Layout 1 INTRODUCTION Analysis of subfossil Cladocera (Crustacea: Bran- chiopoda) is widely used in paleolimnology given its po- tential to reconstruct past environmental conditions (Korhola and Rautio, 2001; Nevalainen and Rautio, 2014; Zawiska et al., 2015). Cladocerans are used as indicators of several abiotic and biotic environmental variables (Rumes et al., 2011; Chen et al., 2014), as they are very sensitive to changes in total phosphorus concentrations (Amsinck et al., 2005; Chen et al., 2010), water depth (Korhola et al., 2005; Nevalainen et al., 2011; Gałka et al., 2014), temperature (Lotter et al., 1997; Korhola, 1999; Mirosław-Grabowska and Zawisza, 2013; Nevalainen et al., 2013; Zawiska et al., 2015), pH (Locke and Sprules, 2000; Zawiska et al., 2013). The crucial step in subfossil Cladocera analysis is the correct taxonomical identification of the remaisns at the species level, which is usually based on the use of light microscope (magnification 100-400x) and several deter- mination keys ( Alonso, 1996; Szeroczyńska and Sarmaja- Korjonen, 2007; Korosi and Smol, 2012). As the light microscopy allows to observe the remains in two dimen- sions, the body sculpture appears to be only a pattern on the surface of the carapace. The microstructural charac- teristics of the chitinous remains can be observed in three- dimensional appearance only by scanning electron micro- scope (SEM), which enables to create images by scanning the surface with a focused beam of electrons (Goldstein et al., 2003). The magnifications obtained with SEM are much greater than those of light microscopy and reach 100,000x. SEM is commonly used in taxonomy of living Clado- cera to describe morphological features such as the limb setae, the lateral pores or the shell denticles (Sinev et al., 2005; Sinev and Elmoor-Loureiro, 2010). Cladocera for SEM observations are usually collected from water by using a plankton net and dried using either a wide range of alcohol percentages (70%, 90%, 95%, 100%) (Duigan, 1992; Nandini et al., 2009), or the strong reagent hexam- ethyldisilazane (Laforsch and Tollrian, 2000; Sousa et al., 2015; Juračka et al., 2016). Saha et al. (2011) recently presented a new simplified procedure, where specimens collected from water samples are washed in distilled water, dried in the room temperature for 30 mins, coated with gold palladium and examined with SEM. SEM images are also frequently used in paleolimnol- ogy to study sediment properties, such as the origin of car- bonates (terrestrial or autogenic), porosity, composition of lamination and microfossil taxa identification (Kemp et al., 2001; Martín-Serrano et al., 2009; Wetzel, 2013; Kirillova et al., 2016). They are also very useful in ob- serving small morphological details of microorganism and Advances in Oceanography and Limnology, 2016; 7(2): 177-183 ARTICLE DOI: 10.4081/aiol.2016.6218 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). Exploring the world of micro sculptures - subfossil Cladocera remains under the SEM Izabela Zawiska,1* Edyta Zawisza,2 Marta Wojewódka,2 Artem Y. Sinev3 1Department of Geoecology and Climatology, Institute of Geography and Spatial Organization, Polish Academy of Sciences, Twarda 51/55, PL-00-818 Warsaw, Poland; 2Institute of Geological Sciences, Polish Academy of Sciences, Twarda 51/55, PL-00-818 Warsaw, Poland; 3Department of Invertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russia *Corresponding author: izawiska@twarda.pan.pl ABSTRACT The scanning electron microscope (SEM) is widely used for the identification of microstructural characteristics and morphology of different microorganisms. Common procedures are based and developed for remains of living species. This paper presents an effective method for drying and preparing subfossil Cladocera remains for SEM observation, which has been recently adapted and tested on several samples originating from different American and European lakes. This method results to be fast and cheap, as it excludes the use of expensive and toxic reagents. Moreover, it allows to recognize the micro sculpture and other species specific characteristics present on the different body parts of the Cladocera remains. The present contribution provides 29 high quality pictures of 12 cladoceran species at magnification between 200x and 11,000x. SEM images reveal that the patterns observed on the shells under the light micro- scope actually are always three dimensional structures. Key words: SEM; subfossil Cladocera; micro sculpture; chitin. Received: August 2016. Accepted: December 2016. No n c om me rci al us e o nly 178 I. Zawiska et al. often help to identify remains such as diatoms (Battarbee, 2001) or testate amoebae (Beyens and Meisterfeld, 2001). Therefore, the methodology for preparing the subfossil remains of these organism for