A ST U D Y OF THE WATER MO L D S OF THE LYDELL STATE
F I S H H A T C H E R Y AT CQiSTOCK PARK, MIC HIG AN
4

I

E DWIN Y. MON SM A
A.B., Calvin College, 1925
S . , University of Illinois, 1927

A THESIS
Submitte d to the Faculty of Mi ch igan State College
in partial fulfillment of the requirements for
the degree of Doctor of Philosophy, 1935.

ProQuest Number: 10008390

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Outline
I

In troduction

II

Des cr ip tion of the Hatchery

III

Methods and Technique

IV

Relative Abundance of Water Molds in Sources,
Ponds, and Soil

V

Classif icatio n and Descript ion of Isolated

Molds

VI

Experiments to Determine the Extent
of Water Molds

VII

Discussion of the Necessity and Possibility of
Control

VIII

Summary

IX

Literature Cite d

ofParasitism

Introduction
It is a matter of common knowledge among mycologists
and ichthyologists that certain water molds are very
prevalent

in fish hatcheries*

Fish and fish eggs are

often attacke d by such molds which effect the destruction
of countless numbers*

It is u su ally assumed that at

least one of the species of Saprolegnia, J3. parasitica
Coker,

is parasitic on fish and fish eggs.

Alt houg h the

degree of parasitism is difficult to determine,
(10) has given some valuable

Kanouse

information on this subject

in her paper on S.. parasitica published in 1932.
Coker (4) in his monog raph on the Saprolegniaceae
also refers to S. parasitica as being associated with
yo ung fish and fish eggs especially in hatcheries,

but

he also reports the collecting of several other species
from fish hat che ry ponds.
Duri ng the summer of 1934,

252 samples of water were

collected from 15 different sources,
Michigan.

chiefly from We st er n

Eighteen samples of water were collected from

ponds of the Lydell State Fish Hatchery at Comstock Park.
12.3$ of the samples from Weste rn Mic higan produced water
molds on hemp seeds,

whereas 55.5$ of the samples

c o l le cted from the hatchery ponds yiel ded molds.

This

great difference could not escape the attention especially
since

it was evident that the molds from the fish hatche ry

2

were not all
species*

parasitica but represented various

In a conver sation with Mr. Claude Lydell,

manager of the hatchery,

to whom the author is indebted for

a great deal of assistance and information,

he was informed

that these troublesome fungi had increased considerably in
the last few years,
each year,

and that great losses were

suffered

presumably due to the fungi.

It was evident that a th oro ug h study of the water
molds of the fish hatchery,
the kinds of molds found,
of water supply,

especially with reference to

their distribution,

the sources

and soil contamination would not only be

interesting and instructive,

but might eventually lead to

some met hod of control.
The writer is greatly indebted to Doctor E. A. Bessey,
of Mi chig an State College, under whose direction this work
was undertaken,

for his helpful advice and encouragement;

and to Doctor R a l p h Stob, president of Calvin College,

who

at all times showed a spirit of helpfulness in provi ding
laboratory facilities.

Descrip tio n of the Hatchery.
The Lydall State Fish Hatchery is located in Comstock
Park, two mile s north of Grand Rapids on Hi ghway 484.
grounds cover an area of about 32 acres.

On this tract

26 ponds of various sizes have been constructed.
one of these pond s have soil bottoms.

Mill Creek,

supplies most of these ponds with water,
the hatchery grounds.

All but
which

winds through

The h a t che ry proper receives its

water from Strawberry Creek,

The

a tributary of Mill Creek.

3
St rawb er ry Creek runs into Mill Cr eek about three-quarters
of a mile north of the hatcher y grounds.
bee n built,

one in Strawberry Creek near its mouth,

other two in Mill Creek.

and the

The accompanying map indicates

the p o s i t i o n of these dams.

From above each dam the water

is piped to the various ponds.
the map.

Three dams have

The pipes are indicated on

Eac h pond receives an inlet pipe and has an overflow

pipe wh ich becomes the inlet pipe for another pond.

The

ponds are thus connected in a series so that the water runs
from one into another and then back into Mill Creek.
ma y be several ponds in a series.
with a drainage pipe which,

There

Each pond is also supplied

when open,

drains the pond and

carries its water directly to Mill Creek.

The ponds are

drained in the fall and the spring of the year.

The exact

course of the waterflow through the ponds m a y be traced by
following the arrows on the map.
The numbers and letters given on the map correspond
to the pond numbers used by the hatchery officials and
employees.

These numbers and letters will be used in

naming the ponds in this paper.

Methods and technique.
In order to study the water mold contamination of this
ha t c h e r y to the best advantage

it was deemed advisable to

make collections of both water and soil and to divide these
collect ion s into the following ma in groups:

4

1*
2.
3.
4.

Water from the sources.
Water from the ponds.
Soil from the bottom of the ponds.
Soil from the hatchery ground in the pr o x i m i t y
of the ponds.

The water from the

sources was collected from four

locations as follows:
Loc ati on A.
Location B*
Locatio n JS.
Location D.

Strawberry
Mill
Creek
Strawberry
Mill
Creek
Mill
Creek

Creek
above
Creek
above
above

just above the dam.
the point where
joins it.
the first dam.
the second dam.

For a more accurate und erstanding as to the positio n
of these locations the map m a y be referred to.

Te n

collections were made from each of the four locations,
is,

that

in all 40 collections from the sources.
Five collections of water were made from each of the

£6 ponds, mak in g 130 collections from the ponds.
The soil collections from the bottoms of the ponds
had to be taken when the ponds were drained.

Since the

water is drained out for only a short time the number of
co llections was n ec essarily limited.

The writer was able

to get 4 soil collections from each of the 12 ponds, making
48 in all.

In addition to this 19 collections were made

from other ponds at random.
For the sake of comparison 48 collections of soil
from the h a t c he ry grounds were taken,

that is,

4 collections

near each of the 12 ponds from which the bottom soil was
taken.
For these collections a collecting belt with 18 screw
top vials of about 20cc capacity was used.
sterilized each time before taking them out.

The vials were
At the point

5
where the c ol le ction was made the entire vial was submerged
and the screw top removed and replaced a few inches below
the surface of the water.

In this manner the po ssibility

of co nta min ation with mold spores from other sources was
eliminated.
Soil collections were made
surface

in similar vials.

The

soil was removed at the point where the sample

was taken and a soil boring was

made with the vial

no thing but the sterile vial touched the soil

sothat

that was

collected.
In the laboratory,

both soil and water samples were

transferred to sterile Soyka dishes.

To the soil sterile

distilled water was added and the two were mixed by agitating
the dish.

Into the Soyka dishes a couple of sterile hemp

seeds and a sterile house fly,

sterilized in a pressure

cooker under 15 pounds of pressure,

were dropped.

If,

mold spores were present the hyphae would usually appear
on the hemp

seed or fly within one week after the

co llectio n was made.
To get these molds

into pure culture,

single hyphal

tips were transferred to potato

dextrose agar plates. This

isolation of single hyphal tips

was performed under a

binocular dissecting microscope and by means of two
specially constructed needles.

The needles were made from

the fine platinum wire of a discarded radio tube.

A little

piece of this wire can easily be fused into the end of an
ordinary piece of glass tube,

this making it easy to handle.

G rowt h on the agar plates was evident very soon after the
is olation of the hyphae.

About two days later a piece of

6
the agar about Jem square and containing hyphal tips was
cut from these plates and transferred to ho meopathic vials
containing about lOcc of sterile distilled water*

For each

form isolated at least 4 of these vials were set up.
Sterile hemp

seeds were dropped into two of these vials,

sterile Dermesti d larva into the third,
fly into the fourth.

a

and a sterile house

The glassware used for these experiments

was sterilized in an electric drying oven and the culture
m e d i a in a pressure cooker at 15 pounds of pressure.

During

warm weather these cultures were kept in an ice box.

During

cold weather they were kept

in a window box which was

specially constructed for this purpose.
inside of the window.

The box was placed

One of the window panes was removed

and replaced by galvanized metal which contained an opening
for a pipe 4 j w in diameter and 14j"long.
the pipe was connected with the box.

The other end of

A damper was placed

in the pipe to shut out some of the cold air during very
cold weather.

The temperature in this box ranged from 10

to 20 degrees centigrade;

the average temperature was about

15 degrees centigrade.
In most cases the characteristics needed for the
classificati on of these molds were obtained from these
single hyphal

cultures.

In a few cases,

however,

single

spore cultures were made to determine characteristics more
accurately.

The technique

followed to isolate single

spores ma y be considered as a modified form of the method
described by Ka uf fman (11).
The writer's method is here given somewhat
1.

in detail:

Wi th fine scissors clip a quantity of mycelium

7
which is producing zoospores from a culture
one of the vials.

in

2.

Mount this bit of mycelium
water on a slide.

3.

Stir the mycelium in the drop of water by means
of fine needles, so as to set free some of the
zoospores that ma y be adhering to the mycelium.

4.

Now, with the needles, remove the mycelium,
being sure to remove all hyphae and hyphal
segments.
(In this way only zoospores will
remain in the water on the slide.)

5.

Invert the slide and place it with the drop
downward on potato dextrose agar in a petri
dish.
(The pressure of the slide will spread
the water between the slide and the agar and
at the same time disseminate the zoospores.)

6.

Leave the petri dish over night.

7.

U p o n microscopic examination the next morning
it will be found that ma ny of the zoospores
have germinated on the agar.

8.

Remove the slide and cut out a bit of agar
containing only one germinating zoospore.

9.

in a drop of sterile

Place this in the center of another agar plate
and in a few days the plate will be covered with
mycelium originating from the single spore.

If the initial culture
and if sterile

is relatively free from bacteria

instruments are used,

it was found that this

process can be used without danger of contamination,

and

with con siderably more ease and less consumption of time
than any other method that was tried.
Of great

aid in the study of the details of the

developmental processes was the use of moist chamber
cultures constructed as suggested by G-iltner (6).

8

Window box used for culturing
water molds during cold weather

/

<

The open box showing interior
con str uc ti on and one of the two
removable shelves with culture
vials*

X

9

Relative Abundance of Water molds in Sources,
Ponds, and Soil.
The degree of water mold contamination of the sources,
ponds,

and soil as indicated by the molds produced in

specimens brought to the laboratory can best be represented
by a series of tables and charts.

TABLE I
Showing number of water molds pro duced in
specimens of surface water from sources.
Number; o f . .Time of
Percentage
j Number
Specimens
Collection ; Producing
Producing
Collected
Mold
Mold

Locatio n
of
Co ll ect io n
!

B

10

10/5/341/2/35

I
1

7

70

10

10/5/34
1/2/35

j

7

70

1

C

10

10/5/341/2/35

!
|

6

60

D

10

10/26/3412/14/34

|
|

8

80

28

Av. 70

! Total

40
.

t-- :
-------------

i

;

10

A

100

B

C

D

Location of sources

.-..

90
Percentage

80

of

;------

samples

produc ing

Chart I

molds

Showing graphically
the percentage of
water samples from
the sources that
were contaminated
with water molds.

