DOI: 10.5061/dryad.wh70rxwz6
title: The influence of artificial light at night (ALAN) on algal phenol concentrations can mediate herbivore-alga interactions

description: 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.
description: Sampling and
 acclimation:<strong> </strong>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 (<em>M. laminarioides</em>; 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 (<em>T. atra</em>, 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:<strong>
 </strong>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
 (Na<sub>2</sub>CO<sub>3</sub>), 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 =
 (E<em><sub>f</sub></em>-E<em><sub>i</sub></em>) – (C<em><sub>f</sub></em>-C<em><sub>i</sub></em>); where E and C stand for experimental and control algae, and <em>f</em> and <em>i</em> represent final and initial algal weights, respectively. This was subsequently expressed as consumption per day (70).  <strong>            </strong>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 <em>sensu </em>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 <em>a-posteriori tests</em>). Lastly, to assess snail preference for algae exposed to two different light treatments we used paired t-tests. Assumptions were assessed as indicated above.
description: # The influence of artificial light at night (ALAN) on algal phenol
 concentrations can mediate herbivore-alga interactions
 [https://doi.org/10.5061/dryad.wh70rxwz6](https://doi.org/10.5061/dryad.wh70rxwz6) ## Description of the data and file structure This study focused on the influence of ALAN on the production of secondary metabolites (phenolic compounds) by the red alga*Mazzaella laminarioides*, and its indirect effects on the feeding behavior of consumers, the snail*Tegula atra*. We followed the variation of phenolic compounds in the alga, and then studied their variation when algal were exposed to: natural daylight/night, ALAN, and darkness. Algae were then used as food sources to assess snails’ food behavior ### Files and variables #### File: DATA.xlsx ##### Description:  **Sheet one:** Consumption assays (without choice of feed). Where we can find the following treatments (Column 2 “Treatment”): LL: ALAN, DD: Complete dark, and LD: Natural light/night cycle During 24 and 48 hours of experiment (Column 1 “Day”), the consumption as the difference in algae weight (Column 3 “difference”) and the total consumption divided by the total number of organisms in the experiment (Column 4 “dif/4”). **Sheet two:** Choice assay. Where we can find the different crosses (Column 3 “Assay”): LL:LD, LL:DD and DD:LD. During 24 and 48 hours of experimentation (Column 2 “Hour”= time). The first column (“Aquaria”) corresponds to the aquaria where they were performed, the fifth column corresponds to the total consumption of the snails on the algae (“difference (g)”), and the sixth column corresponds to the consumption divided by the number of animals per aquarium (difference/4 animals). **Sheet three:** Phenols Oscillation, natural oscillation of phenolic compounds in algae. Test carried out for 2 days (Column 1 “Day”). Where you can find the time in which the sampling was performed (Column 2 “Time”= sampling hour ), the tide that corresponds to these times (Column 3 “Tide”) low tide and and “high tide” and finally the concentration of phenolic compounds in the sampled fronds (Column 4 “Phenols concentration”). **Sheet four**: Phenols concentrations-light treat, effect of ALAN on the concentration of total phenols. Where we can find the different treatments (Column 1 “Treatment”): LL: ALAN, DD: Complete dark, and LD: Natural light/night cycle During both tides (Column 2 “Tide”), low tide and high tide, and the concentration of total phenols (Column 3 “Phenols concentration”). ##### Variables **Treatment** * LL: ALAN * DD: Complete dark * LD: Natural light/night cycle **Tide** * High: High tide * Low: Low tide **Units** * Consumption and selection: grams (g) * Oscillation of total phenols: concentration of total phenols (mg/ 0.1 mg of dried seaweed) * Effect of ALAN on total phenols: concentration of total phenols (mg/ 0.1 mg of dried seaweed)