Gut Bacteria Promote Phosphine Susceptibility of Tribolium castaneum by Aggravating Oxidative Stress and Fitness Costs

Simple Summary The red flour beetle, Tribolium castaneum, has developed widespread pesticide resistance. Knowledge about resistance mechanisms can provide ideas for pesticide resistance management. Since gut microbes can affect host pesticide resistance, we explored gut bacteria-mediated phosphine susceptibility in T. castaneum and its molecular basis. Among five cultivable gut bacteria excised from a phosphine-resistant T. castaneum, only Enterococcus sp. inoculation significantly promoted host susceptibility to phosphine, while inoculation of any other gut bacteria did not. Furthermore, when T. castaneum was exposed to phosphine, Enterococcus sp. inoculation decreased the female fecundity, promoted host oxidative stress, and suppressed the expression and activity of host antioxidant enzymes. In the absence of phosphine, Enterococcus sp. inoculation also elicited overactive host immune responses, including the dual oxidase–reactive oxygen species system. These results indicate that Enterococcus sp. likely promotes host phosphine susceptibility by aggravating oxidative stress and fitness costs. Abstract Knowledge about resistance mechanisms can provide ideas for pesticide resistance management. Although several studies have unveiled the positive or negative impacts of gut microbes on host pesticide resistance, minimal research is available regarding the association between gut microbes and host phosphine resistance. To explore the influence of gut bacteria on host phosphine susceptibility and its molecular basis, mortality, fitness, redox responses, and immune responses of adult Tribolium castaneum were determined when it was challenged by phosphine exposure and/or gut bacteria inoculation. Five cultivable gut bacteria were excised from a population of phosphine-resistant T. castaneum. Among them, only Enterococcus sp. inoculation significantly promoted host susceptibility to phosphine, while inoculation of any other gut bacteria had no significant effect on host phosphine susceptibility. Furthermore, when T. castaneum was exposed to phosphine, Enterococcus sp. inoculation decreased the female fecundity, promoted host oxidative stress, and suppressed the expression and activity of host superoxide dismutase, catalase, and peroxidase. In the absence of phosphine, Enterococcus sp. inoculation also elicited overactive immune responses in T. castaneum, including the immune deficiency and Toll signaling pathways and the dual oxidase–reactive oxygen species system. These results indicate that Enterococcus sp. likely promotes host phosphine susceptibility by aggravating oxidative stress and fitness costs.


Introduction
The red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is one of the most damaging pests, which can infest 246 grain commodities and cause economic losses in warehouses and mills worldwide [1].Product losses posed by T. castaneum infestations are up to 34-40% [2].Owing to the magnitude of such losses, it has been controlled with various pesticides over a long period.However, T. castaneum can easily accommodate to Insects 2023, 14, 815 2 of 14 frequently used pesticides, including contact pesticides and the fumigant phosphine.It has developed diverse strategies, including metabolic resistance, increased efflux capacities, and target site insensitivity, to deal with pesticides [3].Therefore, knowledge of resistance mechanisms is crucial for the development of novel pest management strategies [4].Recently, the association between gut microbes and host resistance to pesticides and its possible mechanisms have been unveiled in many studies [5], which provides a direction for investigating resistance mechanisms.
Phosphine can suppress the activity of electron transfer chain enzymes and antioxidant enzymes such as catalase and peroxidase, thus eliciting massive accumulation of highly reactive oxygen species (ROS), which contributes to phosphine toxicity [3].Additionally, considering that infection with a high load of gut bacteria commonly elicits overactive immune responses, subsequently promoting host oxidative stress via a sudden boost of humoral immune effectors such as ROS [6,7], it is assumed that some bacteria can modify host phosphine susceptibility by intervening in the redox system.Furthermore, tradeoffs exist between physiological traits due to competition for limiting resources, i.e., increases in the fitness value of one trait may lead to a corresponding decline in the fitness value of another [8].Thus, it is speculated that when pests are simultaneously challenged by pesticide exposure and bacterial infection, the tradeoff between immune responses and other fitness traits associated with pesticide resistance will increase host susceptibility to pesticides, as has been found in Diaphorina citri infested with Candidatus Liberibacter asiaticus [9].
Although several studies have unveiled the positive or negative impacts of gut microbes on host pesticide resistance [5,10], minimal research is available regarding the association between gut microbes and host phosphine resistance [11,12].Herein, we aim to explore gut bacteria-mediated phosphine susceptibility in T. castaneum and its molecular basis, with emphasis on host oxidative stress and fitness costs, through a four-stage research process.When T. castaneum was challenged by phosphine exposure and/or gut bacteria inoculation, (1) the mortality of T. castaneum was measured to unveil the impact of gut bacteria on host phosphine resistance and to screen resistance-mediating gut bacteria, (2) the female fecundity of T. castaneum was measured to unveil the impact of resistance-mediating gut bacteria on host reproductive fitness, (3) the level of hydrogen peroxide and malondialdehyde in T. castaneum was measured to unveil the impact of resistance-mediating gut bacteria on host oxidative stress, and (4) antioxidant and immune responses in T. castaneum were measured to unveil the molecular basis for gut bacteria-induced oxidative stress.

