Turkish Journal of Zoology Turk J Zool (2014) 38: 131-135 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1302-47

Morphological examination of the resting egg structure of 3 cladoceran species [Ceriodaphnia quadrangula (O. F. Müller, 1785), longispina (O. F. Müller, 1776), and D. magna Straus, 1820]

1,2, 2,3 Murat KAYA *, Sevil ERDOĞAN 1 Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey 2 Science and Technology Application and Research Center, Aksaray University, Aksaray, Turkey 3 Fisheries Program, Keşan Vocational College, Trakya University, Keşan, Edirne, Turkey

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

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

1. Introduction significance of the resting egg (Brendonck and De Meester, Cladoceran species reproduce asexually with thin-shelled 2003), the importance of the resting egg in terms of summer eggs under normal circumstances (Davison, continuity of zooplankton populations (Jankowski and 1969; Boersma et al., 2000). In unfavorable environmental Straile, 2003; Panarelli et al., 2008; Conde-Porcuna et al., conditions (e.g., low water temperature, predation 2011), the buoyancy of the ephippium (Slusarczyk and pressure, dry periods), males appear, and thick-shelled, Pietrzak, 2008), and the hatching of resting eggs (Rother resistant, fertilized eggs (called ‘resting eggs’) are produced et al., 2010; Haghparast et al., 2012). (Slusarczyk, 2001; Panarelli et al., 2008; Slusarczyk and In this study, we comparatively analyzed size, shape, Pietrzak, 2008). When conditions return to normal, these and color of resting eggs and the eggs enclosed in the resting eggs hatch and create new offspring; thus, these ephippia of 3 different cladoceran species [Ceriodaphnia organisms ensure the continuity of generations by means quadrangula (O. F. Müller, 1785), (O. of these eggs (Panarelli et al., 2008; Vanickova et al., 2010). F. Müller, 1776), and D. magna Straus, 1820] to understand It has been observed in previous studies that the resting if there were significant morphological differences among egg can survive for many decades without decomposing the resting eggs of these 3 cladoceran species. (Caceres, 1998; Rother et al., 2010). Until now, studies about resting eggs have generally 2. Materials and methods focused on subjects such as the genetic characterization Following the winter egg-production time of cladoceran of resting eggs (Reid et al., 2000), maternal effects on the species in Mamasın Dam Lake, large amounts of floating size of resting eggs (Boersma et al., 2000), resistance to resting eggs were observed on the water surface in predation (Slusarczyk, 2001), ecological and evolutionary November 2012. Resting eggs were collected using a * Correspondence: [email protected] 131 KAYA and ERDOĞAN / Turk J Zool plankton net with a mesh size of 100 µm from the surface The ephippium of D. magna is in the shape of a straight of the water and were left to dry at room temperature in letter D. Egg chambers containing 2 eggs are located more the laboratory. Dry resting eggs were examined under or less horizontally at an angle to the spinose dorsal margin the microscope, and 3 different types of resting eggs were of the ephippium. The ephippium is long and has anterior determined. These 3 different types of resting eggs were and posterior projections (Figures 1C and 1F). cultured under laboratory conditions (at a temperature of 28 °C and under a 514-W fluorescent light), and the species 3.2. Resting egg color hatching from the resting eggs were identified according Stereomicroscope analysis indicated that the background to Benzie (2005). After that, the resting eggs were sifted color of the ephippium of C. quadrangula is whitish cream through sieves with mesh sizes of 200 µm and 400 µm, and the margins are transparent. The egg is orange-brown and the resting eggs of the different species were separated (Figure 1D). from each other by size differences. One hundred resting The ephippium ofD. longispina is covered with a eggs from each species were randomly picked, and the transparent outer cover; the background color of the length and width of each ephippium and eggs enclosed ephippium ranges from white to light brown, and egg in the ephippium were measured under a Leica DM 4000 chambers are deep brown (Figure 1E). binocular microscope. In addition, photos of resting eggs The background color of the ephippium of D. magna were taken with a Leica DFC 295 camera attached to a Leica Z6 APO zoom system at a magnification of 32–36×, and appears cream under a stereomicroscope; the dorsal the surface morphology of the resting eggs was examined margin is deep brown or gray, and egg chambers are brown with an EVO LS 10 ZEISS scanning electron microscope (Figure 1F). at magnifications of 70–200–300×. The resting eggs were 3.3. Resting egg size coated with gold for scanning electron microscopy (SEM) As a result of measurements, it was determined that analysis using a sputter coater (Cressington 108 Auto). ephippium sizes vary largely according to species. We applied the normality test to check whether the Ephippia of D. magna are approximately 3 times data followed normal distribution or not. We saw that larger than ephippia of D. longispina and 4 times larger the data did not follow normal distribution; therefore, we used a nonparametric test. We used the Mann–Whitney than ephippia of C. quadrangula. The size difference U test to understand if there were statistically significant between ephippia of D. longispina and C. quadrangula is differences among species in terms of sizes of ephippia approximately 100 µm (Table 1). and eggs enclosed in the ephippia. Analysis of data was The Mann–Whitney U test indicated that there are performed using SPSS 16. statistically significant differences between D. magna and D. longispina, D. magna and C. quadrangula, and D. 3. Results longispina and C. quadrangula in terms of ephippium Identification of the individuals hatched from cultivated length and width according to the minimum P < 0.05. resting eggs showed that these resting eggs belonged to 3 different cladoceran species: Ceriodaphnia quadrangula, 3.3.1. Sizes of eggs enclosed in ephippium Daphnia longispina, and D. magna. It was observed that eggs enclosed in the ephippia of D. 3.1. Resting egg shape magna are much larger than those of the other 2 species. It was observed that the resting eggs of the 3 cladoceran Although eggs enclosed in the ephippia of D. longispina species have different morphological characteristics are slightly larger than the eggs of C. quadrangula, there is (Figures 1A–1F). almost no difference between their sizes (Table 2). The ephippium of C. quadrangula is semicircular and Considering the results of the Mann–Whitney test, symmetrical, and it carries floating cells. An egg chamber it was revealed that there are statistically significant containing only 1 egg is located parallel and close to the differences betweenD. magna and D. longispina, D. magna dorsal margin of the ephippium. The dorsal margin does and C. quadrangula, and D. longispina and C. quadrangula not have anterior or posterior projections (Figures 1A and in terms of width of eggs enclosed in ephippia according 1D). to the minimum P < 0.05. When we considered the length The ephippium of D. longispina is in the shape of the letter D, narrowing at the posteroventral side. Egg of eggs enclosed in ephippia, we observed that although chambers carrying 2 eggs are located perpendicularly there are statistically significant differences between D. to the spinose dorsal margin of the ephippium, and the magna and D. longispina and between D. magna and C. ephippium does not have anterior or posterior projections quadrangula, there is no significant difference between D. (Figures 1B and 1E). longispina and C. quadrangula.

