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
Sponges (Porifera), representing one of the oldest metazoans, are often a prominent component of benthic communities [3
]. They can be found in polar, temperate and tropical regions [4
]. Sponges are sessile filterfeeders which cannot escape or actively fight predators [6
]. Therefore, they developed morphological and chemical defense mechanisms, providing them with protection against various predators [8
Based on their skeletal composition, sponges can be divided into three classes: calcareous sponges (Calcarea), glass sponges (Hexactinellida), and demosponges (Demospongia) [14
]. Recently, a fourth class, the Homoscleromorpha, was phylogenetically defined [15
Demospongiae represent the largest class of sponges [17
]. Their skeletons are made from proteins [18
], polysaccharides [19
], and/or inorganic components such as siliceous spicules [14
]. Demosponges are subdivided into three orders: Dendroceratida, Dictyoceratida, and Verongida. The skeletons of all of these orders exhibit spongin, a collagenous protein [22
Till now, spongin is not really well defined. Nevertheless, it has remarkable properties. Spongin is considered to be a halogenated scleroprotein [22
] of collagenous character. Spongin fibers are more resistant than collagen fibers against enzymatic digestion [23
]. Exposito et al. showed in genomic and cDNA studies that spongin possesses the typical collagen sequence motif Gly-Xaa-Yaa [24
]. Glycine is thus the most abundant amino acid found in spongin. Apart from glycine, the amino acids serine, arginine, lysine, valine and cysteine are reported to occur in spongin [25
]. Additionally, Ehrlich et al. showed that the polysaccharide chitin appears as an integral skeleton component in Verongida sponge skeletons [19
], forming a chitin scaffold of the same overall morphology as the integer skeleton.
It is not yet clear how spongin is connected with these chitin scaffolds in sponge skeletons. However, there are a number of examples of so-called chitin-protein complexes in nature showing that proteins are often strongly—mostly covalently—bound to chitin [27
]. Hackman suggested that proteins are covalently bound to chitin in different insects and crustaceans [27
]. Furthermore, Blackwell and Weih developed a model for the three-dimensional structure of an insect chitin-protein complex with chitin fibrils surrounded by layers of proteins [28
]. These examples lead to the assumption that spongin in the skeletons of the Verongida sponges is also strongly—maybe covalently—bound to the chitin scaffolds.
Furthermore, sponges of the order Verongida are well-known for the biosynthesis of characteristic bioactive natural products, especially brominated tyrosine derivatives like bastadins from Ianthella basta
], aerothionin from Aplysina cavernicola
], or psammaplins from Aplysinella
]. These compounds are biosynthesized from brominated tyrosines [35
]. Initial investigations show that brominated substances are present even in the sponge skeletons [36
]. Since brominated tyrosines can inhibit chitinase activity [38
], these compounds could have a protective effect preventing undesired skeletal degradation.
Until now the full amino acid composition of sponges was only determined for three species. This includes the commercial unbleached sponge Hippospongia equina
) and the bath sponge Spongia officinalis obliqua
(S. officinalis obliqua
). Both of the sponges belong to the order Dictyoceratida [39
]. Furthermore, we have recently determined the skeletal amino acid composition of the Verongida sponge A. cavernicola
]. These studies revealed the presence of halogenated tyrosines in addition to non-halogenated amino acids. 3,5-Diiodotyrosine was found in H. equina
], while 3-Monoiodo-tyrosine, 3,5-Diiodotyrosine, and 3,5-Dibromotyrosine could be detected in S. officinalis obliqua
]. Compared to these two Dictyoceratida sponges, the Verongida sponge A. cavernicola
exhibits a surprising variety of halogenated, i.e., brominated, iodated, and chlorinated amino acids [41
The goal of the present study is to examine the amino acid composition of the sponge skeleton of another Verongida sponge and to compare it with the other three known amino acid compositions of sponges. The results should reveal whether or not there is a significant difference between the Verongida sponges from two different families. This may encourage future investigations of further sponge species.
The order Verongida comprises four families distinguished mainly by the structure and composition of their spongin fibers [42
]. Aplysinidae is the largest verongid family (63 species from three genera: Aplysina
, and Aiolochroia
). This family is defined by an anastomosing fiber skeleton with both pith and bark elements [18
]. Ianthellidae is the second largest verongid family (12 species from three genera: Ianthella
, and Hexadella
). This family is distinguished from other Verongida families by the presence of eurypylous choanocyte chambers. Aplysinellidae consists of nine species from three genera (Aplysinella
, 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
], 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
3. Materials and Methods
3.1. 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
3.2. Extraction of the Skeletons
skeletons were isolated following the protocol described previously [41
]. Small pieces of I. basta
) 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.
3.3. 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.
3.4. 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.
3.5. 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.
3.6. 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.
3.7. 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 HNO3 (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 H3BO3 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 HNO3 (65%) and 2 mL H2O2 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.
3.8. 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 H2SO4 and centrifuged. The supernatant was removed and freeze dried.
3.9. 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 tR 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.