DOI: 10.5061/dryad.brv15dv7g
title: Amphibalanus amphitrite begins exoskeleton mineralization within 48-hours of metamorphosis

description: Barnacles are ancient arthropods that, as adults, are surrounded by a
 hard, mineralized, outer shell that the organism produces for protection.
 While extensive research has been conducted on the glue-like cement that
 barnacles use to adhere to a surfaces, less is known about the barnacle
 exoskeleton, especially the process by which the barnacle exoskeleton is
 formed. Here we present data exploring the changes that occur as the
 barnacle cyprid undergoes metamorphosis to become a sessile juvenile with
 a mineralized exoskeleton. Scanning electron microscope (SEM) data show
 dramatic morphological changes in the barnacle exoskeleton following
 metamorphosis. Energy dispersive x-ray spectroscopy (EDS) indicates a
 small amount of calcium (8%) 1-hour post-metamorphosis that steadily
 increases to 28% by 2-days following metamorphosis. Raman spectroscopy
 indicates calcite in the exoskeleton of a barnacle 2-days following
 metamorphosis and no detectable calcium carbonate in exoskeletons up to
 3-hours post-metamorphosis.  Confocal microscopy indicates during
 this 2-day period, barnacle base plate area and height increases rapidly
 (0.001 mm2/hr and 0.30 µm/hr, respectively). These results provide
 critical information into the early life stages of the barnacle, which
 will be important for developing an understanding of how ocean
 acidification might impact the calcification process of the barnacle
 exoskeleton.
description: <i>Experimental set-up</i>: <b>
 </b><i>Amphibalanus (=Balanus) amphitrite </i>cyprids
 were reared from field-collected adult barnacles at the Duke University
 Marine Lab following methods of (Rittschof et al., J. Exp. Mar. Biol.
 Ecol, 1984; Rittschof et al., Biofouling, 1992). A single cyprid rearing
 was used for confocal experiments, while samples acquired from two
 separate batches were used for Raman and SEM experiments. Cyprids were
 settled in one of three growth environments: 1) glass confocal chamber
 slide; 2) 24-well plate with a silicon wafer at the bottom of each well;
  or 3) 24-well plate with a thin glass coverslip at the bottom of each
 well. The confocal chamber slides were used for confocal microscopy
 experiments while the silicon wafers and glass coverslips held within
 24-well plate environments were used in Raman spectroscopy and scanning
 electron microscopy experiments. Both silicon wafers and glass slides were
 used as substrates to maximize settlement rates, in the case that cyprids
 showed a preference between these substrates. Each of the growth
 environments contained artificial seawater (Instant Ocean, 32 psu). In
 half of the growth environments, a 140 mg/L calcein (Sigma-Aldrich C0875;
 SOM Figure S1) solution was added, prepared per Jacinto et al. 2015. 
 After placement in growth environments, cyprids were kept in a dark
 incubation chamber at 27.2°C. During daytime hours (8 am – 5 pm), the
 cyprids were monitored every hour to check for settlement (i.e. release of
 cyprid cement) and metamorphosis to the juvenile form. For barnacles in
 the 24-well plate environments, after metamorphosis was observed, juvenile
 barnacles were left to develop for specific time periods (1 hour
 post-metamorphosis, 2 hours post-metamorphosis, etc.). After this time
 period, juveniles were removed from solution, washed with ethanol and
 acetone and placed in a -80°C freezer to preserve any amorphous calcium
 carbonate that may be present for future analysis.
 <i>Confocal microscopy</i>: A Zeiss LSM710 laser
 confocal microscope was used to examine three barnacles under a 20x, 0.8
 NA objective following settlement in seawater containing calcein. Two of
 the barnacles were less than 16 hours post-metamorphosis and one had
 committed to settling (i.e. it had secreted cement) but had not yet
 undergone metamorphosis to the juvenile form. Images of the three
 barnacles were acquired every 10 minutes for the first 5.7 hours and every
 30 minutes for an additional 88.5 hours. For each acquisition, a z-stack
 was automatically collected with spacing between each focal plane of 2.297
 µm; x and y scaling were 1.38 µm. 488 nm light was used to obtain calcein
 fluorescence. As confocal imaging can provide highly detailed 3-D
 measurements, base area and height were measured from the image stacks
 taken on each of the 3 barnacles. Base area measurements were calculated
 by first determining the lowest in-focus image of the barnacle
 exoskeleton. The elliptical tool in ImageJ was then used to measure the
 area of the barnacle in the x- and y-plane at constant focal plane (z) and
 successive time points. Error bars for area measurements were calculated
 using 1.38 µm uncertainty in both the x- and y- dimensions. Height
 measurements were determined by calculating the number of z-steps
 in-between the highest in-focus exoskeleton image and the lowest in-focus
 exoskeleton image, and multiplying said number by 2.297 µm, the spacing
 between each z-step; 2.3 µm error bars were used for the height
 measurement based on the z-scaling. Height measurements were made only
 when the entire parietal plate structure was within the depth of
 field. <i>Scanning electron microscopy (SEM):
 </i>Barnacles were imaged with a JEOL JSM636OLV SEM after completion
 of Raman spectroscopy (see below) or after removal from the -80°C freezer.
 At room temperature, barnacle samples from 1-hour post-metamorphosis to
 6-days post-metamorphosis (1 1-hr, 1 3-hr, 3 1-day, 1 2-day, 1 3-day, and
 1 6-day) were thawed, dried, and coated with 10 nm of platinum and imaged
 in backscatter mode; this thawing procedure likely led to collapse of the
 earlier stage exoskeletons. Energy dispersive x-ray spectroscopy (EDS) was
 conducted with an Oxford X-max Silicon Drift X-ray Detector. EDS samples
 were embedded in epoxy resin while still attached to the substrate and
 oriented with their base plate down. Samples were polished with decreasing
 grit size silicon carbide papers until a cross-section of the parietal
 plates were revealed. The final polish was done with 50-nm alumina slurry
 and the samples were coated with 10 nm of platinum prior to EDS. On each
 sample (one individual per age point, with two individuals for the two day
 age point), 26-66 spectra were acquired in spot mode from several
 areas. <i>Raman spectroscopy: </i>Five
 barnacles were selected for analysis with Raman spectroscopy. A 2-day old
 barnacle was used for initial assessments and was thus dry and at room
 temperature for several days prior to analysis. The remaining barnacles
 (1-hour post-metamorphosis, 3-hour post-metamorphosis, 3-days
 post-metamorphosis, 6-days post-metamorphosis) were kept frozen until the
 Raman spectroscopy experiments were conducted; experiments were done at
 room temperature and in air, thus by the time data was collected, samples
 were thawed and dried. Experiments were done at the Cornell Center for
 Materials Research (CCMR) on a Renishaw InVia Confocal Raman microscope
 with a 785 nm laser at 10% power and exposure times between 10-100s. Wire
 4.1 was used to remove the baseline and peaks from cosmic rays. Spectra
 were compared to reference spectra for calcite, chitin, and amorphous
 calcium carbonate.
description: Confocal microscopy data was taken in 3 rounds, with the first
 round labeled as #1. <br> Software:<br> -
 Confocal - Zen Lite -
 https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html<br> - EDS - INCA - the software was made by Oxford and has been replaced by AztecLive - https://nano.oxinst.com/products/aztec/azteclive<br> - Raman - WiRE - https://www.renishaw.com/en/raman-software--9450