Comparison of Surface Sterilization Methods for the Analysis of Insect Gut Microbiota : Solenopsis invicta ( Formicidae ) as an Example

Globally, invertebrate insects dominate our living ecosystems, and their diversity drives the functioning of gut microbiota. An insect’s digestive tract comprises a foregut, crop, midgut, and hindgut. Previous studies have isolated insect gut microbiota samples by dissecting the abdomen under an optical microscope or stereoscope microscope. However, this method has some limitations, for example, He et al. (2014) obtained isolates from the gut microbiota of Camponotus fragilis via traditional dissection methods, and their results showed that the lab-raised ants yielded 12 Abstract The gut microbiota of vertebrates and invertebrates has become the focus of recent research. However, current sterilizing practices need more systematic quantitative methods due to limitations caused by their minute body size. Thus, an effective sterilization process incorporating organic and inorganic methods to obtain invertebrate microbiota, particularly when evaluating smaller insects, has yet to be elucidated. This study investigated if the whole abdomen should be utilized as the material to sterilize and examined whether physical and chemical surface sterilization methods could be combined to facilitate the acquisition of gut microbiota from the imported red fire ant (Solenopsis invicta Buren, 1972). Eight methods were designed by incorporating three chemical reagents (sterile water, 2.0 mg/L NaClO, and 75% ethanol) and one physical treatment (250 nm UV). The length range of the amplified fragment in the red imported fire ant is 401-450 bp. According to the results of the GLM regression model and interaction effect model, none of these factors (sterile water, 2.0 mg/L NaClO, and 75% ethanol, 250 nm UV) were significant for statistical regression of the Chao index, and these factors did not significantly interact with each other. Based on Alpha and Beta diversity analysis, none of the methods significantly affected the diversity of insects’ gut microbiome. Finally, we suggested that it is feasible for different species of small insects to select appropriate methods according to the current situation. Still, it is best to achieve unity in the same group. Sociobiology An international journal on social insects


Introduction
Globally, invertebrate insects dominate our living ecosystems, and their diversity drives the functioning of gut microbiota.An insect's digestive tract comprises a foregut, crop, midgut, and hindgut.Previous studies have isolated species via the use of classical microbial culture methods.Nonetheless, polymerase chain reaction-restriction fragment length polymorphism analysis yielded 11 species.However, these methods are only sometimes reliable as most archaea and bacterial diversity remain uncultured or poorly characterized (Lewis et al., 2020).Moreover, traditional anatomical methods do not apply to tiny insects.Nowadays, with the development of deep sequencing (Urakawa et al., 1999;Koto et al., 2008;Kim et al., 2020), many methods are available for acquiring samples and cultivating bacteria, leading to more significant variations in results.Some of these variations might be because human factors influence gut dissection.
Ultraviolet (UV) is a reliable and environmentfriendly sterilization method for water (Yagi et al., 2007) that can cause significant attenuation of the growth factor release kinetics (Evrova et al., 2019), leading to a sterilization effect on the microbiota (Yung et al., 2020).Yung et al. (2020) found that UV effectively inactivated the Bacillus atrophaeus endospores, and it is now becoming a common sterilization method.Borm et al. (2002) showed the efficacy of this treatment by utilizing UV to sterilize ants for 2 hours to complete a comprehensive screening of all the potential microorganisms inside the bodies of leafcutter ants.
Ethanol, an organic molecule, is a common body surface sterilization reagent used to eliminate pathogens in stool specimens from healthy donors.This reagent is useful for evaluating the safety and efficacy of bacterial spores and is important in preventing recurrent Clostridium difficile infections (Khanna et al., 2016;Afouda et al., 2020).Taylor et al. (2017) used several antimicrobials and surfactants to decrease the three stink bugs.Sodium hypochlorite (NaClO) is one of the most effective disinfectants in endodontics.It can also suppress endosymbionts in stink bugs as a pest control strategy in the field (Kashkouli et al., 2019).
To our knowledge, no method has been developed that combines physical and chemical treatments simultaneously to obtain the gut microbiota of ants.Thus, we select the chemical treatment NaClO and ethanol, which refer to the method that obtains Camponotus fragilis gut microbiology (He et al., 2014), and select the physical treatment UV according to Borm et al. (2002); these methods had been put into gutassociated researches.The current study combines inorganic (NaClO), organic (ethanol), and physical (UV) sterilization treatments to investigate the gut bacteria of very small insects (fire ants) in order to determine the most combined favorable efficacy of these treatments.This study also investigates if the whole abdomen can be utilized as the material required for sterilization procedures.

