Fresh swine manure

Seed stocking (5%)Pupation

Fly oviposition

Larvae inoculation

Larvae vermicomposting

Biomass separation

Separated vermicompost

Harvested larvae

Eclosion

Figure S1 A brief introduction into vermicomposting biotechnology using

housefly larvae (Musca domestica). A) Six key processing modules of

vermicomposting biotechnology, including (1) seed-pupation and eclosion; (2)

housefly oviposition; (3) larvae inoculation in unprocessed manure; (4)

vermicomposting using housefly larvae; (5) larvae-vermicompost separation; and (6)

seed sifting and breeding. B) Photos of this technology in real-world settings

(authorized).

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Harvested larvae Secondary Pupa

Oviposit

Breeding cages

Larvae in manure

Larvae

Concentrated pig barn

(A)

(B)

Figure S2 The larvae vermicomposting module with raw swine manure input during larval gut passage. A)A continuous-feeding

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vermicomposting practice is applied to treat raw fresh manure samples with the aid of housefly (Musca domestica) larvae. Raw swine manure

with a surface density of 11.0 kg m-2 is evenly spread on the ground of cement-block pools (7.2m×2.5m×0.2m; vermireactors) prior to larval

inoculation. The thickness of such unprocessed manure is initially about 5 cm. After that, 12-hour-old larvae are applied to the surface of the

manure with an average population density of 580 thousand per m2. The vermicomposting cycle, following larval inoculation, is 6 days. Twelve

hours after larvae inoculation, raw swine manure is added daily at a rate of 5.56, 15.6, 15.6, and 5.56 kg m -2 (wet weight) on day 1, day 2, day 3,

and day 4, respectively. These additions increase the vermicomposting layer from 5 cm to 8-10 cm in depth. On day 5, manure solids (45%

moisture), after solid-liquid separation (sieve opening: 2.5 mm), are added at rate of 6.5 kg m -2 (wet weight) into and mixed with the stack of

residues in order to improve the permeability and accelerate the biological dehydration before larvae-vermicompost separation on 6th day.

Samples were collected from the exact same locations at 8: 00 am once a day and raw manure addition took place at 2:00 pm. Samples were

taken from three same sampling locations in vermireactors using a cylindrical stainless-steel container (diameter 6 cm). After the whole core was

obtained from each location, we homogeneously mixed the cores before separate storage for further analysis. Besides, a continuous-feeding

composting practice is also applied without larvae introduced as controls, of which the feeding and sampling protocols are completely the same

as vermicomposting. B) The anatomy of the gut transit of housefly larvae (Musca domestica). The ingested manure-borne microorganisms were

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encountered by readily assimilable organic matter (mucus) and excreted enzymes. After digestion, the casts and feces were shed through the gut

as vermicompost, which can then be applied to farmlands as fertilizer. C) The cumulative amount of fresh manure input (blue curve, kg m-2),

remaining residues (red curve) and cumulative manure reduction mass (green curve) after each day, and the cumulative water reduction percent

in residues (bar, %) throughout the 6-day vermicomposting.

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Figure S3 Detecting the absence/presence of antibiotic resistant genes. A) The average numbers of unique resistance genes detected among

all of the samples. Since many resistance genes are targeted with multiple primers, if multiple primer sets detected the same gene, this is only

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counted as the detection of single unique resistance gene. Error bars represent standard deviation of either 3 or 12 field replicates. The different

letters above error bars represent significant differences at p< 0.05 using non-parametric Kruskal-Wallis test with Dunn’s multiple comparison

correction. C0d representing fresh raw manure samples was used as the reference, S1d-S6d represents manure samples during 6 days

vermicomposting, C3d and C6d represents manure samples under traditional composting on day 3 and day 6. The resistance genes detected in

fresh raw manure samples were classified based on B) the mechanism of resistance, and C) the antibiotic to which they confer resistance. MLSB

= macrolide-Lincosamide-Streptoqramin B resistance. The figures labeled in each pie-chart represent the detected genes numbers. The

highlighted numbers (in red color) in pie-charts indicate that the detected numbers of unique genes within such category were significantly (p<

0.05) lower in S6d compared to C0d.

