Marine sponges are classified into three classes: Calcarea, Hexactinellida and Demospongiae containing 25 orders,127 families and 790 genera. There are approximately 15,000 species reported worldwide ^[1,2,3,4]. Almost 1/3 marine natural products come from sponges ^[3,5]. In all the prevalent traditional ways to identify sponges,the most accurate and commonly used method is spicule identification,which examines sponge skeletal structure. This method routinely requires a whole piece of sponge and heavily depends on experts mastering with English,French and German and ages of experience ^[6]. Now the number of experts in the art of sponge identification is greatly reduced. Hence,it is necessary to establish more objective and simpler methods to discriminate marine sponges.

‘‘Multi-step infrared spectroscopy’’ including FT-IR,SD-IR and 2DCOS-IR has the above advantages for sponge identification. FT-IR has been proved to be a quick,simple and effective method with good signal-to-noise ratio and excellent repeatability to investigate complicated mixtures such as herbal medicine (HM) ^[7,8]. To delineate the overlapped spectra in FT-IR,second derivative infrared spectroscopy (SD-IR) is used to improve the apparent resolution ^[9]. If the differences are still too small to discriminate, two-dimensional correlation infrared spectroscopy (2DCOS-IR) can be adopted to illustrate FT-IR spectra in a second dimension to reveal the differences more convincingly ^[10,11].

Ten different sponges collected from Xisha Islands in South China Sea of two classes,five orders and ten species have been studied using the multi-step infrared spectroscopy to establish a new method to discriminate sponges in a more objective,direct and quicker manner. 2. Experimental

2.1. Apparatus

A spectrum GX FT-IR spectrometer (PerkinElmer,UK) equipped with a DTGS detector and a scanning range from 400 to 4000 cm^-1 with a 4 cm^-1 resolution was used. Spectra were calculated from a total of 32 scans at 0.2 cm/s of OPD speed. The interferences of H2O and CO₂ were eliminated during scanning. A CKW-II programmable temperature controller (Beijing Chaoyang Automatic Instrument Co.,China) was employed to perform the thermal perturbation from the range 50°C to 120°C. Spectra were collected at 10°C intervals. The second derivative IR spectra were gained after 13-point smoothing of the IR spectra by Savitzky-Golay polynomial fitting. Two-dimensional IR correlation spectra were obtained after using 2D correlation analysis software (developed by IR Lab,Tsinghua University) to analyze the series of thermo perturbation dynamic spectra. 2.2. Samples and reagents

Sponges (Table 1) were obtained from Xisha Islands in South China Sea in April 2007 and 2009 (kept in Department of Pharmacy, Changzheng Hospital,Second Military Medical University) and were authenticated by Prof. Jinhe Li,Qingdao Institute of Oceanology,Chinese Academy of Sciences. KBr was bought from Sigma (St. Louis,MO,USA).

Table 1

Table 1 Ten China sponges collected from Xisha Islands of South China Sea.

Table 1 Ten China sponges collected from Xisha Islands of South China Sea.

Sponge samples were desiccated in vacuo and then were grounded into powder before IR measurement. Each sponge sample (about 1-2 mg) was mixed with KBr (100 mg),grounded into powder (200 mesh),and then pressed into a tablet. 3. Results and discussion 3.1. Analysis and discrimination of five sponges from two classes

Fig. 1 shows the IR spectra of five sponges belonging to two classes. In these five sponges,Leucandrasp. is a calcareous sponge and is collected for the first time in China. The main composition of Leucandrasp. is CaCO₃ with five peaks at 2523,1803,1418,874, 715 cm^-1 . The other four sponges are demosponges and their main compositions are proteins with two characteristic absorption bands peaks: Amide I (1650 cm^-1) and amide II (1535 cm^-1). The attribution of main absorption peaks of the five sponges is summarized in Table 2.

Fig. 1

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Fig. 1. IR spectra of five sponges from different classes and orders:Aplysinopsissp. (AP),Hyrtiossp. (HY),Hippospongia lachna(HI),Mycale fibrexilis(MY) andLeucandrasp. (LA).

Table 2

Table 2 Preliminary assignment of main peaks in FT-IR spectra of five sponges.

Table 2 Preliminary assignment of main peaks in FT-IR spectra of five sponges.

The IR spectra of the four demosponges,from three orders,four families,are different according to the group-peak matching technique ^[12,13]. The peaks at 1422,875,712 cm^-1 and 1082, 855,712,699 cm^-1 of AP suggest that it contains calcium carbonate of both calcite and aragonite forms. The peaks at 873 cm^-1 and 2522,1468,1082,855,712,699 cm^-1 of HI indicate that HI contains more aragonite than calcite. HY (1080,873,855, 712,699 cm^-1) and MY (878,707 cm^-1) contains both forms of CaCO₃ but with lower contents. Thus,the four demosponges from different five orders can be distinguished readily (Fig. 2).

Fig. 2

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Fig. 2. SD-IR spectra of five sponges from different classes and orders:Aplysinopsissp. (AP),Hyrtiossp. (HY),Hippospongia lachna(HI),Mycale fibrexilis(MY) andLeucandrasp. (LA).

By spicule identification,LA and LO belong to the same order (class Calcarea,order Leucosolenida),but their exact species cannot be identified. CaCO₃ is the main component of the two sponges. LA can be easily differentiated from LO through comparing the peak profiles in the range of 2000-1000 cm^-1 (Figs. 3 and 4). The peaks at 1514 and 1043 cm^-1 of LA are evidently higher than those of LO and the peak at 1412 cm^-1 of LO is stronger than that of LA. Hence,the two specimens can be easily discriminated.

