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EFFECTS OF NUTRIENT ENRICH-MENT
AND A ROUGH FISH POPULATION (CARP)
ON A GAME FISH POPULATlON.
(STMALLMOUTH BASS)

Thesis for the Degree of Ph. D, g?
MICHIGAN STATE UNIVERSITY
TERRY A. HAKNES .
1971

 

 

This is to certify that the

thesis entitled
Effects of Nutrient Enrichment and a Rough
Fish Population (Carp) on a Game Fish
Population (Smallmouth Bass) .

presented by
Terry A. Haines

has been accepted towards fulfillment
of the requirements for

 

Ph.D. degreein Fisheries and Wildlife

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ABSTRACT

EFFECTS OF NUTRIENT ENRICHMENT AND A ROUGH
FISH POPULATION (CARP) ON A GAME FISH
POPULATION (SMALLMOUTH BASS)

BY

Terry A. Haines

This experiment was conducted to test the effects
of nutrient enrichment and a rough fish population,
individually and in combination, on a game fish population.

The rough fish and game fish species used were carp

 

(Cyprinus carpio L.) and smallmouth bass (Micropterus

dolomieui Lacépéde) respectively.

 

Using a series of artificial ponds, two fertility
levels (low and high) were established. Low fertility
ponds were left undisturbed, while inorganic nitrogen and
phosphorus were added to the high fertility ponds at bi-
weekly intervals. Replicates of three fish populations
were established at each fertility level: game fish only,
rough fish only, and game fish and rough fish in combi-
nation. The physical, chemical and biological conditions
in the ponds were sampled regularly. The fish populations
were censused after 15 months. Growth rate was used as

an index of the status of the various fish populations.

Terry A. Haines

RNA-DNA ratios also were measured to test the degree to
which they reflect growth rate.

In low fertility ponds, bass and carp had similar
growth rates and RNA levels. In high fertility ponds the
growth rate and RNA levels of bass were significantly
reduced (p < 0.01), while those of carp increased
(p < 0.01).

The growth rate of each species was not signifi-
cantly (p = 0.5) affected by species interactions at either
fertility level. The RNA data showed no significant
difference (p = 0.4) in the RNA level of bass in low
fertility ponds when carp were present or absent. However,
in high fertility ponds bass had higher RNA levels when
carp were present (p < 0.01). Carp in low fertility ponds
had higher RNA levels when bass were present (p < 0.01),
but carp in high fertility ponds had lower RNA ratios when
bass were present (p = 0.01).

The depression of bass growth rate and RNA concen-
trations in enriched ponds may have been a result of the
increased durnal dissolved oxygen flux, decreased water
clarity and increased ammonia concentrations in these
ponds. Carp apparently were not affected by these factors.

The increase in RNA levels of bass in high fer-
tility ponds when carp were present may be a result of
bass feeding on carp eggs and fry, or to an increased
availability of benthos resulting from the feeding

activity of carp. Social facilitation in feeding response

Terry A. Haines

may also be present. The reasons for the changes in carp
RNA levels observed when bass were present are unknown.
The significant responses found in growth rate
data also were present and significant in the RNA data; in
several cases non-significant trends seen in growth rate
data were present and significant in the RNA data. RNA
concentration may be a more sensitive indicator of the

condition of a fish than is growth rate.

EFFECTS OF NUTRIENT ENRICHMENT AND A ROUGH
FISH POPULATION (CARP) ON A GAME FISH

POPULATION(SMALLMOUTH BASS)

BY

.9
Terry AI Haines

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Fisheries and Wildlife

1971

'7', ________

ACKNOWLEDGMENTS

I wish to express my gratitude to all persons who
helped me in my research. I would particularly like to
thank my major professor, Dr. N. R. Kevern, and my graduate
committee, Dr. W. E. Cooper, Dr. H. W. Johnson and
Dr. E. W. Roelofs, for continuing advice, criticism and
encouragement throughout my study.

I am indebted to the Federal Water Quality Office
(S-Fl-WP-26,225 and STl-WP-109) and the Michigan State
University Agricultural Experiment Station for financial

support, without which I could not have completed my work.

ii

TABLE OF CONTENTS

Chapter
INTRODUCTION . . . . . .
MATERIALS AND METHODS . . .

Experimental Design . .

Description of the Experimental Ponds

Fish Populations . . .

Dissolved Oxygen and Gross Photosynthesis

Temperature . . . . .
Water Chemistry. . .
Benthos and Zooplankton .

Fish Growth Rate and RNA Content

RESULTS 0 O O O O O O 0
Temperature . . . .
Dissolved Oxygen and Gross
Water Chemistry. . . .

Benthos and Zooplankton .
Fish Growth Rate . . .
Fish RNA-DNA Ratios . .

DISCUSSION. . . . . . .

Physical and Chemical Conditions

Gross Photosynthesis . .

Photosynthesis

in the Ponds.

Effects of Fertility Level on Fish Populations
Effects of Species Interactions on Fish

Populations . . . . .
SUMMARY AND CONCLUSIONS . .

REFERENCES CITED. . . . .

iii

Page

50
50
51
52
59
70

73

Chapter
APPENDICES
Appendix

A. Maximum and Minimum Temperatures Recorded
in a Low and a High Fertility Pond . .

B. Method for RNA-DNA Analysis . . . .

iv

Page

81

83

Table

1.

LIST OF TABLES

Page
Analysis of Variance Table for Growth Rate
Data Showing Orthogonal Comparisons
Between Treatments . . . . . . . . . . 41
Comparison of RNA and DNA Concentrations
in Various Species of Fish . . . . . . . 46
Analysis of Variance Table for RNA Data
with Orthogonal Comparisons Between
Treatments. . . . . . . . . . . . . 47

Figure

10.

11.

12.

13.

LIST OF FIGURES

The Experimental Ponds . . . . . . . .

An Experimental Pond Showing the Method of
Division. . . . . . . . . . . . .

Twenty-four Hour Dissolved Oxygen Curves for
a Low and a High Fertility Pond of July 8
and 9, 1970 Respectively . . . . . . .

Gross Photosynthesis Values for the
Experimental Ponds . . . . . . . . .

Photosynthesis to Respiration Ratios for the
Experimental Ponds . . . . . . . . .

Mean Per cent Transmission Values for the
Experimental Ponds . . . . . . . . .

Mean Total Phosphorus Values (as P04) for
the Experimental Ponds . . . . . . . .

Mean Nitrate Nitrogen Values for the
Experimental Ponds . . . . . . . . .

Mean Ammonia Nitrogen Values for the
Experimental Ponds . . . . . . . . .

Mean Standing Crop Biomass of Benthos in the
Experimental Ponds . . . . . . . . .

Mean Standing Crop Biomass of Zooplankton in
the Experimental Ponds . . . . . . . .

Mean Growth Rates of the Fish Populations
from the Various Experimental Conditions . .

Mean RNA-DNA Ratios for Fish Populations
from the Various Experimental Conditions . .

vi

19

21

24

26

28

30

32

35

37

40

45

Figure Page

14. Interaction Graph Showing the Effect of the
Presence of the Other Species on the Growth
Rate of Each Species of Fish . . . . . . 62

15. Interaction Graph Showing the Effect of the
Presence of the Other Species on the RNA-DNA
Ratio of Each Species of Fish. . . . . . 66

A-l. weekly Maximum and Minimum Temperatures
Recorded in the Experimental Ponds . . . . 82

vii

INTRODUCTION

Much attention has been given to artificial
eutrophication in recent years and an extensive literature
is being developed (see National Academy of Sciences,

1969, for a recent review). It is now generally accepted
that eutrophication is the result of excessive inorganic
plant nutrients, especially phosphorus and nitrogen,
entering surface waters from municipal and industrial
wastes, and urban and rural runoff (Mackenthum, 1968; Ohle,
1953; Sawyer, 1954, 1965). The resultant increase in
productivity and excessive plant growths are well documented
(Hutchinson, 1969). The usual effect on fish is a decline
in numbers of species and an increase in numbers of indi—
viduals (Gaufin and Tarzwell, 1952; Hasler, 1947; Thompson
and Hunt, 1930). In lakes that stratify thermally,
hypolimnetic oxygen depletion is commonly associated with
eutrophication. This depletion has been blamed for the
change in fish species by elimination of the habitat
required by cold water species (Hasler, 1947; Larkin and
Northcote, 1969). The same type of change also occurs in

waters which do not stratify, and the species lost are

normally the desirable game fishes, while the rough fishes
increase in population size (Katz and Gaufin, 1953;
Tarzwell and Gaufin, 1953). Instead of hypolimnetic oxygen
depletion, non-stratifying waters exhibit extreme diurnal
variations in oxygen content because of plant activities
(Mackenthum, Ingram and Porges, 1964; Schroepfer, 1942;
Smith, 1934). It has been proposed that the low point in
dissolved oxygen at night may limit fish distributions
(Gaufin and Tarzwell, 1952, 1956).