Insects
The stock of T. castaneum originated from a population sampled from Shantou, Guangdong, China, in June 2015 and was cultured continuously in laboratory conditions for more than seven years.This stock was strongly resistant to phosphine with a resistance ratio of 208.3, determined via the FAO standard method [13].The normal diet for the insects was a blend of wholemeal flour and yeast powder (19:1, w:w) sterilized via exposure to ultraviolet radiation (about 254 nm) for 2 h.Insects were cultured at 28 • C and 65% relative humidity in continuous darkness.The pupae were collected and then separated by sex as previously described for subsequent assays [2].If there were no special instructions, experiments with T. castaneum were performed under the same normal conditions mentioned above.

Isolation and Identification of Gut Bacteria
To isolate gut bacteria, 7-day-old mixed-sex adult T. castaneum were starved for 1 d, then surface disinfected with 70% alcohol, and subsequently rinsed thrice with aseptic water.Gut bacteria were excised and purified as previously described [14].The purified gut bacteria were collected and preserved in Luria-Bertani (LB) broth added with 25% glycerine at −80 • C for subsequent assays.Bacterial isolates were revived in LB broth prior to use.Five cultivable gut bacteria were successfully excised from the tested T. castaneum.Bacterial identification based on 16S rDNA genes was conducted as previously described [15].The 16S rDNA gene primers 27F and 1492R were used to amplify DNA fragments (Supplementary Table S1).Amplified DNA fragments were sequenced and blasted against the 16S rDNA database and submitted to the NCBI database, and their GenBank accession numbers were OM992224.1,OM992227.1,OM992228.1,OM992229.1,and OM992230.1 (Supplementary Figure S1).

Antibiotic Treatment and Gnotobiotic Inoculation
Since antibiotics could reshape and deplete insect gut bacteria [16], newly emerged beetles were cultured with a normal diet containing 20 µg/g antibiotic cocktail (ciprofloxacin:penicillin:vancomycin = 1:1:1) for 3 d to produce germ-free beetles.After antibiotic treatment, 4-day-old germ-free beetles were cultured with a normal diet mixed with a given bacterial isolate for 3 d to produce gnotobiotic beetles [15].All treatments were conducted in an aseptic environment.Germ-free and gnotobiotic beetles were then cultured with a normal diet in an aseptic environment for subsequent assays.The beetles cultured with a normal diet under normal conditions were used as conventional beetles.
To confirm whether gut bacteria were successfully depleted and inoculated, their abundance was determined via real-time quantitative PCR (qRT-PCR) using the 2 −∆∆CT method [17] and normalized by the qRT-PCR data of the β-actin gene of T. castaneum.Gut homogenates of thirty 6-, 8-, 10-, 12-, 14-, and 16-day-old beetles were prepared according to the method described above.Total DNA was extracted from the gut homogenate using an E.Z.N.A. ® soil DNA kit (Omega Bio-Tek, Norcross, GA, USA).qRT-PCR primers are shown in Supplementary Table S1.The qRT-PCR was run in a 20 µL reactive mixture including 10 µL of SYBR Green Mix (TaKaRa, Otsu, Japan), 0.8 µL of each primer (50 µmol/L), and 5 ng of template DNA.Thermocycling conditions were 95 • C for 30 s, and then 35 cycles of 95 • C for 5 s, 60 • C for 30 s, and 72 • C for 30 s.After the thermal cycles, melting curve analysis was conducted to confirm that the reaction produced a single, specific product.The thermocycling programs for the melting curve analysis were the same as those previously described [6].Reactions were conducted in triplicate, and the mean of triplicate analyses represented one biological repetition.Each treatment consisted of five biological repetitions.

