Regulating Soil Bacterial Diversity, Enzyme Activities and Community Composition Using Residues from Golden Apple Snails

Golden apple snails (GAS) have become a serious pest for agricultural production in Asia. A sustainable method for managing GAS is urgently needed, including potentially using them to produce commercial products. In this study, we evaluate the effects of GAS residues (shell and meat) on soil pH, bacterial diversity, enzyme activities, and other soil characteristics. Results showed that the amendment of GAS residues significantly elevated soil pH (to near-neutral), total organic carbon (TOC) (by 10-134%), NO3-N (by 46-912%), NH4-N (by 18-168%) and total nitrogen (TN) (by 12-132%). Bacterial diversity increased 13% at low levels of amendment and decreased 5% at high levels, because low-levels of GAS residues increased soil pH to near-neutral, while high-levels of amendment substantially increased soil nutrients and subsequently suppressed bacterial diversity. The dominant phyla of bacteria were: Proteobacteria (about 22%), Firmicutes (15-35%), Chloroflexi (12%-22%), Actinobacteria (8%-20%) Acidobacteria, Gemmatimonadetes, Cyanobacteria and Bacterioidetes. The amendment of GAS residues significantly increased the relative abundance of Firmicutes, Gemmatimonadetes, Bacterioidetes and Deinococcus-Thermus, but significantly decreased the relative abundance of Chloroflexi, Actinobacteria, Acidobacteria, Cyanobacteria and Planctomycetes. Our results suggest that GAS residues treatment induces a near-neutral and nutrient-rich soil. In this soil, soil pH may not be the best predictor of bacterial community composition or diversity; rather soil nutrients (ie., NH4-N and NO3-N) and soil TOC showed stronger correlations with bacterial community composition. Overall, GAS residues could replace lime for remediation of acidic and degraded soils, not only to remediate physical soil properties, but also microbial communities. Importance The wide spreading golden apple snail (GAS) is a harmful pest to crop productions and could result in soil and air pollutions after death. In the previous study, we developed a biocontrol method: adding GAS residues to acidic soil to mitigate the living GAS invasion and spread, improve soil quality, and reduce soil and air pollution. However, the effects of GAS residues amendment on bacterial diversity and community still remain unclear. This study provided insights into bacterial diversity and community compositions to facilitate the evaluation of GAS residues application.