Rough fish have been accused of interfering with
game fish in a variety of ways, including competition for
food, roiling of the water and spawning interference
(Aldrich, 1943; Bennett, 1943; Krumholz, 1956). Rough fish
also are associated with waters of naturally high nutrient
levels (Moyle, 1956; Moyle, Kuehn and Burrows, 1950).

There is thus some question concerning the reason for the
observed species change in fish populations in eutrophic
waters. The decline of game fish may be due to physical
changes in the environment, to interference from rough fish
which are better adapted to enriched conditions, or to some
combination of these factors. This experiment was designed
to test the effects of nutrient enrichment and a rough fish
population, individually and in combination, on a game fish

population.

MATERIALS AND METHODS

Experimental Design

 

A series of 12 small artificial ponds was used in
this experiment. Two fertility levels were established by
leaving half of the ponds undisturbed (low fertility) and
enriching the others by the regular addition of nitrogen
and phosphorus (high fertility). Within each fertility
level replicate fish populations of each of three types
were established: game fish only, game fish and rough fish
in combination, and rough fish only. This gave eight
experimental conditions:

1. Game fish only in low fertility ponds.

2. Game fish with rough fish present in low
fertility ponds.

3. Game fish only in high fertility ponds.

4. Game fish with rough fish present in high

fertility ponds.

. Rough fish only in low fertility ponds.

6. Rough fish with game fish present in low

fertility ponds.

. Rough fish only in high fertility Ponds.

8. Rough fish with game fish present in high
fertility ponds.

The fish populations were exposed to these con-
ditions for 15 months and were censused at the end of this

time. The growth rate of each species under each of the

experimental conditions was used as an index of the

suitability of that condition for that species. Growth
rate has been reported to be the major mechanism by which
fish adjust to environmental conditions (Allen, 1951;
Backiel and Le Cren, 1967; Ricker, 1958). A biochemical
indicator of growth also was used secondarily in an attempt
to determine the extent to which it agrees with growth rate
in semi-natural conditions. Since growth in fish is due
primarily to protein synthesis, the amount of ribonucleic
acid (RNA), the protein organizer, should be a reflection

of the growth rate of that fish (Leslie, 1955).

Description g£_the Experimental Ponds

 

 

The ponds used in the study were former waste water
treatment ponds located at the corner of Kalamazoo and
Harrison Streets, East Lansing, Michigan. Six identical
ponds (7.2 x/7.2 x 1.8 m deep), arranged in two rows of
three ponds each, were available (Figure 1). To obtain a
sufficient number of ponds for replication of experimental
conditions, the ponds were divided in half by the use of
black polyethylene film reinforced with nylon net (Figure
2). Prior to use in this study these ponds twice had been
allowed to stand full of water for several months, then
were drained and cleaned. To prevent nutrients bound in
the bottom of the pond from entering the water and thus
raising the fertility level of the low fertility ponds, the
bottom of each pond was covered with a layer of polyethylene

film and filled to a depth of 0.3 m with peastone gravel.

 

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The ponds were then filled to a depth of 1.2 m with water
from University wells. This water had a pH of 8.3 and a
total alkalinity in excess of 200 mg/l as CaCOB.

Treatments were assigned to ponds by numbering the
ponds and then drawing numbers from a random number table.
Fertility levels were assigned to the six whole ponds
first, then fish populations were assigned to the half
ponds within each fertility level.

Biweekly applications of 250 g urea (45% N) and
110 g super-phosphate (20% P) were made to the high fer-
tility ponds during the ice-free period of the experiment.
This was approximately 3.5 mg/l N and 0.67 gm/l P, giving
an N:P ratio of 5:1. Fertilization was begun one month
before fish were introduced into the ponds.

Temperature and oxygen profiles made prior to the
introduction of fish and periodically thereafter indicated
that the ponds did not stratify. The maximum temperature
gradient observed from surface to bottom in the ponds was
1 C; the maximum oxygen gradient was 1.5 mg/l 02.
Therefore, it was assumed that the ponds were uniformly

mixed, probably by wind action.

Fish Populations

 

Smallmouth bass (Micropterus dolomieui Lacépéde)

 

and carp (Cyprinus carpio L.) were selected as the game

 

fish and rough fish species. These species are common to

many warm water streams and lakes in Michigan. Attempts

10

were made to collect native fish for use in this study but
were unsuccessful due to weather and equipment problems.
The bass were obtained from a private hatchery in Pennsyl-
vania; the carp were seined from a marsh connected to Lake
Erie near Toledo, Ohio. The literature was consulted to
determine the age structure and standing crop biomasses of
these species occurring naturally (Beckman, 1949; Bennett,
1938, 1945; Carlander, 1955; O'Donnell, 1943; Smith, 1942).
The standing crops of each species which could be supported
in ponds of the size used were then calculated. Since the
ponds were small and older fish were difficult to obtain,
only age 0 and age I fish were used. This allowed a larger
number of fish to be used per pond than if older fish had
been present, and these age classes possess all of the
feeding habits developed in the ontogeny of these species
(Emig, 1966; Burns, 1966). In addition, the literature
(Hubbs and Bailey, 1938; Reynolds, 1965; Swee and McCrimmon,
1966) indicated that neither carp nor bass would spawn
until age III at the earliest. Thus it was believed that
spawning would be eliminated as a variable. This proved

to be an erroneous belief. The biomass calculated above
was reduced by 20 per cent to allow for error and growth,
and then apportioned between the two age classes on the
basis of the relative sizes of these age classes in natural
populations. The naturally occurring populations used in

arriving at this estimate were obtained from mixed species

11

populations. Therefore the same biomass and age structure
were used for a species population regardless of whether
the population was to be alone or in combination with the
other species in the experiment. Each rough fish popu-
lation was composed of approximately 80 g of age 0 carp
and 100 g of age I carp (180 9 total); 20 g of age 0 bass
plus 30 g of age I bass (50 9 total) constituted a game
fish population. The bass were of known age; the carp
were aged by the scale method.

Recently hatched bass feed on microcrustaces for
the first few weeks, then gradually expand their diet to
include larger crustaces and insects. After the first
year, their diet consists of insects, crustaceans and fish
(Emig, 1966; Lachner, 1950, Wickliff, 1920). Young carp
feed in microcrustacea, small insects and plant debris.
Older carp feed primarily on insects (especially midge
larvae). The amount of plant material found in carp
stomachs is quite variable and its importance is unknown
(Burns, 1966, Clemens, Rawson and McHugh, 1939, Moen,

1953).

 

Dissolved Oxygen and Gross Photosynthesis

 

Continuous 24 hour dissolved oxygen measurements
were made on successive days in one high and one low fer-
tility pond, selected at random. Two determinations were
made per month during the ice-free period of the experiment.

