Internat. Rev. Hydrobiol.

87 2002 1 97–105

CÉSAR ALBERTO VELÁZQUEZ-ROJAS, GUSTAVO EMILIO SANTOS-MEDRANO and

ROBERTO RICO-MARTÍNEZ*

Universidad Autónoma de Aguascalientes. Centro Básico. Departamento de Química.

Avenida Universidad 940, Aguascalientes, Ags. C. P. 20100, México.

Sexual Reproductive Biology of Platyias quadricornis

(Rotifera: Monogononta)

key words: invertebrate reproduction, male morphology, invertebrate taxonomy

Abstract
This study examined important aspects of the sexual reproductive biology of the monogonont rotifer
Platyias quadricornis. We made observations on the following: (1) Morphological description of the
male, heretofore unknown. (2) An analysis of male life-span at two temperatures. (3) Morphometric
characterization of the three types of eggs known in this species and determination of hatching percen-
tages at three temperatures. (4) Determination of the cells in the testis vesicle. The mating behavior of
P. quadricornis is similar to those of other brachionids. However, three main differences were found;
(a) Copulation lasts 5–6 times longer than a typical brachionid. (b) His foot is bent to allow penile
attachment. (c) The sites of copulation are similar to those of members of Lecane.

1. Introduction
Mating behavior in monogonont rotifers has been the objective of scientific studies in the
last 37 years (RICO-MARTÍNEZ, 1998), and more than 2,000 species of rotifers have been
described so far (WALLACE and SNELL, 1991). In spite of that, males are known in only 10%
of monogononts and only a few have been properly characterized (PENNAK, 1989). SNELL
and HAWKINSON (1983) described the steps of the mating behavior of B. plicatilis. These
steps correspond to; encounter, circling, coronal localization, sperm transfer, and dissociati-
on (NOGRADY et al., 1993). SNELL et al. (1995) studied the molecular basis of the mating
behavior of male B. plicatilis. They isolated a glycoprotein of 29 kD which is necessary and
sufficient to elicit male mating behavior. A similar glycoprotein is present in nine freshwa-
ter rotifer species (RICO-MARTÍNEZ et al., 1996). Therefore, it is clear that mating behavior
in rotifers represents a highly developed system to ensure proper exchange of gametes be-
tween conspecific mates.
Given the great diversity of rotifers, it is reasonable to expect important differences in the
mating behavior of rotifers. ALOIA and MORETTI (1973) compared the mating behavior of
Asplanchna brightwelli with that of two Brachionus species described by GILBERT (1963).
They concluded that both genera behave similarly, but they found four differences; (a) Dif-
ferences in the sites of initial mating attempts. (b) Asplanchna has more active females. (c)
Differences in the sites where copulation occurs. (d) Penile attachment in Asplanchna ver-
sus penetration in Brachionus. For many years and given the small amount of information
collected on mating behavior, the pattern described for brachionid rotifers has been consi-
dered the standard pattern, and comments comparing the behavior of different species to

* Corresponding author: Phone (49) 108420, Fax (49) 108401, e-mail: Rrico@correo.uaa.mx

© WILEY-VCH Verlag Berlin GmbH, 13086 Berlin, 2002 1434-2944/02/0101-0097 $ 17.50+.50/0

