Various aquatic organisms have different dormant/ resting stages; as a mechanism of adaptation to existence in unstable aquatic habitats, and surviving adverse conditions(Hairston, 1996; Menu et al., 2000; Brendonck and De Meester, 2003; Radzikowski, 2013). Dormant stages are present in representatives of most groups of crustaceans, including the Anostraca, Cladocera, Copepoda, and Ostracoda(Fryer, 1996; Rossi et al., 2004; Radzikowski, 2013). A substantial pool of dormant stages of planktonic organisms in saline lakes is a frequent component of plankton communities; it needs to be taken into account to underst and plankton dynamics(Marcus, 1984; Brendonck and De Meester, 2003). “Dormant” biodiversity is part of the memory of saline lake ecosystems that contribute a large element of choice, allowing alternative stable state of the ecosystem to be chosen when there are strong salinity changes(Shadrin, 2013). Species diversity in active animals in extreme and strongly changing water bodies is usually less than that in the “dormant” part of a community at any given moment(Brendonck and De Meester, 2003; Shadrin, 2013). The importance of the resting-stage pools in the functioning and dynamics of ecosystems in extreme water bodies is diffi cult to overestimate, but at the same time it has clearly been insuffi ciently investigated.

The Crimea is the largest(nearly 26 500 km ²)peninsula in the Black Sea. There are 50 relatively large lakes and numerous small hypersaline water bodies in the peninsula(Shadrin, 2009, 2013). Two hypersaline lake types are present in the Crimea: of marine origin(thalassohaline), and of continental origin(athalassohaline). The latter include sulfate ones in the calderas of ancient mud volcanoes. Long-term ecological study of these lakes was done, and the main results, including data on the crustacean stages has been published(Shadrin, 2009, 2013; Belmonte et al., 2012; Anufriieva and Shadrin, 2014; Anufriieva et al., 2014). To date, only one study has been performed on “dormant-stage” animal species diversity in sediments of the Crimean hypersaline lakes(Moscatello and Belmonte, 2009). These authors reported dormant eggs of Rotifera and Turbellaria as well as Crustacea— Anostraca(Artemia parthenogenetica Bowen and Sterling, 1978, A. urmiana Günther, 1899 and Phallocryptus spinosa Milne-Edwards, 1840), Cladocera(Moina salina Daday, 1888), and Copepoda(Arctodiaptomus salinus Daday, 1885)in bottom sediments sampled from two lakes in 2004. While there were dormant eggs of P. spinosa in bottom sediments sampled from 10 lakes of the Crimea in 2004–2006 no P. spinosa active stages were detected during that study(Belmonte et al., 2012), although in later years they were observed(Shadrin et al., 2009). This shows that, to be complete, in such lakes the identifi cation of the crustacean species diversity in zooplankton must include a study of its “dormant” component.

The aims of the present study have been to examine the emergence of active crustaceans from resting stages in sediments of dried up sites of hypersaline lakes in the Crimea, to assess Artemia cyst-size diversity, and to discuss the ecological role of dormant eggs, adding more details to preliminary published previously(Anufriieva and Shadrin, 2014).

On February 15, 2013 we collected Artemia cysts in Lake Chersonessus at the surface of the water. The cysts were dried at a temperature of 50–60°C. On February 28, 2013 they were used in the following experiment: cysts were added to two vessels with 0.5 L water(salinity 65–70 g/L, 19°C) and incubated under artifi cial lighting. Vessels were examined daily for the appearance of nauplii. Cysts collected from the water surface of Lake Chersonessus on October 3, 2012, and dried as described above were used in a similar experiment set up on June 3, 2013. On February 6, 2012 and August 30, 2012 similar experiments were performed under the same conditions with the cysts collected from water of the Lake Dzharylhatch in August 2009. Before the experiments cysts were stored in a refrigerator(T =-5°C)in water which developed a strong smell of hydrogen sulfi de during the storage.