Ant collection
Twenty-seven samples (540 ants in total, each sample includes 60 ants, three samples of the same prefix as a processing group) were collected from a common nest built in the soil at Guangxi Normal University in Guilin, Guangxi, in southern China.In addition to that, 200 ants were prepared to ensure a suitable number of obtaining gut microbiomes and were divided into ten groups (which was carried out before this study, these 200 ants were washed with sterile water twice, once a minute), group 1-5 represent minor workers, the specific number of each group is in order 10, 15, 20, 25, 30; group 6-10 represent large workers, specific number as same as group 1-5 (Fig 1).Here, the major workers and minor workers were placed in a glass bottle with some soil to keep them alive.Then, these ants were chosen on a clean bench (provide a local dust-free aseptic working environment with one-way flow type air purification equipment).The total length of the large workers chosen was about 5.0 mm, with the abdomen of about 1.5 mm.The total length of the minor workers was about 3.0 mm, with an abdomen of about 1.0 mm.We mixed in the two types of ants randomly.We brought their numbers up to 20 (in our preliminary experiment, both large and minor ants successfully extracted bacteria DNA from their gut).
Surface sterilization: 75% ethanol and 2.0% NaClO were prepared.Surgical blades, beakers, tweezers, 2 ml centrifuge tubes (labeled), and water were sterilized in the autoclave (vapor pressure-103.4kPa, 122 °C, for 30 minutes).Method GT was set as a blank control with 20 ant abdomens.The other eight methods were used to clean the surface; each included 20 ant abdomens.Specific methods are listed in Table 1.In order to ensure that the microbiome is more fully acquired in the host, all of the ants are alive; prior to the experiment, several ants were put into centrifuge tubes and monitored at each step until it was confirmed that the ants would survive all of the steps.

DNA extracted and PCR amplification
According to the steps of the test kit from TIANGEN biotech company, DNA was extracted from 540 ants and stored in dry ice before sending to the Shanghai Magi Biological Company for sequencing.

Establishing Illumina library and sequencing
The "Y" shaped joint was connected, then magnetic bead screening was used to remove the connector self-continuous segment, and the library templates were enriched by PCR amplification; finally, these templates were degenerated with NaOH, resulting in single-stranded fragments of DNA.
One endpoint of DNA fragments was complemented with the primer's basic groups and then fixed into the chip.
Another endpoint of DNA fragments was matched with a nearby different primer to be fixed and form a bridge.Then, PCR amplification was conducted again, which produced DNA clusters.The DNAs were linearized and divided into single strands.DNA polymerases were added along with four dNTPs with fluorescence.The surface of the reaction plate was scanned with a laser, cutting the fluorescence and termination genes chemically, restoring the viscosity of the 3' endpoint, and continuing to aggregate the second nucleotide.Finally, the results of fluorescent signals were collected, and the sequences of the DNA fragments were obtained.

Community richness
The Mothur software was utilized for all the diversity analyses (version v.1.30.1 http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity) (Schloss et al., 2009).The Chao index was used to estimate the species abundance.In addition, we also calculated the Ace, Coverage, Shannon, and Simpson estimators, which enriched the uniformity and sequencing depth indexes of the samples, respectively.