4 1 Introduction 47 Invasive golden apple snails (GAS) Pomacea canaliculata (Lamarck) have 48 become a serious pest for agricultural production in Asia (1). A series of control 49 methods have been developed (2, 3); the most widely used is chemical control by 50 molluscicides (4), which, could harm environmental and human health (5). A better 51 method for sustainable management is urgently needed, potentially by using them to 52 produce commercial products. 53 Studies have reported that GAS contains abundant CaCO 3 and proteins, and 54 could be used as feed for livestock such as pigs and ducks (6). However, only small 55 GAS can be eaten by ducks or pigs, because the hard shells of large adult GAS make 56 them unpalatable. The CaCO 3 in GAS can also neutralize acidic soils, similar to lime. 57 However, lime tends to lead to soil compaction, Si and P deficiency, and reduced soil 58 microbial biomass and diversity. 59 Anthropogenic N inputs to terrestrial ecosystems have increased three-to 60 five-fold over the past century (7). High levels of N fertilization can drive soil 61 acidification both directly and indirectly (8). J. H. Guo et al. (8) found that 62 anthropogenic acidification driven by N fertilization is at least 10 to 100 times greater 63 than that associated with acid rain. The application and deposition of N is expected to 64 continue to increase (9). 65 To alleviate soil acidification and control the invasion of the GAS, we have 66 proposed using powered residues from GAS to mitigate soil acidification 67 (unpublished data). The application of GAS residues can significantly increase soil 68 5 pH and nitrate nitrogen (NO 3 -N) both at a high and low amendment levels, and can 69 increase soil total organic carbon (TOC), total nitrogen (TN), ammonium nitrogen 70 (NH 4 -N) and NO 3 -N compared with controls and with liming, which has no 71 significant effect on soil nutrients (unpublished data). Although our previous research 72 has indicated that the addition of GAS residues could significantly increase soil 73 microbial biomass and regulate microbial community structure, the mechanisms by 74 which GAS residues regulate microbial community composition, relative abundance 75 and diversity remain unknown. 76 Previous studies have proposed soil pH and N input as the main predictors of soil 77 microbial diversity in soils (10-12). However, the responses of soil microbes to 78 elevated N inputs and pH are inconsistent. Numerously studies have revealed that N 79 addition led to significant reductions in soil microbial activity (13), diversity (14) and 80 community composition (15) because of increases in C sequestration and/or decreased 81 soil respiration rates (13). Some studies have suggested that neutral soils support 82 greater bacterial diversity than do acidic soils (10, 11). However, some researchers 83 have suggested the opposite-that forest soils with lower pH support greater 84 microbial diversity than agricultural soils with higher pH values (16).  Here, we conducted a series of greenhouse experiments amending GAS residues 91 and lime to acidic and degraded soils. We hypothesize that GAS residues and 92 lime-both of which increase soil pH-may differ in their regulation of microbial 93 community structure, diversity and microbial enzyme activities. The objectives of this 94 study were: (1) to explore bacterial community composition and diversity in soils 95 neutralized with powered GAS residues, (2) to determine key factors controlling the 96 composition of bacterial communities, (3) and assess differences in bacterial 97 communities in soils treated with GAS residues and soils treated with lime. washed and frozen at -40℃ in a freezer for 24h. Then the dead snails were dried, 105 grounded into powder and stored in a desiccator. A slightly acidic soil was also 106 collected from the paddy fields with the pH levels ranging from 6.25 to 6.53. The soil 107 predominately consists of medium (38%) and fine (22%) sand, silt (36%) and clay 108 (4%) and has 16.38 g kg -1 of TOC, 2.20 g kg -1 of TN and 0.58 g kg -1 of total 109 phosphorus (TP). Agricultural University. We implemented three treatments: (1) the control treatment 113 with soil only (CK); (2) soil amended with GAS residues (i.e., shell and meat) (SR); 114 and (3) soil amended with lime (SL). Each treatment had six levels of amendment: 0.5, 115 1, 2.5, 25, 50, and 100 g kg -1 . Amendments were homogenized with soil in each 116 treatment and carefully packaged in a polyvinyl chloride (PVC) column with a 117 diameter of 180 mm and a height of 260 mm. A filter paper was mounted at the 118 bottom of the column to prevent loss of soil or GAS residues. A base plate was also 119 placed at the bottom of the column to position the column. About 400 ml of deionized 120 water (pH = 7.0±0.1) were sprayed to the column each week to prevent the column 121 from drying. Each treatment was triplicated in this study. After 120 days incubation, 122 the soil samples were collected and stored at 4 o C for about 4h.      Amendments of GAS residues and lime resulted in increased soil pH (Fig. 1a).

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The addition of 1.0-2.5 g kg -1 GAS residue increased soil pH to neutral (7.0) and 215 additional amendments further increasing pH. Even the smallest amendment of lime 216 (0.5 g kg -1 ) changed soil pH sharply from acidic to light alkaline (pH > 7.8) (Fig. 1a).

