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Gut Microbiota of Tenebrio molitor and Their Response to Environmental Change
Gut Microbiota of Tenebrio molitor and Their Response to Environmental Change
Journal of Microbiology and Biotechnology. 2014. Jul, 24(7): 888-897
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : May 08, 2014
  • Accepted : May 26, 2014
  • Published : July 28, 2014
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About the Authors
Jaejoon Jung
Institute of Life Science and Natural Resources, Korea University, Seoul 136-713, Republic of Korea
Aram Heo
Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea
Yong Woo Park
Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea
Ye Ji Kim
Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea
Hyelim Koh
Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea
Woojun Park
Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea
wpark@korea.ac.kr

Abstract
A bacterial community analysis of the gut of Tenebrio molitor larvae was performed using pyrosequencing of the 16S rRNA gene. A predominance of genus Spiroplasma species in phylum Tenericutes was observed in the gut samples, but there was variation found in the community composition between T. molitor individuals. The gut bacteria community structure was not significantly affected by the presence of antibiotics or by the exposure of T. molitor larvae to a highly diverse soil bacteria community. A negative relationship was identified between bacterial diversity and ampicillin concentration; however, no negative relationship was identified with the addition of kanamycin. Ampicillin treatment resulted in a reduction in the bacterial community size, estimated using the 16S rRNA gene copy number. A detailed phylogenetic analysis indicated that the Spiroplasma -associated sequences originating from the T. molitor larvae were distinct from previously identified Spiroplasma type species, implying the presence of novel Spiroplasma species. Some Spiroplasma species are known to be insect pathogens; however, the T. molitor larvae did not experience any harmful effects arising from the presence of Spiroplasma species, indicating that Spiroplasma in the gut of T. molitor larvae do not act as a pathogen to the host. A comparison with the bacterial communities found in other insects ( Apis and Solenopsis ) showed that the Spiroplasma species found in this study were specific to T. molitor .
Keywords
Introduction
The insect gut is a distinctive habitat for microbial colonization. Bacterial communities are thought to perform many roles that are beneficial to their hosts. Insects are often dependent on gut bacteria to perform basic biological functions such as aiding in the digestion of low-nutrient food, protection from disease or predators, mating, and reproduction [2 , 4 , 23] . To understand the relationship between gut microbiota and many aspects of insect life, community analysis has been performed across a diverse range of insects, including the bumble bee (genus Bombus ), honey bee (genus Apis ), leafcutter bee (genus Megachile ), ants (genera Cephalotes and Solenopsis ), Drosophila , and many genera of termites [3 , 5 , 10 , 13 , 14 , 26] . The gut microbiota of insects usually represents a very simple community. This is because the environment of the gut is quite selective for a specific bacterial group; in addition, some insects have a regulatory system for maintaining specific gut communities [21 , 22] .
Spiroplasma (class Mollicutes) is a genus that is often found in the gut microbiota of insects. Spiroplasma species are non-cell-wall bacteria with a spiral cell morphology. There are four clades of Spiroplasma based on the 16S rRNA genes; namely, the Apis, Mycoides, Citri, and Ixodetis clades. The isolation sources of these Spiroplasma species are roughly congruent with phylogenetic trees [8] . Spiroplasma is a major endosymbiont of Drosophila [24] . Endocellular and extracellular associations with a variety of plants and arthropods have also been reported. Several Spiroplasma species have been reported to possess male-killing pathogenic activity of many insects, including Drosophila , ladybird beetles, and butterflies [15] . However, not all Spiroplasma species are pathogens, and infection with Spiroplasma does not always have detrimental effects.
Tenebrio molitor is a species of darkling beetle (order Coleoptera), which produces larvae that are commonly called mealworms. Acting as decomposers in the natural environment, they feed on decaying plant materials and dead insects. In addition, as a result of its easy handling and non-fastidious culture conditions, T. molitor is used as a pet food, an educational material, and as a biological research model [27] . Whereas the relationship between microorganisms and several insects, such as termites and honey bees, has been well-studied by many researchers, the role of the microorganisms residing in T. molitor is rarely understood. To enhance the understanding of the interactions between microbiota and T. molitor , we conducted a bacterial community analysis from the gut of T. molitor larvae. We evaluated the effect of antibiotics (ampicillin and kanamycin) on the gut bacterial community. To our best knowledge, studies on the exposure of gut microbiota of T. molitor to antibiotics has not been reported. To further evaluate the response of the gut microbial community to its environment, we provided the bacterial community with great diversity in its environment by culturing the larvae in a mixture of soil and bran. Finally, we compared the microbial community structure of the gut of T. molitor larvae with those from other insects to provide insight into the insect gut microbiota. This is the first report of a bacterial community analysis in the gut of T. molitor .
