An aluminum shield enables the amphipod Hirondellea gigas to inhabit deep-sea environments

Abstract

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.

Fig 1. SEM/EDS analysis of the exoskeleton of H. gigas.

H. gigas specimens captured from the Challenger Deep were freeze-dried for SEM observations. The SEM observations were conducted without any coating. Calcium (red) and aluminum (green) are mapped on the SEM images (A, a-h). The EDS spectrum includes an annotation of each element with its Kα energy levels (C: 0.284, O: 0.532, Na: 1.071, Mg: 1.253, Al: 1.486, P: 2.013, S: 2.307, Cl: 2.621, Ca: 3.69 (keV)).

Fig 2. SEM/EDS analysis of the exoskeleton of H. gigas after washing with DDW.

Three H. gigas specimens captured from the Challenger Deep were washed 3 times with DDW and freeze-dried for SEM observations. The SEM observations were conducted without any coating. Exoskeleton parts of the head (A, E, I), the body (B, F, J), the back (C, G, K), and the telson (D, H, L) were observed. Each panel shows an SEM image (top), EDS spectrum (middle), and element composition obtained from the EDS spectrum (bottom).

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.

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.

Fig 5. Extraction of aluminum from the sediment of Challenger Deep by H. gigas protein and nonprotein fractions.

Three H. gigas individuals were scrapped and then suspended in 1 ml of (NH₄)₂SO₄ solution at 80% saturation. After incubation at 4°C for 2 h, the H. gigas suspensions were centrifuged at 20,000 x g for 30 min. The supernatants were used as nonprotein fractions, and the precipitates were suspended in 1 ml of DDW to prepare protein fractions. We mixed 300 μl of each fraction with the sediment suspension and added sodium acetate buffer to a final volume of 1.8 ml (final concentration: 50 mM, pH 5.0). The mixtures were then pressurized at 100 MPa and incubated at 2°C. After 1 h incubation, the mixtures were depressurized and centrifuged at 20,000 x g for 10 min. The aluminum content of the supernatants was measured as described in the Materials and Methods section. The error bar shows the S.D. (n = 3).

Fig 6. Extraction of aluminum from the sediment of the Challenger Deep by gluconic acid/gluconolactone.

Sediment samples were washed with DDW five times and then suspended in buffers containing 10 mM sodium gluconic acid/gluconolactone (closed circle) or none (open circle). The extracted aluminum was measured as described in the Materials and Methods.

Fig 7. The release of calcium ion from the exoskeleton of H. gigas under 100 MPa.

The exoskeleton was removed from 5 individuals. After dividing the exoskeleton into two pieces, the wet weight of each piece was measured. One piece was washed with DDW (washed sample), and the other piece was not washed (control). Both samples were pressurized under 100 MPa in artificial seawater at 2°C for 24 h. The released calcium was measured as described in the Materials and Methods. The error bars show the SD (n = 5). The results of the t-test confirmed that the impairment was significant (p<0.005).