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The effect of GAS residues on pH may have been mitigated by the decomposition of Addition of GAS residues also increased soil carbon and soil 227 nutrients-specifically, TOC, TN, NO 3 -N and NH 4 -N (Fig. 1b). TOC and NH 4 -N 228 progressively increased as more GAS residues were added, increasing by 134.28%

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(TOC) and 167.80 % (NH 4 -N) with the amendment of 100 g kg -1 GAS residue. Soil 230 nitrogen, TN and NO 3 -N showed peak values when 25 g kg -1 GAS residues were 231 13 added, a threshold value prior to which proteins in the GAS residues decomposed or 232 dispersed quickly, but after which anaerobic soils limited the activities of soil 233 microbes and the transfer of proteins into small molecular and inorganic matter so that 234 more NH4-N and NOx were produced and released into the air (27). and lime both significantly affected soil bacterial diversity (Fig. 1c). Specifically, 239 amendment of up to 2.5 g kg -1 GAS residues increased diversity, as measured by 240 Shannon diversity index, from 4.76 (CK) to 4.99. The addition of more GAS residue 241 decreased bacterial diversity; the addition 75 g kg -1 GAS residues resulted in the 242 lowest Shannon diversity index value (4.55) across treatments. Similar to GAS 243 residue, the addition of lime had a positive effect on diversity, but had a negative 244 effect when more than 2.5 g kg -1 was added (Fig. 1c).

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Previous studies have proposed that soil pH affects soil bacterial community Gemmatimonadetes, Cyanobacteria and Bacterioidetes (Fig. 3). The amendment of 300 GAS residues significantly increased the relative abundance of Firmicutes,

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For the SR treatments, most significant correlations were found in soil pH; the 370 only groups with relative abundance that was significantly positively related to pH 371 were Bacteroidetes, Firmicutes and Chlorobi with soil pH (Fig. 4a). Fewer phyla 372 showed significant correlations with soil nutrients in the SL treatments compared to 373 the SR treatments. These results suggest that more phyla were affected by C, N (ie.,  (Fig. 5). In SL treatments, soil pH was the factor most strongly related to changes in 381 bacterial community composition (R 2 = 0.90) (Fig. 5a). However, in SR treatments 382 soil nutrients tend to shape changes in bacteria composition (Fig. 5b). Our results are  from low levels of GAS residues amendments, because high levels of GAS residues 407 amendments and low levels of lime amendments showed similar effects on soil pH.

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The results showed that more specialized bacteria phyla were detected in soils treated 409 with high levels of GAS residues amendments, which suggests that GAS residues can 410 shape bacterial community compositions. Overall, in this study, the amendment of GAS residues significantly increased 448 soil pH due to the CaCO 3 component of the GAS's shell. However, GAS residues had 449 weaker effects on soil pH than did lime treatments. For example, at the same amended 450 levels, the pH of soils amended with lime increased sharply, while soils amended with 451 GAS residues rose only to near-neutral pH. In addition, amendment of GAS residues 452 26 resulted in increased levels of soil nutrients, which could in turn lead to increased 453 bacterial diversity at low amendment levels and decreased bacterial diversity at high 454 amendment levels. That likely attribute to the amendment of GAS residues induced a 455 copiotrophic environment in which, the relative abundance of copiotrophic bacterial 456 communities were increased while oligotrophic bacterial communities were reduced.

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What's more, soil pH also responsible for the changes of soil bacterial communities.

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Some of them such as Gemmatimonadetes, Tenericutes, Chlorobi and Bacteroidetes 461 were increased due to their roles in C and N cycling, while some of them were 462 decreased because they were suppressed at a higher pH environment. Most 463 researchers suggested that pH was the best predictor for bacterial diversity and Our study proposed that GAS residues may be appropriate to remediate acidic 478 soil, improve soil quality and reduce GAS populations in areas subject to GAS 479 invasion. In practice, it may not be practical to dry and crush GAS into powder before 480 applying it to soils. Instead, practitioners could create GAS residues at lower costs by 481 collecting living or dead GAS, spreading them on the soil surface and smashing them 482 using high speed rotary tiller. Also, we suggest applying GAS residues to nonirrigated 483 farmland to reduce the potential water pollution. We suggest amending GAS at 2.5 -484 25 g kg -1 , which appears to be better for soil health and bacterial diversity. These