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1 respectable 2019 A brief Description of surface ar Structure and also Composition of the Pseudo-Snail Shell created by a Sea Anemone Stylobates sp. Symbiotic through Hermit Crabs native the Deep-Sea Floor
Akihiro Yoshikawa, Shiori Nakazawa, Akira Asakura
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Akihiro Yoshikawa,1 Shiori Nakazawa,2,* Akira Asakura1 11Seto Marine biological Laboratory, ar Science Education and also Research Center, Kyoto University, 4 22Sugashima Marine organic Laboratory, Graduate school of Science, Nagoya University, 429-63 Sugas *Current affiliation: Hitachi, Ltd. Study and advance Group, 2520 Akanuma, Hatoyama, Hiki, Saitama 350-0395, Japan
Zoological Science, 36(4):284-293 (2019). Https://doi.org/10.2108/zs180167
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Abstract

Sea anemones belonging come the genera Adamsia and Stylobates have a exceptional symbiotic relationship with hermit crabs. This symbiotic sea anemones create a shell-like structure, called a “carcinoecium,” that covers and extends end the gastropod covering of the host hermit crab together hermit crabs grow. This structure has been explained as “chitinous carcinoecium” or “chitinous coating.” A previous research investigated carcinoecia the Stylobates aeneus, the results of which shown that it included at the very least 1.7% chitin, while the remaining contents were unidentified. Moreover, the microscopic structure of a carcinoecium still remains to be detailed. We, therefore, conducted detailed monitorings using a stereoscopic microscope and scanning electron microscope (SEM) and the analyses that the chemical composition the carcinoecia produced by Stylobates sp. (apparently conspecific v Isadamsia sp. “J” report in Uchida and Soyama, 2001) associated with a pagurid hermit crab Pagurodofleinia doederleini collected in the south of the Shima Peninsula, Mie, Honshu Island, Japan at a depth the 294–306 m. Our results indicate that carcinoecia that Stylobates sp. Contain HCl-soluble contents (13%), NaOH-soluble components (38%), chitin (11%) and unidentified remnants (39%). Additionally, our observations display that Stylobates sp. Incorporates dark- and white-colored particles that could be sand and/or mud into the carcinoecium.


INTRODUCTION

Symbiosis is a widespread phenomenon in the sea, and ecological partnership in the synergy is one of major forces of evolution of interspecific interaction and shaping marine communities. Decapod crustaceans room a major component in naval community, and, in particular, hermit crabs are recognized to have actually a wide selection of symbiotic relationships with various other invertebrates (Williams and McDermott, 2004; Goto et al., 2006; Yoshikawa et al., 2018a).

The symbiotic relationship between hermit crabs and also sea anemones is a well-known example of mutualism in the sea. Many species of hermit crabs lug symbiotic sea anemones on their chelipeds or the outside surfaces the the shells castle inhabit (Williams and MacDermott, 2004; Antoniadou et al., 2013). The services for hold hermit crabs are largely protection indigenous predators, such as cephalopods and also fish, by the nematocysts of sea anemones (Ross, 1971; Balasch and also Mengual, 1973; Ross and Boletzky, 1979; McLean, 1983) and use that the sea anemones together stock food (Imafuku et al., 2000). The symbiotic sea anemones additionally benefit from their association through hermit crabs, together the sea anemones gain accessibility to food resources (Stachowitsch, 1979, 1980), substratum accessibility (Conover, 1979; Brooks, 1989), defense from predators, and also increased dispersal (Balss, 1924; Jonsson et al., 2001; McLean and Mariscal, 1973). Due to the fact that both hermit crabs and also sea anemones get benefits from their symbiotic associations, those relationships have been recognized as “mutualism” (Antoniadou et al., 2013). Some species of hermit crabs have the capacity to transport the symbiotic anemones ~ above their new shells once they change shells (Ross, 1974, 1975; Yoshikawa et al., 2018b).

