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This is to certify that the
thesis entitled
ATTEMPTED TETRAPLOIU INDUclION IN DEUEGILL SUNFISH

(LEPOMIS MACROCHIRUS) USING HEAT SHOCKS

 

presented by

Paul D. Wilbert

has been accepted towards fulfillment
of the requirements for

Master of Science Fisheries and Wildlife

 

 

degree in

 

 

Dan: March 23, 1990

 

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MSU is An Affirmative Action/Equal Opportunity Institution
c:\circ\datedm.pm3-p.1

ATTEMPTED TETRAPLOID INDUCTION IN BLUEGILL SUNFISH
(LEPOMIS MACROCHIRUS) USING HEAT SHOCKS

BY
Paul D. Wilbert

A THESIS

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

MASTERS OF SCIENCE

Department of Fisheries and Wildlife

1990

@449” Iowa

ABSTRACT

ATTEMPTED TETRAPLOID INDUCTION IN BLUEGILL SUNFISH
(LEPOMIS MACROCHIRUS) USING HEAT SHOCKS

BY
Paul D. Wilbert

Sterile triploid and fertile tetraploid bluegill have
significant potential for use in fishery management and
aquaculture. Experiments were conducted to develop
techniques to produce tetraploid bluegill sunfish.
Preliminary studies indicated that: the upper lethal heat
shock temperature for bluegill eggs was 40°C for 10 minutes,
bluegill could be spawned out-of—season, bluegill fry could
be raised on brine shrimp nauplii, and bluegill ploidy
levels could be determined by flow cytometry.

Triplicate groups of bluegill eggs were heat shocked at
temperatures of 30, 35, and 40°C for 2.5, 5.0, 7.5, and 10.0
minutes. Three controls groups used. All heat shocks were
initiated 30 minutes after fertilization which is
approximately 5 minutes before cytokinesis. Five fish from
each heat shock treatment and control replicate were
analyzed for ploidy by flow cytometry. Fish tested for
ploidy were 67 to 85 days old.

No tetraploid bluegill were identified from the 180
tested. Tetraploids may have been produced; but, were
either in too low a number to identify by the sampling

procedure or died prior to the swim up stage of development.

ACKNOWLEDGEMENTS

I would like to thank my committee, Dr. Donald Garling,
Dr. Clarence McNabb, and Dr. Patrick Muzzall for their
advice and input into this project. A special thanks goes
to Dr. Garling, my major professor, for his guidance and
never ending patience with me.

Without funding from the American Tackle Manufacturer
Association and the Chicagoland Sport Fishing, Travel and
Outdoor Show this project wouldn't have taken place. I
thank them as well as the Cullerton staff for their
hospitality and support.

I would like to thank Dr. Kathy Brooks of MSU for
allowing me to use her lab and flow cytometer for ploidy
determination. I would also like to thank Vonnie
VanderPloeg for enduring the boredom of running the bluegill
samples.

Many individuals have assisted me in this project that
deserve recognition. I would like to thank Paul Spruell for
his help and support. A special thanks goes to Andy
Westmaas for his help, advice and enthusiasm. Finally, I'd
like to thank Bill Young, Ken Cain, Roger Glass, and Chris

Starr who worked with me to help complete this project.

Last, but definitely not the least, I'd like to thank
my parents. They've always been supportive of my career
goals, and I appreciate that. I never took the "easy" path,

and they are always there for me.

iv

TABLE OF CONTENTS

LIST OF TABLES ......... . ............................. Vi
LIST OF FIGURES ...................................... Vii
INTRODUCTION ......................................... 1
LITERATURE REVIEW ................... . ................ 5
I. Stunting of Bluegill Sunfish .............. 5

II. Naturally Occurring Polyploids ............ 6

III. Induction of Tetraploidy in Fish .......... 7

IV. Induction of Triploidy in Fish ............ 11

V. Ploidy Determination ...................... 13
MATERIALS AND METHODS ................................ 18
RESULTS .............................................. 26
DISCUSSION ........................................... 27
CONCLUSIONS .......................................... 32
APPENDIX A ........................................... 34
APPENDIX B ........................................... 39
APPENDIX C ........................................... 43
APPENDIX D ........................................... 48
REFERENCES ........................................... 51

LIST OF TABLES

 

Number Page
1. Summary of Tetraploid Induction
Experiments in Fishes.... ................... 9
2. Summary of Triploid Induction
Experiments in Fishes ....................... 12
Al. Outline of Pilot Experiment to Determine
the Upper Lethal Temperature for
Bluegill Sunfish Eggs ....................... 37
C1. Steps in Cell Staining Technique used to
Determine Ploidy Level in Bluegill Sunfish.. 44
C2. Composition of Reagents used in Flow
Cytometric Determination of Ploidy Level
in Bluegill Sunfish ......................... 45
i
vi

mtg

1.

Bl.

Cl.

LIST OF FIGURES

Page
Temperature and Duration Regime of
Heat Shocks Applied to Bluegill
Sunfish Eggs ................................ 21
Hot Water Bath used to Heat Shock
Bluegill Sunfish Eggs ....................... 22
Brine Shrimp Incubation Unit ................ 40
Data of Diploid Bluegill and Chinook Salmon
Blood as Generated by the Ortho 2150
Computer System ............... . ............. 47

INTRODUCTION

Stunting of the bluegill sunfish (Lepomis macrochirus;
Fam: Centrarchidae) is a problem that is often encountered
in ponds and small lakes. Stunting is caused by inadequate
amounts of food and/or space that lead to a decrease in
individual growth rate of fish (Murnyak et al., 1984).
Overpopulation is usually the cause of stunting.
Potentially, polyploid bluegills could be used to decrease
the population size and decrease or eliminate stunting.

A polyploid organism is one with three or more complete
sets of chromosomes (Ayala and Kiger, 1984). Triploid fish
have three sets of chromosomes (Ayala and Kiger, 1984) and
are sterile (Purdom, 1983; Thorgaard, 1986). Tetraploid
fish have four sets of chromosomes (Ayala and Kiger, 1984)
and are fertile (Chourrout et al., 1986, Chourrout and
Nakayama, 1987). Normal bluegill sunfish (diploid) have 48
chromosomes (Roberts, 1964); consequently a triploid
bluegill would have 72 chromosomes and a tetraploid bluegill
would have 96 chromosomes.

Artificially induced polyploid fish have many potential
aquaculture and fisheries management applications. Extended

growth and/or survival of mature fish may be expressed by

 

2
triploid fish (Thorgaard, 1986). Sterile triploid grass
carp (Ctenopharyngodon idella) can be used for aquatic weed
control (Wattendorf, 1986) were reproduction is undesirable.
Triploid fishes may be desirable where overpopulation and
stunting occur (Thorgaard and Allen, 1987).

Tetraploid fishes could be crossed with diploid fish to
produce triploid fish (Chourrout et al., 1986; Chourrout and
Nakayama, 1987). This method would have greater efficiency
compared to direct triploid induction using shocks.
Mortalities due to heat shocking and handling of triploids
would be eliminated. All of the fish produced by the cross
of diploid and tetraploid fish would be triploid, so
undesirable diploid reproduction would be eliminated. A
tetraploid broodstock can be easily maintained as tetraploid
fish can perpetuate themselves by normal mating (Chourrout
and Nakayama, 1987).