A single mid-depth measurement was made using a Rustrak

12

Temperature-Oxygen recorder (model 192, Rustrak Instrument
Co., Manchester, N. H.). A small (three gallons per minute
capacity) submersible pump and a length of garden hose were
used to provide a flow of water past the oxygen probe, with
the water being returned to the depth from which it was
taken. The calibration cf the oxygen probe was checked by
taking a water sample from the vicinity of the pump, using
a Kemmerer water sampler, and determining its dissolved
oxygen content with the azide modification of the iodo-
metric method (American Public Health Association, 1965).
Gross photosynthesis values were calculated from the
diurnal oxygen curves following the single station method
of Odum (1956). The values for the gas transfer coefficient
(K) were calculated from the rate of change of oxygen
concentrations for each diurnal curve as described by Odum

(pp, 913.).

Temperature

 

Temperature was recorded continuously in one high
and one low fertility pond, chosen at random, using Ryan
thermographs (model D-8, Ryan Instrument Co., Seattle,

Wash.).

Water Chemistry

 

Chemical determinations were made biweekly
throughout the ice-free period of the experiment and twice

during the period of ice cover. The samples were taken on

l3

alternate weeks from those during which fertilizer was
added to the ponds. The determinations were made from a
single water sample per pond, taken from mid-depth with a
Kemmerer water sampler and transferred to a one liter
plastic bottle. Duplicate determinations of transparency,
ammonia nitrogen and nitrate nitrogen were made the same
day the sample was taken; phosphorus measurements were
performed the following day. The samples were stored
overnight in a refrigerator.

Clarity of the water was estimated by measuring
the per cent transmission of the water sample using a
filter photometer (Klett-Summerson Photoelectric Colori-
meter, Klett Mfg. Co., New York, N. Y.) equipped with a red
filter. Nitrate nitrogen measurements were made using the
brucine method of Jenkins and Medesker (1964). Ammonia
nitrogen was determined with the direct nesslerization
test, and total inorganic phosphorus (as phosphate) was
measured by the stannous chloride method (American Public

Health Association, 1965).

Benthos and Zooplankton

 

To determine the amounts of food organisms present,
and thus possibly detect a competition for food, biweekly
estimates of the standing crop biomass of zooplankton and
benthos were made. Duplicate plankton samples were taken
at night from each pond. A single, vertical net tow

(number 20 mesh, 76 u opening) was used per sample.

14

During 1969 these samples were preserved immediately in

10 per cent formalin. To determine the zooplankton weight
the sample was diluted to a known volume from which two
subsamples were removed. These subsamples and the re-
mainder of the original sample were filtered onto indi-
vidually tared glass fiber filters. The zooplankters were
removed from the subsample filters by picking them off with
forceps. All filters were then dried (105 C) to constant
weight and weighed. It was then possible to calculate the
dry weight of the total sample and the weight of the sample
minus the zooplankton. The weight of the zooplankton then
was obtained by subtraction. (This method was extremely
time-consuming, so a different technique was sought.

During the winter of 1969-70 an easier method was
developed for separating zooplankton from debris and most
phytoplankton. The samples were collected as before, but
now they were carefully transferred to a plastic box filled
with filtered water. An amber light was used to draw the
zooplankters into a glass vial which was attached to the
bottom of the box. After the zooplankters had entered the
vial it was removed from the box and the sample preserved
as before. The sample was later filtered onto dry glass
fiber filters, dried and weighed. In preliminary tests
this method was found to be about 85 per cent efficient at
removing the zooplankton from the sample, and no difference

in response between different species of zooplankton was

15

observed. On several occasions during 1970 large blooms
of Volvox sp. occurred in a few ponds. These organisms
were also attracted to the amber light and were highly
mobile. When these organisms were present in bloom
quantities the previous method of estimating zooplankton
weight was used.

Benthos samples were taken with a 15 x 15 cm
Ekman dredge. Duplicate samples were taken from each pond
and sample sites were selected randomly. The benthic
organisms were concentrated by sugar flotation and then
sorted from the remaining debris by hand. They were
filtered onto dry glass fiber filters, dried and weighed
as described for zooplankton. No correction was made for
any benthos which may have been present on the walls of

the ponds.

Fish Growth Rate and RNA Content

 

 

At the end of September, 1970 the ponds were
drained and all fish recovered. The fish from each pond
were washed, sorted by species and weighed. The growth
rate for each species was calculated in terms of grams of
weight gained per gram initial weight per 15 months (the
term of the experiment).

Four fish of age I and four of age II of each
speCies were randomly chosen from each experimental con-
dition and used in the determination of RNA concentration.

About one gram of dorsal muscle tissue was removed from

16

each fish and frozen for future analysis. Since the amount
of deoxyribonucleic acid (DNA) per cell is relatively
constant within a species, the ratio of RNA to DNA (RNA per
unit DNA) can be used as a measure of the relative amount

of RNA per cell (Hotchkiss, 1955). This ratio automatically
corrects for differences in numbers of cells per tissue
sample. Bulow (1970) has developed a technique for de-
termining RNA and DNA concentrations in fish and applied it

experimentally. His method was followed.

RESULTS

Temperature

 

The temperature data indicate that the average daily
summer temperature was about 23 C in low fertility ponds
and 20 C in high fertility ponds. The maximum temperature
recorded was 26 C; the average daily range was about 2 C.
In autumn, winter and spring there was no difference in

temperature between low and high fertility ponds.

Dissolved Oxygen and Gross Photosynthesis

 

Typical 24 hour dissolved oxygen (D.O.) curves for
a low and a high fertility pond are shown in Figure 3. The
high fertility ponds had wide diurnal variations in D.O.
throughout the ice-free period. Afternoon values often
reached 18 mg/l O2 and pre-dawn lows were commonly below
2 mg/l 02. Low fertility ponds had much less diurnal
variation in D.O., the variation seldom exceeding 3.5 mg/l.
The D.O. in low fertility ponds was at or near saturation
for much of the day, while high fertility ponds varied from
200 per cent saturation to 25 per cent saturation.

The gross photosynthesis values obtained are shown

in Figure 4. The first observation was made immediately

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after nutrients were first added to the ponds. However,
high fertility ponds quickly reached very high levels and
stayed high throughout the study, commonly exceeding

28 mg Oz/mZ/day and reaching a maximum of 35.5 mg OZ/mz/day.
Low fertility ponds never exceeded 13 mg Oz/mz/day.

Ratios of photosynthesis to respiration (P:R ratios) are

shown in Figure 5.

Water Chemistry

 

The water chemistry data are presented in Figures 6
through 9. The standard errors shown are the mean standard
errors for all points indicated. Since there were two
observations per pond and two (or more) ponds per treatment,
these standard errors represent variation due to sampling
error and differences between ponds treated alike. No
attempt was made to separate these sources of error. The
data indicate that widely different conditions were
established in the low and high fertility ponds.

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were arranged to show differences between fertility level
and presence or absence of carp. The values plotted were
obtained by averaging values from all ponds which contained
carp and from all ponds in which carp were absent within
each fertility level. High fertility ponds were lower in
Clarity than low fertility ponds in all cases. There was
no consistent difference between ponds which contained carp

and those which did not at either fertility level.

 

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31

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32

 

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33

During the growing seasons phosphorus averaged
0.5 mg/l as P04 in high fertility ponds and 0.05 mg/l in
low fertility ponds (Figure 7). Nitrate nitrogen values
were quite variable in high fertility ponds but reached
0.7 mg/l in both years, while low fertility ponds averaged
about 0.04 mg/l (Figure 8). Ammonia nitrogen values for
high fertility ponds averaged 0.1 mg/l during the summer
of 1969 (Figure 9), but were as high as 2.05 mg/l in 1970.
This increase was associated with a decrease in nitrate
nitrogen values in these ponds. Low fertility ponds rarely

had any detectable ammonia nitrogen.

Benthos and Zogplankton

 

The benthos and zooplankton standing crop biomass
data (dry weight) are given in Figures 10 and 11. The
standard errors shown were obtained in the same way and
contain the same components of error as those given for the
chemistry data. In general, higher standing crops of
benthos were found in high fertility ponds than in low
fertility ponds. The dominant benthic organism was

Chironomus sp. The same organism was dominant in both high

 

and low fertility ponds. Species of fish present had little
effect on the benthos until mid-summer of 1970. After this
time, high fertility ponds in which carp were present had
lower weights of benthos than those ponds which contained

bass only. The magnitude of the standard errors suggests

- .C: new};

 

Figure 10.

