Methods

Adult specimens of N. heteropoda were decapitated (15 females and 15 males) and fixed with 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (pH 7.4, 4°C, 2h), postfixed in 2% osmium tetroxide in a 0.1 M phosphate buffer (4°C, 1.5 h) and dehydrated in a graded series of concentrations of ethanol (50, 70, 90, 95 and 4x100% each for 15 min) and acetone (15 min). Eventually, the material was embedded in epoxy resin (Epoxy Embedding Medium Kit; Sigma). Semi- (0.8 μm thick) and ultra-thin (70 nm) sections were cut on a Leica Ultracut UCT25 ultramicrotome. Semi-thin sections were stained with 1% methylene blue in 0.5% borax and observed using an Olympus BX60 light microscope. After staining with uranyl acetate and lead citrate, ultra-thin sections were examined using a Hitachi H500 transmission electron microscope. Ultrathin sections were used in order to count the number of cells that had signs of necrosis or autophagy in relation to the total number of midgut epithelial cells. The percentage of necrotic or autophagic cells was determined by randomly counting cells in the intestine and hepatopancreas of 3 adult specimens. Significance of differences in the levels of percentage of necrotic cells and autophagic cells between intestine and hepatopancreas was assessed with the Student’s t-test, p<0.05.

The midguts (intestine and hepatopancreas) from two specimens were fixed in Karnovsky’s fixative (2h, 4°C), washed in a cacodylate buffer 0.1M (15 min, RT), 0.2M Tris-Maleate (10 min, RT) and incubated in the incubation medium– 0.2M Tris-Maleate buffer, 0.1M sodium β-glycerophosphate and 0.2M lead citrate (2h, 37°C). After washing the material with Tris-Maleate (2 x 15 min, RT), it was postfixed in 1% osmium tetroxide and prepared according to the standard method for TEM. The material was then analyzed using a Hitachi H500 transmission electron microscope.

Adult specimens of N. heteropoda (10 males and 10 females) without fixation were embedded in a tissue-freezing medium (Jung). Cryostat sections were cut (5 μm thick) and mounted on slides.

After washing in Tris-buffered saline (TBS) (5 min, RT) and a 0.1N sodium acetate-acetic acid buffer (pH 5.0–5.2, RT), cryosections were incubated in a 0.1N sodium acetate-acetic acid buffer (pH 5.0–5.2, 1,5h, 37°C) containing 0.01% naphtol phosphate AS-BI, 2% N-N-dimethylformamide, 0.06% Fast Red Violet LB, 0.5mM MnCl_2. Negative controls were performed by omitting the specific naphtol phosphate AS-BI substrate. The material was analyzed using an Olympus BX60 light microscope.

This red-fluorescent dye selectively accumulates in acidic organelles and can be used to investigate lysosomes and autolysosomes. The intestine and hepatopancreas isolated from animals body without fixation were incubated in the dark for 15 min in 2.5 mM LysoTracker Red DND-99 (Molecular Probes, L 7528) diluted in 500 ml of PBS (Phosphate-buffered saline) at RT. Next, the material was washed several times with PBS. The nuclei were stained for 10 min in 1 mg/ml Hoechst 33342 diluted in PBS and washed several times in PBS. The slides were analyzed using an Olympus FluoView FV1000 confocal microscope. Excitation at 543 nm was provided by an argon/krypton laser.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is the method that is used to investigate the DNA fragmentation that occurs during apoptosis. The intestine and hepatopancreas isolated from animals body without fixation were washed in TBS (3 x 5 min) and stained with a TUNEL reaction mixture (In Situ Cell Death Detection Kit, TMR red, Roche) (60 min at 37°C in the dark). After washing in TBS, the material was analyzed using an Olympus FluoView FV1000 confocal microscope with 60×/NA 1.35 objectives. Z-stack images were generated using a 405nm laser for Hoechst 33342 dyes and 568nm for TMR red dye (TUNEL reaction mixture). 3D data sets were analyzed as volume-rendered data sets using Imaris (custom software developed by Bitplane Scientific Software, Zurich, Switzerland). For statistical analysis all cells were segmented using the surface modeling that is available in Imaris for Hoechst 33342 dye. The mean fluorescence value for TMR red dye was calculated for each segmented cell. The arbitrary mean intensity threshold for the TMR red dye was used to determine the number of stained cells. As a result, we calculated the mean percentage of the stained cell population. Negative controls without terminal deoxynucleotidyl transferase (TdT) were prepared according to the labeling protocol (In Situ Cell Death Detection Kit, TMR red, Roche). Statistical analyses were performed using the R (ver. 3.0.2) statistical environment. Normality was checked by the Shapiro-Wilk test. Significance of differences in the levels of percentage of apoptotic cells between intestine and hepatopancreas in TUNEL assay was assessed with the Student’s t-test, p<0.05.

Dissected organs (hepatopancreas and intestine that were isolated from five specimens) were crushed mechanically and suspended in 100 μL PBS (pH 7.4). Then, using 0.05% trypsin in EDTA solution with 0.01% collagenase II, enzymatic isolation was carried out for 10 min at 37°C. The cells were suspended in DMEM low-glucose medium (1 g/L) and incubated at 37°C. The cell suspension was washed using centrifugation at 1500 rpm for five minutes and the precipitate was suspended in 100 μL of PBS buffer.

JC-1 (5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethyl-benzimidazolyl-carbocyanine iodide) is a membrane-permeant cationic dye that is widely used in order to monitor the mitochondria in cell death studies. Changes in mitochondrial transmembrane potential (ΔΨm) were monitored using JC-1 cationic dye whose accumulation in mitochondria is dependent on the magnitude of mitochondrial potential. JC-1 differentiates cells with a high mitochondrial potential (orange fluorescence; polarized mitochondria) and a low mitochondrial potential (green fluorescence; depolarized mitochondria) (Salvioli et al., 1997). The intestine and hepatopancreas were isolated from 7 animals body. The cell suspension obtained from these organs without fixation was incubated in the dark with 5 μL of 1.5 mM JC-1 solution in DMSO (99.97%, H₂O < 0.1%) for 15 minutes at room temperature. The cells were analyzed using flow cytometry (Coulter Instrument EPICS XL MLC) with a 488 nm argon laser, using the MXP software Beckman Coulter program and the results were presented as the percentage of cells with depolarized mitochondria. Statistical analyses were performed using the STATISTICA 10.0 software package (StatSoft, Inc. (2010) version 10.0. http://www.statsoft.com). Normality was checked by the Kolmogorov-Smirnov test. The data were tested for homogeneity of variance using Levene’s test of equality of error variances. Significance of differences in the levels of percentage of cells with depolarized mitochondria between intestine and hepatopancreas was assessed with the Student’s t-test, p<0.05.

The prepared intestine and hepatopancreas isolated from animals body were incubated in the dark with 5 μL of 1.5 mM JC-1 solution in DMSO (99.97%, H₂O < 0.1%) for 15 minutes at room temperature without fixation. The material was visualized using an Olympus FluoView FV1000 confocal microscope.