DOI: 10.25338/b8cp96
title: Top-down effects on biological soil crust function

description: Biological soil crusts (BSCs) are communities of microorganisms that
 control ecosystem functions in drylands. Despite their importance, little
 is known about how trophic interactions affect BSC function. We conducted
 a series of mechanistic experiments to tease out the direct (i.e.,
 consumption) and indirect (i.e., fecal and mucus deposition) pathways by
 which crustivore (i.e., consume BSCs) and detritivores affect BSC
 functions— complemented by a manipulative field experiment exploring the
 integrative effect of these pathways. We showed that detritivore feces,
 mucus, and grazing increased the BSCs CO2 efflux. Detritivore feces also
 increased BSC %N. Crustivorous snail feces increased BSC CO2 efflux, and
 their mucus decreased BSC %C and %N. In the field, both snail species
 increased BSC %C and did not affect BSC CO2 efflux. Combined, our findings
 highlight that macro-invertebrate consumers exert top-down regulation on
 BSC function, opening the door for a whole new avenue of trophic research.
description: <strong>Study site and species</strong>
 The Avdat Research station (30°47’02” N, 34°46’09” E; hereafter,
 Avdat) is in the central Negev highland and receives ~93 mm of rainfall
 year<sup>-1</sup> across 19-42 days (Israel Meteorological
 Survey, 2021; Station: 253052).  Avdat is characterized by 1-2 mm thick
 cyanobacteria dominated BSC and has multiple common snail species,
 including <em>Xerocrassa simulate</em> and
 <em>Sphincterochila prophetarum</em>, which reach densities of
 12 snails m<sup>-2</sup> (Rinehart et al. 2021).
  <em>Xerocrassa simulate</em> (hereafter, detritivore)
 consumes plant litter and BSCs (Ward and Slotow 1992), while
 <em>S.</em> <em>prophetarum</em> (hereafter,
 crustivore), feed exclusively on BSCs (Shachak and Brand 1981, Appendix
 S1). A single crustivore can consume 203 – 577 mg of BSC
 day<sup>-1</sup> (Appendix S1). Both snail species break their
 aestivation and become active after substantial precipitation events when
 the ground becomes wet. Then, snails begin foraging on BSCs leaving behind
 feces and mucus trails. <strong>Mechanistic
 laboratory assays </strong> <em>Laboratory
 grown BSCs. </em>We grew laboratory BSCs by adding 25 ± 1.0g of
 soil, followed by 25 ± 1.0g of BSCs to 120 mm Ø petri dishes. We used
 laboratory generated BSCs to minimize natural variation in BSC function.
  This method has been employed previously in BSC experimentation (Doherty
 et al. 2015, Rinehart et al. 2021). Soils and BSCs were collected from
 Avdat. Laboratory-reared BSCs were watered with DI water every-other day
 for 80 d then left dormant in an incubator at 16°C with 40% humidity and a
 16:8 light cycle (light intensity: 240Mmol m<sup>2-1
 </sup>s<sup>-1</sup>) until used.
 <em>F</em><em>ecal deposition. </em>Snail
 crustivores and detritivores collected from Avdat were placed inside
 circular plastic containers in groups of three, by species. All containers
 were provided BSCs and plant litter (a 50:50 mix of <em>Hammada
 scoparia </em>and grasses) <em>ab labium</em> then
 placed in an incubator at 18˚C with 40% humidity and a 16:8 dark: light
 cycle. Every other day, we collected any feces produced by snails and
 refreshed all food resources. Collected feces were freeze-dried and their
 dry mass (DM) determined. Crustivores generated 77 ± 7.9 mg DM (mean ±
 1SE) of feces day<sup>-1</sup>, while detritivores only
 generated 13 ± 0.2 mg DM of feces day<sup>-1</sup>. This
 difference in fecal production is not due to differences in snail biomass
 (crustivores: 1.29 ± 0.1 g WM; detritivores: 1.24 ± 0.2 g WM). We
 allocated fecal samples, by consumer species, to laboratory BSCs in the
 following three consumer treatments: crustivore, detritivore, and no snail
 (n = 15).  We added 130 ± 10 mg DM of homogenized detritivore feces to
 detritivore BSC replicates and 770 ± 10 mg DM of crustivore feces to
 crustivore BSC replicates. We did not standardize the DM of feces added
 across species because we wanted to know the mean impact of feces
 snail<sup>-1</sup>. No feces were added to no snail BSC
 replicates. All BSCs were watered every-other day with 3.5 ml of DI water
 and housed in an incubator at 18˚C with 40% humidity and a 16:8 dark:
 light cycle. Ten days into our watering regime we paused our study for 30
 d due to Covid-19. After the lockdown, we resumed our watering regime for