Materials and Methods
- Culture of T. molitor Larvae and Injection of Antibiotics
T. molitor larvae were purchased from a local market. The body weight of the larvae used in the study ranged from 50 to 60 mg. Antibiotics were either injected directly into the gut or were added to the diet of the larvae. The concentrations of injected antibiotics were 50 and 100 μg/ml for ampicillin, and 1, 5, 10, and 50 μg/ml for kanamycin. In order to add antibiotics to bran, antibiotics dissolved in distilled water were thoroughly mixed with bran and the distilled water was removed by evaporation. The final concentrations of the antibiotics in the bran were 300, 500, 700, and 900 μg/ml for ampicillin (Amp), and 100, 200, 300, and 500 μg/ml for kanamycin (Km). When the larvae were cultured in soil, the soil and bran were mixed at a 1:1 (v/v) ratio. Following their exposure to antibiotics by injection, we incubated the larvae for 5 days and the guts were dissected from 10 individuals. In the antibiotic soil conditions, we incubated the larvae for 5 or 10 days, and the guts were dissected from 10 individuals. For brevity in this manuscript, we have named the samples with an abbreviation of the name of the antibiotics and with the concentration. For example, Amp 100 means the sample was exposed to 100 μg/ml ampicillin. Although there are various modifications that can occur, in general, the insect gut is known to have a three-part structure: foregut, midgut, and hindgut [11] . The structure of the gut of T. molitor larvae was not visibly separated. Consequently, DNA extraction and analysis were performed without considering the gut structure.
- Denaturing Gradient Gel Electrophoresis (DGGE)
To investigate the variation in the bacterial communities of T. molitor larvae, DGGE was performed on samples from nine T. molitor larvae. The gut was dissected from T. molitor larvae and DNA was isolated using the NucleoSpin system (Machery-Nagel; Germany) according to the manufacturer’s instructions. The bacterial 16S rRNA gene was amplified using the 27F and 1492R primers. The quantity of template DNA used in the protocol was 10 ng. The polymerase chain reaction (PCR) protocol utilized included 5 min at 94℃, followed by 20 cycles of 45 sec at 94℃, 45 sec at 55℃, and 45 sec at 72℃. A final extension step was performed for 5 min at 72℃. The PCR products from this reaction were used as template DNA for the second PCR step using the b341GC and 758r primers. The same PCR protocol was used for the second PCR step with the exception of 35 cycles in place of 20. DGGE was performed using the D-Code system (Bio-Rad; USA) for 16 h at 60℃ and 70 V. The denaturants (urea and formamide) range was from 40% to 60%.
- Bacterial Community Analysis Using Pyrosequencing
In order to perform a culture-independent bacterial community analysis, pyrosequencing of the bacterial 16S rRNA gene sequence was performed. Hypervariable regions (V1-V3) were amplified using the 27F and 518R primers containing 10 bp barcode sequences. The construction of a sequencing library and pyrosequencing using the GS-FLX Titanium system were performed by Macrogen Inc. (Republic of Korea). Short (<180 bp) and low-quality reads were eliminated from the raw sequencing data using Mothur software [30] . The demultiplexed sequencing data were normalized by subsampling 8,733 reads from all samples using Mothur. This number was selected because it was the smallest number of reads from the samples. Taxonomic classification and the calculation of diversity indices were performed using the Ribosomal Database Project (RDP) database ( http://pyro.cme.msu.edu/ ) [9] . Sequence alignment, clustering of the sequences using a variety of cutoff values, and the calculation of diversity indices were performed using the RDP database. QIIME was used to compare the bacterial communities and create an OTU network visualized using Cytoscape software [31] . Raw pyrosequencing data from previously published literature were downloaded from the NCBI SRA database and were processed with an SRA toolkit and Mothur software.
