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http://journals.tubitak.gov.tr/zoology/

Turkish Journal of Zoology Turk J Zool(2014) 38: 131-135© TÜBİTAKdoi:10.3906/zoo-1302-47

Morphological examination of the resting egg structure of 3 cladoceranspecies [Ceriodaphnia quadrangula (O. F. Müller, 1785), Daphnia longispina

(O. F. Müller, 1776), and D. magna Straus, 1820]

Murat KAYA1,2,*, Sevil ERDOĞAN2,3

1Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey2Science and Technology Application and Research Center, Aksaray University, Aksaray, Turkey

3Fisheries Program, Keşan Vocational College, Trakya University, Keşan, Edirne, Turkey

* Correspondence: muratkaya3806@yahoo.com

1. IntroductionCladoceran species reproduce asexually with thin-shelled summer eggs under normal circumstances (Davison, 1969; Boersma et al., 2000). In unfavorable environmental conditions (e.g., low water temperature, predation pressure, dry periods), males appear, and thick-shelled, resistant, fertilized eggs (called ‘resting eggs’) are produced (Slusarczyk, 2001; Panarelli et al., 2008; Slusarczyk and Pietrzak, 2008). When conditions return to normal, these resting eggs hatch and create new offspring; thus, these organisms ensure the continuity of generations by means of these eggs (Panarelli et al., 2008; Vanickova et al., 2010). It has been observed in previous studies that the resting egg can survive for many decades without decomposing (Caceres, 1998; Rother et al., 2010).

Until now, studies about resting eggs have generally focused on subjects such as the genetic characterization of resting eggs (Reid et al., 2000), maternal effects on the size of resting eggs (Boersma et al., 2000), resistance to predation (Slusarczyk, 2001), ecological and evolutionary

significance of the resting egg (Brendonck and De Meester, 2003), the importance of the resting egg in terms of continuity of zooplankton populations (Jankowski and Straile, 2003; Panarelli et al., 2008; Conde-Porcuna et al., 2011), the buoyancy of the ephippium (Slusarczyk and Pietrzak, 2008), and the hatching of resting eggs (Rother et al., 2010; Haghparast et al., 2012).

In this study, we comparatively analyzed size, shape, and color of resting eggs and the eggs enclosed in the ephippia of 3 different cladoceran species [Ceriodaphnia quadrangula (O. F. Müller, 1785), Daphnia longispina (O. F. Müller, 1776), and D. magna Straus, 1820] to understand if there were significant morphological differences among the resting eggs of these 3 cladoceran species.

2. Materials and methodsFollowing the winter egg-production time of cladoceran species in Mamasın Dam Lake, large amounts of floating resting eggs were observed on the water surface in November 2012. Resting eggs were collected using a

Abstract: Morphological characteristics of the resting egg, which is important to ensure the continuity of generations, were examined to understand if there were morphologically significant differences among 3 cladoceran species. Resting eggs floating on the water surface were collected by a plankton net with a mesh size of 100 µm from Mamasın Dam Lake in November 2012 and were left to dry at room temperature in the laboratory. In this study, the size, shape, and color of the ephippium and the size of the egg/eggs enclosed in the ephippium were comparatively analyzed by stereomicroscope and scanning electron microscope. It was observed that each resting egg type had its own characteristic shape and color. Results of the statistical analysis showed that there were statistically significant differences between the sizes of the ephippium and egg/eggs enclosed in the ephippium of the resting egg of each species. We concluded that the morphological features of the resting eggs of 3 species are different from each other and that these characters can be used for species identification. In addition, we described morphological features of the resting eggs of the 3 cladoceran species in detail, gave extra information about color and size differences, and contributed to improving the literature knowledge of these species with this study.

Key words: Scanning electron microscopy, resting egg, size, shape, color

Received: 27.02.2013 Accepted: 14.10.2013 Published Online: 17.01.2014 Printed: 14.02.2014

Research Article

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plankton net with a mesh size of 100 µm from the surface of the water and were left to dry at room temperature in the laboratory. Dry resting eggs were examined under the microscope, and 3 different types of resting eggs were determined. These 3 different types of resting eggs were cultured under laboratory conditions (at a temperature of 28 °C and under a 514-W fluorescent light), and the species hatching from the resting eggs were identified according to Benzie (2005). After that, the resting eggs were sifted through sieves with mesh sizes of 200 µm and 400 µm, and the resting eggs of the different species were separated from each other by size differences. One hundred resting eggs from each species were randomly picked, and the length and width of each ephippium and eggs enclosed in the ephippium were measured under a Leica DM 4000 binocular microscope. In addition, photos of resting eggs were taken with a Leica DFC 295 camera attached to a Leica Z6 APO zoom system at a magnification of 32–36×, and the surface morphology of the resting eggs was examined with an EVO LS 10 ZEISS scanning electron microscope at magnifications of 70–200–300×. The resting eggs were coated with gold for scanning electron microscopy (SEM) analysis using a sputter coater (Cressington 108 Auto).