7 0 % of all samples
c ont am inat ed

The table and chart just given show the contami na ti on
of surface water from the

sources indicated since all the

samples were neces sar ily t ak en from near the surface.
Whereas,

the water pipe d to the ponds,

deeper layers,

is taken from the

a question with regard to the accuracy of

the results tabul ated above might arise.
In order to determine the contamination of the deeper
wate r h a w th or n fruits were suspe nde d in the water in ord inary
soap shakers such as are used in kitchens for washing dishes.
These h a w t h o r n fruits were left

in the water for from one •

to two weeks and then examined for water molds.
results

The

for this experiment are here tabulated:
TABLE II

Show ing the re sul ts for the experiment with h a w t h o r n
fruits to determine the mo ld contamination of
the deeper layers of water from the sources.

...... .. ----- ..---- --.<.-

L o c a ti on

No. of h a w ­ Time of
thorn fruits Suspension
suspended
]

A

10

10/13/3410/20/34

j

A

11

!

A

f

No. with
water
molds

Percentage of
fruits with
water mo lds

6

60

10/20/34
11/2/34

11

100

8

11/2/34
11/16/34

5

68.5

D

8

10/13/34
10/20/34

3

37.5

D

8

10/20/3411/2/34

6

87.5

3

11/2/3411/16/34

2

66.6

34

A v . # 69

j

t ....... . -f
D
Total

1■«—----- ••

48

j

|

12
These results are s t r i ki ng ly similar to those obtaine d
with the samples of surface water from locations A and D,
the latter showing a cont am in at ion of 75$ whereas these
results give an approximate c o n t a m i n a t i o n of 69$.

The

difference

that

is negligible.

We conclude,

therefore,

the c o n t a m i n a t i o n of surface water and deeper water is
approx im at el y the

same.

The next table and chart give the results obtained
for the 130 samples of water co lle cte d from the 26 ponds.

13
TABLE III
Sho w i n g number of molds prod uce d in specimens of water
from the ponds*
Pond No. ,No. of
Collect ions

•Time of Co l l e c t i o n
From
To

tfo* P ro du c­ % Produc-|
ing molds 1
ing molds

1

5

9/28/34 - 12/14/34

4

80.

2

5

8/14/34 - 1/2/35

3

60

3

5

9/28/34 - 12/29/34

4

80

5

8/14/34 - 1/2/35

2

40

5

5

8/14/34 - 12/29/34

2

40

6

5

8/14/34 - 12/29/34

5

100

8

5

9/28/34 - 12/29/34

2

40

9

5

9/28/34 - 12/29/34

2

40

10

5

9/28/34 - 12/29/34

3

60

11

5

9/28/34 - 12/29/34

3

60

|

12

5

9/28/34 - 12/29/34

2

40

j

13

5

12/8/34 - 12/29/34

3

60

|

14

5

12/8/34 - 12/29/34

5

100

|

17

5

12/14/34 -12/14/34

5

100

j

18

5

12/14/34 -12/14/34

2

40

19

5

10/26/34 -12/29/34

1

20

20

5

12/29/34 -12/29/34

1

20

22

5

10/26/34 - 1/2/35

0

0

23

5

10/26/34 - 1/2/35

0

0

25

5

10/26/34 - 1/2/35

4

80

24

5

3

60

Island

5

9/28/34 - 12/29/34

1

20

Cement

5

8/14/34 - 12/1/34

2

40

B

5

8/14/34 - 12/1/34

4

80

D

5

8/14/34 - 1/2/34

5

100

X

5

8/14/34 - 1/2/35

3

60

!.

4

Total

!

130

1/2/35

- 1/2/35

71

Av*«£

F14.A

!

j

About

54*6^

of

all

samples

contaminated

Showing graphically the percentage of water samples from
the ponds that were contaminated with water molds

14

15

Table IV and Chart

III show the results obt ai ne d

for the 48 soil collections from the bottom of 12 ponds.
About 4 0 ^ of these collections pr od uc ed water molds.
Ninete en other soil collections from several
different ponds but less than 4 from each pond are not
included in this table and chart*

The percentage of

c o n t a m i n a t i o n was somewhat lower for these 19 samples.

TABLE IV
Showing number of water molds produ ce d in 49
samples of soil collected from the bottoms
of 12 ponds*
Number of No. of
Time of collections No. pr o ­ % p r o ­
pond
collections
From
To
ducing
ducing
molds
molds*
1

4

11/16/34-11/16/34

0

0

2

4

:11/23/34-11/23/34

1

25

3

4

11/9/34 -11/23/34

3

75

5

4

12/1/34 -12/1/34

0

1

6

4

11/16/34-11/16/34

3

| 75

9

-4

11/16/34-11/16/34

0

0

10

4

11/2/34 -12/1/34

!

2

50

11

4

10/12/34-12/1/34

|

3

75

12

4

'11/9/34 -12/1/34

4

100

13

4

10/12/34-12/1/34

2

50

Island

4

11/16/34-11/16/34

1

25

Cement

4

11/23/34-11/23/34

0

0

Total

48

-- — ..-—...

,

-f. 19

Av

0

39.6

16

Soil was also collected from the ha tc he ry grounds in
the pr o x i m i t y of the ponds listed in Table IV and Chart III*
The results o b ta in ed for these

soil samples are listed in

Table V and Chart IV*

TABLE V
Showing number of water molds pro du ced in 48 specimens
of soil col lected from the hatchery grounds*
Pr oxim it y
of Pond Wo.
1
2

4

3/22/35

0

0

3

4

3/22/35

0

0

5

4

3/26/35

0

0

6

4

3/26/35

0

0

9

4

i

4/5/35

1

25

10

4

|

4/10/35

1

25

4

|

4/10/35

1

25

2

50

j

j

11
1

N o . of
Time of Co l l e c t i o n No. produc- % pr o ­
Collections
ducing
ing molds
molds
4
3/ 22/ 35/
0
0

I

12
13,

15

1
\

| Island

4

4/5/35

4

4/10/35

1

25

\

4

!

4/5/35

0

0

4

|

3/22/35

1

25

7

A v.g £ 1 5

i

j Cement

1
|

Total

!

i( 48

r

|

CHART III
Showing results for 48 soil collections
from the bottoms of 12 ponds
Cem*

I si.

11
11

i

A b o u t 4 0 $ of samples contam inat ed with water molds

CHART IV
Sho wing results for 48 soil collections
in proximity of 12 ponds
Cem,

Isl.

O

About

1 5 % of samples contaminated with water molds

18

A co m p a r i s o n of Tables IV and V and Charts III and IV
indicates a striki ng difference betwe en the co nt am ina tion
of soil samples from the bottom of the ponds and that of the
soil from the fish h a tc hery grounds.

It is evident that the

r el a t i v e l y h i g h con ta mina ti on of the soil at the bottoms of
the ponds can be traced to the water which is in the ponds
for the

greater part of the year.

the dat a given above

Further exami nat ion of

indicates a uniform grad at io n of

c on t a m i n a t i o n of the water from the sources to the water in
the ponds and again from the water in the ponds to the soil
from the bottom of the ponds.

In the first the co ntam ina tion

is 15$ higher th an in the second and in the second the
co nt am in atio n is 15$ higher t h a n in the third.

The results

p r e s en te d in graphic form give a straight line curve:
G r a p h rep rese nt in g the relative co ntamination of
sources, water in ponds, and soil from bottom
of ponds.

100—
Pe rc ent ag e
908
of

70..
60L
5C_

Contaminat ion
4C_
3Q_
2Q_

1Q_
Q1

19

It is dou btf ul whether the li nearity of this curve has
an y significance*
however,

The general d i r e ct io n of the curve is,

indicative of the general trend of contamination.

The h i g h per ce ntage of cont am in at ion of the water from
sources is no doubt responsible for the lower but
n e v e r t h e l e s s serious co ntamin ati on of the water and soil
from the ponds.
Since water from all the four sources mentioned above
is h i g h l y contaminated and since 19 of the 26 ponds receive
their water either directly or in directly from source C,
an attem pt to correlate

the conta mina tio n of water from

the sources with the contamin at ion of water from the ponds
fed by them would h a r d l y be of any value.

Suffice it to

point out here that whereas water from source C showed a
c o n t a m i n a t i o n of 60$,

the average contamin ati on of the

19 p on ds fed by it is 55^.

Here again we have the slight

drop in co nt amin at io n from source to ponds as indicated
by the graph given above.

C la ss if i c a t i o n and D e scrip ti on of Isolated Molds.
A s stated in

the secti on on methods and technique

an attempt was made to get the 120 forms p ro duc ed in the
various samples of water and soil into
or single spore

cultures.

single hyp hal-tip

Ninety-four were successfully

iso lat ed and an attempt was made to classify them.
E a c h form was cultur ed o n hemp seed,
a De r m e s t i d larva,

in distilled water.

a house fly,

and

From these three

cu ltu res the characteristics necessary for cla ssi ficati on
were in most cases obtained.

Eight forms could not be

20

c la s s i f i e d because of lack of information.

Three of these

forms were def initely S ap rolegnia but the

species could not

be determined.

Three others were A c h l y a but the

ch ara cteris tic s for species det ermination were lacking.
The remainin g 79 forms were referre d to species by the use
of the k e y s given by Coker (4) and Hu m p h r e y (8).

In each

case the descriptions gi v e n by Coker and Humphrey were care­
fu lly read and compared with the results obtained in the
study of the form under consideration.

The original

d es cr ip tio ns of the species were th e n consulted.

Alth ou gh

in a few cases the writer*s form did not agree in all details
wit h the descriptions give n for the species in which he
p l a c e d it,

these differences he believes were of no great

significance,

since they might have been due to differences

in environmental conditions.
been s ho wn by Klebs

It has repeatedly and clearly

(13), Kauff man (12), Pieters (15) and

others that these fungi are very sensitive to environmental
conditions,

and show differences

in growth and structure

dependent u p o n differences in nutrient media,
and other factors.

temperature,

Taki ng this into consideration,

the

writer believes that the differences referred to above were
of such a nature that they did not warrant the establishment
of any new species.

Some of these differences will

be

m en t i o n e d later in con ne ction with the species concerned.
The

following is a list of the species found and the

number of times each species occurred,
detail ed account

for each species:

followed by a more

21

Name of species

No, of times it
occ urr ed

S a pro le gn ia p a r a si tica

34

Saprole gn ia ferax

17

S a p r o legn ia diclina

4

S a p r o legn ia hypogyna

4

Sa p r o le gn ia anisospora

2

Sa pr ol eg nia asterophora

1

S a pr ol egni a delica

1

Sapr ole gnia mi x t a

1

A c h l y a oblongat a var globosa

4

Ac h l y a K l e bs iana

3

A c h l y a racemos a

3

A c hly a ameri cana

1

A c h l y a hy p o g y n a

1

A c h l y a sp.

4

(sterile)

Sapr o l e g n i a par asitica

Coker

Growth abundant o n most media,
abundant,
clavate,

es pe cia lly on a fly.

Hyphae with gemmae

Gemmae cylindrical,

spherical or subspherical,

often in long chains.

Pr olif er at io n of new hyphae through old zoo sporangia common.
Zo o s p o r a n g i a abundant in vig or ous ly growing mycelium,
cylindrical,

averaging 200x26p,

Oog o n i a few,

not occurring in all cultures;

smooth,

thin,

and unpitted.

