Sexual reproductive biology of Brachionus quadridentatus Hermanns (Rotifera: Monogononta)
Estudio de la biología sexual reproductiva del rotífero Brachionus quadridentatus Hermanns (Rotifera: Monogononta)
Desiree Díaz1, Gustavo E. Santos-Medrano2, Marcelo Silva-Briano3, Araceli Adabache-Ortiz3 and Roberto Rico-Martínez2
1 University of Illinois at Urbana-Champaign. Student of the Program of Biophysics. 607 South Mathews Avenue. Urbana, IL , 61801, USA.
2 y 3 Universidad Autónoma de Aguascalientes. Centro Básico. Departamentos de Biología y Química. Avenida Universidad 940, Aguascalientes, Ags. C.P. 20100, México.
]]>Recibido: 21 de febrero de 2005
Aceptado: 10 de noviembre de 2005
ABSTRACT
This study examined important aspects of the sexual reproductive biology of the monogonont rotifer Brachionus quadridentatus Hermanns. Observations on the following was made: 1) Morphological description of the male, 2) An analysis of mating behavior, 3) An analysis of female and male life-span at 25oC, and 4) Morphometric characterization of the three types of eggs known in this species and determination of hatching percentages of sexual eggs at 20 and 25oC. SEM photographs of the male are included, the female and parthenogenetic and sexual eggs. Some complementary photographs with the light microscope are also included. The mating behavior of B. quadridentatus is similar to those of other brachionids. Attempted copulations lasted on average 12.4 s, and completed copulations lasted on average 71.4 s. B. quadridentatus is the Brachionus species with the longest duration of copulation recorded so far. Photographs showing episodes of mating attempts and copulations are included. Mating attempts and copulations are similar to those of other members of Brachionus. A comparison of the mating of B. quadridentatus with that of other brachionids is also included.
Key words: Rotifer mating behavior, rotifer, evolution, taxonomy.
RESUMEN
Este estudio examinó aspectos importantes de la biología sexual y reproductiva del rotífero monogononte Brachionus quadridentatus Hermanns. Se han hecho las siguientes observaciones: 1) Descripción morfológica del macho, 2) Un análisis del comportamiento sexual 3) Un análisis de la longevidad de hembras y machos a 25oC, y 4) Una caracterización morfométrica de los tres tipos de huevos que se conocen en esta especie, y se han determinado los porcentajes de eclosión de los huevos sexuales a 20 y 25oC. Se ha documentado con fotografías (empleando microscopía electrónica de barrido), al macho, la hembra, y los huevos partenogenéticos y sexuales. Algunas microfotografías complementarias realizadas con el microscopio de luz, también se incluyen. El comportamiento sexual de B. quadridentatus es similar al de otros brachionidos. Los intentos de cópula duraron en promedio 12.4 segundos, y las copulaciones completas duraron en promedio 71.4 segundos. B. quadridentatus es la especie del género Brachionus con la mayor duración de cópula que se haya registrado hasta miembros del género Brachionus. Se incluye una comparación del comportamiento sexual de B. quadridentatus con la de otras especies de ahora. Se ha incluido una secuencia de microfotografías con los diferentes episodios de la cópula. Los intentos de cópula son similares a los de otros brachionidos.
]]> Palabras clave: Comportamiento sexual de rotíferos, evolución, taxonomía.
INTRODUCTION
Rotifers are cyclic parthenogenetic animals that are important components of the zooplanktonic community of a typical lake. Rotifer populations are commonly represented by females with males appearing sporadically (Pennak, 1989). That is perhaps the reason that mating behavior of rotifers is poorly known in spite of being studied for over thirty years (Velázquez-Rojas et al. 2002). Actual understanding of mating behavior in rotifers started with Gilbert (1963) who demonstrated that contact chemoreception is used by male rotifers to identify conspecific females in Brachionus angularis Gosse, and B. calyciflorus Pallas. In 1983, Snell & Hawkinson described the mating behavior of B. plicatilis Müller, and described its different steps. Mating behavior in B. plicatilis is divided into five phases which correspond to: encounter, circling, coronal localization, sperm transfer, and dissociation (Nogrady et al. 1993). Snell et al. (1995) have demonstrated that the mating behavior of male B. plicatilis is based on the recognition of a mate recognition pheromone (MRP) a glycoprotein that is both necessary and sufficient to elicit male mating behavior, representing a highly efficient mechanism to recognize conspecifics.
Given the great diversity of rotifers, it is reasonable to expect important differences in the mating behavior of rotifers (Velázquez-Rojas et al. 2002). Aloia & Moretti (1973) concluded that the mating behavior of Asplanchna brightwelli Gosse is different in four aspects to that of the two Brachionus species described by Gilbert (1963). Velázquez-Rojas et al. (2002) argued that the coronal localization step is irrelevant to several species of rotifers, including some members of the family Brachionidae.