Statistical model comparison
The GLM regression model was designed to assess which factor is significant for the regression of results, and for further exploring these factors and whether they react with each other, the interaction effect model was utilized to verify that.These analyses were completed on the software R 4.0.4.
Table 1.Methods design of surface sterilization.There are 540 ants divided into 27 samples (nine groups as nine methods).The three samples of method GT (Group GT_1, GT_2, GT_3) were not subject to sterilization and were used as controls.All 27 samples included the same amount of workers (n = 20).Each method involved three repetitions (1-3), and each sample was processed following the table from left to right."+" represent perform this operation, "-" represent not.

Sequencing depth and average sequencing length
The dilution curve (Fig 2) randomly extracts a certain number of sequences from the sample and makes statistics on the alpha diversity index of the corresponding samples of these sequences.In our results, we choose the Shannon estimator as an indicator to detect the depth of sequencing; the curve is gentle, which means the amount of sequencing data is sufficient.
We analyzed the 16S rDNA gene sequences of the imported red fire ant gut microbiota from 540 ants treated by these nine methods, the total number of bases reached 1539082, and the average length was about 401-450 bp (Fig 3).

Distribution of phylum taxa level through different methods
Results from all the methods showed that the dominant phyla are p_Proteobacteria and p_Firmicutes.In more than ten groups, the first dominant phylum takes part in more than 80%.The phylum p_Firmicutes occurred in 5 groups, and more than a half.It is confusing that p_Deinococcota occupy groups GT_2 and GT_3 by a significantly greater number of OTUs.The detailed distribution is depicted in Fig 4.

Alpha diversity analysis
Based on the 97% similarity level of OTU, the OTUs (operational taxonomic unit) were used to calculate all these alpha diversity indexes (Table 2).The largest number of OTU (75340) exists in group M5_2, the smallest OTU exists in group GT_3 (33813); the highest Ace index and the Chao index both exist in group M1_3 (803.30,798.01); the maximum value of the Shannon index and the minimum value of Simpson index exist in group M2_1 (3.891, 0.042); and all of the coverages were more than 0.99.

Beta diversity analysis of surface sterilization methods
NMDS (Nonmetric multidimensional scaling) analysis on the OTU level showed the comparison of differences between groups of these methods.Stress (0.14) less than 0.2 means high reliability.In Fig 5, all the groups are not completely separated, R = 0.0482, p = 0.237, which means these nine surface sterilization methods' influence on the OTU level results was insignificant.

Statistical analysis for the GLM regression model and interaction effect model
The results of the GLM regression model showed that none of the three factors contributed significantly to the Chao index's regression; all of the p values were greater than 0.05 (Table 3).In order to explore whether these factors sufficiently interacted with each other to impact the results, the interaction effect model was evaluated (Table 4).This model could assess the influence induced by any two factors; the results proved that no significant interaction effect was found between any two factors (p> 0.05).

