Initiation of feeding
Impacts of water treatments on development of larval particle feeding mechanisms were determined by measuring the proportion of mussel larvae from each treatment that ingested fluorescent beads at 44 h post-fertilization (initiation of feeding, IF). Preliminary experiments demonstrated that at 44 h after fertilization >50% of M. californianus larvae began feeding when reared at ambient PCO2 (~380 ppm) and 18C.

We expanded upon previous IF findings and determine the length of the delay of the onset of feeding and how this delay affected the modeled growth of larvae to 260 um in shell length, the size at which larvae typically develop into pediveligers. We quantified the delay by first determining the relationship between the proportion of larvae feeding under optimal conditions and time since fertilization. This relationship was best described by the following three parameter logistic equations:

% Feeding= 94.1/((1+Exp (-0.74 × (h-45.1))))

where h is the hour post-fertilization. The logistic equation was then rearranged and linearized, enabling us to estimate the functional age of larvae feeding in each ocean acidification (OA) treatment, by comparison with the proportion of larvae feeding under normal conditions.

Particle processing
To assess the effects of OA on particle processing, 48 h old larvae from each treatment were stocked in nine 25 ml VOA vials (10 larvae/ml) containing the same water treatment in which they developed from fertilized eggs. After an acclimation period of one hour, larvae were then exposed to 2 um Fluorescbrite Polychromatic (Polysciences Inc., Warrington, PA) yellow (Y) beads (excitation maxima of 441 nm and emission maxima at 485 nm) at a concentration of 20 beads/ul and allowed to feed on these beads for one hour. A second and equal dose of 2 um red (R) beads (excitation maxima of 491 nm and 512 nm and emission maxima at 554 nm) were added to the vials at a concentration of 20 beads/ul following the hour-long exposure to Y beads. Triplicate vials were assigned to one of three exposure groups (10, 30, and 50 min) after red beads were added to the vials. To terminate feeding activity at the prescribed exposure time and preserve larvae for later analysis, 40 ul (0.2% v/v) of 10% buffered formalin (pH = 8.1-8.2) were added to vials. Later, larvae were crushed under a cover slip to flatten gut contents and allow better enumeration of all ingested beads in larvae under an epifluorescent microscope (objective 20x; Leica DM 1000). Larval sample sizes consisted of greater than or equal to 20 larvae per replicate vial per treatment.

Gut fullness
Gut fullness was defined as the mean total number of ingested beads (Y+R beads) per larva over 10, 30, and 50 min sampling periods.

Ingestion rate
Ingestion rates were estimated by determining the uptake of R beads after the first 10 min of exposure to this bead type. We then doubled the number of ingested beads as larvae were found to consume R and Y beads at equal rates in preliminary experiments.

Standardizing particle processing for shell-length effects
We examined the relationship between larval shell length (SL), gut fullness, and ingestion rate from a subset of treatments spanning the range of experimental omega-aragonite categories (greater than or equal to 10 larvae from 10 different VOA vials). Shell lengths, defined as the longest axis parallel to the shell hinge, were obtained by photographing larvae under a light microscope (50x) and measuring shell lengths using Image-pro (v.7).

After finding a significant relationship between larval shell size and feeding metrics, we applied the following hyperbolic function from Waldbusser et al. (2015), which strongly predicted the shell lengths of these larvae from the omeaga-aragonite for the first 48 h of development, to estimate shell lengths of larvae for all treatments:

SL=(884.378 × OM_ar )/(1+7.691 × OM_ar )

Next, we divided gut fullness values and ingestion rates of each treatment by their shell length estimate using the above equation. We then reexamined the effects of carbonate chemistry parameters on these feeding metrics after accounting for shell length.

OA_feeding_phys.csv