Diameter of oogonia 40-114p,
dark and subcentric,

1-2 0

their di ame ters 19-24p,

zoospores

10.5-12,25p.
their walls

Ant her idia diclinous.
mostl y 60-80p.

Oospores

us ually 6 - 8 in each oogonium,

a few as large as 30p.

22
This species occ urred in the collections as f o l l o w s :
Number in
w r i t e r 's
c o l l ec ti on

Locatio n of C ol le ct ion

285

Water from Pond 11

335

Water from Mill Creek,
Water from Pond 3

347
348

Date of
Collect ion

9/28/34
Loc. C

10/26/34
11/2/34

Water from Pond 4
Wa t e r from Pond 5

11/2/34
11/2/34

350
351
379

Water from Pond 6
Water from Cement Pond

11/2/34
11/2/34

Soil ;
f rom Pond 12

11/9/34

381

Water from Mill Creek,

389

Wa ter from Pond 8

11/16/34

403

Soil ;
f rom iPond €

11/16/34

417

Soil ;from Pond 2

11/23/34

424

Water from Mill Creek,

Loc,

B

11/23/34

425

Water from Mill Creek,

Loc. B

11/23/34

426

Soil ;
f rom ;Pond 10

12/1/34

428

Soil :
f rom Pond 10

12/1/34

435

Soil :
f rom Pond 13,

437

W at er from Mill Creek,

447

Water from Pond 14, 16

12/8/34

452

Esyrt gtom Pond 11

12/8/34

453

Wate r from Pond 10

12/8/34

454

Water from Moll Creek,

Loc.

456

Wate r from Mill Creek,

Loc. C

462

Water from Pond 17

12/14/34

464

Water from Pond 17

12/14/34

466

Water from Pond 17

12/14/34

476

Water from Mill C r e e k , Loc. B.

12/14/34

479

Water from Pond 1

12/14/34

483

Water from Pond 12

12/29/34

485

Water

from Pond 10

12/29/34

501

Water from Pond X

1/2/35

505

Water from Pond 24

1/2/35

506

Water from Pond 24

1/2/35

509

Water from Pond 25

1/2/35

349

Loc. D.

15

11/9/34

12/1/34
Loc. D

C.

12/1/34

12/8/34
12/8/34

23
A glance at the foregoing list will convince one of
the general prevalence of Saproleg ni a parasitica.

This

species was collected from all the sources except Location
A,

Strawbe rry Creek,

and from all but 9 of the 26 ponds.

It was found in soil as well as water and it appeared quite
c o n si stent ly from September to January.
Kanouse

(1 0 )

in 1 9 3 2 for the first time definitely

n o t e d the appearance
in this

species.

and described the structure of oogonia

At least 10 of the 34 forms listed above

p r o d u c e d oogonia in the writer's cultures.

Invariably,

ho w e v e r only a few oogonia were found in any one culture.
The

structure of th e s e

agreed on the whole with the

d e s c r i p t i o n and illustrations of Kanouse.
o b ser ve d had diclinous antheridia,
smooth,

and unpitted.

subcentric.

Kanouse

The oogonia

their walls were thin,

The oospores were very dark and
states that the diameter of the

oo g o n i a is 6 5 -1 3 5 x 6 0 -7 5 p i,

or 65-95^1.

The oogonia here

ob s e r v e d measured 4 0 -1 1 4 p ., mo st ly 6 0 -8 0 jli.

The oospores

also ranged larger than the 1 8 -2 2 p . indicated by Kanouse.
Their me as ure ments ranged from 1 9 -2 4 p .,

and a few

e xc eption all y large ones were 28 and 30 p. in diameter.
Notwi ths tandin g these differences,

these forms are no

doubt Saproleg nia parasitica since the rest of the
d es cr ip tion and the observations of Kanouse agree perfectly
on the oogonia of this species.
In his key to the species of Saprolegnia Coker (4)
remarks that S. parasitica probably belongs to the Ferax
group.

However,

since the oogone walls of the species are

24
thin,

and pits,

if present,

are not conspicuous,

since the an th eri dia at least

and

in the m a i n are diclinous,

this species would naturally fall in the D i clin a group.
Wi th reference to an abundance of Saprolegnia parasitica
at the fish ha tc he ry the following summary can be made:
Of the seventeen identified forms yielded by water
from the sources 8 or approximately 47# were
S. p a r a s i t i c a .
Of the 46 identified forms yielded by water from
the ponds 20 or approxima tel y 43# were j3. p a r a s i t i c a .
Of the 16 identified forms yielded by soil from the
bottoms of the ponds 6 or approximately 4 9 # were
S. parasitica.
From these figures it is safe to conclude that from
4 0 - 5 0 # of all the molds found at the hat chery are
S. p a r a s i t i c a .
Sa pro leg ni a ferax

(Gruith)

Thuret

M o d e ratel y stout hyphae producing a large number of
o o g o n i a and re la tiv ely few gemmae and zoosporangia.
zoo spo ra ng ia form in old ones.
zoospores about 10y* in diameter.

New

Zoospores escape separately;
Oo gon ia have thick walls

with large conspicuous pits, pits 5.25-7:00ji in diameter.
A n t h e r i d i a observe d in only a few cases and then appearing
to be androgynous,

arising from the oogonial stalk.

of oogonia 2 4 - 1 1 5 . 5yi, mostly from 60-80)1.
1 - 20 in an oogonium,
oospores 19-30p,

mostly less tha n 10.

Diameter

Oospores centric,
Diamet er of

usua lly 28-24p often varying in size in

same oogonium.
This species ranks next to _S. parasitica so far as
f r e q ue nc y of occurrence

is concerned.

times as shown in the following list:

It occurred 17

25
Number i n
w r i t e r ’s
collection

l o c a t i o n of Collection

Date of
C o l l ec ti on

250

Wa t e r

from Pond 6

8/14/34

275

Wa ter

from Island Pond

9/28/34

286

Water

from Pond 11

290

W ater

from Mill Creek,

313

Water

from Pond 10

318

9/28/34
Loc. B

10/5/34
10/12/34

Soil from Pond 18

10/12/34

319

Wate r

from Pond 2

10/26/34

352

Water

from Pond D

11/2/34

385

Water

from Str awberry

420

W at er

from Mill Creek

460

Water

from Strawberry

465

Water

from Pond 17

12/14/34

470

Water

from Pond 18

12/14/34

471

Wa t e r

from Pond 18

12/14/34

472

Wat er

from Pond B

12/14/34

474

Water

from Po nd B

12/14/34

493

Creek A
C
Creek A

Water from Island Pond

11/9/34
11/23/34
12/8/34

12/29/34

W i t h the aid of the ke ys and descriptions of Coker

(4)

and Hu mph re y (8) these forms were rather easily identified.
The large number of oogonia and the relatively small number
of gemmae were evident in nearly all cultures.

Wi th a very

few exceptions the measurements of the diameters of oogonia
and oospores fell within the extremes given by Coker.

An

ex cep tional ly small oogonium appearing in a fish hatchery

26
culture h a d a diameter of 24^i; an excep ti ona lly large
oogonium m e a s u r i n g 115.5^ was found,
extremes as 37p. and 97p,

Coker gives the

and while Coker gives 14.8p as

a m i ni mum for the diameter of oospores,

in the cultures

u nd er c o n s i d e r a t i o n an oospore me asu rin g 12.25}i was found.
The diameter of the pits in the oogonial walls is given
as from 4.5ja to 5.5pi.

Several mea su red in these cultures

were at least 7ji in diameter.
deemed to be of sufficient

These differences are not

significance for the

establis hme nt of a new species or variety.

The

pre po nde ra nc e of evidence places all of these 17 forms in
the species S.

ferax.

The conclusion,

therefore,

is that

about 2 1 * 5 % of the identified forms were £>. ferax.

Saprolegnia di c1 ina

Hum phre y

G ro wt h rather abundant.
as

in Saprolegnia.

t h i n walls,

M a n y gemmae and oogonia.

unpitted,

oog o n i a diclinous.

Proliferation of zoosporangia

diameter 45-80p.

Oogon ia with

Ant her idia on all

Oospores m ostly 22-26p, us ually 3 - 1 0

in an oogonium.
This species was found four times:
No.

304

in water from Pond D on

10/12/34

No.

372

in water from Mill Creek,

Loc.

C on 11/9/34

No.

373

in water from Pond 25 on

11/9/34

No.

486

in water from Pond 19 on

12/29/34

These forms were compared with the descriptions and
illustrations of Humph re y (8) and Coker (4) and were found
to agree with them.
identity.

There can be no doubt as to their

27
S a p r o l e g n i a hypo gy na

Pringsheim.

Hyphae with new sporangia proliferating through older
ones.

Zoospores 10-12ju in diameter.

c on s p i c u o u s l y pitted.

Pits 5-7/1.

Many oogonia which are

Most oogonia have a

pro top lasmic pr o t r u s i o n growing up into them from below.
In addition some have androgynous antheridia arising directly
be low the oogonium.
21-26/1;

1 - 1 5

Diameter of oogonia 30-70/2,

Oospores

mo s t l y 3 - 8 in each oogonium.

This species was found four times as follows:
No.

249

in water from Pond 6 o n

8/14/34

No,

314

in water from Pond 9 on

10/12/34

No.

410

in soil from Pond 2 on

11/23/34

No.

504

in water from Pond 24 on

1/2/35

From ^ o k e r 's (4) description of this species it is
evident that it is a species with great variation in form.
Pringsheim

(17) first described the species as a variety of

S.

It was recognized as a species by de Bary (2).

ferax.

M a u ri zio

(14) describes^ at least 5 varieties.

In Ame r i c a

a form of the species has heretofore been found only by
K a u f f m a n in Michigan.
The writer has compared the forms listed above with the
original

illustrations and descriptions of Pringsheim and

de Bary and feels certain that these forms belong to this
species.

Whether or not they are the same form as the one

which K a u f f m a n has found in Michigan has not been determined.

Sa pro leg ni a anisospora

de Bary

Dense m y c el iu m growing well on all media used.

Hyphae

of medium thickness producing zoosporangia whose diameters are

28

a little greater th an the hyph ae bearing them*
r e a d i l y p r o l i f e r a t e d through old sporangia*
zoospores in each zoosporangium.
d is ti nc tly different

New hyphae

R e l a t i v e l y few

There are at least two

sizes of zoospores in this species.

The smaller ones are from 12-14ju in diameter when encysted,
the larger ones up to 17*5ja*

Oo gon ia moder at el y abundant,

30-60^1 in diameter with thin un pit te d walls.
diclinous,

branching,

Ant he ri di a

oospores 1 - 7 in an oogonium,

in diameter, mo s t l y 17-21^i,

17-30 p.

centric but soon breaking down,

some appea ri ng eccentric when old.
This

species was encountered twice,

from Pond 14.
No.

The record shows;

444

No. 8
40

in water from Pond 14 on

in wa t e r

Considerable
species.

The

from Pond 14 on

12/8/34
12/29/34

difficulty was experienced in placing this

"durchwachsung" of the zoosporangia clearly

showed this form
oospores

both times in water

to be a species of Sapro le gn ia . The

howev er appeared to be eccentric, which is not

cha rac teristic for this genus.

