EPA-600/3-77-051
August 1977
Ecological Research Series
CULTURING AND ECOLOGY STUDIES
OF THE ROTIFER POLYARTHRA VULGARIS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-77-051
August 1977
CULTURING AND ECOLOGY STUDIES OF THE
ROTIFER, POLYARTHRA VULGARIS
by
Arthur L. Buikema, Jr.
John Cairns, Jr.
Paul C. Edmunds
Thomas H. Krakauer
Department of Biology
and
Center for Environmental Studies
Virginia Polytechnic Institute and State University
Blacksburg, Virginia 24061
Grant No. R800815
Project Officer
Richard Anderson
Environmental Research Laboratory
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U.S. Environmental Protection Agency, nor does mention of
trade names or commerical products constitute endorsement or recommendation
for use.
ii
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FOREWORD
Our nation's fresh waters are vital for all animals and plants, yet our
diverse uses of water for recreation, food, energy, transportation, and
industry -- physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in water.
The Environmental Research Laboratory in Duluth, Minnesota, develops methods,
conducts laboratory and field studies, and extrapolates research findings
to determine how physical and chemical pollution affects aquatic
life,
to assess the effects of ecosystems on pollutants,
--to predict effects of pollutants on large lakes through use of
models, and
to measure bioaccumulation of pollutants in aquatic organisms that
are consumed by other animals, including man.
This report describes conditions that affect survival and reproduction
Of the freshwater rotifer, Polyarthra vulgaris.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
m
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ABSTRACT
The results contained in this report represent research conducted to iden-
tify variables which affect the survival and reproduction of the rotifer, Poiy-
arthra vulgaris. The following variables were studied: handling stress, con-
tainer size, frequency of changing the culture medium, light quantity and
quality, photoperiod, oxygen and vitamin requirements, fungal parasites, food
preference and concentration, antibiotic effects of bluegreen algae, and tem-
perature.
Temperature had an effect on population dynamics, percent of females with
eggs, number of eggs per female, and sexual reproduction. Egg production rates
were estimated and observations on the duration of egg development were made.
This report also includes a field study of the relation between Poiyarthra
vulgaris and 19 selected chemical and physical parameters.
This report was submitted in fulfillment of Grant Number R800815 by
Virginia Polytechnic Institute and State University, and the research was part-
ically supported by the Environmental Protection Agency. It covers a period
from February 1, 1973, to June, 1974, and the work was completed in June, 1974.
1v
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CONTENTS
Foreword 1-|i
Abstract iv
Figures v11
Tables viii
Acknowledgments x
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Factors Affecting Survival and Reproduction of
Polyarthra vulgaris 6
Materials and Methods 7
Results and Discussion 8
Handling 8
Culture Containers 8
Light 10
Oxygen Requirements 10
Vitamin Requirements 10
Fungi and Bacteria 13
Food 15
Antibiosis 20
Temperature 20
Egg Development 20
5. The Effects of Temperature on Reproduction of
Polyarthra vulgaris 23
Materials and Methods 23
Results 24
Discussion 26
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6. Natural Population Dynamics Of Polyarthra vulgaris. ... 30
Materials and Methods 30
Description of Study Area 30
Sampling and Analytical Techniques 30
Results 32
Pond Chemistry 32
Bacteria 33
Chlorophyll-a 33
Rotifer populations 35
Relation between Polyarthra vulgaris and
Environmental Parameters 35
Discussion 39
References 44
Publications 51
Appendix 52
vi
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FIGURES
Number Page
1 Effect of temperature on populations of Poiyarthra
vulgar is 21
2 Effect of 10 and 30 C temperature on populations of
Poiyarthra vulgaris 25
3 Effect of 20 C and room temperature (21 +2 C) on
populations Of Poiyarthra vulgaris 26
4 Relationship among population numbers, percent
ovigerous females, number of eggs per ovigerous
female and sexual reproduction for one culture of
Poiyarthra vulgaris at 20 C 27
5 Relationship among population numbers, percent
ovigerous females, number of eggs per ovigerous
female and sexual reproduction for one culture of
Poiyarthra vulgaris at room temperature 28
6 Map of Pandapas Pond, Montgomery County, Virginia,
showing the two collection stations 31
7 Seasonal fluctuations of photoperiod, temperature, and
mean number of Poiyarthra vulgaris per liter 37
vii
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TABLES
Number Page
1 Chemical Analysis of Culture Waters ........... 9
2 Effects of Red, Green, and Blue Wavelengths and Full
Spectrum Light on Poiyarthra vulgaris Populations ... 11
3 Vitamin Content of Various Foods and Waters ....... 12
4 Effect of Vitamin Mixtures on Populations of
Poiyarthra vulgaris After One Week .......... 13
5 Effects of Various Foods plus Vitamins and Trace
Metals on Survival and Reproduction of Poiyarthra
vulgaris ....................... 14
6 Elements in Food Media Available to Poiyarthra vulgaris . 15
7 Culture Media Used for Cryptomonas ovata ........ 16
8 Potential Food Organisms Tested on Poiyarthra vulgaris . 17
9 Effect of Various Foods on Survival and Egg Production
for Poiyarthra vulgaris ................ 18
10 Effect of Various Concentrations of Protozoans (No
Vitamins) on Survival and Reproduction of Poiyarthra
vulgaris ....................... 19
11 Significant Correlations Between Environmental
Parameters and Poiyarthra vulgaris at Station 1 .... 33
12 Nonsignificant Correlations Between Environmental
Parameters and Populations of Poiyarthra vulgaris
at Station 1 ..................... 34
13 Significant Correlations Between Environmental
Parameters and Poiyarthra vulgaris at Station 2 .... 35
14 Nonsignificant Correlations Between Environmental
Parameters and Poiyarthra vulgaris at Station 2 .... 36
viii
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Number
15 Stepwise Regression Analysis and Increase in
Coefficient of Determination (R ) for the Total
Number of poiyarthra vulgaris per Liter with No
Lag. Data are for the 35 Micron Net 38
16 Stepwise Regression Analysis and Increase in
Coefficient of Determination (R) for the Total
Number of Poiyarthra vulgaris per Liter with No
Lag. Data are for the 75 Micron Net 39
ix
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ACKNOWLEDGMENT
The facilities of the Biology Department and the Center for Environmental
Studies of Virginia Polytechnic Institute and State University are gratefully
acknowledged. The assistance of Mr. James Geiger was appreciated. Thanks
are due to Dr. Daniel Jones and to Dr. Lawrence Whitford for information on
cryptomonas ovata. Assistance with protozoan identification was provided by
Dr. William H. Yongue, Jr. Drs. Martha Roane and Noel Krieg assisted in the
identification of the fungi and bacteria. Dr. C. Y. Kramer assisted with the
statistical analysis. We are indebted to Dr. Richard Anderson of the Environ-
mental Research Laboratory (formerly National Water Quality Laboratory),
Environmental Protection Agency, Duluth, Minnesota, for his advice.
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SECTION 1
INTRODUCTION
Rotifers are an important component of aquatic ecosystems and their role
varies from herbivores to carnivores to detritivores to combinations of these
types. Rotifers are preyed upon by larval fishes, copepods, other rotifers,
and a variety of other invertebrates including protozoans. As such, they are
important in energy transfer.
The rotifer, Poiyarthra vulgaris, has been classified as a perennial,
eurythermal species which exhibits population maxima in the late spring or
early summer (Carlin, 1943; Pejler, 1957). From the research of Edmondson
(1965), Dieffenbach and Sachse (1911), Pejler (1957), and others, P. vulgaris
has been identified as a herbivore feeding primarily on the alga cryptomonas.
In limited studies rotifers have been used to culture larval fish (Theilacker
and McMaster, 1971; Harada, 1970; Maksinova, 1969). Siefert (1972) studied
the first food of the yellow perch, white sucker, bluegill, emerald shiner,
and the rainbow smelt. Based on electivity indices he concluded that Poiyarthra
were highly selected initial prey for the yellow perch and the bluegill. This
information on feeding was significant for these primary reasons: (1) if the
rotifer Poiyarthra could be cultured, it would facilitate the laboratory
culturing of bluegills or yellow perch, (2) the sensitivity of Poiyarthra to
toxicants in nature could be important in determining a successful year class
of the larval fish that preyed upon it, and (3) the effect of starvation could
be diminished when conducted acute and chronic bioassay studies on fish to
determine application factors.
Poiyarthra vulgaris, while perennial, was most dominant in late spring
and it was never abundant most of the year for research purposes. To our
knowledge, this rotifer has never been cultured in the laboratory. This in-
ability to culture the animal stems from a limited knowledge of the animal and
of the factors which affect its reproductive success.
The objectives of this study were to:
1. Determine what parameters affect survival and reproduction of the
rotifer
2. Determine if healthy cultures of the rotifer could be maintained
in the laboratory
3. Study a field population with emphasis on chemical and physical
parameters.
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This project was supported by a grant (R800815) from the Environmental
Protection Agency. This is the final report for this grant.
The grant was awarded for one year and extended for five months without
additional funds.
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SECTION 2
CONCLUSIONS
With reference to objectives 1 and 2 and the results of this preliminary
research, the following culture conditions were favorable for Poiyarthra
vulgaris:
1. Handling - the rotifers should not be handled any more than
necessary.
2. Containers - glass containers containing a large volume of water,
at least one liter.
3. Culture medium - water from a natural source that contains Poiyarthra
vulgaris. TRe" water should be filtered through a 30 micron mesh net
to remove larger algae and animals.
4. Replacing medium - partially twice a week and totally once a week.
5. Light - an incident illumination of 400 to 500 ft-c, a complete
light spectrum, and a 16L:8D photoperiod.
6. Oxygen - moderate aeration to maintain a concentration near 8 ppm.
7. Vitamins - minimally the vitamins B,2» thiamine, Biotin, and
pantothenic acid may be required by the rotifer. To cultures
containing five liters of water, one-half gram of Vionate was
added after each partial change of culture medium and one gram was
added after each complete change.
8. Food type - Rotifers fed on a mixture of chiiomonas paramecium,
Cyathomonas truncatus, Bodo minimus, B. variabilis, and B. mutabilis
which was raised in a Purina trout chow medium and fortified with
vitamins B,2» thiamine, biotin, and pantothenic acid.
9. Food quantity - 50 ml of this protozoan mixture was added to a
5-11ter culture daily and the protozoan concentration was around
300,000 protozoans per ml.
10. Temperature - within a few degrees of 20 C.
11. Ant1b1ot1c~and parasitic agents - Bacteria of the sphaerotiiis-
Leptothrix complex, fungi, and dense populations of green and
bluegreen algae were detrimental to the rotifer.
12. Population density - if the rotifer density decreased below 40
animals per liter the population usually did not recover.
Objective 2 was partially achieved and cultures were maintained for 70
to 100 days at room temperature (21 +_ 2 C) and at 20 C. Because the popula-
tions cycled and because densities far exceeded natural levels, we had good
culture success but more research is needed.
