pneumostrongy lus tenuis Dougherty of
white-tailed deer. Can. J. Zool. 46:373-383.
Levine, N„D. 1963. Weather, climate, and the bionomics of
ruminant nematode larvae. Adv. vet. Sci. 8:215-261.
_ . 1968. Nematode Parasites of Domestic Animals and of
Man. Burgess Publishing Company, Minneapolis, Minn. 600 p.
_ and K. So Todd, Jr. 1975. Micrometeorological factors
involved in development and survival of free-living stages
of the sheep nematodes Haemonchus contor tus and
Trichostrongy lus colubriformis . A review. Int . J. Biometeor.
19 : 174-183.
Lorentzen, G. and 0. Halvorsen. 1976. The biology of the free-
living first stage larva of Elaphostrongy lus rangif eri
(Nematoda, Metastrongy loidea) . Proc. Scand. Soc.
Parasitology, 20-22 August, 1976. p. 461.
Matekin, P.V. , E.S. Turlygina and N.M. Shalaeva. 1954.
[Contribution to the biology of the larvae of protostrongy lid
of sheep and goats and epizootiology of protostrongylosis
in Central Asia]. Zoologichesky Zhurnal 33:373-394. (cited
by Forrester, D.J. and C.M. Senger 1963) (In Russian) „
Mitskevich, V0Y. 1964. [Cycle of development of Elaphostrongy lus
rangiferi Miz. 1958]. In Boev, S.N. (Ed.). Parazity
Sel'skokh. Zhiv. Kazakh. Izdatel. Akad. Nauk. Kazakh. SSSR.
3:49-60. (In Russian).
Nath, S. 1978. Effect of relative humidity on the free-living
stages of Strongy loides papi 1 losus (Rhabdiasoidea: Nematoda) „
Experientia 34:580-582.
Nettles, V0Fo and AoK0 Prestwood. 1976. Experimental
Par elaphostrongy lus ander soni infections in white-tailed
deer. Vet. Pathol. 13:381-393.
Nickel, S. 1960. Das Verhalten der freilebenden Erstlarve des
kleinen Lungenwurmes Muellerius capil laris in Freiland und
Lobor. Angew. Parasit. 1:88-92. (Abstract).
O'Brien, F.EoM0 1948. The control of humidity by saturated
salt solutions. J. Sci. Instr. 25(3):73-76.
I
123
O'Roke, EoC, 1936. The lungworm situation in the white-tailed
deer (Odocoi leus virginianus borealis) in Michigan.
Proc. North Am. Wildl. Conf. 3-7 February, 1936. pp . 473-478.
Perry, RoN0 1977a. Desiccation survival of larval and adult
stages of the plant parasitic nematodes, Pity lenchus dipsaci
and D. myceliophagus . Parasitology 74:139-148.
_ • 19 77b_. The water dynamics of stages of Pity lenchus
dipsaci and D. myceliophagus during desiccation and
rehydration. Parasitology 75:45-70.
Pigon, A0 and B. Weglarska. 1955. Rate of metabolism in
tardigrades during active life and anabiosis.
Nature 176:121-122.
Pillmore, R.E. 1956. Investigations of the life history and
ecology of the lungworm, Proto strongy lus stilesi . Federal
Aid Div. Quart. Rep. Colorado Department of Game and Fish
47-70.
Platt, T0R0 1978. The life cycle and systematics of
Par elapho strongy lus odocoilei (Nematoda :Metastrongy loidea) ,
a parasite of mule deer (Odocoi leus hemionus hemionus) , with
special reference to the molluscan intermediate host.
Ph. D„ Thesis. University of Alberta. 233 p.
_ and W„M. Samuel. 1978a. A redescription and neotype
designation for Pare laphostrongy lus odocoilei (Nematoda:
Metastrongy loidea) . J. Parasitol. 64:226-232.
)
124
_ and _ . 19 78b. Parelaphostrongy lus odocoi lei : life
cycle in experimentally infected cervids including the mule
deer, Odocoileus h. hemionus. Exp. Parasitol. 46:330-338.
Poole, J.Bo 1954. Studies on the microenvironment of the free-
living stages of nematodes. Ph. D. Thesis. McGill University.
115 p. (cited by Todd, K.S., Jr., N.D. Levine and
C.Co Whiteside 1970).
_ . 1956. Reaction to temperature by infective larvae of
Nematodirus f ilicollis , Trichostrongy lidae (Nematoda) .
Can. Jo comp. Med. 20:169-172.
Prasad, D. 1959. The effects of temperature and humidity on the
free-living stages of Trichostrongy lus re tor taef ormis .
Can. J. Zool. 37:305-316.
