Midgut bacteria in deltamethrin-resistant, deltamethrin-susceptible, and field-caught populations of Plutella xylostella, and phenomics of the predominant midgut bacterium Enterococcus mundtii

Gut bacteria play a significant role in host insect. This study evaluated detail difference of midgut bacteria in deltamethrin-resistant, deltamethrin-susceptible and field-caught populations of diamondback moth, and studied phenomics of the predominant midgut bacterium Enterococcus mundtii. Cultivable bacteria revealed that E. mundtii and Carnobacterium maltaromaticum dominated the bacterial populations from deltamethrin-resistant and deltamethrin-susceptible larval midguts, whereas E. mundtii was predominant in field-caught population. Illumina sequencing analysis indicated that 97% of the midgut bacteria were from the phyla Firmicutes, Proteobacteria and Cyanobacteria. Both resistant and susceptible populations had more Enterococcus and Carnobacterium. Enterococcus, Carnobacterium, Bacillus, and Pseudomonas were predominant in the field-caught population. A phenomics analysis revealed that E. mundtii was able to metabolize 25.26% of the tested carbon sources, 100% of the nitrogen sources, 100% of the phosphorus sources and 97.14% of the sulfur sources, had a wide range of osmolytes and pH conditions, and showed active deaminase activity but no decarboxylase activity. This is the first report regarding different populations of DBM midgut bacteria analyzed using both high-throughput DNA sequencing and cultivation methods, and also first report concerning the phenomics of E. mundtii. The phenomics of E. mundtii provide a basis for the future study of gut bacteria functions.


Results
Gut bacteria isolation. The highest number of bacteria per larval midgut was found in the resistant larval gut (log 6.68 CFU ml −1 of gut suspension) on NA medium, followed by the susceptible larval midgut (log 6.32 CFU ml −1 of gut suspension), the smallest in the midgut of field-caught larvae (log 5.76 CFU ml −1 of gut suspension). However, on LB plates, the highest number was also found in the resistant larval midgut (log 6.17 CFU ml −1 of gut suspension), but this was followed by the midgut of field-caught larvae (log 6.14 CFU ml −1 of gut suspension), and the smallest in the susceptible larval midgut (log 5.70 CFU ml −1 of gut suspension) ( Table 1). After successive purification, a total of 18 purified gut bacterial strains were obtained, of which seven (Br-2, Br-3, Br-4, Br-5, Br-6, NBr-1 and NBr-2) were from the midgut of deltamethrin-resistant larvae, eight (M-2, M-3, M-4, M-5, M-6, NM-1, NM-2 and NM-3) from the deltamethrin-susceptible larvae, and three (T-1, NT-1 and NT-2) from the field-caught larvae ( Table 2).

Insect source
Bacterial counts in each larval midgut (log CFU ml −1 gut suspension)