Single spore cultures were

made by the met hod described in the

section on technique*

Later a tuft of mycelium originating from a single

spore

and growing on a Derme stid larva was mounted in a hanging
drop.

The fungus continued to grow in this drop for at

least 48 hours so that a thorough study could be made of
the development of zoosporangia and the discharge of
zoospores.
Ob s e r v a t i o n soon showed that at least two different
sizes of zoospores were discharged from different zoosporangia,
a characteristic

found in £>. A ni s o s p o r a . On the day fol lowing

29
this obs e r v a t i o n oogo nia were
diclinous antheridia.

starting to form*

These bore

The oospores were present in these

o og onia on the next day and these were centric.
of hyphae,

zoospores,

oospores,

Measurements

antheridia and zoosporangia

were ta ken and these coincided with those given by Coker
for _S. a n i s o s p o r a , with the except ion that the encysted
zoospores v a ri ed from 12.25-17. 5ji.
few, most of the

The smaller ones were

spores were either 14 or 17.5p.

A single

spo rangium gave rise either to the larger or the smaller
forms.

The

swimming spores,

however,

were about 12ji in

their smallest diameter which is nearer to the measurements
given by Coker

(4) who states;

"the smallest spores are

about 8-9ji in diameter,

others from 10.5-ll.5ji, the large

ones from 13.7-14.8ji. "

This plant agrees also with the

descriptions of de Bary (3) who gives no measurements.

The

oospores obs erved in the original culture and which were
apparently eccentric were
de Bary (3).
observations

similar to those illustrated by

These oospores are no doubt

abnormal and our

substantiate Coker's suggestion:

".... one is

inclined to suspect that de Bary who rarely made a mistake,
was in t h i s case wrong in thinking the normal eggs eccentric.
His figures clearly show eccentric eggs,
have been bre ak ing down?
other S ap ro leg nia

but may they not

This seems the more likely as no

has an eccentric eg g . ”

In connection with the study of this species in the
h a n g i n g drop some

interesting facts may be recorded:

Ob servat ion s on an immature zoosporangium were started
at 4:30 P.M., Ma rch 28,

1935.

At 8:00 P.M.

ha d formed at the tip of the sporangium.
knob had take n on an oval

shape.

a spherical knob

At 9:30 P.M.

this

Zoo spores were not yet

30

starting to form in the zoosporangium.
however,

The next morning,

at 9:30 the zoospores had been discharged and could

be s e e n encysted a short
zoosporangium*

distance from the mouth of the

These were 17. 5p in diameter.

Because the discharge of zoospores had not actually
been observed,
observation*

another immature
At 11:00 A.M.

sporangium was found for

this zoosporangium appeared at

the tip of a hypha 12.25p. in diameter.
itself was 26.25xl40p.

The sporangium

Its protoplasm was dense and was

separated from the lighter protoplasm of the hypha by a
distinct wall.

The sporangial protoplasm;, however,

no signs of breaking up into zoospore
P.M.

initials.

showed

At 2:30

the zoospores had been formed and the characteristic

knob had been produced at the apex of the zoosporangium.
From 2:30 to 2:35 there was a squirming movement of zoospores
wi t h i n the sporangium.

At 2:35 the uppermost zoospore

escaped fol lowed immediately by the others,
At first the escape was very rapid,

one at a time.

slowing down as the

escape of the final zoospore neared.

All the 32 large

zoospores ha d left the zoosporangium in less th an a minute
and were

swimming about

independently in quick gyrating

m o t i o n not far from the mouth of the sporangium.
zoospores were all large,

These

oval-shaped and of equal size,

each m eas ur in g about 12x21 p.

A pair of long cilia could

be se en at one end of many of these zoospores.

At

2:45,

/

t e n minut es after the escape of the first zoospore,
of them was found to have encysted.
encystments were closely watched.
timed:

gyrations

one

After that several
In one the process was

stopped and the zoospore lay quivering

31

or v i b r at ing for 30 seconds,

then both ends contracted and

the zoospore rou nded up into a spherical mass.
lasted 30 seconds,
lasted one minute.

This also

so that the whole process of encystraent
At 2:55,

20 minutes after the escape,

all the zoospores h a d encysted.

These encysted forms were

14;00p in diameter.
The rate of growth of a hyphal tip was observed in this
hang ing drop culture.
ocula r micrometer.

The microscope was equipped with an

It was placed in such a position that a

growing hy p h a lay lengthwise directly unde rneath the scale..
The amount of growth was then observed at intervals for a
p erio d of one hour,

after which the slide was accidentally

m o v e d and the readings could not be continued.

The

reading s showed the following results:
from 1:03 to 1:23 the hyph a increased 21:00 microns
in length
from 1:23 to 1:43 the hypha increased 19:25 microns
in length
from 1:43 to 2:03 the hyph a increased 12:25 microns
in length.
The total

increase for the hour was therefore 52.5 microns.

Since the hypha was about 15ji in diameter the increase

in

one hour was about 3j times the diameter of the hypha.

S ap rolegn ia as terophora

de Bary

Hyphae with pr olif era tion of new hyphae through old
sporangia.

On hemp seed and fly numerous small oogonia

m o s t l y with papillae,
in an oogonium.

some smooth.

Oospores most ly one

Diameter of oospores about 22ji.

of oogon ia about 26u not including papillae.

Diameter

32

^his form was found only once:
No.

431

in soil from Pond 12 on

12/1/34

The pr ol i f e r a t i o n of new hyphae through old zoosporangia
and the oogonia with papillae,
oospores,

havi ng mostly one,

seldom two

places this form with £5. as tero pho ra. Measurements

t a k e n agree with those given by de Bary (1) and Coker
Saprolegnia delica

(4).

Coker

Mycelium rather delicate with cylindrical sporangia,
oogonia with thin walls,

pits not conspicuous.

All

oo go ni a with delicate diclinous antheridia sometimes nearly
surrounding oogonium.
Oo sp or es centric,

Oogonia 50-70p in diameter.

mos tly 5 - 10 in an oogonium.

Diameter

of oospores mos tly 24-26ju.
This form was collected once:
No. 272

in water from Pond 1 on

9/29/34

Its oogonia with thin walls and inconspicuous pits
h a v i n g delicate

antheridia which are mostly diclinous

places this form with _S. d e l i c a . > Number of oospores per
oogonium,

size of oogonia and oospores,

and the general

appearance of the plant all coincide with Coker's original
de sc ri pt ion and illustrations.

Saprole gni a m i x t a

de Bary

Hyphae with nu mer ous oogonia and few gemmae.
walls thick and with conspicuous pits about

Oogone

in diameter.

Ma n y of the oogonia have antheridia some apparently
diclinous,

chiefly terminals,

of the oogonis 40-70^i.
Oospores centric,

a few intercalary.

Diameter

Number of oospores mostly 1 - 10.

diameter 19-25p.

33
This form was collected once:
No*

273

in water from Pond 3 on

9/28/34

Thi s form is very mu c h like S3. ferax listed above,

but

because of the ma ny oogonia with antheridia it must
accor din g to keys and descriptions be classed as ^S. m i x t a .
Me asur em en ts agree with those given for that

species.

Coker

(4) calls attention to the similarity of _S. ferax

and

m i xt a .

A c h l y a ob lo ngata var globosa

Humphrey

Mycelium consis ting of long,
num er ous zoosporangia.
older ones.

New zoosporangia produced below

Zoo spores encyst at mouth of zoosporangium.

En c y s t e d zoospores 12-15 p..
spherical,

Oogonia abundant, most ly

50-90^1 in diameter,

on nearly all oogonia,
6 - 2 0

stout hyphae with

a few larger.

diclinous.

Oospores numerous,

mos tly 12 - 15 in an oogonium,

ap pa ren tl y subcentric,

Antheridia

centric,

a few

diameter 22-27pi.

This form was collected under four numbers:
No.

271

in water from

Pond 1

on

9/28/34

No.

282

in water from Mill Creek,

No.

434

in soil from Pond 13

on

12/1/34

No.

500

in water from Pond D

on

1/2/35

Loc. D

11/9/34

The identity of this species cannot very well be
mistaken.

Since the oogonia were nearly all spherical

oospores averaged more than 10 in an oogonium,

and

these forms

are no doubt of the variety g l o b o s a , as indicated above,
v ar iety esta blished by Hu m ph re y (8) on the basis of these
ve ry characteristics.
W h e n this form was first encountered (No.

271) the

a

34
large and long hyphae were very conspicuous on hemp seed.
These hyphae were exceptionally broad at the base and
tap ered to a point

at the top.

One of the hyphae was found

to be 27 1. 25p in diameter at the base.

Wh en the hemp seed

was h e l d out of the water the hyphae stood out straight
1*5 cm perpendicu lar to the surface of the seed.
A c h i y a racemosa
Short,
di am et er

Hildebr and

slender hyphae

with terminal zoosporangia of a

slightly greater than that of hyphae.

Zoospores

encyst at m outh of zoosporangiuip, about 12/a in diameter.
My c el iu m prod uci ng very many oogonia on short racemose
stalks.

The abundance of oogonia make the mycelium appear

as a dense,

white fringe aroung the substratum.

26.25-50.75/1,

with thick,

Oogonia

smooth, yel lowish walls.

oogonium bears one or two short, unbranched,
a n t h er id ia which arise from the oogonial

Each

tuberous

stalk directly

beneath the oogonium.

Oospores are 17.5-33.25/a averaging

about 24/a in diameter,

1 - 3

sometimes 4 and very rar ely

5 in an oogonium.
This species appeared three times:
No. 330

in w a t e r

from Pond 25 on

10/26/34

N o . 492

in water

from Pond 20 on

12/29/34

No . 499

in water

from Pond 4 on

1/2/35

This

species stands out clearly from the other species

A c h i y a because of its numerous oogonia on short racemose
stalks,

and by its short unbranched antheridia arising

from the oogonial

stalk directly below the oogonium.

That

all of the three

forms belong to this species is evident

since they possess all the characteristics up o n which
H i l d e b r a n d (7) bas ed the

species.

Later it was described

for Am e r i c a by Hum ph rey (8) and Coker (4).

As to details

of size in oogon ia and oospores and as to number of oospore
in an oogonium all of these forms agree.
however,
Coker

In this respect,

the y differ somewhat from the forms described by

(4) and Hu mph re y (8).

small 40-70ji",

Coker states

"oogonia rather

Thirty-two oogonia taken at random firom

different cul tur es of our three forms showed that the
oogonia var ie d from 26.25-50*75p.

On the whole,

therefore,

th ey are cons ide rably smaller than those described by
Coker.

Further,

Coker (4) states that the oospores are

"variable in size 16.6-27.
centric,

in diameter, most about

1 -8 in an oogonium,

in most cases 2 - 5 ,

centric.

H u m p h r e y (8) with reference to number and size of oospores
states "1 - 10,
25ja. "

commonly 1 - 6 ,

Hi ld eb r a n d

species states,

(7) in the original description of the
"In den Oogonien haben sich mehrere 3 - 1 2

Be fr uch tu ng sk ugeln gebildet."
oospores,

their diameter averaging

As to the size of the

forty oospores picked at random

from our cultures

va ri ed from 17.50 to 33.25^1 with an average diameter of
about 24^.