A amazing mutualistic partnership is seen in between sea anemones of the genera Adamsia Forbes, 1840, and Stylobates Dall, 1903 and hermit crabs. These anemones work out on gastropod shells occupied by hermit crabs and also produce a bronze-colored shell-like framework that very first covers, and then extends over the host hermit crab covering as the host hermit crab grows (Dunn and Liberman, 1983; Antoniadou et al., 2013). Both parties advantage from this symbiotic relationship; the hermit crab could not need to seek new, larger shells together it grows, and also the anemone does not have actually to change the substrate. The safety “shell” developed by the anemone, the carcinoecium, has actually been described previously together “chitinous carcinoecium” or “chitinous coating” (Antoniadou et al., 2013; Crowther et al., 2011; Dunn et al., 1980; Fautin, 1987). The carcinoecium the Stylobates aeneus Dall, 1903 has actually been report to save chitin, but this represents just 1.7% of the total components (Dunn and Liberman, 1983). Come date, the remaining materials of carcinoecia have remained unidentified, meriting further detailed investigation.

During a survey that was conducted on benthic organisms approximately Kumano-Nada, south of the Shima Peninsula, Mie, Honshu Island, Japan (at a depth the 294–306 m), we accumulated specimens of a species of Stylobates sp. Found on shells provided by a pagurid hermit crab Pagurodofleinia doederleini (Doflein, 1902) (Fig. 1). This sea anemone is conspecific with Isadamsia sp. J reported in Uchida and also Soyama (2001) and also Yanagi (2006) judged native morphological personalities including body shape and also coloration and also direction the the mouth (Fig. 1A). That is unique, unlike various other Stylobates species, in producing only one extension section of the lip of one aperture of a gastropod shell inhabited by a hermit crab (Fig. 1B, C). Because Isadamsia is now a small synonym the Stylobates (Dunn et al., 1980), we hereinafter refer to this sea anemone together Stylobates sp.

Our research concentrated on describing the surface ar structure and the chemical materials of carcinoecia developed by Stylobates sp. We observed the surface framework of carcinoecia making use of stereoscopic microscope and scanning electron microscopic lense (SEM). This is the very first description that the surface structure of the carcinoecia component with high magnification pictures from SEM. In addition, we likewise investigated the components by extract minerals and proteins from shells based on the methods for chitin isolation native crustacean shells (Younes and Rinaudo, 2015). We then evidenced the visibility of proteins and chitin in carcinoecia by protein analysis and chitinase treatment.


MATERIALS and also METHODS


Field sampling and also preparation of product for analysis

Specimens the the hermit crab P. Doederleini were built up by beam trawl from the southern of the Shima Peninsula, Mie, Honshu Island, Japan, on 7 November 2017 (depth 294–306 m; 34°07.56N, 136°40.47E come 34°07.91N, 136°39.44E), and those bearing Stylobates sp. Sea anemones were offered for chemical analysis.

In the laboratory, specimens that Stylobates sp. Were eliminated from the gastropod shells. Three gastropod shells, every bearing a carcinoecium, were dried at room temperature (25°C) for 12 h.

Once dry, the bronze-colored carcinoecia, which solely contain the prolonged parts the carcinoecia, were carefully removed native the gastropod shells and powdered utilizing a pestle and mortar. Because Stylobates sp. Expand the section of the lip indigenous the aperture the a snail shell (Fig. 1B, C), we offered the extended parts for experiments to avoid pollution by the snail shell part. Roughly 100 mg the carcinoecium flour was divided into 4 1.5-ml tubes.

Table 1.

Specimens the Stylobates sp. Used for structural observation and evaluation of the chemical composition that carcinoecia, and also their host hermit crabs and gastropod shells. Every specimens were built up from Kumano-Nada, southern of the Shima Peninsula, Mie, Honshu Island, Japan (294–306 m deep) ~ above 7 Nov 2017 by beam trawl. * indicates the specimens provided for chemistry analysis. ** suggests the specimen offered for structural observation.

*

The surface framework of both inner and also outer sides and also partial cross-section were observed through the stereoscopic microscope. Additionally, the high-magnification photos of surface framework of carcinoecium were acquired by using a scanning electron microscopic lense (SEM). For SEM imaging, we fixed the dried carcinoecia to a phase with adhesive tape and coated them because that 1 min v platinum utilizing an auto-fine ion coater (QUICK AUTO COATER SC-701AT, Sanyu Electron Co., Ltd., Tokyo, Japan). SEM images were derived by an SS-550 scanning electron microscopic lense (Shimadzu) in ~ an increased voltage that 15 kV under high vacuum.


