Determination of the Halogenated Skeleton Constituents of the Marine Demosponge Ianthella basta

Demosponges of the order Verongida such as Ianthella basta exhibit skeletons containing spongin, a collagenous protein, and chitin. Moreover, Verongida sponges are well known to produce bioactive brominated tyrosine derivatives. We recently demonstrated that brominated compounds do not only occur in the cellular matrix but also in the skeletons of the marine sponges Aplysina cavernicola and I. basta. Further investigations revealed the amino acid composition of the skeletons of A. cavernicola including the presence of several halogenated amino acids. In the present work, we investigated the skeletal amino acid composition of the demosponge I. basta, which belongs to the Ianthellidae family, and compared it with that of A. cavernicola from the Aplysinidae family. Seventeen proteinogenic and five non-proteinogenic amino acids were detected in I. basta. Abundantly occurring amino acids like glycine and hydroxyproline show the similarity of I. basta and A. cavernicola and confirm the collagenous nature of their sponging fibers. We also detected nine halogenated tyrosines as an integral part of I. basta skeletons. Since both sponges contain a broad variety of halogenated amino acids, this seems to be characteristic for Verongida sponges. The observed differences of the amino acid composition confirm that spongin exhibits a certain degree of variability even among the members of the order Verongida.


Introduction
The oceans represent the biggest habitat on earth, which is however, not yet fully explored. Consequently, the marine habitat is one of increasing interest for the research and further exploration of this understudied environment. For example, a huge diversity of bioactive natural products is found in marine organisms. To date, approximately 28,000 of these marine natural products have been discovered, and sponges are the main producers of such compounds [1,2].
Verongida families by the presence of eurypylous choanocyte chambers. Aplysinellidae consists of nine species from three genera (Aplysinella, Porphyria, and Suberea). It is defined by a dendritic fiber skeleton with both pith and bark elements. Pseudoceratinidae consists of four species from a single genus (Pseudoceratina) and is defined by a dendritic fiber skeleton with only pith elements. We have chosen I. basta as a characteristic representative of the Ianthellidae family, which represents the second largest among the four Verongida sponge families.
Based on previous work on A. cavernicola [41], we optimized the methods for the effective isolation of the sponge skeletons of I. basta and the complete extraction of amino acids from the skeletons with Ba(OH) 2 . The determined amino acid composition of I. basta was finally compared with the amino acid composition of A. cavernicola skeletons as well as with the amino acid composition of the Dictyoceratida sponges H. equina and S. officinalis obliqua. pith elements. We have chosen I. basta as a characteristic representative of the Ianthellidae family, which represents the second largest among the four Verongida sponge families. Based on previous work on A. cavernicola [41], we optimized the methods for the effective isolation of the sponge skeletons of I. basta and the complete extraction of amino acids from the skeletons with Ba(OH)2. The determined amino acid composition of I. basta was finally compared with the amino acid composition of A. cavernicola skeletons as well as with the amino acid composition of the Dictyoceratida sponges H. equina and S. officinalis obliqua.