 eight days before measuring the CO<sub>2</sub> efflux, %C, and
 %N of each BSC. This delay should not affect our results as BSCs are
 inactive when dry (Weber et al. 2016). <em>Mucus
 deposition. </em>Snail crustivores and detritivores collected from
 Avdat were randomly assigned to plastic containers in groups of 14
 individuals by consumer species (n = 7). Containers with snails were
 maintained in a climate-controlled room at 14-17°C with an 11:13 light:
 dark cycle. Snails were incubated in an 11:13 hour light cycle, rather
 than the 16:8 hour light cycle used for BSC incubations, to further
 promote movement and mucus deposition. Snail consumers in all containers
 were provided BSCs and <em>H. scoparia ad labium</em>. Mucus
 was extracted from snails weekly for three weeks. During each extraction,
 snails in each container were housed on a transparent plastic sheet and
 wetted with 1ml of DI water daily. After 48 hour, we scraped mucus off the
 transparent plastic sheets with razor blades— using 14 ml of DI water per
 container to facilitate scraping. Collected mucus was stored at -80°C
 until all extractions were complete. After the third extraction, we
 freeze-dried 1.0 ml mucus samples to determine their DM. The remaining
 mucus was homogenized by snail consumer species and allocated to
 laboratory BSCs in the following three consumer treatments: crustivore,
 detritivore, and no snail control (n = 20-21). Mucus harvested from snails
 was allocated, by species, to laboratory BSCs in the following three
 consumer treatments: crustivore, detritivore, and no snail control (n =
 20-21). We watered crustivore and detritivore BSCs with 1.5 ml of
 homogenized crustivore or detritivore mucus, respectively, and 8.5 ml of
 DI water every-other day. No snail BSCs were watered with 10 ml of DI
 water every-other day. All BSCs were housed in an incubator set at 18˚C
 and 40% humidity with a 16:8 dark: light cycle. After 14 d, we quantified
 the CO<sub>2</sub> efflux, %C, and %N of each BSC.
 <em>Grazing and mucus deposition. </em>We manipulated
 snail consumer treatment (crustivore, detritivore, or no snail) within
 circular plastic containers (Ø = 25 cm; height = 15 cm). In crustivore and
 detritivore treatments, we included three snail crustivores and
 detritivores, respectively, all collected from Avdat and starved for 6 d
 prior to the assay. No snail treatments received no snails. All containers
 received a single pre-weighed laboratory reared BSC. Containers were then
 placed in a climate-controlled room at 18˚C with a 16:8 dark: light cycle.
 We watered all treatments with 3 ml of DI water daily to promote snail
 activity. All feces generated by snails were removed daily before watering
 occurred. After 4 d, we measured the biomass loss of BSCs,
 CO<sub>2</sub> efflux, %C, and %N of each BSC.
 <strong>F</strong><strong>ield
 experiment</strong> At Avdat, we deployed ten
 blocks of three 0.25 m<sup>2</sup> circular enclosures on
 natural BSCs. In each block, we added five crustivore snails to a randomly
 chosen enclosure, five detritivore snails to another, and the third
 enclosure was a no snail control. The enclosures were constructed from
 flexible PVC tubing (40 mm Ø) anchored in place with metal pegs and
 painted with antifouling paint. Antifouling paint is used to restrict
 gastropod movement in marine communities (Bracken et al. 2011) and
 effectively restricted snail movement in our system. We used antifouling
 paint covered enclosures to minimize climatic effects of typical
 enclosures. All enclosures started with no vascular plants but were
 equipped with 1) a fake plant constructed of plastic-coated wire and
 plastic leaves, 2) 1.5 ± 0.1g of <em>H. scoparia</em> litter,
 and 3) a rock of standard size (~ 6 cm long, ~ 4 cm wide). The fake plant
 and rock were included to provide refuge and aestivation sites for the
 snails. We monitored all enclosures weekly and replaced any snails that
 escaped the enclosure (no ‘wild’ snails entered the enclosures). After 113
 d, we collected five, 120 mm Ø BSCs samples from each plot. BSCs samples
 were housed in an incubator at 16°C with 40% humidity and a 16:8 light:
 dark cycle until CO<sub>2</sub> efflux, %C, and %N were
 quantified.<strong> </strong>For all our BSC responses we
 averaged the five subsamples collected from each enclosure.<strong> 
  </strong> <strong>BSC function
 measurements</strong> <em>Biomass loss.