Stylobates sp. And its carcinoecium connected with Pagurodofleinia doederleini built up by beam trawl from the south of the Shima Peninsula, Mie, Honshu Island, Japan, on 7 November 2017 (depth 294–306 m; 34°07.56N, 136°40.47E to 34°07.91N, 136°39.44E). (A) the gastropod snail covering covered through Stylobates sp.; (B) and also (C) the gastropod covering (pale fancy portion) and also the carcinoecium section as an expansion of a lip of the aperture that the gastropod shell (black portion) (B, Voucher ID, SMBL-V00002; C, Voucher ID, SMBL-V00003). Range bar = 5 mm.


Investigation the carcinoecium components

For demineralization (Younes and also Rinaudo, 2015), 1000 µl of 1 M HCl was added to the powdered carcinoecium, the samples were centrifuged in ~ 3000 × g because that 1 min, and also the supernatant to be discarded. This procedure was recurring until the supernatant became strongly acidic. ~ confirming that the pH the the supernatant had dropped come pH 1 or 2 using indicator paper (UNIV, 07011030, Toyo Roshi Kaisha, Ltd., Tokyo, Japan), the powdered samples to be resuspended in acetone and also dried in a centrifugal concentrator because that 10 min. Finally, the load of the sample to be measured.


Surface and also partial cross-section the the carcinoecium developed by Stylobates sp. (part of SMBL-V00002) observed by the stereoscopic microscope. (A) photo of the external surface; (B) pictures of the within surface; (C) unidentified biological-organisms suggested by “I” in (B); (D) had foraminiferans, Elphidiidae gen. Et sp. Indet., suggested by “II” in (B); (E) fragment of diatoms, Coscinodiscaceae gen. Et sp. Indet., in the carcinoecium; (F) the partial cross-section of the carcinoecium; (G) the enlarged see of the partial cross-section shown by white square in (F).


For protein contents analysis, 1000 µl 1 M NaOH was included to the HCl-extraction residue to extract crude proteins (Younes and also Rinaudo, 2015). The samples to be centrifuged in ~ 3000 × g for 1 min and the supernatant to be discarded; 1000 µl NaOH was added and the sample to be agitated at 60°C because that 60 min with moderate mixing. After 60 min, the samples to be centrifuged at 3000 × g for 1 min and also the supernatant was discarded. This process was repeated 5 times. The samples were then resuspended in 1000 µl deionized water (DW) to wash them, centrifuged at 3000 × g because that 1 min, and the supernatant was discarded. After washing with DW, the samples to be resuspended in acetone and dried in a centrifugal concentrator because that 10 min. Finally, the to decrease in load was measured.


Biuret test and also xanthoproteic reaction

The NaOH extract was neutralized with 35% hydrochloric acid and desalted utilizing a gel filtration shaft (PD-10, 17085101; GE Healthcare, Chicago, IL, USA), follow to the manufacturer"s gravity circulation protocol. It was eluted v 3.5 ml that ultrapure water, every 1 ml and the critical 0.5 ml that which to be pooled together fractions 1, 2, 3, and 4. Ultrapure water and 0.1% skimmed milk systems (Morinaga Milk industry Co., LTD., Tokyo, Japan) fountain were based on a biuret test by mixing 100 µl sample equipment with 5 µl 1 M salt hydroxide and 2 µl 1% copper (II) sulfate. To intensify the color, the mixtures to be condensed through a centrifugal concentrator.


Scanning electron microscopic lense (SEM) images of the external surface the the carcinoecium produced by Stylobates sp. (part that SMBL-V00002). (A) surface structure (× 20); (B) and also (C) surface structure (× 50); (D) and (E) included-sand top top the carcinoecium (× 200); (F) included-materials covered by the substance created by Stylobates sp. (× 100).


For the xanthoproteic reaction, 50 µl of the eluates indigenous the gel filtration were blended with one autumn of nitric acid (approximately 60%), heated in ~ 95°C for 15 min, then neutralized through 50 µl 10 M sodium hydroxide.