Artemia cysts were separated from sediments as described by Moscatello and Belmonte(2009) and then they were hydrated in distilled water subject to air bubbling agitation for 2–3 h, until they were completely spherical. Then a few dozens of cysts were placed into a drop of water on an excavated glass slide, and covered with a thin cover glass slide in order to keep them hydrated and immovable. Cysts were measured under a microscope with a calibrated 100-division eyepiece inserted in the ocular(10×), and working with a 10× objective lens. For each sample 200–400 cysts were measured.

Sediment samples were taken in dried-up parts of 9 lakes, collecting the top 5 cm(Anufriieva and Shadrin, 2014). We used fourteen sediment samples and one sample of precipitated salt in the experiments. One of these samples was taken from an active mud volcano situated on the dried-up site of the bottom of Lake Tobechikskoye. Information on the sampling sites and the experiments is given in Table 1. We placed sediment samples(50–90 g)on the bottom of the vessel and added 100–400 mL distilled water, and the suspension was stirred. In experiments with salt, 5 g of lake salt was added to the experimental vessel. To adjust the salinity of bottom sediments or salt solutions we added smaller or larger amounts of distilled water. For each sample 2 versions of experiments with different salinities were performed. All experimental vessels were placed under artifi cial light at 18–19°C. Almost daily we measured salinity(portable h and -held salinity refractometer Kelilong WZ212), temperature(рН/temperature meter PHH-830), and looked for any presence of living moving organisms. If they presented we measured their length. On average, our experiments lasted for one month. Live specimens were caught and transferred to other glass vessels to grow them to the adult stage.

Table 1 Information on sampling sites and experiments with bottom sediments from the Crimean hypersaline lakes

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In the fi rst experiment with cysts collected on February 2013 in Lake Chersonessus, the first Artemia nauplii appeared on the 4th day of the experiment in both experimental vessels. All nauplii hatched during 2–3 subsequent days. In the second experiment with cysts collected in Lake Chersonessus(October 2012), the fi rst Artemia nauplii appeared on day 5 in both experimental vessels, and on day 13 in both experimental water vessels massive presence of harpacticoid copepodids 0.25–0.4 mm in size was noted.

In two experiments with Artemia cysts, collected in Lake Dzharylhatch(August 2009) and stored for 3 years in water with hydrogen sulfi de, we did not observe an appearance of nauplii after 12 or 22 days.

In all hatching experiments of Artemia(parthenogenetic Artemia, A . urmiana), ostracods(Eucypris mareotica Fischer, 1855), harpacticoids(Cletocamptus retrogressus Schmankevitsch, 1875) and cladocerans(Moina salina)were found. There were only a few cases of hatching of the two last species. These are therefore not considered in later analyses.

Artemia . Emerging of nauplii occurred in 80% of all trials under salinity of 35–100 g/L. In two cases they were A . urmiana, but in others—they were parthenogenetic females. In some cases we failed to grow the crustaceans to adults. Artemia urmiana came from Lake Marfovskoye sediments and from the salt of Lake Koyashskoye.

On average, the time of appearance of the fi rst nauplii from sediment was 14.9 days(CV=0.45). For sediments from different lakes this time varied widely—from 10 to 31 days. In two experiments with sediment taken from the dry bed of Lake Tobechikskoye, near siphons of an active mud volcano near the village Kostyrino, this time was very different from other results(31 days). In this part of the lake we did not observe development of Artemia during of recent years(5–6 years)because salinity was above 340 g/L or this part was dried during most of the time. No animals emerged from sediment samples taken from the active mud volcano located nearby(temperature 25°C). If we exclude the different results of those two experiments, the average time of occurrence of the fi rst nauplii from sediments was 12.4 days(CV=0.13). Nauplii fi rst appeared faster from salt collected on Lake Koyashskoye—on day 6 at a salinity of 100 g/L. At a salinity of 100 g/L the fi rst nauplii(5 individuals)appeared on day 6, 29 more nauplii on day 9, and one nauplius on day 10. The emergence of nauplii from salt was quite synchronous. At 150 g/L salinity monitored for 10 days, no nauplius emerged. After dilution of the salinity in the same sample to 80 g/L, the fi rst nauplii(21 individuals)appeared on day 6, on day 7 we found 15 additional individuals, on day 9, 11 nauplii, on day 11, 3 nauplii, on day 14, 2 nauplii, on day 16, 2 nauplii, on day 18, 2 nauplii, and on day 19, 1 nauplius. Thus, in the fi rst case 35 nauplii emerged throughout the experiment from 5 g of salt, and in the second, 54 nauplii emerged. Averaged over both experiments, 8.9 nauplii emerged per g of salt. The total duration of a period during which nauplii emerged from the sediments of different lakes(fi rst nauplius to the last nauplius)varied from 2–3 days to 14–15 days.