Bioassay of Phosphine Susceptibility
Phosphine susceptibility of conventional, germ-free, and gnotobiotic beetles was measured according to the FAO standard method [13] to unveil the impact of bacteria inoculation on host phosphine resistance.Thirty 10-day-old beetles were exposed to phosphine at the concentrations of 0, 1000, 1250, 1500, 1750, and 2000 mL/m 3 for 20 h.After exposure to phosphine, the beetles were ventilated and then cultured under normal conditions.Their mortality was assessed after 14 d of culture and corrected for control mortality [18].Each treatment consisted of five biological repetitions.Since only Enterococcus sp.significantly influenced host phosphine susceptibility, it was used in subsequent assays to explore the molecular basis for gut bacteria-mediated phosphine susceptibility in T. castaneum.

Determination of Female Fecundity
Female fecundity of conventional, germ-free, and gnotobiotic beetles was measured to assess the impact of Enterococcus sp.inoculation on reproductive fitness of T. castaneum after exposure to phosphine.After exposure of 20 pairs of 10-day-old beetles to 1250 mL/m 3 phosphine for 20 h, the beetles were ventilated and then cultured under normal conditions.Pairs were separately cultured with a normal diet in a culture plate with a diameter of 15 mm and checked daily for 45 d, on which most females did not lay eggs anymore.The diet was changed every 2 d.Freshly laid eggs were transferred to a new culture plate and monitored for 10 d for emergence.Female fecundity was defined as the number of viable eggs laid per female.The average fecundity of 20 pairs represented one biological repetition.Each treatment consisted of five biological repetitions.

Qualification of MDA and H 2 O 2
Since malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) are commonly used as biological indicators of oxidative stress [19,20], the level of MDA and H 2 O 2 in conventional, germ-free, and gnotobiotic beetles was measured to assess the impact of Enterococcus sp.inoculation on oxidative stress in T. castaneum after exposure to phosphine.After exposure of 10-day-old beetles to 1250 mL/m 3 phosphine for 0, 6, 12, and 24 h, the level of MDA and H 2 O 2 was determined using BC0020 and BC3590 assay kits (Solarbio, Beijing, China), respectively.MDA reacts with thiobarbituric acid to produce tridione with a maximum absorbance at 532 nm, and H 2 O 2 reacts with titanic sulfate to produce a titanium peroxide complex with characteristic absorbance at 415 nm [21].The level of MDA and H 2 O 2 was calculated based on the absorbance according to the assay kit protocol.The samples were conducted in triplicate, and the mean of triplicate analyses represented one biological repetition.Each treatment consisted of five biological repetitions.

Determination of SOD, CAT, and POD Activities
The activity of three antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), in conventional, germ-free, and gnotobiotic beetles was measured to assess the impact of Enterococcus sp.inoculation on the antioxidant system of T. castaneum after exposure to phosphine.After exposure of 10-day-old beetles to 1250 mL/m 3 phosphine for 0, 6, 12, and 24 h, the activity of SOD, CAT, and POD was determined using BC0170, BC0200, and BC0090 assay kits (Solarbio) according to the protocols of the manufacturer, respectively.
SOD activity was determined by detecting superoxide radicals generated by xanthine oxidase and hypoxanthine and monitoring absorbance at 560 nm [22].One unit of SOD activity refers to the quantity of enzyme necessary to display 50% dismutation of the superoxide radical.CAT activity was determined by detecting the decomposition rate of H 2 O 2 and monitoring absorbance at 240 nm [23].One unit of CAT activity refers to the quantity of CAT necessary to decompose 1 µmol of H 2 O 2 per min per g of sample.POD activity was determined by detecting guaiacol oxidation and monitoring absorbance at 470 nm [24].One unit of POD activity refers to a 0.01 change in absorbance at 470 nm per min per g of sample per mL of the reactive mixture.The samples were conducted in triplicate, and the mean of triplicate analyses represented one biological repetition.Each treatment consisted of five biological repetitions.