- Quantitative Real-Time PCR (qPCR) for Estimating the 16S rRNA Gene Copy Numbers
The bacterial 16S rRNA gene copy numbers were estimated using qPCR. The DNA template was the same DNA that was used for pyrosequencing. Bacterial 16S rRNA genes were amplified with the b341 and b534 primers as previously described [17] . The quantity of template DNA used in the experiment was 10 ng. The PCR protocol used was 90 sec at 94℃, followed by 35 cycles of 20 sec at 94℃, 20 sec at 60℃, and 20 sec at 72℃. PCR specificity was checked using melting curve analysis. PCR data analysis was performed using an iCycler system (Bio-Rad, USA). The standard curve used was prepared according to a previously established method [17] .
- Phylogenetic Analysis of Spiroplasma-Associated Sequences
Representative sequences associated with Spiroplasma were selected and re-evaluated using the EzTaxon-e database [20] to manually confirm that the classification of the selected sequences was affiliated with Spiroplasma . Spiroplasma -associated sequences and the 16S rRNA gene sequences of Spiroplasma type species were aligned using MEGA 6.0 software [32] . After trimming the unaligned regions, a neighbor-joining phylogenetic tree was constructed using MEGA software.
- Enumeration of the Culturable Isolates in Different Oxygen Conditions
We evaluated the number of culturable isolates in microaerobic and aerobic conditions using Luria-Bertani (LB) agar, nutrient agar (NA), and R2A agar. The mean mass of the dissected gut was approximately 12 mg (approximately 1/10 of larval body weight). The gut homogenate was serially diluted and spread on agar media. A microaerobic condition was achieved using a Gas Pak system (BD, USA). Following a 5 day incubation period at room temperature (~25℃), the number of colonies was counted.
Results
- Bacterial Community of T. molitor Larvae and Variations Between Individuals
We observed that the bacterial communities of the two control samples (Amp 0 and Km 0) were different ( Fig. 1 ). The bacterial community found in Amp 0 was primarily composed of three phyla, Tenericutes (36.6%), Proteobacteria (34.1%), and Firmicutes (26.2%). Unclassified bacteria accounted for 3.1% of the bacterial community. At the genus level, Spiroplasma (38.7%) was the most prevalent genus, with unclassified Enterobacteriaceae species also comprising a large portion of the genera (36.4%). However, the bacterial community of Km 0 was primarily composed of the Tenericutes phylum (99.8%) and Spiroplasma genus (99.8%). The presence of many novel species in the samples was expected, because many sequences were not taxonomically affiliated at the gene level. The presence of a large number of sequences that are taxonomically poorly identified provides evidence that the taxonomic study of gut bacterial communities of T. molitor larvae requires further research. It is interesting to note that two different bacterial communities were identified from the control samples, even though both controls were treated in the same manner and were grown in an identical environment. Community analyses performed in previous studies have indicated that bacterial communities can differ between individuals that grow in the same environment [25] . We could not identify sex, age, and other information of the larvae, and hence, it was difficult to determine any biological characteristic for variance. To identify if this was the case in this study, bacterial communities from nine T. molitor larva individuals were analyzed using DGGE ( Fig. 2 ). The DGGE band patterns generated showed that the bacterial communities were not identical across all individuals. In fact, there was not a common band found across all nine individuals included in the experiment. This result demonstrated that the differences in bacterial communities of the two control samples resulted from individual variation rather than experimental error. These data also suggested that experimental subject individuality should be taken into consideration when examining the microbiome of insect samples.
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Bacterial community composition of the gut of Tenebrio monitor larvae. Illustration of the bacterial community composition of the gut of T. molitor larvae exposed to ampicillin, kanamycin, and grown in the mixture of soil and bran (Soil+Bran) at the phylum (A) and genus (B) levels. Antibiotics were injected directly or orally digested. Bacterial communities were statistically compared using three-dimensional principal coordinate analysis (C) and unweighted pair group method with arithmetic mean (D).
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Bacterial communities of nine individual gut samples.
