Nauplii were rinsed several times in Phosphate-Buffered Saline (PBS) 1× solution and frozen in liquid nitrogen to fracture the carapace and left at −80 °C for one night. Animals were then incubated for 1 h 30 min in 0.5 U mL−1 chitinase enzyme (EC126.96.36.199; Sigma–Aldrich) to permeabilize the chitinous wall (Buttino et al., 2004). After rinsing in PBS OSI-906 nmr 1×, samples were incubated in 0.1% Triton x-100 for 3 min at room temperature, and then washed twice in PBS and once in PBS+1% Bovine Serum Albumin (BSA) buffer. Animals were incubated in TUNEL for 1.5 h at 37 °C following the manufacture’s instructions. Samples were rinsed again in PBS and observed with the Zeiss fluorescence microscope using 10× and 20×
objectives equipped with Green Fluorescent Protein (GFP) filter to detect TUNEL green fluorescence which reveals apoptosis. Experiments were performed in a transparent PVC vessel 32 cm (length) 13 cm (width) and 10 cm (height), equipped with two 2-cm high vertical bars placed in the middle and separated Caspase inhibitor in vivo by a 3-cm wide space. Two agarose gel blocks incorporating DD or methanol (as control), were placed at the opposite sides of the vessel. Agarose gels
(0.6%) were prepared by adding 0.3 g of agarose powder (Applichem) to 50 mL of bi-distilled water (BDW), followed by heating. After cooling, 1 mL of DD (Sigma) at 0.5 mg/mL in methanol was added, to obtain a final DD concentration of 10 μg/mL in agarose. One milliliter of methanol was also added to another agarose gel preparation, which was used as a control.
Agarose gels were then poured into two 9-cm wide Petri disks, left to harden and stored overnight at 4 °C. Experiments were performed the next day by placing half of each agarose disk (A = 32 cm2 × h = 0.8 cm) on the bottom of the container, at opposite sides of the vessel. We then identified an area of the vessel with the DD-incorporated agarose block (+), an area with the methanol-incorporated agarose block (−) (control), and an area in the SPTLC1 middle (0), where the copepods were released at the beginning of the experiment. The experimental method of using agarose blocks incorporating a known toxin or metabolite is similar to that described in Jüttner et al. (2010) and differs from the Y-shaped choice chambers where copepods are provided with the option of clean seawater or seawater containing test compounds such as in Brooker et al. (2013). T. stylifera specimens were sorted from zooplankton samples collected in the Gulf of Naples from October to November 2012, using routine procedures previously described in the methods section. About 50 ripe females were sorted, incubated into two 1-L stericups containing 50-μm natural filtered seawater, and kept in a temperature-controlled room at 20 °C and 12:12 Light:Dark cycle. After 24 h, the experiment was started by filling the vessel with 2.5 L of 0.