## 3 Discussion  
The headspace VOCs were monitored from _M. gaditana_ being actively grazed by the marine rotifer, _B. plicatilis_. During grazing, a single rotifer can consume ~200 algal cells per min [27] and the ultimate depletion of the algal culture is dependent on the initial concentration of the algal inoculum and the number of rotifers added. In addition to algae undergoing active grazing, viable and actively growing algae were also present in these cultures. Thus, algal growth rates and VOC emissions were detected during this time for both healthy and grazed algae. The initial algal concentrations were chosen to allow the grazing to take place over several days for the collection of multiple time-resolved VOC samples. Algal cell concentrations were monitored for cultures of _M. gaditana_ exposed to three successive freeze-thaw cycles. During this process, algal cells are physically disrupted and rendered non-viable through the formation of ice crystals. This is evidenced by the lack of growth and release of photosynthetic pigments from the cells. All algae cells were simultaneously frozen and lysed within a comparatively shorter time frame compared to rotifer grazing, preventing active growth after freeze-thaw procedure was complete. The direct enumeration of the FTA cultures, by particle counting, showed no decrease in particle number but FTA cultures displayed a rapid loss of chlorophyll color and fluorescence due to a release and apparent degradation of chlorophyll from disrupted cells when compared to time-matched healthy A controls. Additionally, since the FTA cultures never increased in algal concentration over the course of the FTA experiments, it can be inferred that the entire algal culture was, upon treatment, disrupted and rendered nonviable. Thus, we utilized chlorophyll fluorescence to demonstrate the lack of algal culture growth [28]. The cellular disruption of _M. gaditana_ cells was accomplished via repetitive freeze-thaw cycles to abiotically result in algal cell death via rapid ice crystal formation. The freezing process quickly affects the entire algal culture, simultaneously, compared to gradual algal crop damage from rotifer grazing. During FTA Experiment 1, measurements of FTA VOCs at 24 and 48 h indicated that VOCs generated by the freeze-thaw cycle were mostly depleted by 24 h and not detected at 48 h post-inoculation. After the FTA Experiment 1 (Figure 1D), the experimental design was altered to collect VOCs at additional timepoints (e.g., 1 h and 6 h) post algal inoculation (Figure 1E,F). In doing so, we observed a higher diversity of VOCs generated by the freeze-thawed algal samples in subsequent Experiments 2 and 3. The fundamental difference between the two crop damage mechanisms is that freeze thaw is strictly a physical process whereas grazing has a biological component in the rotifer digestive tract. The digestive processes of the _B. plicatilis_ are not well characterized. It is known that at higher prey concentrations, as would be expected in high density algae production ponds, ingested particles travel faster thought the rotifer digestive track and may not be completely digested. Although relatively little is known of the rotifer digestive process along with those of other microzooplankton grazers it is not unexpected that the VOCs released from grazing would differ from those released from purely physical disruption. However, our data reveals that some VOCs are released by both processes. \(\beta\)-ionone, generated from the breakdown of carotenoids, was the only biomarker identified as a product of both freeze-thaw disruption and rotifer-grazing (Compound R4). \(\beta\)-ionone was seen previously in rotifer-grazed cultures of _M. salina_[24]. \(\beta\)-ionone gradually decreased (<96 h) with rotifer grazing and rapidly decreased (<6 h) after initial freeze-thawing. The prolonged detection of \(\beta\)-ionone in the rotifer-grazing is consistent with algal cells being gradually individually consumed by rotifers with concomitant oxidative degradation of caroten.