*4.2. Pigment Extraction*

Worms were immobilized by hypotonic shock and microdissected to collect proboscis and skin (factually the body wall containing epidermis plus underlying musculature). The organs were then homogenized separately with a pestle in cold Dulbecco's phosphate-bu ffered saline (PBS), pH 7.5 to extract the hydrophilic fraction of pigments. Samples were afterwards centrifuged for 5 min, 5000 *g* at 4 ◦C. The supernatant containing the pigments was collected and immediately placed on ice and in the dark. The pellet was subjected to repeated extractions until the supernatant was visibly devoid of pigments. The supernatants were then pooled. Each pooled sample contained extracts from approximately five animals.

The crude extracts of either organ were then analyzed spectrophotometrically at 440 and 700 nm, the maxima described by Martins et al. [20]. Extracts were then diluted in PBS, with the absorbance at 440 being used to normalize the dilutions until samples yielded an absorbance value of 1 (D1), corresponding to the 100% concentrated extract. This solution was necessary to overcome the absence of suitable standards for the mixtures of pigments in the extracts from either organ. Serial dilutions D1 (100%) were then produced, termed D2 and D3, corresponding to 50% and 10% of D1, respectively. Pigment extraction was performed in a dim-lighted environment to avoid photodegradation of pigments. Exposure of worm tissues and extracts to ambient light and air was kept to the minimum. Analysis and toxicity testing involved only fresh extracts.

### *4.3. Chemical Characterization of Pigments*

Crude extracts of the two organs (skin and proboscis) were filtered with a GHP filter before analysis. Pigments were analyzed based on the protocol for separation and quantification of human bilins developed by Woods and Simmonds [32], with many optimizations. In brief, high-performance liquid chromatography (HPLC) analyses were conducted on a Merck-Hitachi instrument equipped with a diode array detector (DAD), scan range 200–800 nm (Merck-Hitachi L-4500 Diode Array Detector, Merck, Poole, UK), operating at 20 ◦C, using a reversed-phase analytical column (RP-HPLC, Onix® Monolithic C18 column, 100 × 4.6 mm i.d., 13 nm and 2 μm). Samples were prepared in MeOH and the injection volume was 20 μL. Preliminary assays showed that optimal peak separation was obtained with sodium phosphate buffer 10 mM, pH 3.5 (solvent A) and pure MeOH (solvent B) at a flow rate of 2 mL/min: linear gradient from 45% to 95% B for 10 min and 95% B for 2 min. The total run time excluding equilibration was 12 min. Linear gradient from 30% to 60% B for 5 min and linear gradient from 60% to 95% B for 10 min. The total run time excluding equilibration was 15 min. Throughout analysis, the pressure was maintained at about 67 bar and the temperature column kept at environmental temperature (20 ± 2 ◦C).