Polyploid bluegills could be used to control or
eliminate stunting in small lakes or ponds. In new ponds,
or ponds where the resident fish have been removed, sterile
triploid bluegills could be stocked directly. A continued
stocking program would be required to maintain the
population. Tetraploid bluegills of one sex and diploid
bluegills of the other sex could be stocked into these types
of ponds instead to produce triploid offspring. Finally,
tetraploid bluegills could be introduced into a resident

population of bluegill sunfish and over time a larger

3
percentage of the total offspring produced would be sterile
triploid bluegills. Male triploid rainbow trout (Benfey et
al., 1986) and grass carp (Allen et al., 1986) have produced
aneuploid sperm, and eggs fertilized with this sperm were
not viable. Triploid bluegill may show spawning behavior,
but fertilization by triploid sperm would yield inviable
eggs and would reduce the bluegill population. Initially,
artificial induced tetraploid bluegills would have to be
produced to establish a tetraploid broodstock.

Polyploid bluegill have a great potential for
aquaculturists as a food fish. The number of triploid
bluegill stocked in a pond would be absolute. The rate of
stocking triploid bluegill could be calculated so the
carrying capacity of the pond would be attained when the
fish reach market size. The market size of bluegill would
be determined by fish processors.

Artificially induced polyploid fish have been produced
by applying a temperature, pressure, or chemical shock to a
fertilized egg at the appropriate time in development. In
producing triploid fish, the shock must be applied early in
development prior to the second meiotic division (Purdom,
1983). Tetraploidy has been induced when the shock was
applied just prior to the first cellular division (Purdom,
1983). Shocks that have been used include temperature
(Swarup, 1959a; Lincoln et al., 1974; Valenti, 1975; Lemoine

and Smith, 1980; Thorgaard et al., 1981; Chourrout, 1982;

 

4
Benfey and Sutterlin, 1984; Solar et al., 1984; Bidwell et
al., 1985; Johnstone, 1985; Arai and Wilkins, 1987; Thompson
et al., 1987; Don and Avtalion, 1988), pressure (Benfey and
Sutterlin, 1984; Chourrout, 1984; Lou and Purdom, 1984), and
chemical (Smith and Lemoine, 1979; Refstie et al., 1977;
Allen and Stanley, 1979; Refstie, 1981; Shelton et al.,
1986; Johnstone et al., 1989).

A number of important preliminary studies were
completed in 1988-89 prior to tetraploidy induction
experiments. A pilot study was completed to determine the
upper lethal temperature for bluegill sunfish eggs. This
study is outlined in Appendix A. Techniques to raise
bluegill fry with brine shrimp is outlined in Appendix B.
Ploidy of the bluegill obtained from heat shock experiments
was analyzed using flow cytometry; the techniques are
presented in Appendix C. Tetraploid induction experiments
were performed on the bluegill sunfish during the summer of
1989. The body of this thesis describes attempted

tetraploid induction in bluegill sunfish using heat shocks.

LITERATURE REVIEW

Stunting of Bluegill Sunfish

Stunting in bluegill sunfish is caused by inadequate
amounts of space and/or food that lead to a decrease in
individual growth rate of fish (Murnyak et al., 1984). In
fish, a marked negative correlation has been observed
between population density and the rate of body growth
(Smith, 1980). The density of bluegill in a given body of
water will determine the size of bluegill found. Predators
are necessary to lower bluegill numbers, so the population
remains normal (Mittlebach, 1981).

Aquatic vegetation also has been shown to influence the
level of stunting observed in lakes and ponds. As the area
of submersed vegetation increased, the predation of bluegill
sunfish by largemouth bass (Micropterus salmoides) decreased
(Savino and Stein, 1982) and growth of bluegill sunfish
decreased (Crowder and Cooper, 1982). The decrease in
growth of the bluegill sunfish is due to reduced predation
efficiency by the largemouth bass (Crowder and Cooper,

1982).

 

6

Reducing the number of stunted fish in lakes and ponds
has increased the size of the remaining fish (Beckman, 1941)
and condition factor (Beckman, 1943). The condition factor
is used as an index of the length and weight of an
individual fish (Piper et al., 1983). When stunted bluegill
sunfish are placed into a body of water containing a normal
bluegill population, they grew; but not as well as the
unstunted bluegills (Murnyak et al., 1984). Potentially
triploid fish could be utilized where overpopulation and

stunting occurs (Thorgaard and Allen, 1987).

Natural Occurring Polyploids

Naturally occurring polyploidy has been relatively
common in plants, but rarely found in animals, occurring
only in lower forms such as annelids, insects, crustaceans,
fish, and amphibians (Ayala and Kiger, 1984). Natural
triploid fish have been identified from several species.

In salmonids, naturally occurring triploids have been
found in brook trout (Salvelinus fontinalis) (see Allen and
Stanley, 1978) and rainbow trout (Oncorhynchus mykiss) (see
Cuellar and Uyeno, 1972; Thorgaard and Gall, 1979). Single
triploid fish have been found in natural populations of
California roach (Hesperoleucus symmetricus) (see Gold and
Avise, 1976) and fathead minnow (Pimephales promelas) (see

Gold, 1986). Naturally occurring triploids have been

 

 

7
reported by Schultz (1967) in a population of hybrid
poeciliids (Poeciliopsis lucida X Poeciliopsis latidens).
Triploid fish were probably produced by triploid eggs being
elevated to a hexaploid (6N) level by an endomitotic
division and the sperm stimulated the egg to develop without
fertilization (Schultz, 1967). Electrophoresis could be
used to confirm sperm stimulation, if a suitable isozyme
marker is found. Naturally occurring triploids have also
been found from the hybrid Poecilia latipinna and Poecilia

mexicana (see Menzel and Darnell, 1973).

Induction of Tetraploidy

Tetraploidy in fishes has been artificially induced in
two ways by applying a shock just prior to the first mitotic
division of the developing egg. Tetraploid fish have been
produced when the separation of chromatids (karyokinesis)
was interrupted, or the separation of cytoplasm
(cytokinesis) was interrupted (Chourrout, 1984). A triploid
egg shocked prior to the second meiotic division will also
result in a tetraploid offspring (Chourrout et al., 1986).

A triploid egg has been produced by fertilizing a normal
haploid (1N) egg with diploid (2N) sperm from a tetraploid
male. Heat shocks may denature the spindle apparatus
(Briggs, 1947; Fankhauser and Godwin, 1948) and this could

lead to polyploid induction. Rieder and Bajer (1979)

8
showed that heat shock depolymerizes the microtubules in
cultures of newt lung cells. Tetraploidy has been induced
in amphibians in newts (Fankhauser, 1945; Fischberg, 1958)
and frogs (Gurdon, 1959). Experiments involving tetraploid
induction in fish are summarized in Table 1.

