Bacterial communities varied in different Coccinella transversoguttata populations located in Tibetan plateau

Coccinella transversoguttata is an important predatory beetle in Asia and America. Currently, few studies have investigated C. transversoguttata in China especially in the Tibetan plateau. In this study, full-length 16 s rRNA sequencing and qPCR experiment were performed on eight C. transversoguttata populations collected from Tibet to analyze their bacterial communities and bacteria abundance. In summary, our results revealed the microbial compositions, diversities and bacterial titers in the bacterial communities in C. transversoguttata populations in the Tibetan plateau. In future, there is a need to explore the differences in microbiota among various C. transversoguttata populations collected from different locations. These results add to our understanding of the complex bacterial communities of C. transversoguttata and their utilization as potential biocontrol factors.


Full-length 16 s rRNA sequencing of 8 C. transversoguttata populations
To identify differences in microbial communities among different C. transversoguttata populations, full-length 16 s rRNA gene sequencing was performed.Notably, C. transversoguttata DNA amplification, sequencing, library construction, and 16 s rDNA data analysis were carried out using the Biomarker Technologies Corporation, Beijing, China.DNA extract of C. transversoguttata were performed by TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 with instructions by TaKaRa Co., Ltd.(Dalian, China).Briefly, the V3-V4 region of the whitefly's bacterial 16 s rRNA gene was first amplified with a pair of primers: forward primer 27f.(5′-ACT CCT ACG GGA GGC AGC A-3′) and reverse primer 1492r (5′-GGA CTA CHVGGG TWT CTAAT-3′).In this test, a combination of barcode sequences and adapter sequences was implemented.The PCR amplification experiment was carried out as previously described 21 , and Quant-iT™ dsDNA HS Reagent was used to quantify the PCR products.Next, all products were pooled and sequenced on the sequencing platform of PacBio SMRT RS II DNA (Pacific Biosciences, Menlo Park, CA, USA) to construct a sequence amplified library.Off-target sequences and low-quality sequences were filtered using the PacBio circular consensus sequencing technology 31 .The raw reads generated from sequencing were filtered and demultiplexed using the SMRT Link software (version 8.0) (https:// www.pacb.com/ suppo rt/ softw are-downl oads/) with the minPasses ≥ 5 and minPredictedAccuracy ≥ 0.9, in order to obtain the circular consensus sequencing (CCS) reads.Subsequently, the lima (version 1.7.0) was employed to assign the CCS sequences to the corresponding samples based on their barcodes.CCS reads containing no primers and those reads beyond the length range were discarded through the recognition of forward and reverse primers and quality filtering using the Cutadapt quality control process (version 2.7).The UCHIME algorithm (v8.1) was used in detecting and removing chimera sequences to obtain the clean reads.Sequences with similarity ≥ 97% were clustered into the same operational taxonomic unit (OTU) by USEARCH (v10.0), and the OTUs with rebundace < 0.005% were filtered.Unassigned OTU refers to taxa that cannot be matched to the database, are not annotated, or are unclear in terms of species identification.Others OTU refers to species with lower abundances.
The phylogenetic tree; diversity indexes, including alpha diversity (Ace, Chao1, Shannon, and Simpson) and beta diversity indexes (PCoA analysis and heatmaps based on Bray-Curtis similarity analysis); and function of bacterial communities (analyzed by PICRUSt, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) were all analyzed using the Usearch, mothur v1.30.0 (to obtain the OTU and taxonomy matrices), and QIIME v2.0 software (https:// qiime2.org/) on the BMK Cloud (www.biocl oud.net) (to analyze the beta diversity indexes).The Shapiro-Wilk test (SPSS 21.0) was conducted to assess whether the alpha diversity index