General Chemical Characterization of the I. basta Skeleton and Comparison with A. cavernicola
Morphological differences between the skeletons of the two Verongida sponges I. basta and A. cavernicola are shown in underwater and light microscopic images (Figure 1a-d). I. basta grows in large fan or funnel shapes ( Figure 1a) which is reflected in its planar, netlike skeleton structure ( Figure  1b). A. cavernicola grows thick and incrusting with numerous short tubes (Figure 1c), which is supported by its three-dimensional skeleton (Figure 1d). The SEM images of the isolated I. basta skeletons ( Figure 2) reveal a fiber system. These fibers consist of several concentric layers similar to the structural organization previously described for A. cavernicola skeletons (see [44]). The central channel of the fiber is filled with the pith, a porous material. Table 1 summarizes various chemical parameters from the two sponges. Interestingly, the amount of skeletal material related to the entire sponge is more than one order of magnitude higher The SEM images of the isolated I. basta skeletons ( Figure 2) reveal a fiber system. These fibers consist of several concentric layers similar to the structural organization previously described for A. cavernicola skeletons (see [44]). The central channel of the fiber is filled with the pith, a porous material.
in I. basta than in A. cavernicola. An explanation for the higher amount of skeletal material in I. basta might be a need for mechanical stability because of its two-dimensional skeleton structure and the large size of the sponges (more than 2 m maximum diameter) [45].
The chitin content in I. basta skeletons is also higher than in A. cavernicola. Campbell [46] showed that chitin is responsible for the flexibility and not for the strength of chitin-protein complexes of insects. If this conclusion also applies here, it would lead to the assumption that the sponge skeleton of I. basta has to be more flexible than the skeletons of A. cavernicola. Given that the sponges can be found at depths as shallow as 5 m, where wave exposure certainly affects large sponges, a flexible skeleton might be a prerequisite to successfully colonize and persist on shallow reef slopes.   [44] 51 ± 4 mg/g 40 ± 3 mg/g Furthermore, the percentage of saccharides in the skeletons other than chitin (estimated by the so-called resorcin method [47], see Materials and Methods) is also higher in I. basta, but in general much lower than the amount of chitin.
Ehrlich et al. [48] reported the presence of amorphous silicate and crystalline calcium carbonate in the form of aragonite as skeletal components of the Verongida sponge Verongula giganteum. In order to test this possibility for I. basta, the calcium and silicon contents of the skeletons were determined by ICP-OES. The calcium concentrations are low. Assuming that the calcium in the skeletons occurs as calcium carbonate, an amount of ca. 4% for I. basta and ca. 1% for A. cavernicola can be estimated. The silicon content is below the detection limit. These results show that both sponges, I. basta and A. cavernicola, do not exhibit the silica-aragonite-chitin-biocomposites found in Verongula giganteum.
The content of sulfur is of the same order for the skeletons of both samples. EDX measurements confirm that chlorine and iodine are present in both skeletons in addition to bromine. The bromine content (determined as described previously in [44]) is significantly higher in I. basta skeletons than in A. cavernicola.  Table 1 summarizes various chemical parameters from the two sponges. Interestingly, the amount of skeletal material related to the entire sponge is more than one order of magnitude higher in I. basta than in A. cavernicola. An explanation for the higher amount of skeletal material in I. basta might be a need for mechanical stability because of its two-dimensional skeleton structure and the large size of the sponges (more than 2 m maximum diameter) [45].  15.5 mg/g 3.5 mg/g estimated content of calcium carbonate in the skeleton 3.9 wt. % 0.9 wt. % content of silicon in the skeleton <1.7 mg/g <1.7 mg/g estimated content of protein in the skeleton ≤77 wt. % ≤90 wt. % content of sulfur in the skeleton 14.4 mg/g 11.8 mg/g halogens present in the skeleton Br, Cl, I Br, Cl, I bromine content in the skeleton [44] 51 ± 4 mg/g 40 ± 3 mg/g The chitin content in I. basta skeletons is also higher than in A. cavernicola. Campbell [46] showed that chitin is responsible for the flexibility and not for the strength of chitin-protein complexes of insects. If this conclusion also applies here, it would lead to the assumption that the sponge skeleton of I. basta has to be more flexible than the skeletons of A. cavernicola. Given that the sponges can be found at depths as shallow as 5 m, where wave exposure certainly affects large sponges, a flexible skeleton might be a prerequisite to successfully colonize and persist on shallow reef slopes.
Furthermore, the percentage of saccharides in the skeletons other than chitin (estimated by the so-called resorcin method [47], see Materials and Methods) is also higher in I. basta, but in general much lower than the amount of chitin.
Ehrlich et al. [48] reported the presence of amorphous silicate and crystalline calcium carbonate in the form of aragonite as skeletal components of the Verongida sponge Verongula giganteum. In order to test this possibility for I. basta, the calcium and silicon contents of the skeletons were determined by ICP-OES. The calcium concentrations are low. Assuming that the calcium in the skeletons occurs as calcium carbonate, an amount of ca. 4% for I. basta and ca. 1% for A. cavernicola can be estimated.
The silicon content is below the detection limit. These results show that both sponges, I. basta and A. cavernicola, do not exhibit the silica-aragonite-chitin-biocomposites found in Verongula giganteum.
The content of sulfur is of the same order for the skeletons of both samples. EDX measurements confirm that chlorine and iodine are present in both skeletons in addition to bromine. The bromine content (determined as described previously in [44]) is significantly higher in I. basta skeletons than in A. cavernicola.
Finally, we estimated the protein content of the skeletons as a remaining fraction after the consideration of the other analyses. It is significantly higher in A. cavernicola than in I. basta-mainly due to the higher amount of chitin in I. basta. Since I. basta also exhibits a higher content of bromine, we have to conclude that I. basta skeletons contain a higher overall number of brominated compounds than A. cavernicola. The question arises whether or not these brominated compounds are the same for both Verongida sponges. In order to answer this question, GC-MS analyses were performed.