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Figure S4 Attenuation or enrichment of antibiotic resistance genes. A) The descriptive statistics for attenuated resistance genes (the relative abundance, expressed as S6d/C6d when <1). The box plots denote 25th to 75th percentile; horizontal line, median; and whiskers, 10th and 90th

percentile. Symbols along X-axis indicate genes conferring resistance to aminoglycoside (Am), beta_lactamase (Be), (flor)/(chlor)/(am)phenicol (Ph), (flouro)quinilone (Qu), MLSB (Ml), Multidrug (Mu), Tetracylines (Te), Vancomycin (Va), and Sulfonamide (Su). B) The descriptive statistics for enriched resistance genes (S6d/C6d >1). Fold change values are considered significant only if the range created by standard deviations of the average are entirely < 1 (attenuated) or entirely > 1 (enriched)(see Table S4 for the datasets).After 6-day larvae treatment, 113 unique genes were attenuated (p< 0.05) in vermicompost (S6d) by 79.3% when compared to controls (C6d), which mainly encoded for antibiotic deactivation and cellular protection, conferring resistance to beta_lactamase, MLSB, tetracyclines, and vancomycin. Meanwhile, 16 unique ARGs were enriched (p< 0.05) by 5.6 fold. See Fig. 1 for attenuated or enriched ARGs expressed as S6d/C0d.

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Figure S5 Rarefaction curves and species rank-abundance curves for detected

sequences within each sample. Rarefaction curves of A) the numbers of derived

OTUs, and B) PD_whole_tree for all studied samples by randomly selecting

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sequences with step size of 1000. C) The species rank-abundance curves are

represented as means for triplicates of the samples with wider distribution of data

points along X-axis indicating higher abundances, and flatter shapes of curves along

Y-axis indicating higher evenness. Samples titled C stand for manure under traditional

composting on 0, 1st, 3st, 6th day, samples titled S stand for manure under

vermicomposting during 6 days, samples titled L stand for larval gut during 6 days.

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Figure S6 Alpha-diversity measurement among studied samples. Five calculated indices includes A) The numbers of derived OTUs, B)

Chao1, C) ACE, D) Shannon, E) Simpson, and F) Good_coverage for samples collected under traditional composting (Green in color), under

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vermicomposting processes using housefly larvae (Red in color), and larval gut samples (Blue in color). We randomly subsampled 25,000

sequences per sample ten times to correct for differences in sequencing depth, and then calculated these index. In the box plots, the symbols

indicate the following: box, 25th to 75th percentile; horizontal line, mean values; whiskers, 10th and 90th percentile. The circle above or below the

box plots indicates the maximum and minimum values, respectively.

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Figure S7 Comparison between 16S rRNAand metagenomic sequencing analysis for bacterial community composition. A) The relative

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abundance (%) of bacteria for each phylum by 16S rRNA analysis. B) The relative abundance (%) of bacteria for each phylum by metagenomic

analysis. The samples highlighted in red color in A) are further analyzed by metagenomic analysis. C) The relative abundance (%) for each

dominant genus as determined by 16S rRNA analysis without the inclusion of Ignatzschineria. D) The relative abundance (%) for each dominant

genus as determined by metagenomic analysis.

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Figure S8 Comparison of functional profile at hierarchy level IIIbetween raw manure (C0+S0) and larvae gut, as well as vermicompost

(S6) using 16S reads predicted by PICRUSt. A) Comparison of relative abundance of sequences involved in cell motility, B) Lipid

metabolism, and C) Xenobiotics biodegradation and metabolism. The numbers in the plot are indicated as follows: 1- 1,1,1-Trichloro-2,2-bis (4-

chlorophenyl) ethane (DDT) degradation, 2-Aminobenzoate degradation, 3-Atrazine degradation, 4- Benzoate degradation, 5- Bisphenol

degradation, 6- Caprolactam degradation, 7- Chloroalkane and chloroalkene degradation, 8- Chlorocyclohexane and chlorobenzene degradation,

9-Dioxin degradation, 10- Drug metabolism - cytochrome P450, 11- Drug metabolism - other enzymes, 12- Ethylbenzene degradation, 13-

Fluorobenzoate degradation, 14- Metabolism of xenobiotics by cytochrome P450, 15-Naphthalene degradation, 16-Nitrotoluene degradation, 17-

Polycyclic aromatic hydrocarbon degradation, 18- Styrene degradation, 19- Toluene degradation, 20- Xylene degradation. Differences (p< 0.05)

are represented as means (black dot) plus 95% confidence intervals. See Table S13 for other functional categories by PICRUSt. Shifts in

functions between raw manure and larval gut are similar to shifts in functions between raw manure and vermicompost.

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Figure S9 Contigs with syntenous location of ARG and transposase. Two contigs were found in both vermicompost and larvae gut with the

percent identity more than 98%.

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Figure S10 Codon substitution patterns between orthologous genes of

reconstructed population genomes and their closest phylogenetic neighbors. The

average dN/dS ratio (y-axis) is plotted against the synonymous substitution rates (dS)

(x-axis). Enterococcus, Providencia and Providencia, genomes were reconstructed

across larvae gut samples. Bacteroides (_MnS1 and _MnS2) and Alcanivorax

genomes were reconstructed across raw manure and vermicompost samples,

respectively.

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