98 C. A. VELÁZQUEZ-ROJAS et al.

such pattern are found in the literature (GILBERT and WILLIAMSON, 1983). However, such
pattern as depicted in NOGRADY et al. (1993) is different in many species (RICO-MARTÍNEZ
and SNELL, 1997). For instance the coronal localization step is irrelevant to several species
where mating attempts and copulations do not occur here (RICO-MARTÍNEZ et al., 1996;
RICO-MARTÍNEZ and SNELL, 1997). Even in the case of some brachionids they prefer the foot
opening or other regions of the lorica to attempt mating or to copulate (RICO-MARTÍNEZ and
SNELL, 1997). Therefore the diagram shown in NOGRADY et al. (1993) is incomplete. It may
not even apply to all members of the genus Brachionus since some authors consider Pla-
tionus patulus to be in reality Brachionus patulus (KOSTE, 1978; RAO and SARMA, 1985;
KOSTE et al., 1993a and b).
Previous observations of mating behavior in six different families of monogononts, eight
genera and about twenty species (RICO-MARTÍNEZ, 1998) have indicated that the two species
best studied are B. plicatilis and B. rotundiformis. In these two species, male preferences for
females of the same strain have been detected (RICO-MARTÍNEZ and SNELL, 1995a and b).
In only three genera (Asplanchna, Brachionus, and Lecane) observations are there on inter-
specific crosses.
Little is known about the reproductive system of male monogononts. In studies of mating
behavior the information regarding reproductive system and germinal cells is scarce. The
primitive reproductive system of the males occupies most of the pseudocoel. The testis is
pear-shaped or globular, and followed by a short vas deferens which may or may not end
in a penis (PENNAK, 1989). Atypical rod-like cells are commonly found in the primitive
vesicles of the single testis of all males (GILBERT, 1983). In fact, in three species of rotifers
the typical germ cells are twice as abundant as rods (GILBERT, 1983).
Platyias quadricornis (EHRENBERG, 1832) is a member of the family Brachionidae whose
male has not been described (KOSTE, 1978). Here we describe several aspects of the sexual
reproductive biology of the brachionid Platyias quadricornis, making a detailed description
of the male, and discuss the different patterns of mating behavior observed in brachionids
in view of the observations included in this work.

2. Materials and Methods

2.1. Rotifer Culture

Platyias quadricornis was collected in a pond at the Botanical Garden of the Universidad Autónoma
de Aguascalientes. The approximate geographical coordinates of this sampling site are 21° 53′ 10′′ N
and 102° 28′ 54′′ W (geopositioner GPS 4000 XL Satellite Navigator, Magellan Inc., 1997). P. quad-
ricornis was cultured in EPA medium (192 mg NaHCO3, 120 mg CaSO4 · 2H2O, 246 mg
MgSO4 · 7H2O, and 8 mg KCl in 2 L) prepared with deionized water (16–18 megaohms) from a Water
Pro PS deionizer (Labconco Co., USA), and fed the green algae Nannochloris oculata (UTEX No.
LB2194) grown in Bold’s Basal Medium (NICHOLS, 1973).

2.2. Mating Behavior Test

Cross-mating assays were done according to SNELL and HAWKINSON (1983) with some modifications.
Briefly, this assay involves placing two neonate females and one neonate male (both < 18 hours old),
into 50 µl of EPA medium. Then, the number of male mating attempts and completed copulations in
five minutes was recorded in each of seven replicates using different males and females. A mating
attempt was recorded if a male circled around a female maintaining contact with his corona. A copula-
tion was recorded when a male attached his penis to a female. We also videotaped many complete
sequences of the mating behavior of this species (n = 25) and recorded; a) duration of copulation, b)
sites of mating behavior initiation, c) sites of copulation, and d) swimming speed. Additionally, we
 Sexual Reproductive Biology of a Rotifer 99

obtained photographs of several steps of the mating behavior, the sexual egg, and the male and female
of P. quadricornis by using a Hitachi Color Video Printer (HITACHI Co., Japan).

2.3. Swimming Speed Estimation

Swimming speed was estimated by videotaping ten males at 75X magnification for several minutes.
Then, the video was replayed with a clear acetate sheet taped to the monitor and the swimming path of
a rotifer was traced for ten seconds. A cartometer was then used to measure the length of the path.

2.4. Life History Data of the Male

The life span of the males and the hatching percentages of male eggs were recorded at two different
temperatures (20 and 25 + 2 °C). Newly born males and male eggs were placed in 300 µl of EPA medi-
um in individual wells of a 24-well culture polystyrene plate (Costar Co, USA) kept in a bioclimatic
chamber (REVCO Co., USA) with fluorescent light (600–1100 lux) and a light/darkness cycle of 16: 8
hours, and male eggs were review each 2 hours before hatching, then male eggs and males were obser-
ved each 12 hours.

2.5. Morphology of Males, Eggs and Rod-Like Cells

Morphometric characterization of males and the three types of eggs known in this species; a) male
unfertilized sexual egg, b) female asexual egg, and c) female fertilized sexual egg was performed by
direct observations at 40X in a light microscope with a micrometer (Olympus Co., USA). Determinati-
on of the number of sperm and atypical rod-like cells in the vesicle of the testis of virgin males was
done according to SNELL and CHILDRESS (1987).

