Community structure of gut bacteria of Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae) larvae during overwintering stage

Survival rate at low temperature becomes a crucial strategy since temperature change often leads to fluctuations in the insect population. Microbes play important roles in the process of resisting low temperature. In this study, we analyzed gut bacterial communities from Chinese white pine beetle Dendroctonus armandi which remained overwintering process under natural conditions from October 2015 to January 2016, monthly, in the Qinling Mountains, Shaanxi, China using Illumina MiSeq sequencing. A total of 835,227 high-quality sequences and 48 singleton operational taxonomic units were obtained. Gut bacterial communities showed variation in relative abundance during the overwintering stage. As ambient temperature declined, Proteobacteria (mostly γ-proteobacteria) became the predominant phylum in the larvae guts, and followed with Actinobacteria and Firmicutes, respectively. In particular, there was no Deinococcus-Thermus in January 2016. Thermoleophilia appeared in November and December 2015, but not for October 2015 and January 2016, nor did δ-proteobacteria. By contrast, gut bacterial community compositions increased in relative abundance in November and December 2015. This study provided initial evidence that gut bacterial communities were associated with the larvae overwintering process at low temperature. Moreover, no complementary studies combining overwintering process of Coleoptera insect and high-throughput sequencing were carried out, paying particular attention to insect in cold season.

The insects' survival rates at low temperatures influence their population dynamics 8,17 and geographical distribution 8,18 .
Gut microbiota play important roles in insects' life histories contributing to effective reproduction rate, community interactions and survival rates by metabolizing toxins and providing protection against a variety of environmental stresses [19][20][21][22] . Bacteria can contribute to the insects which feed on a nutrient-poor food source by nutritional supplements [23][24][25][26] . Insects that exploited woody substrates are particularly nutrient poor and available to competitors once the woody substrate are depleted [27][28][29] . Bacteria can provide nutritional supplements such as amino acids 23 , essential vitamins 24 , nitrogen and carbon compounds 25,26 . Such relationships seem to be widespread among insects that exploit woody substrates. Host insects show slow growth and high mortality if they are short of symbiont 30 . Most of studies focus on the variation of endosymbionts during the insect development stage [31][32][33] ; however, it is unknown if the composition of endosymbionts varies during the overwintering stage at low ambient temperature. A certain type of bacteria was first reported in stinkbugs (Plataspidae) via a "symbiont capsule" which was transmitted vertically 34 . We hypothesized that some relationships existed between gut bacterial community structure and the overwintering process of D. armandi larvae at low temperature. In order to understanding the roles of endosymbiont community members in bark beetles, the community composition should be defined in the first step 35,36 .
In our study, gut bacterial communities of D. armandi larvae at low temperatures were investigated using high-throughput sequencing method during overwintering stage. We quantified the composition and structure of gut bacterial communities in each month from October 2015 to January 2016 in winter.

Results
Environmental conditions. The average of daily total sunshine duration started with 5 Overview of sequencing analysis. The proportion which equaled the number of high quality sequences/ valid sequences (Shared reads/Total reads) was over 90% in each month, expect for December 2015 (89.57%) in the current study (Table 2). After sequence trimming, quality filtering and removal of chimeras, 835,227 high-quality sequences remained, with an average length of 273 bases (Fig. 1). The mean number of sequences per sample was 208,807 ± 9,698 (s.d.). The rarefaction curves in four months had approached the plateau phase, and they were unlikely that more bacterial communities composition would be detected with additional sequencing efforts (Fig. 2). These high-quality sequences were clustered into different operational taxonomic units ( Bacterial diversity during overwintering period. The community richness and community diversity for D. armandi larvae rapidly increased in November and December 2015 compared with the larvae collected in October 2015 and January 2016 (Table 3). A Venn diagram was used to compare the similarities and differences between the communities in the different months (          showed a distinct clustering of gut bacterial communities every month from overwintering larvae at low temperature ( Fig. 9) PCoA revealed the overwintering pattern in four months for the unweighted and weighted unifrac distances (Fig. 9). According to the unweighted unifrac PCoA, larvae collected from November and December 2015 formed a unique cluster, separated from the other two months (Fig. 9). According to principal coordinate 1 (PC1) and PC2 analysis (69.61% and 30.15% of variance explained, respectively), the differences of gut bacterial communities at low temperature during overwintering stage were great between November, December 2015 and October 2015, January 2016, while the differences were little in November 2015 compared with December 2015 (Fig. 9). According to weighted unifrac PCoA, the larvae collected in November and December were separated and the gut bacterial communities (collected in October 2015 and January 2016) were clustered based on PC1 and PC2 analysis (79.95% and 16.74% of variance explained, respectively) ( Fig. 9). At the genus level, the larvae collected in November and December 2015 generally also had more OTUs than those in October 2015 and January 2016, when the relative abundances of the top 50 OTUs were compared with Z-score method (Fig. 10). This analysis revealed similar results with

