DOI: 10.25338/b8nk9n
title: Snail mucus increases the CO2 efflux of biological soil crusts

description: Biological soil crusts (hereafter, biocrusts) are communities of
 microorganisms that regulate key ecosystem processes such as water
 distribution, soil erosion, and nutrient cycling in drylands worldwide.
 The nature of biocrust function can be influenced by multiple
 environmental factors, including climatic conditions (e.g.,
 precipitation), interactions with plants, and anthropogenic disturbances.
 Animal regulation of biocrust function has received less research
 attention, focusing primarily on livestock trampling and to a much lesser
 extent on biocrust consumption by mesofauna. Deposition of animal waste
 products, carcasses, and other body secretions such as mucus may also
 affect biocrust function. Yet, this novel regulatory pathway, to our
 knowledge, has never been empirically tested. Our goal was to begin
 bridging this knowledge gap by exploring how snail mucus affects biocrust
 CO2 efflux— using two distinct biocrust communities and three snail
 species. We found that snail mucus increased the CO2 efflux of both
 cyanobacteria- dominated and lichen/moss- dominated biocrusts. However,
 the magnitude of snail mucus effects on biocrust CO2 efflux varied between
 snail species— possibly due to species-level differences in snail diet.
 Our study highlights a novel interaction between animals and biocrusts and
 suggests that even small quantities of animal-derived nutrients can have
 important consequences for biocrust carbon dynamics. Manuscript Highlights
 Mucus increased the CO2 efflux of cyanobacteria-dominated biocrusts by
 &gt;20%. Mucus enhanced the CO2 efflux of moss/lichen-dominated
 biocrusts by &gt; 86%. Dietary differences likely underlie
 species-specific effects of mucus on biocrusts.
description: <b><i>Study system</i></b>
 We focused our investigation
 on two LTER sites in the Negev desert that differ in climatic conditions
 and biocrust composition. 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 per year spread across 19-42 days (Israel
 Meteorological Survey, 2021; Station: 253052). Avdat is characterized by
 1-2 mm thick cyanobacteria dominated biocrusts (also containing bacteria,
 algae, and fungi) that have a distinct flat, solid surface. This solid
 surface is due to cyanobacteria secreting polysaccharides that adhere soil
 particles (Mazor and others 1996). Sayeret Shaked Park (31°16’16” N,
 34°39’03” E; hereafter, Shaked Park) is in the Northern Negev and receives
 190 mm of rainfall per year spread across 25-52 days (Israel
 Meteorological Survey, 2021; Station: 251691). Shaked Park is
 characterized by moss/lichen dominated biocrust that also contain
 bacteria, cyanobacteria, algae, and fungi. The moss/lichen dominated
 biocrusts at Shaked Park reach thicknesses of 8-10 mm on south-facing
 slopes and 10-15 mm on north-facing slopes (Zaady and others 1996; Zaady
 and others 1998). At Avdat and Shaked Park, snails
 are relatively abundant— reaching densities of 12 and 89 snails per
 m<sup>2</sup>, respectively. Avdat has multiple common snail
 species, including <i>Sphincterochila zonata
 </i>(SZ)<i>, Xerocrassa simulata </i>(XS), and
 <i>Sphincterochila</i> <i>prophetarum</i> (SP)
 that overlap in their distribution and use of biocrust surfaces (Bar,
 1975; Degen and others 1992; Genot-Lahav, 1986). Meanwhile, Shaked Park is
 mainly dominated by XS, but also has a small population of SZ. The diets
 of all three snail species include biocrusts (Yom-Tov and Galun 1971;
 Shachak and Steinberger 1980; Shachak and Brand 1981). However, XS
 preferably consumes plant litter (Yom-Tov and Galun 1971). The activity of snail species in
 the Negev is regulated by moisture, with all species only becoming active
 when the ground is damp (Yom-Tov, 1971; Hermony and others 1992).
 Consequently, snail movement along biocrusts in search of food, mates, and
 egg laying habitat is limited to short bouts following substantial
 precipitation events  (Shachak and Steinberger 1980). For example,
 <i>S. zonata</i> are active for only 8-27 days annually
 (Shachak and Steinberger 1980). These short bouts of snail activity likely
 have important consequences for biocrust function, as snails deposit
 considerable nutrient-rich mucus while moving along the biocrust surface—
 covering approximately 15% of the biocrust surface in mucus trails (Fig.