We applied the normality test to check whether the data followed normal distribution or not. We saw that the data did not follow normal distribution; therefore, we used a nonparametric test. We used the Mann–Whitney U test to understand if there were statistically significant differences among species in terms of sizes of ephippia and eggs enclosed in the ephippia. Analysis of data was performed using SPSS 16.

3. Results Identification of the individuals hatched from cultivated resting eggs showed that these resting eggs belonged to 3 different cladoceran species: Ceriodaphnia quadrangula, Daphnia longispina, and D. magna. 3.1. Resting egg shape It was observed that the resting eggs of the 3 cladoceran species have different morphological characteristics (Figures 1A–1F).

The ephippium of C. quadrangula is semicircular and symmetrical, and it carries floating cells. An egg chamber containing only 1 egg is located parallel and close to the dorsal margin of the ephippium. The dorsal margin does not have anterior or posterior projections (Figures 1A and 1D).

The ephippium of D. longispina is in the shape of the letter D, narrowing at the posteroventral side. Egg chambers carrying 2 eggs are located perpendicularly to the spinose dorsal margin of the ephippium, and the ephippium does not have anterior or posterior projections (Figures 1B and 1E).

The ephippium of D. magna is in the shape of a straight letter D. Egg chambers containing 2 eggs are located more or less horizontally at an angle to the spinose dorsal margin of the ephippium. The ephippium is long and has anterior and posterior projections (Figures 1C and 1F). 3.2. Resting egg color Stereomicroscope analysis indicated that the background color of the ephippium of C. quadrangula is whitish cream and the margins are transparent. The egg is orange-brown (Figure 1D).

The ephippium of D. longispina is covered with a transparent outer cover; the background color of the ephippium ranges from white to light brown, and egg chambers are deep brown (Figure 1E).

The background color of the ephippium of D. magna appears cream under a stereomicroscope; the dorsal margin is deep brown or gray, and egg chambers are brown (Figure 1F).3.3. Resting egg size As a result of measurements, it was determined that ephippium sizes vary largely according to species.

Ephippia of D. magna are approximately 3 times larger than ephippia of D. longispina and 4 times larger than ephippia of C. quadrangula. The size difference between ephippia of D. longispina and C. quadrangula is approximately 100 µm (Table 1).

The Mann–Whitney U test indicated that there are statistically significant differences between D. magna and D. longispina, D. magna and C. quadrangula, and D. longispina and C. quadrangula in terms of ephippium length and width according to the minimum P < 0.05.3.3.1. Sizes of eggs enclosed in ephippium It was observed that eggs enclosed in the ephippia of D. magna are much larger than those of the other 2 species. Although eggs enclosed in the ephippia of D. longispina are slightly larger than the eggs of C. quadrangula, there is almost no difference between their sizes (Table 2).

Considering the results of the Mann–Whitney test, it was revealed that there are statistically significant differences between D. magna and D. longispina, D. magna and C. quadrangula, and D. longispina and C. quadrangula in terms of width of eggs enclosed in ephippia according to the minimum P < 0.05. When we considered the length of eggs enclosed in ephippia, we observed that although there are statistically significant differences between D. magna and D. longispina and between D. magna and C. quadrangula, there is no significant difference between D. longispina and C. quadrangula.

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Figure 1. Resting egg photos of 3 species (Daphnia magna, D. longispina, Ceriodaphnia quadrangula). A) SEM photo of C. quadrangula, B) SEM photo of D. longispina, C) SEM photo of D. magna, D) light microscope photo of C. quadrangula, E) light microscope photo of D. longispina, F) light microscope photo of D. magna.

4. DiscussionMany studies have revealed that the resting eggs of cladoceran species might be used for species identification (Brendonck and De Meester, 2003; Vandekerkhove et al., 2004). However, there are only 2 recently published studies describing the resting eggs of Cladocera (Vandekerkhove et al., 2004; Benzie, 2005), and the information presented

in this literature is brief and not detailed. In addition, there are only 1 or 2 sentences about the resting eggs of Daphnia magna and D. longispina in the guide book by Benzie (2005). This study describes the resting eggs of 3 of about 800 cladoceran species (Kotov et al., 2013) in detail and reveals that there are significant morphological differences among the resting eggs of cladoceran species.