SDS-PAGE and also Coomassie brilliant Blue (CBB) staining

The NaOH extracts were neutralized with 35% hydrochloric acid, desalted by including an equal volume the water and also a 6-fold volume the ice-cold acetone, incubated in ~ –20°C because that 2 h, and also centrifuged at 15,000 rpm because that 10 min for acetone precipitation. Desalted NaOH extracts were dissolved in 6 M urea, combined with sodium dodecyl sulfate (SDS) sample buffer (final concentration: 10% glycerol, 2% SDS, 0.1% bromophenol blue, and also 50 mM Tris-HCl at pH 6.8) through or there is no 1% by volume 2-mercaptoethanol, heated at 95°C, and also subjected come SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 5%–20% gradient polyacrylamide gelatin (E-T520L; Atto Corporation, Tokyo, Japan) with a protein molecular load marker (26614; Thermo Fisher Scientific, Waltham, MA, USA). The gelatin was then equilibrated through 10% acetic acid and 50% methanol, stained in 0.25% CBB-R250, 5% acetic acid, and also 50% methanol because that 2 h, and finally destained in 7% acetic acid and also 25% methanol.


Liquid chromatography/mass spectrometry (LC/MS)

CBB-stained protein bands were excised from the polyacrylamide gel and also subjected come in-gel digestion, LC/MS, and a database search, as defined previously (Nakazawa et al., 2015). Briefly, samples were reduced with dithiothreitol and also carbamidomethylated and carbamidomethylated, digested v modified trypsin (V511A; Promega, Madison, WI, USA), be separated in one octadecyl silica gel pillar over a gradient the acetonitrile containing 0.1% formic acid, and analyzed through a fixed spectrometer (LTQ-XL; Thermo Fisher Scientific) equipped with an electrospray ionization unit. The spectra to be compared against a database the 105,046 Actiniaria protein sequences retrieved from the NCBI Protein database on January 16, 2018, and a database that 556,388 SwissProt database entries retrieved on December 26, 2017, utilizing Mascot ver 2.4.1 software. Hits through protein scores the 40 or much more were pertained to as “detected.” Tryptic digests that 100 fmol bovine serum albumin (BSA) offered protein scores the 522 and 683 in two repeated scans performed just prior to the evaluation of the NaOH extracts.


Scanning electron microscopic lense (SEM) images of the inner surface ar of the carcinoecium created by Stylobates sp. (part that SMBL-V00002). (A) surface framework (× 20); (B) and also (C) surface structure with included sand top top the carcinoecium (× 50); (D) included-sand on the carcinoecium; (E) included-materials covered by the substance produced by Stylobates sp. (× 100); (F) semi exposed-materials native substance produced by Stylobates sp. (× 200).


Chitinase treatment

For the chitinase treatment, us used component of the residue after ~ NaOH treatment and also divided the samples into four 1.5-ml tubes to boost reaction efficiency. Two of the aliquots to be treated through chitinase and also two were chitinase-free blanks ( Supplementary Table S1 (10.2108.zsj.36.284.s1.pdf)). First, the samples were made colloidal with 35% hydrochloric acid and neutralized v 10 M NaOH. Then, 1000 µl DW was added, the samples to be centrifuged at 3000 ×g for 1 min, and also the supernatant to be discarded. We changed the supernatant with 1000 µl chitinase buffer A (25 mM Tris-HCl pH 7.5, 1 mM Ethylene-diaminetetraacetic mountain (EDTA), 25 mM sodium chloride) (Oku and Ishikawa, 2014). Then, 150 µl chitinase (Thermostable, Recombinant, Solution. 034-19891 Wako, lot of WDP4902 , activity 140 units/ml) was included to one of the split samples, and nothing to the other. We maintained both samples at 85°C because that 8 d. After 8 d, we resuspended the samples in acetone and also dried them making use of a centrifugal concentrator because that 10 min. Finally, the load was measured.


Sample deposition

All Stylobates sp. Specimens, along with their hold hermit crabs and also shells as well as the residual water from every experiments, to be deposited in the Seto Marine biological Laboratory, Kyoto University, v the catalog numbers (Table 1 and Supplementary Table S1 (10.2108.zsj.36.284.s1.pdf)).


