The influence of artificial light at night (ALAN) on algal phenol concentrations can mediate herbivore-alga interactions

Artificial light at night (ALAN) is a human-induced factor affecting various biological complexity levels and species. Research on ALAN impact has focused on vertebrates and invertebrates, with less attention on primary producers like marine algae. The aim of our study was to evaluate the effect of ALAN on production of phenolic compounds in the red alga, Mazzaella laminarioides, and their indirect impact on the feeding behavior of the marine snail Tegula atra. Algae were exposed to the following treatments: natural day/night cycles, ALAN, and continuous darkness. We observed a higher phenolic concentration during high tide, in according with periods of feeding activity of herbivores. In comparison to algae exposed to natural day/night conditions, those exposed to ALAN showed the lowest concentrations of phenols. Tegula atra consumed significantly more algae than those exposed to ALAN, a result that is consistent with the preference trials, where algae exposed to ALAN was consumed more than algae maintained in natural conditions or continuous darkness. This evidence suggests that ALAN can impact on the production of phenolic compounds and, indirectly, on algal-herbivore interactions.

Methods
Sampling and acclimation: Experimental organisms were collected during spring 2024 at “Las Conchitas” beach, Isla Negra, Valparaíso-Chile (33º26'19.8" S, 71º41'19.9" W). Blades of red algae (M. laminarioides; hereafter algae) of similar size were collected by hand and quickly brought to the laboratory. To avoid potential biases associated with reproductive stage or sex (65 - 67), and possible fragmentation, all the algae used were non-reproductive (i.e., vegetative thalli) and individual blades came from separate holdfasts. Black snails (T. atra, hereafter snails) were also collected manually, targeting a small size range (1.75 ± 0.19 cm shell height), which prevented potential biases associated with reproductive status. Snails were placed in coolers with seawater and contact aeration. Both organisms are abundant and can be found in the mid-intertidal zone, the algae typically on top of the rocks whereas the snails typically at the base of rocks, cracks and pools. In the laboratory, algal blades were carefully washed with distilled water to remove biofouling and then rinsed with seawater to avoid osmotic shock damage. Both algae and snails were maintained in an automated tidal system for a five-day acclimation period at 15°C, 33 ppm of salinity, and a 12:12 (light:dark) photoperiod that reflected field conditions. Daylight conditions were set between 8:00 AM and 8:00 PM, with an intensity of 1,200 lux and a 82 PAR (Photosynthetically Active Radiation) full spectrum light normally used for Aqua Brand aquatic plant growth. Night conditions were set at 0 lux. The system was set to change tides every six hours follow field conditions, with high tide (HT, algae completely submerged) set at 1:00 PM and 1:00 AM, and low tide conditions (LT, algae completely uncover) set at 7:00 AM and 7 PM.

Variation of total phenols and the influence of light treatment: After acclimation, we examined the variation in total phenolic compound concentrations in the blades of the algae (in the absence of snails), by taking samples every 3h for 36 h (12 sampling points). Each time, a ~10 mg tissue sample was collected from five independent algae (no thalli were sampled twice), for a total of 60 samples. The samples were individually frozen in liquid nitrogen and stored at -80ºC. Subsequently, the "Folin-Ciocalteu" spectrophotometry method for laminarian algae was used to measure the concentration of total phenolic compounds in the tissues of the alga. In brief, this method used dried tissue of the homogenized entire thallus of the algae. The resulting powder was placed in 1 mL of water on a water bath at 45 °C for 10 min with constant stirring. Subsequently, the samples were centrifuged until a pellet was observed. The supernatant was collected and mixed with Folin-Ciocalteu (Sigma-Aldrich) and distilled water. The solution was incubated in the dark at room temperature, followed by the addition of 20% sodium carbonate (Na2CO3), and incubated again in the dark at room temperature. Finally, the concentrations of total phenols were determined colorimetrically, using a calibration curve and spectrophotometry with an absorbance at 765 nm (68). 

