Isodictya pacmata (Ellis & Solander, 1786)

Pettersen, Ragnhild, Johnsen, Geir, Bruheim, Per & Andreassen, Trygve, 2014, Development of hyperspectral imaging as a bio-optical taxonomic tool for pigmented marine organisms, Organisms Diversity & Evolution (New York, N. Y.) 14 (2), pp. 237-246 : 241-243

Isodictya pacmata

Five pigments (Fig. 3a) detected with LC-MS analysis of I. pacmata are listed in Table 1. Four of the five pigments were tentatively identified by DNP search while the fifth (Pigment 3, Table 1) did not get any hits and was presumed unknown. The DNP database returned three hits for the first of the five pigments detected: 2,6-benzothiazolediol and two derivatives of the same component (Pigment 2, Table 1, Fig. 3a). The ppm error for this pigment was higher than the 10 ppm window usually used for DNP searches, but the pigment was illustrating that the OD(λ) spectra are inversely related to the R(λ) spectra. The in vitro OD(λ) curve has been interrupted from 630 nm – 642 nm (marked with: = =) due to saturation of the detector in the spectrophotometer. The spectra have been scaled to 1 at their highest OD (λ) and R(λ) for easier comparison considered interesting for this organism (see following “Results and Discussion”).

Derivatives of 2,6 benzathiazolediol are known to be produced by the bacteria Micrococcus sp. , which has been isolated from the tropical sponge Tedania ignis ( Stierle et al. 1988) . Sponges are known to be hosts for symbiotic microorganisms and, since all sponges are filter feeders, they filtrate and extract microorganisms from the water. Microorganisms that resist the digestive process and immune response of the host sponge can successfully inhabit the sponge ( Wilkinson 1987; Lee et al. 2001). Some species of Micrococcus are pigmented and a large number of heterotrophic bacteria are also able to synthesise carotenoides ( Hailian et al. 2006) and may therefore contribute to the colour of the host organism. Further analysis is needed to make a definite identification of the pigment found, but it is highly likely that 2,6 benzathiazolediol and derivatives can be found in I. pacmata . DNP search of pigment 4 returned five hits ( Table 1), of which the purine pigment erinacean with an absorption max at 272 nm in methanol is the most likely identity. The pigment detected from I. pacmata had an OD(λ)max at 278 nm (Fig. 3a). Erinacean was isolated for the first time from the Antarctic sponge Isodictya erinacean (Topsent 1916; Moon et al. 1998) and found to be bioactive by being weakly antibiotic and cytotoxic ( Moon et al 1998; Bandaranayake 2006). Cytotoxic substances in sponges are presumed to be a part of the defence mechanism to prevent predation from other benthic species ( Moon et al. 1998), indicating that I. pacmata has both physical (spicules) and chemical defence mechanisms. The other hits from the DNP were Asperopterin B isolated from Aspergiccus oryzae; 1,3,9-trimethyl-8- nitrosoisoxanthine isolated from Cucumaria frondosa ; and

Fig. 3 a, b Bio-optical a b

signatures from I. pacmata . a 1.4

Specific absorbance spectra of 1.4 1.4 tentatively identified pigments 1.2

and unidentified pigments 2–6 1.2 1.2 ( Table 1) detected by LC-MS 1 R) analysis. b The absorbance 1 1 (

OD spectra spectra (λ) plotted in in vivo vivo against OD R(λ () λ)) and and reflection in in vitro situ OD

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OD 0.8 0.8 Reflectance

R the related

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the the spectra

R

HI

(λ

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illustrating are spectra inversely

. The that

0.4

0.6 0.6

0.4

0.6

0.4

Relative spectra have been scaled to 1 at highest OD(λ) and R(λ) for easier 0.2 0.2 0.2 comparison

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230 330 430 530 630 300 350 400 450 500 550 600 650

Wavelength (nm) Wavelength (nm)

two derivatives of Pyrimido[5,4- e]-1,2,4-triazine-3,5,7-triol: 4-Methylfervenulone isolated from Streptomyces spp. and 2- methylfervenulone. Also for this pigment, further analysis is needed for definite identification, but it is highly likely that the pigment is erinacean because it has been isolated previously from a species in close relation to I. pacmata . Calicogorgin B (Pigment 5) isolated from the gorgonian Cacicogorgia sp. was also identified tentatively in I. pacmata ( Table 1, Fig. 3a). In Cacicogorgia sp., calicogorgin B has been reported to possess lethal and repellent activities against predatory gastropods ( Ochi et al. 1992); this pigment might be one reason why I. pacmata has few predators. The absorption of calicogorgin B in the visible part of the wavelength spectrum with a OD(λ)max at 404 nm, indicates that this pigment is a main contributor to the orange colour of the sponge. A derivative of aspergamide B was the last pigment detected from I. pacmata (Pigment 6, Table 1, Fig. 3a). One biological source of aspergamide B is the fungus Aspergiccus sp., which is found everywhere in the marine and terrestrial environment and is probably taken up by filtration of water.

The in vivo and in vitro OD(λ) spectra showed absorbed in the UV region (<389 nm) (Fig. 3b), corresponding to the specific absorption of the pigments detected in Fig. 3a. There was also a broad absorbance band in the visible part of the wavelength spectra at 400–450 nm that corresponded to the specific absorption of pigment 5 (Fig. 3a). OD(λ) spectra decreased in the 450–500 nm areas and, correspondingly, the in vivo and in situ R(λ) spectra from HI measurements increased (Fig. 3b), making the OD(λ) and R(λ) spectra from I. pacmata inversely related. The in vivo and in situ HI R(λ) spectra were measured with different distances and medium between the sponge and the HI detector; in vivo measurements were taken in air with a microscope and in situ measurements were taken under water at a distance of 1.35 m between OOI and UHI. The shape of the R(λ) spectra show that the distance and inherent optical properties in the water did not have a significant impact on the reflection signature when a reference plate was used for correction. These results are in accordance with those of Johnsen et al. (2013b) and R. Pettersen et al. (unpublished) and show that HI and UHI has the potential to be an accurate taxonomic tool.