SEM observation is well established (Jiang et al., 2015). On the contrary, Cladocera subfossil remains are still rarely observed under the SEM (Kirillova et al., 2016). In fact, although Cladocera skele- ton is composed of fairly hard chitin, the typical thickness variation depending on the species, body part and lake en- vironment conditions, make the Cladocera preparation for SEM more complicated and time-consuming (Andrade- Morraye et al., 2004). The sediment samples should be firstly prepared according to standard procedure (Frey, 1986), then remains have to be picked up from the sam- ples and washed several time with distilled water. After that remains should be put to osmium tetroxide for 2 h, washed in distilled water again and submitted to the ethanol dehydration sequence (Andrade-Morraye et al., 2004). When the remains are acquired from unconsoli- dated sediments they have to be submitted to dehydration in graded alcohols solutions (Kirillova et al., 2016). In our research we aimed at testing whether the sim- plified method proposed by Saha et al. (2011) for aquatic samples could be applied also to subfossil Cladocera re- mains in order to obtain good quality pictures. METHODS Subfossil Cladocera remains for SEM observation were obtained from sediment samples using two ap- proaches. In the first one fresh sediment from different lakes located in Central and South America was analysed (i.e. from Lake Comendador, Lake Chicabal, Lake Quexil (Guatemala), Lake Emiliano Zapata (Yucatan Peninsula, Mexico), Lake Los Negritos, Lake Verde (Salvador), Lake Madre Vieja, (Honduras), Lake San Martin, (Argentina). Remains were picked directly from the unconsolidated upper first cm of surface sediments (1 cm3), diluted with distilled water and put into a petri dish. Cladocera remains were pick out using a pipette (in the drop of water) under the dissecting microscope and directly put on the SEM microscope stubs covered with a carbon adhesive tape. In the second approach the sediment samples for SEM observation were obtained from European Lakes, i.e. Atnsjøen (Norway), Czechowskie (Poland) and Suchar IV (Poland). Samples were taken from sediment cores and chemically prepared for subfossil Cladocera analysis ac- cording to standard procedures (Szeroczyńska and Sar- maja-Korjonen, 2007). The amount 1 cm3 of fresh sediment was treated with hot 10% KOH for 20 min using a magnetic stirrer in order to deflocculate the material and remove humic substances. Thereafter the carbonates were removed using 10% HCl. The remaining material was sieved through 33 µm mesh and diluted in 10 cm3 distilled water. The subfossil remains were removed consecutively with a pipette from the cleaned sediment and directly put on the SEM microscope stubs covered with a carbon ad- hesive tape. The remains obtained from both superficial fresh sed- iments and cleaned core material were left to dry at the room temperature for 48 hs. When dried they were put into the sputter coater SC7620 for 120 s and coated with a gold-palladium. The sample coating with an electric conducting material is necessary in order to avoid the ac- cumulation of electrostatic charge at the sample surface (Sinev et al., 2005; Sinev and Elmoor-Loureiro, 2010). After the specimens were coated they were put into the scanning electron microscope (Jeol JSM-6610LV) cham- ber and observed in high vacuum, using SEI mode, volt- age 20 kV. RESULTS Figures 1-8 show 29 good quality images of subfossil Cladocera remains belonging to 12 Cladocera species. The pictures were taken at magnification ranging from 200x to 11,000x. The remains from both fresh sediments and from the sediment cores have different state of preser- vation, independently from the age of the sample. The re- mains of Chydorus spp. (Leach) preserved well and therefore easier to be photographed, compared to the other observed Cladocera species (Figs. 1 and 2). The SEM pic- tures allowed to observe magnificent sculpture of Cerio- daphnia spp. (Dana) and Simpocephallus ephippia (Schoedler) (Figs. 3 and 4). The delicate structure of Alonella excisa (Fischer) shell and Leydigiopsis ornata (Daday) (Fig. 5), the deep carvings of Graptoleberis tes- tudinaria (Fischer) and Monospilus dispar (Sars) (Fig. 6), as well as characteristic triangle on the Paralona pigra (Sars) shell are well documented (Fig. 1). The SEM pic- tures revealed the three-dimensional aspect of the head pores of Alona ossiani (Sinev), Bosmina (E.) coregoni (Baird) and Bosmina (E.) longispina (Leydig) (Fig. 7). On the contrary, the specimens form lake Suchar IV showed high level of degradation, and diminished sculpture of the remains (Fig. 8). This might be possibly due to the fact that these sediment samples were prepared for subfossil Cladocera analysis already five years ago. DISCUSSION The simplified procedure