Early attempts at tetraploid induction in fish leaves
some question as to whether the fish obtained were
tetraploids. Valenti (1975) attempted to induce tetraploidy
in Tilapia aurea using heat shocks; but appears to have
induced triploidy, tetraploidy, and mosaics. Mosaics are
fish with triploid and tetraploid cells. Refstie (1981)
induced polyploidy in rainbow trout and concluded that the
fish he produced were "probably tetraploid."

Several methods have been used to induce tetraploidy.
Tetraploid fish have been produced using temperature shocks.
Heat shocks have been used to produced tetraploid coldwater
fish (Thorgaard et al., 1981; Chourrout, 1982) and warmwater
fish (Valenti, 1975; Bidwell et al., 1985). Heat shocks
also have been used to induce tetraploid rainbow trout by
shocking a developing triploid egg (Chourrout et al., 1986;
Blanc et al., 1987). Cold shocks have been used to produce
tetraploid Tilapia aurea (see Valenti, 1975).

Hydrostatic pressure has been used extensively in
inducing tetraploidy in rainbow trout (Chourrout, 1984;
Chourrout et al., 1986; Myers et al., 1986). Low levels of

tetraploid induction were found when hydrostatic pressure

 

 

Table 1. Summary of Tetraploid Induction Experiments
in Fishes

 

 

Shock Primary Author

Species Method and Year (1900)
Atlantic salmon cytochalasin b Allen 79
brook trout cold Lemoine 80
channel catfish heat Bidwell 85
Chinook salmon pressure Myers 86
coho salmon pressure Myers 86
coho x Atlantic salmon pressure Myers 86
rainbow trout heat Thorgaard 81
Chourrout 82

pressure Chourrout 84

Chourrout 86

Myers 86

cytochalasin b Refstie 77

Refstie 81

tilapia heat Valenti 75
cold Valenti 75

pressure and cold Myers 86

 

 

10
shocks were used with different salmonid species — Chinook
salmon (Oncorhvnchus tshawvtscha), coho salmon (Oncorhvnchus
kisutch), and the hybrid female coho x male Atlantic salmon.
(Salmo salar) (see Myers et al., 1986). Myers (1986) used a
combination of hydrostatic pressure and cold shock to
produce tetraploidy in two tilapia, Oreochromis niloticus,
Oreochromis mossambicus, and their hybrid.

Induction success has varied among the different
treatments. The rate of temperature shock induction has
varied from 16 percent (Thorgaard et al., 1981) to 75
percent (Valenti, 1975). Hydrostatic pressure treatments
produced 90 percent (Myers, et al., 1986) to 100 percent
(Chourrout, 1984; Chourrout et al., 1986; Blanc et al.,
1987) success in tetraploidy inducement. Treatments using
hydrostatic pressure and cold shock in tilapia yielded up to
8.3 percent induction.

Survival of the shocked fish has varied by species.

The survival of rainbow trout have been 10 percent
(Chourrout et al., 1986), 4.1 percent (Myers et al., 1986)
or approximately 30 percent when produced by heat and
hydrostatic pressure shocks (Thorgaard et al., 1981;
Chourrout, 1982; Chourrout, 1984; Blanc et al., 1987).
Tetraploid rainbow trout induced by cytochalasin b had only
a 12 percent survival rate (Refstie, 1981). Other salmonid
tetraploids have had very poor survival rates, never

exceeding 1.8 percent (Myers et al., 1986). Tetraploid

 

 

 

 

ll
tilapia produced by heat shocks and cold shocks had a 50
percent and 90 percent survival rate respectively (Valenti,
1975). Myers (1986), observed up to 8.3 percent survival of
tetraploid tilapia in two species and their hybrid using
hydrostatic pressure and cold shock. Survival of tetraploid

channel catfish was 40 percent (Bidwell et al., 1985).

Induction of Triploidy in Fish

While the induction of tetraploidy in fish is a
relatively new tool for fisheries managers, triploidy has
been utilized for several years. Triploidy induction
experiments have provided the basis for attempts in
tetraploid induction. Heat shocks have been the most
successful method of induction in several species of fish
(Chourrout, 1984). Triploid induction experiments in fishes

have been summarized in Table 2.

 

12

 

 

Table 2. Summary of Triploid Induction Experiments in
Fishes

Shock Primary Author

Species Method and Year (1900)
Atlantic salmon heat Benfey 84
Johnstone 85

pressure Benfey 84

anaesthetics Johnstone 89

brook trout cold Lemoine 80
brown trout heat Arai 87
carp cold Gervai 80
channel catfish cold Wolters 81
heat Bidwell 85

Chinook salmon heat Utter 83
Westerhof 88

Spruell 89

coho salmon heat Utter 83
grass carp heat Thompson 87
pink salmon heat Utter 83
rainbow trout heat Chourrout 80
Thorgaard 81

Lincoln 83

Lou 84

Solar 84

Bye 86

Thorgaard 86

pressure Lou 84

nitrous oxide Shelton 86
stickleback heat Swarup 59a
cold Swarup 59a

tilapia heat Valenti 75
Don 88

cold Valenti 75

Don 88

 

 

13

Ploidv Determination

Karyotyping

Karyotyping has been the most common way of determining
the ploidy level of a fish. A karyotype is the
characterization and analysis of a chromosome complement at
metaphase within the nucleus of a given species (Blaxhall,
1983). Kligerman and Bloom (1977) developed a simpler
chromosome preparation technique for karyotyping. The
technique has been used in the identification of polyploids
in several fishes which have included: rainbow trout
(Thorgaard et al., 1981; Chourrout, 1982), channel catfish
(Wolters et al., 1981; Bidwell et al., 1985), triploid
hybrid grass carp (Ctenopharyngodon idella X
Hypophthalmichthys nobilis) (see Cassani et al., 1984), and
various tilapia species (Myers, 1986).

Staining of blastodiscs that have been removed from
the egg has been used in determining ploidy of fertilized
eggs (Lincoln et al., 1974; Refstie et al., 1977). Baksi
and Means (1988) developed an inexpensive technique of
preparing larval fish for chromosome examination. They
noted however, as well as Kligerman and Bloom (1977), that
multiple samples would be very time consuming. Another
difficulty in studying fish chromosomes by karyotyping

techniques is their small size and the relatively large

 

 

14

numbers of chromosomes present (Blaxhall, 1983).

Cvtophotometric DNA Measurement

Another method of determining ploidy levels in fish has
been the use of a microdensiometer to measure the amount of
DNA in the nucleus cytophotometrically. Blood was Feulgen
stained (Johnstone, 1985) and the DNA content was measured
in the erythrocytes using a microdensiometer (Gervai et al.,
1980). This technique has been used successfully in
triploid carp, Cvprinus carpio (Gervai et al., 1980),
rainbow trout (Shelton et al., 1986) and triploid Atlantic

salmon (Johnstone, 1985; Johnstone et al., 1989).

