vvEPA
United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
EPA/600/4-86/032
September 1986
Research and Development
Taxonomy of
Cerio daphnia
(Crustacea:
Cladocera) in
U.S. Environmental
Protection Agency
Cultures
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EPA/600/4-86/032
September 1986
TAXONOMY OF CERIODAPHNIA ( CRUSTACEA :CLADOCERA)
IN
U.S. ENVIRONMENTAL PROTECTION AGENCY CULTURES
Dorothy B. Berner
Department of Biology
Temple University
Philadelphia, PA 19122
P.O. # C2357 TTST
Project Officer
William B. Horning, II, Chief
Aquatic Biology Section
Biological Methods Branch
Environmental Monitoring and Support Laboratory - Cincinnati
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
Environmental Monitoring and Support Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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DISCLAIMER
The information in this document has been funded wholly or in part by
the United States Environmental Protection Agency (USEPA) under Purchase
Order No. C2357TTST with the author. It has been subject to the Agency's
peer and administrative review, and it has been approved for publication
as an USEPA document. The mention of trade names or commercial products
does not constitute endoursement or recommendation for use.
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FOREWORD
Environmental measurements are required to determine the quality of
ambient water, the character of effluents, and the effects of pollutants
on aquatic life. The Environmental Monitoring and Support Laboratory -
Cincinnati (EMSL-Cincinnati) conducts research to develop, evaluate, and
promulgate methods to:
Measure the presence and concentration of physical, chemical, and
radiological pollutants in water, wastewater, bottom sediments,
and solid waste.
Concentrate, recover, and identify enteric viruses, bacteria, and
other microorganisms in water.
Measure the effects of pollution on freshwater, estuarine, and
marine organisms, including the phytoplankton, zooplankton,
periphyton, macrophyton, macroinvertebrates, and fish.
Automate the measurement of physical, chemical, and biological
quality of water.
Conduct an Agency-wide quality assurance program to assure
standardization and quality control of systems for monitoring
water and wastewater.
Ceriodaphnia is a small relative of Daphnia that is currently being
used to evaluate the chronic toxicity of pollutants to freshwater
organisms. A chronic toxicity test employing this organism was included
in the EMSL-Cincinnati manual, "Short-Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters to Freshwater
Organisms," EPA-600/4-85-014, which went to press in December, 1985.
During the initial development and field validation of the
Ceriodaphnia chronic toxicity test, the organisms being cultured in the
various Agency and private sector laboratories were tentatively
identified as Ceriodaphnia reticulata. but as the use of the test became
more widespread, there was increasing uncertainty and controversy about
their true identity. Since the correct identification of test species is
vital to the toxicity evaluation program, it was important to examine the
issue promptly. To resolve this problem, Dr. Berner, the leading U.S.
expert on Ceriodaphnia taxonomy, was engaged by EMSL-Cincinnati to
examine organisms from Agency cultures. It was determined that the
correct identification was Ceriodaphnia dubia.
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This Report contains many excellent scanning electron micrographs and
drawings of the specimens examined, and will prove very useful to
biologists in confirming the identity of the organisms used In toxicity
tests.
Robert L. Booth
Director
Environmental Monitoring and Support
Laboratory - Cincinnati
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ABSTRACT
This study investigated the taxonomy of three groups of the
cladoceran genus Ceriodaphnia in cultures being used by the
U.S. Environmental Protection Agency. One taxonomic group, having heavy,
triangular denticles in a pecten on the postabdominal claw and very short
male antennules, was identified as £. reticulata (Jurine 1820). The
second group, with a heavy, setulated pecten on the claw and long male
antennules was identified as C. dubia Richard 1894. The third group was
taxonomically nearly identical to £. dubia except that the claw pecten of
females sometimes had ovate, sharp teeth rather than comb-like setules,
depending upon culture conditions. This was determined to be a hitherto
undescribed phenotypic variant of C. dubia, and is designated as C.
dubia, toothed-pecten variety. Specimens of this form have been found in
populations of £. dubia collected in the U. S. west of the Mississippi
River.
Similarities in the general morphology, postabdomens, and ephippia of
(3. reticulata and JC. dubia suggest that they are evolutionarily closely
related and might be able to hybridize and produce offspring having an
ovate-toothed pecten like that of the £. dubia variant. Experiments
designed to test this possiblity were inconclusive although two
successful interspecific matings were observed. It is suggested that the
relationship between these two Ceriodaphnia could be further elucidated
by study of more extensive field samples, and by interspecific breeding
experiments that include hatching of hybrid young from ephippia and study
of their taxonomy and fertility.
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CONTENTS
Forward ii:L
Abstract v
Figures vii
Acknowledgements • viii
Introduction 1
Conclusions 3
Materials and Methods • • 4
Source of Specimens .. ........ 4
Preservation of Specimens 5
Microscopy ........ 5
Mating Experiments 6
Results 7
Taxonomy 7
1. Ceriodaphnia reticulata .... 8
2. Ceriodaphnia dubia 14
dubia.
Ceriodaphnia
toothed-pectt
4. Comparison of EPA Ceriodaphnia with other populations
and descriptions
Mating Experiments.
20
25
Discussion and Recommendations • .... 26
Literature Cited
Appendix A
30
32
vi
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FIGURES
Number
Figure
Page
1. Composite drawing of the lateral aspect of a parthenogenetic
female Ceriodaphnia illustrating typical morphological features . . 2
2. Ceriodaphnia reticulata; 'parthenogenetic and sexual females;
male 10
3. Ceriodaphnia reticulata postabdomens ... 11
4. Shape changes during growth of Ceriodaphnia reticulata females . . 12
5. SEM of Ceriodaphnia reticulata 13
6. Ceriodaphnia dubia; parthenogenetic and sexual females; male . . 16
7. Ceriodaphnia dubia; postabdomens . . 17
8. Shape changes during growth of Ceriodaphnia dubia females .... 18
9. SEM of Ceriodaphnia dubia , 19
10. Ceriodaphnia dubia, toothed-pecten variety: parthenogenetic and
sexual females; male 21
11. Ceriodaphnia dubia, toothed-pecten variety: postabdomens .... 22
12. Shape changes during growth of Ceriodaphnia dubia, toothed-pecten
variety females .23
13. SEM of Ceriodaphnia dubia, toothed-pecten variety 24
vii
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ACKNOWLEDGMENTS
This work was supported, in part, by a contract from the U» S. Environ-
mental Protection Agency, Environmental Monitoring and Support Laboratory,
Newtown Facility, Newtown, OH, and by a Faculty Research Grant-in-Aid from
Temple University. It was mostly done while the author was a Visiting
Scholar at Harvard University, where H. Levi kindly provided her with
laboratory space in the Department of Invertebrates, Museum of Comparative
Zoology.