Discussion
The PCR amplification results showed that the amplification fragment was about 401-450 bp (Fig 3), and the primer 338F-806R amplified the DNA fragments successfully.In this study, the suitable number of ants required to be sequenced was successfully determined; the design of each group with 20 ants is believable for Shannon curves are gentle and electrophoresis bandings are clear; this provided us a view of sample number for acquiring gut microbiome in this tiny insect (Fig 1,Fig 2).It is worth noting that though the electrophoresis banding "1" (Fig 1, which represents the ten ants) is as clear and bright as banding "3" (which represent the 20 ants), according to the conclusion of Lee et al. (2008), we speculated it might be due to their own rare and unstable bacterial composition.Our results supported it (20 ants) as a more stable and accurate value because each method was designed with 20 ants in this study, and the intestinal flora was successfully extracted just once.
Our sequenced bacterial clones from the guts were determined to be predominantly from the phyla p_Proteobacteria and p_Firmicutes (Fig 4), which is consistent with Lee et al. (2008).They provided powerful evidence to confirm it was possible to use the whole abdomen as the material to sterilize.
It seems the Alpha diversity index cannot reveal the effect of sterilization.The large Shannon index difference of group M5_1 (0.158) (or group M5_2, 0.104) and group M5_3 (3.53) treated by UV may suggest the limitation of using the alpha diversity index to evaluate the surface sterilization intuitively.Because they are collected from the same nest and treated under UV at the same time.In addition to the results of Alpha diversity, NMDS analysis further confirms that the effect of surface sterilization is not obvious between different methods (Fig 5).Statistical analysis (GLM regression model and interaction effect model) approves the above results by more intuitive data (p> 0.05).In other words, our results seem to fit well with the experimental results of Hammer et al. (2015).The different surface sterilization methods did not significantly affect the diversity of the insect gut microbiome, suggesting that no single method or combination of methods is best.However, we believe that the process of body surface sterilization cannot be ignored; a strong piece of evidence is that ethanol and bleach-treated specimens showed major differences in internal microbial diversity in ticks (Binetruy et al., 2019).Long-term field collection experience tells us that most ants live in the soil with a wide variety of bacteria.Although cleaning the body surface for studying the gut microbiota is important, unifying the sterilization method within an insect taxa is more important than selecting different sterilization methods for the body surface.For example, aphid surface sterilization is almost always a combination of 70% ethanol and sterile water (Qin et al., 2021).At the same time, we should pay attention to insects' different growth and development stages or the material sources of different parts.
Some outstanding research has been reported recently, which has confirmed that a primate's gut microbiota could change with seasons (Li et al., 2020); they also determined the dominant microbial community and predicted its function.As for Solenopsis invicta, a recent study by Xiao et al. (2023) demonstrated that gut microbiota composition and diversity varied among populations due to differences in host genotype and geographical distribution.They pointed out that population-level differences in S. invicta gut microbiota may depend more on environmental factors than host genotypes.Previous studies have reviewed the gut microbiota of bees (Voulgari-Kokota et al., 2019).However, the testing methods available could not completely define the core microbial community or determine the impact on the host.Therefore, since more extensive testing methods are currently available, future research should focus on the microbial community, the impact on the host, seasonal variation, living habitats, and feeding habits.

Conclusion
Regarding the ideal material to obtain gut microbiome from ants, the whole abdomen is worthy of adoption, and the number of extraction units (20 ants) could be used as a numerical reference for other tiny insects (species similar in size to the red fire ant).Different surface sterilization methods did not significantly affect the diversity of insect gut microbiome, but choosing a suitable method for better comparison of published research is indispensable.The scientific community working with ant gut microbiota must unify sterilization methods as best as possible within the same species.

Fig 1 .
Fig 1.The pre-experimental design for detecting optimal ant populations, stripes 1-5 represent five groups of different quantitative gradients of minor workers (The specific numbers are respectively 10, 15, 20, 25, 30), stripes 6-10 represent the same situation but of minor workers.

Fig 2 .Fig 3 .
Fig 2. Dilution curve of Shannon estimator.The abscissa represents the amount of data extracted; the y-coordinate represents the value of the Shannon estimator.

Fig 4 .
Fig 4. The composition percent of red imported fire ants gut microbiota in the phylum taxa.Abscissa represents these sterilization methods; ordinate shows the percent of each phylum.

Fig 5 .
Fig 5.NMDS analysis of all the surface sterilization methods.The confidence ellipse of the same shape and color represents a method.

Table 3 .
Effect of three factors on the Chao index in the GLM regression model.

Table 2 .
The sequencing information of the eight samples.Number of OTUs, Alpha diversity index: Ace index, Chao index, Coverage index, Shannon index, and Simpson index are included.

Table 4 .
The interaction effect model of any two factors on the Chao index.Three models are designed to detect whether any two of the three factors interact with each other or not.