Comparison of Erythrocyte Volumes

Significant differences have been found between the
nuclear volume of erythrocytes in polyploid and diploid fish
(Swarup, 1959a; Purdom, 1972; Valenti, 1975; Allen and
Stanley, 1978; Lemoine and Smith, 1980; Refstie, 1981;
Wolters et al., 1982; Lou and Purdom, 1984; Johnstone and
Lincoln, 1986). Erythrocyte volume was determined by the
equation V = (4/3)ab?,‘were (a) equalled the major semiaxis
and (b) equalled the minor semiaxis of the erythrocyte
(Valenti, 1975). Lou and Purdom (1984) measured

erythrocytes, cartilage, brain, and epithelial cells; but,

 

 

15
obtained their best results using erythrocytes. When
compared to karyotyping, erythrocyte volumes were less
tedious and highly (92.65 percent) accurate (Wolters et al.,

1982).

Use of Coulter Counter and Channelyzer

The use of a Coulter Counter and Channelyzer to measure
erythrocyte volume has proven to be a rapid and accurate
method for identification of polyploids (Benfey et al.,
1984). While a Coulter Counter and Channelyzer are
expensive, they are cost efficient, since large numbers of
fish can be done rapidly (Wattendorf, 1986). In Florida,
all triploid grass carp must have their ploidy confirmed by
the Coulter Counter or similar instrument before they can be
stocked for aquatic weed control (Cassani and Canton, 1986;
Thompson et al., 1987).

The Coulter Counter can be used to measure the actual
erythrocyte volume of any aged fish. The Channelyzer
accumulates individual erythrocytes into size intervals
(Benfey and Sutterlin, 1984). Ploidy levels have been
calculated from mean and median erythrocyte volumes. This
method has failed to identify mosaics that have been
identified using other methods. The accuracy of a small
number of chromosome counts or erythrocyte nuclear volume

measurements to identify mosaic polyploid fish has been

 

l6

questioned (Benfey et al., 1984).

Flow Cvtometrv

Measurement of erythrocyte DNA content by flow
cytometry has been a very practical method for rapid and
accurate identification of polyploid fish (Thorgaard et al.,
1982). Erythrocytes were stained with a fluorescent stain.
Stained single cells were suspended in an aqueous solutions
and delivered, single file, to a signal detection center by
vacuum or air pressure at rates of thousands of cells per
second. As each cell traversed the laser beam, it scattered
fluorescent light which was filtered and collected by a
fluorescent detector. The light signals were converted into
electric signals, measured, digitized, and sent to a
computer for analysis, display and storage (Downing et al.,
1984).

Two fluorescent stains have been used in the analysis
in fish DNA: 4'-6-diamidino-2 phenylindole (DAPI)
(Thorgaard et al., 1982; Utter et al., 1983; Solar et al.,
1984) and propidium iodine (Allen, 1983). Fish species that
have been tested by flow cytometry have included rainbow
trout (Thorgaard et al., 1982; Solar et al., 1984), Atlantic
salmon (Allen, 1983; Graham et al., 1985), coho salmon
(Utter et al., 1983; Johnson et al., 1984; Johnson et al.,

1986), Chinook salmon (Johnson et al., 1986; Westerhof,

17
1988; Spruell, 1989), grass carp (Allen et al., 1986) and
hybrid grass carp X bighead carp (Allen, 1983). Johnson et
a1. (1984) found that the flow cytometer is more accurate
(100 versus 89 percent) than the Coulter counter in ploidy

measurements.

Miscellaneous Methods

Electrophoresis has been used in determining the ploidy
of grass carp (Wiley and Wike, 1986) and soft-shell clams
(Allen et al., 1982). Silver staining has been used in
identifying triploid fish cells (Phillips et al., 1986).
Chinook salmon, coho salmon, and rainbow trout ploidy levels
were determined by counting the number of nucleoli per cell.
Diploid fish had one or two nucleoli per cell while triploid
fish had one, two, or three nucleoli per cell. Multiple
cell samples were since only triploid fish had three
nucleoli per cell (Phillips et al., 1986). This method does
not provide absolute identification of diploid fish, and

would limit its use in most applications.

MATERIALS AND METHODS

In a pilot study, it was determined that the upper
lethal temperature for heat shocking bluegill eggs was 40%:
for 10 minutes (Appendix A). This was comparable to
tetraploid induction experiments done with tilapia (Valenti,
1975) and channel catfish (Bidwell et al., 1985).
Tetraploidy induction has been induced when a shock is
applied to eggs just prior to the first cleavage (Thorgaard,
1986). The first cleavage has been reported to occur 35
minutes after fertilization in the bluegill sunfish (Morgan,
1951). Shocking of bluegill eggs beginning 30 minutes after
fertilization should result in the disruption of the first
cleavage. Shocks lasting up to ten minutes cover the
development of the bluegill egg through first cleavage.

Spawning bluegill were collected by hook and line from
Lake Lansing (near Haslett, Michigan) from June 28 to July
28, 1989 and transported to the Michigan State University
Aquaculture Laboratory (East Lansing, Michigan) in aerated
163 liter coolers. Bluegill were identified in the field
and verified at the laboratory (Eddy and Underhill, 1978).
The bluegill were transferred to 38 liter aquaria maintained

at 20W3, 0.5 liter/minute water flow and equipped with an

18

19
external standpipe (Garling and Wilson, 1976) to maintain a
constant water level.

Bluegill were hand spawned after being allowed to
acclimate at least 30 minutes. Female bluegill were
considered ripe and ready to spawn when gentle squeezing of
both sides of the abdominal area resulted in egg flow from
the genital pore. Female bluegill were dried around the
genital opening and eggs were stripped onto a clean, dry
watch glass. Bluegill eggs were stripped from the fish by
applying firm, gentle pressure to both sides of the
abdominal area of the female bluegill. One or two female
bluegill were stripped into each watch glass depending on
the amount of eggs obtained per female. Since percentage of
tetraploid bluegill surviving to 3-4 cm in length was to be
analyzed, the actual number of bluegill eggs shocked were
not counted. Egg batches were discarded if the eggs had
malformed oil globules, physical deformities, or cloudiness.
Malformed oil globules indicated under-ripeness and
cloudiness indicated over-ripeness (Banner and Hyatt, 1975).

Male bluegill were dried around the genital opening and
were spawned by applying firm, gentle pressure to both sides
of the abdominal area. Milt was collected using a 5 3/4
inch pasteur pipet and pipet bulb (VWR Scientific). Milt
viability was confirmed by taking a small sample of the milt
collected, making a smear on a slide, and examining it under

a compound microscope (American Optical) at 450x power. If

20
individual, mobile sperm were observed, the milt was
considered viable and added to the eggs. Inviable milt was
discarded. Viable milt from at least two male bluegill was
added to each batch of bluegill eggs to ensure
fertilization. Stirring with a finger mixed the milt and
the eggs. When the milt was added to the eggs, a stopwatch
(Micronta) was started to time egg development.

Stripped female bluegill were placed in a concrete pond
(10' x 15' x 12' deep) located adjacent to the laboratory
for use in future experiments. Male bluegill were held in a
1900 liter holding tank maintained at 20°C and 2
liter/minute flow. Male bluegill could be hand spawned
repeatedly throughout the experiment period.