The difference

is more marked, however,

respect to number of oospores per oogonium.

with

All of the

three investigators cited above placed the oospore number
hig her than was found to be the case in any of these
cultures.

The oospores in one hundred oogonia in a

my celi um growing on a Dermestid larva were counted.

This

appeare d to be a representative culture so far as this

36

point is concerned.
21
37
36
5
1

The result was as follows:

oogonia contai ned 1 oospore
oo g o n i a contained 2 oospores
oogon ia contained 3 oospores
oo gon ia contained 4 oospores
oogoni um contained 5 oospores

For these forms,

therefore,

the oospore number is 1 - 3,

sometimes 4 and very seldom 5.

At no time was an oogonium

with more than 5 oospores observed.
One might conclude

from these data that a new and

distinct variety of A* racemosa has been found.

However,

here again the differences recorded above m a y not be
essential but may possibly be due to a nutritive factor.
We again refer to the work of Pieters (15) on the vegetative
vigor and reprod uctio n in S a pr ol eg niace ae .

Special reference

is made to his Table V (p. 544) where size and number of
oogonia,

and number of oospores in an oogonium are shown

to vary with different percentages of peptone in the medium.
Achlya Kl e b s i a n a

Pieters

Mycelium consisting of moderately stout hyphae.
hyphae
just

New

and zoosporangia branching off from older hyphae

below a zoosporangium.

diameter,

about 12p in

encysting at mouth of zoosporangium.

walls unpitted.
63-10lji.

Zoospores,

Antheridia diclinous.

Oospores eccentric,

17.5-24.5^1.

Oogonial

Diameter of oogonia

3 - 15 in oogonium,

diameter

^

A. Kl e b s i a n a was collected on three occasions;
No.

290 in

water from Mill Creek Loc.

B. on 10/5/34

No.

334 in

water from Mill Creek Loc.

C on

No.

429 in soil from Pond 11 on

10/26/34
12/1/34

37

Thi s
Piet ers
Coker

species was first described from Mic hi gan by

(16).

It is no doubt a truly M i chiga n species.

(4) has,

however,

found it in North Caroli na in 1921.

The forms listed above agree with the descriptions given by
Pi e t e r s and Coker in the essential characteristics,
di clinous antheridia,
oospores.

u n p it te d oogonial walls,

These forms seem to differ,

such as

and eccentric

however, from the

p r e v i o u s l y described forms in size of oogonia and number of
oospores per oogonium.
the

Pieters does not

give the

size of

oogonia but in C o k e r ’s form they are usually from

48-62^1.

In these forms they vary from 63-lOlp,.

Pieters

gives 1 - 10 as the number of oospores per oogonium.
states 1 - 8

usuall y 6.

oospores in an oogonium,
number of oospores

Coker

In our forms there were from 3 - 1 5
averaging 9.

The larger average

is perhaps due to the larger size of the

oogonia since the average oospore size is the same as that
given by Coker.

Notwithstanding

the differences given

above there can be no doubt as to the identity of the species.

Ac h i y a ame ricana

Humphrey

Long tapering hyphae bearing oogonia having eccentric
oospores.

Walls of oogonia smooth, hyaline,

and pitted.

A n t h er id ia androgynous arising from the ma i n hypha near
the

oogonial

stalk.

7 - 15 in oogonium,

Diameter of oogonia 70-90p. Oospores
22-26p in diameter.

Collected as:
No.

422

in water from Strawberry Creek,

Loc. A on 11/23/34

38

This species has not been reported from M i c h i g a n
before.

Since this form agrees wit h the descri pti ons

and

il lustr ati ons of both H u mphre y (8) and Coker (4) there

can

be no doubt as to its identity,

Achlya hypogyna

Coker and P e m b e r t o n

A c h l y a wit h o o g o n i a of various
papillae.

shapes,

Di a me te r of oog onia m o s t l y 50-80}i,

26-30}i, m o s t l y 28p,

1 - 13 in an oogonium.

m a n y with
oospores

Antheridia

androgynous.
C o l le cted as
No. 432

in soil from Pond 12 o n

12/1/34

Except for the number of oospores in an oogonium which
is given by Coker as 1 - 7 us ually 3 - 5 ,

this form agrees

entirely wit h descriptions and illustrations given for
A. hypogyna.

In the oogones found in this culture there

are from 1 - 1 3

oospores,

the

average number being 5.

It

must without doubt be assigned to this species.

Achlya s p .
In his monogra ph on the Saprolegniaceae Coker (4)
describes and illustrates an Ach ly a species which remained
sexually sterile

for several months and in which one could

not induce the formation of oogo nia by means of culture
m e d i a of various compositions.

Among our collections were

found four forms which agree with the description and
illustrations of Coker.

They were cultured for from three

to four months without producing any sexual organs under
conditions in w hi ch the other A ch lya species produced
abundant oogonia.

We have come to the con clusion that this

39

is most likely the
by Coker*

same A c hl ya species as the one referred

It occurred in our collections as follows:

No-

384

in soil from Pond 3 on
cu ltured until

11/9/34
3/13/35

No.

391

in soil from Island Pond
cultured until

11/16/34
3/16/35

No.

405

in soil from Pond 6 on
cultured until

11/16/34
3/15/35

No.

416

in soil from P o n d 3 on
cultured until

11/23/34
3/15/35

The

accompanying chart

shows graphically the relative

abundance of the identified species described on the
prece din g pages,
from sources,
ponds:

as well as the relative number collected

water from ponds,

and soil from bottom of the

UHART S H O W I N G RELATIV E ABU NDANCE OF DIF FE RE NT
SPECIES
(Based on the 79 species identified)
i
ft
r
a
1
i
1
d
0 1
i
I cct
o
a
i
1
0
0
0
0
d
Q
d o
ft
i •H 0 rH
P 0 0 1—1 I P
O 0
0
ft
0 -h
X >» -H 0 d d 0
0 0 d r*»■rH rQ 0
£ rH 0 B 0
O XJ ftp
d 0 o X*
0
4
3
0
x
l
P 0 Q rH d O bO
0 £2 d 0 d 43 0
0 0
•H
d
d •rH ft 0
0
0
X £3 XI 0
d
O
d
0
o 01
rH • 0 » O ••rH # ft o p • o • 0 • •pH • o • >>
0 • >>
Is; 0 0 CQ aJ M «h CO CiDCQ O
o CQ O CQ •H CQ £ CQ '©■!<2 0 < •H C d < 0 <C •rH <2 f
ciO
36

40

35

34
33
32
31
30
29
28
27
26
25
24
23

22
21
20
19
18
from soil
at bottom
of ponds

17
16
15
14
13

from ponds

12
11
10
9

8
from sources

7

6
5

r>:-;

3S>3

4
3

2
1

y t4 *> ;w

41

In con side ri ng the distribution of any particular
species with reference

to sources,

ponds,

that this distri but ion is very general.
pa r t i c u l a r
ponds,

and soil,
That

is,

we find

any

species does not confine itself to sources,

or soil.

For instance,

species are found in all three

the most representative
situations.

Those that were

en co un te red at least four times are likely to come from at
least two locations.

Seldom is a species found more than

once or t w i c e at any particular location.
From water from the
.§>•
A.

f e r a x ,S * d i c l i n a , A.

sources we obtained:

_S. p a r a s i t i c a ,

oblongata var g l o b o s a , A. K l e b s i a n a ,

americana.
From water from the ponds we collected;

£!. p a r a s i t i c a ,

S.

f e r a x ,_S. d i c l i n a , S.

h y p o g y n a , S. a n i s o s p o r a , S. d e l i c a ,

£5.

m i x t a ,A. oblon ga ta var g l o b o s a , A. r a c e m o s a , A. K l e b s i a n a .
From soil from the bottom of the ponds we collected:

S.

p a r a s i t i c a , S.

f e r a x , S. h y p o g y n a , S. a s t e r o p h o r a ,

A. h y p o g y n a , Ac h l y a sp.

(sterile)

T h i s general distribution of the species indicates a
close c on necti on between the contamination of water in the
sources,

water in the ponds,

and soil at the bottom of the

ponds.
Experiments to Determine the Extent of Parasi tism
of Water Molds.
Because of the abundance of these various mold species
at the fish hatchery,
reference

the problem of their parasitism with

to fish and fish eggs becomes important.

Kanouse

(9)

42

has con sidere d this problem with reference to _S. parasitica
Coker.

U p o n the basis of her experiments and observations

she concludes that the
means of zoospores

infection of living eggs and fry by

is not likely,

but "that the pressure of

growing hyphae on the egg membranes when the eggs are held
tightly together is sufficient to allow pe netrat ion of the
my c el iu m

into the living eggs."

(p. 449)

Huxley (8) more or less assumed the parasitism of a
form of fungus which no doubt was _S. parasitica Coker, upon
the basis of the numerous infections of living fish in
B r it is h rivers.
It was deemed advisable,

in connection with this

problem to determine more definitely the probable parasitism
of £>• par asitica and other species found in these collections
from the hatchery.

Accordingly the following experiments

were undertaken.
1.

Experiment with wall-eyed pike eggs.

Living eggs of the wall-eyed pike,

Stizostedion v i t r e u m ,

were obtain ed from a shipment received at the hatchery on
April 20,

1935.

Four apparently living eggs were placed in

each of 63 vials,
water.

each containing lOcc of sterile distilled

Into 7 of these vials Dermestid larvae on which the

mycelium of £>. parasitica was growing,
water with the eggs.

were placed in the

Seven other vials were infected similarly

with S. f e r a x . Still others were infected with S>. d i c l i n a ,
S. anisospora,

A.

oblongata var globosa,

and Achlya sp.

43

(sterile),

se ven vials for each form.

To determine whether

the m y c e l i a thus pla ce d in the vials were actually pr od uc ing
zoospores,

sterile Dermestid larvae were placed in each of

these 42 vials.
and were mot
larvae.

These larvae floated on top of the water

in contact with the mycelia growing on the other

A n y subsequent

infection of these

sterile larvae

must therefore be due to zoospores set free by the introduced
mycelia.

All the molds used were thus shown to be producing

zoos por es in the water which contained the eggs.

Ma ny of the

eggs adhe red to the sides of the vials and with very few
except ion s none of the eggs were
at the outset of this experiment.
were not

in contact with the mycelium
The remaining 21 vials

infected and were used as controls.

This was deemed

nec es sa ry since as a matter of course the eggs could not
be sterilized and some other check was needed in order to
determine the likelihood of previous infections.

All the

vials were pl a c e d in an icebox where the temperature ranged
from 10 to 15 degrees C . , mos tly 12 or 13 degrees.
The first careful examination of these vials was made
two days,

or about 48 hours,

after the experiment was

started.

From then on the vials were examined every day and

the observations carefully recorded.
The first examination showed that about 60# of the
eggs

in the cultures and about 50# of the eggs in the

control vials were dead.

The high mortality must be

r e g ar ded as due to factors involved in the

sudden changes

to which these eggs were subjected whe n the experiment

44

was started.