Molecular characterization of culturable larval midgut bacteria from the DBM. A 16S rRNA
analysis revealed that the isolates obtained from the midgut of the three DBM larval were mostly belonging to three different genera (Enterococcus, Enterobacter and Carnobacterium) in Firmicutes and Proteobacteria ( Table 2). The nucleotide sequences of the bacterial strains were subjected to homology searches in DNA databases. The results revealed that the sequences of two strains (NBr-1 and NBr-2) from the midguts of resistant larvae, four strains (M-3, M-4, NM-1, NM-3) from the midguts of susceptible larvae, and three strains (T-1, NT-1 and NT-2) from the midguts of field-caught larvae had a 99% or 100% similarity with the 16S rRNA gene sequences of E. mundtii. The sequences of two strains (Br-2 and Br-3) from the midguts of resistant larvae and four strains (M-2, M-5, M-6, NM-2) from the midguts of susceptible larvae had a 99% or 100% similarity with the 16S rRNA gene sequences of C. maltaromaticum. The sequences of three strains (Br-4, Br-5 and Br-6) from the midguts of resistant larvae showed a 99% or 100% similarity with the 16S rRNA gene sequences of E. amnigenus. E. mundtii was the predominant larval midgut bacterium obtained from the midguts of all three DBM populations.
Sequencing results and microbial diversity in the DBM larval midgut. A total of 112,321 reads and 143 OTUs were obtained from three samples through the MiSeq sequencing analysis. Each library contained 29,280 to 47,882 reads, with different phylogenetic OTUs ranging from 31 to 77. All the rarefaction curves tended to approach the saturation plateau, indicating that the data volume of the sequenced reads was reasonable, and the discovery of a high number of reads made a small contribution to the total number of OTUs. This rarefaction curve indicated the presence of a large variation in the total number of OTUs from the different samples ( Fig. 1). Compared with the samples from the field-caught larvae, the samples from resistant larvae and susceptible larvae had a lower OTU density. The OTUs of sample m1 had the lowest value (31), followed by sample Br1 (35), whereas the highest was in sample T1 (77) ( Table 3). The alpha diversity species richness (Chao), evenness (ACE), and Shannon index all confirmed the highest diversity in sample T1 from the field-caught larvae and less diversity in the samples from resistant (Br1) and susceptible larvae (m1) ( Table 3). The Shannon diversity indices of the three samples were 2.56 (T1), 0.69 (m1), and 0.56 (Br1), indicating that the Shannon diversity of the sample from field-caught larvae was significantly higher than that of the other two samples.
Taxonomic composition of the samples from the DBM larval midgut. Sequences that could not be classified into any known group were unclassified. The bacterial OTUs were assigned to 11 genera, 5 different phyla. Two phyla (Firmicutes and Proteobacteria) out of 5 total phylotypes were common to the three samples, which comprised more than 95% of the total reads in every library. Firmicutes was the most abundant group (Fig. 2a)    Additionally, Bacteroidetes was detected only in a sample (T1) from field-caught larvae and not in the other two samples. Cyanobacteria (4.17%, 6 OTUs), Bacteroidetes (7.64%, 11 OTUs), and Actinobacteria (3.47%, 7 OTUs) comprised 1.62% (1,787 reads), 0.95% (1,046 reads) and 0.48% (533 reads) in all libraries, respectively. Two genera (Enterococcus and Carnobacterium) of the eleven detected genera were common to all three samples, comprising more than 95% of the total reads in the libraries from samples Br1 and m1 and approximately 20% of the total reads in the library from sample T1. For sample m1 from susceptible larvae and sample Br1 from resistant larvae, Enterococcus was the most abundant group (Fig. 2b), comprising approximately 3.01% (2) of the OTUs and 80.15% (50,878) of the reads across all samples; Carnobacterium, the second most abundant genus (3.01%, 2 OTUs), comprised 17.11% (10,863 reads) in all libraries. The read proportions of Enterococcus in samples T1, m1 and Br1 were 2.55%, 75.60% and 83.99%, respectively, whereas the read proportions of Carnobacterium were 19.62%, 21.07% and 13.77%, respectively (Fig. 2b). Additionally, the read proportions of Bacillus, Pseudomonas, Lactococcus, Oceanobacillus, Chloroplast_norank, Psychrobacter, Myroides, Brochothrix and Arthrobacter in sample T1 were 5.19%, 3.90%, 2.60%, 1.20%, 2.60%, 2.60%, 2.60%, 1.30% and 1.30%, respectively, whereas much lower read proportions of these genera were detected in samples m1 and Br1.
Differences between the bacterial genera in different midgut samples. At the genus level, the differences between the communities from different gut samples were depicted with a Venn diagram. A total of 84 genera were discovered, and 27.38% of them were shared genera (Fig. 3). Sample T1 contained more bacterial varieties (77 genera) than samples Br1 (36 genera) and m1 (31 genera), as shown in Fig. 3. An overlap between the genera detected in the three samples was also observed. The largest overlap was found between samples Br1/ T1 (29 genera), followed by samples m1/T1 (28 genera) and samples m1/Br1 (26 genera).
Based on the relative abundance of the genera as shown in Fig. 4, genera with an average abundance of >1% in at least one sample were defined as predominant. The Venn diagram indicates that four dominant genera belonged to the genera shared by three samples (m1, Br1 and T1), including Carnobacterium, Pseudomonas, Enterococcus and Chloroplast_norank. In sample Br1, the genus Pantoea was also predominant, whereas for sample T1, seven other predominant genera were found, including Bacillus, Oceanobacillus, Lactococcus, Myroldes, Brochothrix, Psychrobacter and Arthrobacter. The relative abundances of the genera Bacillus, Pseudomonas, Lactococcus, Oceanobacillus and Psychrobacter in sample T1 were much higher than in samples m1 and Br1. Additionally, the relative abundance of the genus Enterococcus in samples m1 and Br1 was greater than 3% and significantly higher than in sample T1.  (Fig. 5). The sole sulfur source that could not be metabolized by E. mundtii was thiophosphate (PM4, Well F05). Additionally, the species contained none of the biosynthetic pathways tested (0/94 in plate PM5).
The PM3 plate tested isolate NT-1 of E. mundtii for its ability to grow on 95 different nitrogen sources (amino acids) (Fig. 5). All these compounds were utilized by E. mundtii, including L-cysteine, uric acid, etc. (Fig. 5). Meanwhile the PM6, PM7 and PM8 plates tested E. mundtii for its ability to grow on 285 different nitrogen pathways. All the tested compounds could also be utilized by E. mundtii (Fig. 5).
Plates PM9 and PM10 were used to test for growth under various stress conditions. E. mundtii showed active metabolism with up to 8% sodium chloride, 6% potassium chloride, 5% sodium sulfate, 20% ethylene glycol, 6% sodium formate, 7% urea, 4% sodium lactate, 200 mM sodium phosphate (pH 7.0), 200 mM sodium benzoate (pH 5.2), 100 mM ammonium sulfate (pH 8.0), 100 mM sodium nitrate, and 100 mM sodium nitrite, but it could not metabolize sodium lactate, which ranged from 5% to 12% (plate PM9, Well F05 to F12) in our analysis (Fig. 5, Table 5). When combined with various osmolytes in 6% sodium chloride, E. mundtii grew well in all tests (plate PM9, Well B01 to B12, and C01 to C12). The pH range for the active growth of E. mundtii was between 5 and 10, with an optimal pH of approximately 10.0. When combined with various amino acids at a pH of 4.5, E. mundtii showed no growth in any test except when combined with the amino acid L-norvaline (plate PM10, well D03) (Fig. 5, PM10). In comparison, when combined with various amino acids at a pH of 9.5, E. mundtii grew actively in all tests. PM10, wells B1-D12 and E1-G12, tested the decarboxylase and deaminase activities of E. mundtii in the presence of amino acids at pH 4.5 and pH 9.5, respectively (Table 6). In the presence of most the amino acids, E. mundtii showed active deaminase activity but no decarboxylase activity (Fig. 5, PM10).