Determination of SOD, CAT, and POD Gene Expression
The expression levels of SOD, CAT, and POD genes in conventional, germ-free, and gnotobiotic beetles were measured to explore the molecular basis for the impact of Enterococcus sp.inoculation on the antioxidant system in T. castaneum after exposure to phosphine.After exposure of 10-day-old beetles to 1250 mL/m 3 phosphine for 0, 6, 12, and 24 h, the total RNA of 50 beetles was extracted using TRIzol ® reagent (Accurate Biotechnology, Changsha, China).Contaminating genomic DNA was depleted using DNase I (Accurate Biotechnology).Subsequently, cDNA libraries were constructed from 1 µg of RNA using M-MLV Reverse Transcriptase (Accurate Biotechnology).The relative gene expression data were assessed using the 2 −∆∆CT method and normalized with the qRT-PCR data of the Rps18 gene of T. castaneum [25].qRT-PCR primers are shown in Supplementary Table S1.The 20 µL qRT-PCR reactive mixture included 10 µL of SYBR Green Mix (TaKaRa, Otsu, Japan), 0.8 µL of each primer (50 µmol/L), and 2 µL of cDNA (diluted 1:10).qRT-PCR conditions were the same as those described above.Reactions were conducted in triplicate, and the mean of triplicate analyses represented one biological repetition.Each treatment consisted of five biological repetitions.

Measurement of Immune Responses
The expression levels of PLCβ, Atf2, and DUOX from the dual oxidase-reactive oxygen species (DUOX-ROS) system, IMD from the immune deficiency (IMD) signaling pathway, and Toll from the Toll signaling pathway in conventional, germ-free, and gnotobiotic Insects 2023, 14, 815 5 of 14 beetles were measured to assess the impact of Enterococcus sp.inoculation on host immune responses in the absence of phosphine.The total RNA of fifty 6-, 8-, 10-, 12-, 14-, and 16-day-old beetles was sequentially extracted, processed, and analyzed according to the method described above.qRT-PCR primers are shown in Supplementary Table S1.Reactions were conducted in triplicate, and the mean of triplicate analyses represented one biological repetition.Each treatment consisted of five biological repetitions.

Statistical Analysis
Datasets were tested for normality using the Kolmogorov-Smirnov test.All datasets were normally distributed and statistically compared with one-way analysis of variance (ANOVA) and Tukey's honest significant difference (HSD) test.All statistics were conducted using SPSS Statistics 22.0 (IBM, NY, USA), and differences among means were considered significant at p < 0.05.
Insects 2023, 14, x FOR PEER REVIEW

Impact of Bacteria Inoculation on Host Phosphine Susceptibility
The impact of five gut bacterial isolates on host phosphine susceptibility was assessed to screen resistance-mediating bacteria.Enterococcus sp.inoculation significantly promoted host susceptibility to phosphine, and the mortality of the beetles inoculated with Enterococcus sp. was substantially higher than those of conventional and germ-free beetles after exposure to Insects 2023, 14, 815 6 of 14 1000, 1250, 1500, 1750, and 2000 mL/m 3 phosphine for 20 h (F 6,28 = 4.09, p = 0.005; F 6,28 = 42.72,p < 0.001; F 6,28 = 13.21,p < 0.001; F 6,28 = 15.54,p < 0.001; F 6,28 = 4.26, p = 0.004, respectively) (Figure 2).Moreover, inoculation of any other gut bacterial isolate had no significant effect on host phosphine susceptibility, and no significant difference was found in the mortality between germ-free and gnotobiotic beetles.Therefore, Enterococcus sp. was used in subsequent assays to explore molecular mechanisms of gut bacteria-induced phosphine susceptibility in T. castaneum.