It is also possible that m a n y of the eggs

were not f e r t i li ze d and con sequently died.
cannot be ascribed to fungus infections

This mortality

since the

m o r t a l i t y in the controls was nearly as high as that
observed

in the cultures.

We conclude that only the eggs

with the greatest vitality survived the sudden changes.
These,

therefore,

were fit subjects for the object of

this experiment.
A further general statement may be made w it h reference
to the first observations,

namely,

that none of the living

eggs showed any signs of mold infections,

whereas,

49#

of the dead eggs in the cultures were definitely infected.
None of the dead eggs in the controls were infected.
^he observations for each of the mold species were
r e c o r d e d separately and can best be reproduced by a series
of graphs.

The graphs show the number of eggs that were

dead and the number of eggs that were infected on each
successive

day.

A curve for mortality and one for infection

were draw n in each case.
to the

The proxi mity of the first curve

second indicates how readily dead eggs are infected

by the particular species of mold.
at any point lies above,
curve

If the infection curve

or to the right of,

the mo rtality

it indicates that living eggs were found infected.

45

Graph showing Mortality Curve and
Infection Curve for S_» parasitica
HI
qJ cH 00 tO ^

lO O

o h w to ^ 10 o ^ co o o h w to ^ ^ o ^ cp N o .
OQ <j> i— I i— | i— I rH i— 1 r—( i— ) i— I i— i rH CO CO CO CO CO 00 CO CO CO

of

eggs

Q
1
a

z
3
4
5

*T-

6
7

G r a p h showing Mortality Curve and
Inf ection Curve for S>. ferax
to.
oHW«^ino^cooOHWto^ioo^«)
r_ l ( \ ) f O ^ I O ^ o f ' C O ( J > H H H H H H H H H H ( M ( M W W W 0 3 0 J N ( J 3 N •
Q
1

2
3
4
5
6

7

f PjTp-s
§&

46

Graph showing Mortality Curve and
Infection Curve for S. diclina

^ c o c n o rH
H W t o ^ i o ^ ^ c o No. of eses
CN2DO<^lO<O|>C0cr> HO HH WHi Ho ^H iH oHt o
H H H W W W W O J e a W W W

W
to
^
LO
tO
SA'SQ
•
1

2
3
4

5
6
7

G ra ph showing Mor ta lity Curve and
Infection Curve for _S. anisospora

47

Graph Showing Mortality Curve and
Infection Curve for A. oblongata var globosa
01
t>>
ctf

—| W

IV

U ) u ; C > UU

o#HCvJto-«?i*int£>i>cocr»Or-i<MtO'4«ioiot>oo

r i f— | 1— 1 r-1 r— I I— 1 I— 1 I
— I rl H

W

W

W

W

w

w

W

W

W

No.

of eggJ

Gr a p h
Showing Mo rt al it y Curve and
Infe ct io n Curve for Achlya sp.
OHNtO^lOONOaO^OHWlO^inONCO,,
tO

tO cOt>*00CT>pHr—I i—I i—I i—I i—I *—I rH i—I <
—I OJ

W CV} CXJ 0 3 CVj (X} (X?

n f p n-o-q
&o

48
A n ex a m i n a t i o n of the foregoing graphs brings to light
c e r t a i n facts wh i c h are here summarized:
1.

All of the species used in the experiment
infect dead fish eggs nore or less readily*

2.

S* par asi tica appears to infect dead fish
eggs more readily th an do the other Species
us e d in this experiment*

3.

S. p ar asi tica is parasitic on fish egg s to a
ce rt ai n extent.

T
■‘‘his last statement requires further explanation and
amplification*

It was observed that in no case was a living

©Eg lying out of contact with the mycelium infected.

Such

an infection of dead eggs, however,

was observed repeatedly

i n cultures of all of these molds.

It appears,

therefore,

that none of these molds are parasitic on fish eggs so far
as infections by isolated zoospores are concerned.
In a few cases

living eggs were observed to lie in

contact with growing mycelium.
enw ra ppe d the eggs

In those cases the hyphae

so that they could not easily be dislodged

from the meshes of the mycelium.

These cases were

studied

ca refu ll y with the aid of dissecting and compound microscopes.
The my celium was removed from these eggs in so far as this
was possible without
isolated and put

injuring the egg.

in a moist chamber.

These eggs were then
In this way subsequent

development of the my cel ium on the egg membranes and of the
eggs could be watched.
manner.

Five eggs were isolated in this

Three of these were surrounded by hyphae of

S. p a r a s i t i c a , one by hyphae of _S. d i c l i n a , and one by
hyphae of A. oblon gata var globosa.

In all cases the

49
hyphae a t t ac he d themselves to the egg membranes very firmly
so that

it was ha rd to remove them.

seem to be due to any physical

This phenom eno n did not

factor such as pressure since

in all cases the eggs lay close to the mycelium but the
hyphae were free to grow around them without becoming
attached.

Rather,

positi ve tropism
could hyphae
this time

the phe n o m e n o n appeared to be due to a

in the nature of t h i gmotr op is m.

In no case

be observed penetrating the egg membranes at

as was the case in the

infected dead eggs.

These

iso la ted eggs were kept in the ice box with the other cultures.
Five days after the isolation of the egg surrounded by
the hyghae of A. oblongata var g l o b o s a , the egg was still
alive and developing,
The hyphae

containing a living and active embryo.

showed no further development.

The observations on the egg surrounded by hyphae of
• diclina coincided with those on A. oblongata var g l o b o s a .
Three days after the

isolation this egg was still alive

c on ta in ing a living embryo and showing no infection.
Of the three eggs isolated with hyphae of _S. parasitica
one was dea d the following day and was severely infected,
a tuft of new hyphae extending from the focus of infection.
The other two eggs were still alive but
foci of infection in each case.

they showed distinct

These eggs were dissected

and the foci of infection carefully examined under the
compou nd microscope.

It was observed that at these foci

of infection small rhizoid-like hyphae were clumped together
on the surface of the egg and rhizoids also extended into
the egg.

A m o n g these clumps of hyphae several germinating

50
zoospores were also observed.
tubes,

however,

The pe netra tio n of the germ

could not be determined.

It is due to these observations that in the graph for
S. p a r a s i t i c a the infection curve is to the right of the
m o r t al it y curve on the last day of these observations.
These obs erv ations also would lead one to conclude that
.S* p arasi ti ca is parasitic on fish eggs at least to some
extent.

This parasitism

and due to some external
anJactive parasitism.

is not merely incidental or passive,
force

such as pressure,

It is limited,

however,

but it is

to this extent

that living fish eggs do not appear to be readily infected
by single zoospores of £>• parasitica,

but that due to the

abundant growth of the hyphae on the surface of the egg the
resistance of the egg is broken down and an avenue of
entrance

for hyphae and perhaps also for germ tubes becomes

establi shed.
During the five week period of our observations the
eggs in the controls showed no mold infection whatsoever.
The mort al it y rate

of the controls was also much lower than

that of the cultures.

At our first observation-two days

after the start of the experiment 41 of the 86 control eggs
were dead.

Seven days after the start of the experiment

44 of the 86 eggs were dead.

It may safely be concluded on

the basis of these observations that the eggs used for this
experiment were not previously infected.

The greater rate

of m o r t a l i t y in the cultures may be due to the fact that
these cultures had actively growing mycelia in them.
of course,

This,

diminished the relative amount of free oxygen in

51
the

culture water.

It is possible,

therefore that these

culture eggs died for lack of oxygen rather than because of
any direct effect of the

fungi.

This conclusion is

substa n t i a t e d by our observations on one of the seven vials
contai ni ng _S. d i c l i n a . The vial contained a very weak growth
of this fungus as indicated by the shight growth of mycelium.
All the eggs in this vial remained alive during the
day pe r i o d of observation.
the water was indicated,
test larva on the

2.

seven

That zoospores were present in

however,

by the infection of the

surface of the water.

Exper ime nts with eggs of black bass.

In the preceding experiment no difficulty was
encountered so far as mold contamination of the experimental
eggs was concerned.

This was found to be an exceptional

condition and yet one which is quite necessary in order to
determine with any degree of certainty the parasitism of a
particular

species of mold.

ne c e s s a r i l y ruled out.

All methods of sterilization are

The experimental eggs used in this

experiment were taken from a nest found in Pon d 2 at the
hatchery.

Th e y were brought into the laboratory in water

with a considerable amount of debris and also with eggs
that were already infected with water mold.

This mold

was very e v i de nt ly a species of Saprolegnia and most likely
_S. parasit i c a .

From the material brought into the laboratory

250 eggs were carefully selected.
out one by one,

were alive,

any mold contamination.

These eggs were picked

and were apparently free from

Twenty-five of the eggs were placed

52

in each of 10 petri

dishes con taining water from Strawberry

Cr eek which had pr ev io usly been sterilized,
a e rat ed by agitation.

The eggs were

cooled and

spaced so that no two

eggs were in contact with each other.

Hemp seeds with

wate r m ol ds growing on them were placed in 8 of these 10
pe tri

dishes.

E a c h of the 8 dishes was thus infected with

a diff ere nt species of mold*

The remaining 2 dishes were

not inf ec ted and were used as controls.
day,

however,

On the following

the eggs in the control dishes were as badly

infected as those in the other dishes.

This showed

d e f i n i t e l y that alt houg h apparently free from mold infection,
the

selected eggs were not actually free from contamination.

The experiment as set up could not give any information in
reg ard to the par asitis m of any particular species of mold.
However,

it could still throw light on the general

“Does any mold zoospore,

regardless of species,

infect living fish eggs?"

question,

actually

Here were 250 living eggs of the

small-mo ut he d black bass, Micropterus dol om ie u, with no
apparent mold infection,

all carefully selected,

in water from their natural habitat.

and spaced

Any infection that

would subsequently take place must necessarily be due to
zoospores set free in the water or to those that were
o ri g i n a l l y clinging to the sticky surface of the egg
membrane.

These eggs were carefully watched to determine

w he ther mold zoospores would infect living fish eggs.

The

results of the observations are recorded in the following*
table•

53
TABLE VI
Show ing the results on observations of 250 small-mouthed
bass eggs sub je ct ed to the z>ospores of va rious
water molds.
*Date of N o . of
o b s e r v a ­ living
tio n
eggs not
infected

No. of
living
eggs
infected

N o . of
de ad
eggs not
infected

No • of
dead
eggs
infected

Total
No. of
dead
number
larvae
not
infected

6/6/35

250

0

0

0

0

250

6/7/35

98

2

66

84

0

250

6/8/35

22

3

49

176

0

250

0

0

0

239

11

250

6 /I0/35

W h e t h e r in this table an egg was listed as dead or alive
dep en ded ent ir el y upo n its tr ans parency to light from the
substage lamp of the microscope.
that

It is considered likely

some of the eggs recorded as dead and infected may have

been alive,
column.

since all doubtful cases were placed in this

The five eggs recorded in Column 2 contained embryos

$hat were def initel y alive.
examined.

These eggs were mic ros copically

It was found in each case that the infection had

not pe netrat ed beyond the egg membrane.
r e a d i l y be remov ed with fine needles.