Prestwood, A»K„ 1972. Parelaphostrongy lus ander soni sp. n.
(Metastrongyloidea : Protostrongy lidae) from the musculature
of white-tailed deer (Odocoileus virginianus) .
J. Parasitol. 58:897-902.
_ and V.F0 Nettles. 1977. Repeated low-level infection of
white-tailed deer with Parelaphostrongy lus ander soni .
J. Parasitol. 63:974-978.
_ , _ and F.E0 Kellogg. 19 74. Distribution of
muscleworm, Parelaphostrongy lus ander soni , among deer of
the southeastern United States. J. Wildl. Dis. 10:404-409.
and J.F. Smith. 1969. Distribution of meningeal worm
(Pneumostrongy lus tenuis) in deer in the southeastern United
States. J. Parasitol. 55:720-725.
125
Pursglove, S.R., Jr. 1977. Helminth parasites of white-tailed
deer (Odocoileus virginianus) from New Jersey and Oklahoma.
Proc. helminthol . Soc. Wash. 44:107-108.
Rogers, W„PC and R.I. Sommerville. 1963. The infective stages
of nematode parasites and its significance in parasitism.
Adv. Parasitol. 1:109-177.
Rojo-Vazquez, F.A. 1976. Effect of temperature on the development
of pre-parasitic stages of Trichostrongy lus axei and
T. colubriformis. Rev. Iber. Parasitol. 36:249-255
Rose, J0Ho 1957. Observations on the bionomics of the free-
living first stage larvae of the sheep lungworm, Muellerius
capillaris. J. Helminthol. 31 : 17-28.
Rdssner, J0 and R.N. Perry. 1975. Water loss and associated
surface changes after desiccation in Roty lenchus robustus .
Nematologica 21:438-442.
Rowe, J.S„ 1972. Forest Regions of Canada. Department of
Fisheries and the Environment, Canadian Forest Service.
Publ c No. 1300. 172 p.
Samuel, W.M. and J.C. Holmes. 1974. Search for elaphostrongy line
parasites in cervids from Alberta. Can. J. Zool. 52:401-403.
_ and D.O. Trainer. 1969. A technique for survey of some
helminth and protozoan infections of white-tailed deer.
J. Wildl. Manage. 33:888-894.
Schmidt, J.M. , K.S. Todd Jr. and N.D . Levine. 1974. Moisture
stress effects on survival of infective Trichostrongy lus
colubriformis larvae. J. Nematol. 6:27-29.
I
126
Senger , C.M. 1964. Lethal effect of ultraviolet radiation on
free-living nematodirid larvae.
J. Parasitol . 50 (3, Sec. 2):56.
Smith, Ho Jo and R0 McG. Archibald. 1967. Moose sickness, a
neurological disease of moose infected with the common
cervine parasite, Elaphostrongy lus tenui s . Can. Vet.
Jour. 8:173-177.
Snedecor, G0W0 and W.G. Cochran. 1967. Statistical Methods.
The Iowa State University Press, Ames, Iowa. 593 p.
Sokal, R0R0 and F.J. Rohlf. 1969. Biometry. W.H. Freeman and
Company, San Francisco. 776 p.
Stock, T0Mo 1978. Gastro-intestinal helminths in white-tailed
deer (Odocoileus virginianus) and mule deer (Odocoileus
hemionus) of Alberta: a community approach. M. Sc. Thesis.
The University of Alberta. Ill p.
Thurston, D0Ro and R„G. Strout. 1978. Prevalence of meningeal
worm (Par elaphostrongy lus tenuis) in white-tailed deer from
New Hampshire. J. Wildl. Dis. 14:89-96.
Todd, KoSo, Jr., N0D. Levine and P.A. Boatman. 1976. Effect of
desiccation on the survival of infective Haemonchus contor tus
larvae under laboratory conditions. J. Parasitol. 62:247-249.
_ , _ and B0N. Wagher. 1977. Effects of repeated
desiccation on survival of infective Cooper ia punctata
larvae. J. Parasitol. 63:956-957.
127
_ , _ and C.C. Whiteside. 1970. Moisture stress
effects on survival of infective Haemonchus contor tus larvae.
Jo Nematol. 2:330-333.
Turner, J0H0 1953. Viability studies on eggs and infective
larvae of Nematodirus spathiger (Railliet, 1896), a
tr ichostrongy lid nematode of ruminants.
Parasitology 39:589-598.
Uhazy, L„S., J.C. Holmes and J.G. Stelfox. 1973. Lungworms in
the Rocky Mountain bighorn sheep of western Canada.
Can. J. Zool. 51:817-824.
Winston, P.W. and D.H. Bates. 1960. Saturated salt solutions
for the control of humidity in biological research.