 </em> We obtained the initial dry biomass of BSCs added to each
 experimental replicate. Upon completion of the study, we let BSCs dry for
 24 hours in an incubator at 18˚C with 40% humidity and a 16:8 dark: light
 cycle (light intensity: 240Mmol m<sup>2-1
 </sup>s<sup>-1</sup>) before obtaining the final dry
 biomass of each BSC. Biomass loss was then calculated for each BSC
 (Initial Dry Biomass – Final Dry Biomass = Biomass Loss).
 <em>CO<sub>2</sub> efflux. </em>We
 quantified the BSC CO<sub>2</sub> efflux by placing watered (3
 ml of DI water) BSCs in airtight plastic chambers [155mm x 155mm x 61mm
 (length x width x height) Lock & Lock HPL 823; www.s-d.co.il]. We
 flushed chambers with CO<sub>2</sub>-fress air at a rate of 2L
 minute<sup>1</sup> for a total of 5 minutes. The BSCs were
 then incubated in their flushed, airtight chambers at 18°C with no light
 for a total of 48 hours. After the dark incubation, we used a LI-7000
 CO2/H2O infrared gas analyzer (IRGA; LI-COR, Inc.) with a designated self-
 manufactured injection system to quantify the amount of
 CO<sub>2</sub> released by each BSC sample. We quantified the
 CO<sub>2</sub> efflux of BSCs used in our fecal deposition,
 mucus deposition, and grazing laboratory assays at the start and end of
 each assay, allowing us to calculate the consumer-mediated change in BSC
 CO<sub>2</sub> efflux (i.e., Final CO<sub>2</sub>
 efflux – Initial CO<sub>2</sub> efflux = Change in
 CO<sub>2</sub> efflux). We used the change in
 CO<sub>2</sub> efflux for all statistical analyses of our
 laboratory assays. In our field study (see “<em>Long-term field
 study</em>” in Methods section of main text), we only quantified BSC
 CO<sub>2</sub> efflux at the end of the study (i.e., final BSC
 CO<sub>2</sub> efflux) since our method of measuring
 CO<sub>2</sub> efflux required destructive sampling. Because
 we collected five BSC sub-samples from each of our experimental plots, we
 pooled the results of these sub-samples and used these pooled means for
 all our statistical analyses. <em>Total carbon
 and nitrogen. </em>After completion of CO<sub>2</sub>
 efflux assays, we homogenized five, 0.8 cm<sup>3</sup>
 systematically collected (“x” shape with samples at the ends of each arm
 and center) sub-samples from each BSC sample. We randomly selected 12 of
 our 20 BSCs in our Grazing and mucus deposition laboratory assay for total
 carbon and nitrogen analysis, in all other laboratory assays we quantified
 total carbon and nitrogen in all BSCs (Table S1). For our field study, we
 further homogenized subsamples collected from each of the five BSC samples
 harvested within each plot to generate a single homogenized sample from
 each field plot. All homogenized BSC samples were then freeze-dried for 48
 hours and packaged (29-30ug sediment sample<sup>-1</sup>) for
 analysis of total C and N at the University of Georgia’s Center for
 Applied Isotope Studies. We then calculated the %C and %N of each samples
 and used these percentages for all statistical analyses.
description: Treatments are consistant across all datasheets, where Crustivore
 is biological soil crusts exposed to crustivorous snails and their
 relative byproducts (i.e., feces or mucus), Deteritivore is biological
 soil crusts exposed to deteritivorous snails and their relative nyproducts
 (i.e., feces or mucus), and No Snail are biological soil crusts exposed to
 no snail consumer byproducts (i.e., served as our control for snail
 effects).  Missing Values:  In the
 "Feces Nutrient Composition" dataset, the %N values for
 Crustivore are listed as "Below Detection Limit" because their
 %N content was below 0.2%-- the detection limit of University of Georgia
 Isotope Laboratory's equipment.  In the
 "Grazing Assay" dataset, we report biomass loss and
 CO<sub>2</sub> efflux for 20 replicates and %C, %N, and C:N
 for 12 replicates. We do not have all 20 values for our stoichiometry
 measurements because one of our plates was unreturned from the University
 of Georgia Isotope Laboratory.