The author is indebted to P. Lewis for suggesting this study, for
patiently maintaining cultures, and, for supplying materials for the
experiments at the Newtown EPA Facility. W. B. Horning, II, of the same
facility, kindly arranged for funding and gave encouragement. W. Peltier
and K. Lamott were especially helpful in culturing and providing samples
of the morph that turned up at the Athens, GA, EPA laboratory. D. Mount
and T. Norberg are thanked for sending specimens from the EPA Duluth
laboratory.
All those investigators who sent samples or let the author search
through their collections in the course of this study are especially
appreciated. Thanks go to the curators who loaned specimens from the
Idlljeborg Collection, Uppsala; the British Museum (Natural History); and
the U.S. Natural History Museum.
Lastly, E. Seling and K. Moskowicz are appreciated for their expertise
in operation of the scanning electron microscope used in this study.
viii
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Introduction
Cladocerans from the family Daphniidae are ubiquitous in temperate
freshwaters. Numerous field and laboratory studies in the past have focused on
Daphnia, which often are abundant in limnetic communities and are large enough
to be easily distinguished from other zooplankton. Consequently, techniques
have been developed for utilization of Daphnia magna and Daphnia pulex in
water quality testing (Buikema et al 1980).
However, because of their relatively large body size, these cladocerans
do not reproduce until the 4th to jjth instar after birth (Anderson et al 1937;
Anderson and Jenkins 1942) and are best employed in tests with a duration
greater than a week.
Ceriodaphnia, daphnids of smaller size and shorter generation times, are
also amenable to laboratory culture (Burgess 1967), producing 3-4 broods a
week under optimal conditions. Taxonomically, Ceriodaphnia resemble Daphnia
(Fig. 1) except that they are more rotund and lack the prominent rostral
projection typical of that genus. They exhibit some cyclomorphism, but do not
develop the dorsal helmets and long posterior spines often seen in Daphnia.
Since 1969, the U.S. Environmental Protection Agency (EPA) Environmental
Research Laboratory-Duluth and other EPA and private laboratories have been
exploring the suitability of Ceriodaphnia for short-term (7-10 day) toxicity
testing.
Initial Ceriodaphnia stock cultures were established during 1969 in the
Duluth EPA laboratories with animals obtained from fish ponds at the Newtown
Fish Toxicology Station, Newtown, OH (D. Mount 1983 .in litt.). The stocks
were identified as Ceriodaphnia reticulata. In 1982-83, subtle differences in
the appearance of the cultured animals suggested that the stocks comprised
more than one species (D. Mount 1983 in_ litt.). Subsequent microscopic examin-
ation in December, 1983, by myself and P. Lewis, of the EPA Environmental
Monitoring and Support Laboratory-Cincinnati, Newtown Facility, revealed that
their cultures contained two, possibly three, species of Ceriodaphnia. Two
were tentatively identified as C._ reticulata and £._ dubia (or C._ affinis
because a problem in synonomy exists) but the third was unidentifiable and
appeared to have characteristics of both the other two.
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15
16
19
321
1. Antennule with anterior sense hair and terminal aesthetascs (fine
2. Ocellus sensory hairs)
3. Frons
4. Supraocular depression
5. Fornix
6. Fenestra (headpore)
7. Cervical notch
8. Cardiac bulge (heart shown as dotted line beneath)
9. Ecdysial line
10. Exopod of antenna
11. Endopod of antenna
12. Brood chamber
13. Abdominal appendage
14. Abdominal seta
15. Posterodorsal angle
16. Postabdomen
17. Anal denticles
18. Postabdominal claw with a pecten
19. Reticulations
Figure 1—Composite drawing of the lateral aspect of a parthenogenetic
female Ceriodaphnia illustrating typical morphological features.
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The goals of the study reported herein were: 1. to verify the identifica-
tion of C. reticulata and C. dubia in the Newtown Fieldsite (and other) EPA
cultures, and 2. to try to determine, by taxonomic comparison and by inter-
specific matings, if the unidentifiable animals were hybrids of the two former
species, a morph of one of those species, or a new species.
Conclusions
1. Nearly all Ceriodaphnia cultures at the EPA Newtown Facility and
Duluth laboratories, and other cultures derived from them, were C._ reticulata
or C^ djubi_a_.
2. A third Ceriodaphnia found in a few EPA cultures, particularly those
from the Athens, GA, laboratory, was a morphological variant of £._ dubia,
which it resembles almost completely. The morph differs mainly in that the
females have a heavy-toothed pecten, somewhat reticulata-like, on the claw.
This pecten was reversibly altered to the dubia form with heavy setules when
the animals were cultured in reconstituted, rather than well water. The males
are indistinguishable from C._ dubia males, regardless of culture medium.
Specimens of this variant have been found in natural populations of C,_ dubia
west of the Mississippi River (D. Berner 1985, unpublished observations). It
is designated in this report as C._ dubia, toothed-pecten variety.
3. Experiments attempting to hybridize C. reticulata and C. dubia were
inconclusive, although two successful interspecific matings occurred. Taxo-
nomically, these species appear to be closely enough related that males might
mistake a female of the other species as their own. To ascertain whether
hybridization is possible, more experiments of the kind attempted in this
study would have to be carried out. Furthermore, ephippia of successful
matings should be gathered and hatched to see if viable populations of hybrids
can be produced. Lastly, the morphology of such hybrids should be compared
with specimens from field populations in which C^ reticulata and £._ dubia co-
exist, to see if hybrid forms occur naturally.
4. Comparison of the EPA C. dubia with N. American and European popula-
tions designated C. dubia or C. affinis revealed no significant differences
among them. This study therefore supports Johnson's (1956) conclusion that the
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two names are synonomous, and that C. dubia Richard 1894 takes precedence over
C. affinis Lilljeborg 1901. It is likely, however, that other species of
Ceriodaphnia exist that have a heavy, fine-toothed central pecten on the claw
similar to that of dubia. Therefore, that character alone should not be used
to identify animals found in natural populations.
Materials and Methods
Source of specimens
Most of the Ceriodaphnia examined in this study were from cultures being
maintained at the EPA Environmental Monitoring and Support Laboratory, Newtown
Facility, OH. Some were from the EPA laboratories in Athens, GA, and Duluth,
MN. The following also provided specimens for identification from their
cultures, which had originally come fron the Duluth laboratory? P. Dorn, Shell
Development, Houston TX; J. Fava, Ecological Analysts, Inc., Sparks, MD; D. R.