Microscopic structure of a carcinoecium

The carcinoecium was thinner 보다 the snail covering from i beg your pardon it prolonged (approx. 0.5 come 1.0 mm) (Fig. 2F, G), and also the development rings were recognized on the surface. The carcinoecium included many dark- and also white-colored particles consisting of sand and foraminiferans (Elphidiidae Galloway, 1933 gen. Et sp. Indet.) (Fig. 2A, B, D), fragment the unidentified biology (Fig. 2C) and diatoms (Coscinodiscaceae Küt-zing, 1844 gen. Et sp. Indet.) (Fig. 2E). These materials were uniformly scattered quite than gift patchy top top the carcinoecium. Layered frameworks as it was observed in gastropod snail shells were absent in the carcinoecium (Fig. 2F, G). In the SEM images, the external surface of carcinoecium was smoother than the inner surface was. SEM photos showed the the consisted of materials were covered by the substances developed by Stylobates sp. (Figs. 3, 4). In the within surface, an ext exposed particles were observed 보다 in the outer surface of the carcinoecium (Fig. 4).


Carcinoecium components

We obtained a full of 385.36 mg that carcinoecium powder. The quantity of carcinoecium powder and the to reduce in weight after each treatment are shown in Supplementary Table S2 (10.2108.zsj.36.284.s1.pdf). The complete decreases in weight after HCl treatment for demineralization and also NaOH therapy for crude oil protein extraction to be 49.27 mg and 144.79 mg, respectively, leaving a residue that 191.30 mg. Thus, the carcinoecium consisted of 13% HCl-soluble component and also 38% NaOH-soluble ingredient (crude protein). The carcinoecium vigorously released bubbles when exposed come HCl. The residual 49.64% to be examined next.

Of the 191.30 mg residue derived after NaOH treatment, 55.01 mg was provided to test for the presence of chitin (28.69 mg because that chitinase treatment and 26.32 mg for chitinase-free control). The weights that the samples before and after chitinase therapy are shown in Supplementary Table S1 (10.2108.zsj.36.284.s1.pdf). The decreases in sample weights ~ chitinase therapy were 7.12 mg (chitinase 1), 0.94 mg (chitinase-free 1, blank), 0.86 mg (chitinase 2, 10-fold dilution), and also 0.74 mg (chitinase-free 2, blank) ( Supplementary Table S1 (10.2108.zsj.36.284.s1.pdf)). Therefore, the 28.69 mg the residue after ~ NaOH treatment had at least 6.30 mg (22%) chitin. Thus, the carcinoecium had at the very least 11% of chitin and also 39% of various other unidentified components. The unidentified components included insoluble dark- and also white-colored corpuscle (Fig. 5A, B).

To summarize, our preliminary investigation indicated that the carcinoecium consisted of 13% HCl-soluble component, 38% NaOH-soluble ingredient (crude protein), 11% chitin, and also 39% other unidentified components.


Protein analyses

The biuret test and also the xanthoproteic reaction caused reddish-purple and yellow coloration, respectively (Fig. 6A, B), denote the visibility of proteins in the NaOH extracts. The extracts to be desalted and subjected to SDS-PAGE to assess the component proteins. Most influential were two bands at around 10 × 103 Da and 15 × 103 Da, attach by a long smear (Fig. 7). The two bands were subjected to LC/MS, back no proteins other than the trypsin offered in the in-gel digestion procedure to be detected.


Photographs of the unidentified remnants ~ HCl treatment, NaOH treatment, and chitinase treatment. (A) remnants on a black color background; (B) remnants ~ above a white background (white and also black bars show 500 µm).


Results the protein analyses. (A) Biuret test of alkaline extracts gave reddish-purple colors, denote the visibility of proteins. Ultrapure water and 0.1% skimmed milk solution were used as negative and positive controls, respectively; (B) xanthoproteic reaction that the alkaline extracts gave yellow colors, denote the visibility of proteins. Ultrapure water was used as a an unfavorable control and 0.1% BSA solution and 0.1% skimmed milk equipment were used as optimistic controls.


Presence of protein

The NaOH carcinoecium extract registered optimistic for proteins/peptides to the same level as 0.1% skimmed milk solution and also 0.1% BSA solution in biuret test, xanthoproteic reaction, and CBB staining. This clearly indicates the the carcinoecium created by Stylobates sp. Has a comprehensive amount the protein (maximum 38%) and is the an initial evidence of the existence of protein in the carcinoecium, supporting the forecast of Dunn and also Liberman (1983) based upon research ~ above S. Aeneus.