3. Results

3.1. Description of Male Platyias quadricornis

The body consists of three regions; a) a retractile head where the ciliar corona is located,
b) the cylindrical trunk with a vestigial digestive system lacking a mastax and a primitive
reproductive system with a single testis with two prostate glands and a short penis, c) the
foot with two separate and mobile toes (Fig. 1).

3.2. Mating Behavior

We observed and videotaped more than 35 complete episodes (total observation time was
about fifty hours during one month) of mating behavior in Platyias quadricornis. In less than
10 % of these episodes we observed the coronal localization step. Instead, in most of the
cases the circling step was not observed and the males went directly to the juncture between
the ventral and dorsal plates in 92 % of the observed copulations (Tab. 1). A copulation was
videotaped and then a photograph taken “in vivo” of this step (Fig. 2). This photograph was
taken from a videotape containing the whole episode of mating behavior. This species is also
the brachionid with the longest copulation recorded so far (Tab. 1). The percentage of
encounters becoming mating attempts in five-minute mating tests (n = 7) was 19.3 (Tab. 1).
This is the one of the lowest percentages registered for any brachionid male copulating with
conspecific females, and only comparable to that of Keratella americana (RICO-MARTÍNEZ
and SNELL, 1997). No copulation was recorded during the five-minute mating tests (Tab. 1).
100 C. A. VELÁZQUEZ-ROJAS et al.

Figure 1. A photograph of
the male of P. quadricornis.
Bar = 25 µm.

However we recorded 25 copulations in our videotape observations. The mean duration of

copulation that we recorded is the longest for a brachionid, although is only half of that of
Lecane quadridentata (RICO-MARTÍNEZ and SNELL, 1997). In general, P. quadricornis prefer
the gap between the junctures of the ventral and dorsal plates (Fig. 2) and the foot opening
to initiate mating and to copulate. The corona is rarely chosen (Tab. 1).

3.3. Swimming Speed

Males swam faster than females (p = 0.007, n = 10; female swimming speed data from
SANTOS-MEDRANO et al., 2000), but the swimming speed of P. quadricornis males (Tab. 1)
falls in the range reported for other brachionid males (RICO-MARTÍNEZ and SNELL, 1997).

3.4. Life History Data of the Male

Male rotifers live longer at 20 °C than at 25 °C (p < 0.001; see Tab. 1). Life span ranged
from about 35 hours at 25 °C to almost 54 hours at 20 °C. This life span is half of that of
the females (RICO-MARTÍNEZ unpublished data).
 Sexual Reproductive Biology of a Rotifer 101

Table 1. Characteristics of the mating behavior and life span and hatching percentages of
males of Platyias quadricornis. Numerical data correspond to the mean ± one SD.
Duration of copulation 339.6 ± 396.0 (n = 25)
Number of spermatic cells in virgin males 10.0 ± 5.5 (n = 20)
Number of atypical rod cells in virgin males 10.8 ± 10.0 (n = 20)
Male swimming speed (mm/s) 0.8 ± 0.2 (n = 10)
Percentage of encounters becoming mating attempts 19.3 ± 10.9 (n = 7)
Percentage of mating attempts becoming copulations 0 (n = 7)
Sites of initiation of mating attempts on females Corona (32 %)
Foot opening (68 %) n = 25
Sites of copulation on females Gap between dorsal and ventral
juncture (92 %)
Corona (8 %) n = 25
Life span of males at 20 °C (h) 53.7 ± 12.8 (n = 24)
Life span of males at 25 °C (h) 34.9 ± 9.9 (n = 33)
Duration of hatching after deposition of male eggs (h) 11.6 ± 3.0 (n = 23)
at 20 °C
Duration of hatching after deposition of male eggs (h) 10.7 ± 2.5 (n = 57)
at 25 °C
Duration of hatching after deposition of male eggs (h) 18.3 ± 3.4 (n = 10)
at room temperature (approximately 28 °C)

Figure 2. A male of Platyias quadricornis copulating with a conspecific female at the gap between
the junctures of the dorsal and ventral plates of the lorica. Note the bending of the foot to allow better
penile penetration. Bar = 25 µm.