- Bacterial Community Composition in the Presence of Varied Antibiotic Conditions
Bacterial communities in all the experimental conditions were found to be primarily composed of species within the Tenericutes phylum and the Spiroplasma genus ( Fig. 1 ). The predominance of the Spiroplasma genus was not the result of the application of antibiotics, because the control sample, Km 0, also contained a large portion of Spiroplasma . There was no concentration-dependent effect of antibiotics on the community composition. In the study, antibiotics were either directly injected into the gut at a low concentration, or orally digested at a high concentration (See Materials and Methods section for details). However, no differences were found to result from the application of the two methods. Bacterial communities were compared using three-dimensional principal coordinate analysis (3DPCoA) and unweighted pair group method with arithmetic mean (UPGMA) ( Figs. 1 C and 1 D). As Figs. 1 A and 1 B imply, the community of Soil+Bran and Amp 0 samples was distinguished, while all the other samples were closely related in the 3D-PCoA and UPGMA tree.
- Decreased Bacterial Diversity Caused by Ampicillin
The diversity of the bacterial communities was estimated using the Chao1 and Shannon-Weaver indices. Prior to ampicillin being injected or ingested, the Chao1 and Shannon-Weaver indices were 1,024 and 5.023, respectively. These values are considered very low in comparison with the results of our previous study using soil microcosms [18] . Exposure of these communities to ampicillin reduced the biodiversity, which was demonstrated by a negative correlation between ampicillin concentration and diversity indices (R 2 = 0.6379 and 0.7647 for Chao1 and Shannon-Weaver indices, respectively) ( Fig. 3 ). This reduced diversity was also reflected in rarefaction curves (data not shown). As the ampicillin concentration increased, the slope of the rarefaction curve became increasingly flat. Phylogenetic diversity accumulation curves also indicated a reduction in diversity ( Fig. 4 ). However, a significant decrease in diversity was not observed in the Km samples. As a result, even though the application of antibiotics did not change the composition of the bacterial community, diversity was decreased by ampicillin in a concentration-dependent manner.
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Analysis of the bacterial community diversity. Plots of the negative correlations between Chao1 (A) or Shannon-Weaver (B) diversity indices, and ampicillin. (C) Plot of the negative correlation between the ampicillin concentration and the bacterial community size.
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Phylogenetic diversity accumulation curves. (A) Ampicillin-treated insects. (B) Kanamycin-treated insects. (C) Soil+Bran-fed insects.
- Reduction of Bacterial Community Size by Ampicillin
The bacterial 16S rRNA gene copy number in the gut of T. molitor larvae was 2.54 ± 0.33 × 10 13 copies/mg gut. Gut microbiota exposed to ampicillin showed a decrease in the 16S rRNA gene copy number as the concentration of ampicillin increased. The 16S rRNA gene copy number in the Amp 700 sample was 1.82 ± 0.53 × 10 11 copies/mg gut. It is possible that the estimation of community size using the 16S rRNA gene copy number could be inaccurate; however, the copy number in the Amp 700 sample represented only 0.6% of the copy number from the Amp 0 sample, implying a significant reduction in the community size as a result of treatment with ampicillin. There was a negative correlation between the 16S rRNA gene copy number and the ampicillin concentration (R 2 = 0.9399) ( Fig. 3 ). We could not determine any relationship with the Km samples because of a lack of DNA following construction of the sequencing library. It is important to note that a high concentration of ampicillin (>300 μg/ml) was added to bran, and that it was not possible to measure the exact quantity of ampicillin ingested by the T. molitor larvae. However, our results showed that the oral administration route did result in effective ampicillin uptake. T. molitor growing in a natural environment may orally ingest a diverse range of compounds that may have antibiotic activity. Likewise, these data suggest that the bacterial community in the T. molitor gut may experience dramatic environmental changes due to feeding. Thus, ampicillin reduced the bacterial community size and diversity, indicating that the bacterial community of the gut of T. molitor larvae is vulnerable to antibiotics.