34

Mean standing crop biomass of benthos in
the experimental ponds.

Mean dry weight per sample in grams

 

High

on”. Doss alone
e—--e Bass and carp
0—-o Carp alone

Fish Introduced

l969

35

Fertility Ponds

Mean standard error
for all points

l970

 

 

 

0.0
1.
i
0.4 -
Q
E
O
'5
.s
0.3 -
.2
O.
E
O
O
i
_ 0.2 -
S
.9
O
l
r
U
0.: .
5
O
2
o l
0.10

h.-

Lou Fertility Ponds

e—-e Boss and carp
o--o Carp alone

6

Fish Introduced

I969

Mean standard error
for all points

|97O

 

 

 

L.-

Figure 11.

36

Mean standing crop biomass of zooplankton
in the experimental ponds.

 

Mean dry weight per sample.

in grams

Mean dry weight per sample, in grams

 

37

High Fertility Ponds

Mean standard error

‘c‘ 80.. 0.0". Ifof a" po'n'.

e—-e Boss and corp
o--o Corp alone
1 Fish Introduced

l969 |97O

 

 

 

0.05
0.04
0.03 t
0.02
0.0I
0
Low Fertility Ponds
on. Bass atone
e—e Boss and com Mean standard error
one Corp alone I for all points
l Fish introduced
l969 l970
0.02
0.0 l
I _ ’4.
o s L 4 _1

 

 

 

38

that this may be a significant difference. There was no
such difference in low fertility ponds.

Similar results are evident for zooplankton
standing crops, except that differences related to the
presence or absence of carp were found in low as well as
high fertility ponds in late 1970. Again, the weights were

lower in those ponds which contained carp.

Fish Growth Rate

 

The fish growth rate data are shown in Figure 12.

3 factorial

These data were analysed using a replicated 2
analysis of variance test. The standard error for treatment
means shown in Figure 12 was calculated from the error mean
square. The treatment sums of squares were partitioned
into orthogonal single degree of freedom components. The
results of this analysis are given in Table 1. It can be
seen that carp had a significantly (p < 0.01) greater
growth rate than did bass when all treatments are pooled
for each species. This is likely a result of the greater
growth of carp in the high fertility ponds. Carp spawned
successfully in the high fertility ponds, and therefore
part of the increased biomass observed was due to repro-
duction and not to body increment alone. Since it was
impossible to determine what the growth rate of the older
carp would have been if the age 0 fish had not been
present, no attempt was made to separate reproductive

increase from body increment. Carp did not spawn in low

39

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40

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41

 

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Hmcomonuuo mCH3onm mump mumn nuBoum mom OHnmu OOCMHHM> mo mHmemcé .H OHQmB

42

fertility ponds and there was no evidence of bass spawning
in any pond. It was not possible to design a treatment
comparison contrasting bass growth rate with that of carp
at each fertility level, which would be orthogonal to the
other comparisons desired. This should be considered for
future experiments. The magnitude of the standard error
assigned to treatment means makes it reasonable to conclude L
that the growth rates of the two species are probably not
significantly different at low fertility levels. The
difference in growth rate found between the two species,
then, is most likely a difference in response to fertility
level and not an intrinsic difference between the species.

When growth rates in low and high fertility ponds
are compared within each species, it can be seen that bass
have a significantly (p ~ 0.01) lower growth rate at high
fertility levels than at low fertility levels. The
opposite is true for carp (p < 0.01).

The presence or absence of carp had no significant
effect (p = 0.5) on the growth rate of bass at either
fertility level. An inspection of Figure 12 shows that the
mean growth rate of bass in high fertility ponds is
slightly higher when carp are present than when they are
absent. There was no apparent difference at low fertility
levels. Although not significant, it is interesting to
note that this response is exactly opposite to that

expected. A slight increase also was noted when the growth

43

rate of carp alone in enriched ponds was compared to that
when bass were present but the difference was not signifi-

cant (p = 0.5).

Fish RNA-DNA Ratios

 

The RNA/DNA ratios obtained for fish from the
various treatments are shown in Figure 13. The standard
error for treatment means shown was calculated from the
error mean square as found below. These ratios are
considerably larger than those obtained by Bulow (1970)

for the golden shiner (Notemigonus crysoleucas). However,

 

the RNA and DNA concentrations found in this study, and
used to calculate the ratios given in Figure 13, agree well
with those reported for a variety of other fish (Table 2).
The data were analyzed in essentially the same way as the
growth rate data. An additional factor, age, was added to
the factorial analysis, and two subsamples were taken from
each fish. The analysis of variance table with single
degree of freedom comparisons is given in Table 3. With
this design it is possible to separate sampling error
(among samples within fish) from experimental error (among
fish within treatment). These errors are significantly
different (p < 0.01), indicating that fish subjected to the
same treatment did not respond exactly alike. Therefore
experimental error (which includes sampling error) was

used to calculate all remaining F ratios, and the degrees

 

44

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Cmpcoa quHHuuom 30H u mg "m3oHHom mm mH mpoo 0cmmoH one .mcoHUHpcoo
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46

 

 

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47

 

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mv.m esmH.m0 mm.mmv H mm.m00 .uuom 20H: 0000 m> .uumm 30H 000m
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mH.~ .000.v~ NH.me mH mn.Hmm.~ 0005000He
mm.o 0 m2 mo mm mousom

 

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48

of freedom for experimental error were used to determine
the critical F values.

iAll references to RNA amount which follow concern
the RNA-DNA ratios. Responses obtained with the RNA data
are similar to those obtained with the growth rate data.
Carp had a higher RNA value than did bass when all
treatments were pooled. Again, this difference probably
is due to the large differences between bass and carp from
high fertility ponds. In low fertility ponds the RNA
values obtained for bass and carp were similar and, judging
from the magnitude of the standard error obtained, probably
not significantly different. Bass from high fertility
ponds had significantly lower RNA values than those from
low fertility ponds (p < 0.01); carp from high fertility
ponds, on the other hand, had significantly higher RNA
values than those from low fertility ponds (p < 0.01).
These are the same responses observed with growth rate data.

Whereas no significant differences were observed
in growth rate data with regards to the effect of the
presence or absence of one species of fish on the other,
such differences do occur in the RNA data. The presence
or absence of carp does not result in a significant
difference in the RNA level of bass under low fertility
conditions (p = 0.3). In high fertility ponds, however,
bass have a higher amount of RNA when carp are present

than when carp are absent (p < 0.01). This was observed

49

as a trend in the growth rate data but was not significant.
In low fertility ponds carp had a significantly greater
amount of RNA when bass were present than when bass were
absent (p = 0.01). An opposite response was obtained in
high fertility ponds; that is, carp had a significantly
lower RNA level when bass were present (p = 0.01). The
growth rate data for carp from low fertility ponds gave a
non-significant trend parallel to that obtained in the RNA
data, but no such trend was noted in the carp from high
fertility ponds.

A significant effect was also obtained for age
differences. Age II fish had lower levels of RNA than
Age I fish (p = 0.01) except for carp alone in low fer-

tility ponds (p = 0.3).

DISCUSSION

Physical and Chemical Conditions
TEE the Ponds

 

 

The physical and chemical conditions observed in
the high fertility ponds are typical of eutrophic waters.
Nutrient levels established are within the range of values
reported for enriched waters (Edmondson, 1969; Sawyer,
1965). The decrease in transparency was the result of an
increase in the amount of phytoplankton present. The
slightly lower temperatures recorded for high fertility
ponds probably resulted from the shading effect of the
phytoplankton as described by Smith (1934). The diurnal
oxygen cycles observed are similar to those observed in
other eutrophic waters which do not stratify (Gaufin and
Tarzwell, 1952; Odum, 1956; Olson, 1932).