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Studies on poiyarthra vuigaris population dynamics and reproduction at
different temperatures have been evaluated and the following conclusions
reached:
1. Reproduction does not occur at 10 and 30 C but it does at 20 C
and room temperature (21 +2 C).
2. At or near 20 C population densities may exceed 20,000 per liter.
3. The percent of ovigerous females was lowest when population densities
were highest.
4. Ovigerous females usually carried one egg but they could exceed
three per female. Multiple eggs usually occurred after the popula-
tion peak.
5. Sexual eggs appeared after a population peak.
6. Estimated egg production rate varied from 0.08 to 0.25 eggs per
day.
7. Time for egg development may exceed 24 hr at room temperature and
this may be due to a fluctuating temperature.
In fulfillment of objective 3, the following parameters significantly
correlate with field populations Of Polyarthra vuigaris:
1. Photoperiod - positive correlation
2. Temperature - positive correlation
3. Oxygen - negative correlation (illusory)
4. Ammonia - negative correlation
5. Nitrate - positive correlation (possibly illusory)
6. Orthophosphate - positive correlation (possibly illusory)
7. Sodium - negative correlation
8. Total filtrable solids - negative correlation
Significant variables identified by the stepwise regression analysis were
temperature, photoperiod, orthophosphate, silicon, magnesium, and nitrates.
The other parameters were variable in their effect and not significant.
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SECTION 3
RECOMMENDATIONS
Much more research is needed to optimally culture Poiyarthra vulgaris
for future studies. These needs are:
1. Poiyarthra should be cultured, if possible, in an artificial
medium. This would reduce the antibiotic effects of algae and
protozoans found in pond water and the negative effects of
naturally occurring chemicals such as high ammonia and sodium
concentration.
2. The vitamin requirements of zooplankton are poorly understood.
The results of this study suggests that the B vitamins in solutions
were a major factor controlling successful reproduction and growth.
3. Because the vitamin enriched water also enhanced the growth of
fungi and bacteria, research needs to be conducted on the possible
use of antibiotics to reduce infestations that reduce population
success.
4. The food organisms (protozoans) used in this study were adequate
for culturing. However, more research is needed to identify and
culture more suitable food organisms. In recent work oinobryon sp.
may be the major natural food for Poiyarthra. (Buikema, unpublished),
5. Because natural systems are thermally labile, research is needed
to determine if Poiyarthra reproduction is enhanced by fluctuating
temperatures rather than by static temperatures.
6. Studies should be conducted on the effects of periodic harvesting
of Poiyarthra to determine optimum harvesting rates for minimizing
population fluctuations.
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SECTION 4
FACTORS AFFECTING SURVIVAL AND REPRODUCTION
OF POLYARTHRA VULGARIS
Rotifers have been cultured in filtered natural waters (Edmondson, 1960,
1964a_; Hal bach, 1970a_, 1970b; etc.), in defined media (Gilbert, 1963, 1970;
Maly, 1969; King, 1967; Laderman and Gutlman, 1963; Meadow and Barrows, 1971;
Lynch and Smith, 1931; Shull, 1911; Finesinger, 1926; Buikema, Cairns, and
Sullivan, 1974), and in undefined media (Pennak, 1953; Dougherty, 1960, 1963;
Maksinova, 1969; Halbach, 1970a_) which generally were natural waters innocu-
lated with milk, dried greens, etc. Algal media were not satisfactory (Adachi,
1964; Lansing, 1942, 1947). The rotifers which were cultured were both
littoral and limnetic, and commonly cultured genera include Aspianchna, srachio-
nus, ttonostyla, Kerateiia, and Keiiicottia (Edmondson, 1960, 1964; Gilbert,
1963, 1968; Chu, 1934; Halbach, 1970a_, 1970^, 1972; Maly, 1969; Laderman and
Gutlman, 1963; Dewey Bunting, pers. comm.; etc.).
Numerous problems culturing Kerateiia and Keiiicottia were reported by
Edmondson (1960). These problems included vessel size, shape, and material.
Food was an extremely important variable and two factors were important:
(1) the size of the food particle, and (2) the nutritive value of the food.
Particle size is important for Poiyarthra (Edmondson, 1965; Gossler, 1950)
and populations of Poiyarthra have been positively correlated with the cryp-
tophyte cryptomonas (Edmondson, 1965; Pejler, 1957; Dieffenbach and Sachse,
1911; Pourriot and Hillbricht-Ilkowska, 1969) but Carlin's data (Figures 50
and 101 in 1943) show considerable variation between populations of cryptomonas
and Poiyarthra. This alga varies from 15 to 80 y in length and 8 to 18 y in
width (Prescott, 1951) while the rotifer is 130 to 150 y in length (Bartos,
1959). The feeding observations by Dieffenbach and Sachse (1911) were for
Poiyarthra platyptera and P. euryptera (Hutchinson, 1967) and Cryptomonas
ovata. The studies by Pourriot and Hillbricht-Ilkowska (1969) were for
Poiyarthra trigla and Cryptomonas curvata. The correlation of Edmondson (1964bj
was for Poiyarthra vulgaris and smaller species Of Cryptomonas (14 X 31 y).
Smaller sized organisms probably were not eaten by Poiyarthra vulgaris
(Edmondson, 1965). Food quality and quantity are known to influence population
dynamics of rotifers (Pourriot, 1957; King, 1967; Halbach, 1972).
Supplemental vitamins added to the culture medium may be necessary for
rotifer culture. Vitamin supplementation enhanced egg production of the cope-
pod, rigriopus (Shiraishi and Provasoli, 1959) and of oaphnia (Fritsch, 1953).
Dougherty, Solberg, and Harris (1960) suggest that bacteria may provide
essential nutrients for rotifers. But algae may also because they produce
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vitamins B,2» biotin, and thiamine (Carlucci and Bowes, 1970a_, 1970b).
Temperature is probably the most important factor affecting rotifer re-
production and development. Positive correlations between temperature and
limnetic rotifers have been observed by many workers (Kolisko, 1938; Edmondson,
1960, 1964, 1965; King, 1967; Halbach, 1970a_; Pourriot and Hillbricht-Ilkowska,
1969; Tauson, 1926; and others). The temperature optimum for Poiyarthra vui-
garis is probably between 10 and 20 C (Edmondson, 1965; Carlin, 1943) although
Carlin suggested that there is a population peak in the fall when the temper-
atures are between 5 and 10 C. Duration of life span is also temperature de-
pendent and rotifers generally live longer at lower temperatures (Kolisko,
1938; Edmondson, 1945; etc.). Egg development occurs faster at higher temper-
atures and for Poiyarthra vulgaris it varies from 70 hr at 10 C (Edmondson,
1965) to 23 hr at 20 C. Acclimation also has an effect (Hillbricht-Ilkowska,
1969).
Light may also be an important factor. Long photoperiod or increased in-
tensity may affect populations of Keilicottia (Edmondson, 1965), and rotifers
such as Poiyarthra behaviorally respond to light (iaud, 1943; Hutchinson,
1967; etc.). Light intensity may be an important factor affecting organisms
as it has been suggested for Daphnia pulex (Buikema, 1972, 1973a_, 1973b_, 1975).
Hutchinson (1967) summarized the possible chemical variables that may
affect rotifer populations. The pH may affect rotifer populations (Edmondson,
1944; Adachi, 1964; Harring and Meyers, 1928; Lansing, 1942; Myers, 1931;
Ahlstrom, 1940), and Edmondson (1944) suggests that there may be more than one
factor which produces the apparent limitation due to pH. Pejler (1957) con-
cluded that pH was of no real importance in the distribution of rotifers from
northern Sweden. Not very much is known about planktonic organisms (Hutchinson,
1967). Oxygen concentration may affect populations (Whitney, 1917, 1919;
Tauson, 1925; Adachi, 1964) and egg hatching (Lite and Whitney, 1925) although
some species are able to live in oxygen deficient water (Pejler, 1957; Beadle,
1963). Carbon dioxide, bicarbonate concentration, and calcium can affect re-
production (Tauson, 1925, 1926, 1927; Lansing, 1942) and distribution of some
species (Hutchinson, 1967).
Birky and Gilbert (1971) summarize the literature of variables which con-
trol rotifer sexuality, and temperature, photoperiod, dissolved oxygen, pH,
carbonate, food concentration, diet and population density can be controlled
to limit mictic and amictic organisms.
MATERIALS AND METHODS
The initial series of experiments that were conducted with Poiyarthra
vulgaris were begun by estimating those variables (from the preceding litera-
ture) that could affect reproduction and survival and observing animals under
these conditions in the laboratory in which these were varied.
The animals usually were collected from Pandapas Pond located 6 km north-
west of Blacksburg, Montgomery County, Virginia, and on occasion from Carvins
Cove Reservoir, Botetourt County, located 4 km north of Roanoke, Virginia.
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Field collections were made with a 35 micron mesh net towed through the
surface water. Concentrations of zooplankton were transported back to the
laboratory in 18 liter polypropylene containers of pond or reservoir water.
Cultures of animals were isolated from other zooplankton, placed in filtered
pond or reservoir water, and slowly warmed to the experimental temperature.
Preliminary experiments were conducted at room temperature (21 +_ 2 C) and
at a long photoperiod (16L:8D). Later experiments were conducted in Scherer-
Gilette CEL 4-4 growth chamber.
To our knowledge, this rotifer has never been cultured. Our preliminary
experiments were based on simple observations of the rotifer under various
conditions, and three criteria were used to indicate favorable conditions:
(1) survival, (2) appearance of eggs, and (3) hatching of eggs. The follow-
ing results and discussion summarize this preliminary research.
RESULTS AND DISCUSSION
Handling
This rotifer was quite sensitive to handling and death usually occurred
within 8 to 12 hr after transfer. If the animal survived the first 12 hr 1t
may survive for as long as 8 days. Handling with larger bore pipettes (1 mm)
reduced death rate to a certain extent. Handling of the rotifers also in-
creased the release of the egg (or eggs) which were carried by the female.
Culture Containers
Rotifers were placed in 0.5 mm depression slides, 5.0 ml shallow spot
plates, 10.0 ml dish, 15.0 ml test tubes, 125.0 ml Erlenmeyer flasks, and 5.0
liter containers. Survival and egg production increased as the container size
increased; this was most notable 1n the 5.0 liter container. All containers
were glass except the 5.0 ml spot plate which was a polycarbonate plastic.
The small glass containers were pyrex and the largest one was soft glass. A
relationship between large containers and culture success has been demonstrated
for the copepod Diaptomis ciavipes (Robertson, Gehrs, Hardin and Hunt, 1974)
and for rotifers (Edmondson, 1960). For Poiyarthra the minimum size for a
container appeared to be 10 ml.
Culture Water
Two sources of water were used for culture experiments (Table 1). This
water came from sources that had Poiyarthra vulgaris populations. Water was
collected in 18-liter polypropylene containers and stored at room temperature
for no more than 2 weeks. The water was filtered through a 35 micron mesh net
and/or glass fiber filters prior to use. Pandapas Pond water was usually used.
The filtered water was used for setting up all the culture experiments.
