The amphipod Hirondellea gigas inhabits the deepest regions of the oceans in extreme high-pressure conditions. However, the mechanisms by which this amphipod adapts to its high-pressure environment remain unknown. In this study, we investigated the elemental content of the exoskeleton of H. gigas specimens captured from the deepest points of the Mariana Trench. The H. gigas exoskeleton contained aluminum, as well as a major amount of calcium carbonate. Unlike other (accumulated) metals, aluminum was distributed on the surface of the exoskeleton. To investigate how H. gigas obtains aluminum, we conducted a metabolome analysis and found that gluconic acid/gluconolactone was capable of extracting metals from the sediment under the habitat conditions of H. gigas. The extracted aluminum ions are transformed into the gel state of aluminum hydroxide in alkaline seawater, and this gel covers the body to protect the amphipod. This aluminum gel is a good material for adaptation to such high-pressure environments.

Copyright: © 2019 Kobayashi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this manuscript, we examined the chemical components of the exoskeleton of H. gigas specimens, which are affected by extra-high pressure. We identified aluminum and calcium carbonates in the exoskeleton though electron microscopy analysis. Aluminum is rarely present in seawater but present in large amounts in sediments. Thus, we examined the means by which H. gigas obtains aluminum from the sediment and found that gluconolactone/gluconic acid acts as the extractant of aluminum. Through a pressure experiment using H. gigas exoskeletons, we found that the aluminum protected calcium carbonate from the effects of high pressure.

The deepest bottom of the ocean is an extreme environment characterized by extra-high pressures, low temperatures, and oligotrophy, and few animals can adapt to such extreme environments [ 1 – 3 ]. The amphipod Hirondellea gigas is a resident of the deepest points of the Mariana Trench (Challenger Deep), the Philippine Trench, the Izu-Ogasawara Trench, and the Japan Trench, where it inhabits depths greater than 8,000 m [ 4 – 8 ]. We attempted to widely capture marine creatures using baited traps along deep-sea points, but these amphipods were the only catch [ 5 , 6 ]. H. gigas produces a number of polysaccharide hydrolases as digestive enzymes and survives in oligotrophic environments by obtaining sugars from degradable plant debris by using such enzymes [ 6 , 7 ]. However, the adaptation system to extra-high pressure is still unknown. The extra-high pressure in the deep sea affects the various chemical components of organisms. Calcium carbonate is an important component of crustacean exoskeletons; however, this component dissolves in seawater deeper than approximately 4,000–5,000 m (carbonate compensation depth, CCD) [ 9 ]; furthermore, crustaceans cannot migrate on the deep-sea floor below the CCD [ 10 , 11 ]. Recently, a few species of crustaceans and foraminifera have been found in regions slightly deeper than the CCD [ 12 – 14 ]. Moreover, many foraminifera found at the bottom of the Challenger Deep are organic-walled allogromiids, which do not have a calcareous wall [ 15 ]. Because the habitable zone of H. gigas is at depths greater than 8,000 m, this species uses very little calcium carbonate in its exoskeleton. However, the mechanisms by which H. gigas adapts to its high-pressure environment remain unknown.

We obtained permission from the government of the Federated States of Micronesia to capture animals from the Mariana Trench (PERMIT NO. FM09-RV00083RS-01). Because the Izu-Ogasawara Trench is located in the exclusive economic zone (EEZ) of Japan, permission to catch animals at this Trench was not necessary.

We cut out the exoskeleton from a H. gigas body and divided the exoskeleton into two pieces. One piece was washed twice with ice-cold DDW to remove adhered Al gel. The other piece was washed twice with ice-cold artificial seawater (NaCl, 20.7 g; MgCl 2 6H 2 O, 9.4 g; CaCl 2 2H 2 O, 1.3 g; Na 2 SO 4 , 3.5 g; KCl, 0.6 g; NaHCO 3 , 0.17 g; KBr, 85 mg; Na 2 B 4 O 7 10H 2 O, 34 mg; SrCl 2 , 12 mg; NaF, 3 mg; LiCl, 1 mg; other trace elements, <0.1 mg per liter) (Nihon Pharmaceutical Co., Ltd, Tokyo, Japan). A piece of exoskeleton was placed into a 1.6 ml plastic tube filled with ice-cold artificial seawater. After sealing with Parafilm M (Bemis Flexible Packaging, Neenah, WI, USA), we pressurized the packed samples to 100 MPa for 24 h at 2°C. Then, we measured the Ca ion concentration dissolved in the artificial seawater with Ca ion assay kit (OCPC) (Metallogenics Co., Ltd, Chiba, Japan) [ 17 ].

A sample volume of 0.1 ml was mixed with sediment from the Mariana Trench (0.1 g dry weight) in 0.1 M sodium acetate buffer (pH 5.0). We incubated the mixture for 2 h at 4°C and 100 MPa. After centrifugation of the mixture (5,000 x g, 10 min, 4°C), the aluminum content of the supernatant was measured.

