Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Abstract

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid-base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H(+)-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH(e) and pH(i)) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO(2) conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO(2). Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH(e) whenever seawater pH changes. However, measurements of pH(i) demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na(+) and HCO(3)(-), suggesting a bicarbonate buffer mechanism involving secondary active Na(+)-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH(i) enables calcification to proceed despite decreased pH(e). However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.

Fig. 1.

(A) S. droebachiensis pluteus larvae attached to the holding pipette and the tip of the measuring electrode inside the primary body cavity (PBC). (B) Relationship between seawater and the PBC pH (NBS scale) during acute and chronic environmental hypercapnia. Bars represent ±SD; n = 4–5.

Fig. 2.

Determination of epithelial permeability in pluteus larvae in vivo using FITC dextran conjugates of varying molecular weights and confocal microscopy. (A) Transmission image of pluteus larva; (B) confocal image of larva in a similar position as in A exposed to 4-kDa FITC dextran in seawater for 60 min; the green color in the extracellular matrix (ECM) indicates equilibration of FITC dextran between surrounding seawater (SW) and ECM; (C) confocal image of larva exposed to 40 kDa for 60 min; the dark color indicates lack of equilibration of FITC dextran between ECM and SW. (D) Equilibration of FITC dextrans of varying molecular weights between SW and ECM following 15- and 60-min incubation. Equilibration is represented as the ratio of FITC fluorescence within the ECM and the SW surrounding the larva. Low values indicate a low permeability. (E) Corresponding confocal (FM1-43-stained) and transmission images of the pluteus larva outer epithelium. Bars represent ±SD; n = 9.

Fig. 3.

In vivo confocal images of pluteus larvae using the vital dye FM1-43 that stains membranes. Primary mesenchyme cell (PMC) syncytia and their sheaths, as well as filopodia connecting them with each other and epithelial cells are visible, as are vesicles within cells and filopodial connections. A is a close-up confocal image of the region depicted in the Inset in the transmission image (B). C depicts a similar region from a different larva as shown in A and B, but rotated by 90° clockwise.

Fig. 4.

Ratiometric fluorimetry in PMCs using the pH-sensitive dye BCECF-AM. (A) Schematic illustration of a recording trace including ratio images (Top: the dashed lines represent the orientation of spicules). Data were obtained from the control period (control), after addition and removal of NH₃/NH₄⁺ (alkalosis and acidification; ammonium pulse), and during pH_i recovery. (B) Calibration curve of BCECF-AM in PMCs fitted by a function that flattens toward more acidic or alkaline conditions allowing the translation of ratios to pH values.

Fig. 5.

Acid–base regulatory abilities of PMCs in the presence of low (5 mM) Na⁺, without HCO₃⁻ and with 100 µM amiloride. (A) Original recordings of control and treatment traces representing average values of four to five single measurements. Presence of NH₃/NH₄⁺-induced alkalosis and the washout-induced acidosis under the three experimental conditions. (B) The recovery given in percentage of the initial value, before alkalosis in response to control and treatment conditions. (C) Recovery rate represented by the compensatory slope after acidosis given in percentage of the control (100%) value, in response to control and treatment conditions. Bars represent ±SD; n = 4–5.

Fig. 6.

Schematic model summarizing the interplay of calcification, pH regulation, and energetic costs in sea urchin larvae during environmental acidification. The primary body cavity (PBC) pH conforms to the surrounding seawater pH (pH_sw). A decrease in pH_e directly affects the calcifying primary mesenchyme cell (PMC) syncytia challenging the intracellular pH (pH_i) regulatory machinery due to decreased proton gradients. The vesicular precipitation of amorphous calcium carbonate (ACC) within PMCs is intrinsically linked to pH_i regulation. pH homeostasis is maintained by ion transporters, which either directly or indirectly depend on the consumption of energy. Increased energetic costs due to decreased proton gradients lead to a shift in the larvae’s energy budget, which decreases scope for growth and may also translate into juvenile impaired fitness. ST, stomach; putative transporters are in gray.