Two initial experiments were conducted to determine if the culture medium had
to be replaced to insure culturing success. Survival and egg production
8
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TABLE 1. CHEMICAL ANALYSIS OF CULTURE WATERS
(Except for pH, all values are in mg/1)
Parameter
Total Hardness (CaC03)
Total Alkalinity (CaC03)
PH
co2
Iron
Manganese
Nitrate
Sulfate
Silicon
Magnesium
Calcium
Orthophosphate
Total Phosphate
Potassium
Sodium
Ammonia
Total Filtrable Solids
Pandapas Pond
Seasonal Range of Means
9.00 - 22.1
6.20 - 31.4
6.40 - 8.30
0.00 - 17.30
0.04 - 2.15
__ _
0.00 - 0.57
0.40 - 4.80
5.00 - 11.70
1.08 - 2.11
0.96 - 4.86
0.00 - 0.09
0.002- 1.99
0.99 - 1.86
2.95 - 6.22
0.04 - 0.70
17.20 -143.20
Carvins Cove*
42 - 56
48.0
8.0
1.0
0.02
0.01
0.0
16.0
3.4
1.9
20.04
0.01
1.18
2.0
2.0
90.0
*December, 1972, from Roanoke City Water Authority.
increased if the water was replaced periodically. In our subsequent culture
experiments, one-third of the culture water was changed every two days. The
water was passed through a 30 micron net and the animals were returned to the
aquarium. Once a week the entire water was replaced in each culture.
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Three lighting variables, photoperiod, light intensity, and wave length
range, were tested for their effect on rotifer survival and reproduction. All
experiments were conducted using GE cool white fluorescent bulbs (20 and 40 W).
Three photoperiods were set up, 8L:16D, 12L:12D, and 16L:8D. The results
appeared better under the longer photoperiod, but there were not statistically
significant differences among the results.
The effect of light intensity on population numbers was also examined.
The light intensities studied were 1500, 500, and 100 incident ft-c measured
with a GE photometer. No detectable differences in population numbers were
noted among the light intensities. However, when observations were made of
the rotifers behavioral response in a light intensity gradient, maximum aggre-
gration occurred at approximately 400 ft-c.
Wavelength effects also were examined using Rohm and Haas plexiglass fil-
ters and unfiltered light at 400 ft-c. Specifically the filters were #2400
(red), #2092 (green), and #2264 (blue). Respectively, the filter transmissions
were 5.7%, 21.4%, and 2.9%. There were some differences among the light con-
ditions (Table 2) and among the wavelengths; red was more favorable. In com-
paring the data between partial and full spectrum, population numbers increased
more rapidly under the full spectrum.
Buikema (1973a_) found that reproduction of Daphnia pulex was inhibited by
red wavelengths and stimulated by blue wavelength. These results were opposite
those obtained for Poiyarthra. In comparing the effects of wavelength and
light intensity on reproduction of Daphnia pulex the results were variable de-
pending on the intensity examined and 14 ft-c was the best. These differences
in numbers between light quality for Poiyarthra may reflect light effects in
survival as they may for Daphnia pulex (Buikema, 1973a_).
Oxygen Requirements
The rotifers were very sensitive to oxygen deficiency and aeration was
mandatory. Survival of the animals was less than 12 hr under low oxygen con-
ditions. These results are consistant with field observations of the rotifers
sensitivity to low oxygen (Abel, 1972; Pejler, 1957; this study).
Vitamin Requirements
Egg production was not observed in the laboratory until vitamin mixtures
were added to the protozoan food medium which already contained No. 3 Purina
Trout Chow (Table 3). The vitamins B,2, pantothenic acid, thiamine, and biotin
promoted egg production (Table 4) and survival (Table 5) whether added as a
mixture (#1) or as Vionate, a commercial pet food supplement.
Dieffenbach and Sachse (1911), Pejler (1957), and Edmondson (1965) have
noted positive correlations between Poiyarthra vulgaris populations and the
alga cryptomonas. We observed (see below) that Poiyarthra vulgaris would not
feed on cryptomonas ovata - even though it meets the criteria of size and
10
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TABLE 2. EFFECT OF RED, GREEN AND BLUE WAVELENGTH AND
FULL SPECTRUM LIGHT ON POLYARTHRA VULGARIS POPULATIONS
Day
0
1
3
6
8
10
13
15
17
19
21
23
25
29
31
33
35
Red
200
120
40
80
0
80
80
160
40
40
<40
120
200
760
2480
3320
3080
Number
Green
200
80
160
160
80
120
200
200
40
40
40
<40
160
<40
<40
0
0
of Rotifers/Liter
Blue Full
200
320
80
0
120
100
200
160
160
<40
40
<40
120
<40
40
0
0
Spectrum
200
160
120
160
120
40
560
680
920
1000
1920
3680
5080
3600
8080
4280
600
11
-------
TABLE 3. VITAMIN CONTENT OF VARIOUS FOODS AND WATERS
Vitamin
A
D,
Vionate*
(dry
per gram)
220.75 units
22.07 units
Vionate in
pond water
(cone. /liter
of culture water)
44.15 units
4.41 units
Purina #3 Mixture #1
Trout food (cone. /liter
(dry per of
pound)t culture water)
-
B-j (Thiamine)
B2 (Riboflavin)
Bg (pyridoxine)
B12
Pantothenate
Niacin
Folic Acid
Choline Chloride
C
E
Biotin
0.0397 mg
0.0795 mg
0.01 mg
0.000155 mg
0.110 mg
0.276 mg
0.0022 mg
5.7395 mg
2.503 mg
0.120 units
0.0079 ygram
0.0159 pgram
0.002 pgram
0.00031 pgram
0.022 pgram
0.0552 pgram
0.00044 pgram
1.1480 pgram
0.500 pgram
0.024 units
33 ppm 0.20 mg
18 gram 0.001 mg
160 ppm 0.20 mg
1.1 ppm 0.20 mg
*Vionate is made by Squibb and Sons, Inc. The diluent is soy grits, gelatin,
sucrose, dried skim milk and corn germ meal.
tThese are added amounts and they do not include natural concentrations in
the peruvian menhaden fish meal.
12
-------
TABLE 4. EFFECT OF VITAMIN MIXTURES ON POPULATIONS
OF POLYARTHRA VULGARIS AFTER ONE WEEK
(Rotifers were fed vitamin enriched cultures of chiiomonas
paramecium, Cyathomonas truncata, Bodo minimus, B. variabilis
and B. mutabilis)
Population after
Initial population one week
Vitamins (number/liter) (number/liter)
None 200 <40
3 cc of Mixture #1 200 <300
3 cc of Mixture #1
plus mud 200 <40
2 gm of Vionate 200 300-600
3 cc of Mixture #1
and 2 gm of Vionate 200 <2000*
*eggs present
presumed food. Cryptomonads require B,2 and thiamine for optimum growth
(Hutchinson, 1967; L. Whitford, pers. comm.) and presumably so do the rotifers.
It is possible that these two organisms occur together because of common re-
quirements and not solely because Poiyarthra is feeding solely on cryptomonas.
The presence of trace elements may also have an effect because many are
cofactors for enzyme activity. The concentrations present in Vionate and a
mixture (#1) added to the water are in Table 6. There were no apparent effects
when the mixture #1 trace elements were added to the culture. Data for Purina
Trout Chow were not available.
Fungi and Bacteria
Unfortunately the high vitamin content of the culture water stimulated
the growth of a chytridiaceous fungus and a sheath forming bacterium of the
sphaerotiius-Leptothrix complex. It was not uncommon to find adult rotifers
and eggs enmeshed in fibers. Once enmeshed, rotifer death was certain. Con-
tact with the fungi or bacterium was enhanced by the fact that the female
rotifers released the developing eggs 12 to 24 hr prior to hatching. These
eggs fell to the bottom of the culture container where the fungus or bacteria
were growing on the debris or where there were spores.
13
-------
TABLE 5. EFFECTS OF VARIOUS FOODS PLUS VITAMINS AND TRACE
METALS ON SURVIVAL AND REPRODUCTION OF POLYARTHRA VULGARIS
Food
Vitamins and Average number surviving per day
trace metals Day 0 T"^ 23
A. Cryptomonas ovata
Cryptomonas ovata
B. Ochromonas
Ochromonas
C. Bodo sp.*
D. Chilomonas
paramecium and
Cyathomonas truncata*
E. 3 species of Bodo,
Pleuromonas and
Oikomonas*
F. no food
no
yes
no
yes
no
yes
no
no
yes
yes
no
no
yes
yes
yes
10
10
10
10
10
10
10
10
10
10
10
10
10
10
0
6
0
6
2
7
2
6
2t
8
8
14
6
9
0
3
0
10
It
6
*protozoans were raised on trout chow
teggs present
One experiment was conducted with the wide spectrum antibiotic kanamycin
sulfate. It was used at a concentration of 0.01 mg/liter which is 1/100 the
level for treating Phiiodina acuticomis eggs (Meadow and Barrows, 1971). Even
at this low concentration the antibiotic was toxic to the rotifer.
Fungal and bacterial growth was reduced if filtered Vionate solution was
used rather than the straight compound in the culture water, but they were
not eliminated.
Rotifer eggs are parasitized by fungi (Paterson, 1958; Seymour and
Johnson, 1973) and one chytrid, oipidium gregarium, has been identified from
rotifer eggs (Paterson, 1958). Paterson also found that the rotifers Nothoica
and poiyarthra were parasitized by saprolegnianceous fungi. Fungi, but not
bacteria, have been suggested for the decline in natural populations of
poiyarthra (Beach, 1960; Pejler, 1961).
14
-------
TABLE 6. ELEMENTS IN FOOD MEDIA AVAILABLE
TO POLYARTHRA VULGARIS
(Data for Purina trout chow are not available)
Vionate Mixture #1
Element (per gm dry) weight Medium (ygm/1)
Ca
P
Na
I
Fe
Co
Cu
Mg
Mn
Zn
Mo
89.9 - 107.9
47.94
5.0 - 15.0
0.022
0.552
0.0055
0.0552
0.5298
0.0759
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0022
0.0040
0.0
0.0041
0.0200
0.0019
Food
Because of the correlations between poiyarthra and the alga, cryptomonas,
initial experiments were conducted with this alga. Survival was poor indi-
cating that the animals were not feeding on it. Six different culture media
were used to raise cryptomonas ovata (Table 7) at a recommended light inten-
sity of 200 ft-c (Daniel Jones, pers. comm.). Basically the media were
Bristol's and Chu's with different concentrations of nitrate and peptone. In
all Instances survival of the rotifers was still poor. Observations under the
microscope indicated the the rotifer would grasp cryptomonas ovata but then
the rotifer would reject it. It was never observed eating c. ovata, even
when there was no other choice. Also in subsequent studies Poiyarthra was
never observed to feed on c. erosa (W. Yongue, pers. comm.).