We crushed and mashed an H. gigas individual with a BioMasher II (Nippi Inc., Tokyo, Japan). The mashed sample was centrifuged (3,000 x g, 10 min 4°C), and a body fluid sample was collected. The precipitate was washed with 0.2 ml of DDW, and the supernatant was collected and combined with the body fluid sample after centrifugation (3,000 x g, 10 min 4°C). The combined sample was filtered to remove protein using an Amicon Ultra 3K device (Merck, Darmstadt, Germany). The pH and volume of the filtered sample were adjusted to 8.0 with 0.1 N NaOH and 0.5 ml, respectively. A sample volume of 0.1 ml and the same volume of enzyme reaction solution consisting of 0.2 M Tris-HCl buffer containing 20 mM ATP and 5.0 mM NADP were mixed, and then, 1 U of gluconate kinase and 10 U of 6-phosphogluconate hydrogenase (R-Biopharm AG, Darmstadt, Germany) were added. The enzyme reaction was carried out at 25°C for 20 min. The reaction was stopped by filtration with a 3K Amicon Ultra 0.5 ml filter. The pH of the filtrate was adjusted to approximately 5.0 by the addition of HCl. A control reaction was also carried out without enzymes.

The aluminum contents of the amphipod extract and water were measured using fluorometric analysis with 8-quinolinol [ 16 ]. We suspended samples in DDW, added 0.2 ml of 1% (wt./vol.) 8-quinolinol (Nacalai Tesque, Kyoto, Japan) and 0.2 ml of 2 N CH 3 COONa, and then added DDW to a volume of 5 ml. After mixing well, the aluminum 8-quinolinol complex was extracted with 1 ml of chloroform. The aluminum content was measured by the fluorescent intensity (excitation: 360 nm, emission: 535 nm). An aluminum chloride solution (Wako Pure Chemical Industries, Ltd.) was used as the reference. The iron content of the extracted sediment or soil was measured at an absorbance of 510 nm using a Pack Test Fe (Kyouritu Chemical-Check Lab. Co., Tokyo, Japan) based on the reaction of the Fe 2+ ion and o-phenanthroline after reduction. An iron(II) chloride solution was used as a reference. The D-gluconic acid/gluconolactone content of the amphipods was measured using a “gluconic acid/D-glucono-∂-lactone” E-kit (R-Biopharm AG, Damstadt, Germany). Sodium gluconic acid (Wako Pure Chemical Industries, Ltd.) was used as a reference. We calculated the total amount of aluminum or gluconic acid/gluconolactone in each amphipod from the measured contents and volumes of the amphipod extracts.

To measure the content of metal ions and gluconolactone/gluconic acid, each component was extracted from the amphipods. We carefully removed the exoskeletons from the amphipods with tweezers and dissecting scissors. Then, we subdivided the exoskeleton in 0.1 N sodium acetate buffer (pH 4.0) and stirred this suspension with a vortex mixer to extract the metal ions and gluconolactone/gluconic acid from the exoskeleton. After centrifugation (2,000 x g, 10 min, 4°C), the supernatant was collected, and the remaining parts of exoskeleton were suspended in the same buffer. Then, we repeated the stirring and centrifugation of the suspension. Both supernatants were collected and used for the measurement of metal ions and gluconolactone/gluconic acid in the exoskeleton. The remaining body was also subdivided in 0.1 N sodium acetate buffer (pH 4.0) and mashed by a BioMasher II (Nippi Inc, Tokyo, Japan) to extract metal ions and gluconic acid/gluconolactone. A small amount of the solution that had leaked from the amphipod in the process of removing the exoskeleton was also added. After centrifugation (5,000 x g, 10 min, 4°C), the supernatant was divided into two layers of oil and water. Each layer was collected separately. Then, the precipitate was suspended in the same buffer and mashed again. After centrifugation (5,000 x g, 10 min, 4°C), each oil and water layer was collected separately. We combined each of these layers with the corresponding layers obtained from the first extraction and used the results for measurements.

The exoskeletons of H. gigas were removed from individuals with tweezers and dissecting scissors and washed with methanol and chloroform. After drying the exoskeletons, we cut and crushed them into powder for X-ray powder diffraction (XRD) analysis. The exoskeleton powder was analyzed by an X-ray diffractometer (SmartLab, Rigaku) with a Cu radiation source (Kα = 1.5418 Å) at 45 kV and 200 mA. The 2θ scan speed, step width, and range were 21.6746 deg/min, 0.02 deg, and 20 to 50 deg, respectively. Calcite was identified in the exoskeleton powder through a database (International Center for Diffraction Data (ICDD)) search of the obtained peak positions.

We removed exoskeletons from H. gigas and washed them with deionized distilled water (DDW) and ethanol to avoid interference from oil components during electron microscope observations. Pieces of H. gigas exoskeleton obtained after milling were placed on a transmission electron microscopy (TEM) grid (200 mesh Cu Formvar/carbon-coated grid, JEOL) and observed by TEM (JEM-2100, JEOL) with an accelerating voltage of 200 kV. Scanning TEM (STEM)-EDS analysis was performed at 200 kV with an accumulation time of 60 s.