Carp have been reported to be responsible for
roiling of the water in some lakes (Black, 1946; Burns,
1966; Sigler, 1955, 1958). This did not occur in the
experimental ponds. Although the bottom of the ponds
consisted of gravel a considerable amount of fine silt also
was present. In addition, much organic debris was quickly

deposited on the gravel after the ponds were filled. When

50

 

51

benthos samples were taken with the dredge a large amount
of sediment was stirred up. Thus the sediment was there,

the carp just did not disturb it.

Gross Photosynthesis

 

The increased nutrient supply in the high fertility
ponds resulted in higher gross photosynthesis values than
those observed in low fertility ponds. Compared to the
values reported by others (Odum, 1956; Welch, 1968) the
gross photosynthesis values obtained in the experimental
ponds are quite high, although not unreasonably so. The
gas transfer coefficients (K) found ranged from 0.13 to
0.50 and thus fall well within the range of values reported
by Odum (1956) for standing waters. The variation in the
K values is believed to be due to differences in wind
action (Welch, 1968) and surface disturbances by small carp
(McConnell, 1962). In any event, the effect of the cor-
rection for diffusion on the gross photosynthesis values was
relatively minor, never exceeding 10 per cent in my data.

Welch (1968) believes that available carbon may
limit primary production in most waters to 12-15 mg
Oz/mz/day. Since the water in the experimental ponds was
highly alkaline, it is doubtful that carbon limited the
production in low fertility ponds. Phosphorus was probably
not limiting, but nitrogen may have been. It is difficult

to determine what may have limited production in high

rpm

52

fertility ponds. Phosphorus was probably not limiting,
but nitrogen, carbon or a micronutrient may have been.

The water in high fertility ponds had a lower
transparency than that of low fertility ponds throughout
the winter. This indicates that the enriched ponds had
higher standing crops of phytoplankton. Since light and
temperature conditions were the same for all ponds, they
could not have been limiting in low fertility ponds. It
is more likely that the loss of nutrients by the deposition
of dead plankton organisms was an important limiting factor.

Since all P:R ratios were greater than one, it is
apparent that the communities in all ponds were autotrophic
and not heterotrophic, by Odum's (1956) definition. The
decline in P:R ratios in the summer of 1970 may be a
reflection of increased bacterial decomposition of organic
matter deposited in the ponds. This may also account for
the increase in ammonia nitrogen observed at this time.

Effects of Fertility Level
an Figh Populations

 

 

The data for growth rate and RNA-DNA ratio both
demonstrate that carp are able to take advantage of the
high productivity in the enriched ponds and grow at a
significantly greater rate than in the low fertility ponds
(p < 0.01). The bass, on the other hand, are inhibited
by the conditions in the high fertility ponds and grow at

a slower rate than they do in the low fertility ponds

53

(p < 0.01). The fact that the carp spawned in the high
fertility ponds tends to confound the results somewhat,

but the magnitude of the differences in response between
bass and carp are such that the outcome would probably have
been the same had spawning not occurred. English (1951)
and Rehder (1959) indicate that carp may spawn as early as
age II. This is apparently what happened in the enriched
ponds. Successful spawning was observed only in 1970 when
the carp introduced into the ponds as age I in 1969 were
then at age II. Since these fish were aged by the scale
method it is possible that some mistakes were made and fish
actually older than age I were put into the ponds. However,
verification of the aging technique by experienced person-
nel and the sizes of the fish used indicate that this was
not so. Carp in high fertility ponds reached a much larger
body size than those from low fertility ponds (maximum
total length 35 cm vs 22 cm). This may be an important
factor in explaining the lack of spawning in the low fer—
tility ponds.

While gross photosynthesis was higher in high
fertility ponds than in low fertility ponds by a factor of
three (9 mg Oz/mZ/d vs 30 mg/mZ/d), carp growth rate was
higher by a factor of four (400 g/g/15 months vs 1650
g/g/15 months) and bass growth rate was lower by a factor
of two-thirds (600 g/g/15 months vs 200 g/g/15 months).

The carp increase indicates that the consumer food chain

54

is still functioning efficiently and that there is no
imbalance between production and consumption as described
for eutrophic waters by Odum (1961) and Ryther (1954).

The decline in bass growth indicates that the bass cannot
efficiently use the available food in high fertility ponds.

The reasons for the difference in response to high
fertility conditions between bass and carp cannot be
determined with certainty, but inferences can be drawn
from the conditions in the ponds. The high and low fer-
tility ponds differed with respect to nutrient level,
transparency, temperature and diurnal oxygen changes.

No toxicity has ever been reported for the con-
centrations of phosphorus and nitrate compounds which
occurred in the enriched ponds (Abegg, 1950; Fuller, 1949;
McKee and Wolf, 1963). The toxicity of ammonia nitrogen
concentrations as reported in the literature is variable.
Ellis (1937) reports that ammonia levels of 2.0-2.5 mg/l
were toxic to goldfish. McKee and Wolf (1963) cite
references that report 3.4 mg/l ammonia toxic to bluegills
in soft water and 24.4 mg/l in hard water. There is no
information available concerning the sublethal effects of
ammonia on fish. Therefore ammonia levels in the high
fertility ponds may be an important factor.

The temperature reached in the experimental ponds
were close to the preferred temperatures of both species

of fish. Bennett (1965) reports a preferred temperature

55

of 28 C for smallmouth bass, but acknowledges that this is
higher than that normally observed in smallmouth bass
streams and lakes. Ferguson (1958) reports that smallmouth
bass preferred temperatures of 20.3 to 21.3 C in natural
lakes in northern Wisconsin. Beamish (1964) reports that
carp reached a peak in spontaneous activity at 27 C, while
Pitt, Garside and Hepburn (1956) found a final preferrendum
of 32 C. Although the enriched ponds were slightly cooler
in summer than the non-enriched ponds, the difference was
small (about 2 C), and on the basis of preferred temper-
ature alone it would be expected that bass would grow
better in high fertility ponds than in low, while carp
would do best in the low fertility ponds. This is the
opposite of what occurred.

It has been proposed by Bennett (1965) that since
centrarchids are sight feeders, a decrease in water clarity
could impair their feeding ability. Since the enriched
ponds in this experiment experienced a decrease in clarity,
this may have had some effect on the growth rate of the
bass. It is difficult to imagine a depression of growth
rate as severe as that observed resulting solely from a
decrease in water clarity. In addition, several authors
(Langlois, 1936; Surber, 1939) have reported higher growth
rates for smallmouth bass from turbid environments as

compared to relatively clear environments.

56

A likely reason for the difference in response to
high fertility conditions between bass and carp is a
difference in response to the oxygen conditions in those
ponds. Sustained low 0.0. values were never observed in
the ponds, but the diurnal variations were quite marked.
In high fertility ponds the oxygen content was frequently
at or below 2.0 mg/l 02 for four hours per day, and below
4.0 mg/l for eight hours each day. The oxygen content in
the low fertility ponds never dropped below 6.0 mg/l. At
average summer temperatures oxygen saturation was about
9 mg/l. Although 24 hour D.O. measurements were not made
during the period of ice cover (November to March) spot
checks of oxygen content were made on randomly selected
ponds at two week intervals. These checks were usually
made in mid-afternoon, and the oxygen levels found were
usually at or near saturation. No evidence of winterkill
conditions was ever observed. The wind kept the snow
Cleared off of the center of the ponds so that light
penetration was not impaired.

Sustained low oxygen levels as the result of
organic pollution have frequently been cited as a cause of
undesirable changes in fish populations (Ellis, 1937;
Hynes, 1960; Thompson, 1925), and the importance of a
lethal low point in a diurnal oxygen cycle has been
mentioned (Gaufin and Tarzwell, 1952, 1956). However, the

effect of a diurnal oxygen cycle with a sublethal low

57

point has not been investigated in natural systems.