GC-MS Analysis of the Skeletal Amino Acid Composition
The isolated skeletons of I. basta were treated with a saturated Ba(OH) 2 solution to extract the skeleton-bound amino acids and allow their analysis by GC-MS. The protocol for this extraction was initially developed for the sponge A. cavernicola [41]. It had to be adapted and optimized for the more resistant skeletons of I. basta. In contrast to A. cavernicola, the complete extraction of I. basta skeletons required a significantly higher temperature (100 • C versus 37 • C) because of the stronger hydrolysis resistance of the I. basta spongin fibers. This emphasizes the different characters of the two different sponge skeletons.
The dissolved amino acids were derivatized using MTBSTFA and analyzed as described for A. cavernicola [41]. The derivatization method was optimized such that a maximum number of amino acids with their broad range of structures is completely converted. The same method as for A. cavernicola was then applied for I. basta. Since the analysis methods for both sponges were exactly the same, the results are well comparable.
The chromatogram of the GC-MS measurements is shown in Figure 3. The assignment of the peaks is given in Table 2. It was carried out as described in detail in [41], using the information in EI mass spectra and confirmed for most of the peaks by retention times of standard compounds. The structure of a few halogenated amino acids could not be verified with pure standards due to the lack of availability of these reference compounds. For these compounds, the identification was based on their mass spectra and is also described in detail in [41]. Finally, we estimated the protein content of the skeletons as a remaining fraction after the consideration of the other analyses. It is significantly higher in A. cavernicola than in I. basta-mainly due to the higher amount of chitin in I. basta. Since I. basta also exhibits a higher content of bromine, we have to conclude that I. basta skeletons contain a higher overall number of brominated compounds than A. cavernicola. The question arises whether or not these brominated compounds are the same for both Verongida sponges. In order to answer this question, GC-MS analyses were performed.

GC-MS Analysis of the Skeletal Amino Acid Composition
The isolated skeletons of I. basta were treated with a saturated Ba(OH)2 solution to extract the skeleton-bound amino acids and allow their analysis by GC-MS. The protocol for this extraction was initially developed for the sponge A. cavernicola [41]. It had to be adapted and optimized for the more resistant skeletons of I. basta. In contrast to A. cavernicola, the complete extraction of I. basta skeletons required a significantly higher temperature (100 °C versus 37 °C) because of the stronger hydrolysis resistance of the I. basta spongin fibers. This emphasizes the different characters of the two different sponge skeletons.
The dissolved amino acids were derivatized using MTBSTFA and analyzed as described for A. cavernicola [41]. The derivatization method was optimized such that a maximum number of amino acids with their broad range of structures is completely converted. The same method as for A. cavernicola was then applied for I. basta. Since the analysis methods for both sponges were exactly the same, the results are well comparable.
The chromatogram of the GC-MS measurements is shown in Figure 3. The assignment of the peaks is given in Table 2. It was carried out as described in detail in [41], using the information in EI mass spectra and confirmed for most of the peaks by retention times of standard compounds. The structure of a few halogenated amino acids could not be verified with pure standards due to the lack of availability of these reference compounds. For these compounds, the identification was based on their mass spectra and is also described in detail in [41].  Components with * could not be verified with pure standards due to the lack of availability of these reference compounds. Table 2. Amino acids detected as TBDMS-derivatives in the Ba(OH) 2 extract of isolated I. basta sponge skeletons. Components with * could not be verified with pure standards due to the lack of availability of these reference compounds.