Folley, N.C. Department of NRCD, Gary, NC; R. Keen, Michigan Technological
University, Houghton, MI; A. V. Nebeker, Western Fish Toxicology Station,
Corvallis, OR; D. Nimmo, Colorado State University (Region V11I, USEPA), Ft.
Collins, CO; J. Owsley, Tennessee Department of Health and Environment,
Nashville, TN; R. Rupp, Southern Experimental Streams Facility, NCASI, New
Bern, NC; C. N. Scott, Environmental Laboratories, Burlington Research, Inc.,
Burlington, NC; M. Taylor, Environmental Safety Department, Proctor and Gamble
Ivorydale Technical Center, Cincinnati, OH; C. D. Webster, Ohio EPA, Columbus,
OH; and J. B. Whittaker, Biological Monitoring, Inc., Blacksburg;, VA.
EPA Ceriodaphnia were compared with specimens from the author's personal
collection and ones from S. Cooper, Santa Barbara, CA; D. G. Frey, Bloorn-
ington, IN; T. Edmonson, Seattle, WA; J. Korstad, Tulsa, OK; W. Murdock, Santa
Barbara, CA; W. Nelson, Ft. Collins, CO; and W. Hollwedel, Varel, W. Germany.
Specimens were also borrowed from the Riser collection at the U.S. Natural
History Museum (Smithsonian), Washington, DC; the British Museum (Natural
History), London; and the Lilljeborg Collection, Uppsala, Sweden.
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Preservation o£ specimens
All museum specimens were preserved in alcohol or mounted on slides.
Those from laboratory cultures and personal field collections were in formal-
dehyde solution or formaldehyde-sucrose (Haney and Hall 1973). Fixation often
distorts Ceriodaphnia. especially those cultured in reconstituted water, which
have particularly soft exoskeletons. To avoid 'ballooning1 of the carapace and
retraction of the postabdomen up against the body during fixation of live
specimens for this study, animals were first concentrated in a small volume of
water. Ninety-five percent ethanol saturated with sucrose (table sugar) was
gradually added until the animals ceased swimming and relaxed so that their
postabdomens could be seen at 30X under a dissecting microscope. A propor-
tionate amount of 37-40? formaldehyde solution saturated with sucrose was then
added to achieve a final 2-4% formaldehyde concentration.
Microscopy
Specimens to be examined by light microscopy (LM) were transferred to 1:1
glycerol-water solution in a deep depression slide and allowed to clear for an
hour or more. Small chambers for microscopy were made of two parallel strips
of plastic coverslip enclosing a small volume of 100% glycerol. Individual
animals were moved into this with an etched tungsten wire loop. Sometimes they
were propped into position in the chamber by snips of strands from a cotton
ball. Frequently the swimming antennae of animals to be viewed laterally were
first dissected away with tungsten wire needles; this made it easier to orient
them and view the head and antennules. No coverslips were used on whole mounts
in order to avoid distortion. Appendages to be examined in detail, such as
swimming antennae and postabdomens, were first dissected free of the body in
1:1 glycerol-water, then mounted under a coverslip in a drop of 100% glycerol
or CMC9-AF water-miscible medium (Masters Chemical Company).
An Olympus BH-2 microscope equipped with phase-contrast optics and an
Olympus LB drawing tube (camera lucida) was used for observations and
drawings. Most structures in whole mounts could be visualized best with direct
illumination and the condenser diaphram stopped down to provide oblique light.
Phase-contrast was used only for appendages mounted under coverslips. Whole
animals were drawn with direct illumination; postabdomens were drawn with both
phase-contrast and direct illumination. Measurements were made with ocular and
stage micrometers and a millimeter rule on the drawing surface.
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Specimens for scanning electron microscopy (SEM) were dehydrated in two
changes of 30, 50, 80, and 95% ethanol for at least 30 minutes at each step.
Final dehydration was in 2-3 changes of 10055 ethanol for several hours or
overnight. Specimens were transferred to small, capped carriers made from #10
plankton netting and size 00 Beem capsules (Ernest F. Fullam, Inc.), and
critical point dried from C02 in a Tousimis drier. An eyelash mounted on a
probe was used to mount the animals on double-stick tape affixed to a stub.
Specimens were spatter coated with gold-palladium. tScans were made with an
"Ameray" AMR model 1000 microscope and photographed with Polaroid 55 negative-
positive film.
Most of the specimens used for LM drawings and SEM in this study were
from cultures at the Newtown Facility. A few were from cultures sent to the
author directly from EPA laboratories at Duluth, MN and Athens, GA.
Mating experiments
Gamogenetic Ceriodaphnia (males, ephippial females, and sexual females in
the sterile, pre-ephippial instar) can, with some difficulty, be isolated from
living cultures. Sexual females are recognizable in the sterile,, pre-ephippial
instar because they have a dark egg mass in only one ovary and the dorsal
carapace is compressed laterally. In late sterile instar and unmated ephippial
females the clear or orangish lateral bulges and greyish borders of the ephip-
pium are visible in addition to the single, dark ovary. A single, dark egg is
evident in the ephippium of a sexual female who has already mated (Fig. 10..2).
Males resemble juvenile females, especially in earlier instars. More mature
males can be identified by their posteriorally tapered bodies, extended anten-
nules and claspers, reddish-pink color, and restless swimming behavior.
Garaogenetic individuals were sometimes located by examining cultures in
fingerbowls with a binocular microscope and direct illumination. More often,
glass beakers containing cultures were placed directly on a light table and
examined with a head or ring-mounted magnifier. Individual animals were
removed with a largemouth pipet and isolated in small Stender dishes. Male C._
reticulata were particularly difficult to identify because of their small size
and insignificant antennules. An attempt to isolate them was made by pouring
cultures through a fine sieve, resuspending the animals in a small amount of
medium, then anaesthetizing them with C02 (Club Soda) in order to sort them
under a binocular microscope. This technique yielded a number of males. How-
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ever, although all the animals resumed swimming following their return to
culture water, only females survived. The males died within 12 hours and this
technique had to be abandoned. Thus very few male £._ reticulata were available
for use in this study.
Animals to be mated were kept in pond or culture water and fed one or
two drops of cerophyll-yeast-trout chow medium (formula supplied verbally to
the Newtown Facility by the EPA Environmental Research Laboratory-Duluth, T.
Norberg 1985 personal communication). The planned procedure for these experi-
ments was to place a mature male with one or more sexual females of the same
species and to make observations at periodic intervals until one or more
females had an egg in her ephippium, evidence of succesful mating (see
Results, below). The male would then be isolated with sexual females of
another species and observed to see if mating occurred. If none was seen after
two or three days, the male would again be placed with sexual females of his
own species as a control to see if he was still fertile. Because of culture
conditions, this procedure could not be carried out fully (see Results). The
males and females used in these experiments were preserved in formalin-sucrose
for later examination.