Several laboratory studies have been made and are of
interest here. Diurnally fluctuating oxygen concentrations
have been shown to cause reduced growth rates in brook

trout, Salvelinus fontinalis (Dorfman and Whitworth, 1969;

 

 

Whitworth, 1968), striped bass, Morone saxatilis (Dorfman ‘
i

and Westman, 1970), largemouth bass, Micropterus salmoides ‘

 

(Stewart, Shumway and Doudoroff, 1967) and coho salmon,

Oncorhynchus kisutch (Doudoroff and Warren, 1957). The

 

reduced growth rate was correlated with a reduced food
intake and not with a reduction in metabolic efficiency
(Stewart ep'al., 1967). Similar data are not available
for any species of rough fish. Bouck and Ball (1965)
observed a change in blood serum proteins in largemouth
bass and bluegills subjected to diurnally fluctuating
dissolved oxygen levels. The change, a reduction in the
low mobility fraction of the protein, was interpreted as
an indication of physiological stress. Such a change was
not found in the yellow bullhead, a fish common to low
oxygen content waters.

Sustained low but sublethal D.O. concentrations
have been shown to reduce the swimming performance in
several centrarchids and salmonids (Dahlberg, Shumway and
Doudoroff, 1968; Katz, Pritchard and Warren, 1959). Grahm
(1949) found a reduced scope for activity in brook trout

which were exposed to low oxygen concentrations. Similar

58

studies have not been done for diurnally fluctuating D.O.
concentrations, but it is reasonable to presume that the
nocturnal low D.O. values could affect activity levels of
some fish and this could possibly reduce feeding ability.

Carp are commonly found in waters with low oxygen
contents (Burns, 1966; Mccrimmon, 1968). Carp hemoglobin
has been shown to have a high oxygen affinity and a small
Bohr effect, thus carp are able to saturate their blood
with oxygen in water which has a low oxygen and a high
carbon dioxide content (Basu, 1959; Fry, 1957; Riggs,
1970). Garey and Rahn (1970) have shown that tissue oxygen
concentrations in carp are independent of diurnal fluctu-
ations in the oxygen content of the water in which the carp
are maintained. Muennich (1958) found that carp blood
sugar levels were not affected by the oxygen content of the
water when the D.O. was varied from supersaturation to very
low levels.

Smallmouth bass are associated with clean, well
aerated aquatic environments (Emig, 1966; Hubbs and Bailey,
1938). Detailed information on their blood oxygen
characteristics is lacking but they are probably similar
to those found for other centrarchids. In order to be able
to saturate their hemoglobin with oxygen, centrarchids
generally require water with a higher 0.0. concentration
than do carp. They also show a significant Bohr effect

(Fry, 1957; Riggs, 1970).

59

Bouck and Ball (1967) determined the blood serum
protein distribution in a variety of fish. They found
that fish associated with polluted environments had high
levels of low mobility proteins, while those associated
with clean environments had low levels of these proteins.
In their experiment, carp had 20.2 grams of low mobility
protein per 100 g of sample protein, while smallmouth bass
had 2.7 g/100 g. The biological significance of this
protein fraction is unknown, but the amount present in a
species appears to be genetically determined.

From the above information it can be concluded that
the physical and chemical conditions associated with
eutrophication, i.e., decreased light penetration, increased
diurnal oxygen flux, increased decomposition and ammonia
byproducts, are quite likely to have had an adverse effect
on smallmouth bass and are unlikely to have affected carp.
The reduced growth rate and RNA content of the bass from
high fertility ponds was an expression of this effect.
Carp, being unaffected, could use the increased production
in the enriched ponds to obtain an increased growth rate
and RNA content.

Effects pf Species Interactions
gp_Fish POpulations

 

 

The expected inhibition of bass by carp did not
occur. There was no significant difference in growth rate

of bass when carp were or were not present (p = 0.5).

60

There also was no significant difference in the growth rate
of carp when bass were present or absent (p > 0.5). There
are some trends in the data, however, and these are best
seen in an interaction graph (Figure 14). This graph is
obtained by pairing the growth rate of a population of one
species alone with the growth rate of that species when

the other species was present. This was done for both
species of fish and for both fertility levels. The popu-
lations were paired on the basis of "potential for growth."
This potential was estimated from the amount of food
organisms present in the experimental ponds before the fish
were introduced. An index of the potential for growth was
obtained by summing the standing crop biomass estimates of
zoOplankton and benthos from the July, 1969 samples for
each pond. Fish populations from the ponds were then
paired on the basis of similarity of this index. If there
was no effect on the growth rate of a species due to the
presence or absence of the other species, then the points
plotted should be equidistant from each axis, falling on a
line drawn through the origin at a 45 degree angle. If

one species inhibits the other these points would fall
below this line, and if a beneficial interaction were
present, the points would fall above the line. Distance
from the origin is an indication of the magnitude of the
growth rate. An inspection of Figure 14 shows that the

growth rate of carp in high fertility ponds was not

 

61

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0:0 00 monomOHa 0:0 00 000000 0:0 mCHzoCm na0Hm coHuomumucH

 

 

.0H OHDmHm

62

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0000 000. 000. 00! 000. 000. 000 000 00... 000 0 . .

 

 

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63

affected by the presence of bass, and that the growth rate

of bass in low fertility ponds was not influenced by the

presence of carp. However, there is a slight increase in

the growth rate of carp in low fertility ponds when bass

are present, and a similar increase in the growth of bass

in high fertility ponds when carp were present. Since 3
these effects were not significant their importance is ‘
doubtful, but it is interesting to see that they are
opposite of what was expected. Since growth rate de-
pression due to species combination did not occur, food
competition can be ruled out even though the ponds in which
carp were present had lower standing crOps of benthos and
zooplankton in the summer of 1970. An examination of the
stomach contents of selected fish at the termination of

the experiment indicated that fish of all ages and both
species were feeding heavily on midge larvae of about the
same size. Midge larve comprised the greatest portion of
the invertebrate biomass throughout the study. The fact
that the carp did not cause an increase in turbidity of

the water could be important. While this factor could
explain the absence of a depression of bass growth in the
presence of carp, it cannot explain the increase which was
found. The lack of any significant species interaction

may have resulted because the conditions necessary for

such an interaction were not created.

64

If the RNA data are examined, it is seen that bass
in high fertility ponds had higher RNA levels when carp
were present than when carp were absent (p < 0.01). The
same is true for carp in low fertility ponds when bass
were present. The same trends were found in the growth
rate data but were non-significant. Bass in low fertility
ponds still show no species interaction effect (p = 0.4).
Carp in high fertility ponds show a significant (p = 0.01)
depression in RNA level when bass are present. An inter—
action graph shows these effects very well (Figure 15).
The graph is plotted the same as that for the growth rate
data (Figure 14). Each point represents the mean of the
subsamples from a single fish. The fish populations were
paired as they were for growth rate data; fish of the same
age were paired randomly between the populations.

The increased growth rate and RNA level shown by
bass in high fertility ponds when carp were present may be
a result of the bass feeding on the eggs and fry of
spawning carp. This would be a plentiful and easily
available food supply. The feeding actions of carp may
also have dislodged benthic organisms, making them easy
prey for the bass. A type of social facilitation of
feeding could also be operating (Baerends, 1957). That is,
the presence of fish that are actively feeding may trigger
the feeding response in other fish. The relatively low

standing crops of invertebrates in low fertility ponds and

Figure 15. Interaction graph showing the effect of
the presence of the other species on the
RNA-DNA ratio of each species of fish.

 

 

RNA/DNA ratio, each species with

RNA/DNA ratio, each species with

other species present

other species present

66

o corp,low fertility ponds

0 car ,hi h tertilit onds
AGE 1 O 00.5.09. fertilityyrfonds
I hass,hi htertilit ponds
o /
22 - . /,
20 - /
l8 i- 2’ a
00 D
.. L . . . .
I4 - 7:
'2 "" . /
t0 .. e /
'l /
0 - /
/
" /
4 i- /
/
2 »- /
o l 4 l L l i 1 l J 1 l J

 

 

02468

l0 l2 l4

IS 20 22 24

RNA/DNA rati0,eoch species alone

AGED
24.
22.
20,
la-
l6-

i4~ 0/

l2- o
IO- /o

 

010305
T
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02468

1 l l l l l l

l0 l2 l4

0 corp,low fertility ponds

Cl carp, high fertility ponds

e boss,low fertility ponds

I boss, high fertility ponds
/

l l l J

l8 20 22 24

RNA/DNA ratio, each species alone

67

absence of spawning carp may have made the effects of
these mechanisms unimportant in those ponds and thus
resulted in no effect on the growth of the bass. I

The increase in the growth rate and RNA level of
carp from low fertility ponds when bass were present is
more difficult to explain. A social facilitation in
feeding response seems unlikely but should not be ruled
out. Carp are able to absorb organic molecules from the
water (Brown, 1957) and therefore could make use of some
of the waste products of the bass plus their own to obtain
additional energy.