Peak
Amino Acid Proteinogenic Halogenated Oxoproline 10a Hydroxyproline (2 TBDMS) 11 Methionine X 6b Serine (3 TBDMS) X 12a Threonine (3 TBDMS) X 12b Threonine (3 TBDMS) X 13 Phenylalanine X 14 Aspartic Acid X 10b Hydroxyproline (3 TBDMS)  15 Glutamic Acid X  16  Ornithine  17  Lysine  X  18  Arginine  X  19  Histidine  X  20  Tyrosine  X  21  Tryptophan  X  22  Hydroxylysine  23 3-Monochlorotyrosine X 24 * Monobromotyrosine X 25 * Dichlorotyrosine X 26 3-Monoiodotyrosine X 27 * Monobromo-monochlorotyrosine X 28 3,5-Dibromotyrosine X 29 * Monochloro-monoiodotyrosine X 30 * Monobromo-monoiodotyrosine X 31 3,5-Diiodotyrosine X Obviously, the isolated I. basta sponge skeletons contain a variety of amino acids. The gas chromatogram can be divided into two parts: below a relative retention time of t R,rel = 0.85, twenty-two different amino acids could be observed. Seventeen of these amino acids could be assigned to proteinogenic amino acids. Some of the amino acids give rise to multiple peaks. This is caused by different reasons. Serine (peaks 6a, 6b) and hydroxyproline (peaks 10a, 10b) exhibit two peaks due to different degrees of derivatization. In each case, the first peak is caused by the incomplete derivatization of the molecule while the second peak is caused by the completely derivatized amino acid. Isoleucine (peaks 7a, 7b) and threonine (peaks 12a, 12b), however, yield two peaks of the same TBDMS derivative. As described previously (see [41]), the existence of two stereocenters in isoleucine as well as in threonine leads to two different diastereomers [49,50], which can be separated by gas chromatography.

Comparison of the Found Amino Acid Composition with the Literature
The found amino acid composition of the I. basta skeletons was compared with the previously determined amino acid composition of A. cavernicola skeletons [41] from the order Verongida, and with that of the sponges H. equina [39] as well as S. officinalis obliqua [40], both belonging to the order Dictyoceratida. All these sponges are from the Demospongia class. The results are given in Table 3.
Since the extraction of the amino acids was carried out with an alkaline Ba(OH) 2 solution in all cases, the amino acid compositions are well comparable. First of all, it can be stated that the skeletal amino acid composition of the Verongida sponges I. basta and A. cavernicola is relatively similar. There are, however, also some differences: I. basta skeletons exhibit the non-halogenated amino acids hydroxylysine and isoleucine, which cannot be found in A. cavernicola skeletons. Monobromohistidine, in turn, cannot be detected in I. basta skeletons but are in the skeletons of A. cavernicola. Striking differences occur between the amino acid composition of the two Verongida sponges and the Dictyoceratida sponges H. equina and S. officinalis obliqua, especially with respect to halogenated amino acids. Most of the characteristic halogenated amino acids are only observed in the skeletons of the Verongida sponges I. basta and A. cavernicola. The halogenated amino acids monobromotyrosine, 3-monochlorotyrosine, monochloro-monoiodotyrosine, monobromomonochlorotyrosine and dichlorotyrosine could not be identified in H. equina and S. officinalis obliqua. Interestingly, the amino acid monobromo-monochlorotyrosine is occurring in the gastropod mollusk Buccinum undatum [51].
In addition, the results of the analysis of I. basta skeletons support the hypothesis [41] that the presence of AABA or GABA is species-specific. H. equina is the only sponge in which GABA occurs. In the other sponges AABA was found instead of GABA. Moreover, cystine only occurs in H. equina but not in the other examined species. The proteinogenic amino acids alanine, aspartic acid, glutamic acid, glycine, leucine, lysine, proline, tryptophan, tyrosine, valine and the halogenated amino acid 3,5-diiodotyrosine have been identified in the skeletons of I. basta as well as in A. cavernicola, in the sponge H. equina and in the sponge S. officinalis obliqua. Moreover, the non-proteinogenic amino acids hydroxyproline, oxoproline and ornithine were found in all four sponge samples. The presence of hydroxyproline is well known for collagen [52]. This observation, together with the very high amount of glycine in both sponge skeletons, emphasizes the collagenous character of spongin. Oxoproline and ornithine were also found in Aplysina aerophoba [53], which is the closest relative to A. cavernicola.
Since both H. equina and S. officinalis obliqua are members of the order Dictyoceratida while I. basta and A. cavernicola belong to the order Verongida it is quite possible that the observed amino acid composition is genus specific.
For further comparison, the relative amounts of the detected amino acids were estimated for I. basta and A. cavernicola which were analyzed exactly by the same method by referring the individual peak intensities to that of the most intensive peak (cf. Table 4). Glycine was the most abundant amino acid in both sponge skeletons. Alanine, lysine, ornithine, proline, and tyrosine were present in large amounts in both samples. In contrast, phenylalanine and valine occurred in smaller amounts in both cases. Methionine, arginine and tryptophan were only detected in minute amounts. Table 4. Comparison of the relative amino acid amounts in I. basta and A. cavernicola skeletons. Relative amounts were estimated referring the peak heights in the gas chromatogram to the most intense peak (++++: very large amount (100%-80%); +++: large amount (80%-30%); ++: small amount (30%-10%); +: smallest amount (<10%); -: not present). Amino acids only found in I. basta are marked in orange, those only found in A. cavernicola are marked in green.