A few of these mating experiments were carried out. in the author's
laboratory; the rest were done during a week's stay at the Newtown Facility.
Results
Taxonomy
The Ceriodaphnia examined in this study fell into three groups. Two could
clearly be classified as C._ reticulata and C^ dubia. The third, at first
thought to be a hybrid of the first two, or even a new species, has been
determined to be a variety of C._ dubia. Descriptions of the two known species
and the new variety are presented separately and then their similarities and
differences are considered.
For those unfamiliar with cladoceran taxonomy, Fig. 1 presents most of
the morphological characters used in this study. A general description of
Ceriodaphnia morphology is given in Appendix A. In most of the drawings
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accompanying the descriptions, the swimming antennae, which vary little in
detail, have been omitted for the sake of simplification.
1. Ceriodaphnia reticulata
Parthenogenetic female. Adult length, to 0.77 mm. Height, from 0.6 to 0.8
times length, depending upon maturity and number of eggs in brood chamber.
Shape, oval to almost round (Fig. 2.1, 4.4-.5, 5.1). Head, quite depressed,
with the frons at or below the level of the ventral carapace margin. A
distinct supraoptical depression on the anterior head surface bordered by
arched lateral margins. Ventral margin of head curved, with little or no angle
anterior to antennule but sometimes with protruded edges of reticulations that
look like tiny spines (Fig. 4.2, 4.3). Cervical notch broad and shallow with
oval fenestra on anterior margin. Dorsal margin of carapace arched, terminat-
ing in a distinct point at the posterodorsal angle. Lateral bulge of brood
chamber extends anteriorally into posterior headshield (Fig. 4.4, 5.1) and may
rise above the mid-dorsal line (Fig. 4.4). Individuals with many young may be
more rounded, shorter, and have a more distinct, lower posterodorsal point
than females with fewer eggs (Fig. 4.5). Antero- and posteroventral carapace
margins evenly and equally curved. Posterior carapace margin sometimes with
one or more tubercles that mark pores connected to glands. Fornix has a low
arch, sometimes with a small spine on the fold dorsal to the antenna. Reticu-
lations on carapace small in area, with distinct but not prominent edges. Head
and headshield usually smooth (in EPA cultures) with a single, raised edge
running from the anterior fornix ventrally to the base of the antennule.
Eye fairly large, nearly filling anterior-ventral portion of head. Ocel-
lus small and triangular, located close to base of antennule. Antennule cylin-
drical and short, not extending beyond line of margin of head,, Nine aesthe-
tascs, about equal in length to length of antennule. Anterior sensory seta
arising from a small peduncle near apex of antennule. Antenna is of typical
morphology, with setae that do not extend as far back as the posterior cara-
pace margin (Fig. 2.1, 5.1).
Postabdomen (Fig. 3.1) long, narrow (length about 3X width) and gently
tapered with a slight, mid-dorsal inflection. Abdominal process present,
sometimes long and tapered, separated from abdominal setae by three dorsal
rows of long, fine setae or hairs. Patches of short setae and very fine hairs
8
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on lateral surface anterior to abdominal setae. Anal denticles about 8 in
number, long, fine, and recurved, decreasing in length proximally except for
most distal 1 or 2, which are short and straight (Fig, 3.1, 5.2). Two rows of
fine setules on lateral surface adjacent to anal denticles; proximally, these
break up into scalloped clusters, with heavier spinules adjacent to the dorsal
inflexion. Postabdominal claw long, slightly recurved, with three distinct
divisions of setules and denticles on the lateral surface (Fig. 3.2, 5.2).
Setules of most proximal set number about 16, are short and distinctly heavier
than ones in the distal group. Denticles in the middle set form a pecten of 2-
8 heavy, sharp, triangular teeth that are the outstanding characteristic of
this species. They are separated by distinct gaps from the sets to either
side. The distal group of fine, short setules runs nearly to the tip of the
claw, terminating in one heavier spinule.
Juvenile females resemble adults except that the dorsal margin of the
carapace is flattened and the posterodorsal point is higher (Fig. 4.1-4.3).
The pecten on the postabdominal claw is visible at 200X magnification in in-
tact specimens of the second instar juvenile (Fig 4.2).
Gamogenetic female (Fig. 2.2, 5.3). Length, about 0.73mm. Height, about
0.76 times length. Shape rounded, flattened dorsally along top of ephippium.
Lower borders of ephippium forming a rounded curve or a broad V. Ephippium
exhibits three distinct regions: a flattened border region lacking cellular
outlines, a raised, semicircular region of deep polygonal cells having
slightly domed surfaces, and the dorsal locule, which is covered with small,
circular bumps (Fig. 5.3). These become more prominent as ecdysis (moulting)
approches. The ephippial surface is decorated with extremely short, stubby
hairs (Fig. 5.4). A single, dark egg forms (in either ovary) in the sexual
female; it moves into the brood chamber only as a consequence of mating. Color
of the ephippium is usually reddish orange.
Other characteristics are as in the parthenogenetic female.
Male. Length, about 0.58mm. Height, about 0.55 times length. Shape,
elongate oval, flattened dorsally and ventrally (Fig. 2.3). Head, larger in
proportion to body than female, and not as fully depressed, with a distinct
dorsal fenestra and supraocular depression. Antennule only slightly longer
than that of female, with short aesthetascs and a very short, straight term-
inal male seta, equal or shorter in length than the body of the antennule.
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Figure 2—Ceriodaphnia retlculata;
(ephippial) female; 3. male.
1. parthenogenetic female; 2. sexual
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O.05 mm
Figure 3—Ceriodaphnia reticulata postabdomens: 1. parthenogenetic female;
2. detail of claw of (1.); 3. male.
11
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o.
Figure 4—Shape changes during growth of Ceriodaphnia reticuXata
parthenogenetic females: 1.-3. juveniles; 4., 5. adults.
12
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Figure 5—SEM of Ceriodaphnia reticulata; 1. parthenogenetic female;
2. detail of postabdominal claw; 3. ephippial female; 4. detail of
ephippial surface. Bar measure: in 2 = 0.01 mm; in others = 0.1 mm.
13
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First thoracic appendage with a typical clasper that terminates in a right-
angled hook. Postabdorainal size proportions and characteristics as in the
female but lacking an abdominal process. Termination of the sperm duct later-
ally near distal denticles was not observed in this study.