The size of the food particle was important for rotifers (Gossler, 1950;
Edmondson, 1965), and other potential food sources were tested. Observations
15
-------
TABLE 7. CULTURE MEDIA USED FOR CRYPTOMONAS OVATA
Medium
Chu's #10
ChiTs #10
Chu's #10
Bristol s
Bristol s
Bristol s
Additional
Nitrogen mg/1
750
750
250
0
250
750
Peptone
mg/1
0
1
1
0
1
1
PH
7.8
7.0
7.0
7.2
7.1
7.0
were made on the rotifer's selectivity for food under the microscope and on
rotifer survival and appearance of eggs when the rotifer was placed in a 10
ml microcosm with various foods. Seventeen possible foods were made available
to Poiyarthra vulgaris (Table 8). Of these 17 foods the rotifer was observed
eating 7 of them. Most other foods were ignored except cryptomonas ovata
which was grasped but then rejected by the rotifer. The incidence of feeding
on Euglena viridis and Chlamydomonas reinhardii was much lower than it W3S
for the non-chlorophyll containing forms.
The survival of Poiyarthra increased markedly when they were raised on
mixtures Of Chilomonas paramecium and Cyathomonas truncata or three Species
of Bodo (Table 9). Interestingly, chiiomonas and Cyathomonas are also cryp-
tophytes as are cryptomonas and Rhodomonas, possible foods suggested for
Poiyarthra (Edmondson, 1965; etc.). Additionally egg production and limited
egg hatching occurred with these food mixtures even though no vitamins were
added to the food. Survival was also best if vitamins were present with the
food (Table 5).
Food concentration had some effect on survival (Table 10). Generally
survival was better at the lower food concentration. At these lower food
concentrations the number of protozoan per ml of food stock varied between
80,000 to 350,000. The study of Erman (1962) suggest that high food concen-
trations are necessary. Brachionus caiydfiorus eats up to 180% of its wet
weight in wet food per day (Erman, 1962). Unfed Poiyarthra usually died with-
in 8 to 12 hr although some lived for up to 24 hr (Table 10). Food concen-
tration does affect reproduction of Euchianis (King, 1967), Brachionus (Halbach,
1972), and other rotifers (Edmondson, 1965; etc.).
16
-------
TABLE 8. POTENTIAL FOOD ORGANISMS TESTED
ON POLYARTHRA VULGARIS
Taxa
Size (in microns)
Selected
by animal
1. Euglena viridis
2. Cryptomonas ovata
3. Paramecium aurelia
4. Paramecium bursaria
5. Phacus sp.
6. Trachelomonas sp.
7. Chlamydomonas reinhardii
(#89,+)
8. Chlamydomonas moewusii
9. Cyathomonas truncata
10. Chilomonas paramecium
11. Bodo variabilis
12. Bodo minimus
13. Bodo mutabilis
14. Pleuromonas jaculans
15. Oikomonas termo
16. Ochromonas sp.
17. Chlorella sp.
14-20 x 40-65
508 x 20-80
50-60 x 120-180
50-60 x 100-150
c. 25 x 50
c. 15 x 30
3-5 x 10-15
10-15 x 15-20
10-15 x 15-25
10-15 x 15-25
5-15
c. 5
5-15
5 x 10
5 - 10+
5 - 30
5 - 10
yes
no
no
no
no
no
yes
no
yes
yes
yes
yes
yes
no
no
no
no
These data on food selectivity, survival, and reproduction of poiyarthra
do not agree with the observations of Dieffenbach and Sachse (1911), Gossler
(1950), Pejler (1957), or Edmondson (1965). For one, Poiyarthra vulgaris did
not feed on Cryptomonas and in its presence the rotifer did not survive for
17
-------
TABLE 9. EFFECTS OF VARIOUS FOODS ON SURVIVAL
AND EGG PRODUCTION OF POLYARTHRA VULGARIS
Food
Cryptomonas
Cryptomonas
Cryptomonas
Cryptomonas
Cryptomonas
Cryptomonas
Chilomonas and
Cyathomonas
Cryptomonas ,
> Chilomonas, and
Cyathomonas
Chlamydomonas moewusii
Euglena viridis
Trachelomonas
3 species of Bodo,
Pleuromonas and
Oikomonas
Ocromonas
Ochramonas and Bodo sp.
Bodo sp.
Day 0
10
10
10
10
10
10
10
12
10
10
10
10
10
10
10
10
10
10
10
10
1
-
3.0
1.0
1.5
8.0
3.0
2.0*
3.0
9.0
3.0
2.0
2.0
Averaqe Number Surviving Per Day
2 3 4 5 6 7 8 10 11 15 17
1.5 1.0 ----- - - -
1.5
2.0
4.0 3.0 1.0 - -
0.3------ - - --
1.0
- 11 - 4.0 2.0* 3.0* 2.0 1.0
3.0------ - - -_
2.0 - - -
------- _ _ __
4.0 - -
------- _ _ __
6.0* 5.0* 4.0* 2.0 2.0 1.0 - - -
------_._ _ __
1.0
2.0--
*Eggs present; development occurred in some
-------
TABLE 10. EFFECT OF VARIOUS CONCENTRATIONS OF PROTOZOANS
(NO VITAMINS) ON SURVIVAL AND REPRODUCTION OF
POLYARTHRA VULGARIS
Food
Relative
Concentrations
Averae Number Surviving per Day
~ ~ ~
A. None
B. Euglena
2X
5X
10X
C. Chilomonas and 2X
Cyathomonas 4X
(Run 1) 5X
10X
C. Chilomonas and
Cyathomonas
(Run 2)
IX
2X
3X
5X
10X
20X
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
7.5
2
7
6
8
9
8
0
0
3
1
2.5
2*
0
0
5.5 5.5 6*
11
8
3
4
0
3
3
3
3.5
2
2
0
2
0
0
2
1
1
1
0
0
D. 3 species of
Bodo, Pleuromonas IX
and Oikomonas 2X
3X
5X
10X
20X
10
10
10
10
10
10
0
0
2
1
1
2
_
-
2 20
0
0
0
_ -
-
-
-
-
*eggs present
long periods of time much less produce eggs. Earlier we proposed an alterna-
tive hypothesis for the field correlations between cryptomonas and poiyarthra
vulgaris based on their common requirements for vitamins B,2 and thiamine.
Secondly, Poiyarthra will feed on small sized protozoans such as Bodo in con-
trast to the observations of Edmondson (1965) although he acknowledges that
Poiyarthra W3S "not Utterly dependent on Cryptomonas."
19
-------
In our study the rotifers fed most frequently on the colorless flagellates
and reproduction occurred when this food source was available. Edmondson (1965)
did not find a significant correlation between Poiyarthra vulgaris and colorless
flagellates. There are two possible reasons for this: (1) the colorless
flagellates he examined were less than 10 y long and 2 of our food organisms,
chiiomonas and cyathomonas, were larger; and (2) his analysis of the colorless
flagellates was not species specific but rather a composite of various proto-
zoans. In both instances we would not expect a significant correlation to
appear in his analyses.
Antibiosis
Possible inhibitory effects of algae on Poiyarthra populations were ob-
served. Concurrent with declines in populations of Poiyarthra there were in-
creases in numbers of specific algal genera. In five of the seven instances
recorded the dominant genera were bluegreen algae. These included Anajbena,
spiruiina, phormidium, and osdiiatoria. In two instances the dominant genera
were green algae, especially Ankistrodesmus, Eudorina, Pandorina and Pediastrum.
Direct or indirect antibiotic effects of chioreiia have been suggested for
populations Of Kellicottia longispina (Edmondson, 1965), Brachionus plicatus
(Hirayama, Watanabe, and Kusano, 1973) and B. caiydfiorus (Halbach, 1972;
Halbach and Halbach-Keup, 1974) and similar relationships may exist with other
rotifers and algae. In comparing Carl in's data for osdiiatoria and Poiyarthra
vulgaris populations (Figures 145 and 146 in Hutchinson, 1967) there may be an
inhibitory effect because the rotifer population was low when the osdiiatoria
population was high. Similar inhibitory effects may occur for the bluegreens,
Aphanizomenon and Lynbya (Figures 54, 56, and 101 in Carlin, 1943). Bluegreen
algae may even affect the vertical distribution of Poiyarthra (Figures 53, 55,
57, and 100 in Carlin, 1943).
Temperature
Preliminary experiments were conducted on temperature and survival.
Poiyarthra vulgaris seems to be resistant to wide temperature fluctuations.
For example, animals can be taken from the field at 3 C and slowly warmed to
20 C within 5 hr with no apparent effects. Some rotifers lived as long as
48 hr.
Initial population experiments were conducted at 10 and 20 C and at room
temperature (21 + 2 C). These population experiments were conducted prior to
refinement of cuTture techniques, but they provided some insight into tempera-
ture effects. Two cultures were begun at 10 C and three each at 20 C and room
tempeature. The rotifers were obtained from 25 to 26 C pond water. These
data are summarized in Figure 1. All populations died within 40 days.
Population success was best at 20 and 21 C and was least at 10 C. These
observations were consistent with the field observations of Carlin (1943),
Pejler (1957), and Edmondson (1965).
Egg Development
Incomplete observations were made on egg development at room temperature
20
-------
n
o
ro
0>
_0
E
3
z
A 10 C
O 70 C
Room Temperature
Figure 1. Effect of Temperature on Populations of poiyarthra vulgaris.
-------
(21 +_ 2 C) incidental to our other work. Pourriot and Hillbricht-Ilkowska
(1969) demonstrated that egg development of poiyarthra vulgaris can occur with-
in 24 hr at 20 C and that it can take up to 70 hr (Edmondson, 1965). Such
rapid development is commonly accepted for parthenogenetic zooplankton, espe-
cially the rotifers. In our observations we did not notice such rapid devel-
opment. At room temperature (21 +_ 2 C) development took much longer than 24
hr since observations were begun with ovigerous females. In one instance a
female already bearing an egg was observed in a 10 ml microcosm and the egg
hatched 8 days later. The young rotifer was morphologically similar to the
female with distinct paddles and it appeared to be healthy.
There are many possible reasons for this extended development time of the
egg. Maternal nutrition may be an important factor. Edmondson (1965) suggests
that egg laying of rotifers is influenced by light conditions and perhaps the
same may be true for egg development. Another hypothesis, on constant versus
fluctuating temperatures on egg development, is proposed in Section 5.
Edmondson's (1965) calculations of duration of egg development were in part
based on the assumption that the animals were found at a fixed temperature,
and his duration studies were conducted at the "same temperature in which the
animal has been living." Because the surface water temperatures where the
animals were found fluctuated daily and because the rotifers respond to changes
in light intensity (=vertical migration), we would expect that the rotifers
were subjected to a range of temperatures each day even if this range were
small.
The egg was attached to the female during part of the development time.
It has been our experience that the egg was released by the female about 12
hr prior to hatching. The egg settled to bottom of the container where hatch-
ing occurred. In a lake it probably would remain in suspension. This egg
release may have an effect on the calculation of egg ratio and reduce estimates
of reproductive rates (Edmondson, 1960). The handling of Poiyarthra also
causes a release of egg, and the egg ratio would be further reduced.