Halogenation State
Amino Acids

Ianthella basta Aplysina cavernicola [41]
Non-halogenated  However, the compositions of the sponge skeletons from I. basta and A. cavernicola also exhibit interesting differences with respect to the non-halogenated amino acids. Histidine, serine, and threonine were present in much larger amounts in A. cavernicola skeletons than in I. basta. For aspartic acid, glutamic acid and phenylalanine the situation is opposite. Much larger amounts of these amino acids were detected in the skeletons of I. basta than in A. cavernicola. Moreover, the amino acids hydroxylysine and small amounts of isoleucine were found in the I. basta skeletons, with hydroxylysine supporting the collagenous nature of I. basta spongin. These amino acids were not identified in the skeletons of A. cavernicola. Furthermore, the amount of leucine is much higher in the skeletons of I. basta than in A. cavernicola.
Within the group of halogenated amino acids, 3,5-dibromotyrosine and monobromomonochlorotyrosine were most abundant. Smaller amounts were detected for all iodated tyrosines. The major difference was recognized in the dichlorotyrosine amounts, which were larger in the skeletons of A. cavernicola.

Sponge Samples
Sponges were collected in July 2013 while snorkeling at depths ranging from 8 to 12 m at Western Shoals, Apra Harbor, Guam (13.27.018 N; 144.39.120 E). Specimens were inspected in regards to their health and only healthy and intact specimens were collected. Sponges were kept in large Ziploc bags during collection and transported in a cooler to the Guam marine laboratories. Specimens were frozen at −20 • C and freeze-dried prior to their transport to the laboratories in Dresden, Germany (see also [20]).

Extraction of the Skeletons
I. basta skeletons were isolated following the protocol described previously [41]. Small pieces of I. basta (5-10 cm 2 ) were soaked in 40 mL of distilled water for two weeks. Subsequently, the samples were transferred into freshly distilled water for 24 h. This procedure was repeated two times under continuous shaking.

Light Microscopy
Small pieces of the purified chitin-scaffolds were put on a sample holder. Microscopic studies were carried out on a Keyence BZ-8000K microscope (Keyence, Osaka, Japan). The exposure time was 1/230 s.

Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX)
Dried pieces of the skeletons were fixed on a sample holder and coated with carbon. The SEM images were recorded on a ZEISS DSM 982 GEMINI field emission scanning electron microscope (ZEISS, Oberkochen, Germany) using an acceleration voltage of 2 kV. Furthermore, EDX spectra were recorded with an acceleration voltage of 15 keV.

Extraction of the Chitin-Based Scaffold
The pure chitin skeletons were uncovered by an alkaline extraction according to [26]. Five to Thirty-five mg of isolated and freeze dried samples were treated with 2.5 M NaOH at 37 • C for 7 days. The remaining fibrous skeletal material was neutralized. In a second step, the samples were treated with 20% acetic acid at 37 • C for 24 h. Subsequently, the remaining fibrous skeleton material was neutralized and freeze dried again. The percentage of chitin in the skeletons was determined gravimetrically.

Estimation of the Content of Other Saccharides
The content of saccharides beside chitin in the skeletons was estimated using the resorcin method [47]. Therefore, 4 mg of A. cavernicola or 1.5 mg of I. basta were soaked with 1 mL pure water, 1 mL of a solution of resorcin (6 mg/mL) and 5 mL of sulfuric acid (75%) for 40 min at 95 • C and then quenched for 30 min in a darkened water bath. For calibration, soaked standard solutions of a 1:1:1 mixture of glucose, glucuronic acid and mannose containing defined amounts of chitin and protein (lysozyme) to simulate the matrix of skeletons were used. For quantification, the UV/Vis spectrometer Cary 50 (Varian, Palo Alto, CA, USA) was used with the following conditions: wavelength 300-800 nm, 0.0125 s per scan, data interval 1 nm, scan rate 4800 nm.