2. Ceriodaphnia dubia
Parthenogenetic female. Adult length, to 0.88mm. Height, about 0.6 times
length. Shape, roundish oval (Fig. 6.1, 8.4, 8.5). Head not fully depressed,
with frons not as low as ventral margin of carapace. Anterior surface of head
with distinct supraorbital depression having arched lateral borders. Ventral
margin of head smoothly curved with a slight protuberance or distinct angle
anterior to antennule; raised edges of reticulations in this region may appear
from lateral aspect as short spines (Fig. 6.1, 8.3). Cervical notch broad with
a distinct oval fenestra on the anterior surface (Fig. 9.2). Heaclshield flat
on either side of cardiac bulge, appearing shelf-like in living specimens
(Fig. 6.1, 9.1). Dorsal margin of carapace broadly arched, with a medial ridge
that is apparent in living specimens viewed posteriorally. Posterodorsal angle
blunt, sometimes broadly pointed in mature individuals, located high above the
body axis. Anteroventral carapace margin more broadly curved than posteroven-
tral margin, which arches dorsally in a circular curve. Posteror margin some-
times with tubercles at the orifice of pores leading to glands. Fornix with a
low arch, not (in EPA cultures) expanded laterally into a wing. Reticulations
evenly sized on carapace, sometimes with heavy edges, and with dotted surfaces
(Fig. 9.2). Headshield and head usually reticulated, but sometimes smooth,
usually with a distinct row of elongate polygons extending from the fornix to
the antennule.
Eye large, nearly filling the anterior-ventral portion of the head, with
pigment mostly obscuring the crystalline lenses. Ocellus small, roundish,
located 1/3 to 1/2 the distance from antennule to eye. Antennule cylindrical
and long, extending beyond the line of the head. Aesthetascs 9, as long as
antennule. Anterior sensory seta long, arising from a distinct peduncle 1/3
distance above apex of antennule. Antenna of usual character, with terminal
setae reaching nearly to posterior margin of carapace (Fig. 6.1).
Postabdomen (Fig. 7.1) moderately long and wide (about 2X as long as
wide), tapered, with a slight mid-point inflexion. Abdominal process usually
moderate in size (Fig. 7.1), sometimes long (Fig. 8.5). Three rows of fine
14
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dorsal setae between abdominal process and abdominal setae; small patches of
short hairs on lateral surface anterior to the latter. Anal denticles 7-8 in
number, most distal two short, others longer and diminishing in size proximal-
ly. Denticles with a stout base, tapered, and only slightly recurved. One row
of setules on lateral surface at base of denticles, continuing proximally to
the mid-point inflexion as two to three rows of crescent-shaped clusters, with
heavier spinules along the dorsal margin. Postabdominal claw moderately re-
curved, with three subdivisions of the lateral setules. Setules of proximal
group short and slightly lighter in weight than those of distal set. Those of
middle set number from 18-24 and are heavier, forming a fine comb or pecten;
height of pecten varies among individuals and populations, making it more or
less prominent. It is not visible at 200X magnification in intact specimens.
Juvenile females have the same characters as the adult except that their
shape is more elongate and the dorsal carapace margin is flattened and even
slightly depressed in the first two instars (Fig. 8.1-8.3).
Gamogenetic female. (Fig. 6.2, 9.3). Length, about 0.71mm. Height, about
0.72 times length. Shape, rounded, flattened dorsally along margin of ephip-
pium. Head quite depressed, with frons at level of ventral carapace margin.
Ephippium of usual shape. Marginal cells of ephippium with flat surfaces,
semicircular band of deep, polygonal cells slightly rounded on top. Surface of
locule nearly smooth in early ephippium (Fig. 9.4), becoming irregularly
slightly bumpy in mature ephippiura (cf. Fig 13.4). Short, stubby hairs decor-
ate the ephippial surface (Fig. 9.4). ^Ephippium has a greyish coloration,
sometimes with an orangeish locule, depending on culture conditions. Single
ephippial egg develops in ovary and moves to brood chamber following mating.
Male. (Fig. 6.3). Length, to 0.66mm. Height, about 0.57 times length.
Shape, quadrangular. Head, noticeably large, not fully depressed, with a
supraorbital depression and slightly inflated appearanc'e. Cervical notch
broad, with a distinct fenestra on the anterior border. Eye larger than in a
female of comparable size. Antennule long and cylindrical with terminal male
seta 1.5 times length of antennule, terminating in a curved hook. Clasper on
second thoracic appendage long and thin, curving to a small terminal hook.
Postabdomen (Fig. 7.3) with denticulation and setulation like that of the
female, but somewhat narrower, proportionally, and lacking an abdominal
appendage.
15
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Figure 6—Ceriodaphnia dubia:
(ephippial) female; 3. male.
1. parthenogenetic female; 2. sexual
16
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0.05 mm
Figure 7—Ceriodaphnia dubia postabdomens:
2. detail of claw of (1.); 3. male.
17
1. parthenogenetic female;
-------
Figure 8—Shape changes during growth of Ceriodaphnia dubia parthenogenetic
females: 1.-3. juveniles; 4., 5. adults.
18
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Figure 9—SEM of Ceriodaphnia dubia; 1. parthenogenetic female;
2. detail of dorsal fenestra; 3. ephippial female; 4. detail of ephippial
surface. Bar measure: in 2 = 0.01 mm; in others = 0.1 mm.
19
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3. Ceriodaphnia dubia, toothed-pecten variety
Parthenogenetic female. Length, to 0.90 ram. Height, about 0.7 times
length. Shape and other characteristics of adult and juveniles like those of
C^ dubia (Fig 10.1, 12.1-.5), with the following exceptions:
Edges of polygons on carapace and head heavy, so that reticulations show
clearly (Fig. 13.1).
Postabdorainal claw with either of two forms of central pecten, depending
on culture medium. In one form, pecten is a comb of heavy, fine setules
slightly longer than adjacent groups; claw is indistinguishable from that of
C. dubia. In the second form, setules have been transformed to 7-14, close-
set, ovately tapered denticles that appear (in SEM) to be somewhat flexible
(Fig. 11.1, 11.2, 13.2).
Gamogenetic female. Length, to 0.9mm. Height, about 0.77 times length.
Except for heavier reticulation of the carapace (Fig. 13«3)» and possible
presence of a heavier pecten on the claw (Fig. 10.2) characteristics are the
same as for C^ dubia, including ornamentation of the ephippial surface (Fig.
13.4).
Male. Length, to 0.66mm. Height, about 0.56 times length. In all its
characteristics, male cannot be distinguished from a male C._ dubia (Fig.
10.3). Postabdominal claw always has a central, comb-like pecten, even in
cultures where females have the heavier form of pecten.