Impact of Bacteria Inoculation on Host Phosphine Susceptibility
The impact of five gut bacterial isolates on host phosphine susceptibility was assessed to screen resistance-mediating bacteria.Enterococcus sp.inoculation significantly promoted host susceptibility to phosphine, and the mortality of the beetles inoculated with Enterococcus sp. was substantially higher than those of conventional and germ-free beetles after exposure to 1000, 1250, 1500, 1750, and 2000 mL/m 3 phosphine for 20 h (F6,28 = 4.09, p = 0.005; F6,28 = 42.72,p < 0.001; F6,28 = 13.21,p < 0.001; F6,28 = 15.54,p < 0.001; F6,28 = 4.26, p = 0.004, respectively) (Figure 2).Moreover, inoculation of any other gut bacterial isolate had no significant effect on host phosphine susceptibility, and no significant difference was found in the mortality between germ-free and gnotobiotic beetles.Therefore, Enterococcus sp. was used in subsequent assays to explore molecular mechanisms of gut bacteria-induced phosphine susceptibility in T. castaneum.

Impact of Enterococcus sp. Inoculation on Host Fecundity
Enterococcus sp.inoculation significantly reduced the female fecundity of T. castaneum.After exposure of beetles to 1250 mL/m 3 phosphine for 20 h, the number of eggs laid per female of gnotobiotic beetles was substantially lower than that of conventional and germ-free beetles (F 2,57 = 106.20,p < 0.001) (Figure 3), suggesting that Enterococcus sp.inoculation results in fitness costs in T. castaneum after exposure to phosphine.

Impact of Enterococcus sp. Inoculation on Host Fecundity
Enterococcus sp.inoculation significantly reduced the female fecundity of T. castaneum.After exposure of beetles to 1250 mL/m 3 phosphine for 20 h, the number of eggs laid per female of gnotobiotic beetles was substantially lower than that of conventional and germ-free beetles (F2,57 = 106.20,p < 0.001) (Figure 3), suggesting that Enterococcus sp.inoculation results in fitness costs in T. castaneum after exposure to phosphine.Insects 2023, 14, 815 7 of 14
Similar variation trends in CAT and POD gene expression occurred in T. castaneum when challenged by phosphine exposure and Enterococcus sp.inoculation.The expression level of CAT3 first increased and then decreased in conventional and gnotobiotic beetles as the phosphine exposure period was extended (F 3,16 = 9.12, p = 0.001; F 3,16 = 4.60, p = 0.017, respectively).The expression level of PRDX6 first increased and then decreased in conventional and germ-free beetles as the phosphine exposure period was extended (F 3,16 = 219.17,p < 0.001; F 3,16 = 174.68,p < 0.001, respectively).The expression levels of CAT3 and PRDX6 reached the maximal values after exposure to phosphine for 6 h.Enterococcus sp.inoculation led to downregulation of the expression of CAT3 and PRDX6 in T. castaneum after exposure to phosphine.The expression level of CAT3 in gnotobiotic beetles was substantially lower than that in germ-free beetles after exposure to phosphine for 12 h (F 2,12 = 11.03,p < 0.001), and the expression level of PRDX6 in gnotobiotic beetles was substantially lower than that in germ-free beetles after exposure to phosphine for 0, 6, and 12 h (F 2,12 = 6.91, p = 0.010; F 2,12 = 176.51,p < 0.001; F 2,12 = 5.29, p = 0.023, respectively).