 1). <b><i>General experimental
 approach</i></b> We used three complementary
 laboratory experiments to reveal how desert snails affect biocrust
 activity. To achieve a comprehensive answer, we used two different
 biocrust types, and three species of snails.  We also used two common
 biocrust cultivation methods to control for variation in biocrust
 performances that may reflect specific rearing conditions (Doherty and
 others 2015).  In <i>experiment 1,</i> we explored how a mix
 of mucus from three abundant snail species affects laboratory grown
 cyanobacteria-dominated biocrusts from Avdat. In <i>experiment
 2</i> we tested how <i>X. simulata</i> (XS) mucus from
 the Avdat and Shaked Park populations affect field collected moss/lichen
 dominated biocrust from Shaked Park. In <i>experiment 3</i> we
 assessed how the mucus of each of the three common snail species at Avdat
 affect field collected cyanobacteria dominated biocrusts from Avdat. In
 all three experiments we measured the CO<sub>2</sub> efflux as
 a measure of biocrust activity.
 <b><i>Experiment 1</i></b>
 We collected biocrust (top 2
 mm) and sediment (2-10 cm depth) from Avdat. After sieving the biocrust
 and sediment with a 2 mm metal sieve to remove rocks and plant litter, we
 added 63.7 ± 0.3g (mean ± SE) of sediment topped with 50.1 ± 0.2g (mean ±
 SE) of biocrust to 50, 145 mm (diameter) x 20mm (depth) plastic petri
 dishes. We grew these crusts in growing chambers at 16°C with 70% humidity
 and a 16:8 light: dark cycle for 111 days. For this first experiment, we
 chose to cultivate biocrusts using techniques designed for biocrust
 restoration (see Doherty and others 2015). This method may be less
 realistic but reduces variation within and between our biocrusts. Then, we
 randomly allocated 25 of our laboratory-grown crusts to mucus and no mucus
 (i.e., mucus- free) treatments. In the mucus treatment, we watered
 biocrusts with 1ml of our diluted snail mucus mixture (equal to 1 days’
 worth of snail mucus) and an additional 2.5ml of DI every other day for 13
 days. In the no mucus control, we repeated the same watering protocol but
 with 3.5ml of DI water. We chose this arbitrary protocol for logistical
 reasons. Yet, both the overall water addition and the distribution are
 well within the range of natural precipitation events in our study site,
 as the median number of rain events from 2008-2020 at Avdat in the wettest
 months (January and February ) is seven (Israel Meteorological Survey,
 2021; Station: 253052). To produce the mucus, we collected
 wild snails (Species: XS, SP, and SZ; n = 20 individuals/species) from
 Avdat and randomly placed groups of 10 individuals by species (n = 2
 containers per species) in plastic containers (249 mm length x 190.5 mm
 width x 94.0 mm height). The containers were placed in a room, maintained
 at 14-17°C with a 11:13 light: dark cycle. We extracted mucus from housed
 snails twice weekly by placing single species groups of 10 snails on a 280
 mm x 216 mm transparent plastic sheet (3M Write-on Overhead Projector
 Transparency Film) covered by a 140 mm x 115 mm x 50 mm height plastic
 lid. We wetted the snails daily with DI to ensure their activity. After 48
 hours, we scrapped the mucus off the transparent plastic sheets and
 homogenized all collected snail mucus (across all species) in 60 ml of DI,
 generating a mixed mucus solution containing the equivalent of 60 days’
 worth of snail mucus production (i.e., 60 snail<sup>-1</sup>
 days<sup>-1</sup>, 1ml equals 1 snail<sup>-1</sup>
 day<sup>-1</sup>).  Mixing all species’ mucus is
 representative of mucus deposition in nature, where all species co-exist
 on moist biocrusts (Yom-Tov 1971). Our mucus harvesting technique should
 capture mainly the water-soluble components of snail mucus. Diluted mucus
 was then frozen at -80°C until use. All snail species studied consume
 biocrusts. Thus, we chose to use pre-extracted mucus (rather than have
 snails directly deposit mucus trails on biocrusts) to isolate the effects
 of mucus deposition from biocrust consumption. At the completion of the 13 days,
 we measured the effects of snail mucus on biocrust activity by measuring
 biocrust CO<sub>2</sub> efflux. We placed watered (3 ml of DI
 water) mucus and control laboratory grown crusts 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 the chambers with
 CO<sub>2</sub>-free air at a rate of 2L
 minute<sup>-1</sup> for a total of 5 minutes. Laboratory grown
 crusts were incubated in the flushed, airtight chambers at 16°C with no
 light for a total of 24 hours. After the dark incubation, we used a
 LI-7000 CO<sub>2</sub>/H<sub>2</sub>O 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 biocrust. <b><i>Experiment
 2 </i></b> The goal<b><i>
 </i></b>of this experiment was to explore how XS<i>
 </i>mucus from two distinct populations affect<i>
 </i>field collected moss/lichen-dominated biocrust from Shaked Park.
