A sea urchin Na+K+2Cl- cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na+K+2Cl- cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50 = 6.5 µM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50 = 26.4 µM) and furosemide (IC50 = 315.4 µM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.

Archive of the PASATA (PASsive Acoustic Tracking of Antarctic marine mammals) field documentation

Recordings from the PerenniAL Acoustic Observatory in the Antarctic ocean (PALAOA) show seasonal acoustic presence of 4 Antarctic ice-breeding seal species (Ross seal, Ommatophoca rossii, Weddell seal, Leptonychotes weddellii, crabeater, Lobodon carcinophaga, and leopard seal, Hydrurga leptonyx). Apart from Weddell seals, inhabiting the fast-ice in Atka Bay, the other three (pack-ice) species however have to date never (Ross and leopard seal) or only very rarely (crabeater seals) been sighted in the Atka Bay region. The aim of the PASATA project is twofold: the large passive acoustic hydrophone array (hereafter referred to as large array) aims to localize calling pack-ice pinniped species to obtain information on their location and hence the ice habitat they occupy. This large array consists of four autonomous passive acoustic recorders with a hydrophone sensor deployed through a drilled hole in the sea ice. The PASATA recordings are time-stamped and can therefore be coupled to the PALAOA recordings so that the hydrophone array spans the bay almost entirely from east to west. The second, smaller hydrophone array (hereafter referred to as small array), also consists of four autonomous passive acoustic recorders with hydrophone sensors deployed through drilled holes in the sea ice. The smaller array was deployed within a Weddell seal breeding colony, located further south in the bay, just off the ice shelf. Male Weddell seals are thought to defend underwater territories around or near tide cracks and breathing holes used by females. Vocal activity increases strongly during the breeding season and vocalizations are thought to be used underwater by males for the purpose of territorial defense and advertisement. With the smaller hydrophone array we aim to investigate underwater behaviour of vocalizing male and female Weddell seals to provide further information on underwater movement patterns in relation to the location of tide cracks and breathing holes. As a pilot project, one on-ice and three underwater camera systems have been deployed near breathing holes to obtain additional visual information on Weddell seal behavioural activity. Upon each visit in the breeding colony, a census of colony composition on the ice (number of animals, sex, presence of dependent pups, presence and severity of injuries-indicative of competition intensity) as well as GPS readings of breathing holes and positions of hauled out Weddell seals are taken.