Mating behavior is known in six different families of monogononts (Asplanchnidae, Brachionidae, Epiphanidae, Euchlanidae, Lecanidae, and Trichocercidae), eight genera and about twenty species (Rico-Martínez & Snell, 1997). Interspecific crosses have been performed among members of three families (Asplanchnidae, Brachionidae, and Lecanidae), but only in one family (Brachionidae), have crossmating tests been made between members of different genera. In fact, the genus Brachionus is the most studied regarding sexual reproductive behavior. Several species of the genus have been studied; B. angularis (Gilbert, 1963), B. bidentatus Anderson (Rico-Martínez, 1999), B. calyciflorus (Gilbert, 1963), B. patulus Müller (Rao & Sarma, 1985), B. plicatilis (Snell & Hawkinson, 1983; Rico-Martínez & Snell, 1995a & b), B. quadridentatus (Ruttner-Kolisko, 1969), B. rotundiformis Tschugunoff (Snell & Hawkinson, 1983; Rico-Martínez & Snell, 1995a & b), B. rubens Ehrenberg (Halbach & Halbach-Keup, 1972; Pilarska, 1972), and B. urceolaris Müller (Ruttner-Kolisko, 1969).
The only data about the mating behavior of Brachionus quadridentatus in the literature is from the work of Gómez & Serra (1995) where they used a euryhaline strain of B. quadridentatus to compare mating with several clones of B. plicatilis. In this work they reported a 34.6 percentage of attempted mating for B. quadridentatus homogamic mating. Also, Ruttner-Kolisko (1969) reported the presence of hybrid females between B. quadridentatus and B. urceolaris. However no description of the mating or of the sexual reproductive characteristics of B. quadridentatus were included in these works.
Several aspects of the sexual reproductive biology of Brachionus quadridentatus are described making a detailed description of the male, and the mating behavior. A brief discussion of the mating behavior in the genus Brachionus is also included.
MATERIALS AND METHODS
]]> Rotifer Culture. Brachionus quadridentatus was collected in a pond at the Water Treatment Plant of the Universidad Autónoma de Aguascalientes. The approximate geographical coordinates of this sampling site are 21o 53' 10'' N and 102o 28' 54'' W (geopositioner GPS 4000 XL Satellite Navigator, Magellan Inc., 1997). B. quadridentatus was cultured in EPA medium (192 mg NaHCO3, 120 mg CaSO4.2H2O, 120 mg MgSO4.7H2O, and 8 mg KCl in 2 L; USEPA, 1985) prepared with deionized water (16-18 megaohms) from a Water Pro PS deionizer (Labconco Co.), and fed the green algae Selenastrum capricornutum Printz (strain UTEX No. 1648) grown in Bold's Basal Medium (Nichols, 1973).Mating behavior test. Crossmating assays were done according to Snell and Hawkinson (1983) with some modifications. Briefly, this assay involves placing two neonate females and two neonate virgin male (both < 18 hours old), into 50µl of EPA medium. Then, the number of male mating attempts and completed copulations in five minutes was recorded in each of five replicates using different males and females. A mating attempt was recorded if a male circled around a female maintaining contact with his corona. A copulation was recorded when a male attached his penis to a female. Many complete sequences of the mating behavior of this species were video-taped (n = 16) and recorded: a) duration of copulation, b) sites of mating behavior initiation and c) sites of copulation. Additionally, we obtained photographs of several steps of the mating behavior using a Hitachi Color Video Printer (Hitachi Co.).
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.
Life span data of the female and male. The life-span of the females and males (virgin and non-virgin) was recorded at 25oC. Parthenogenetic eggs were placed in 500 µl of EPA medium in individual wells of a 24-well culture polystyrene plate (Costar Co.) kept in a bioclimatic chamber (Revco Co.) with fluorescent light (600-1100 lux) and a light/darkness cycle of 16:8 hours, and were review each 2 hours before hatching, then newly born females were kept in an individual well with 1 ml of EPA medium with 1X106 cells/ml of Selenastrum capricornutum, placed in the same conditions of the eggs (25oC, 600-1100 luxes, 16:8 of L:D cycle) and observed each 12 hours. Similarly, unfertilized sexual eggs were placed in 300 µl of EPA medium in individual wells and kept under the same conditions of the females, male eggs were review each 2 hours before hatching, then newly born males were observed each 12 hours under the same conditions. The non-virgin male treatment consisted of a set of two newly born females and a male per each individual well. The hatching percentages of 10 sets of 10 sexual eggs were recorded at two different temperatures (20 and 25 ±2oC), under the same conditions of light and L:D cycle. These temperatures were selected because our particular strain grew well under these conditions.
Morphology of males, females and eggs. Morphometric characterization of males and the three types of eggs known in this species: a) male unfertilized sexual egg, b) female parthenogenetic egg, and c) female fertilized sexual egg was performed by direct observations at 40X in a light microscope with a micrometer (Olympus Co., USA). SEM photographs of the female, male and the sexual and parthenogenetic eggs were taken with a JEOL LV 5900 Scanning Electron Microscope at the Universidad Autónoma de Aguascalientes. Samples were prepared for SEM as follows: the animals are placed in a small 1-cm X 1-cm vial with a 54 µm mesh size. Samples were dehydrated by 10 ml gradual changes in alcohol (70, 80, 90, 96º), and then samples were left 24 to 48 hr in absolute alcohol. Later, the vial with the sample was placed in the critical point chamber (Tousimis Co.), to remove any humidity leftover, using liquid CO2. Once all humidity has been removed from the sample, the later is mounted in small metal cylinders stubs where the sample has been previously adhered with tape. Finally the sample is covered with gold and observed at the electron microscope.
