Skip to main content
Dryad logo

Amphibalanus amphitrite begins exoskeleton mineralization within 48-hours of metamorphosis

Citation

Metzler, Rebecca (2020), Amphibalanus amphitrite begins exoskeleton mineralization within 48-hours of metamorphosis, Dryad, Dataset, https://doi.org/10.5061/dryad.brv15dv7g

Abstract

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.

Methods

Experimental set-up: Amphibalanus (=Balanus) amphitrite 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.

Confocal microscopy: 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.

Scanning electron microscopy (SEM): 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.

Raman spectroscopy: 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.

Usage Notes

Confocal microscopy data was taken in 3 rounds, with the first round labeled as #1.


Software:
- Confocal - Zen Lite - https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html
- EDS - INCA - the software was made by Oxford and has been replaced by AztecLive - https://nano.oxinst.com/products/aztec/azteclive
- Raman - WiRE - https://www.renishaw.com/en/raman-software--9450

Funding

National Science Foundation, Award: 1905619

National Science Foundation, Award: 1905466

Cornell Center for Materials Research

National Science Foundation CCMR, Award: 1719875