Ostracods. In 6 experiments, we observed that eggs of the ostracod E . mareotica emerged from sediments of Lakes Achi and Aktashskoye at salinities 35–50 g/L. In 4 cases active stages appeared after Artemia, 20–22 days from the beginning of the experiment. Furthermore, in two experiments with sediments of Lake Aktashskoye only, ostracod eggs hatched on day 11 and 16 from the beginning of the experiment. On average the fi rst active ostracods appeared in water after 18.5 days. We observed that active ostracod juveniles emerged from sediments were 0.25–0.30 mm in size.

Data for the parthenogenetic cyst sizes are presented in Table 2. Growth experiments showed that the individuals producing bigger cysts(283 μm)were longer(long form), while those producing smaller cysts were shorter(short form). A database on cyst diameter of parthenogenetic cysts of different ploidy is available(F. Amat, unpublished data): cysts of diploid parthenogenetic strains from 30 populations had as average size of 245(±13)μm(range 210– 250 μm), while tetraploids from 12 populations averaged 278(±11)μm with a range of 264–298 μm. The short form probably belongs to diploid parthenogens, and the long form to tri- or tetraploids.

Table 2 Sizes of the parthenogenetic cysts in some Crimean lakes

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Harpacticoids may be in a resting state at various stages of their development—from egg to adult(Champeau and Francezon, 1991; Dahms, 1995; Vopel et al., 1998). Sudden appearance of harpacticoid copepodids in the absence of adults indicates dried resting eggs of harpacticoids or their nauplii/early copepodids were collected and dried together with Artemia cysts.

Emerged active ostracods were 0.25–0.30 mm in size, and their presence indicates that resting ostracod eggs had been present in the sediment. Adult ostracods sometimes eat Artemia nauplii(our unpublished observations). The question must therefore be asked whether the presence of resting eggs in sediments of both ostracods and Artemia manifests an interaction between these organisms. The presence of Cyclopida species, including alien species, in hypersaline temporally dried-up lakes in the Crimea(Anufriieva et al., 2014)has led us to conclude that cyclopid species also have dormant stages. From our data and information available in literature(Moscatello and Belmonte, 2009), it seems likely that in the hypersaline lakes of Crimea all the Crustacea, except Malacostraca, have resting stages that can persist for long periods after drying of the water bodies. The diversity of crustacean resting stages in bottom sediments is a common feature of all water bodies, which are subject to rapid fl uctuations in salinity and /or desiccation(Hairston, 1996; Moscatello and Belmonte, 2004, 2009; Shadrin, 2013).

In our experiments, no nauplii emerged from cysts stored for 3 years in an environment in the presence of hydrogen sulfi de. Clegg(1997)previously observed, however, that nauplii hatched from cysts stored for four years in an environment without oxygen or hydrogen sulfi de. In our case the presence of hydrogen sulfi de is believed to be the factor that led to the death of the Artemia cysts. There is also another difference between our experiments and Clegg’s; he fi rst dried cysts, stored them, and then put them into water without oxygen; we did not preliminarily dry cysts before putting them in the storage environment. Further research on cyst resistance to anoxic conditions and to hydrogen sulfi de are needed because there are anoxic sediments with hydrogen sulfi de present in many hypersaline lakes.