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This study also shows that the shapes of the ephippia of the 3 different species are different from each other. While the ephippium of C. quadrangula is semicircular, the ephippium of D. longispina narrows at the posteroventral side. D. magna has an ephippium in the shape of a straight letter D. Although C. quadrangula has 1 egg, which is located parallel to the dorsal margin of the ephippium, the other 2 species carry 2 eggs perpendicular and horizontal to the dorsal margin. In addition, D. magna differs by having anterior and posterior projections, unlike the other 2 species. Vandekerkhove et al. (2004) stated that the morphology of the ephippium is predominantly determined by the shape of the active animal. Our findings also indicate that resting egg shapes differ according to species.

Stereomicroscope analysis indicated that the background colors of ephippia of these 3 species also differ from each other. The resting egg colors of C. quadrangula (whitish-cream ephippium background and orange-brown egg) are completely different from those of the other 2 species. The ephippium of D. longispina is covered with a transparent outer cover. While the background color of the ephippium of D. longispina ranges from white to light brown, the background color of the ephippium of D. magna is cream. Egg chambers of the resting egg of D. longispina are also a darker brown than those of D. magna.

As mentioned above, the colors of ephippia and the eggs enclosed in ephippia of these 3 species differ from each other. These differences may help to identify cladoceran species through resting eggs.

Benzie (2005) indicated that the ephippium of D. magna is 1556 µm in length and 815 µm in width, and the ephippium of D. longispina is 794 µm in length and 550 µm in width. While the ephippium size of D. magna in that study was similar to our findings (1237 ± 119 µm, mean ± SD), the ephippium size of D. longispina was greater than our values (251 ± 22 µm). This is probably because the measurements specified in the book belong to only 1 individual of each species. Jankowski and Straile (2003) emphasized that ephippium size decreases due to predation pressure. Boersma et al. (2000) also stated that larger and older females produce larger ephippia because ephippial eggs hatch in spring when the abundance of size-selective predators is low.

Considering the results of our study, it is seen that different cladoceran species produce resting eggs of different sizes. The Mann–Whitney U test indicated that there are statistically significant differences between D. magna and D. longispina, D. magna and C. quadrangula, and D. longispina and C. quadrangula in terms of ephippium length and width according to the minimum P

Table 1. Resting eggs sizes of 3 species (Daphnia magna, D. longispina, Ceriodaphnia quadrangula).

Resting eggs Min Max Mean SD

Daphnia magna Length (µm) 1013 1526 1237 119

Width (µm) 479 817 625 74

Daphnia longispina Length (µm) 310 539 405 45

Width (µm) 260 410 330 29

Ceriodaphnia quadrangula Length (µm) 267 381 317 23

Width (µm) 184 276 222 17

Table 2. Size of eggs enclosed in ephippia of 3 species (Daphnia magna, D. longispina, Ceriodaphnia quadrangula).

Eggs enclosed in ephippia Min Max Mean SD

Daphnia magna Length (µm) 390 550 472 40

Width (µm) 247 375 315 30

Daphnia longispina Length (µm) 206 294 251 22

Width (µm) 98 151 135 11

Ceriodaphnia quadrangula Length (µm) 200 287 245 20

Width (µm) 77 156 121 13

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< 0.05. The Mann–Whitney U test also revealed statistically significant differences between D. magna and D. longispina, D. magna and C. quadrangula, and D. longispina and C. quadrangula in terms of width and length of eggs enclosed in ephippia according to the minimum P < 0.05. For that reason, we suggest that size differences might be used for identification of cladoceran species.

Because there has been no previously conducted study relevant to the size of eggs enclosed in the ephippia of these 3 species, data obtained from this study will shed light on future works.

Consequently, in this study, it was revealed that the ephippia and the egg/eggs enclosed in the ephippia of D. magna, D. longispina, and C. quadrangula have different characteristics in terms of shape, color, and size. Some information about shape and size of the ephippia of these 3 species has been reported in previous taxonomical

studies (Vandekerkhove et al., 2004; Benzie, 2005). However, the information provided was brief and not detailed. This study supplies detailed information about these features and statistical analysis that indicates that the sizes of ephippia of these 3 species are statistically different from each other. In addition, the colors of ephippia and colors of eggs enclosed in the ephippia of these 3 species were described in detail for the first time in this study, and information about the size of the eggs enclosed in the ephippia was also revealed for the first time. Results of this study indicated that these features differ according to species, and thus they can be used to distinguish these cladoceran species.

AcknowledgmentsWe thank Dr Fatih Duman (Erciyes University, Biology Department) for statistical analysis.

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