Discussion
To understand the contribution of gut bacteria to host processes, it is necessary to determine the bacterial structure and diversity and the metabolic phenotypic characterization of some of the predominant bacteria in the host insect gut environment. This study investigated the larval midgut bacteria from deltamethrin-resistant, deltamethrin-susceptible and field-caught DBM populations and also characterized the metabolic phenotypes of the dominant midgut bacterial species E. mundtii.
A statistical analysis of the cultivable bacterial populations obtained on two different media did not show significant differences in the bacterial populations, indicating that the media composition did not appear to affect the cultivable bacterial strains, as previously demonstrated 27,36 . Only three bacterial genera were obtained in the present study, indicating that cultivation-dependent methods have limitations for bacterial diversity studies and do not reflect the actual quantitative relationships in the DBM larval midgut. Similar findings have also been previously reported 27,29 . Meanwhile, the bacterial genera obtained by the cultivation-dependent method used in this study have also been documented as present in the gut of the DBM 27,29,37 and other Lepidopteran families as well 25,34,35,38 . The field-caught population of DBM larvae harbored single phylotypes of E. mundtii, and similar bacterial monoassociations have been reported in some other insect orders, including Orthoptera, Thysanoptera, and Hemiptera 36,39,40 . A monoassociation of predominant bacteria might eliminate or prevent the colonization of other competitive micro-organisms.
The sequencing results in this study showed that DBM midgut bacteria were diverse, but only two microbial phyla (Firmicutes and Proteobacteria) were predominant, as were three genera (Enterococcus, Carnobacterium, and Bacillus). Similar results have also been found in eight species of mosquitoes 41 and the midguts of other Lepidoptera, including Lymantria dispar, Helicoverpa armigera, and Bombyx mori 25,26,38 . The most abundant genera in the DBM larval midgut were Enterococcus and Carnobacterium. It has been reported that the capacity of these genera to degrade carbohydrates could be useful to the digestion of the host insect 42 , and this function should be tested in the DBM.
Larval midgut bacteria of the deltamethrin-resistant, deltamethrin-susceptible and field-caught DBM populations had different structures and diversity patterns according to Illumina sequencing in this study. They should have had a similar structure prior to insecticide exposure. The midgut bacteria from field-caught populations were more diverse, and this structure should be much nearer to the actual midgut bacterial structure of the DBM in the field in Guizhou province in China. The structure and diversity of midgut bacteria from the deltamethrin-resistant and deltamethrin-susceptible populations were similar in some cases, possibly due to nearly 20 years in a similar rearing environment in our laboratory 30 ; however, the differences at the genera level were large. Although these could be a consequence of exposure to insecticides that had differential toxicities to different bacterial taxa, it is important to consider the possibility of the role of certain larval midgut bacteria related to deltamethrin resistance. The susceptible population exhibited a higher proportion of bacteria from the phylum Cyanobacteria than the resistant population. The resistant population had a higher proportion of bacteria from the genus Pseudomonas than the susceptible population. Additionally, some of the less abundant bacteria varied markedly between the susceptible and resistant populations. These differences might be due to the chemical environment in the gut. Similar findings have been reported by others. Symbiont-mediated insecticide resistance has been demonstrated in stinkbugs 18 . Symbiotic Burkholderia from the soil have been shown to enhance the resistance of Riptortus pedestris 43 . The differences in bacterial taxa in the larval midgut from three different DBM populations in this study indicate that further work to identify the possible reasons for deltamethrin resistance in the DBM is warranted.  Although a few studies have been performed on the gut bacteria of Lepidoptera, they have dealt with only the isolation and characterization of the microbial flora 26,27,29 . However, this study involved the characterization of the metabolic phenotype of the predominant midgut bacterium E. mundtii, revealing significant metabolic diversity. Many carbon compounds could be utilized, and most nitrogen, sulfur, and phosphorus sources were also metabolized. These data indicate the great versatility of E. mundtii in the DBM gut environment. The most informative plates for E. mundtii were PM1/PM2 (carbon sources), PM9 (osmolyte conditions) and PM10 (pH conditions). The most informative utilization patterns for carbon sources were saccharides and for nitrogen sources were various amino acids and peptides. These compounds are commonly found in many plant leaves. They might play a key role in the survival of E. mundtii and thus in supporting the digestion of food in the DBM. Additionally, E. mundtii had wide range of tolerance to various osmolytes and pH conditions, as indicated by plates PM9 and PM10. Bacterial deaminases generate acid via the catabolism of amino acids, which help counteract an alkaline pH 44,45 . The phenotypic diversity of E. mundtii can be explained by considering the seasonal variation in osmolytes and gut pH due to dietary variation of the DBM. Consequently, phenotypic characteristics for the utilization of those sources and the wide range of tolerances of E. mundtii could have a high adaptive value in host-microbe interactions and the survival of the bacterium in the DBM gut.
In conclusion, although there is insufficient evidence to demonstrate whether certain bacterial taxa are responsible for conferring DBM deltamethrin resistance and whether such a mechanism works together with other mechanisms (insect physiology changes or gene mutation), the data obtained in this study still provide useful information about the molecular characterization of insect midgut bacteria and its relationship with the important phenomenon of insecticide resistance. The phenotypic characterization of the dominant midgut bacterium E. mundtii could also help us understand its potential role in the DBM midgut. Given the significant damage that the DBM causes worldwide and the difficulty in controlling it due to insecticide resistance, the roles of some  predominant bacteria and the possibility that microbial symbiont-mediated resistance is active in this insect should be further investigated.