Impact of Enterococcus sp. Inoculation on Host Immune Responses
To assess the impact of Enterococcus sp.inoculation on host immune responses, the gut load of Enterococcus sp. and the expression levels of PLCβ, Atf2, DUOX, IMD, and Toll in gnotobiotic T. castaneum were evaluated on days 0, 2, 4, 6, 8, and 10 after inoculation of Enterococcus sp. to germ-free beetles.After Enterococcus sp. was inoculated into germ-free beetles, the gut load of Enterococcus sp. in gnotobiotic beetles (Figure 7A) was significantly higher than that in conventional beetles during the first two days after bacteria inoculation (F 1,8 = 313.73,p < 0.001; F 1,8 = 11.77,p = 0.009) and recovered to the normal level from the fourth day after bacteria inoculation (F 1,8 = 0.89, p = 0.374 for the fourth day;

Discussion
Five cultivable gut bacteria, namely, Brevibacterium sp., Enterococcus sp., Microbacterium sp., Micrococcus sp., and Staphylococcus sp., were excised from phosphine-resistant T. castaneum.Gut microbes may increase host pesticide resistance by directly degrading pesticides [10], modulating the host immune system [26], and producing nutrients and other beneficial chemicals [27,28].However, Enterococcus faecalis inoculation impaired phosphine resistance of adult T. castaneum [12].In this study, inoculation with another Enterococcus sp. also promoted host phosphine susceptibility, while inoculation of any other gut bacteria had no significant effect on host phosphine susceptibility.The negative influence of gut microbes on host pesticide resistance to contact pesticides, which has also been found in other studies [29,30], will provide ideas for pesticide resistance management by integrating pesticides with biocontrol agents containing resistance-mediating bacteria [31,32].
Phosphine exposure elicited an increased level of MDA and H 2 O 2 , indicating high oxidative stress in T. castaneum.On the other hand, inoculation with a high load of Enterococcus sp.upregulated the expression of PLCβ, Atf2, and DUOX from the DUOX-ROS system in T. castaneum, further aggravating oxidative stress in T. castaneum [7].Furthermore, when T. castaneum was exposed to phosphine, Enterococcus sp.inoculation suppressed the activity of host antioxidant enzymes, including SOD, CAT, and POD, which play a vital role in scavenging ROS [33].Additionally, since SOD is responsible for the transformation of active superoxide anions into stable H 2 O 2 , a decreased level of H 2 O 2 formation in T. castaneum due to the suppression of SOD activity by Enterococcus sp.inoculation will further aggravate oxidative stress.Considering that phosphine-induced oxidative stress and its damage are considered to be among the primary mechanisms of phosphine toxicity [34], it is not difficult to infer that increased mortality rates of T. castaneum simultaneously challenged by phosphine exposure and Enterococcus sp.inoculation could be attributed to overgeneration of ROS, which will cause damage to essential cellular building blocks, including DNA, proteins, and lipids [35].
Besides the DUOX-ROS system, other immune responses, including IMD and Toll signaling pathways, were also activated by Enterococcus sp.inoculation.As an essential life-preserving process, the immune response is privileged over other processes regarding energy supply [36].The tradeoff between immune responses and other fitness traits associated with pesticide resistance because of alternative allocation of limiting energetic resources will increase host susceptibility to pesticides.For example, infection of Culex pipiens with the bacterium Wolbachia increased the fitness cost of resistance to pesticides, rendering hosts more susceptible to pesticides [37], and parasitism by Vavraia culicis caused fitness costs in Culex pipiens quinquefasciatus bearing organophosphate resistance alleles, potentially altering the strength and direction of selection against resistance mutations in untreated environments [38].Since both antioxidant and immune responses are energetically costly [8,39], it is speculated that tradeoff between them will lead to Enterococcus sp.-induced phosphine susceptibility in T. castaneum.
During the initial stage of phosphine fumigation, T. castaneum attempted to mitigate oxidative stress from phosphine by upregulating the expression of SOD1a, SOD1b, SOD1c, SOD2, CAT3, and PRDX6 and promoting SOD activity, as has been found in other phosphine-resistant insects [40].However, Enterococcus sp.inoculation constantly suppressed the expression and activity of SOD, CAT, and POD in T. castaneum after exposure to phosphine, demonstrating that beetles inoculated with Enterococcus sp. could not allocate enough resources to contradict oxidative stress due to tradeoff between immune and antioxidant responses.Similarly, infection of D. citri with Ca.Liberibacter asiaticus reduced the activity of host detoxifying enzymes such as glutathione-S-transferase (GST) and cytochrome P 450 [31], and infection with a high titer of Phytoplasma inhibited the activity of host detoxifying enzymes such as β-esterase and GST in Amplicephalus curtulus [41].Meanwhile, Enterococcus sp.inoculation decreased reproductive fitness of T. castaneum after exposure to phosphine, suggesting that beetles could not allocate enough resources to conduct normal physiological activities.Increased mortality, reduced antioxidant enzyme activity, and low fecundity indicate increased fitness costs in T. castaneum simultaneously challenged by phosphine exposure and Enterococcus sp.inoculation.From an evolutionary perspective, gut microbe-induced fitness costs will delay the development of phosphine resistance among insect populations.
Previous research showed that bacteria inoculation can activate the Toll signaling pathway and subsequently suppress the insulin signaling pathway [36].The low expression of AGC kinase (Akt), an element in the insulin signaling pathway, can inhibit the activity of the downstream transcription factor erythroid 2-related factor 2 (Nrf2).Thus, Nrf2 cannot translocate into the nucleus to activate antioxidant enzyme expression [42].Therefore, it is speculated that Enterococcus sp.regulates the expression of antioxidant enzymes by sequentially activating the Toll-Akt-Nrf2 pathway.The expression level of Toll was upregulated when T. castaneum was inoculated with a high load of Enterococcus sp., partially illustrating the possibility of this regulatory mechanism.Future studies of the molecular mechanisms behind gut microbe-mediated host pesticide resistance with an emphasis on the immune system will inform development of new pesticide targets.For example, the miR-34-5p target gene in the immune pathway of Spodoptera frugiperda has been developed as a target of nucleic acid pesticides [43].