4. Comparison of EPA Ceriodaphnia with other populations and descriptions
The EPA C. reticulata did not differ noticeably from populations col-
lected in Wisconsin, New York, and Massachusetts, and also were similar to two
northern European populations that were examined. None of tVie males from
these populations had an antennule as long as shown by Lilljeborg (1901) in
his classical description. All N. American gamogenetic females had ephippia
with circular bumps and tiny spines but the ephippia of European specimens
lacked such bumps and looked more like C._ dubia ephippia. In all of the
specimens examined, the denticles of the middle claw pecten numbered from 2 to
8; they were straight-edged, heavy, sharp, and separated slightly from each
other and from the spinules to either side.
The EPA C. dubia, in general, were similar to populations collected in
California, Colorado, Louisiana, Mississippi, and Oklahoma, and to ones from
England, and N. Germany. Although no morphometric analysis was done, no
20
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Figure 10—Ceriodaphnia dubia, toothed-pecten variety:
female; 2. sexual (ephippial) female; 3. male.
21
1. parthenogenetic
-------
O. O5" m m
O.OS
Figure 11—€eriodaphnia dubia, toothed-pecten variety postabdomens:
1. parthenogenetic female; 2. detail of female claw; 3. male; 4. detail of
claw of (3.).
22
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O.3- rr\rr\
Figure 12—Shape changes during growth of Ceriodaphnia dubia, toothed-
pecten variety parthenogenetic females: 1.-3. juveniles; 4., 5. adults.
23
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Figure 13—SEM of Ceriodaphnia dubia, toothed-pecten variety:
1. parthenogenetic female; 2. detail of postabdominal claw; 3. ephippial
female; 4. detail of ephippial surface. Bar measure: in 2 = 0.01 mm; in
others s 0.1 mm.
24
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obvious differences were observed among the EPA animals, Lilljeborg's (1901)
type population of C^ affinis, and specimens labeled either 'affinis* or
'dubia' in the British Museum (Natural History) collection. The only signifi-
cant way in which the EPA C. dubia differed from other populations was that
they did not exhibit the degree of polymorphism (widened, spined fornices and
heavily reticulated raorphs) that often occurs in natural populations (Johnson
1956 and this author, unpublished observations).
There are no published descriptions of a Ceriodaphnia with a pecten like
that of the CU dubia variety described herein. It does occur in nature,
however, and has been found in populations of C._ dubia from Colorado,
Oklahoma, Oregon, and California (D. Berner, unpublished observations).
Mating Experiments
The Ceriodaphnia in the EPA Newtown Facility responded differentially to
the culture conditions of reconstituted water and a trout chow-yeast-Cerophyl
food mixture. C^ reticulata cultures steadily produced a small percentage of
sexual females, but very few males. The C^ dubia cultures, on the other hand,
rarely had sexual females but always produced some males. C._ cf. dubia
cultures, which had recently arrived from the Athens, GA, EPA facility, had
few gamogenetic individuals of either sex. Therefore, the original plan of
using adult males for consecutive intra- and interspecific mating could not be
carried out because of lack of sufficient gamogenetic individuals.
Nevertheless, enough C^ dubia males were found to attempt matings with
gamogenetic females: 20 with (^ dubia and 17 with C^ reticulata. Of the
former, 3 were successful; of the latter, 2 were successsful. A successful
mating was judged to have occurred when a dark resting egg was observed in the
ephippium. This criterion was based on two observations. Firstly, no egg was
subsequently observed in the ephippium of a female who had been carrying the
egg in her ovary at the time she was isolated from males. When these females
moulted, empty (sterile) ephippia were released. Secondly, one pair of £._
dubia in a culture was discovered mating. They separated after several minutes
and were isolated into individual dishes. When the female was observed twenty
minutes later, the egg had already moved into the ephippial chamber. It was
concluded that an egg is found in the ephippium only as a consequence of
copulation.
25
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In a few cases, matings were unsuccessful because animals died, perhaps
because of poor nutrition. In six cases, the males were probably not mature.
Two C. dubia females turned out to be parthenogenetic females bearing single
eggs rather than sexual females in the pre-ephippial instar. However, in most
of the unsuccessful pairings the selected females had well-developed ephippia
that were moulted within 24hr of isolation with a male. Four of the successful
raatings occurred with females that moulted from the sterile instar to the
ephippial instar while with the male. One female that had just moulted from
the sterile instar before isolation with a male also copulated. It thus
appears that there is only a relatively brief period of time following the
sterile instar moult during which copulation occurs.
Discussion and Recommendations
The EPA and N. American C^ reticulata observed in this study do not
appear to differ significantly from northern European specimens except that
the surface of the ephippium has distinct bumps, or broad tubercles, in the
former but not in the latter. This may be a matter of interpopulational
difference rather than an indication of interspecific difference. Therefore,
it is concluded that the species in some of the cultures at the EPA Newtown
Facility and the EPA Environmental Research Laboratory-Duluth was Ceriodaphnia
reticulata (Jurine 1820).
The second species being cultured in these two laboratories and found in
most cultures derived from the Duluth cultures was identified as Ceriodaphnia
dubia Richard 1894. This study supports the view of Johnson (1956) that £._
affinis and C»_ dubia are synonomous, with the latter name taking precedence.
Johnson had worked extensively with European populations of £._ affinis Lill-
jeborg 1901 prior to examining specimens from Richard's type locale, and gave
good arguments in support of his opinion. This synonomy was accepted by Scour-
field and Harding (1958). Nevertheless, the name affinis has come into general
usage because many temperate latitude investigators use Lilljeborg's (1901)
text for identification. The identification of this species in N. America is
further confused by the fact that Brooks (1959) commonly used key does not
describe it under either synonym. It perhaps is frequently mis-identified as
26
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C.. quadrangula. It should be cautioned that probably not all Ceriodaphnia with
a long, finely setulated middle pecten on the claw are C._ dubia; at least two
populations have been found along the NE American coast that resemble C._ dubia
in this respect but have markedly different ephippial morphology; they almost
certainly are new, undescribed species of Ceriodaphnia (Berner, unpublished
observations).
In many respects, C^ reticulata and C._ dubia are very similar: except for
pectenation of the claw, their postabdomens are quite alike. The only other__
striking differences are in the morphology of the antennules, especially in
the males. Their ephippia are alike in having tiny spinules over the surface,
while the bumpy tubercles obvious on the mature reticulata ephippium are not
prominent during the period in which the females will copulate. Goulden (1966)
has suggested that male cladocerans explore the ephippial surface prior to
coplation as a means of mate identification. This has not been observed in
Ceriodaphnia. but if it occurs, one can imagine, given the similarity in
ephippial surfaces, that these species might mismate. In addition, the morpho-
logical similarities between C._ reticulata and dubia could be interpreted as
evidence that they share a common ancestor in Ceriodaphnia evolution. If so,
there would be a greater probability of their producing viable, fertile
hybrids as a consequence of interspecific matings.