of preparing specimens for SEM observation proposed by Saha et al. (2011) was tested on different remains of Cladocera species from sev- eral American and European lakes. This procedure is based on the concept developed for the remains of living species (Sinev et al., 2005; Van Damme and Dumont, No n c om me rci al us e o nly Exploring the world of micro sculptures - subfossil Cladocera remains under the SEM 179 Fig. 1. SEM images of Cladocera remains. A) Chydorus sphericus shell, magnification 270x (Lake Comendador, Guatemala). B) Chy- dorus sphericus shell sculpture, magnification 4000x (Lake Comendador, Guatemala). C) Paralona pigra shell, magnification 330x (Lake San Martin, Argentina). D) Paralona pigra shell, triangular anterior accessory flange on the anteriror-ventral margin, magnification 950x (Lake San Martin, Argentina). E) Chydorus spp., magnification 500x (Lake Comendador, Guatemala). F) Chydorus spp., magni- fication 270x (Lake Comendador, Guatemala). Fig. 2. SEM images of Cladocera remains of Ceriodaphnia spp. ephippium (Lake Emiliano Zapata, Yucatan Peninsula, Mexico). Mag- nification: A) 160x; B) 650x; C) 900x; D) 4000x. No n c om me rci al us e o nly 180 I. Zawiska et al. 2007; Sinev and Elmoor-Loureiro, 2010; Kotov, 2013; Sousa et al., 2015). The time for drying the remains sug- gested by Saha et al. (2011) was not long enough in the case of fragmented parts of the cladoceran body found in the studied samples. Since most of them were very small and difficult to pick out from the sediment sample with a needle, they were put on the stage with a pipette, in a fairly large drop of water. Therefore the prolongation of the drying time was necessary. From all types of examined Cladocera remains, ephip- pia showed the best reservation of structure and ornamen- tation, as their chitinous envelope is thick and less prone for mechanic destruction. It was also noted that not all subfossil Cladocera remains were suitable for SEM ob- servation, as some were so thin that the specimens were barely visible. In addition, it was recognized that the sub- fossil material for SEM observation should be pick out from the freshly prepared sediment sample, as the remains slowly degrade and the chitin structure become less prominent after sediment preparation (Fig. 8). The SEM images clearly showed that the patterns on the shells ob- served under the light microscope always correspond to three dimensional structures. CONCLUSIONS The simplified method of preparing subfossil Clado- cera was applied on different samples from several lakes. The presented method resulted to be simple, cheap and allowed to create high quality images of all types of re- Fig. 3. SEM images of Cladocera remains of Simpocephalus spp. ephippium (Lake Chicabal, Guatemala). Magnification: A) 150x; B) 950x; C) 3500x. Fig. 4. SEM images of Cladocera remains. A) Alonella excisa shell, magnification 400x (Lake Quexil, Guatemala). B) Alonella excisa shell sculpture, magnification 1300x (Lake Quexil, Guatemala). C) Leidigiopsis ornata head, magnification 230x (Lake Los Negritos, Salvador). D) Leidigiopsis ornata head sculpture, magnification 1600x (Lake Los Negritos, Salvador). No n c om me rci al us e o nly Exploring the world of micro sculptures - subfossil Cladocera remains under the SEM 181 mains even in fairly high magnifications up to 11,000x. Although the procedure developed for the remains of liv- ing species revealed to be effective also for subfossil Cladocera, it appeared necessary to prolong the drying time when working with subfossil remains. Moreover, the samples should be prepared just before the SEM analysis, in order to prevent the degradation of the micro sculpture after the cleaning procedure. Fig. 5. SEM images of Cladocera remains. A) Graptoleberis tes- tudinaria shell, magnification 300x (Lake Verde, Salvador). B) Graptoleberis testudinaria head, magnification 370x (Lake Verde, Salvador). C) Monospilus dispar head, magnification 400x (Czechowskie Lake, Poland). Fig. 6. SEM images of Cladocera remains of Alona ossiani head (Lake Madre Vieja, Honduras). Magnification: A) 190x; B) 1600x; C,D) 11,000x. Fig. 7. SEM images of Cladocera remains from Lake Atnsjøen, Norway. A) Bosmina E.coregoni head, magnification 500x. B) Bosmina E.coregoni head pore, magnification 3300x. C) Bosmina E. longispina head, magnification 800x. Fig. 8. SEM images of Cladocera decaying remains from sedi- ment from Lake Suchar IV (Poland) prepared for subfossil Cla- docera analysis 4 years ago. Magnification: A) 500x; B) 330 x. No n c om me rci al us e o nly 182 I. Zawiska et al. ACKNOWLEDGMENTS The research was founded by Polish National Sci- ence Centre, grant NCN 2014/13/B/ST10/02534 and by the EEA and Norway Grants (Grant no. 459 FSS/2013/IIC/W/0022). 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