3.5. Morphology of Males, Eggs and Rod-Like Cells

Figures 3a, b, and c, show the unfertilized sexual male egg, the asexual female egg, and
the fertilized sexual egg of this species respectively. Note the thick outer coat surrounding
the sexual egg (Fig. 3c). The fertilized sexual egg of P. quadricornis is similar to the ferti-
lized sexual egg of Brachionus polyacanthus regarding the shape and the presence of an
outer coat (see KOSTE, 1978; Taf. 8, Fig. 4g). The female asexual egg of P. quadricornis is
longer and wider than the male asexual egg (p < 0.001), but it is smaller in size than the coa-
102 C. A. VELÁZQUEZ-ROJAS et al.

Table 2. Morphometric characterization of male, female, and the three types of eggs; male,
female and sexual egg of Platyias quadricornis. Abbreviations correspond to the following;
ML =Maximum length, MW = Maximum width. All measurements are in mm. N = 20 in all
cases.

Characteristic Mean SD

ML unfertilized sexual egg 85.4 7.2

MW unfertilized sexual egg 72.8 4.6
ML asexual egg 117.0 10.9
MW asexual egg 93.9 4.4
ML complete fertilized sexual egg 128.6 13.1
MW complete fertilized sexual egg 99.6 6.0
ML internal perimeter of fertilized sexual egg 108.5 9.8
MW internal perimeter of fertilized sexual egg 82.5 5.7
ML male 215.8 22.2
MW male 105.1 16.9
ML foot male 21.5 3.1

Fig. 3A Fig. 3B

ted sexual egg (p = 0.003). The male egg is the smallest of all eggs (p < 0.001). For com-
parison of the sizes of males, and of the three types of eggs presented in this species, see
Tab. 2.

4. Discussion
The male of P. quadricornis has a true penis and a two-toed foot. During mating, the foot
is bent to allow better contact between the penis and the lorica surface of the female and
 Sexual Reproductive Biology of a Rotifer 103

Fig. 3C

Figure 3. The three different types of eggs known in Platyias quadricornis so far. A) Unfertilized
sexual egg. B) Asexual egg. C) Fertilized sexual egg. The bars at the bottom of the figures represent
20 µm for A, 30 µm for B, and 25 µm for C.

then copulation can occur (Fig. 2). The male also lacks a digestive system as most mono-
gonont males do (PENNAK, 1989). The single testis is globular and has a vesicle containing
equal amounts of atypical rod-like germinal cells and sperm. In contrast, many of the spe-
cies observed so far have twice more sperm than rods (GILBERT, 1983).
The mating behavior of P. quadricornis is in many aspects similar to that of other
brachionids. The female assumes a passive role throughout the process, the circling step is
present, males are fast swimmers and 0.74 times smaller than females, initiation of mating
behavior and copulation occurs at corona. However, there are several differences from the
typical pattern thought to occur in all brachionids (NOGRADY et al., 1993). The coronal loca-
lization step very rarely occurs in P. quadricornis and instead the gap between the junctu-
res of the dorsal and ventral plates and the foot opening are the preferred places for copu-
lation and mating initiations respectively. The circling step, which is so remarkable in the
Brachionus species studied so far, it is not so evident in P. quadricornis. In fact, most of
the time there is no complete circling by the male around the female. This is the case of
other non-brachionid species (RICO-MARTÍNEZ and SNELL, 1997). Therefore, we recorded a
mating attempt whenever a male contacted a female with its corona and reacted to this
contact by keeping close contact to the female in longitudinal or transversal planes. The
sexual egg of P. quadricornis is different from that of several Brachionus species, like
B. angularis, B. calyciflorus, etc. However, it bears a great resemblance to that of B. poly-
acanthus (KOSTE, 1978). The presence of a thick outer layer in the egg perhaps gives this
species an extra advantage against other species during desiccation conditions. It is import-
ant to mention that this strain was collected in a pond which dries completely two times a
year.
104 C. A. VELÁZQUEZ-ROJAS et al.