qPCR experiment of bacteria in 8 C. transversoguttata populations
The qPCR primers of bacteria in C. transversoguttata were designed using Primer Premier 6.0 based on the 16 s rRNA gene sequence.GAPDH gene served as the reference gene in C. transversoguttata (Table S1).qTOWER 2.0/2.2Real Time PCR Systems (Jena Bioscience GmbH, Thüringen, Germany) with SYBR Premix Ex Taq (Takara Bio Inc., Dalian, China) were utilized to run the qPCR reactions.Regarding the qPCR results of bacteria abundance in C. transversoguttata (Fig. 6), if the data exhibited normal distribution, one-way ANOVA with post-hoc Tukey HSD analysis was performed.If the bacteria abundance and diversity data did not conform to normal distribution, they were analyzed using the Kruskal-Wallis test and Dunn's test with Bonferroni correction for multiple comparisons.All statistical analyses were performed using SPSS 21.0.All figures were drafted using GraphPad Prism 9.0.0.

Abundance of different bacteria in various C. transversoguttata populations
Sequencing data of the 16S rRNA genes of eight C. transversoguttata populations were shown in Table 2.In total, 32 bacteria species were detected in all C. transversoguttata populations, and most of them belonged to the filum Proteobacteria (Fig. 1).PCoA analysis revealed no significant differences among all the bacterial communities in all C. transversoguttata populations (Fig. 2).In terms of bacterial genus abundance, Wolbachia was detected in three populations (BS, GC and ZN), Serratia was found in three populations (LS, MLrc and SH) and Rickettsia was found in MLgmmc and MR populations (Fig. 3).

Bacterial diversity and functional analysis in C. transversoguttata populations
The alpha diversity indices of bacterial communities in all C. transversoguttata populations were compared using One-Way ANOVA in SPSS 21.0.The results revealed significantly higher ACE and Chao1 indices in the SH population compared to the MLgmmc, MLrc, MR, and ZN populations, while the Shannon and Simpson indices were similar across all populations (Fig. 4).Functional analysis revealed that genes related to metabolism were the most enriched in all bacterial communities of C. transversoguttata populations (Fig. 5A).In comparison, the LS population had significantly higher levels of genes involved in energy metabolism than the SH population (Fig. 5B).

Bacteria abundance varied among different C. transversoguttata populations
Analysis of the qPCR results revealed that the Wolbachia abundance varied significantly different among the C. transversoguttata populations (P < 0.01, Kruskal-Wallis tests), while the post-hoc of Dunn's tests with Bonferroni correction indicated found no significant difference between any 2 populations (Fig. 6A).The abundance of Buchnera in BS population (17.97 ± 8.48, Mean ± SEM) was significantly higher than that in other populations  (P < 0.01, Kruskal-Wallis tests) (Fig. 6B).The abundance of Rickettsia in MR population (22.61 ± 15.69, Mean ± SEM) was significantly higher compared with that in other populations (P < 0.05, Kruskal-Wallis tests) (Fig. 6C).The Serratia abundance varied significantly different among the C. transversoguttata populations (P < 0.01, Kruskal-Wallis tests), but post-hoc of Dunn's tests with Bonferroni correction indicated that there was no significant difference among the paired comparisons (Fig. 6D).The abundance of Stenotrophobacter in MLgmmc population (60.87 ± 49.04, Mean ± SEM) was significantly higher relative to that of other populations  (P < 0.05, Kruskal-Wallis tests) (Fig. 6E).Notably, the abundance of endosymbiont of Liposcelis decolor varied significantly among the C. transversoguttata populations (P < 0.01, Kruskal-Wallis tests), while the post-hoc of Dunn's tests with Bonferroni correction found no significant difference in the paired comparisons (Fig. 6F).