Figure 1 .
Figure 1.Mean ± SE relative gut load of Brevibacterium sp., Enterococcus sp., Microbacterium sp., Micrococcus sp., and Staphylococcus sp. in adult Tribolium castaneum with different treatments of gut bacteria (n = 5).Germ-free means that gut bacteria are successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that a given gut bacterium is successfully inoculated into germ-free beetles.The same letters indicate no significant difference among the means in the same cluster (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 2 .
Figure 2. Mean ± SE mortality of adult Tribolium castaneum after exposure to different concentrations of phosphine for 20 h (n = 5).Germ-free means that gut bacteria are successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that a given gut bacterium is successfully inoculated into germ-free beetles.The same letters indicate no significant difference among the means in the same cluster (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 2 .
Figure 2. Mean ± SE mortality of adult Tribolium castaneum after exposure to different concentrations of phosphine for 20 h (n = 5).Germ-free means that gut bacteria are successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that a given gut bacterium is successfully inoculated into germ-free beetles.The same letters indicate no significant difference among the means in the same cluster (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 3 .
Figure 3. Mean ± SE number of eggs laid per female after exposure of adult Tribolium castaneum to 1250 mL/m 3 phosphine for 20 h (n = 5).Germ-free means that Enterococcus sp. is successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that Enterococcus sp. is successfully inoculated into germ-free beetles.The same letters indicate no significant difference among different treatments (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 4 .
Figure 4. Mean ± SE level of malondialdehyde (MDA, A) and hydrogen peroxide (H2O2, B) in adult Tribolium castaneum after exposure to 1250 mL/m 3 phosphine for different periods (n = 5).Germ-free means that Enterococcus sp. is successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that Enterococcus sp. is successfully inoculated into germ-free beetles.The same uppercase letters indicate no significant difference among the means with the same treatment of gut bacteria across different exposure periods, and the same lowercase letters indicate no significant difference among the means of conventional, germ-free, and gnotobiotic beetles within the same exposure period (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 4 .
Figure 4. Mean ± SE level of malondialdehyde (MDA, A) and hydrogen peroxide (H 2 O 2 , B) in adult Tribolium castaneum after exposure to 1250 mL/m 3 phosphine for different periods (n = 5).Germ-free means that Enterococcus sp. is successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that Enterococcus sp. is successfully inoculated germ-free beetles.The same uppercase letters indicate no significant difference among the means with the same treatment of gut bacteria across different exposure periods, and the same lowercase letters indicate no significant difference among the means of conventional, germ-free, and gnotobiotic beetles within the same exposure period (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 5 .
Figure 5. Mean ± SE activity of superoxide dismutase (SOD, A), catalase (CAT, B), and peroxidase (POD, C) in adult Tribolium castaneum after exposure to 1250 mL/m 3 phosphine for different periods (n = 5).Germ-free means that Enterococcus sp. is successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that Enterococcus sp. is successfully inoculated into germ-free beetles.The same uppercase letters indicate no significant difference among the means with the same treatment of gut bacteria across different exposure periods, and the same lowercase letters indicate no significant difference among the means of conventional, germ-free, and gnotobiotic beetles within the same exposure period (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 5 .