The number of sperm found in virgin males of P. quadricornis (10.0) is lower than the
30–40 reported by some authors in Brachionus plicatilis (RUTTNER-KOLISKO 1983; SNELL
and CHILDRESS, 1987). The proportion between sperm and atypical germinal rod cells (1/1)
is also quite different in P. quadricornis than in other species where 2/1 proportions have
been found (GILBERT, 1983). These findings in this species bring new emphasis to the que-
stion put forward by SNELL and CHILDRESS (1987) about the possible limitation of cyst
production by sperm availability. In spite of having high number of males and females, we
very rarely observed sexual eggs (cysts) in our cultures. However, we did not count the num-
ber of sperm in the pseudocoelom of females. Therefore, this question would remain
unanswered.
As a member of the family Brachionidae, Platyias quadricornis has several differences
from other brachionids regarding mating behavior. Its duration of copulation (Tab. 1) is 5–6
times longer than an average brachionid (RICO-MARTÍNEZ and SNELL, 1997), the male bent
its foot to allow better penile attachment (Fig. 2) and the sites of copulation are similar to
those of the genus Lecane (RICO-MARTÍNEZ and SNELL, 1997; SEGERS and RICO-MARTÍNEZ,
2000), which curiously has the longest duration of copulation recorded for a rotifer (RICO-
MARTÍNEZ and SNELL, 1997). Although the family Brachionidae is the most studied regar-
ding mating behavior (RICO-MARTÍNEZ, 1998), we know the characteristics of mating beha-
vior for less than 10 % of the members of this family, and we know nothing of the mating
behavior of any species of several genera (for instance; Notholca, Kellicottia). Therefore, we
caution against misconceptions that can be created through generalizations based on the
investigation of a small number of species.
The data presented here together with previous observations (RICO-MARTÍNEZ and SNELL,
1997) make a strong argument for the fact that there are important differences in the mating
behavior pattern among freshwater species of rotifers. The circling and the coronal loca-
lization steps may not occur in some rotifers species. Figures published (NOGRADY et al.,
1993) illustrating rotifer mating behavior represent generalizations that do not apply to all
brachionids, and certainly not to all rotifers.
Why are there differences in the mating behavior of different rotifer species? The answer
to this question has multiple components; 1) Differences have been found in the size and
swimming speed of male and female rotifers in many species (RICO-MARTÍNEZ and SNELL,
1997). According to GILBERT and WILLIAMS (1983) these differences can be explained by
two divergent lines of evolution; one that favored fast, small and non-feeding males (such
as the males found in the families Brachionidae and Trichocercidae), other that favored big-
ger males to avoid cannibalism (Asplanchnidae) or to accomodate more sensory receptors
to recognize conspecifics (Epiphanidae, Euchlanidae and Lecanidae). 2) Differences in sites
of initial mating attempts can be explained in terms of Mate Recognition Pheromone con-
centration (RICO-MARTÍNEZ et al., 1996). 3) Differences in the sites of copulation may be
related to softer places in the body of female rotifers. This would explain why Asplanchna
males copulate anywhere in the surface of the body wall (ALOIA and MORETTI, 1973),
and why brachionids prefer the corona and foot opening to copulate (RICO-MARTÍNEZ and
SNELL, 1997). However, Platyias quadricornis copulates mostly at the gap at the junction
between the dorsal and ventral plates. Perhaps the hard lorica in this species covers the coro-
na more efficiently than in other brachionids becoming an obstacle for copulation. 4) The
differences in the duration of copulation can be related to; a) Efficiency and number of rod-
like structures and other accessory structures to attach the male copulatory organ to the
female body. In that respect P. quadricornis showed a remarkable low ratio of sperm/rod-
like structures which would contradict this relationship. b) Hardness of the female body wall.
This view is supported by the fact that the two species with the longest copulation recorded
(Lecane quadridentata and P. quadricornis) have females with hard lorica. These arguments
fairly explain some of the mating behavior differences found so far in rotifers. However, this
view could change, as more patterns of mating behavior in rotifers are unveiled.
 Sexual Reproductive Biology of a Rotifer 105

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Manuscript received August 10th, 2000; revised November 27th, 2000; accepted January 7th, 2001