22
-------
SECTION 5
THE EFFECTS OF TEMPERATURE ON
REPRODUCTION OF POLYARTHRA VULGARIS
The previous research on Poiyarthra vulgaris had been concerned with the
identification of variables and their effects on survival, egg production, and
egg hatching. The purpose of this experiment was to optimize all the known
variables except the temperature and study the effect of temperature on popu-
lation dynamics. Temperature was chosen because it was a significant variable
identified in this research and that of others (Edmondson, 1965; Edmunds, 1974;
and Pejler, 1957).
MATERIALS AND METHODS
Populations of Poiyarthra vulgaris were obtained from Pandapas Pond during
October, 1973, when the water temperature was between 15 to 20 C. The animals
were concentrated with a 35 micron mesh net and then added to the aquaria at an
initial concentration of approximately 1000 rotifers per liter.
Filtered Pandapas Pond water was used for the cultures and five liters
were placed in six-liter soft glass aquaria (Carolina Biological Supply Company,
Burlington, North Carolina). One and one-half to two liters of the culture
water were replaced twice a week and the water was completely replaced once a
week. During the changes the rotifers were collected in a Wisconsin net
bucket with a 30 micron mesh bolting cloth and returned to the aquarium. The
debris at the bottom of each aquarium was removed each week and discarded.
Vitamins were added to the cultures in two ways. First, after each com-
plete water change 1.0 gm of Vionate (Squibb and Sons, Inc.) was added to the
water. An additional 0.5 gm of Vionate was added after each partial change of
water. Secondly, vitamins were supplied through the protozoan cultures. Proto-
zoan food organisms were cultured on dilute #3 Purina Trout Food which also
contained vitamin mixture #1 (Table 3).
The protozoan cultures were mixed and contained chilomonas paramecium,
Cyathomonas truncata, Bodo mutabilis, B. variabilis, and B. minimus. Fifty
ml of this protozoan mixture was fed to each culture each day. The concen-
tration of protozoans was about 300,000 protozoans per ml.
Concurrently three cultures were placed in each of four temperature con-
ditions: 10, 20, 30 C, and room temperature (21 + 2 C). The first three
temperatures were regulated in Scherer-Gilette CeT 4-4 growth chambers and
23
-------
temperature was regulated within 0.75 C.
The aquaria were aerated moderately to maintain the dissolved oxygen con-
centration in excess of 8 ppm. The photoperiod was a 16L:8D cycle with the
incident light intensity between 100 and 500 ft-c (from one end of the aquarium
to the other). The light sources were 20 and 40 watt 6E cool white fluorescent
bulbs.
Five 5-ml subsamples were removed every 2 to 3 days from each aquarium
with a pipette and counted. The population counts are probably underestimated
because 22% of Poiyarthra can escape a glass tube such as an eye dropper
(Szlauer, 1965). If available, 10 to 20 freeswimming females were examined
for eggs, and notes were made on the percent of females carrying eggs and the
number of eggs per ovigerous female. The occurrence of sexual eggs was noted.
After counting the eggs, 10% formalin was added to each subsample and the total
number of rotifers was counted. Formalin caused egg release so it was necessary
to count eggs on living rotifers.
RESULTS
The population data for the 4 temperatures are depicted in Figures 2 and
3. Population success was poor at 10 and 30 C, and there was more variation
among the cultures at 30 C than at 10 C (Figure 2). Reproduction was depressed
at 10 C and no rotifers were present after 13 days. The populations at 30 C
exhibited reproduction within 5 days with a decline in population numbers until
day 10. Population maxima exceeded 10,500 rotifers per liter. No rotifers
were present after day 10. There was a significant bloom of bluegreen algae
in the 30 C cultures and the dominant genera were Anabena, spiiuiina, phomddivm,
and osdiiatoria. An antibiotic effect has been suggested earlier (Section 4).
No eggs were observed in the 10 and 30 C cultures.
The best results were obtained at 20 C and room temperature (Figure 3).
There was considerable variability among the cultures at both of these tem-
peratures, and only the mean values for three cultures are presented. While
mean population numbers were as high as 7,500 rotifers per liter, individual
cultures at 20 C were above 11,000 rotifers per liter and at room temperature
rotifer concentration exceeded 13,000 per liter. In other experiments at 20
C populations greater than 20,000 per liter were observed.
The populations at 20 C exhibited 3 major peaks which were 22 and 28 days
apart, while only 2 major peaks, 37 days apart, were observed at room tempera-
ture (Figure 3). The data in Figure 3 are for 69 days, but 1 rotifer culture
at room temperature lasted longer than 100 days. Five of the 6 cultures crashed
after the water was changed on the 70th day, and 1t was believed that a toxic
substance was present in the pond water. Usually the population numbers did
not rebound if the rotifer density fell below 40 animals per liter.
The data in Figures 4 and 5 illustrate representative patterns between
population numbers, percent of ovigerous females, number of eggs per ovigerous
female, and appearance of sexual eggs. The percent of ovigerous females was
24
-------
e Culture A
Culture B
A Culture C
10 c
Figure 2. Effect of 10 and 30 C Temperatures on Populations of Poiyarthra
vulgaris.
always greatest when population numbers were low with a general decrease after
population peaked. There were large fluctuations in the percent of ovigerous
females, and it varied from 0 to 70% when population numbers were low (Figure
4). Similar fluctuations occurred in other populations. Generally the fluc-
tuations in percent of ovigerous females was regular (up and down) in 20 C
cultures but not in room temperature cultures. In these cultures dips may per-
sist from two to five days (e.g., Figure 5, days 22 to 27).
Ovigerous females usually carried one egg at a time. Two eggs per female
were not uncommon and rarely did a female carry three eggs. In one instance
a female with 5 eggs was observed in a 20 C culture. The average number of
eggs per ovigerous female usually increased after a population peak (Figure
4). This phenomenon was observed five times. Only once was the average number
of eggs per ovigerous females greather than one prior to a population peak
(Figure 5).
egg,
When there were multiple eggs they usually were smaller than the single
but not as small as would be expected for male producing eggs. Males
25
-------
1O
S> 8
s4
|3
I2
1
0
D 20 C
Room Temperature
10 20 30 40
Days
50
60
70
Figure 3. Effect of 20 C and Room Temperature (21 +_ 2 C) on Populations
Of Polyarthra vulgaris.
were not observed, but their presence was indicated by the appearance of sexual
eggs. These eggs were large and darkly pigmented. Sexual eggs were only ob-
served twice (Figures 4 and 5), and in both cases it was after a population
peak.
DISCUSSION
In comparing the data at 10, 20, and 30 C the results are comparable to
data of others. Reproduction of rotifers is greatest at higher temperatures
(Edmondson, 1960, 1965; etc.). The 30 C may be above the critical thermal
maxima even though Polyarthra vulgaris was reproducing in 26 C water (Section
6, this study). Suppression of reproduction at 10 C corresponds favorably with
Edmondson's (1965) observations on reproductive rate.
26
-------
30 _ 40
Days
70
Figure 4. Relationship among Population Numbers, Percent Ovigerous Females,
Number of Eggs per Ovigerous Female and Sexual Reproduction for One Culture
Of Polyarthra vulgraris at 20 C.
The variation in data obtained at 20 C and room temperature was interest-
Ing. Small sample size definitely influences our data such that differences
in time between population peaks may not be significant. However, an analysis
of the data suggest real temperature effects. Even though the cultures were
conducted concurrently at similar temperatures with similar treatment, there
was one difference that could have affected the results. The temperature
fluctuation of the 20 C cultures was about 0.75 C or a range from 19.25 to
20.75 C. The room temperature cultures varied from 19 to 23 C over a 24 hr
period for a 4 C range. The effect of a 4 C oscillation in temperature may
have had an effect on rotifer reproduction and development. Halbach (1973)
noted that variable temperature had the following Impact on populations of
Brachlonus caiydfiorus: immaturation time decreased; life duration increased;
the intrinsic rate of population increase was larger, the environmental
capacity was higher, and population fluctuations were more severe.
27
-------
10
60
70
Figure 5. Relationship among Population Numbers, Percent Ovigerous Females,
Number of Eggs per Ovigerous Females and Sexual Reproduction for One Culture
of poiyarthra vulgaris at Room Temperature.
Egg production of plankton rotifers has been studied by Edmondson (1960).
Values for Kerateiia cochiearis may vary from 0.077 to 0.267 eggs per day. The
higher values were obtained at higher temperatures. While daily observations
on specific females were not possible at this stage of the research, an analysis
of the percent of ovigerous females provides a crude index (Figures 4 and 5).
At 20 C the time between peaks was from 4 to 7 days. Because most females
carried 1 egg, the rate of egg production at 20 C probably varies from 0.14 to
0.25 eggs per day. At room temperature the peaks were 7 to 12 days apart and
the rate ranged from 0.08 to 0.14 eggs per day. These figures were similar
to the values obtained by Edmondson (I960), but they also identify another
difference between the 20 C and the room temperature cultures.
Our observation that the time for egg development at room temperature ex-
ceeded 24 hr (Section 4, this study; Edmondson, 1965; Pourriot and Hillbricht-
Ilkowska, 1969) also suggests that the oscillating temperature may retard
development. Acclimation of female Poiyarthra vulgaris definitely has an effect
28
-------
on egg development (Pourriot and Hillbricht-Ilkowska, 1969) which almost doubled
when unacclimated animals were taken from 8 C and placed at 20 C. The data
suggest that the temperature interaction was via the female prior to or during
egg laying. Perhaps an oscillating temperature prior to egg laying may also
prolong development.
Fluctuating temperatures are known to reduce the oxygen consumption of
oncopeitus eggs to levels below those of eggs held at an equivalent constant
mean temperature (Richards and Suanraksa, 1962). Lower metabolic rate would
suggest a retardation of development. Unfortunately, oncopeitus eggs reared
at a fluctuating temperature hatched 10% faster, but this same effect may not
be true for parthenogenetically reproducing females subjected to a fluctuating
environment. Hutchinson (1967) suggests that "maternal influences of a bio-
chemical kind may have a greater effect on development in the rotifers..." and
he cites data from Lansing (1942) on calcium and rotifer aging.
The decrease in the percent of ovigerous females when population numbers
were high illustrates the classic textbook pattern of a feedback mechanism
controlling population size and reproduction.
While sexual eggs were observed twice, males were not. Carlin's (Figure
101 in 1943) data for Poiyarthra vulgaris show that in natural populations
males appear only in the autumn and not during or after populations peaks as
they do in other species of Poiyarthra. From our data we suggest that males
may appear after very large populations blooms. Our laboratory population
densities were over 40 times greater (Figure 5) than the natural populations
studied by Carl in (1943) and up to 13 times greater than the maximum recorded
densities in our pond studies (1115/liter). At lower natural population levels,
environmental parameters such as decreasing photoperiod or temperature may be
more appropriate triggers for male formation.
29
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SECTION 6
NATURAL POPULATION DYNAMICS OF POLYARTHRA VULGARIS
The laboratory culture research was based on the data from many literature
sources. These data Included information on a few possible chemical and phys-
ical parameters and food organisms. Because of the limited information avail-
able, we conducted a field investigation to (1) substantiate the observations
by earlier works, and (2) examine additional chemical and physical parameters
of the water that may effect populations of Poiyarthra vulgaris.