Figure 5. Mean ± SE activity of superoxide dismutase (SOD, A), catalase (CAT, B), and peroxidase (POD, C) in adult Tribolium castaneum after exposure to 1250 mL/m 3 phosphine for different periods (n = 5).Germ-free means that Enterococcus sp. is successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that Enterococcus sp. is successfully inoculated into germ-free beetles.The same uppercase letters indicate no significant difference among the means with the same treatment of gut bacteria across different exposure periods, and the same lowercase letters indicate no significant difference among the means of conventional, germ-free, and gnotobiotic beetles within the same exposure period (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 6 .
Figure 6.Mean ± SE relative expression levels of SOD1a (A), SOD1b (B), SOD1c (C), SOD2 (D), CAT3 (E), and PRDX6 (F) in adult Tribolium castaneum after exposure to 1250 mL/m 3 phosphine for different periods (n = 5).Germ-free means that Enterococcus sp. is successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that Enterococcus sp. is successfully inoculated into germfree beetles.The same uppercase letters indicate no significant difference among the means with the same treatment of gut bacteria across different exposure periods, and the same lowercase letters indicate no significant difference among the means of conventional, germ-free, and gnotobiotic beetles within the same exposure period (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 6 .
Figure 6.Mean ± SE relative expression levels of SOD1a (A), SOD1b (B), SOD1c (C), SOD2 (D), CAT3 (E), and PRDX6 (F) in adult Tribolium castaneum after exposure to 1250 mL/m 3 phosphine for different periods (n = 5).Germ-free means that Enterococcus sp. is successfully depleted from beetles via antibiotic treatment.Gnotobiotic means that Enterococcus sp. is successfully inoculated into germ-free beetles.The same uppercase letters indicate no significant difference among the means with the same treatment of gut bacteria across different exposure periods, and the same lowercase letters indicate no significant difference among the means of conventional, germ-free, and gnotobiotic beetles within the same exposure period (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 7 .
Figure 7. Mean ± SE relative gut load of Enterococcus sp.(A) and relative expression levels of PLCβ (B), Atf2 (C), DUOX (D), IMD (E), and Toll (F) in adult Tribolium castaneum after inoculation of Enterococcus sp.into germ-free beetles for different periods (n = 5).Gnotobiotic means that Enterococcus sp. is successfully inoculated into germ-free beetles.The same uppercase letters indicate no significant difference among the means of conventional and gnotobiotic beetles within the same period (one-way ANOVA, p > 0.05), and the same lowercase letters indicate no significant difference among the means with the same gut bacteria treatment across different periods (one-way ANOVA and Tukey HSD test, p > 0.05).

Figure 7 .
Figure 7. Mean ± SE relative gut load of Enterococcus sp.(A) and relative expression levels of PLCβ (B), Atf2 (C), DUOX (D), IMD (E), and Toll (F) in adult Tribolium castaneum after inoculation of Enterococcus sp.into germ-free beetles for different periods (n = 5).Gnotobiotic means that Enterococcus sp. is successfully inoculated into germ-free beetles.The same uppercase letters indicate no significant difference among the means of conventional and gnotobiotic beetles within the same period (one-way ANOVA, p > 0.05), and the same lowercase letters indicate no significant difference among the means with the same gut bacteria treatment across different periods (one-way ANOVA and Tukey HSD test, p > 0.05).