An hypothesis at the start of this study was that the third Ceriodaphnia
in EPA cultures, which has an ovate-toothed pecten, is such a hybrid. Its
close similarity to C._ dubia, especially in the male characters, and the fact
that females can express either the toothed pecten or a finely-setulated
pecten argues against that possibility. Discovery of this form in field popu-
lations of C. dubia lacking co-existing C. reticulata suggests that it is a
naturally occurring phenotypic variant of that species. A conclusion of this
study, therefore, is that this form is not a hybrid, but is probably a pheno-
typic variant of C. dubia Richard 1894, herewith designated as the toothed-
pecten variety.
It seems quite likely that the toothed-pecten morph arrived along with C._
dubia as a contaminant of the original C._ reticulata cultures at the EPA
Laboratory-Duluth. From there it was disseminated to the Athens, GA, EPA
laboratory, and to ones at Shell Development and Ecological Analysts, Inc. The
morphology of the pecten in this variety of £._ dubia appears to be under
27
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environmental control: females had ovate-toothed pectens when cultures from
the Athens, GA, laboratory arrived at the Newtown Facility. After several
weeks in reconstituted water, the animals all had a dubia-like pecten. When
the animals were returned to culture in well water at Athens, with a similar
diet, the female pectens reverted to the ovate form. It is possible that
differences in salts or trace minerals in the culture media effected the
change. (Complete analyses of the culture waters involved were not available).
As both forms of the pecten occur simultaneously, but to greater and lesser
degrees in natural populations (D. Berner, unpublished observations), this
phenomenon deserves further study.
It is possible that the toothed-pecten rnorph occurs rather widely across
the continent but is not recognized or identified correctly. Murdoch et al
(1984), for instance, reported the use of C^ reticulata in field and labor-
atory experiments in southern California. Recent examination of their samples
by this author revealed that they had, instead, populations of C.. dubia, many
of which were the toothed-pecten morph and easily mistaken for C.. reticulata.
As an outcome of this study, the following recommendations are made:
1. There needs to be a nationally organized and funded program for
sampling freshwaters, accompanied by support for systematic studies, develop-
ment of reference collections, and the publication of adequate taxonomic keys.
Difficulties encountered by EPA personnel in identifying the species in their
cultures reflect the inadequacy of the keys (Brooks 1959 and Pennak 1978)
commonly used in N. America to identify zooplankton. Furthermore, original
species' descriptions are frequently in Latin, German, or French, are not
readily available, and may not be applicable to N. America because they are of
taxa from other continents. Reference field populations were obtained for this
study largely because individual investigators were kind enough to share their
research materials. This author feels strongly that, as the need to monitor
freshwaters increases, so does our need for contemporary, comprehensive
reference collections and taxonomic information.
2. To determine whether or not C. reticulata and C. dubia can hybridize
successfully, more experiments of the type proposed in this study should be
done. The techniques described by Ivleva (1969) could be used to obtain more
abundantly gamogenetic populations. In addition, ephippia resulting from
28
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interspecific matings should be collected, hatched and reared (Leonhard
and Lawrence 1981) to see if viable, reproductive hybrids can be
produced. The hybrids morphology should be compared with that of the
parent species.
3. It is suggested that the form of pecten in C. dubia is under
nutritional control. This could possibly be tested by rearing them in
defined medium and feeding them algae also grown in defined medium
(Keating and Dagbusan 1984). This would make it possible to manipulate
the presence and concentrations of micronutrients, which seem likely
candidates for such control. Such studies might contribute to our
knowledge of some of the factors affecting polymorphism in the
Daphniidae.
29
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Literature Cited
Anderson, B. G., H. Luraer, and L. J. Zupancic, J*-. 1937. Growth and variability
in Daphnia pulex. Biol. Bull. 73: 444-463.
Anderson, B. G. and J. C. Jenkins. 1942.A time study of events in the life span
of Daphnia magna. Biol. Bull. 83: 260-272.
Brandlova, J., Z. Brandl, and C. H. Fernando.1972.The Cladocera of Ontario
with remarks on some species and distribution. Can. J. Zo'pl. 50: 1373-
1403.
Brooks, J. L. 1959. Cladocera In Ward and Whipple, Freshwater Biology 2nd ed»,
W. T. Edmondson, ed. John Wiley and Sons, Inc. New York. p. 587-656.
Buikeraa, Jr., A. L., J. G. Geiger, and R. L. Lee. 1980. Daphnia toxicity tests.
Ill Aquatic invertebrate bioassays, A. L. Buikema, Jr. and J. Cairns, Jr.,
eds. Amer. Soc. Test. Mat., Special Technical Publication 715. p. 48-69.
Burgis, M. J. 1967. A quantitative study of reproduction in some species of
Ceriodaphnia (Crustacea:Cladocera). J. Animal Ecol. 36: 61-75.
Goulden, C. E. 1966. Co-occurrence of moinid Cladocera and possible isolating
mechanisms. Verh. int. Ver. Limnol. 16: 1669-1672.
Haney, J. F. and D. J. Hall. 1973. Sugar-coated Daphnia; a preservation
technique for Cladocera. Limnol. Oceanogr. 18: 331-332.
Iveleva, I. V. 1969. Mass culture of invertebrates. Biology and methods. Acad.
Sci. USSR, Ail-Union Hydrobiol. Soc., Moscow. Israel Program for
Scientific Translation, Jerusalem, 1973.
30
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Johnson, D. S. 1956. Systematics and ecological notes on the Cladocera of Lake
Toba, and the surrounding country, North Sumatra. J. Linn. Soc. 43: 72-
91.
Keating, K. I. and B. C. Dagbusan. 1984. Effect of selenium deficiency on
cuticle integrity in the Cladocera (Crustacea). Proc. Nat. Acad. Sci.,
USA 81: 3433-3437.
Leonhard, S. L. and S. G. Lawrence.1981.Daphnia rnagna Straus and Daphnia pulex
(Leydig) Richard. In Manual for the culture of selected freshwater
invertebrates, S. G. Lawrence, ed. Canadian Special Publication of
Fisheries and Aquatic Sciences 54: 412-42. Department of Fisheries and
Oceans, Ottawa.
Lilljeborg, W. 1901. Cladocera Sueciae. Nova Acta Reg. Soc. Sci. Upsal. Ser.
Ill: vi. 701pp.