 We used mucus from XS because this is the main snail species found at
 Shaked Park. To create the laboratory biocrusts, we collected 30 intact
 biocrusts from Shaked Park using 120 mm diameter x 20 mm deep plastic
 petri dishes (following Weber and others 2016). We housed the field
 collected biocrust in the lab for 25 days at 18°C, with 60% humidity and a
 8:16 light: dark cycle. During this time, we watered our field collected
 biocrusts daily with 3.6-4.2 ml of DI water, using a hand-held spray
 bottle. Using field-collected biocrust for experiments 2 and 3 may provide
 more realistic, but more heterogeneous, results compared to those obtained
 from laboratory cultivated early successional biocrusts, complementing the
 approach used for experiment 1. We randomly allocated field
 collected Shaked Park biocrusts (n =10 per treatment) to one of three
 treatments 1) XS-AV mucus, 2) XS-SS mucus, and 3) mucus-free control. In
 the XS-AV mucus treatment, we watered biocrusts with 1.5ml of the diluted
 mucus (equal to 1 days’ worth of snail mucus) extracted from XS snails
 from Avdat and 2.0 ml of DI water every other day for 16 days (until 01
 April 2019). In the XS-SS treatment, we repeated the same procedures but
 with extracted XS mucus from Shaked Park. In the mucus-free control, we
 watered biocrusts with 3.5ml of DI water every other day for 16 days.
 Here, we chose the same arbitrary protocol as in experiment 1 but with
 eight watering days, which corresponds with the median number of rain
 events in January and February (the wettest months) from 2008-2020 in this
 region (Israel Meteorological Survey, 2021; Station: 251691).
 To produce the mucus, we
 collected snails (Species: XS; n = 30 individuals per site) from both
 Avdat and Shaked Park. We randomly placed snails in 249 mm length x 190.5
 mm width x 94.0 mm height plastic containers [in groups of 10 individuals
 by site (n = 3 containers per site)]. We placed all containers in a
 climate-controlled room, maintained at 15 ± 1.5 °C with a 11:13 light:
 dark cycle. Snails were fed biocrusts (Avdat and Shaked Park),
 <i>Hammada scoparia </i>litter, and <i>Atractylis
 serratuloides </i>litter <i>ad libitum. </i>We extracted
 snail mucus from housed snails once a week for two weeks, using the same
 protocol described for Experiment 1. However, at the end of each
 extraction session, we scrapped the mucus off the transparent plastic
 sheet and the small plastic container using 15 ml of DI water per box and
 homogenized all collected mucus (across boxes) in 50 ml falcon tubes.
 Falcon tubes containing diluted mucus were then frozen at -80°C until use.
 We did experience snail death during the snail extraction process (~10%
 mortality per week). When snails died, we replaced them with a new snail
 that was collected from the appropriate field site. We quantified snail mucus
 production between XS populations (i.e., Avdat vs. Shaked Park) by
 creating six, 1.5ml pseudo-replicated samples of homogenized snail mucus.
 We then freeze-dried the mucus samples for 24 hours and weighed the
 remaining dried material. To account for natural variation in
 field collected biocrust CO<sub>2</sub> efflux we wanted to
 quantify the pre-experimental CO<sub>2</sub> efflux of each
 biocrust but, due to incubator malfunctions, we were unable to measure
 biocrust CO<sub>2</sub> efflux prior to experimental
 manipulations. We quantified the CO<sub>2 </sub>efflux using
 the same protocol as for Experiment 1.
