Marked variations in diversity and functions of gut microbiota between wild and domestic stag bettle Dorcus hopei hopei

doi:10.21203/rs.3.rs-3326985/v1

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Research Article

Marked variations in diversity and functions of gut microbiota between wild and domestic stag bettle Dorcus hopei hopei

https://doi.org/10.21203/rs.3.rs-3326985/v1

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published 18 Jan, 2024

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Background Although spade beetles are a popular saprophytic insect, their gut microbiome has been poorly studied. Here, 16S rRNA gene sequencing was employed to reveal the gut microbiota composition and functional variations between wild and domestic Dorcushopei hopei larval individuals.

Results The results indicated a significant difference between the wild and domestic Dhh gut microbiota, the domestic Dorcus individuals contained more gut microbial taxa with xenobiotic degrading functions, such as genera Ralstoniaand Methyloversatilis, while the wild Dorcus possesses gut microbiota compositions more appropriate for energy metabolism and potential growth, for instance Turicibacter and Tyzzerella. This study furthermore assigned all Dhh individuals by size into groups for data analysis; the results indicated limited disparities between the gut microbiota of different-sized Dorcus hopei hopei larvae.

Conclusion The outcome of this study illustrated that there exists a significant discrepancy in gut microbiota composition between wild and domestic Dorcus hopei hopeilarvae, and the assemblage of gut microbiome in Dorcus hopei hopei was primarily attributed to environmental influence instead of Dorcus individuals varied developmental potential and size. These findings will provide valuable theoretical foundation for the protection of wild saprophytic insects and the development and utilization of the insect-associated intestinal microbiome in the future.

Dorcus hopei hopei

habitat ecology

gut microbiota

16S rRNA gene sequencing

bioinformatics

Dorcus is a genus of the Lucanini tribe, belonging to the Lucaninae subfamily and the Lucanidae (Stag beetle) family (superfamily Scarabaeoidea, infraorder Scarabaeiformia, suborder Polyphaga, order Coleoptera, class Insecta, phylum Arthropoda, kingdom Animalia). The specific classification, identification method, and the current knowledge of the specific species and subspecies within the Dorcus genus are critically under-researched; thus, information such as the specific molecular phylogenic outline and nomenclature are severely lacking in detail. While, some research about Dorcus ecological functions [1] niches [2], and physical mechanisms [3] were roughly conducted. The most commonly researched species in Dorcus genus is Dorcus rectus, a native species widespread in East Asia, which was thoroughly studied from its larval feeding diet [4, 5], larval resources competition [6], to adults' physical features [7, 8] even extended to ecological functions [1] and potential medical values [9], symbolizing the importance of the Dorcus genus as an example and highlighting the genus latent potential.

Dorcus hopei hopei (Dhh in short, AKA the Giant Chinese Stag Beetle), one subspecies of Dorcus hopei, is predominantly distributed in southern mainland China's broad-leaf forests at an altitude of approximately 200–2000 meters, also widely distributed in East Asia [10]. The adult male measures around 40–75 mm in length and 15–30 mm in width, while the female measures around 31–52 mm in length and 13–20 mm in width. It is one of the most abundant subspecies in Dorcus and one of the most common stag beetles in mainland China. Because the larvae are exceptionally efficient in the decomposition of timber & fungus and the adults are important consumers of plant detriment and exudate, Dhh plays a vital role in the carbon cycle and is a critical species in the maintenance of healthy forest flora. Still, Dhh itself was rarely researched, but Dorcus hopei binodulosus, another subspecies of Dorcus hopei was researched from an evolutionary perspective [11, 12]. Although Dhh gut microbiota was significantly under-researched, there are potential usages of its gut microbes in delignification due to the similar role it played compared to termite gut microbiome [13, 14], which are enriched with plant cell wall degrading enzymes and xenobiotic genes [15, 16], shows the underlying practical application of insects gut microbiome in yielding biofuels [17, 18] and other potential commercial values. As a result, we came up with the idea of conducting this research.

In this study, we aim to outline distinct differences between the gut microbiota composition of wild and domestic larval Dhh individuals, as well as to touch on gut microbiota disparities between different-sized larval Dhh, to establish relationships between larval health/larval size and specific microbial categories, which could provide information for future studies oriented towards the biochemistry and microbial conditions of stag beetles, especially of the Dorcus genus.