One minute after fertilization, 20°C water was added to
the bluegill eggs and milt to water activate the bluegill
eggs (Piper et al., 1983). Fertilized bluegill eggs were
maintained at 20°C to ensure accurate timing of egg
development for heat shocking. At thirty minutes post-
fertilization, heat shocks were applied to the bluegill
eggs.

Heat shocks were performed at 30, 35, and 40%: for 2.5,
5.0, 7.5, and 10.0 minutes (Figure 1). Thirty minutes after
fertilization the bluegill eggs were submersed in a hot
water bath (Figure 2). The temperature of the hot water
bath was maintained, at heat shock temperatures (+/- 0.25%»

by a 75 watt submersible aquarium heater (Dan-sea).

21
Figure 1. Temperature and duration regime of heat

shocks applied to bluegill sunfish eggs.

Duration (min.)
2.5 5.0 7.5 10.0

 

 

30°C 30°C 30°C 30°C
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40°C for for for for VA
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23

Bluegill eggs were heat shocked at the appropriate
temperature for the allotted amount of time and then
carefully transferred without acclimation to a 38 liter
aquaria maintained at 26W3, 0.5 liter/minute flow and
equipped with an external standpipe (Garling and Wilson,
1976) to maintain a constant water level. Control groups of
eggs were held at 20°C for 35 minutes after fertilization
and then placed into an aquaria. One replicate of each
individual treatment was completed before additional
replicates were completed. A total of three replicates were
performed for each treatment. Three control groups were
used, each being performed before new treatment replicates.
A total of 36 individual bluegill egg heat shocking
treatments and three controls were completed (Figure 1).

Bluegill eggs were incubated at 26°C to speed
development and decrease the potential growth of fungus on
dead bluegill eggs. Secondary temperature shock, the return
from hot to cold water, was minimized and chemical treatment
of bluegill eggs for fungus was unnecessary. Morgan (1951)
noted that bluegill eggs hatched between 32 and 62 hours
after fertilization when incubated at 72°F (22°C). At 26%:,
hatching time would possibly be accelerated and should occur
between 30 and 62 hours after fertilization.

Bluegill fry were found to swim up nine to ten days
after fertilization when incubated at 20°C (Appendix A).

Fry needed to be fed immediately at swim up. It had been

24
estimated that at 26%:it.would take six to seven days after
fertilization for the bluegill fry to swim up. Six days
after fertilization the bluegill fry were fed newly hatched
brine shrimp nauplii and continued to be fed four times
daily until tested for tetraploidy (Appendix B).

Aquaria water temperature was decreased from 26%:113
20°C after swim up over a six hour period. Water
temperature was monitored in the morning and maintained at
ZOWS. Photoperiod was maintained at 18 hours light and 6
hours dark by an automatic timer (Intermatic Incorporated).

Beginning on the fourth day of feeding, aquaria were
siphoned each morning to remove feces and uneaten brine
shrimp which accumulated the previous day. Solid wastes
were not removed by siphoning until three days after the
first feeding due to the small size of the bluegill fry and
their susceptibility of being caught in the siphon action.

Approximately 65 days after fertilization, bluegill
attained a size suitable for tetraploidy analysis. Bluegill
needed to reach a size where a half drop of blood could be
drawn. Bluegill to be tested for tetraploidy were
anesthetized using tricane methane sulfonate (Finquel MS-
222, Argent Chemical Laboratories). Blood was sampled from
individual bluegill by cardiac puncture using a 1 cc syringe
with a 25 gauge needle (Becton Dickinson). Blood samples
were taken from five bluegill from each heat shock treatment

replicate and were tested for ploidy using flow cytometry

25
(Appendix C).

The procedure for testing bluegill blood using flow
cytometry was similar to the procedure used for testing
Chinook salmon (Oncorhynchus tshawytscha) (see Spruell,
1989). Fifteen bluegill were sampled from each heat shock
treatment group. If tetraploidy had been detected from any
heat shock treatment replicate an additional ten bluegill
would have been tested to determine the percentage of
tetraploid fish in that treatment group. Treatments
containing tetraploid bluegill would have been retained and

raised for future experiments.

 

 

RESULTS

As expected based on preliminary experiments, no
bluegill hatched from the 40°C heat shock treatment groups
at ten minutes duration. Qualitative observations were made
on the egg and fry survival of the remaining treatments.
Bluegill egg mortality appeared to increase as the heat
shock temperature and duration increased. Survival of eggs
in the 30 and 35°C treatments for 2.5 minutes appeared to be
comparable to controls. Hatching of bluegill eggs occurred
between 30 and 48 hours after fertilization. Heat shocked
eggs treated at 35°C for 7.5 minutes hatched in relatively
large numbers; however, two days after hatching large die
offs of the bluegill fry occurred in two of the three
treatments. Mortality data was not collected, since this
study was concerned with the percent of tetraploid bluegill
surviving to 3-4 cm in length. At least five bluegill
survived to be analyzed for ploidy in all remaining
treatments.

After swim up, mortality was extremely low for all
groups. Swim up fry were observed to feed aggressively on
newly hatched brine shrimp nauplii. Brine shrimp nauplii

were offered in large quantities and the majority were not

26

 

27
eaten and accumulated on the bottom of the tank. Daily
siphoning kept the water quality suitable for the growing
fry. Few bluegill were killed by siphoning.

Blood samples from bluegill were tested for tetraploidy
on 25, 26, September and 5, 6, 9, 10 October, 1989, using
flow cytometry. Blood samples had been taken from 65 day
old bluegill (Appendix A). Some bluegill were held past 65
days and they were tested with heat shock treatment groups
performed later in the summer. Bluegill were 67 to 85 days
old when analyzed. Five fish were sampled from each of the
33 treatments and 3 controls. This would allow a detection
of tetraploidy at 6.66 percent (1 out of 15) for any heat
shock treatment. No tetraploid bluegill were found in 180
bluegill sampled. Diploid bluegill blood histogram peaks
were consistently 25-30 units of DNA fluorescence and
Chinook salmon blood (internal standard) was 82-92 units of
DNA fluorescence.

Some bluegill were retained after ploidy analysis and
fed a ground commercial trout feed (Zeigler, 5/32 inch
pellet). After four to five days the bluegill began to feed

aggressively on this feed.

 

 

 

DISCUSSION

Several procedural problems were encountered while
conducting this study. Completion of the pilot study to
determine the upper lethal heat shock for bluegill eggs was
delayed when ripe bluegill were not available during the
summer of 1988 due to record high temperatures (Appendix A).
Many bluegill were collected, but few were ripe. Techniques
were developed during the winter of 1988-89 to induce
bluegill to spawn out of season (Appendix D) and the pilot
study was completed prior to the spring of 1989. During the
pilot study, zooplankton was not a reliable feed source for
bluegill sunfish in the laboratory. Zooplankton were
collected from ponds behind the laboratory and from Lake
Ovid. Zooplankton were available to the bluegill fry, but
no feeding was observed. It was speculated that the
ZOOplankton were too large to be eaten by bluegill fry.
During the winter of 1988-89 bluegill were successfully
raised on newly hatched brine shrimp nauplii (Appendix B).