Separate samples were used to measure phenolic compounds in algal blades exposed to three different light treatments for a period of four consecutive days (a period deemed representative and short enough to prevent any algal decay due to light exposure). These light treatments included a) L:D or light : dark conditions (as in the acclimation period), b) D:D or continued dark conditions (0 lux at day and night) as a negative control, and c) L:L or ALAN conditions (1200 lux during day hours and 30 lux at night). ALAN (30 lux) reflects light intensities measured in nearby Chilean shorelines (e.g., 12) and are well within the range of ALAN intensities used in other experiments (69). After the exposure period, five ~10 mg tissue samples from separate algae were collected during high tide, and other five samples during low tide from other five algae (no alga was sampled twice), for a total of 30 samples (n=5 samples per treatment and tide level). Samples were stored and phenol concentrations estimated following the protocol described above.

Influence of light treatment on snails’ feeding rates: A first set of experiments measured the consumption rates by snails on algal blades offered without a choice, i.e., providing the snails with an algal blade collected during high tide exposed to one of the light treatments described above (either L:D, D:D or L:L). In these experiments, three snails were placed at the center of a 30 × 20 × 15 cm tank filled with seawater, and one algal blade from one of the light treatments was then placed at one of the sides of the tank. That side (left or right) was alternated between replicates to avoid any positional bias. The snails were allowed to graze for 24 h (n = 5 tanks for each light treatment). Based on availability of specimens and tanks, we conducted two separate experiments (Trials 1 and 2), starting at high tide and with identical duration and replication levels, by using different snails and algae (no organisms were used twice). The weight of individual algal blades was measured before and after the exposure to the snails using a FA2204E digital scale (+/- 0.0001 g). To account for potential changes due to factors other than herbivory, the trials were matched with parallel 24 h trials in which algal blades were not exposed to the snails. Weight differences in these “autogenic” control trials were then incorporated in the calculations using Roa (1992)’s protocol: Consumption = (Ef-Ei) – (Cf-Ci); where E and C stand for experimental and control algae, and f and i represent final and initial algal weights, respectively. This was subsequently expressed as consumption per day (70). 

            A second set of experiments was conducted to assess the consumption rates of snails when two algae (previously exposed to different light treatments) were simultaneously offered to them (i.e., preference experiments sensu 71). These trials were conducted in the same tanks described above and were designed as pairwise-choice experiments (three snails were placed in the middle and one alga of each treatment was placed randomly at each side of the tank) (71). Combinations of algae were as follows: a) L:D vs D:D (alga previously exposed to control vs alga exposed to constant darkness), b) L:D vs L:L (control vs ALAN), and c) D:D vs L:L (dark vs ALAN). These pairwise comparisons (n = 9 tanks for each type of comparison) were conducted in two separate (independent) experiments (Trials 1 and 2) lasting 24 h each. Neither algae or snails were used twice. We assessed preference by comparing the consumption rates of each of two algae offered to each snail. Consumption rates on individual algae were calculated by weight difference, controlling for changes not associated with herbivory (70), as described above.

Statistical analysis: A two-way ANOVA was performed to evaluate the effect of tide level (HT vs LT) and light treatment (L:D vs D:D vs L:L) (both fixed factors) and their potential interaction on the concentration of phenolic compounds in the algal tissues. Since samples were collected from separate algae in all instances, factors (tide level and light treatment) were independent. In addition, assumptions of homoscedasticity and normality were previously checked using the Levene Test and the Shapiro test, respectively, with no violations being detected. This was done using the R Studio software (version 4.3.1) with the CarData packages (72). A same type of two-way ANOVA was performed to assess the influence of trial (1 vs 2) and light treatment (L:D vs L:L vs D:D) on the snail consumption rates without a choice (factors were again independent). For both ANOVAs, differences among light treatments were further examined with Tukey a-posteriori tests). Lastly, to assess snail preference for algae exposed to two different light treatments we used paired t-tests. Assumptions were assessed as indicated above.