However, ours data disclose that the relationship of protein is much less than the 98% guess by Dunn and also Liberman (1983) and also that the carcinoecium consists of at the very least 23% non-protein components (HCl-extractable components and chitin). One possible reason for the discrepancy in the contents of chitin and proteins in this study and also in that carried out by Dunn and also Liberman (1983) may be as result of the newly cloned chitinase, which was unavailable at the time, and/or the radical conditions for HCl treatment and NaOH treatment. These various approaches would have enabled much more detailed elucidation of the ingredient of the carcinoecium. However, because our qualitative evaluation could only suggest that NaOH-extractable contents contain some amount of proteins, future investigation of the considerable profiling that the small compounds that carcinoecia including alkaline-soluble link is also needed.


Protein identification

Likely due to the bad solubility the NaOH-extracted proteins under neutral and acidic conditions, virtually no peptides to be detected through LC/MS; the proteins and also peptides would have actually aggregated during desalting, in-gel digestion, or LC and also thus, would have been diminished to very low levels because that mass spectrometry. The smear signal at low molecular weight can have been the protein degraded during HCl- and NaOH-extraction. The solubility the the peptides in the SDS-PAGE sample buffer, the in the in-gel digest extraction equipment (3% formic acid/50% acetonitrile), and that in the resolubilizing equipment for LC (0.1% formic acid/water) entirely differ from every other. Therefore, it is possible that the proteins to be soluble during electrophoresis but insoluble at the phase of LC/MS.

Otherwise, there was no synchronized protein through the referral protein databases, except trypsin; the might also be since one or the 2 low molecular mass bands room the unidentified proteins. Therefore, top-down proteomics or protein sequencing if the amino terminus of the protein is exposed in ~ pH 8 may be options for identifying the compo-nent proteins.


Gel photo of carcinoecium be separated by SDS-PAGE and also stained with CBB-R250. Arrowheads suggest bands.


Presence the chitin

The current study indicates that the carcinoecium that Stylobates sp. Includes 11% chitin, despite Dunn and Liberman (1983) uncovered that the carcinoecium that S. Aeneus had at least 1.7% chitin. Dunn and also Liberman (1983) confirmed the straight evidence that the existence of chitin in the carcinoecium with infrared absorption spectra, yet the section of chitin stays as a issue to it is in discussed. They conducted ultrasonic washing in sodium dodecyl sulfate (SDS) equipment to remove proteins, to wash the residues through deionized water and dried lock on a filter paper at 100°C to attain chitin which gave in 1.7% through weight. Deacetylation the chitin would not define this discrepancy due to the fact that they plainly detected the characteristics infrared absorption of the amide groups at about 1640 cm-1 and near 1540 cm-1 as observed in standard chitin, describe the acetyl group, i m sorry accounts for 19% that the molecular load of chitin, to be mostly current on your sample. Notably, materials like the unidentified remnants in our examine were not pointed out to be existing in their samples, return muds and diatoms shells must be resistant to SDS extraction. The absence of this materials suggests either that they may have actually lost a substantial amount that water-insoluble components through the attempt to gain chitin at an infrared-grade purity, or that the carcinoecium the S. Aeneus consists of different proportions the chitin, muds and other insoluble particles. Thus, the is feasible that the discrepancy is defined by species differences, and further research on carcinoecia of other Stylobates varieties is necessary to compare the contents of the chitin. However, our study has confirmed that, at the very least to some extent, the term “chitinous” is correct when offered to describe the carcinoecium.


Presence the HCl-soluble component

The carcinoecium formed by Stylobates sp. Contains 13% that HCl-extractable components. Although the exact composition of the HCl-soluble materials remains undefined, it would certainly contain a comprehensive amount that carbonate(s), offered that the carcinoecium strongly released bubbles once exposed to HCl and also that calcium lead carbonate is a common seawater-insoluble compound developed by animals. The ahead study also detected the existence of mineral (Ca, Si, Mg, Al Cu, and also several various other metals) (Dunn and also Liberman, 1983). In enhancement to that, our observation shows that dark/white-colored particles, foraminiferans, diatoms, and fragment the unidentified biological-organisms were consisted of on the surface of carcinoecium (Fig. 2); Stylobates sp. May construct the carcinoecium in part by incorporating products from outside environments.

There room no documents of mineralized hard skeletal axes and/or difficult materials from Actiniaria species, although part members that the phylum Cnidaria are able to produce hard, mineralized skeleton axes (e.g., some species of Octo-corallia and also Scleractinia) (Cairns and Macintyre, 1992; Dauphin, 2006; Martin and also Le Tissier, 1991; Tor, 1976). Further investigation will certainly be needed to understand the origin of HCl-soluble components of the carcinoecium part.