MATERIALS AND METHODS
Description of Study Area
Populations of p. vulgaris were studied in Pandapas Pond, a shallow man-
made impoundment located in the Jefferson National Forest, Montgomery County,
6 km northwest of Blacksburg, Virginia, at an altitude of 664.8 m above sea
level (Figure 6). The maximum depth of the pond was 3 m?adjacent to the 100 m
earthen dam. The surface area is approximately 32,376 m and the volume is
estimated at 37,000 m (U. S. Soil Conservation Service, pers. comm.). The
pond is surrounded by steep (15 to 45 degree) slopes which are heavily forested
with second growth pine hardwood forest overlaying a shallow, acidic shaley
loam. Several small spring-fed streams feed Pandapas Pond, but the majority
of the water is from runoff. The pond was generally devoid of aquatic insects
and aquatic vegetation. The dominant fish was the bluegill, Lepomis macrochirus,
and the smallmouth bass, Micropterus doiomieui, was present. In late spring
the pond was the breeding place for large numbers of the newt Notophthaimus
viridescens.
Sampling and Analytical Techniques
Studies began four months before the grant began and continued for eight
months during the grant period. Samples were obtained monthly at two stations
in the pond (Figure 6). Station One was located at the shallow end and Station
Two was near the earthen dam.
An ITT JABSCO electric pump was used to collect all samples through a hose
with a bell-ended "T" apparatus (Welch, 1948). Samples were taken for plankton,
water chemistry, bacteria, and chlorophyll a.
Twenty liters of water were concentrated through 35 and 75 micron mesh
nets for plankton. The 75 micron mesh net was used throughout the study, and
30
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PANDAPAS POND
UPPER
POND
POVERTY
CREEK
Figure 6. Map of Pandapas Pond, Montgomery County, Virginia Showing the Two Collection Stations (S,
S). Also Indicated are a Bed of spirogyra (Stippled) and Beds of niteiia (Hatched).
and
-------
the 35 micron net was used for the last 7 months of the study. The samples
were preserved in 10% formalin and counted at a later date with a Sedgewick-
Rafter counting cell.
Nineteen chemical and physical parameters were determined for each sample.
Temperature was measured with a YSI model 54 oxygen-temperature meter. Light
penetration was obtained with a G.M. Submarine Photometer (GM Manufacturing
and Instrument Corporation, Bronx, New York). Dissolved oxygen was measured
with azide modification of the Winkler method. Oxygen samples were fixed in
the field and titrated immediately upon return to the laboratory. All other
water samples were iced and analyzed in the laboratory.
The pH was measured with a Corning model 109 meter. Conductivity was
measured by a YSI conductivity meter. Nitrate and ammonia nitrogen, ortho-
and total phosphate, sulfate, alkalinity, and total filtrable solids were
measured by the methods outlined in Standard Methods for the Examination of
Water and Wastewater (American Public Health Association, 1971).Calcium,
magnesium, iron, sodium, and potassium were measured on an Unicam Model SP90
Atomic Absorption Spectrophotometer. Total hardness was calculated from the
calcium, magnesium, and iron values (APHA, 1971), and silica was measured by
a Hach Chemical Kit (Hach Chemical Company, Ames, Iowa).
Samples for bacterial analysis were collected in sterile flasks and iced
immediately. In the laboratory these samples were diluted and incubated as
outlined in the Standard Plate Count method (APHA, 1971). The plates were
counted after 24 hr incubation at 35 C using a Quebec darkfield colony counter.
The number of colonies per ml of pond water was calculated.
Chlorophyll a samples were buffered with magnesium carbonate and iced.
In the laboratory they were extracted and analyzed by the methodology of APHA
(1971) and Strickland and Parson (1968).
The above data were subjected to correlation and multiple regression
analyses using the Statistical Analyses System (SAS) of Barr and Goodnight
(1972) on an IBM 370 Computer. These analysis were with no lag, which is
appropriate for parthenogenetically reproducing animals (Angino, Armitage and
Saxena, 1973).
RESULTS
Pond Chemistry
A detailed discussion of the yearly trends in pond chemistry can be found
in a MS thesis by Edmunds (1974), and the discussion is summarized in Tables
11, 12, 13, and 14. Pandapas Pond was essentially a low hardness, low alka-
linity system with the pH varying from 6.1 to 8.7 over the year. Yearly
temperatures ranged from 0.1 to 26.0 C, and there was evidence of summer
stratification at Station Two. Oxygen varied from 0 to 15.4 mg/1 with obvious
depletion during the summer at Station Two. Nitrate levels varied from 0 to
1.59 mg/1 and ammonia values varied from 0.04 to 1.27 mg/1. Orthophosphate
varied from 0.08 to 0.24 mg/1 and the total phosphate was usually below 0.5
32
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TABLE 11. SIGNIFICANT CORRELATIONS BETWEEN
ENVIRONMENTAL PARAMETERS AND POLYARTHRA VULGARIS
AT STATION 1
Environmental
Parameter
Photoperiod
Temperature
Nitrate
Orthophosphate
Oxygen
Net Mesh
35
+0.664*
+0.510*
+0.463**
+0.422**
-0.407tt
(microns)
75
+0.365*
+0.461*
NSt
NSt
-0.366tt
mean
12.14
12.73
0.21
0.03
9.33
range
low
9.67
0.10
0.00
0.00
4.90
high
14.73 hr
26.00 C
0.65 mg/1
0.14 mg/1
12.80 mg/1
Significant levels:
*.005
**.01
tnot significant
tt.05
mg/1 except in June when a high value of 12.6 mg/1 was found at 3.0 m in Sta-
tion Two. Sulfates ranged between 1.4 to 4.81 mg/1. Calcium levels (0.96
to 3.82 mg/1) were similar to magnesium levels (1.10 to 3.07 mg/1) while iron
values varied between 0.04 to 2.15 mg/1. Total hardness ranged from 7.49 to
22.08 mg/1. Sodium values varied (2.95 to 6.22 mg/1) while potassium values
were usually between 1.0 to 2.0 mg/1. Total alkalinity varied from 5.1 to
31.4 mg/1. The concentration of total filterable solids fluctuated irregu-
larly from 17.2 to 143.2 mg/1. Silicon was always present in concentrations
above 4.7 mg/1, and the values were as high as 19.0 mg/1. Carbon dioxide
varied from 0 to 100 mg/1. Conductivity was measured 3 times and it ranged
from 28 to 39 micromohs/cm. Surface penetration of light ranged from 19.8
to 91% and because of high turbidity the percent penetration dropped markedly
by 1.0 m.
Bacteria
The results of the bacterial analysis were too variable and unreproducible
to be included in this study.
Chlorophyll a
Mean chlorophyll a concentration varied widely over time with a general
33
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TABLE 12. NONSIGNIFICANT CORRELATIONS BETWEEN ENVIRONMENTAL
PARAMETERS AND POPULATIONS OF POLYARTHRA VULGARIS at STATION 1
Environmental
Parameter
Carbon dioxide
Total Flltrable
Solids
Sulfate
Sodium
Total Alkalinity
Potassium
Ammonia
Magnesium
Calcium
Total Hardness
PH
Chlorophyll a
Total Phosphate
Iron
Silicon
Nitrate
Orthophosphate
Net Mesh
35
-0.259
-0.271
-0.241
-0.233
-0.266
-0.226
-0.214
+0.197
+0.117
+0.112
+0.086
-0.082
+0.061
-0.031
+0.007
-
-
75
-0.147
-0.171
-0.234
-0.233
+0.169
+0.019
-0.117
+0.113
+0.227
+0.134
+0.031
-0.111
-0.078
-0.237
-0.104
+0.242
+0.153
mean
3.24
52.40
2.93
5.04
9.40
1.23
0.24
1.43
2.06
11.80
7.01
16.6
0.19
0.47
8.70
0.21
0.03
Concentration
high
0.00
4.00
0.00
4.20
4.00
0.97
0.00
1.13
0.92
7.56
6.3
0.005
0.02
0.00
4.70
0.00
0.00
low
11,00 mg/1
192.00 mg/1
6.14 mg/1
8.35 mg/1
18.00 mg/1
1.96 mg/1
0.98 mg/1
2.56 mg/1
7.73 mg/1
25.94 mg/1
8.7
169.00 mg/1
1.03 mg/1
2.91 mg/1
12.00 mg/1
0.65 mg/1
0.14 mg/1
seasonal pattern from a low of 1.18 mg/1 in January to a high of 45.48 mg/1 in
August. Large variances in chlorophyll a concentrations were observed in the
water column, and much of this variance was due in part to the large growths
of spirogyra growing on the bottom of the pond.
34
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TABLE 13. SIGNIFICANT CORRELATIONS BETWEEN ENVIRONMENTAL
PARAMETERS AND POLYARTHRA VULGARIS AT STATION 2
Environmental Net Mesh (Microns) Concentration
Parameter 35 75 mean low high
Photoperiod
Temperature
Ammonia
Sodi urn
+0.
+0.
-0.
522*
497**
385+
+0
+0
-0
-0.298+
.281**
.400*
.272t
NStt
12.
11.
0.
4.
14
99
38
60
9
0
0
1
.67
.20
.00
.72
14.
26.
1.
6.
73
00
27
12
hr
C
mg/1
mg/1
Total filtrable oir nA
solids -0.269t NStt 48.06 1.00 216.00 mg/1
Significant levels:
*.005
**.01
t.05
ttnot significant
Rotifer Populations
A total of 29 species of zooplankton were identified during the study
(Edmunds, 1974) and 21 of these were rotifers. Kerateiia cochiearis was the
dominant rotifer species. K. cochiearis, Kellicottia bostoniensis, and
poiyarthra vulgaris were the most common rotifer species and they were present
all year.
poiyarthra vulgaris fluctuated seasonally (Figure 7). Populations were
low from December through April with a major peak between May and July and a
minor peak in the fall. In May it was the most prevalent rotifer. The highest
density observed was 1115 poiyarthra per liter and this occurred at the 1.5 m
depth in June. The lowest values were between 0 and 1 animal/liter. The
population increased in May within 3 weeks after the temperature exceeded 15
C. When the temperature was below 10 C, populations were very low (Figure 7).
Relation between Poiyarthra vulgaris and Environmental Parameters
In correlating chemical and physical data on water quality to poiyarthra
collected with the 35 micron mesh net there were significant positive corre-
lations with temperature, nitrate, and orthophosphate (Tables 11 and 13).
Additionally there was a signficiant positive correlation with photoperlod.