Ecology 41:232-237.
Worley, D.E. and C„D. Eustace. 1972. Prevalence of helminth
parasites in mule deer from eastern Montana.
Proc. helminthol. Soc . Wash. 39:135-138.
Appendix I. History of first-stage larvae used in this study.
Information is arranged in the following order:
1) experiment number; 2) species of Par elaphostron -
gy lus and source; 3) host animal from which larvae
on feces were obtained; 4) date feces were collected
from the host animal;
5)
the temperature
at which
the feces were stored;
6)
the date larvae
were
recovered from the fee
es ;
and 7) the date
the experi-
ment was initiated.
128
I
129
Experi¬
ment #
Species
Host
animal
Collected
from deer
Temperature
stored (°C)
Recovered
from feces
Exp ' t
started
1
P. o. ( J)
MD-7
29 -Nov -77
8
29 -Nov -77
30 -Nov -77
2
P.o. (J)
MD-6,8
14 -Dec-77
8
15 -Dec -77
16 -Dec-77
3
P. o. (J)
MD-?
?
8
22 -Feb -78
1 -Mar -78
P.o. (V)
BTD-?
?
8
22 -Feb -78
1 -Mar -78
4
P.o. (J)
MD-?
?
-25
10 -Mar -78
15 -Mar -78
P.o.(V)
BTD-?
?
-25
10 -Mar -78
15 -Mar -78
5
P.o. (J)
MD-1
Feb-76
-25
25 -Oct -78
25 -Oct-78
MD -3
Dec -75
-25
26 -Oct -78
26 -Oct -78
6
P.o.(J)
MD-4
Mar-77
-25
25 -Apr -79
1 -May -79
P.t.
WTD
early-78
-25
25 -Apr -79
1 -May - 79
7
P.o. (J)
MD-?
?
-25
7
10 -F eb -78
8
P.o. ( J)
MD-?
?
-25
7
22 -Mar -78
9
P.o. (J)
MD-1
Feb-76
-25
26 -Apr -78
1 -May -78
p.t.
WTD
early-78
-25
26 -Apr -78
1 -May -78
10
P.O.(J)
MD-?
?
-25
7
21 -Feb -78
11
P.o. (J)
MD-?
?
-25
7
1 -Mar -78
12
P.O.(J)
MD-?
?
-25
7
23 -Mar -78
13
P.o. (J)
MD-1
Feb-76
-25
26 -Apr -78
3 -May -78
p.t.
WTD
early-78
-25
26 -Apr -78
3 -May -78
14
P.o. (J)
MD-1
Feb-76
-25
26 -Jun-78
14-Aug-78
p.t.
WTD
early-78
-25
26 -Jun-78
14 -Aug-78
15
P.o. (J)
MD-?
7
-25
7
16 -Mar -78
I
1
130
Experi -
ment #
Species
Host
animal
Collected
from deer
Temperature
stored (°C)
Recovered
from feces
Exp 1 1
started
16
P.o. (J)
MD -1
Feb-76
-25
26 -Apr -78
2 -May -78
17
P.o. (J)
MD -1
Feb-76
-25
26 - Jun -78
14 -Aug -78
18
P.o. (J)
MD -20
12 -Dec -78
8
13 -Dec -78
22 -Dec -78
MD-20
14 -Nov-78
-25
13 -Dec -78
22 -Dec -78
P.t.
WTD -25
12-Dec-78
8
13 -Dec -78
22 -Dec-78
WTD -25
14 -Nov -78
-25
13 -Dec -78
22 -Dec -78
Abbreviations used:
P.o.(J)- P. odocoilei of Jasper Park origin
P.o.(V)- P. odocoilei of Vancouver Island origin
P . t . - P. tenuis
MD - captive mule deer; number follows
BTD - captive blacktailed deer; number follows
WT - captive white-tailed deer; number follows
WTD - wild white-tailed deer
?
information not noted
1
Appendix II. Mean percent survival with 957o confidence limits
for first-stage larvae of P_. odocoilei at a variety
of temperature and moisture combinations (Exp'ts
1,2).
