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The study of cyst hatching, performed on 5 cyst types under lab conditions, …


Biology Articles » Hydrobiology » Freshwater Biology » Egg banks in hypersaline lakes of the South-East Europe » Methods

Methods
- Egg banks in hypersaline lakes of the South-East Europe

Study site

The research interested, from April 2004 to September 2006, Khersonesskoe (44°35'12"N; 33°24'00"E) and Koyashskoe (45°02'31"N; 36°12'20"E) lakes in the Crimea peninsula (Ukraine), Nartë (40°32'13"N; 19°25'48"E) saltworks (Southern Albania), Vecchia Salina (40°18'06"N; 17°43'56"E) at Torre Colimena (Gulf of Taranto, Apulia, Italy), Pantano Grande and Pantano Roveto (36°48'29"N; 15°06'02"E) at Vendicari Nature Reserve (Sicily, Italy) (Figure 5).

Collections of resting stages from the sediments of the 2 contiguous lakes at Vendicari (Sicily, Italy) allowed to compare results coming from nearby habitats with those coming from very distant ones (e.g. Italian and Crimean lakes).

Sampling procedures

Samplings were collected during summer months corresponding to the minimum water level, or even its absence to take only samples not submerged. Three replicate sediment cores (diameter, 7.5 cm; depth, 6 cm) were obtained from each lake by using a core sampler (20 cm length) and stored at 4°C in a refrigerator for 1 year. For the analysis, each sediment core was cut into 3 cm thick layers. Each layer was ultrasonified to break the larger particles of sediment and then sieved at two mesh sizes (212 and 45 μm). The sediment collected by both sieves was centrifuged at 1,090 g in a 1:1 sucrose-distilled water solution for 3 min. The supernatant derived from the centrifugation of the two sieve fractions was analysed to separate cysts.

Cysts were reported as number per 100 cc of sediment. The most abundant resting stages (2 types for the fraction >212 μm; 3 types for the fraction >45 μm) were used in hatch experiments in the laboratory. Sets of 30 cysts of each morphotypes, taken from each layer (from the superficial to the deepest one), were stored in 3 cc wells raised with 2 cc of original water filtered at 0,45 μm. To avoid bacterial growth, in each well, 20 μl of an antibiotic mix (streptomycin/penicillin 1:1) was added. Resting stages were submitted to different storage conditions in thermostatic rooms (an "equinox" simulation, with 13°C and 12 hL:12 hD photoperiod and a "early summer" simulation, with 24°C and 14 hL:10 hD photoperiod) at 4 different salinity values (46‰, 36‰, 26‰, freshwater) obtained by diluting the original-site water. Hatching plates were checked daily to test the presence of active stages, which were counted and removed for identification.

The related water column, on each lake, has been sampled in different periods of the year (at least 2 seasons), and in different years (between 2002 and 2006) to compare its faunal composition to each other. Zooplankton samples were collected monthly (three replicates), with two plankton nets (mouth diameter, 25 cm; length, 65 cm; mesh size, 200 μm and 50 μm) towed horizontally, equipped with a water-flow meter at the mouth. The Italian hypersaline lake Vecchia Salina and Crimean ones were already studied in the past (see data in [10] and [16], respectively).

Data analysis

Data were analysed by multivariate statistical techniques with a non-parametric approach because of the wide disparity in density of some cysts in different lakes. The significance of the spatial variation in "cyst banks" composition was tested using a One-Way Analysis of Similarities for replicated data (ANOSIM) routine in PRIMER (Plymouth Routines In Multivariate Ecological Research) version 6β R6 (PRIMER-E) [21].

For multivariate analyses, the absolute densities of each morphotypes were fourth root transformed, to severely down-weight the importance of the very abundant species so allowing the less dominant, and even the rare morphs, to play some role in determining similarity among samples.

Stress values were shown for each MDS plot to indicate the goodness of representation of differences among samples [22]. A One-Way similarity percentages procedure (PRIMER SIMPER routine, Clarke [22]) was used in order to obtain the percentage contribution that each taxon provided to Bray-Curtis similarities measures. A cut-off criterion was applied to allow the identification of a subset of species whose cumulative percentage contribution reached 80% of similarity value. SIMPER analysis consented to identify the species responsible for the biological characterisation of the "seed banks" stored in each investigated lake.


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