- Exposure of Diverse Soil Bacterial Community to the Gut of T. molitor Larvae
T. molitor larvae were grown in a mixture of soil and bran (indicated as Soil+Bran). Soil+Bran has a bacterial community that is more diverse than does bran alone. We expected that the larval gut bacterial community would take on a different composition owing to diet, and that there could be differences in community composition between T. molitor larvae grown in the Soil+Bran in comparison with larvae grown in bran alone. The bacterial community of the Soil+Bran larvae was primarily composed of Firmicutes (85.5%), followed by Proteobacteria (11.6%) at the phylum level. At the genus level, Bacillus (62.8%) and Enterococcus (11.0%) were the two major constituent groups. After the larvae had been incubated for 5 days, we identified Spiroplasma (81.3%), Lactococcus (14.6%), Lactobacillus (1.6%), and Weissella (1.5%) in the gut bacterial community. However, after 10 days of incubation, Spiroplasma took over almost the entire community (99.8%). A phylogenetic diversity accumulation curve indicated that the culture diversity at day 5 was higher than that in the control samples; however, after 10 days of incubation, the number of OTUs returned to a value similar to those found in the control samples. Therefore, we suggest that the environment in the gut of T. molitor larvae selected a specific group of bacteria, such as Spiroplasma , and that as a result, the bacterial community of a T. molitor larva is very stable regardless of the external environment.
- Phylogenetic Analysis Within the Genus Spiroplasma
The composition of the bacterial community of the gut of T. molitor larvae was found to be very simple. However, the reductions in community diversity and the community size led us to perform a detailed analysis of the Spiroplasma genus. RDP classification did not provide information at the species level; consequently, we selected the representative sequences (sequence identity >97%) and then extracted the sequences that can be taxonomically associated with Spiroplasma . Following this, we performed a phylogenetic analysis of Spiroplasma -associated sequences with the 16S rRNA gene sequences from the Spiroplasma type species. Construction of a neighbor-joining phylogenetic tree indicated that the Spiroplasma -associated sequences found in this study were distinct from previously identified Spiroplasma species, based on the tree morphology ( Fig. 5 ). The sequence identity based on the analysis of the 16S rRNA gene sequences of Spiroplasma species was not higher than 97%, which is a conventional cutoff for detecting novel species. Therefore, we suggest that the Spiroplasma community of the gut of T. molitor larvae contains many novel species and requires further research.
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Neighbor-joining phylogenetic tree of Spiroplasma type species and representative sequences associated with Spiroplasma. The isolation source of the Spiroplasma type species is indicated. The names of representative sequences contain the source sample name and a random number. Species with a bootstrap value below 50 were not included in the tree. The scale bar indicates the expected number of substitutions per site.
- Oxygen Conditions of the Gut of T. molitor Larvae
The oxygen conditions of the insect gut vary between insect species and the gut of T. molitor , which may be in an anaerobic state [7] . Therefore, interactions between the gut microbiota and the insect would be expected to occur under anaerobic conditions. The number of culturable isolates in the microaerobic experimental condition were 4.91 ± 1.60 × 10 7 , 3.58 ± 0.27 × 10 7 , and 3.23 ± 0.48 × 10 7 CFU/g of the gut when grown on LB, NA, and R2A, respectively. Under aerobic conditions, the number of CFUs was slightly lower than when grown in microaerobic conditions, with 1.88 ± 0.32 × 10 7 , 3.01 ± 0.20 × 10 7 , and 2.54± 0.06 × 10 7 CFU/g of the gut when grown on LB, NA, and R2A, respectively. From these results, we conclude that the aerobic condition may be unfavorable for the growth of bacterial cultures from the gut of T. molitor larvae. However, the physiology of the isolates in various concentrations of oxygen is a topic that requires further research.
- Comparison of the T. molitor Bacterial Community with Gut Microbiota from Other Insects
To compare the bacterial community of the gut of T. molitor larvae with those from other insects, we constructed an OTU network containing our data and previously published data from Solenopsis species [16] ( Fig. 6 ). This publication reported that one Solenopsis individual’s bacterial community was dominated by Spiroplasma while the others were not. The OTU network results showed that the Spiroplasma species identified from our data were specific to T. molitor and that the Spiroplasma OTUs were not shared between T. molitor and Solenopsis species. There were some shared OTUs that affiliated to the genera Pantoea , Bradyrhizobium , Solirubrobacter , Shigella , Pseudomonas , Bacillus , and Erwinia .