 <b><i>Experiment 3</i></b>
  The goal of this experiment
 was to reveal how interspecific variation in snail mucus affects field
 collected cyanobacteria dominated biocrusts from Avdat.  We collected 40
 field collected biocrusts from Avdat and reared them in the lab using the
 exact same protocol as for Experiment 2. We randomly allocated 10 field-
 collected biocrusts to each of the four treatments: 1) XS mucus, 2) SP
 mucus, 3) SZ mucus, and 4) mucus-free control. In each mucus treatment, we
 watered field- collected Avdat biocrusts with 1ml of the corresponding
 diluted mucus (equal to 1 days’ worth of snail mucus) and an additional
 2.5ml of DI water every other day for 16 days. In the mucus-free control,
 we watered Avdat biocrusts with 3.5ml of DI water every other day till the
 end of the experiment. We used identical watering protocol as for
 Experiment 2 to allow better comparisons of the XS mucus effect on
 biocrust CO2 efflux between biocrust types.   To harvest mucus, we collected
 snails (XS, SP, and SZ; n = 60 individuals per species) and reared them
 using the same protocol as for Experiment 2. During the mucus excretion
 period, all snail species were able to feed <i>ad libitum</i>
 on biocrusts and <i>Hammada scoparia </i>litter collected from
 Avdat.<i> </i>We extracted snail mucus from housed snails once
 weekly for two weeks using a similar extraction protocol as in Experiment
 2. At the end of each extraction, we scrapped the mucus off the
 transparent plastic sheet and the small plastic container and diluted the
 collected mucus from each box in 10ml of DI water and homogenized all
 mucus by snail species in 50ml falcon tubes. Falcon tubes containing
 diluted mucus were then frozen at -80°C until use. We did experience snail
 death between snail extractions (~10% mortality per week). When snails
 died, they were replaced with new snails also collected from Avdat. We
 quantified the production of snail mucus between the three species of
 snails (XS, SP, and SZ) by creating three, 1.5ml pseudo-replicated samples
 of homogenized snail mucus per species. We then freeze-dried the mucus
 samples for 24 hours and weighed the remaining dried material. We measured
 the CO<sub>2</sub> efflux of field collected biocrusts at the
 beginning and end of the two-week mucus addition period using the protocol
 described in Experiment 1. <b><i>Data
 analysis</i></b> In Experiment 1, we compared the
 final CO<sub>2</sub> efflux (ug C day<sup>-1
 </sup>g<sup>-1</sup>) of laboratory grown biocrusts
 between mucus treatments using a two-sample T-test. In Experiment 2, we
 compared the dry mass of snail mucus between snails from Avdat and Shaked
 Park using a two-sample T-test. We compared the final
 CO<sub>2</sub> efflux of biocrusts between treatments using a
 Generalized Linear Model (GLM) with a Tukey’s HSD test. We used a GLM
 because they accommodate variance in heterogeneity, non-normal
 distributions, and uneven sample sizes (Venables and Dichmont 2004; Bolker
 2008). Additionally, we used goodness-of-fit statistics to determine the
 best distribution for each model. Prior to data analysis, we removed two
 outliers that were 1.5-times the interquartile range above the third
 quartile [No mucus (n = 8); XS-AV (n = 10); XS-SS (n = 10)]. In Experiment
 3, we compared the snail mucus dry mass between our three snail species
 using a one-factor ANOVA with snail species (i.e., XS, SP, and SZ) as a
 fixed factor. <i> </i>We compared the change in
 CO<sub>2</sub> efflux of Avdat biocrusts between treatments
 using a GLM and a Tukey’s HSD test. Additionally, we used goodness-of-fit
 statistics to determine the best distribution for each model. Prior to
 data analysis, we removed five outliers that were 1.5-times the
 interquartile range above the third quartile or below the first quartile
 [No mucus (n = 7); XS mucus (n = 9); SP mucus (n = 9); SZ mucus (n = 10)].
 We ran all our statistical analysis in jamovi version 1.217 using the
 “jmv” and “gamlj’ modules (Gallucci 2019; R Core Team 2019; The jamovi
 project 2020).  We
 calculated the effect sizes of mucus impacts on biocrusts to compare
 across snail and biocrust type using OpenMee software (Wallace and others
 2017). Specifically, we calculated the Hedges <i>d </i>by
 comparing the means of the mucus treatment(s) to the mean of the
 mucus-free control in each of our experiments. We interpreted our effect
 sizes using the benchmarks set by Cohen (1988), who suggested that an
 effect size of 0.2 is small, 0.5 is moderate, and anything greater than
 0.8 is large. While Cohen’s benchmarks are general, we have no other
 standard at which to compare our effect sizes too—as mucus effects on
 biocrusts are a novel interaction in the literature. Thus, we have chosen
 to use Cohen’s benchmarks to simply consider the relative magnitude of
 each interaction in our study.
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