Unfortunately, no tetraploids were found in the ploidy
analysis of 165 fish from 36 heat shock treatments.
Tetraploid bluegill may have been produced, but died prior
to ploidy analysis. The survival of tetraploid salmonid

eggs to hatching has been very low (Thorgaard et al., 1981;

28

29
Chourrout, 1982; Chourrout, 1984; Myers et al., 1986;
Chourrout et al., 1987). Tetraploid tilapia were subvital,
showed abnormalities in development, and died within seven
days after hatching (Myers, 1986). Ploidy determination at
an earlier time in development would be desirable to
identify effective tetraploid inducing shocks. Development
of a technique to determine ploidy of bluegill tissue
samples using flow cytometry would be the next logical step
for future research in this area. Thorgaard et a1. (1982)
suggested that fish tissue treated with non-ionic detergent
may be analyzed by flow cytometry. Flow cytometry is
clearly the most accurate and time efficient methods of
ploidy analysis available.

Death of tetraploid fish may be due to abnormalities
caused by shocking. Myers (1986) suggested that shocks
applied prior to cytoplasmic division (cytokinesis) leads to
developmental abnormalities in the egg and fry. The
abnormalities could be attributed to aneuploidy in the cells
of the developing egg (Myers et al., 1986). Aneuploidy is
the condition of a cell in which one or more whole
chromosomes of a normal set are present more than once
(Ayala and Kiger, 1984). Mortalities of tetraploid fish
have also been associated with yolk resorption (Chourrout et
al., 1986).

Tetraploid bluegill could have been produced; but

failed to survive. Heat shock treatment of bluegill eggs of

30
35%: for 7.5 minutes produced a high number of hatching fry,
but few survived to swim up. The surviving bluegill were
diploids; perhaps the moribund bluegill had been
tetraploids. The moribund fish were not tested as percent
tetraploid induction at 65 days of age was desired. The
heat shock treatment of 35%: for 7.5 minutes could be
repeated and a tissue sample from the newly hatched fry
analyzed using flow cytometry. Slower cooling of the
bluegill eggs after shocking could reduce mortality
associated with secondary temperature shock. While survival
of tetraploids to swim up has been low, survival after swim
up has been comparable to diploid fish (Chourrout, 1984;
Chourrout et al., 1986).

Heat shocks have been the most common shock used in
tetraploid induction, and should have produced tetraploid
bluegill. Perhaps the high temperatures were lethal to
induced tetraploid eggs. Potentially pressure shocks could
be used to prevent these mortalities. Pressure shocks have
been efficient in inducing tetraploidy in rainbow trout
(Chourrout, 1984), but these experiments have been difficult
to repeat and further tetraploid induction experiments using
pressure shocks have been abandoned in favor of heat shocks
(Seeb, pers. comm.). Expensive equipment is needed for
pressure shocks and only a small number of eggs can be
shocked at one time. Cold shocks would be another

possibility for tetraploid induction in bluegill.

31

Myers (1986) suggests that tetraploids shocked at
karyokinesis may have a better survival than those shocked
at cytokinesis. Possibly the bluegill that died in the 35%:
heat shock treatment for 7.5 minutes were tetraploids
produced by interruption of cytokinesis. An earlier shock
in development may inhibit karyokinesis in bluegill and
produce viable tetraploids.

It has been argued that time in development is
important when inducing tetraploidy. Myers (1986) suggests
that proper timing is essential to ensure that tetraploid
induction occurs at karyokinesis and not at cytokinesis.
Tetraploids induced at karyokinesis may exhibit better
survival to swim up than fish induced at cytokinesis (Myers,
1986). The optimal induction time may vary with eggs from
female to female (Chourrout et al., 1986) and from fish
collected in different water temperatures. Nearly ripe
bluegills could be held in the laboratory for several days
prior to hand spawning to allow acclimation and The
temperature of the fish brought into the laboratory may have
an effect on the timing of egg development. However; other
researchers have noted that precise timing of egg
development was unnecessary for the induction of tetraploidy
(Thorgaard, 1981; Bidwell et al., 1985).

Tetraploidy may have been induced in this study with
bluegill sunfish. First cleavage of bluegill sunfish eggs

has been observed at 35 minutes post fertilization (Morgan,

32
1951). Consequently, the heat shock treatments performed at
30 minutes post fertilization should have produced
tetraploid bluegill. Survival of tetraploid bluegill
produced by heat shock appears to be less than 6.66 percent
by our analysis.

Bluegill blood sample preparation techniques were
developed during the winter of 1987-88. Ploidy
determination of Chinook salmon blood has been utilized for
several years at Michigan State University using flow
cytometry (Westerhof, 1988; Spruell, 1989). Analysis of
bluegill blood by flow cytometry required only a few

modifications (Appendix C).

CONCLUSIONS

The aquaculture and fisheries management potential for
polyploid bluegill are too great to abandon future induction
experiments. Tetraploidy analysis of bluegill fry heat
shocked at 35%: for 7.5 minutes should be attempted first.
Timing of developmental stages over a range of temperatures
up to first cleavage would identify karyokinesis; therefore,
increase precision of shocking and potentially higher
survival of tetraploid bluegill. Tetraploid bluegill would
have many applications, and tetraploid induction experiments
should be continued. Cold shock treatments seem like a
logical low cost method to evaluate for induction of
tetraploidy in bluegill sunfish. Pressure shocks may hold
more promise, but may be cost prohibitive for researchers

and potential future users of the technology.

33

 

APPENDICES

APPENDIX A

The maximum temperature that could be used to heat
shock bluegill sunfish eggs for ten minutes was determined
prior to initiating tetraploidy induction experiments. In
other warm water fishes, researchers have used heat shock
temperatures of 38°C to 43°C (Valenti, 1975; Bidwell et al.,
1985) to induce tetraploidy. Bidwell et a1. (1985) noted
that heat shocks above 41°C lasting longer than one minute

were fatal to developing channel catfish eggs. Trial heat

 

shocks were conducted at 40°C for 7.5 minutes and 35°C for
5.0 minutes to observe the lethal effects of heat shocks on
bluegill eggs. Controls were run to insure that mortality

was caused by the heat shock exclusively.

MATERIALS AND METHODS

Bluegill were collected by hook and line from Lake Ovid
(Sleepy Hollow State Park near Ovid, Michigan) from June 24
to July 14, 1988 and transported to the Michigan State
University Aquaculture Laboratory (East Lansing, Michigan)
in aerated 163 liter coolers. Bluegill were identified in
the field and identification was confirmed at the laboratory
(Eddy and Underhill, 1978). Bluegill were transferred to 38 5

liter aquaria maintained at 20°C, 0.5 liter/minute flow, and

34

35

equipped with an external standpipe (Garling and Wilson,
1976) to maintain a constant water level. The bluegill were
acclimated to these conditions for at least 30 minutes. Due
to record high summer temperatures, a low number of spawning
bluegill were collected in summer 1988. Additional bluegill
were artificially spawned (Appendix D) during winter 1988—
89 to complete this pilot experiment.