RESULTS
Description of male Brachionus quadridentatus
The body consists of three regions: a) a retractile head where the ciliar corona is located (Figure 1B), b) the cylindrical trunk with a 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 (Figure 1A).
Mating behavior
]]> More than 30 complete episodes were observed and video-taped (total observation time was about twenty hours during one month) of mating behavior in Brachionus quadridentatus. Males initiated matings at the corona and foot opening. Attempted copulations lasted 12.4 s on average and copulations lasted 71.4 s on average, this difference is significant (p = 0.038; df = 7). Average 36.9 encounters every five minutes was observed (Table 1). Most copulation occurred at the corona, but almost all attempted copulations started at the foot opening. Curiously from the five replicates of the mating behavior test in this work, all attempted matings (18) occurred at the foot opening and none of them resulted in a completed copulation. The percentage of encounters becoming attempted matings was 7.86 and attempted mating becoming completed copulations was 0% (Table 1)As in all brachionid species, the B. quadridentatus female assumes a passive role during mating while the male circles around the female, the coronal localization step is clear in this species and copulation occurs at the corona and foot opening.
Swimming speed
Males swam faster than females (p = 0.003, n = 10), but the swimming speed of B. quadridentatus males (Table 1) falls in the range reported for other brachionid males (Rico-Martínez & Snell, 1997; Velázquez-Rojas et al., 2002).
Life history data of the female and male
Female rotifers live longer than both virgin and non-virgin male rotifers at 25oC (p<0.001; see Table 1). There was no significant difference between the life-span of virgin and non-virgin males (p = 0.4738, df =19).
Morphology of females, males, and eggs
Males of B. quadridentatus are produced from sexual unfertilized eggs, as male brachionids are produced in general. Figure 2A shows a female carrying a fertilized sexual egg (or cyst). The cyst is longer and wider than the parthenogenetic eggs (p < 0.01, n = 20 in both cases). Figure 2B shows a female carrying three parthenogenetic eggs which are longer and wider than the male-producing eggs (Table 2). Figure 2C shows a female carrying a sexual unfertilized egg. This is the smallest of all eggs ranging from 40-60 mm long and 20-30 µm wide. The figure 2D shows a SEM photograph of a female. Females are longer and wider than males (Table 2; p < 0.001, n = 20, in both cases). Figure 3A shows the sexual unfertilized egg. Figures 3B and D shows the parthenogenetic egg with both light and scanning electron microscopy respectively. Figures 3C and 3D shows the fertilized sexual egg in light and scanning electron microscopy respectively.
DISCUSSION
]]> The mating behavior of B. quadridentatus showed most of the characteristics of that of brachionid rotifers: a) Females are two to three times bigger than males, b) Males are faster swimmers than females, c) Copulation lasts from 25 to 340 seconds, d) The preferred sites of attempted matings and copulations are the corona and foot opening. However, B. quadridentatus showed the lowest percentage of encounters becoming mating attempts (7.86%) recorded so far for a brachionid rotifer attempting to copulate with conspecific females. Gómez & Serra (1995) reported a 34.6% value. However, they used a different technique to record mating behavior (one male and 25 females in 50 µl of Instant Ocean Medium), and they used a euryhaline strain of B. quadridentatus. Keratella americana Carlin with 14% (Rico-Martínez & Snell, 1997) and Platyias quadricornis Ehrenberg with 19.3% (Velázquez-Rojas et al. 2002) are the freshwater brachionid species with the lowest percentage after B. quadridentatus. In contrast percentages as high as 66% have been reported for B. plicatilis (Snell et al., 1995).The secuence of mating behavior for B. quadridentatus is the typical complete sequence of mating behavior in brachionid rotifers. However, the authors caution about the meaning of "typical" since we know the mating behavior in as few as ten species of brachionids. Among rotifers, this family represents the best studied regarding mating behavior.
Regarding duration of copulation, B. quadridentatus represents the Brachionus species with the longer period (71.38 seconds). It is know that Platyias quadricornis is an "atypical" brachionid (regarding mating behavior), with copulation taking place mostly at the juncture of ventral and dorsal plates (as in Lecane), and duration of copulation is over 300 seconds (in all other brachionids this duration is less than 100 seconds). Then again, only Lecane spends more time copulating than Platyias (Rico-Martínez & Snell, 1997). Velázquez-Rojas et al. (2002) argued that the duration of copulation is related to the hardness of the lorica. This observation comes from the fact that both Lecane and Platyias females have a very thick lorica. Perhaps, the lorica of B. quadridentatus females is thicker than that of other Brachionus species. However, we can not rule out other factors like the number of sperm cells or efficiency of other copulatory structures that can influence the duration of copulation.
ACKNOWLEDGEMENTS
To Martha Evelia Pérez-Reyes for help in general laboratory work.
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