131
132
Percent Temperature Days
RH (°C)
Number of Mean percent
samples survival
95% confidence
limits
Lower
Upper
5
6
4
96.8
93.2
99.1
12
4
97.7
95.6
99.1
124
2
71.1
0
100
14
16
8
75.9
64.5
85.7
32
4
70.5
33.9
96.0
56
4
57.7
29.1
83.8
87
4
41.5
6.7
82.3
121
2
41.1
0
100
26
4
8
91.7
83.4
97.3
12
8
75.8
53.5
92.4
31
4
48.4
22.7
74.7
38
3
0.9
0
22.3
36
2
4
96.3
90.7
99.4
4
12
30.2
8.0
59.1
7
4
0.2
0
4.2
9
4
0
0
0
48
1
4
57.4
0.3
100
1.5
4
0
0
0
5
6
4
65.9
62.4
69.3
14
8
80.7
71.8
88.3
32
8
56.3
38.3
73.5
72
4
62.0
34.7
85.7
124
4
11.2
2.3
25.6
142
4
7.9
3.7
12.9
14
4
4
87.2
78.6
93.8
14
4
77.5
70.6
84.0
24
4
79.1
67.1
89.0
33
4
54.9
35.2
73.8
56
4
53.7
39.8
67.3
87
4
41.9
33.9
50.2
121
4
11.9
0
44.7
154
4
1.2
0
10.9
26
4
8
51.5
32.7
70.0
10
4
4.1
3.4
4.8
13
4
0
0
0
36
2
4
0.2
0
2.8
4
4
0
0
0
133
Percent
RH
Temperature Days Number of Mean percent
(°C) samples survival
95% confidence
limits
Lower Upper
95
48
1
5
0.7
0
5.7
(cont ' d)
2
4
0
0
0
85
5
6
4
89.0
71.3
98.8
12
4
79.5
71.5
86.4
32
8
47.1
31.5
62.9
72
4
45.0
26.0
64.9
124
4
18.7
9.4
30.2
142
4
8.2
4.9
12.3
175
4
1.5
0
6.7
36
2
4
1.1
0
4.8
4
4
0
0
0
48
1
5
1.0
0
4.9
2
4
0
0
0
75
5
9
8
88.2
59.2
100
28
4
83.2
67.3
94.5
75
4
59.5
36.4
80.6
124
4
49.6
11.7
87. 7
175
4
24.7
1.5
63.5
306
4
9.2
3.2
17.8
14
14
4
84.0
75.6
90.9
32
4
62.4
50.4
73.7
59
4
57.8
43.1
71.8
87
4
47.9
36.5
59.5
121
4
20.2
0
69.7
154
4
31.1
8.4
60.3
194
4
12.1
6.9
18.4
289
4
0
0
0
26
4
8
67.2
58.5
78.0
12
8
33.3
18.4
50.2
18
4
34.6
1.8
81.3
24
4
18.7
0
63.6
31
4
7.1
0.7
19.3
38
4
1.4
0
10.7
36
2
4
1.0
0
4.5
4
4
0
0
0
48
1
5
0.1
0
1.0
3
4
0
0
0
134
Percent
RH
Temperature Days Number of Mean percent
(°C) samples survival
95% confidence
limits
45
20
5
14
26
36
48
14
26
Lower
Upper
6
4
98.8
82.8
100
12
4
98.5
97.6
99.2
47
4
99.0
95.5
100
124
4
96.5
91.4
99.3
306
3
45.2
12.1
80.9
14
4
91.5
87.4
94.9
32
4
93.5
86.0
98.3
87
4
94.2
89 .9
97.4
125
4
91.1
78.9
98.4
194
4
50.9
45.3
56.4
289
4
16.6
3.5
36.8
3
4
85.2
74.1
93.6
10
4
63.9
26.8
97.2
14
8
73.1
55.1
87.8
31
4
45.1
28.4
62.5
46
4
26.8
9.3
49.4
58
4
3.9
0
27.0
68
4
0
0
0
2
4
95.7
89.6
99.2
4
8
24.7
11.6
40.8
7
4
9.0
0.8
24.8
9
4
0
0
0
1
4
93.8
84.4
99.1
3
4
11.4
1.8
27.8
4
4
0.1
0
2.2
5
4
0
0
0
14
4
90.7
85.3
94.9
32
4
93.7
90.7
96.1
87
4
95.6
88.7
99.4
125
4
92.9
85.9
97.6
194
4
52.2
44.9
59.5
289
4
19.1
12.7
26.5
3
4
94.4
87.7
98.5
10
4
90.4
79.9
97.3
14
8
74.2
58.8
87.1
31
4
52.6
33.0
71.8
46
4
30.7
20.2
42.5
58
4
8.7
0
37.4
68
4
0
0
0
135
Percent
RH
20
(cont ' d)
Temperature Days Number of Mean percent
(°C) samples survival
95% confidence
limits
Lower Upper
36
3
4
89.4
84.3
93.6
5
4
71.1
46.4
90.4
7
4
52.7
5.1
97.1
9
4
2.6
0
15.4
12
4
0
0
0
48
1
4
96.5
93.8
98.5
3
4
32.2
13.5
54.5
4
4
5.8
0
21.6
5
4
5.3
0.8
13.4
7
4
0.5
0
4.2
.
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11