Bluegill were spawned and eggs were heat shocked as
described above, except that the bluegill eggs were placed

in aquaria maintained at 20°C. Heat shock treatment

 

durations were limited to 5.0 and 7.5 minutes at 35 and
40°C. Two replications were completed for each heat shock
treatment. Three controls were used to ensure that
mortality was caused by heat shock exclusively.

Zooplankton was collected from a pond adjacent to the
laboratory and from Lake Ovid and offered to bluegill fry in
summer 1988. In winter 1988-89, bluegill fry were fed newly
hatched brine shrimp nauplii (Appendix B). Valenti (1975)
had success raising newly hatched tilapia on brine shrimp
nauplii.

Bluegill fry were raised to a size where a small blood
sample could be taken for ploidy analysis by flow cytometry
(Appendix C). Five bluegill were analyzed from each
treatment replicant. If tetraploid bluegill were found in a
treatment, ten additional bluegill would have been tested to

i
i
determine the percentage of tetraploid fish in that heat F

36
shock treatment. Tetraploid bluegill would have been

retained and raised for further experiments.

RESULTS

Qualitative observations were make on the survival of
heat shock treatment groups. Survival to hatching was
estimated to be less than ten percent with bluegill eggs
that were shocked at 40°C for 7.5 minutes. Bluegill eggs

shocked at 35°C for 5.0 minutes survived comparably to

 

controls. Swim up occurred 9 to 10 days after fertilization
at 20°C. All bluegill that were offered zooplankton died
within 4 days after swim up (Table A1). No bluegill offered
zooplankton were observed feeding and it was speculated that
the zooplankton were too large to be eaten by bluegill fry.
Use of progressively smaller meshed nets may have captured
adequately sized zooplankton for bluegill to eat, but the
use of these nets would be time consuming. 3
Two of three aquaria of bluegill fed brine shrimp
nauplii survived and were tested for tetraploidy by flow
cytometry (Appendix C) on April 17, 1989. A half a drop of
blood was necessary to analyze bluegill ploidy. Bluegill of
3 cm or longer, were suitable for blood sampling. All five
surviving bluegill from the 40°C for 7.5 minute treatment
and five control bluegill were tested for tetraploidy. All

ten bluegill tested were diploid. !

 

 

 

37

Table A1. Outline of pilot experiment to
determine the upper lethal
temperature of bluegill eggs.

 

 

Treatment
Temperature Duration
oC) (min.) Date Feed Type
20 control 6/24/88 plankton
40 7.5 6/24/88 plankton
35 5.0 7/01/88 plankton
20 control 7/14/88 plankton
20 control 1/22/89 brine shrimp
40 7.5 2/11/89 brine shrimp
35 5.0 3/14/89 brine shrimp

 

 

38

CONCLUSIONS

A heat shock of 40%:applied for 10 minutes should be
lethal to bluegill sunfish eggs and should be the greatest
temperature and longest duration used to heat shock eggs.
Zooplankton was unsuitable for feeding newly hatched
bluegill fry, and newly hatched brine shrimp should be used.
Zooplankton appeared to be too large for the bluegill fry to
eat. It was also determined that bluegill could be tested
for ploidy when they reached a size about 3 cm in length;

which is approximately 65 days after fertilization.

APPENDIX B

In the pilot experiment used to determine the upper
lethal heat shocking temperature for bluegill eggs, plankton
were unsuitable for raising bluegill fry (Appendix A).
Valenti (1975) used newly hatched brine shrimp nauplii to
feed tilapia. Bluegill fry were successfully raised on
brine shrimp nauplii. The following is a description of how
bluegill fry were raised on brine shrimp nauplii in our
laboratory.

Fishes must feed at the swim up stage of development.
Swim up occurs when a fish absorbs all of its yolk sac,
begins swimming to the surface of the water column, and
begins eating. Bluegill fry reached the swim up stage seven
days after fertilization when incubated at 26%3. Newly
hatched brine shrimp nauplii were used to feed the bluegill
fry. The brine shrimp nauplii must be newly hatched as
brine shrimp nauplii grow rapidly and become too large to be
eaten by the first feeding bluegill fry.

Brine shrimp nauplii were raised in modified 7.6 l
Nalgene jugs (Figure B1). Each jug cap was fitted with a t-
adaptor with stopcock. An air hose was attached to the t-
adaptor above the stopcock. Half of the jug bottom was cut
out so brine shrimp eggs and water could be added. The jugs
were inverted and placed on an iron ring that was attached

to a ring stand. Air was supplied by an aquarium pump

39

40

Figure Bl. Brine shrimp incubation unit.

 

 

7.6 l Nalgene Jug

Ring Stand

 

 

 

[_ Iron Ring 1

r

 

 

 

 

 

 

Stopcock

 

Aquarium

 

 

' Pump

 

 

 

41

(Second Nature Whisper 1000). Seven liters of water
containing seven tablespoons of salt were added to the jug.
The water pump was started and the appropriate amount of
brine shrimp eggs were added. Brine shrimp eggs (San
Francisco Bay Brand) were weighed on a balance (Fisher
Scientific, Model 7303DA) to the nearest tenth of a gram.
Water temperature was maintained between 27°C and 30%:.
Artificial light was maintained for 24 hours per day. At
these temperatures, brine shrimp eggs would hatch at 24
hours.

Each tank of bluegill fry was fed four times daily at
8:00 a.m., 11:30 a.m., 3:00 p.m., and 6:00 p.m. Four jugs
were utilized to ensure that newly hatched brine shrimp
nauplii were available at each feeding. Each tank of
bluegill fry was fed the equivalent of 0.9 grams dry weight
brine shrimp eggs each feeding. The air source to the jug
being used to feed the bluegill fry was turned off 2-3
minutes before feeding. This allowed the brine shrimp
nauplii to settle to the bottom. Brine shrimp nauplii were
drained onto a piece of tightly woven cotton cloth by
opening the stopcock on the bottom of the jug. The cloth
would allow the saltwater to pass through while retaining
the brine shrimp nauplii. The brine shrimp nauplii would be
rinsed with freshwater and added to the tank of bluegill fry
to be fed. Immediately following feeding the bluegill a new

brine shrimp culture would be started for the following day.

42
During several days the air temperature, and

consequently the water temperature of the brine shrimp
culture rose significantly above 30°C (up to 38°C) . Under
these circumstances the starting time of the new brine
shrimp culture was delayed, as the amount of time necessary
for hatching the brine shrimp eggs was reduced. It is
extremely important that the brine shrimp nauplii are newly
hatched, if the brine shrimp eggs hatch 3-4 hours early they
may be too large for the bluegill fry by feeding time,

especially if the bluegill fry are young.

APPENDIX C

The procedure used for testing the ploidy of bluegill
blood was very similar to the procedure used at Michigan
State University for testing the ploidy of Chinook salmon
(Oncorhynchus tshawytscha) (see Westerhof, 1988; Spruell,
1989). This procedure has been used in testing experimental
Chinook salmon, and triploid Chinook salmon for the Michigan
Department of Natural Resources. Modified techniques were
developed for bluegill blood during winter 1987-88 and the
procedure is outlined below since it has not been used for
bluegill sunfish.