In earlier experiments(Moscatello and Belmonte, 2009)maximum hatchability of Artemia cysts isolated from the Crimean lake sediments was observed at a salinity of 36, after 3 days, which is close to the results of our experiments with cysts(3–5 days). In our experiments with a salinity of 100 g/L production of nauplii occurred on day 6. No nauplii were produced at 150: too high salinity blocks or slows down hatching of cysts. In the experiments carried out with sediments, the average time of appearance of the fi rst nauplii was 15 days at the salinity range 35–100 g/L. Comparing the results of our experiments with dried brine shrimp cysts and sediments, we conclude that nauplii from cysts were produced much faster than from the sediments. Resting eggs of cladocerans and copepods also demonstrated a similar phenomenon: the total number of active crustaceans that hatched in 36 days was about the same, but nauplii from extracted cysts were produced more quickly and synchronously than from sediments(Vandekerkhove et al., 2004). We conclude that some components in bottom sediments slow down and desynchronize the emergence of active forms from resting eggs in different groups of crustaceans. The effect of this varied in sediments of different lakes; bottom sediments inhibited the output of juveniles from Artemia and ostracod resting eggs to varying degrees. More data are needed before we can discuss the possible reasons and mechanisms of this inhibition. The asynchronous output of active stages from resting ones in the bottom sediments is an adaptation that will allow animals to exist in the extreme and unpredictably changing environment. This would be a bet-hedging(risk-avoiding)strategy, reducing risk that all may hatch under unsuitable conditions(Philippi and Seger, 1989; Evans and Dennehy, 2005). As example, this may prevent emergence of active stages from all animal resting stages after a small single rain event when all water evaporates after a few days or mass development of predators presents.

Resting eggs of different groups of crustaceans are highly resistant to digestive enzymes of birds and other vertebrates, and they can survive in guts and feces of different vertebrates(Proctor and Malone, 1965; Lopez et al., 2002; Radzikowski, 2013; Vandekerkhove et al., 2013). Birds and other vertebrates may carry the crustacean dormant stages over long distances. Hence high tolerance of resting stages may also be regarded as an adaptation to rapid colonization of temporary and new water bodies(Frisch et al., 2007)as in the case of alien cyclopoids in the Crimea(Anufriieva et al., 2014).

Resting eggs of crustaceans can accumulate and survive in lake sediments at least for decades, and sometimes up to hundreds of years(Hairston et al., 1995; Radzikowski, 2013). Upon the occurrence of favorable conditions for development, only a fraction of the resting eggs in bottom sediments is activated, the remainders remain in the bottom sediments in a “sleeping” state(Hairston, 1996). Accumulated over decades to centuries, a pool of crustacean resting stages in bottom sediments is a memory of the crustacean taxocene about past environmental changes. Active A . urmiana was not found in Lake Marfovskoye previously, so this is the fi rst evidence of the presence of this “sleeping” species in the lake. The genetic diversity of a resting stage bank of a single crustacean species is also signifi cantly higher than that of the active part. In the Artemia cyst bank in Lake Koyashskoye there are cysts of both parthenogenetic diploids and bisexual A . urmiana, but we never observed their adults together; their occurrence was separated in time(Abatzopoulos et al., 2009). In some lakes there were Artemia cysts of strains with different ploidy at the same time as we observed only one size group of adults in the plankton. The bank of resting stages is an essential mechanism for the maintenance of species and genetic diversity of crustaceans needed for their existence in an extreme and changeable environment. A hypothesis has been proposed that selective activation of a resting stage bank is possible; there is the existence of alternative metagenome expressions of taxocene and populations in the ecosystem(Shadrin, 2012). This may be one mechanism that provides a multiplicity of alternative taxocene/community states. However, more experimental and fi eld data are required to support this hypothesis.

In hypersaline lakes of the Crimea, Anostraca, Cladocera, Copepoda, and Ostracoda have resting stages that may accumulate and persist for long periods after drying of water bodies. This is a general phenomenon common to hypersaline and temporal water bodies. Accumulated over decades to centuries, a pool of crustacean resting stages in bottom sediments is a memory by the crustacean taxocene of past environmental changes. Resting eggs can also be considered as an adaptation enabling rapid colonization of temporary and new water bodies. Some components in the bottom sediments slow down and desynchronize emerging of active individuals from resting eggs in different groups of crustaceans. The asynchronous output of active forms from resting stages in the bottom sediments is an adaptation that allows crustaceans to exist in an extreme and unpredictably changing environment, avoiding the risk that all may emerge under unsuitable conditions.