Determination of Calcium, Silicon and Sulfur Contents by ICP-OES
Digestion for calcium and sulfur determination: 2-18 mg of the skeleton were weighted into a micro vessel and digested with 450 µL of a mixture of HNO 3 (65%), HF (47%-51%) and HCl for 15 min using microwaves at 1600 W and stepwise heating to 130 • C.
Complexation: 1.5 mL of a saturated solution of H 3 BO 3 was added to the digested sample for complexing fluorides. Complexation was accomplished as follows: power 800 W, temperature 110 • C, time 10 min.
Digestion for silicon determination: 3 mL of HNO 3 (65%) and 2 mL H 2 O 2 were added to 30 mg of sponge skeletons. Microwave digestion was accomplished using the following parameters: at the beginning 400 W at 50 • C, followed by stepwise heating to 180 • C using 800 W in 60 min.
Finally, all digested samples were filled up to 10 mL with ultrapure water. Measurement: The samples were measured with an ICP optical emission spectrometer Perkin-Elmer Optima 7000 DV using the analytical lines at 317.933 nm for calcium, at 180.669 nm for sulfur and at 212.412 nm, 251.611 nm and 288.158 nm for silicon. The following operating parameters were used: plasma argon 15 L/min, auxiliary argon 0.2 L/min, nebulizer argon 0.65 L/min, RF-power 1300 W and pump rate 1.3 L/min.

Ba(OH) 2 Extraction of the Amino Acids
30 mg of the dried isolated skeletons were treated with 7.5 mL of saturated Ba(OH) 2 solution containing 2 mg 5-bromotryptophan as internal standard at 100 • C for 5 days. Subsequently, the Ba(OH) 2 solution was neutralized with H 2 SO 4 and centrifuged. The supernatant was removed and freeze dried.

GC-MS Measurements of the Skeleton Extracts
One mg from each of the dried Ba(OH) 2 extracts was soaked in 20 µL 2.5 M HCl and dried under a gentle stream of nitrogen. Subsequently, the residues were soaked twice in 40 µL EtOH and dried under nitrogen. Fifty µL acetonitrile and 50 µL MTBSTFA were added to the dry residues. The mixtures were sonicated for 30 s and then heated to 70 • C for 30 min. One µL of the resulting solution was injected into the GC-MS.
Analyses were carried out on an Agilent Technologies 6890N gas chromatograph directly coupled to an Agilent Technologies 5973N mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). GC separations were performed on a SPB ® -5 capillary GC column (Sigma-Aldrich, St. Louis, MO, USA).
The flow of helium as carrier gas was 1 mL/min. The injector temperature was 300 • C. Split/Splitless injection was used (splitless time 1 min). The column temperature was programmed as followed: isothermal 115 • C for 3 min, then heating up to 300 • C at a rate of 4 K/min, then isothermal 300 • C for 30 min. The ion source temperature was 250 • C and the transfer line temperature was 300 • C. The mass spectra were recorded in the electron impact (EI) ionization mode at 70 eV, m/z range 70-850. The chromatograms were recorded after 3 min solvent delay. The retention time t R was normalized to the second peak of the internal standard. The intensity was normalized to the weighted sample and the total intensity of both peaks of the internal standard.

Conclusions
The isolated skeleton of the marine demosponge I. basta was analyzed with respect to its amino acid composition. These investigations revealed that the skeleton of I. basta contains 17 proteinogenic amino acids as well as five different non-proteinogenic amino acids. Moreover, we detected nine halogenated amino acids, all of them are halogenated tyrosines.
In conclusion, we can state the following: 1.
The composition of non-halogenated amino acids in I. basta is similar to that of A. cavernicola.
Abundant amino acids such as glycine and hydroxyproline confirm the collagenous nature of the I. basta spongin in analogy to all other investigated sponges.

2.
I. basta exhibits a similar variety of halogenated amino acids as already observed for A. cavernicola-in contrast to the Dictyoceratida sponges H. equina and S. officinalis obliqua. It is, therefore, tempting to speculate that this variety of halogenated amino acids is characteristic for the order Verongida. 3.
The differences in amino acid composition in the sponge skeletons of I. basta and A. cavernicola clearly show that the spongin in the skeletons of Verongid sponges is similar, but also exhibits some characteristic differences.

4.
Further investigations of the amino acid composition of other sponge samples should be performed in the future to include a larger set of different sponge species into this comparison.