The procedure for preparing bluegill red blood cell
samples was modified from Spruell (1989) and is summarized
in Tables C1 and C2. Bluegill ploidy was determined using
an Ortho Diagnostics Systems, Inc. Model 50-H dual laser
Cytofluorograph located in Giltner Hall on the MSU campus.
An Ortho 2150 computer system was coupled to the
cytofluorograph and the Ortho Cytofluorograph Analysis for
Cellular DNA Content of Fixed Cells with DNA Doublet
Discrimination program was used to analyze the data.
Bluegill red blood cell samples were run at an argon-ion
laser setting of 488 nm with a 0.5 W output.

Pulse—height histograms were generated by an Ortho 2150
computer system, coupled to the cytofluorograph, based on

DNA measurements from 10,000 cells per bluegill blood

43

44

Table C1. Steps in the cell staining technique used to
determine ploidy level in bluegill sunfish.

 

Step Description

 

1. Blood was drawn from each bluegill using a 25
gauge needle and a 1 ml syringe rinsed with sodium
citrate buffer solution (CBS) (Appendix Table 3).

1a. Chinook salmon blood was obtained by drawing blood
by a cardiac puncture using a 25 gauge needle and a
1 ml syringe rinsed with CBS. The Chinook salmon
blood was added directly to the bluegill sample.

2. Blood was added to a 12 x 75 mm plastic test tube
containing 0.5 ml of CBS by submersing the needle
in the buffer and applying slight pressure to the
hypodermic. Blood was slowly added until samples
were a faint pink color.

3. Samples were stored on ice or refridgerated until
further processing was completed.

4. Samples were centrifuged at 2500 rpm at 10°C for 5
minutes and the supernant was discarded leaving a
cell pellet.

5. Cells were resuspended in 0.5 ml of the sodium
citrate buffer solution and vortexed until all
clumps disappeared.

6. Cells were fixed for approximately 15 minutes
using 70 percent ethanol. Ethanol was stored on
ice prior to use.

7. Samples were centrifuged as described in Step 4,
and the supernant was discarded.

8. Cells were resuspended in 1.5 ml of propidium
iodide solution (Appendix Table 3).

9. 0.5 ml of RNAse-A solution was added to each tube.

10. Samples were run on the Ortho Cytofluorograph.

 

45

Table C2. Composition of reagents used in flow
cytometric determination of ploidy level in

bluegill sunfish.

 

Reagent Composition

Citrate Buffer Solution:

(CBS)
8.55 g
1.17 9
100.00 m1
RNAse-A Solution:
1.0 mg
5.0 ml
Propidium Iodide Solution:
2.5 mg
0.5 ml
1.85 mg
50.0 ml

sucrose
trisodium citrate
distilled water

RNAse-A
Phosphate buffer
solution (1X)

Propidium Iodide

Triton-X

EDTA

Phosphate buffer
solution (1X)

 

46
sample. Chinook salmon blood was used as an internal
standard to analyze bluegill red blood cells. If histogram
peaks shift, an internal standard will discriminate
accidental shifts from shifts due to a tetraploid bluegill
samples. The flow cytometer was adjusted so that peaks
resulting from Chinook salmon blood ran at 85-90 units of
DNA fluorescence and diploid bluegill peaks ran at 25-28
units of DNA fluorescence (Figure C1). Tetraploid bluegill
peaks were expected to run at 48-52 units of DNA
fluorescence. The position of peaks may vary slightly due
to sample preparation or machine settings. A known diploid
bluegill blood sample was run before experimental bluegill
blood samples were run to establish the peak position of
diploid bluegills. This peak, along with the Chinook salmon
blood internal standard, were compared to the test bluegill

blood sample.

Figure C1.

 

47
Data of diploid bluegill and Chinook salmon

blood as generated by the Ortho 2150
computer.

Bluegill Blood
Histogram Peak

__i2212

f Chinook Salmon Blood
Histogram Peak

  

 

 

‘l’

 

49 I so 128 t 168 I 268
21 nun-FL, H/O DOUBLETS

APPENDIX D

During the summer of 1988, the upper lethal temperature
limit for heat shocking bluegill eggs was determined
(Appendix A). Unfortunately, only a small number of
bluegill eggs were collected due to record high summer
temperatures and the pilot experiment could not be
completed. Only three treatments of eggs were obtained.
Three additional treatments were needed. Tetraploid
induction experiments were scheduled for the summer of 1989,
so it was desirable to complete the pilot experiment prior
to the spring of 1989. Attempts were made in the winter of
1988-89 to induce bluegills to spawn in our laboratory using
modified techniques of Banner and Hyatt (1975). Bluegill
were held at 13°C, while Banner and Hyatt held fish at 23°C.
Female bluegill were given a 1000 I.U. injection of human
chorionic gonadotropin (HCG), while Banner and Hyatt used a
400 I.U. injection.

Bluegills used for artificial spawning experiments were
caught by hook and line from Lake Ovid (Sleepy Hollow State
Park, near Ovid, Michigan) during winter 1987-88. Bluegills
were transported back to the Michigan State University
Aquaculture Laboratory (East Lansing, Michigan) in 163 liter
aerated coolers. Bluegills were identified in the field and
identifications were confirmed at the laboratory (Eddy and

Underhill, 1978). Bluegill were placed in a 2015 liter

48

49
holding tank maintained at 13°C.

Bluegill were transferred to a 1900 liter holding tank
and artificial inducement of spawning was begun. On
December 19, 1988 the temperature was raised from 13°C to
22°C at a rate of one degree centigrade per hour.
Photoperiod was increased from 12 hours light and dark to 18
hours light and 6 hours dark over a period of 14 days.

The bluegill were fed twice daily with commercial trout
feed (Zeigler, 5/32 inch pellets). Each week the bluegills
were checked for ripeness. Fish were considered ripe when
the eggs flowed easily from the genital pore when gentle
pressure is applied simultaneously to both sides of the
female. Female bluegills began showing signs of ripeness 19
days after the beginning of temperature and photoperiod
manipulation.

Each female bluegill that showed signs of ripeness was
transferred to a 38 liter aquaria. The aquaria were
maintained at 22°C, 0.5 liter/minute flow and equipped with
an external standpipe (Garling and Wilson, 1976) to maintain
a constant water level in aquaria. The female bluegills
were observed twice daily for ripeness.

Female bluegill that had a distended abdomen and
swollen genital pore were expected to spawn in three to four
days. These female bluegill were given an injection of HCG
(Sigma Chemical Co.). HCG was dissolved in distilled water

at 1000 I.U./0.1 ml. This solution was injected using a 1

50
cc syringe and 25 gauge needle (Becton Dickinson).
Injections were intermuscularly at mid—body, just above the
lateral line.

Female bluegill were usually ready to spawn two days
after injection of HCG. Male bluegill were ready to spawn
at 19 days after temperature and photoperiod manipulations.
The bluegill were spawned in the laboratory using the same
techniques described for naturally ripened bluegill. While
these methods were used to complete the pilot study to
determine the upper lethal temperature for heat shock
treatments of bluegill eggs, the success rate of inducing

female bluegill to spawn was approximately 25 percent.

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