The decrease in RNA level but not growth rate which
was found in carp in enriched ponds when bass were present
is unexplained. This is the only experimental condition in
which the RNA level and growth rate data do not agree.
Creelman and Tomlinson (1959) determined the quantity of RNA

and DNA in the muscle tissue of sockeye salmon (Oncorhynchus

 

pegka) at three points in their spawning migration. They
found a progressive decline in the RNA content of the
flesh. Since carp spawned in the high fertility ponds,

the decrease in RNA level recorded might be associated with
spawning. However, a similar decline did not occur in the
high fertility ponds from which bass were absent, where the
carp also spawned successfully. In fact, the carp from

every high fertility pond showed increased RNA levels as

68

compared to all low fertility ponds, regardless of the
presence or absence of bass.

When comparing growth rates with RNA changes in
the fish, it is important to note that the growth rates
were determined over a period of 15 months. RNA ratios,
on the other hand, are known to change drastically in a
matter of days when fish are fed after being starved or
deprived of food after being fed regularly (Bulow, 1970).
Thus the RNA ratios probably represent only the growth
rate of the last week or so before the populations were
censused.

Since spawning was intended to be excluded from
this experiment, no information on the effects of
eutrophication or species interaction on the reproductive
success of either species was colleCted. Given the high
reproductive potential of warm water fish (Carlander,
1953) and the widely different spawning habitats preferred
by smallmouth bass and carp (Hubbs and Bailey, 1938;
McCrimmon, 1968) a serious effect on population size
because of spawning interference is relatively unlikely to
occur. Reynolds (1965) correlated annual recruitment in a
population of smallmouth bass with mean summer water
temperature and not number of adult spawners. There is
one recorded case (Latta, 1963) where the activity of carp
roiled the water in a shallow cove and spawning smallmouth

bass deserted their nests.

69

The general agreement of responses in growth rates
and RNA levels tends to substantiate the hypothesis that
RNA level is a reflection of recent growth in fish (Leslie,
1955). The fact that most non-significant species inter-
action trends seen in the growth rate data were present
and significant in the RNA data suggests that RNA level may
be a more sensitive indicator of the status of a fish

population than is growth rate.

SUMMARY AND CONCLUS IONS

This experiment was designed to test the influence
of nutrient level and a rough fish population (carp) on a
game fish population (smallmouth bass). Two nutrient
levels (high and low) were used, with high levels created
by addition of inorganic nitrogen and phosphorus. Growth
rate and RNA levels were used as indicators of the status
of the fish populations.

Both bass and carp populations had approximately
the same growth rates in low fertility ponds. The average
RNA concentrations for the two species also were similar
under these conditions. In high fertility ponds, both the
growth rate and RNA content of the bass declined severely.
Under the same conditions carp experienced a large increase
in growth rate and RNA level.

The growth rate data show no significant species
interactions at any fertility level. Bass had slightly
higher growth rates in high fertility ponds when carp were
present than when the bass were alone, and carp in low
fertility ponds also showed a slight increase in growth

when bass were present. These trends were not

70

71

statistically significant. The RNA data, however, showed
the same trends and here they were significant. There was
no effect on the RNA levels of bass in low fertility ponds
when carp were present. However, in high fertility ponds,
carp showed a significantly reduced RNA level when bass
were present. Growth rate data showed no indication of [A
this effect.

The depression of growth rate and RNA level in
bass in high fertility ponds was attributed to the large
diurnal changes in oxygen content of the water in these
ponds, to decreased clarity of the water caused by
phytoplankton blooms, and to increased ammonia concen—
trations due to increased bacterial decomposition of organic
matter. The slight increase in growth and large increase
in RNA levels noted in bass when carp were present in high
fertility ponds may have been a result of the bass feeding
on carp eggs and fry. There was no evidence of food
competition between bass and carp. The changes in carp
growth rate and RNA levels in the presence of bass have not

been explained.

It is concluded that, under the conditions which
were generated in the experimental ponds, the decrease in
smallmouth bass observed in the enriched ponds was the
result of the physical and chemical changes in the water
due to increased plant and bacterial activity and not to

interference by carp. Carp could not be shown to interfere

72

with the smallmouth bass under any conditions employed.
Extensive testing under varied conditions should be

performed to determine the validity of these conclusions.

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_...'

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APPENDICES

APPENDIX A

MAXIMUM AND MINIMUM TEMPERATURES RECORDED

IN A LOW AND A HIGH FERTILITY POND

81

Figure A—l. Weekly maximum and minimum temperatures
recorded in the experimental ponds.

 

Temperature, degrees C

24

20

Temperature, degrees C.

N

6

2

O)

m

.—

 

 

82

High Fertility Ponds

 

 

a- - 4 Maximum
e—e Minimum

|970

I“ I“
I “‘0" 3‘ lope-k

 

 

Low Fertility Ponds
one Maximum
e—e Minimum

l969

 

 

 

 

  

APPENDIX B

METHOD FOR RNA-DNA ANALYSIS

METHOD FOR RNA-DNA ANALYSIS

The following is the RNA and DNA analysis technique
as developed by Bulow (1970). It is reproduced here in
detail for the benefit of those who may wish to use this

technique.

Preparation g£_Fish Tissue Samples

 

 

About one gram of dorsal muscle tissue was removed
from each fish and placed in an individual glass vial of
about 50 m1 capacity. The vials were capped and immedi-
ately frozen. After one week of freezing the fish samples
were defatted and dried by a chloroform-methanol-ether
process and stored for subsequent nucleic acid analysis.
This was done by removing the vials of fish samples from
the freezer and adding a 2:1 mixture of chloroform and
methanol to each vial. About 35 ml of solvent were used
for each extraction. The vials were then capped and
agitated periodically with an automatic shaker. After a
12 hour period the solvent was changed and the process
repeated for an additional 12 hours. The solvent was then
replaced with ethyl ether for an additional 24 hour
extraction period. The ether was then poured off and the
samples left to air dry for 24 hours. Dry, fat-free
samples were ground to a powder in a Wiley mill equipped

with a number 40 sieve and stored in capped vials.

83

 

 

84

Preparation pf RNA and DNA Standard
Stock SolutiOns

 

 

 

One hundred mg of RNA powder (Nutritional Bio-
chemicals Corp.) were placed in a large test tube and
suspended in 5 ml of distilled water. The mixture was
warmed and agitated until it appeared to go into solution.
Ten m1 of 5 per cent trichloroacetic acid (TCA) was then
added and the solution heated at 100 C for 60 minutes.

(In heating tubes in the constant temperature bath it was
found desirable to cover them with large marbles to prevent
loss through evaporation and yet allow for gaseous ex-
pansion.) The solution was then cooled and brought to a
volume of 200 ml with 5 per cent TCA.

The DNA standard stock solution was prepared in
the same way. Stock solutions were quite stable when
stored under refrigeration.

Determination 9£_Total Phosphorus Content
pf Standard Stock Solutions

 

 

 

To accurately establish the concentrations of
standard stock solutions of nucleic acids, total phosphorus
content was determined colorimetrically using the procedure
of Fiske and Subbarow (1925). All colorimetric determi-

nations were made on a Beckman model DK spectrophotometer.

Preparation of reagents and solutions.
Sulfuric acid: Stock solutions of 5 N and 10 N

were prepared.