Discussion
This study analyzed the gut bacterial communities structure and relative diversity with low temprature in each month from October 2015 to January 2016 during overwintering stage of D. armandi larvae. Low temprature is a leading factor of winter survival for most insects in frigid and temperate zones 13,17,18 . Insects survived at low temperature can keep vital bodily functions well and provides tolerance to low temperatures in adverse low ambient environment 37 . Freezing-tolerant insects can survive after the formation of internal ice, whereas most insect are freeze susceptible (freezing-intolerant) species which cannot tolerate the internal ice formation. These insects improved their supercooling capacity by increasing the contents of polyols or other forms of cryoprotectants 8,38 . Whether the gut bacterial communities composition and structure is vital for insects during winter is still unknown. In our study, the dominant phyla during overwintering stage were Proteobacteria, Firmicutes and Actinobacteria (Supplementary Tables S1 and S2), and the dominant classes were γ-proteobacteria, Actinobacteria, Bacilli and α-proteobacteria (Supplementary Tables S1, S2 and S3). The changes of gut bacterial relative diversity accorded with the OTU richness estimation on the whole. The special phyla was Deinococcus-Thermus which just existed in October, November and December 2015. Furthermore, the unique families were Desulfovibrionaceae, Vibrionaceae, Streptomycetaceae, Patulibacteraceae, Sphingobacteriaceae, Carnobacteriaceae and Mogibacteriaceae in November and December 2015. We hypothesized these families were associated with the insect overwintering process and resisting to low temperature.
The present results illuminated that gut bacterial communities differed during each month from October 2015 to January 2016. While, there were still some obvious similarities of insect communities as the result of similar environmental variables such as temperature 36 . So far, few studies analyzed the diversity of gut bacterial species from overwintering insects. Mostly, researchers coped with the gut bacterial communities composition in insects' development stages 33 . Even further, there was no complementary researches combining overwintering process of Coleoptera insect and high-throughput sequencing to our knowledge, especially for insect in cold season. Gut bacterial communities composition and diversity showed differences at low temperatures of Chinese white pine beetle larvae. The phyla of Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Acidobacteria and Tenericutes appeared in each month from October 2015 to January 2016, when the mean monthly ambient temperatures declined from 11.0 °C to 0.5 °C (Table 1). Meanwhile, gut bacterial communities from D. armandi larvae could be monitored immediately, annotating into 7 phyla, 15 classes, 28 orders, 54 families, 64 genera. Their appearances were possibly in correlation with overwintering process and environmental low temperature changes. Among these identified bacteria, γ-proteobacteria from Proteobacteria and Bacilli from Firmicutes exhibited higher presence at low ambient temperature during overwintering period.
Environment changes including altitude, latitude, and ambient temperature influences insects' survival rate in frigid and temperature zones because they are poikilothermic animals 54,55 . According to Bryant et al. 56 and Singh et al. 57 , bacterial diversity was significantly correlated with elevation. This was not surprising that the larvae overwintering process was a temperature dependent biochemical process. This result supported our previous hypothesis that low temperature had an effect on larvae gut bacterial communities diversity on the Qinling Mountains due to the high altitude. Lastly, we attempted to identify the environmental variables which controlled larvae gut bacterial communities in order to understand their survival rates in winter. Low ambient temperature was previously shown to be the first priority predictor of variation in bacterial diversity. Therefore, studying the relationship between low ambient temperature and bacterial diversity during overwintering stage of Chinese white pine beetle larvae was the next purpose. Insect collection and dissection. Chinese white pine beetle (D. armandi) larvae were collected from host trees Chinese white pines (P. armandi) in each month from October 2015 to January 2016. The overwintering larvae were collected from three sample plots, 20 m × 20 m in each one, from the above site in each month. Five trees were selected by five-point sampling method in each sampling plot. The phloem, 20 cm × 20 cm, was peeled off from four directions. All overwintering D. armandi larvae were gathered into glass culture dishes with sterile moist paper using fine forceps and directly transported to the laboratory 33 . 150 larvae samples in each month were gathered for high-throughput sequencing analysis from attacked Chinese white pine (P. armandi).
