Introduction

Globally, more than 6 000 ladybeetle species (Coccinellidae: Coleoptera) have been reported1. Although Coccinella transversoguttata is a ladybeetle native to North America, it has spread to many other areas including Central America, Mexico, Europe as well as the Tibetan plateau in China2,3,4,5,6,7. C. transversoguttata has been found to be an important predatory ladybug that feeds on many agricultural pest including aphids, scale insects and other small arthropods8,9,10. In many locations including America, C. transversoguttata is considered a notable predator that can be exploited in the biological control of many pests mainly aphids11,12.

In Tibet area, which has the highest plateau in the world, many agricultural pest species have been reported including aphids, whiteflies, phylloxeras, scale insects, spider mites and many other pests. They cause enormous losses in terms of crop yield in the Tibet area13,14,15,16,17. Metopolophium dirhodum and Rhopalosiphum padi were reported to be the dominant aphid species in Tibet area farmland of highland barley which are difficultly to control15. Besides C. transversoguttata, several other predatory ladybeetles exist in Tibet such as Hippodamia variegate, Harmonia axyridis, Coccinella septempuntata, which may be used as biotic factors to control agricultural pests in Tibet plateau, especially aphids5. This calls for development of strategies to improve the control efficiency of predatory ladybeetles on pests.

Bacterial communities have important regulatory roles in the growth, reproduction, digestion, thermotolerance, resistance to adverse factors and other important processes in the host insects18,19,20,21,22 as well as other arthropods including spider mites23,24,25. In Drosophila melanogaster, more than 10 different lysozymes have been detected in the midgut; which also harbors a transporter with high affinity for d-amino acids26. In crickets, bacteria in the anterior hindgut degrade various classes of soluble polysaccharides27. Another study showed that the bacterial community in bumblebees protect the host against highly virulent natural parasite (Crithidia bombi)28.

The microbiome of Coleoptera encompasses a complex assemblage of mainly bacteria and other microorganisms inhabiting various niches within and on the beetle's body, including the gut, integument, and reproductive organs, which play diverse and often essential roles in beetle biology, influencing nutrient acquisition, digestion, immunity, reproduction, and defense against pathogens and predators18,29,30, Understanding the microbiome of Coleoptera is essential for unraveling the intricate interactions between beetles and their microbial partners, shedding light on fundamental ecological and evolutionary processes.

Currently, few studies have characterized C. transversoguttata in aphids in Tibet areas. In this study, we collected C. transversoguttata from 8 locations in the Tibetan plateau and found that feeding habits affected the structure and diversity of bacterial communities in beetles. However, we did not identify the factor that influence the bacterial structures. These results add to our understanding of the complex bacterial communities of C. transversoguttata and their utilization as potential biocontrol factors.

Materials and methods

Coccinella transversoguttata population collected in Tibetan area

Eight C. transversoguttata populations were collected from Tibetan plateau, each population contains 3 replicates, and every replicate includes combined 5 C. transversoguttata adults, in the range of latitude N 29.07°–N 30.01° and longitude E 88.28°–E 96.69°, and all the samples were collected at an altitude of 2 942 m above sea level. The host plants of C. transversoguttata were mainly apple, highland barley and alfalfa, and many aphids in these plants can be preyed by C. transversoguttata such as Aphis gossypii, Macrosiphum avenae, Schizaphis graminum, Acyrthosiphon dirhodum (Table 1). Once C. transversoguttata were collected, they were put in abosolute alcohol until sequencing. Total C. transversoguttata adults were used for 16 s rRNA sequencing without dissection, as microbiota could exist within any organ of insects, including gut, gonad, salivary glands or cells. Before sequencing, all C. transversoguttata adults were washed by 75% alcohol to wipe off the microbe in the surface of C. transversoguttata.

Table 1 Information about Coccinella transversoguttata populations collected in Tibetan plateau.
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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′-ACTCCTACGGGAGGCAGCA-3′) and reverse primer 1492r (5′-GGACTACHVGGGTWTCTAAT-3′). In this test, a combination of barcode sequences and adapter sequences was implemented. The PCR amplification experiment was carried out as previously described21, 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 technology31. The raw reads generated from sequencing were filtered and demultiplexed using the SMRT Link software (version 8.0) (https://www.pacb.com/support/software-downloads/) 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.biocloud.net) (to analyze the beta diversity indexes). The Shapiro–Wilk test (SPSS 21.0) was conducted to assess whether the alpha diversity index data followed a normal distribution. Given that the data exhibited a normal distribution, Student's t-test (SPSS 21.0) was employed to analyze the significant differences in symbiont density between the two treatment groups.

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 2.0/2.2 Real 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.

Results

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).

Table 2 Full-length 16 s rRNA sequencing results of all Coccinella transversoguttata samples collected in Tibetan plateau.
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Figure 1
Figure 1
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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.microbesonline.org/fasttree). Bacteria related to different phylum are in different colors.

Figure 2
Figure 2
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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
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Relative abundance of top 10 bacterial genera in Tibetan plateau Coccinella transversoguttata by full-length 16 s rRNA gene sequencing.

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).

Figure 4
Figure 4
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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
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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.biocloud.net); (B) significant differences of functional analysis of bacterial communities between SH and LS populations.

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).

Figure 6
Figure 6
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Relative amount of symbiont density in different Coccinella transversoguttata populations in Tibetan plateau, a total of 6 bacteria including Wolbachia (A), Buchnera (B), Rickettsia (C), Serratia (D), Stenotrophobacter (E) and endosymbiont of Liposcelis decolor (F) were shown.

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 biology32. Wolbachia exhibited mitochondrion–like function in some nematodes, generating ATP for their hosts33, in other Coleoptera species dominate bacteria were also detected, such as Spiroplams would dominate the microbiome of khapra beetle, Trogoderma granarium34, while Wolbachia and Rickettsia were dominated in cereal leaf beetle, Oulema melanopus35. 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 microbiota36.

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 communities37. Rearing environment has also been shown to structure microbial communities in Tenebrio molitor38. 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 communities40. 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.