85

Molybdate reagent I (Acid): Ammonium molybdate
salt (25 g) was dissolved in 200 ml of distilled water.
The solution was then rinsed into a one liter volumetric
flask containing 500 ml of 10 N sulfuric acid, and diluted
to the mark with distilled water. The reagent was stored
in a plastic bottle under refrigeration.

Molybdate reagent III (Water): Ammonium molybdate
salt (25 g) was dissolved to a volume of one liter with
distilled water. The reagent was stored in a plastic
bottle under refrigeration.

Standard phosphate: In a one liter volumetric
flask, 0.3509 g of monOpotassium phosphate was dissolved
in a small amount of distilled water. Ten ml of 10 N
sulfuric acid were added and the solution brought to the
mark with distilled water. This standard stock solution
contains 80 ug of phosphorus per ml. From this stock
solution additional dilutions to 40 ug/ml and 20 ug/ml
were made. Since 5 m1 of stock solution contains 400 ug
of phosphorus, these dilutions became 4, 2 and 1 ug of
phosphorus per ml when 5 m1 is brought up to 100 ml in
subsequent phosphorus analysis.

Amino-napthol-sulfonic acid reagent (ANSA): This
reagent was purchased in pre-mixed dry form (Hartman-
Leddon Co., Inc., Philadelphia, Penna.). It was prepared
according to the manufacturer's instructions and stored

in an amber bottle.

86

Determination of standard curve

Three standards (4, 2 and 1 ug of phosphorus per
m1) and a blank were prepared in four 100 m1 volumetric
flasks. Five ml of standard phosphate solution (replaced
with 5 m1 of distilled water for the blank), 65 m1 of
distilled water, 10 m1 of molybdate reagent I and 4 ml
ANSA were added to each flask. The contents were then
mixed, diluted to the mark with distilled water and again
mixed. After standing for five minutes the optical density

was measured against the blank at 700 mu.

Determination of nucleic acid phosphorus content

The procedure, which was identical for both RNA
and DNA standard stock solutions, was as follows:

1. Five m1 of nucleic acid solution and 5 ml of
5 N sulfuric acid were added to a Pyrex test tube (200 x
25 mm).

2. Several glass beads were added to the mixture
which was then carefully boiled over a microburner.
Caution: This procedure should be performed under a hood
with the solution being constantly agitated. If not
agitated it will superheat and spurt out. When the
solution had boiled down to a small amount of fluid, dense
fumes appeared and the solution turned a dark yellow. At
this time, the flame was turned down and the solution kept
boiling. The solution became progressively darker until

no further darkening occurred. At this time the solution

87

was decolorized by letting one or two drops of concen-
trated nitric acid run down the wall of the test tube.
Boiling was continued for about 30 seconds more to remove
the nitric acid.

3. The test tube was removed from the heat,
allowed to cool slightly and placed under cold tap water
to further cool it.

4. Contents of the tube were emptied into a 50 ml
volumetric flask. The tube was rinsed three times with
about 10 ml of distilled water, the washings being poured
into the flask.

5. Five m1 of molybdate reagent III and 2 m1 of
ANSA reagent were added to the flask.

6. The solution was mixed, brought to the mark
with distilled water and again mixed.

7. After standing for five minutes, the blue color
was measured against a blank at 700 mu. (The blank was
prepared by mixing the same reagents, but replacing the
5 m1 of nucleic acid solution with 5 ml of 5 per cent TCA.
The boiling procedure was eliminated.) Comparison of the
optical density measured in the nucleic acid solution with
the phosphate standard curve gave the concentration of
phosphorus in the nucleic acid standard stock solution.
RNA and DNA standards were then prepared by diluting the
nucleic acid standard stock solutions with 5 per cent TCA.

Standard curves were then run with each batch of unknowns.

88

Extraction pf Total Nucleic Acid

 

 

Nucleic acid was extracted from fish tissue samples
by the methods of Webb and Levy (1955). The procedure was
as follows:

1. Samples of dry, fat-free fish powder were
weighed and suspended in 3 m1 of 5 per cent TCA in 15 ml
centrifuge tubes. Fifty mg of fish powder was used per
sample.

2. Suspensions were allowed to stand at room
temperature until the powder became saturated and fell to
the bottom of the tube.

3. The tubes were covered with glass marbles and
placed in a boiling (100 C) water bath for exactly 30
minutes.

4. At the end of 30 minutes the rack of tubes was
immediately placed in a cold water bath.

5. After being cooled, each tube received an
additional 3 ml of 5 per cent TCA. The tube contents were
mixed thoroughly by shaking.

6. Each tube was centrifuged for about 15 minutes
and the supernatant fluid carefully drawn off with a
pipette, leaving the insoluble tissue protein in the tube
bottom.

7. Supernatant fluid, containing the combined
nucleic acids, was stored under refrigeration in capped

glass vials.

89

Colorimetric Determination
g£_RNA Content

 

 

Analysis of RNA content followed the orcinol method
described by Schneider (1957) with a modification by

Lusena (1951).

Preparation of reagents?

Orcinol reagent: The orcinol should be a perfectly
white crystalline product. Orcinol was purified by
boiling in glass distilled benzene, decolorizing with
charcoal and crystalizing after adding hexane. The process
was repeated twice and was performed under a hood using a
heater which could be regulated to hold the benzene at
boiling temperature (80 C). One gram of purified orcinol
was dissolved, immediately before use, in 100 ml of

concentrated HCl containing 0.5 g of FeCl3.

Method of color development:

1. Duplicate 0.5 ml aliquots of nucleic acid
extract as well as 0.5 ml samples of RNA and DNA standard
solutions were transferred to centrifuge tubes.

2. Each tube received 1.5 ml of distilled water
and 2 ml of orcinol reagent. The contents were mixed.

3. A blank of 2 ml distilled water and 2 ml
orcinol reagent was prepared.

4. The tubes were covered with marbles and heated

in a water bath at 100 C for 60 minutes.

90

5. After cooling the tubes in a cold water bath,
optical density was measured against the blank at 660 mu.

Since orcinol reacts with DNA as well as RNA, a
correction must be made for the fraction of optical density
resulting from the DNA present in each sample of nucleic
acid extract. This was done through use of the DNA I
standards in the orcinol reaction. Calculations were made
as described by Schneider (1957). *

Colorimetric Determination
9f DNA Content

 

 

DNA was determined using the Burton modification

of the diphenylamine reaction (Burton, 1956).

Preparation of reagents:

Diphenylamine reagent: Fisher special indicator
grade diphenylamine (1.5 g) was dissolved in 100 m1 of
glacial acetic acid and 1.5 ml of concentrated sulfuric
acid. Just prior to use, 0.5 ml of aqueous acetaldehyde
(2.1 ml acetaldehyde per 100 m1 glacial acetic acid) was
added to the reagent. This reagent was prepared just
prior to each use. Acetaldehyde is extremely volatile and
extreme care should be exercised in its use.

Perchloric acid (1 N): About 800 ml of distilled
water was placed in a one liter volumetric flask and 83.5
ml of concentrated (70 per cent) perchloric acid was
added. The total volume was then brought up to one liter

with distilled water.

91

Method of color development:

1. Duplicate 1 ml aliquots of nucleic acid extract

were combined with 1 ml of l N perchloric acid and 2 ml
of diphenylamine reagent. Samples of DNA standards were
similarly prepared.

2. A blank was prepared by substituting 1 m1 of
5 per cent TCA for the nucleic acid extract.

3. The contents were stirred and the tubes heated
at 30 C for approximately 18 hours.

4. Intensity of color was measured at 600 mu
against the blank and compared with values obtained with
the DNA standards.

Results were reported as concentrations of RNA and
DNA. The values obtained in the above analysis are

concentrations of RNA and DNA phosphorus. Since the

phosphorus content of both RNA and DNA is about 10 per cent

(Leslie, 1955), the values obtained are multiplied by a
factor of 10 to obtain RNA and DNA values. If only
RNA/DNA ratios are desired, this calculation is not

necessary.

.5'. h)-