150 larvae guts in a month were transferred into three 1.5-mL microcentrifuge tubes (fifty larval guts in each tubes). These three 1.5-mL microcentrifuge tubes were homogenized several times before grinding in liquid nitrogen, and then vortexed at the speed of 2500 r/min for 3 min with 500 mL Tris-EDTA (Sangon Company, Shanghai, pH 8.0). The microbial cells were separated from gut wall tissues after centrifuging at the speed of 4000 r/min for 15 s. The supernatants contained gut bacteria and fungi were totally transferred into three new 1.5-mL microcentrifuge tubes for gut bacterial DNA extraction. All of the steps above were accomplished in a sterile environment with biological air clean bench (Suzhou Antai Airtech, Jiangsu, China).
Bacterial DNA extraction. The E.Z.N.A. Bacterial DNA Kit (Omega Biotech, USA) was used to extract overwintering D. armandi larval gut bacterial deoxyribonucleic acid (DNA) following the instruction booklet. The gut bacterial DNA was stored at −20 °C before using. DNA samples were mixed in equal concentrations, and the mixed DNA specimens were sent to JBYH Biotechnology Co.,Ltd (Wuhan, China) for analysis by MiSeq sequencing.
Bioinformatics and statistical analysis. Paired-end reads were truncated by cutting off the barcode and primer sequence based on their unique barcodes. Then, the single, longer sequences were merged by these paired-end reads using FLASH (Version 1.2.7) 58 . High-quality clean tags performed by quality filtering on the raw tags under specific filtering conditions by QIIME (Version 1.7.0) quality controlled process 59,60 . Chimeric sequences were detected and removed using the UCHIME algorithm 61 .
Clustering was performed using the UPARSE pipeline (version 7.0.1001) 62 , and similar sequences were assigned to OTUs using the threshold of 97% identity. A typical sequence was picked by selecting the longest sequence that had the largest number of hits to other sequences in the OTU. The RDP classifier (version 2.2) 63 was used to annotate taxonomic information for each representative sequence. The typical sequences were aligned using the Greengenes database 64 , with a minimum identity of 80%. The differences in the dominant species were conducted using MUSCLE (Version 3.8.31) in order to study the phylogenetic relationships of different OTUs of different samples, multiple sequence alignments 65 .
The alpha diversity analysis included observed species, Ace and Chao estimators, Simpson and Shannon diversity indices estimate of coverage. Rarefaction curves were generated based on observed species. The beta diversity among overwintering D. armandi larval gut bacterial communities was evaluated using both weighted and unweighted unifrac distances 66 . UPGMA was used to accomplish the hierarchical clustering of samples. The Z-score method was used for heat maps. PCoA was performed to make the differences in larval gut bacterial community composition and structure concrete on the unifrac distances of the unweighted and weighted distance matrices, respectively. Statistical analyses of numbers of OTUs (expressed as the mean ± SE) in each month from October 2015 to January 2016 in D. armandi larval gut bacteria were performed using ANOVA followed by Tukey's tests for multiple comparisons to detect significant differences performed with SPSS 18.0 (IBM SPSS Statistics, Chicago, IL, USA) and Sigma Plot 12.5 sofware (Systat Sofware Inc., San Jose, CA, USA).
Ethics approval and consent to participate. Our manuscript report data collected from insect named Chinese white pine beetle. The larvae Chinese white pine beetles were collected in the Huoditang Experimental Forest Station of Northwest A&F University. Huoditang Experimental Forest Station agreed us to collect insects for experiments, in order to find a way of controlling the damage.
Availability of data and materials. The datasets supporting the conclusions of this article included within the article (and its additional files) are available in the repository.