This membrane could
The embryo was then

set free app ar ent ly alive and not at all infected.
of the dead and

infected eggs were also examined.

Several
In these

cases the egg membr ane s could not be removed because of the
pe n e t r a t i o n of hyphae into the embryo.
On June 15,

1935,

a collection of eggs of the large­

m o u t h e d black bass, Micropte rus s a l m o i d e s , was brought into
the lab ora tory from Lake No.

2, of M o r g a n ’s chain of lakes

in n o r t h e r n Kent Co*
©oritaminated.

This batch of eggs was also badly

On l y 53 living and apparently non-infected

eggs could be isolated*

These were put into 4 petri-dishes

carefu ll y spaced as in the preceding experiment
ob se rva ti ons were made*

The results were much as those

the prec eding experiment.
start of the experiment,
infected,

without

17 of the eggs were dead and

34 were apparently living and not infected,
These

and the embryos were

One of these eggs was in an advanced

stage of development;
was beating,

and 2

2 eggs were again carefully

the me mbr an es were removed,

infection*

in

Twenty-four hours after the

eggs were alive and infected.
examined,

and

the larva was fully formed,

its heart

and the blood could be seen circulating through

the vessels.
E l e v e n of the 53 eggs used in this experiment hatched.
The larvae were observed for several days.
died in a few days.
were

Most of them

Five became infected after death.

None

infected while alive.
From these experiments it appears that

zoospores of at

least cert ai n water molds settle upo n the sticky membranes
of bass eggs and there germinate and penetrate the membrane.
The living embryo is not penetrat ed but due to the thick
meshe s of hyphae on the membrane

the embryo eventually dies

and is th en pene trate d by the hyphae.

3.

Experiment with eggs of the bluegill.

Whet her one or more

species of water molds are capable

of infecting living fish eggs was not determined in the

f o r e g o i n g experiments.

An attempt to determine this was made

wi t h eggs of the bluegill,
ta k e n from Lake No,

Lepomis p a l l i d u s .

These eggs were

2 of Morgan's chain on June

12,

1935,

T h e y were taken to the laboratory and e x p e r im en ta tion was
st art ed the same day.
gravel and shells.

The eggs were found in a bed of fine

None of them showed any mold infection.

Four hu nd re d eggs were separated out,
several changes of sterile water.

cleaned,

The eggs were then

isolated as in the previous experiments,

25 in a petri-dish,

wi t h sterilized water from Strawberry Creek.
ing 16 dishes,

2 were

was he d in

Of the resul t­

infected with zoospores of j3. parasitica

by the metho d described Tor the experiment with the
small-mo ut he d bass eggs.

Similarly,

wi th zoospores of S_. ferax,
ob l o n g a t a var* gjLobosa, A.
2 dishes

for each mold.

dishes were infected

S_. d i c l i n a , S. a n i s o s p o r a , Achlya

a m e r i c a n a , and A. K l e b s i a n a ,
The remaining 2 dishes were not

infected and were used as controls.

Sterile hemp seeds

were d r o p p e d into each petri-d ish to test the presence of
zoospores.

Although the eggs appeared to be entirely free

from molds

at the beginning of the experiment and although

they were thoroughly washed,

it was soon evident from the

in fec tion of control eggs that water mo ld zoospores were
carried over into the experiment
experiment,
definite

therefore,

by the eggs used.

This

again falls short in giving us

information with respect to the parasitism of any

partic u l a r

species of mold.

However,

the results do give

some valuable information as to the tendency of these
species toward infecting the membranes of living fish eggs.
The results are tabulated below.

56
TABLE VII
S h o w i n g results for bluegill eggs exposed to zoospores
of various water molds.
Name of N u m b e r of infected eggs Four days after start Total
mo ld
1 day
2 days
3 days
of experiment
species after
after
No.
Eggs
after
No.eggs
start
start
dead
start
eggs not inf.
of exp. of exp. of exp
inf. and ha t­ not
ched
inf.
S. p a r ­
asit ica

14

19

20

22

26

2

50

S.ferax

20

22

23

23

25

2

50

S.diclina 11
S. anisospora
0
A.oblong­
ata v gl
1
A. americana
1
A* Klebsiana
3

18

20

20

22

8

50

13

17

17

30

3

50

6

11

11

31

8

50

12

18

18

29

3

50

14

19

24

26

0

50

2

12

13

27

10

50

Control

1

It will be noticed that the preceding table does not
rec or d whether the infected eggs were dead or alive.
in fo rm at ion was purposely omitted in the table.
bluegill

Since

eggs are small it is difficult to determine whether

an egg is dead or alive,
light,

This

even with the use of transmitted

es pecially when the egg membrane

small p ar ticles of fore ign matter.
certainty,

however,

is covered with

There is reasonable

that all or nearly all of the eggs found

infected one day after the start of the experiment contained
living embryos.

Several of the infected egg membranes were

removed and living embryos were found inside.

One of the

14 eggs infected presumably by _S. parasitica hatched a few
hours after the

infection was observed.

All of the infected

57

eggs were remov ed from the petri-dishes and placed in other
dishes as soon as infecti on was observed.

These

eggs were kept under obs erva ti on for some time.
cases except the one ment ioned above,

infected
In all

the growth of the

mold in cre as ed and the embryo was killed.

Since after one

day the controls were only lightly infected it is evident
that the h e a v y infections found in the dishes inoculated
with S_. p a r a s i t i c a , _S. f e r a x , and S. diclina were due to the
zoospores of these species.

In each case the test hemp seed

was inf ected and the eggs were surrounded by active zoospores.
This experiment leads to the conclusion that the outer
me mbr anes of living fish eggs are readily infected by
zoospores of ce rta in water mold species including £5.
p ar as it ica and jS. ferax,

the two

species found in such large

numbers in the water and soil of the fish hatchery.

As a

result of such an infection the egg dies, unless the embryo
is in an adva nce d stage of development at the time of
infection,

in which case the egg will hatch,

the larva

e vi dently being unharmed.
4.

Experiment with bluegill fry.

In the p r e c e di ng experiments the parasitism of certain
water molds on fish eggs has been definitely shown.
que st ion to be considered is,

"Are living fish fry

suscepti ble to water mold infections?"
to this question the very young bluegill
the pr ec edin g experiment were used.
dishes none of the

Another

To obtain an answer
fry produced in

While

in the petri-

living fry became infected though they

were surrounded by an abundance of mold zoospores.

In order

58
t>o get these fry into conditions for better observations
they were

transferred from the petri-dishes to vials

s u p p l i e d with fresh,
hemp

sterile Str awberry Creek water,

and

seeds with zoospores producing molds of the same

species as those u s e d in the preceding experiment.

Most

of the fry lived for a week or more under these conditions.
None of the living fry were infected.

The results of the

ob se r v a t i o n s are here tabulated:
TABLE VIII
Sh owing the results of Experiment 4, in which
bluegill fry were exposed to various
kinds of m ol d zoospores.
St ar te d June 20
Name
pate o:" obsei~vatio n
of
6/24
6/85
6/26/
6/21
6/22
de ad
de ad dead
dead
mold
DEAD
Ito ',ni i pi
i ni
i pi .
i ni
species
S. p a r a ­
sitica
1
1
S. ferax
S.
diclina
S. anispspora
A. obi.
v glob.
A. araericana
A. Klebsiana

1

6/29
dead
i ni
9

1

Totals
i

ni
2 11

2

19

0 21

2 2

16

0 20

1

1

12

10

3

11

15

6

1
2

6/27
dead
i ni

6 1

■to 1 - infected

2 17
2 24
0 22

1
2

3
1

Controls

4

2

1

ni- not infected

The results for this experiment

6

1
1

3 18
1

Totals

7

2

9

11 142

show that of the 153

bluegill fry exposed to zoospores of water molds none were
infect ed while alive,
when dead.

and bnly 11 were found to be infected

The dead fry were removed at each observation.

Since so large a majo rity of the fry were found dead without
in fection it can be assumed that the few which were found

59

in fect ed die d from reasons other than the mold infection.
This experiment,

therefore,

leads to the conclusion that

waiter mo l d s are not parasitic on fish fry.

5.

Experiment

with small-mouthed bass fry.

A n oth er experiment to determine the parasitism of water
m ol ds o n fish fry was conducted at the hatchery with fry of
the

small mo ut hed bass.

Approxima te ly one hundred fry were

put

into each of eleven battery jars such as are us ed in the

h a t c h e r y for the ha tch in g of eggs.
of these

At the bottom of eight

jars zoospore producing molds were placed.

species used were S. parasitica,
£>. a n i s o s p o r a , A.

pr od uc ing zoospores
o n hemp seeds,

S. f e r a x , £1. d i c l i n a ,

oblongata var g l o b o s a , A.

A. K l e b s i a n a , and A. r a c e m o s a .

Two small dead fish,

They were growing

and pieces of boiled fish.

picked from the hatchery tanks and

badly inf ect ed with mold,

were placed in the ninth jar.

The other £ jars were left as controls.
wh ich © a m e

a m e r ic an a,

All of these molds were

in the laboratory.

dermestid larvae,

The

Hatchery water

directly from Strawberry Creek was used for

this experiment.

This water was kept running through the

jars as it is ordi na rily done when eggs are being hatched.
The fry were,

therefore, under conditions similar to

those that obtained when they were hatched.
One day after the start of the experiment all of the
fry were alive.

About

wi th y e l lo wi sh spots.
possible

10# of them, however,

were covered

These were at first thought to be

foci of mo l d infection.

On the following day the

60

number of fry ha ving such spots had increased but no mold
g r o w t h was in evidence.
these

Up on microscopical examination

spots proved to be the cysts of a protozoan parasite,

_Ichthyophtherius multifilius , an infusorium rather
abundant at fish hatcheries and causing a disease kn o w n
to ha tc hery m e n as the ’'itch" •

No mold growth was observed

on the second day nor was there any dying off of the fry.
The start of mol d growth was looked for especially at the
points of p r o t o z o a n infection but no such growth appeared
even unde r the microscope.
for 2 more

days.

Similar observations obtained

On the fifth day after the start of the

experiment ma n y of the fry had died.

All

the dead

specimens were removed and microscopically examined for
m o l d infection.
seventh days.
alive.

The same was done on the sixth and
On the seventh day only 108 fry were still

The experiment was terminated at this time and

the living specimens were also microscopically examined for
mold infections.

The observations for the 3 days were as

follows:
Number
Number
Total
Number
Total

of dead specimens infected
of dead specimens not infected
nu m b e r of dead specimens examined
of living specimens examined and
found not infected
number of dead and living specimens
examined

411
406
817
108
925

Infected dead specimens were found in all of the 11 jars
during the 3 day period that dead fry were found.

This

indicates that m o l d zoospores were present in all of these
jars and that the living fry were exposed to them.
Since the control

jars were shown to contain mold

zoospores by the i nf ection of dead fry,

this experiment

does not serve to show parasitism for any particular

61
species of mold*

However,

since no living specimens were

se en wit h infections in any of the jars,

the conclusion'

that none of the mo l d species use d in this experiment
are par as itic on fry of the small mouthed bass is
warranted*

This conclusion is similar to that reached for

the fry of bluegills in the precedin g experiment.