Materials and Methods
Collection and mass rearing of insects. Deltamethrin-resistant and -susceptible populations of DBM larva (Br1, m1), able to tolerate >1000 μg ml −1 and 3 μg ml −1 of deltamethrin, respectively, were obtained in a previous study 30 . The field-caught population of DBM larva (T1) was collected from a cabbage field at the Guizhou Academy of Agricultural Sciences in Guizhou province in China, where no insecticides had been applied to control the DBM. This population was also able to tolerate 3 μg ml −1 of deltamethrin, and was chosen as a control population when compared with the laboratory populations. Larvae from the three populations were reared in a sterile acryl cages (45 × 45 × 50 cm) with cabbage (Brassica oleracea L.) and maintained at 18-25 °C and a 50-60% relative humidity under 16 h of light and 8 h of darkness. The cabbage leaves were washed with 70% ethanol for 60 s followed by 5% NaOCl (60 s), thoroughly rinsed with distilled water to remove the disinfectant, then air dried and used to rear the insects.
Midgut sampling and the isolation of cultivable bacteria. The most destructive third instars of the four larval stages of the DBM were selected for the isolation of midgut bacteria 27,46 . A total of 40 third instars from each population were selected and starved for 24 h. The starved larvae were surface disinfected with 70% ethanol for 60 s followed by 5% NaOCl for 60 s, thoroughly rinsed with distilled water to remove the disinfectant, and the midgut contents of the larvae were isolated and were homogenized with 2 ml of 0.1 M phosphate buffer (pH 7.0). Portions (0.1 ml of each midgut suspension (diluted 10 −1 ) were transferred to 4.5 ml of sterile distilled water and subsequently diluted to 10 −2 , 10 −3 , 10 −4 and 10 −5 . One hundred microliters of aliquots of the 10 −3 to the 10 −6 midgut dilutions were inoculated onto the surface of Luria Bertani (LB) and nutrient agar (NA) plates 27,36 . After inoculation, the Petri dishes were placed at 30 °C in the dark for 48 h. The number of colonies formed on each Petri dish was counted. The number of cultivable bacteria for each DBM midgut population was calculated. Bacteria of different colors, growth rates and morphologies were selected from the agar plates, and a single representative isolate of each morphotype was transferred to a new plate. After five to six successive passages, the purified strains were maintained in 30% glycerol at −20 °C for long term storage. The bacteria were revived on LB agar before use in a study.

Molecular identification of culturable midgut bacteria.
A loopful of each midgut bacterial colony from an LB agar plate was picked, re-suspended in 150 µl of distilled water, boiled for 12 min in an Eppendorf tube, cooled to room temperature on ice for 8 min, centrifuged at 9000 g for 2 min and the supernatant was utilized for PCR. The 16S rRNA gene of each bacterium was amplified via PCR using the forward primer 27F