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Figure 1. Resting egg photos of 3 species (, 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. Discussion in this literature is brief and not detailed. In addition, there Many studies have revealed that the resting eggs of are only 1 or 2 sentences about the resting eggs of Daphnia cladoceran species might be used for species identification magna and D. longispina in the guide book by Benzie (Brendonck and De Meester, 2003; Vandekerkhove et al., (2005). This study describes the resting eggs of 3 of about 2004). However, there are only 2 recently published studies 800 cladoceran species (Kotov et al., 2013) in detail and describing the resting eggs of (Vandekerkhove reveals that there are significant morphological differences et al., 2004; Benzie, 2005), and the information presented among the resting eggs of cladoceran species.

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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

This study also shows that the shapes of the ephippia As mentioned above, the colors of ephippia and of the 3 different species are different from each other. the eggs enclosed in ephippia of these 3 species differ While the ephippium of C. quadrangula is semicircular, the from each other. These differences may help to identify ephippium of D. longispina narrows at the posteroventral cladoceran species through resting eggs. side. D. magna has an ephippium in the shape of a straight Benzie (2005) indicated that the ephippium of D. letter D. Although C. quadrangula has 1 egg, which is magna is 1556 µm in length and 815 µm in width, and located parallel to the dorsal margin of the ephippium, the the ephippium of D. longispina is 794 µm in length and other 2 species carry 2 eggs perpendicular and horizontal to 550 µm in width. While the ephippium size of D. magna the dorsal margin. In addition, D. magna differs by having in that study was similar to our findings (1237 ± 119 µm, anterior and posterior projections, unlike the other 2 species. mean ± SD), the ephippium size of D. longispina was Vandekerkhove et al. (2004) stated that the morphology of greater than our values (251 ± 22 µm). This is probably the ephippium is predominantly determined by the shape of because the measurements specified in the book belong the active animal. Our findings also indicate that resting egg to only 1 individual of each species. Jankowski and Straile shapes differ according to species. (2003) emphasized that ephippium size decreases due to Stereomicroscope analysis indicated that the predation pressure. Boersma et al. (2000) also stated that background colors of ephippia of these 3 species also differ larger and older females produce larger ephippia because from each other. The resting egg colors of C. quadrangula ephippial eggs hatch in spring when the abundance of size- (whitish-cream ephippium background and orange- selective predators is low. brown egg) are completely different from those of the Considering the results of our study, it is seen that other 2 species. The ephippium of D. longispina is covered different cladoceran species produce resting eggs of with a transparent outer cover. While the background different sizes. The Mann–Whitney U test indicated that color of the ephippium of D. longispina ranges from white there are statistically significant differences between D. to light brown, the background color of the ephippium of magna and D. longispina, D. magna and C. quadrangula, D. magna is cream. Egg chambers of the resting egg of D. and D. longispina and C. quadrangula in terms of longispina are also a darker brown than those of D. magna. ephippium length and width according to the minimum P

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< 0.05. The Mann–Whitney U test also revealed statistically studies (Vandekerkhove et al., 2004; Benzie, 2005). significant differences between D. magna and D. longispina, However, the information provided was brief and not D. magna and C. quadrangula, and D. longispina and C. detailed. This study supplies detailed information about quadrangula in terms of width and length of eggs enclosed these features and statistical analysis that indicates that in ephippia according to the minimum P < 0.05. For that the sizes of ephippia of these 3 species are statistically reason, we suggest that size differences might be used for different from each other. In addition, the colors of identification of cladoceran species. ephippia and colors of eggs enclosed in the ephippia of Because there has been no previously conducted study these 3 species were described in detail for the first time relevant to the size of eggs enclosed in the ephippia of in this study, and information about the size of the eggs these 3 species, data obtained from this study will shed enclosed in the ephippia was also revealed for the first light on future works. time. Results of this study indicated that these features Consequently, in this study, it was revealed that the differ according to species, and thus they can be used to ephippia and the egg/eggs enclosed in the ephippia of D. distinguish these cladoceran species. magna, D. longispina, and C. quadrangula have different characteristics in terms of shape, color, and size. Some Acknowledgments information about shape and size of the ephippia of these We thank Dr Fatih Duman (Erciyes University, Biology 3 species has been reported in previous taxonomical Department) for statistical analysis.

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