The freeze-dried amphipod sample was set on the stage of a scanning electron microscope, which was covered with a silicon plate and carbon tape to avoid energy-dispersive X-ray spectroscopy (EDS) signals from the stage. The exoskeleton of the amphipod was observed with scanning electron microscopy (SEM) (SU6600, Hitachi High-Technologies Co., Tokyo, Japan) under an accelerating voltage of 20 kV, and the elementary components were analyzed by EDS (X-Max N , Oxford).

The deep-sea amphipod Hirondellea gigas was captured from the Challenger Deep in the Mariana Trench (11°22.11N, 142°25.86E, depth of 10,897 m) and the Izu-Ogasawara Trench (32°12.5766N, 142°08.0411E, depth: 9,450 m), as described in a previous manuscript [ 6 , 7 ]. H. gigas is >3 cm from head to tail. We also purchased amphipods from Yokoebi-ya (Fukui, Japan). The coastal amphipods were captured from the seashore of Maizuru Bay (35°47.4331N, 135°39.5497E) in Japan, and their size was <2–3 mm from head to tail. All H. gigas and coastal amphipod specimens were stored in storage bags at -80°C without any selection. We randomly selected amphipods for all analyses.

Results

STEM/EDS analysis of H. gigas exoskeleton We also observed crushed exoskeletons through STEM/EDS to find aluminum in the internal exoskeletons of H. gigas captured from the Challenger Deep (Fig 3). We found calcium in all crushed exoskeleton, and nickel or copper in a few pieces of exoskeleton. Copper and nickel are minor elements in marine sediment, and H. gigas does not accumulate sufficient amounts for detection in SEM/EDS analysis. Aluminum was not contained in the internal exoskeleton. Silicate and molybdenum were observed as background signals from sample holder. We also observed the internal exoskeletons of amphipods captured from the Izu-Ogasawara Trench and did not found any aluminum peak in STEM/EDS analysis (S6 Fig). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. STEM/EDS analysis of pieces of H. gigas exoskeleton. Exoskeletons of H. gigas captured from the Challenger Deep were removed from the individuals, freeze dried, and then scraped. Bright-field STEM observations were conducted for pieces of the exoskeletons (A, C, E, G). Characteristic X-ray patterns were collected over 60 s (B, D, F, H), and the major metal signals from panels E and G were mapped (I, J). The observed Cu and Mo signals were caused by the TEM grid. The Si signal was background. https://doi.org/10.1371/journal.pone.0206710.g003

XRD analysis of H. gigas exoskeleton Through EDS analysis, we found that calcium was the major metal in the exoskeleton of H. gigas. Generally, calcium occurs as calcium carbonate and calcium phosphate in crustaceans [20–24]; however, a much lower peak of phosphorus than that of calcium was detected in the exoskeletons of H. gigas whose habitat is much deeper than the CCD (Table 1). This result indicates the possibility of the existence of calcium carbonate in the exoskeleton. Thus, we carried out XRD analysis of the exoskeletons of 5 randomly selected individuals to examine the chemical and physical state of calcium (Fig 4). The diffraction peak positions in the 5 samples were the same except for one unknown peak. The 2θ positions of the 7 main peaks were 23.1±0.1, 29.5, 36.0, 39.5, 43.2, 47.7, and 48.6. The observed peaks were indexed to calcite according to the standard ICDD card No. 00-005-0586. Then, the crystal material in the exoskeleton was suggested to be trigonal calcium carbonate (calcite). In contrast, one unknown peak (2θ = 44.6) was found in one sample and suggested to be AlO(OH) (1 1 1) (panel d). Since we could not find the largest peak of AlO(OH) (1 1 0) (2θ = 22.3), we did not conclude that this peak originated from AlO(OH). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. XRD analysis of H. gigas exoskeleton. Exoskeleton samples prepared from 5 individuals of H. gigas were used for XRD analysis as described in the Materials and Methods (panel A-E). The annotations of peaks were obtained from a database search (panel F). The arrow in panel d indicates an unknown peak, which was suggested to be AlO(OH) from a library search. We could not identify the peak as AlO(OH) because other minor peaks were not found. https://doi.org/10.1371/journal.pone.0206710.g004

Aluminum content in H. gigas Next, we measured the content of aluminum in H. gigas individuals. To avoid the influence of aluminum oxide originating from sediment, we used the 8-quinolinol method and applied an 8-quinolinol-aluminum ion complex as a fluorescent label [16]. We selected three individuals captured from the Challenger Deep in the Mariana Trench (S7 Fig). The exoskeletons of the three H. gigas individuals contained over 50% aluminum and exhibited only minor differences in content (S1 Table), whereas the body fluid presented varying aluminum contents among the 3 individuals. Because crustaceans inhabiting contaminated areas accumulate metals in their gut [25, 26], the presence of aluminum in the exoskeleton was not a result of simple accumulation.