6.

Experiment with Lake Mi ch ig an Shiners

In order to determine the effects of water molds on
older fish an experiment with Lake Michigan shiners was
undertaken.

These fish were chosen because of their

convenient size and because they are extensively used at
fish hat cherie s as food for larger fish.

A battery of

eleven jars was set up as in the preceding experiment.
Into each of these jars 6 shiners were placed,

three of

which were injured on the side by means of a scalpel.
Da i l y observations for molds were made.

These

observa ti on s were carried on over a period of nearly
two weeks.

The dead fish were removed as soon as they

were found and notes with regard to infection or
no n -i nf ec ti on were taken.
period,

At the close of this observation

6 of the 66 fish were still alive and showed no

mold infection,

26 had died from the effects of molds,

and 34 ha d died from other causes.
Of the living fish one was an injured specimen.
was taken from the jar supplied with £3. f e r a x .

It

The other

5 ha d not been injured and were taken as follows;
3
1
1

from the jar
from the jar
from the jar

containing A. oblongata var globosa
containing A. Klebsiana
containing the infected fish.

62
Of the 34 that died from c a u s e s other than mol d infection
5 escaped from the top of the aquaria and were found on the
floor.

The other 27 e vi dently could not adjust themselves

to the confines of aquaria as well as some of their mates.
Ma n y of them, however,

lived for nearly two weeks.

Special importance is attached to those 26 that died
from mold infections.

In n earl y all cases the infection

co uld be seen on the living fish one or two days before it
died*
rapid.

Once the growt h got started its^ spread was very
Not one of the fish lived more than two days after

the in fe ct io n was first observed.

In 16 of the 26 cases

that died from mo ld the infection started at the point of
injury o n the side.

In the others the infection started

either at the tail or in the gills and head region.
is significant,

This

since it is well kno wn that fish are

ea s i l y injured during transportation and handling,

especially

at the anterior and posterior ends. These injuries may
be very small and not noticeable

to casual observation.

It is ve ry likely that not only on the intentionally
injured specimens but also on the others the infection
started at a point of injury.

Some of the infected fish

were examine d mi cr oscopi cal ly and the infection was in
e ve ry case found to be superficial,

the hyphae never

p en et rat ing deeply into the body tissues.
appears

In fact,

it

that the mold establishes itself at the point

of injury and from there

spreads as a thick mat of

m y c el ium over;,the rest of the body,
the tissues.

but not penetrating

D e a d and infected fish, were found in all the jars
u s e d in this experiment,

controls as well as the others.

This was as expected since

in the preceding experiment

the water from Strawbe rry Creek was shown to contain
mo l d zoospores.
do not

The results of this experiment,

therefore,

indicate that any particular species of mold is

the offender.

If, however,

we consider the rate of

m o r t a l i t y due to water mo ld in the various jars it does
point

to the fact that £>. parasitica

if not the only offender.

ma y be the chief

For this reason,

the

in fo rm at ion that has bearing on this point is here given.
TABLE IX
Showi ng the mortality rate of the fish
that died from mold infections
No. of spec­
imens dying
from mol d

In jar
cont aining:

Rate of mold
No. of day
on which this mortality
mortality
per day
occurred

S. p ar asit ic a

4

3

1 1/3

S. ferax

1

3

1/3

S. dicl ina

3

7

3/7

S. a ni sosp or a
A. obl ong at a
var gl obo sa

3

3

2

10

1/5

A. K l e b s i a n a

3

6

1/2

A. rac emosa

1

8

Inf.

1

6

1/6

fish

1

.

.. 1 / 8

Cont rol No.

1

3

9

1/3

Control

2

4

12

1/3

No.

26

64

O n l y the jar containing _S. anisospora showed a
m o r t a l i t y rate that was at all close to that of
p a r a s i t i c a as indicated in the column to the right.
Since S. anisospora was not found in abundance at the
h a t c h e r y it is doubtful whether this species contributes
towa rd infections found in the ponds.

The foregoing experiments lead to the following
conclusions:
1.

One or more species of water molds readily infect
the membranes of living fish eggs by means of
zoospores as well as by their hyphae.
Such an
infection nearly always results in the death of
the embryo and further penetra tio n of the hyphae.

2.

None of the mold species used in these experiments
are cajpable of infecting living fish fry.

3.

S a pr ol eg nia parasitica and perhaps also other
species of water molds, by means of their
zoospores infect living fish at injured surfaces
and continue to grow from that point until a
large part of the body of the fish becomes
covered with a mat of mycelium as a result of
which the fish dies.

D i s c u s s i o n of the Necessity and Possibility of Control.
A t t e n t i o n has bee n called in this paper to the
general prevalence of several species of water molds in
the ponds of the Lydell State Fish Hatchery.

It; has

further been sh own that the zoospores of many of these molds
infect dead fish eggs readily and that one or more species
of water molds will

infect the membranes of living fish

eggs by means of their zoospores as well as by their
hyphae.

There is no doubt that the death of many

65

thousands of fish eggs

in these ponds annually is at

least in part due to the prevalence of these molds.
is evident

It

that the elimination of water molds from chis

and similar situations would be of great benefit to
fi sh culture.
The problem of control, however,
It is not sufficient,

is a difficult one.

as is frequently suggested,

the ponds free from dead fish and spawn,

to keep

and other

organic debris on which these fungi thrive,

so long as

the water supply from the sources is highly contaminated
with mo ld zoospores.

The problem resolves itself into

the p u ri fi cati on of the water from the sources and this
makes it still more difficult.
to keep the streams,

It is out of the question

which supply the ponds with water,

free from the organic matter which supports the life of
these P h y c o m y c e t e s .
the water

If,

as is the case

in this hatchery,

is conveyed from the sources to the ponds by

means of pipes,

it would seem that the proper time to rid

the water of these dangerous zoospores would be when it
passes th rough the pipes.

If at this stage the water were

trea ted with copper sulphate,
ce rtai nl y be eliminated.
for two reasons:
kills

first,

the zoospores would

But this treatment is ruled out
because the copper ion which

the spores is also detrimental to the life of fish

eggs and young fish,

and secondly,

because there is a

continual flow of water through the pipes and the ponds
so that thousands of gallons of water would have to be
tr eated daily.

It is doubtful whether the expense of

66

such an u n d e r t a k i n g would warrant

its usefulness.

If

copper ions could be set free in the water at one point
and ag ain isolated and taken out of the water at a point
further do w n before it reaches the ponds,
the zoospores might

the killing of

be effected and the problem solved.

Whether this would be possible through some method of
electroly sis

or some chemical process is a question

which requir es further experimentation and study.
so l u t i o n of the problem lies,

perhaps,

of physics and chemistry and beyond the
intentions of this paper.

The

along the lines
scope and

Until such a purification

process has been devised the usual precautions to free
the ponds from all organic matter supporting the life
of fungi

should strictly be adhered to.

A periodic

draining and cleaning of the ponds and the subsoil is at
present the

best method to keep the fungi under control.

Summary
1.

130 samples of water from 26 ponds,

of water from sources,

40 samples

and 115 samples of soil were

collected from the Lydell Fish Hatchery with the purpose
of determin ing

the water mo ld contamination of this

hatchery.
2.

A new me t h o d for obtaining single-zoospore

cultures of water molds
3.

is described.

Fourtee n species of water molds were isolated

from the water and soil samples.

These were identified

and described.
4.

It is found the _S. parasitica Coker is more

67

abundant in this situation t h a n any other species,
isolated from the samples.
5«
were

The streams which supply the ponds with water

found to be hi g h l y contaminated with mold spores

of various species,

so that the contamination of the

ponds and the soil of the hatchery could be traced to
these sources.
6.

Experiments to determine the extent of parasitism

of _S. parasit ic a Coker and other molds were conducted
with the result that certa in of these molds were found
to infect membranes of living fish eggs.

They were

found not to be parasitic on the fry of fish.

One or

more of these molds were shown to infect older fish at
injured points and to effect the death of such infected
f i sh.
7.

The necessity and possibility of water mold

control at the hatchery is considered.

LITERATURE CITED
1.

Bary,

A.

2.

Bary,

A, de

3.

Bary, A.

4.

Coker,

5.

de

de

W. C.

Einige neue Saprolegnieen.
Jahrb. f. wiss. Bot. 2:169-192.
Pis 19-21.
1860
Zu P r i n g s h e i m ’s neuen Beobachtungen
iiber den Befruchtungact der Gattungen
Achlya und Saprolegnia.
Bot. Zeit.
41:48-54. 1883
Species der Saprolegnieen.
Bot. Zeit.
46:597-645, Pis 9-10. 1888.
The S a p r o l e g n i a c e a e , with notes on
other water molds.
201 pp. 63 pis.
Un iv ers it y of North Carolina Press,
Chapel Hill, N.C. 1923.

Coker, W. C. and Pemberton, J. D.
A New Species of
Achlya.
Bot. Gaz. 45:194-196. figs 1-6.
1908.

68
6.

Giltner,

W.

7.

Hildebrand,

8.

Humphrey,

9.

Huxley,

Lab orat or y Manual in General M ic ro ­
biology, 472 pp. 72 figs. John Wiley
& Sons, Inc. Ne w York. 1926.
F. Myc ol og ische Beitrage I. Ueber einige
neue Saprolegnieen.
Jahrb. f. wiss.
Bot. 6:249-269.
Pis 15-16. 1867-68.

J. E. The Saprolegniaceae of the United States
with notes on other Species.
Trans. Am.
Phil. Soc. 17:63-148. Pis. 14-20. 1892,
1893.

T. H.

10. Kanouse,

B. B

Saprolegnia in Rel atio n to Salmon Disease.
Quart. Jour. Mic. Soc. 22:311.
1882.
A Physiological and Morphological Study
of Saprolegnia parasitica.
Myco lo gi a
27:431-4-52.
Pis 12 and 13.
1932.

11. Kauffman,

C. H. A Contribution to the Physiology of
the Saprolegniaceae with Special
Reference to the Variations of the
Sexual Organs.
Annals of Botany 23:
362-387.
1908.

12. Kauffman,

C. H. K l e b s * T h eo ry of the Control of
Developmental Processes in Organisms,
and its Application to Fungi.
Proceedings
of the International Congress of Plant
Sciences 2:1603-1611.
1929*

13. Klebs,

Zur Physiologie der Fortpflanzung einiger
Pilze II.
Jahrb. f. wiss. Bot. 33:513593.
1899.

G.

14. Maurizio, A.

Zur Entwickelungsgeschichte und Systematik
der Saprolegnieen.
Flora 79:109-158.
Pis. 3-5.
1894.

15. Pieters,

A. J. The Rel at ion between Vegetative Vigor
and the Re pr oduction in some
Saprolegniaceae.
Am. Jour, of Bot.
2:529-576.
Text fig. 1, 2. 1915.

16. Pieters,

A. J. New Species of Achlya and of Saprolegnia.
Bot. Gaz. 60:483-490. PI. 21. 1915.

17. Pringsheim,

N. Weitere Nachtrage zur Morphologie und
Systematik der Saprolegnieen.
Jahrb, f.
wiss. Bot. 9:191-234*
Pis 17-22. 1873.