Possible components of unidentified remnants

After conducting the methods of prepare the pure chitin indigenous crustaceans (Younes and also Rinaudo, 2015), 39% that the shell continued to be unidentified in this study. The insoluble dark- and also white-colored particles (Fig. 5) and the surface framework (Fig. 2) likewise indicated that Stylobates sp. Incorporate sand, mud and also the remains of other organisms (foraminiferans, diatoms and fragment the unidentified organisms) right into the carcinoecium during formation. This silicate products would account for component of the unidentified remnants the were resistant to HCl, NaOH and chitinase.

Additionally, the unidentified remnants after this treatments could be likewise explained through incomplete extraction of supposed components and/or existence of contents we did no test for. One opportunity is that one or an ext treatments to be incomplete, leaving minerals, proteins, or chitin in the sample. An additional possibility is that the N-acetylglucosamine residual water in chitin were partly deacetylated in the alkaline therapy (Younes and also Rinaudo, 2015), bring about the ns of communication with chitinase. This would result in few of the chitin continuing to be undigested, and also although we demonstrated the the carcinoecium consisted of at least 11% chitin, the quantity of chitin in the unidentified fraction should be investigated further.


Surface framework of carcinoecium

The outer surface the the carcinoecium to be smoother than the within surface, and also many the the included-particles were covered by brown materials produced by the sea anemone. Thus, Stylobates sp. May produce the carcinoecium based on chitin and also proteins, proactively or passively combine other facets from the sea bed.

In other Stylobates species, many individuals totally cover the gastropod shells and extends the lip of that by a slim or thick carcinoecia (Crowther et al., 2011; Dunn et al., 1980; Fautin, 1987), when Stylobates sp. Just extends the part of the lip of one aperture the a gastropod covering by the reasonably thin carcinoecia (Uchida and also Soyama, 2001; Yanagi, 2006) with plenty of particles (Figs. 2–4). Perhaps, together the results of integrated particles, this species could strengthen very own carcinoecium.


The future of research on interspecific sport in the carcinoecium

Stylobates types live in a wide variety of habitats, native the continent shelf to deep sea environments (100 m to approximately 1000 m) in the Indo-Pacific Ocean, and also each species exhibits types specificity or preference for hold hermit crabs (Fautin, 1987; Crowther et al., 2011; Uchida and Soyama, 2001; Yanagi, 2006). The shell utilization pattern of hermit crabs different among varieties (Reese, 1969; Fotheringham, 1976; Imazu and also Asakura, 1994). The rigidity the the carcinoecium, which would certainly be impacted by the composition, for this reason may vary amongst Stylobates species. In addition, ours structural observation showed that carcinoecium contains many particles probably from the sea floor. It could work as strengthening the extension component and/or speeding up the creating speed, otherwise simply reflecting the environmental difference by passively combine the substrates ~ above the sea floor. Therefore, the intraspecific differences of carcinoecium depending on the benthic substrate may also exist. Future study in this field is essential to combine and/or verify (or to refute and extend) our observation of the chemical composition and also structural differences of carcinoecia created by different Stylobates species in various regions and at miscellaneous depths.

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ACKNOWLEDGMENTS

We room grateful come Captain Yoichi Maekawa, chief Officer, Toru Nakamura, and the crew the TRV Seisui-maru, Mie University, for their help in collecting the specimens. Us gratefully thank the following persons who supported our sampling and also field survey: Dr. Taeko Kimura and Mr. Shoichi Kimura (Graduate school of Biore-sources, Mie Univ.), and all participants to the joint survey for benthic biology conducted about Kumano-Nada, south of the Shima Peninsula, Mie, Honshu Island, Japan ~ above November 7–14, 2017. Us are likewise grateful to every one of the staff in Sugashima Marine biological Laboratory, Nagoya University and also for their assist in our experiments. We space grateful come Dr. Keisuke Yanagi (Coastal Branch the Natural background Museum and also institute, Chiba) for identification the the sea anemone provided in this study. We also gratefully thank to Mr. Kazuhisa Fujimoto (Wakayama Prefectural Educational center Manabi-no-Oka) for observation of surface structure of carcinoecia v a scanning electron microscopic lense (SEM).