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OTU network of four bacterial communities from T. molitor larvae, the mixture of soil and bran, and from four individuals of Solenopsis species. Red and green dots indicate the OTUs and Spiroplasma-associated OTUs, respectively.
Discussion
Some Spiroplasma species ( e.g., S. poulsonii ) have been identified as insect pathogens. However, the T. molitor larvae included in this study did not show any symptoms in response to the presence of Spiroplasma ; they successfully molted into adults and repeated their life cycle. An absence of harmful effects of Spiroplasma has also been confirmed in some other insect species, including Cephalotes species (ants) [1 , 19] . Spiroplasma species have been isolated from many insects [6 , 12] . They are considered to be opportunistic symbionts owing to the low numbers of Spiroplasma within a given insect population. We were unable to find any bacterial community analysis results showing that Spiroplasma , or any species from the phylum Tenericutes, was a major member of insect gut bacterial communities. Several individuals of Polyrhachis (a genus of Formicidae, ant family) and Lepidoptera (butterfly order) have been associated with Spiroplasma ; however, the abundance of Spiroplasma within these insects was found to be very low [29] . Taken together, we believe that these data indicate that Spiroplasma is a common bacterial member of the insect gut microbiota. Therefore, our study on T. molitor larvae may represent a very interesting case.
Consistent with previous reports in many insects, the bacterial community of the gut of T. molitor was simple. Our study can be considered as an extreme case of simple gut microbiota, in which Spiroplasma constituted almost the entire community, with the exception of the Amp 0 sample. However, we could not overlook the presence of the minor elements of the community, as indicated by the community variation between individuals ( Fig. 2 ) and the number of OTUs ( Figs. 4 and 6 ). The percentage of Spiroplasma -associated OTUs was 26% and 76% of the total number of OTUs in the Amp 0 and Amp 900 samples, respectively. This OTU ratio of Spiroplasma and non- Spiroplasma OTUs indicated that there were many bacterial species present at very low abundance.
Symbiotic characteristics of the gut bacterial community have been identified from many insects and bacterial species. In an effort to investigate the symbiotic relationship between T. molitor larvae and their gut bacterial communities, we attempted to amplify the nifH gene encoding for nitrogenase (which possesses functions in nitrogen fixation) using qPCR and then to isolate pectin- or cellulosedegrading bacterial strains (data not shown). However, we have not yet succeeded in establishing this symbiotic relationship. Our failure does not necessarily mean that T. molitor does not benefit from its gut microbiota. However, when we consider T. molitor ’s detritivore feeding behavior, T. molitor might be able to satisfy its nutritional requirements from its natural environment without the aid of its gut microbiota. If this is true for T. molitor , then, unlike termites and aphids, it is not dependent on its gut microbiota [29] . The presence of nitrogen-fixing bacteria has been suggested to provide nutritional support for some insects; consequently, the detection of nifH , a gene encoding the enzyme nitrogenase, was performed via qPCR using a previously utilized primer [7 , 17] . Other symbiotic characteristics such as cellulase and pectinase activity were also evaluated in T. molitor gut isolates. However, we failed to identify nifH or any other symbiotic characteristics from DNA isolated from the gut of T. molitor larvae.
Based on the isolation source of Spiroplasma , we can expect that the transmission of Spiroplasma species occurs across diverse environments, such as phylogenetically diverse insects and plants, regardless of endo- and extracellular conditions. However, based on the absence of Spiroplasma -associated OTUs in the sequencing and OTU network data from the soil and bran experiments, the transmission of Spiroplasma from soil or bran seems unlikely. Based on our current knowledge, we speculate that the Spiroplasma community exists only in the T. molitor population. The occurrence of lateral or heredity transfer of the bacterial community in T. molitor populations may be an area for further research. Although T. molitor is a wellknown insect species, a detailed microbiological analysis of this insect has never been performed previously. To the best of our knowledge, this is the first study to investigate the gut microbiota of T. molitor larvae. This study has provided fundamental information on the bacterial community of the gut of T. molitor , which may lead to additional investigations into the interactions between the gut microbiota and their responses to environmental changes.
Acknowledgements
This work was supported by a Korea University Grant (G1300105).
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