Significant negative correlations was obtained with dissolved oxygen, ammonia,
35
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TABLE 14. NONSIGNIFICANT CORRELATIONS BETWEEN ENVIRONMENTAL
PARAMETERS AND POLYARTHRA VULGARIS AT STATION 2
Environmental
Parameter
Silicon
Iron
Oxygen
Carbon Dioxide
Orthophosphate
Sulfate
Hardness
Chlorophyll a
Total Alkalinity
PH
Total Phosphate
Magnesium
Potassium
Calcium
Nitrate
Total Filtrable
Soil Solids
Sodium
Net Mesh
35
-.215
-.180
-.168
-.145
+ .096
+ .092
-.088
-.081
-.074
+ .070
-.046
-.036
-.034
-.029
+ .022
-.161
-
(micron)
75
-.138
-.095
-.168
-.077
+ .093
-.104
-.022
-.031
-.026
-.050
-.028
+ .037
-.025
+ .027
+.117
-
-.141
Concentration
mean
9.05
0.73
7.64
6.02
0.04
2.60
12.50
19.30
12.47
7.02
0.30
1.41
1.25
2.02
0.23
48.06
4.60
low
5.00
0.00
0.00
0.00
0.00
0.00
7.12
0.38
5.00
6.10
0.02
1.07
0.92
0.92
0.00
1.00
1.72
high
19.00 mg/1
5.95 mg/1
13.00 mg/1
100.00 mg/1
0.24 mg/1
5.16 mg/1
38.82 mg/1
150.70 mg/1
123.50 mg/1
8.40
12.60 mg/1
3.07 mg/1
3.06 mg/1
6.99 mg/1
1.59 mg/1
216.00 mg/1
6.12 mg/1
sodium, and total filtrable solids (Tables 11 and 13). Similar correlations
were obtained for the 75 micron mesh net except the effects of nitrate, ortho-
phosphate, sodium, and total filtrable solids were not significant. There were
no significant correlations with pH, total phosphate, sulfates, calcium, mag-
nesium iron, total hardness, potassium, silicon, carbon dioxide, total alka-
linity, or chlorophyll a (Tables 12 and 14).
36
-------
800
a Station one
a Station two
f M9 M21 A M
MONTH
Figure 7. Seasonal Fluctuations of Photoperiod, Temperature and Mean Number
Of Polyarthra vulgaris Per Liter.
Using stepwise regression analysis the relationship between independent
and dependent variables were identified (Tables 15 and 16). Data varied bet-
ween stations and with net size. Five variables were identical from each
station and net size. These were photoperiod, temperature, oxygen, nitrate,
and total filtrable solids. In comparing the two tables, photoperiod accounts
for 9.1 to 40.8% of the variation with better results obtained with the 35
micron mesh net. Variation due to temperature ranged from 11.0 to 21.1%.
Oxygen varied from 0.3 to 13.0% and usually was under 4.0%. Nitrate accounted
for 1.5 to 10.1% of the variation and total filtrable solids accounted for
2.6 to 20.4% of the variation.
Three relationships were identified only for the 35 micron mesh net.
Orthophosphate accounted for 0.00 to 9.0% of the variation. Variation due to
ammonia ranged from 4.6 to 14.8% and that for sodium ranged from 0.17 to 3.0%.
37
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TABLE 15. STEPWISE REGRESSIONJ\NALYSIS AND INCREASE IN
COEFFICIENT OF DETERMINATION (R^) FOR THE TOTAL NUMBER OF
POLYARTHRA VULGARIS PER LITER WITH NO LAG
(Data are for the 35 micron net)
Station 1
Variable
Station 2
Variable
Photoperiod
Total Filterable
Solids
Temperature
Nitrate
Orthophosphate
Ammonia
Oxygen
Sodium
0.408 Photoperiod
Total Filterable
0.204 Solids
0.110 Temperature
0.101 Ammonia
0.090 Sodium
0.046 Nitrate
0.015 Oxygen
0.002 Orthophosphate
0.224
0.179
0.164
0.148
0.030
0.016
0.003
0.00001
Total
0.726 Total
0.459
At the 0.05 level of significance the important parameters associated with 35
micron mesh net data were photoperiod and Orthophosphate at Station One and
Photoperiod and temperature at Station Two. The eight variables identified in
Table 15 account for only 45.9 to 72.6% of the variation.
Relationships between depth, alkalinity, magnesium, and silicon were asso-
ciated with the 75 micron net data. Depth accounted for 4.7 to 5.4% of the
variation while alkalinity ranged from 0.06 to 2.7%. Magnesium ranged from
0.14 to 1.1% of the variation. Silicon accounted for 1.1 to 1.9% of the vari-
ation. At the 0.05 level of significance temperature and silicon were most
important at Station One, and temperature, magnesium, and nitrate were at
Station Two. The nine variables identified in Table 16 accounted for 28.6 to
44.1% of the variation.
Potential invertebrate predators were identified and they include the
rotifers Synchaeta sp. and Asplanchna sp., the COpepod Mesocyclops edax, and
the dipteran chaoborus sp.
38
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TABLE 16. STEPWISE REGRESSION ANALYSIS AND INCREASE IN
COEFFICIENT OF DETERMINATION (R^) FOR THE TOTAL NUMBER OF
POLYARTHRA VULGARIS PER LITER WITH NO LAG
(Data are for the 75 micron net)
Station 1
Station 2
Variable
Variable
Temperature
Oxygen
Photoperlod
Nitrate
Depth
Total Filterable Solids
Total Alkalinity
Magnesium
Silica
0.211
0.130
0.130
0.057
0.054
0.028
0.027
0.011
0.011
Temperature
Photoperlod
Depth
Oxygen
Total Filterable Solids
Silica
Nitrate
Magnesium
Total Alkalinity
0.158
0.091
0.047
0.037
0.026
0.020
0.015
0.001
0.001
Total
0.441
Total
0.286
DISCUSSION
poiyarthra vuigaris has been classified as a perennial, eurythermal species
(Carlin, 1943; Pejler, 1957) which usually has a population maximum in late
spring or early summer. The spring dominance of p. vuigaris in this study was
similar to the observations of Carl in (1943), Pejler (1957), Beach (1960), and
Abel (1974). Carl in (1943) and Edmondson (1965) concluded that the maximum
occurs at a temperature range from below 15 to about 20 C and our data support
these observations. Populations increased after the temperature reached 15 C
and the maximum was observed in June when the temperature at the surface was
26 C. Carl in also suggested that there may be an autumnal maximum between 5
and 10 C. While there was an autumnal maximum observed 1n October this oc-
curred when the mean temperature exceeded 10 C and as the temperature fell be-
low 10 C so did the populations. Our laboratory studies also showed an inhib-
ition of reproduction at 10 C (Figures 1 and 2) and Edmondson (1965) showed a
depression of reproductive rate below 10 C. Temperature accounted for between
11.0 and 21.1% of the population variation. Positive relationships between
temperature and the rotifers Keratella cochlearis, Kellicottia longispina,
39
-------
and Polyarthra vulgaris (Edmondson, 1965) and the Cladoceran Daphnia ambigua
(Angino, Armitage and Saxena, 1973) have been found. Temperature explained
14.4% of Polyarthra variation (Edmondson, 1965), a value very similar to ours.
Because the populations of Polyarthra fell while temperature was conducive
for reproduction other factors which could affect population dynamics were
examined. One possibility is the delayed hatching of eggs. Another possibility
is photoperiod which accounted for 9.1 to 40.8% of the population fluctuation.
The positive correlation of Polyarthra populations and photoperiod may explain
in part the June population peak, but it does not explain the smaller peak in
the fall. In our culturing studies survival and egg production were enhanced
by a photoperiod greater than 12 hr. Long photoperiods were associated with
populations of Daphnia schtfdieri (Parker, 1966) and other species of Daphnia
Angino, Armitage and Saxena, 1973).
Oxygen correlated negatively with Polyarthra populations and oxygen ac-
ccounted for 0.3 to 13% of the variation. Pejler (1957) suggests that P. vul-
garis is more "sensitive to deficiency of oxygen (and/or the conditions con-
nected with it) than are other commoner eurythermal species." Initially, these
results tend to be contradictory. The negative correlation in part may be illu-
sory since higher oxygen concentrations were found during the cooler months
when Polyarthra were in low numbers. Angino, Armitage, and Saxena (1973) found
a similar negative correlation with Daphnia ambigua while Hazelwood and Parker
(1961, 1963) found a positive correlation with D. schijzWieri. Oxygen probably
affects the survival of the early developmental stages of cladocera (Terao and
Tanaka, 1928) and hatching of the rotifer Brachionus (Lite and Whitney, 1925).
Angino, Armitage and Saxena (1973) suggested that low oxygen did not affect
signficiantly the survival of D. ambigua because it only accounted for 0.5% of
the variability. Our data demonstrated a variance as great as 13%. Polyarthra
was rarely found in zones where the oxygen content was less than 5 mg/1. Aer-
ation was important in culturing success and the animals died in stagnant con-
ditions where oxygen was depleted. Our data support Pejler's (1957) observations
on the sensitivity of P. vulgaris to low oxygen.
All correlations between nitrate concentration and Polyarthra numbers were
positive and nitrate accounted for 1.5 to 10.1% of the variation. Nitrate did
not show up as a significant variable until after a three-week lag with Daphnia
ambigua (Angino, Armitage and Saxena, 1973) and after a one-week lag with
Diaptomus paiiidus (Armitage, Saxena and Angino, 1973). Our results suggest
that nitrate may have an effect on Polyarthra but again the effect may be illu-
sory. Polyarthra peaks close to spring overturn when there was an increase in
the nitrate content of the water.
Total filtrable solids (TFS) were negatively correlated with Polyarthra
numbers and TFS explained 2.6 to 20.4% of the variation. Seasonally the mean
values for TFS were highest in late March (143.2 mg/1) and June and July (67.5
and 62.5 mg/1 respectively) corresponding to periods of higher rainfall. The
values of TFS increased near the bottom where there were few Polyarthra. Again
the effect may be more illusory than real. Polyarthra are positively photo-
tactic to weak light (Viaud, 1943) and as turbidity increases they probably
40
-------
would move up the water column away from heavier particles and higher TFS values.
Lastly, it is possible that TFS may effect light quality and quantity and its
effect on the biology of the animal (see below).
Total alkalinity, magnesium silicon, and depth contributed to the vari-
ability of population data obtained with the 75 micron mesh net only. None of
these were significantly correlated and their relationships varied from positive
to negative (Tables 11, 12, 13, 14, 15 and 16). Borecky (1956) concluded that
bicarbonate affected cladocera through its effect on food levels. Since
Poiyarthra may not eat many algae, the low variability for alkalinity and the
insignificant correlations were not surprising.
Magnesium contributed 3.4% of the variation in populations of Daphnia
ambigua (Angino, Armitage and Saxena, 1974) and 0.1 and 1.1% for Poiyarthra
(Table 16, this study). Magnesium is an essential enzyme activation (Prosser,
1973), and concentrations in Pandapas Pond probably were low enough to be
favorable for survival and nontoxic as has been demonstrated by others for
cladocera (Taub and Dollar, 1964; Crosby and Tucker, 1966). Only one slightly
negative correlation with magnesium was found.
Silicon correlations were usually negative and they attributed between 1.1
to 1.95% of the variation. Silicon is related closely to diatom production and
it is possible that dense diatom populations may inhibit Poiyarthra populations.