Fig. 3

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Fig. 3. IR spectra of two calcareous sponges from the same order Leucosolenida: Leucandrasp. (LA) andLeuconiasp. (LO).

Fig. 4

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Fig. 4. SD-IR spectra of two calcareous sponges from the same order Leucosolenida:Leucandrasp. (LA) andLeuconiasp. (LO).

HA belongs to the same order with PA,AC1 and AC2. PA is in the same family with AC1 and AC2. AC1 and AC2 are in the same genus (Table 1). The exact species of AC1 and AC2 cannot be discriminated for the inherent limitations of spicule identification ^[14,15]. Two strong absorption bands,amide I (1649 cm^-1) and amide II (1536 cm^-1) (Fig. 5),suggest the existence of abundant proteins. The peaks at 1113 and 874 cm^-1 of HA clearly differ from those of PH,AC1 and AC2. The peak at 1081 cm^-1 of PH is stronger than that of AC1 and AC2. AC1 and AC2 have similar spectral profile in the range of 1800-1300 cm^-1 with small variations: a sharp peak at 1238 cm^-1 in AC1 can be seen clearly other than that in AC2. Thus,these four sponges can be distinguished and even their species can be accurately identified if standard specimens are available.

Fig. 5

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Fig. 5. IR spectra of four sponges from the same order Halichondrida: Halichondrisp. (HA),Phakellia fusca(PH),Acanthellasp1 (AC1) andAcanthellasp2 (AC2).

Mistakes can occur in traditional spicule identification when the characteristics of sponge spicules are similar ^[14]. With regards to IR identification,if two sponges have close IR spectra,second derivative IR spectroscopy (SD-IR) with higher resolution can be applied to show the minor differences in FT-IR spectra ^[9]. The IR spectra of AC1 and AC2 are similar except for the absorption band around 1238 cm^-1 . In order to discriminate the two sponges more clearly,SD-IR and 2DCOS-IR are used to analyze AC1 and AC2 ^[15]. In SD-IR spectra,overlapped peaks can be divided into two or more peaks and thus hidden peaks can be observed. Specifically,two peaks at 1236 and 875 cm^-1 of AC1 are stronger than those of AC2 in SD-IR (Fig. 6).

Fig. 6

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Fig. 6. Second derivative IR spectra of four sponges from the same order Halichondrida:Halichondrisp. (HA),Phakellia fusca(PH),Acanthellasp1 (AC1) andAcanthellasp2 (AC2) in the range of 1800-600 cm-1 .

A more accurate approach,2DCOS-IR synchronous spectroscopy is used to clearly differentiate the two sponges in the ranges of 900-1300 cm^-1 and 1300-1500 cm^-1 . The auto-peaks on the diagonal line in synchronous IR spectrum show the susceptibility and self-correlativity of certain absorption bands,which cause the change of spectral intensity with thermal treatment. While the cross-peaks at the off-diagonal locations display the relativity of intensity variations of a pair of group vibrations corresponding to their frequencies. The closer the correlativity is,the stronger the intensity of cross-peak will be ^[16]. Positive correlation (red/green area) in 2DCOS-IR spectra indicates that a group of absorption bands change simultaneously (either stronger or weaker),while negative correlation (blue area) suggests the opposite.

For easy comparison,detail information (positions,relative intensities and correlations) of the autopeaks of AC1 and AC2 in Fig. 7 is summarized in Table 3. Six strong autopeaks with the strongest at 980 cm^-1 can be observed in the 2DCOS-IR spectrum of AC2 in the range of 900-1300 cm^-1 while only two peaks with 1110 cm^-1 being the strongest can be seen in AC1 spectrum. However,AC1 and AC2 both have two strong autopeaks despite of small differences in the range of 1300-1500 cm^-1 . Based on above observations,the unique fingerprints of AC1 and AC2 in the range of 900-1300 cm^-1 can be used as exclusive features to discriminate the two sponges.

Fig. 7

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Fig. 7. 2DCOS-IR synchronous spectra of (a)Acanthellasp1 (AC1) and (b)Acanthellasp2 (AC2) in the range of 900-1300 cm-1 (I) and 1300-1500 cm-1 (II).

Table 3

Table 3 Strong auto-peaks in 2DCOS-IR synchronous spectra ofAcanthellasp1 (AC1) and (b) Acanthellasp2 (AC2).

Table 3 Strong auto-peaks in 2DCOS-IR synchronous spectra ofAcanthellasp1 (AC1) and (b) Acanthellasp2 (AC2).

The multi-step infrared spectroscopy allowed ten sponges to be discriminated rapidly. Sponges from different genus,different classes and different orders have their unique IR macro-fingerprints. If the IR spectra of standard specimens could be acquired, even the exact species of sponges could be accurately identified. Meanwhile,SD-IR and 2DCOS-IR have been applied to successfully distinguish the subtle differences of sponges with similar chemical profiles. It has been demonstrated that the multi-step infrared spectroscopy can be a promising objective method for rapid and accurate identification of marine sponges.

Acknowledgments

This research was supported by the National Natural Science Fund for Distinguished Young Scholars of China (No. 81225023), the National Natural Science Fund of China (Nos. 41476121, 81302691,81172978),the Innovation Program of Shanghai Municipal Education Commission (No. 14YZ037) and partially supported by Shanghai Subject Chief Scientist (No. 12XD1400200). We are also grateful for the financial support of the National High Technology Research and Development Program of China (863 Projects,No. 2013AA092902).