Discussion
In this study, we explored the influence of different locations on bacterial communities of C. transversoguttat in Tibetan plateau.Through full-length 16S rRNA gene sequencing and qPCR experiment, it is to be noticed that microbiome of 8 C. transversoguttata populations were complex and abundant.The OTU number and alpha-diversity index of different C. transversoguttat populations bacterial communities were diverse.The abundance of bacteria, including many symbionts, was significantly different among all C. transversoguttat populations.Factors contributing to this phenomenon in C. transversoguttat need to be further explored.
In many host insects, some bacteria are considered essential, and hence are widely spread among hosts.For example, Gammaproteobacteria are considered as essential primary symbiont in the pentatomid bug Graphosoma lineatum because they have important regulatory roles in the host biology 32 .Wolbachia exhibited mitochondrion-like function in some nematodes, generating ATP for their hosts 33 , in other Coleoptera species dominate bacteria were also detected, such as Spiroplams would dominate the microbiome of khapra beetle, Trogoderma granarium 34 , while Wolbachia and Rickettsia were dominated in cereal leaf beetle, Oulema melanopus 35 .However, in this study, although numerous bacteria were detected in many C. transversoguttata populations (such as Serratia, Wolbachia and Rickettsia), no dominant or essential bacteria were observed.In contrast to nematodes, Wolbachia or Spiroplasma were not essential for T. truncatus.These results are similar to those observed in Drosophila: Drosophila species harbor diverse microbiota 36 .
To date, researchers have explored factors influencing bacterial community of insects.Host diet is regarded as an important factor affecting the structure of insect gut bacterial communities 37 .Rearing environment has also been shown to structure microbial communities in Tenebrio molitor 38 .Host-endosymbiont interactions are regulated by environmental factors, including climatic and other geographical factors 39 .Geographic distance may also be an important factor affecting the bacterial community structure.It has been shown that the bacterial community structure was similar within Aphis gossypii obtained from the same province, but it was distinct among those from different provinces, indicating a strong effect of geographic distance on aphid bacterial communities 40 .In the study, despite the varied diets observed among different populations of C. transversoguttata (Table 1), the available data alone are insufficient to ascertain the significance of dietary effects on the microbiome of C. transversoguttata.
In summary, our results revealed the microbial compositions, diversities and bacterial titers of bacterial communities in different C. transversoguttata populations in Tibetan plateau.Further studies are advocated to explore differences in microbial communities including symbionts among different C. transversoguttata populations collected from different locations.These results expand our understanding of the complex bacterial communities in C. transversoguttata and provide ideals to accelerate the utilization of C. transversoguttata as a potential biocontrol factor.

Figure 1 .
Figure 1.Phylogenetic tree of all detected bacteria in different Coccinella transversoguttata populations in Tibetan plateau by full-length 16S rRNA genes based on Probabilistic Methods of Phylogenetic Inference constructed by FastTree 2.0.0 software (http:// www.micro beson line.org/ fastt ree).Bacteria related to different phylum are in different colors.

Figure 2 .
Figure 2. Beta diversity analysis of bacterial communities in different Coccinella transversoguttata populations in Tibetan plateau, the results of PCA analysis was shown.

Figure 3 .
Figure 3. Relative abundance of top 10 bacterial genera in Tibetan plateau Coccinella transversoguttata by fulllength 16 s rRNA gene sequencing.

Figure 4 .
Figure 4. Alpha diversity index of bacterial communities in different Coccinella transversoguttata populations in Tibetan plateau, the four diversity indices including ACE (A), Chao1 (B), Shannon (C) and Simpson (D) were shown, respectively.

Figure 5 .
Figure 5. Functional analysis of bacteria in Coccinella transversoguttata.(A) Functional analysis of bacterial communities in different Coccinella transversoguttata populations in Tibetan plateau based on PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) at BMK Cloud (www.biocl oud.net); (B) significant differences of functional analysis of bacterial communities between SH and LS populations.

Table 1 .
Information about Coccinella transversoguttata populations collected in Tibetan plateau.

Table 2 .
Full -length 16 s rRNA sequencing results of all Coccinella transversoguttata samples collected in Tibetan plateau.CCS: circular consensus sequences generated by PacBio platform; Raw CCS: Counts of identified CCS reads in the sample; Clean CCS: Counts of clean CCS reads (post primer removal and length filtration); Effective-CCS: Counts of effective CCS reads after chimeric reads removal; Avglen (bp): Average length reads in the sample; Effective (%): Percentage of effective CCS reads in raw reads.