Light affects the vertical distribution of Poiyarthra and they are commonly
found in the epilimion near the surface of a body of water (Berzins, 1958;
Pejler, 1957; this study), and they are positively phototactic to moderate
light (Viaud, 1943). Several hypothesis have been proposed for this vertical
distribution Of Poiyarthra vulgaris.
Both Pejler (1957) and our study note that the rotifer is sensitive to
low oxygen and Poiyarthra may be near the surface because of higher oxygen
concentration.
Pejler (1957) noted that the vertical distribution of Poiyarthra may also
parallel the vertical distribution of cryptomonas in nature (Rodhe, 1955) and
Carlin's data (Figures 49 and 100 in 1943) show variation. Edmondson (1965)
obtained a significant positive correlation between cryptomonas and Poiyarthra.
Based on our laboratory research this correlation may be illusory, cryptomonas
requires B,? and thiamin for growth (Hutchinson, 1967), and our culturing
success of Poiyarthra was impeded until B,2, thiamine, and other vitamins were
added to the culture water. Perhaps both organisms were found together because
of specific vitamin requirements.
Research on light effects on populations of Daphnia pulex (Buikema, 1972,
1973a_, 1973b_, and 1974) suggest that light itself may interact with biological
processes and affect reproduction and growth. The vertical distribution of
field rotifer population was always at moderate light intensities and labora-
tory populations aggregated at a light intensity of approximately 400 ft-c.
It is possible that light intensity also has an effect on the biological pro-
cesses Of P. vulgaris.
41
-------
Orthophosphate, ammonia, and sodium contributed to the variation of Poiy-
arthra populations collected with the 35 micron mesh net (Table 15). Ortho-
phosphate was positively correlated with poiyarthra populations and the effect
was significant at Station One (Table 11). Again this correlation may be
illusory because the rotifer peaks in spring when nutrients are released into
the water. A negative correlation existed between phosphate and Daphnia ambigua
(Angino, Armitage, and Saxena, 1973) and they suggest a possible toxic effect.
Ammonia accounts for 4.6 to 14.8% of the variation (Tables 15 and 16) and
the correlation was significantly negative at Station Two. Ammonia was toxic
to aquatic animals and this may alone explain its effect. An opposite effect
was observed with Daphnia ambigua (Angino, Armitage, and Saxena, 1973).
The effect of sodium was negative and it accounted for 0.2 to 30% of the
variability (Tables 11, 12, 13, 14, and 15). This relationship also may be
illusory because sodium was highest in the winter when the rotifers were at a
minimum. Sodium had no effect on populations of Daphnia ambigua (Angino,
Armitage and Saxena, 1973) and its effect on the copepod Diaptomus paiiidus
occurred after a four-week lag (Armitage, Saxena, Angino, 1973).
Carbon dioxide, sulfates, potassium calcium, total hardness, pH, total
phosphate, and iron were not significantly correlated nor did they appear in
the stepwise regression analysis. Apparently poiyarthra vulgaris was insensi-
tive to changes in these parameters over the range recorded in Pandapas Pond.
The lack of a response to pH agrees with the conclusions of Pejler (1957) that
pH was not an important determinant of Poiyarthra distribution.
Pejler (1962) suggests that the appearance of Poiyarthra may be explained
by the abundance of algae in late spring or early summer. There was no signif-
icant correlation between chlorophyll a and numbers of Poiyarthra. Our lab-
oratory results were contrary to the observations of Edmondson (1965),
Dieffenbach and Sachse (1911) and others since Poiyarthra rarely ate algae.
Interestingly, when the mean chlorophyll a values were the highest in July,
August, and September the rotifer populations were declining. Also, phyto-
plankton biomass did not correlate with the population density of Brachionus
caiydfiorus (Halbach, 1972).
The possibility exists that there may be an antibiotic effect of algae
on Poiyarthra. In laboratory cultures if the bluegreen or green algae increased
there was usually a concurrent decrease in Poiyarthra population. If one com-
pares Carlin's data for bluegreen algae and poiyarthra vulgaris an inhibition
may exist (Figures 53 to 58, 100 and 101 in Carl in, 1943; Figures 145 and 146
in Hutchinson, 1967). An antibiotic effect between chioreiia and jceiiicottia
longispina has been suggested (Edmondson, 1965). Branchionus plicatus ex-
hibits a decrease in survival in dense populations of chioreiia (Hirayama,
Watanabe, and Kusano, 1973). Dense populations of chioreiia pyrenoidosa have
a negative effect on Branchionus caiydfiorus (Halbach, 1972; Halbach and
Halbach-Keup, 1974).
Beach (1960) and Pejler (1961) note that declines in Poiyarthra popula-
tions were concomitant with the appearance of a fungal parasite which penetrates
the animals and eggs (Paterson, 1958, pers. observ.). Our observation suggests
42
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that the sheath forming bacterium of the sphaerotiiis-Leptothrix complex also
may have a negative effect on Poiyarthra. While field animals were not ob-
served to be parasitized, pond water returned to the laboratory would develop
fungi and bacteria which hindered culture work. The potential impact when the
female drops the egg prior to hatching is important. If spores contact the
egg it will become infected. Efforts to identify the fungus beyond the chytrid
group have not been successful (Martha Roane, pers. comm.).
Four potential invertebrate predators, synchaeta sp., Aspianchna sp.,
Mesocyclops edax, and Chaoborus Sp., were identified in the Study. Synchaeta
were present from February through May when Poiyarthra populations were their
lowest. None were collected when Poiyarthra population peaks were dominant.
Aspianchna sp. were only found in June at a density of six per liter at Station
One and of two per liter at Station two. Average Mesocyclops edax densities
were lowest (10-20/1iter) during June through September when Poiyarthra peaked.
Chaoborus was seen only in August and September at densities less than 2/1iter.
The breeding season for the bluegill Lepomis macrochirus was not known
for Pandapas Pond but the impact of the bluegill larvae could be significant
since they feed specifically on Poiyarthra (Siefert, 1972). It is believed
that predator impact was minimal on populations of Poiyarthra vulgaris.
43
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50
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PUBLICATIONS AND DISSERTATIONS
Edmunds, P. C. 1974. Seasonal Fluctuations of Rotifer Populations Related to
Selected Biological, Chemical and Physical Parameters In a Small Mountain
Pond, Jefferson National Forest, Virginia. Virginia Polytechnic Institute
and State University, Blacksburg. M.S. Thesis. 109 pp.
Buikema, A. L., Jr., J. Cairns, Jr., and T. H. Krakauer. 1974. Preliminary
studies on the culture methods for Poiyarthra vulgaris (Rotifera). ASB
Bulletin. 21:43 (abstract).
Edmunds, P. C., A. L. Buikema, Jr., and J. Cairns, Jr. 1974. Preliminary
limnological investigation on a spring-fed impoundment, Pandapas Pond,
Jefferson National Forest. ASB Bulletin. 21:52 (abstract).
Buikema, A. L., Jr., P. C. Edmunds, and J. Cairns, Jr. Factors affecting pop-
ulations of the rotifer, Poiyarthra vulgaris. Abstracts of Paper Submitted
to the Thirty-eight Annual Meeting American Society of Limnology and
Oceanography.
51
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APPENDIX
Preliminary procedures for batch culturing of Poiyarthra vulgaris.
Because the rotifer has not been continuously cultured in the laboratory
it will be necessary to periodically obtain animals from the field.
1. Collection - rotifers should be concentrated with a 35 micron mesh
net. Cladocera and copepods should be removed as much as possible
with a larger mesh net. Cultures should be started with an inoculum
of about 1,000 rotifers per liter.
2. Handling - The rotifers should be handled with a 1 mm bore pipette
or larger, and they should not be handled any more than necessary.
3. Containers - Glass containers with a minimum volume of one liter and
a large surface to volume ratio should be used.
4. Culture medium - Use water from a natural source that contains
arthra vulgaris. The water should be filtered through a 10 or 35
micron mesh net to remove larger algae and animals.
The culture medium should be partially replaced twice a week and
totally replaced once a week.
5. Light - An incident illumination of 400 to 500 ft-c, a complete light
spectrum and a 16L:8D photoperiod should be provided. G.E. cool
white fluorescent bulbs are satisfactory.
6. Oxygen - Containers should be moderately aerated to maintain an
oxygen concentration near 8 ppm.
7. Vitamins - Minimally the vitamins B,2> thiamine, biotin, and pan-
tothenic acid should be supplied to the rotifer food organisms and
added to the culture water. Commercial vitamin mixtures for pets,
such as Vionate, can be used. To cultures containing five liters of
medium, add one-half gram of Vionate after each partial change of
culture medium and one gram after each complete change.
8. Food type - Feed a protozoan mixture Of Chilomonas paramecium, Cya-
thomonas truncatus, Bodo minimus, B. variabilis, and B. mutabilis.
These protozoans can be raised in a Purina Trout Chow medium that is
fortified minimumally with vitamins B19, thiamine, biotin, and pan-
tothenic acid. '*
52
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9. Food quantity - Feed 50 ml of this protozoan mixture to a 5-liter
culture daily and the protozoan concentration should be around 300,000
protozoans per ml.
10. Temperature - Optimum temperatures were not specifically determined.
Reproductive success was best at 20 - 22 C.
11. Antibiotic and parasitic agents - Observations should be made for
fungi and bacteria on the rotifers and the presence of dense growths
of green or bluegreen algae. Both were detrimental to the rotifer.
These cultures should be restarted.
12. Population density - If the rotifer density decreases below 40 ani-
males per liter the population may not recover. New cultures should
be started.
53
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing]
1. REPORT NO.
EPA-600/3-77-051
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Dulturing and Ecology Studies of the Rotifer Poiyarthra
vulgaris
5. REPORT DATE
August 1977 issuing date
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
Arthur L. Buikema, Jr., John Cairns, Jr., Paul C.
Edmunds and Thomas H. Krakauer
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biology and Center for Environmental
studies
/irginia Polytechnic Institute and State University
Jlacksburg, Virginia 24061
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
R800815
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Dul., MN
Office of Research and Development
J.S. Environmental Protection Agency
Xiluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final 2/1/73 tn fi/^n/7/L
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The results contained in this report represent research conducted to identify
variables which affect the survival and reproduction of the rotifer, Poiyarthra
uigaris. The following variables were studied: handling stress, container size,
Frequency of changing the culture medium, light quantity and quality, photoperiod,
>xygen and vitamin requirements, fungal parasites, food preference and concentration,
mtibiotic effects of bluegreen algae, and temperature.
Temperature had an effect on population dynamics, percent of females with
;ggs, number of eggs per female, and sexual reproduction. Egg production rates
i/ere estimated and observations on the duration of egg development were made.
This report also includes a field study of the relation between Poiyarthra vulgari.
nd 19 selected chemical and physical parameters.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Ecology, Zooplankton, Cultures,
Population
Aquatic populations,
zooplankton, fish
diet Rotifers
06F
8. DISTRIBUTION STATEMENT
el ease Unlimited
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OP PAGES
64
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
54
U.S. GOVERNMENT PRINTING OFFICE: 1977- 757-056/65U Region No. 5-11
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