**2. Results**

### *2.1. Chemical Characterization of Pigments*

In accordance with Martins et al. [20], the crude extracts in cold Dulbecco's phosphate-buffered saline (PBS) from the proboscis and skin, being yellowish and greenish in color, respectively, were comprised of multiple pigments. Both extracts of skin and proboscis tissues were analyzed by HPLC-DAD and recorded at 280 and 440 nm (exemplified in Figure 1). For simplification purposes, this distinction based on visual inspection will be retained. Albeit variable among individual pigments, the absorption spectra of either extract had maxima within the UV range (ca. 280 nm), plus Soret bands between 350 and 500 nm and the characteristic Q bands (580–750 nm) of porphyrin-like pigments, which is particularly obvious for the main pigments fractionated from the skin (Figure 2). Among the

main pigments in skin extracts that better fit the expected porphyrin signature, we found a yellowish pigment that was exclusive to this organ, with a retention time of 1.8 min. To this substance a greenish pigment was added with retention time 3.43 min and two yellow pigments with retention times 0.95 and 4.40 min (Figure 3). The latter, in particular, was found to be the most abundant pigment in skin extracts, as inferred from its higher absorbance (Figure 3D).

**Figure 1.** Representative HPLC-DAD profiles at 440 and 280 nm (black and red line, respectively). Pigments extracts from (**A**) skin, and (**B**) proboscis. Compounds with \* did not present the characteristic absorption of porphyrin pigments and were not further addressed.

**Figure 2.** Absorption spectra of the four main pigments in *Eulalia viridis* skin crude pigment extracts spectra retrieved from HPLC-DAD analyses (each pigment's spectrum is identified by its retention time). The Soret (350–500 nm) and Q (580–750 nm) bands typical of porphyrinoids are highlighted.

**Figure 3.** Spectra of the principal pigments in the crude extracts from the skin of *Eulalia viridis* fractionated by HPLC-DAD. The skin yielded mainly one greenish and three yellowish pigments. (**A**) yellow pigment (0.95 min); (**B**) yellow pigment (1.8 min); (**C**) green pigment (3.43 min); (**D**) yellow pigment (4.40 min). The pigments detected at retention times 1.80 and 3.34 min are specific to the skin.

The proboscis extract yielded two green and two yellowish main pigments with porphyrinoid signatures (Figure 4). Among the four main compounds, two yellow pigments were detected at retention times of 0.95 and 4.40 min, presenting maxima at both 282 and 286 nm, respectively. To this pigment two greenish pigments detected at retention times of 0.82 and 1.34 min were added, with absorbance maxima at 272 and 257 nm, respectively, which were exclusive to this organ (Figure 5). The yellow pigments detected in the proboscis are similar to those described earlier in the skin, judging from similar retention times (0.95 and 4.40 min) and absorbance magnitudes. As previous, the yellow pigment shown in Figure 5D with the 4.40-min retention time was most abundant, not only in extracts from the proboscis but also comparatively to the skin. Table 1 summarizes the main similarities and differences between pigment extracts from the two organs.

**Figure 4.** Absorption spectra of the four main pigments in *Eulalia viridis* proboscis crude pigment extracts spectra retrieved from HPLC-DAD analyses (each pigment's spectrum is identified by its retention time). The Soret (350–500 nm) and Q (580–750 nm) bands typical of porphyrinoids are highlighted.

**Figure 5.** Spectra of the principal pigments in the crude extracts from the proboscis of *Eulalia viridis* fractionated by HPLC-DAD. The proboscis yielded mainly two yellowish and two greenish pigments. (**A**) green pigment (0.82 min); (**B**) yellow pigment (0.95 min); (**C**) green pigment (1.34 min); (**D**) yellow pigment (4.40 min). The pigments detected at retention times of 0.82 and 1.34 min are specific to the proboscis.