Murdoch, W. W., M. A. Scott, and P. Ebsworth. 1984. Effects of the general
predator, Notonecta (Hemiptera) upon a freshwater community. J. Animal
Ecol. 53: 791-808.
Pennak, R. W. 1978. Freshwater invertebrates of the United States, 2nd ed.
Ronald Press Co., New York. 769pp.
Richard, J. 1894. Entomostraces recuillis par M. E. Modigliani dans le lac Toba
(Sumatra). Ann. Mus. Gen. 34: 556-578.
Scourfield, D. J. and J. P. Harding. 1958. A key to the British freshwater
Cladocera, with notes on their ecology. Freshwater Biological Association
Scientific Publication 5, 2nd ed. Ambleside, Westmorland. 55pp.
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APPENDIX A
Taxonoraic Characters of Ceriodaphnia Used in This Study
Parthenogenetic female (Illustrated in Figure 1)
From the lateral aspect, the body form is rounded to oval, sometimes
slightly flattened dorsally and ventrally. The head is depressed; the frons in
front of the compound eye is rounded and occasionally angular anterior to the
antennule. The ocellus (simple eye) is small, lying between the eye and
antennule. There may be a supraoptical depression on the anterior surface of
the head below the origin of the fornix, a lateral extension of the headshield
which arches posteriorally above the antenna. Dorsally, a conspicuous cervical
notch separates the head from the posterior headshield; on its anterior
surface an oval headpore, or "fenestra", is frequently visible. The posterior
headshield bulges where it overlies the heart (cardiac bulge) and is separated
from the dorsal carapace by a usually distinct ecdysial line, which splits
when the animal moults.
The carapace is more or less strongly arched along the dorsal margin
where the two shells are fused. Dorsolaterally, it bulges out to accommodate
eggs in the brood chamber. Posteriorally, the dorsal margin extends to the
dorsoposterior angle, where the shells separate. This angle forms a blunt
point or pronounced spine, and may lie well above or close to the horizontal
axis of the body, depending upon species and maturity of the individual. The
carapace and, to varying extent, the head and headshield are "reticulated" by
5- to 7-sided polygons with raised edges and minutely patterned surfaces. The
anterior, ventral, and posterior free margins of the carapace curve into each
other without abrupt angles or distinguishing landmarks. Just inside the
carapace, close to the margin, runs a row of fine spines and spinules which
enlarge to form plumose setae toward the posterior portion of the ventral
edge.
The antennule (first appendage) is small and raoveable, varying in size
and shape with species. It has a terminal cluster of 9 aesthetascs (fine
sensory setae) and a longer, anterior sensory seta located slightly proximal
to the apex. The antenna (second appendage, used for swimming) has a basal
32
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segment and two rarai of 4 and 3 segments. The latter bear long, 2-segmented,
plumose setae according to the formula: exopod (dorsal ramus) 0-0-1-3; endo-
pod (ventral ramus) 1-1-3. Hidden under the carapace there are five pairs of
branchial appendages for feeding and respiration which were not examined in
this study except in the male (see below).
The posterior, dorsal portion of the body bears a single abdominal
appendage which helps retain eggs in the brood chamber and may be
insignificant in size or long and tapered. Posterior to it, a pair of long,
plumose abdominal setae protrude from under the carapace and mark the
beginning of the tapered postabdomen, which terminates with a pair of curved
claws. Near its midpoint, the dorsal margin has a slight or strong inflection,
depending on species. Posterior to this, it splits to form the margins of the
anal opening, then curves , sometimes with a flare, ventrally to the claw. The
spinulation of the postabdomen is significantly different among species.
Denticles arm the anal margins and the posterior curve of the dorsal margin;
they are generally longest along the curve and vary in length, weight, and
curvature. Just ventral to them, on the lateral surfaces of the postabdomen,
are rows and clusters of fine spinules that run proximally toward the dorsal
inflection where, depending on species, they become heavier and more scat-
tered. Each claw has a row of fine spinules on its medial and lateral
surfaces. The medial row is undifferentiated among species. The lateral row
consists of three sections, or pectens, that vary in length, weight, and size
among certain species.
Sexual (ephippial) female (Refer to Figures 2.2, 6.2, 10.2)
In the sexual female, the upper half of the carapace is modified to form
an ephippium, a saddle-shaped protective case for the single 'resting' embryo
resulting from mating. The ephippium develops during a 'sterile1 instar in
which no parthenogenetic eggs develop in the brood chamber; during this stage
the dark, haploid egg can be seen developing in one ovary lateral to the
gut and the dorsal margin of the carapace appears somewhat raised and pinched
laterally. Development of the ephippium, which has a somewhat triangular shape
and a straight dorsal margin, pushes the ecdysial line forward and depresses
the head more ventrally than in the parthenogenetic female.
The margin of the ephippium is composed of shallow cells. Above these is
33
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a semicircular collar of deep, polygonal cells enclosing a region of shallower
cells that bulges out to form a 'locule', in which the embryo is encased after
the carapace is moulted and the ephippium pulls free. Among species, the
surfaces of the polygonal cells and the locule differ in decoration by tufts,
hairs, tiny spinules and other modifications. In addition, the dorsal ridge
where the two sides of the ephippium meet may vary in height and prominence
among species. Depending on species, the ephippium of living specimens fre-
quently has an orange-greyish color.
The antennule and postabdomen are as in the parthenogenetic female.
Male (Refer to Figures 2.3, 6.3, 10.3)
Males strongly resemble immature females in size and shape: from lateral
aspect they are almost quadrangular in shape, with a flat dorsal margin
between cervical notch and posterodorsal angle. The latter is extended into a
distinct point which sometimes bears small spines. The head and compound eye
are larger than in juvenile females, and the posterior portion of the body
is more compressed laterally. The fornix, antenna, and postabdomen are usually
like those of the female.
Aside from the head and large eye, males are distinguished by modificat-
ions of the antennule and first thoracic appendage. The antennule is longer
than in the female and bears at its apex a special, 2-segmented 'male' seta.
The aesthetascs lie slightly proximal to this on the posterior surface of the
antenna and the longer, sensory seta is proximal to these on the anterior
surface. The size and shape of the male antennule and its distal seta varies
among species and may be quite distinctive in some. The first thoracic
appendage bears a small, inconspicuous hook, hard to see in undissected speci-
mens, and a long, thin, 2-segmented clasper. This reaches nearly to the post-
eroventral curve of the carapace when folded inside the shell and is very
conspicuous when extended outside the animal.
In culture, males are often recognizable by their rapid, erratic swim-
ming habits, their denser coloration than the females, and their extended
antennules and claspers.
34
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