Sequencing Information

A total of 25 samples, including 13 domestic insect gut samples and 12 wild insect gut samples, were sequenced and obtained 1,888,381 raw tages and 1,667,599 solid tages (Table 1). Total 4,013 ASVs was clustered, in which we identified 33 phyla, 92 classes, 205 orders, 331 families, 732 genera and 1,072 species. The number of observed ASVs varied from 74 to 848 per sample, with an average of 316 ASVs per sample Among the 4,013 ASVs, 2,406 ASVs were solely found in the intestinal of Dhh_W, 1,048 ASVs in the intestinal of Dhh_D, 559 ASVs were shared by two Dhh groups (Fig. 2A). Rarefaction curves plotted from observed OTUs have reached plateau with a high Good’s coverage index (> 99%) that indicated that the sequence depth was able to represent the majority of the ASVs present in all the 25 fecal samples (Fig. 2B, C)

Table 1

Summary of sequencing information
Sample	Raw_Tags	Raw_Bases	Valid_Tags	Valid_Bases	Valid%	Q20%	Q30%	GC%	Counts	ASV_number
Dhh_D1	84812	42.41M	73467	30.29M	86.62	91.30	80.40	53.39	40372	209
Dhh_D10	69037	34.52M	53430	22.58M	77.39	95.74	89.45	54.14	45338	146
Dhh_D11	83237	41.62M	74313	31.40M	89.28	95.95	89.73	53.33	64667	299
Dhh_D12	61070	30.54M	49742	21.17M	81.45	95.30	88.43	51.93	42071	102
Dhh_D13	87220	43.61M	80503	34.23M	92.30	95.74	89.20	54.05	71077	206
Dhh_D14	48490	24.25M	42414	17.95M	87.47	95.93	89.78	54.16	36152	142
Dhh_D2	83566	41.78M	73514	30.84M	87.97	96.81	91.42	52.89	66852	74
Dhh_D3	75021	37.51M	63152	26.46M	84.18	95.44	88.78	52.90	54036	106
Dhh_D5	63897	31.95M	57960	24.59M	90.71	95.50	88.81	54.09	49393	225
Dhh_D6	86454	43.23M	80613	34.18M	93.24	95.69	89.18	53.85	70537	208
Dhh_D7	49376	24.69M	44539	18.74M	90.20	94.79	87.05	53.76	35651	221
Dhh_D8	51993	26.00M	44317	18.62M	85.24	95.67	89.42	53.78	36370	319
Dhh_D9	54100	27.05M	48940	20.70M	90.46	95.67	89.29	54.05	41158	304
Dhh_W10	80614	40.31M	72689	30.14M	90.17	94.86	87.48	52.12	58475	387
Dhh_W11	83606	41.80M	75211	30.96M	89.96	96.05	89.91	53.64	64845	848
Dhh_W13	82205	41.10M	73898	30.41M	89.89	95.53	88.94	52.26	62242	772
Dhh_W15	80764	40.38M	71674	29.80M	88.74	95.90	89.71	53.34	62534	281
Dhh_W16	81554	40.78M	67680	28.46M	82.99	96.42	90.46	52.75	60917	378
Dhh_W17	82634	41.32M	75282	30.57M	91.10	95.36	88.33	53.36	60672	811
Dhh_W3	87446	43.72M	78913	32.39M	90.24	96.35	90.55	53.06	69403	392
Dhh_W4	79638	39.82M	71327	30.14M	89.56	95.35	88.61	52.72	61053	330
Dhh_W5	85254	42.63M	77693	32.95M	91.13	95.22	88.37	51.83	66993	291
Dhh_W6	81515	40.76M	69685	29.54M	85.49	95.82	89.21	52.51	61489	278
Dhh_W7	82333	41.17M	73747	31.08M	89.57	95.58	89.04	52.69	63677	301
Dhh_W9	82545	41.27M	72796	30.92M	88.19	96.50	90.66	52.87	66206	266
SUM	1888381	944.22M	1667599	699.11M	/	/	/	/	1,412,180	4013
AVG	75535	37.77M	66700	27.96M	88.14	95.54	88.89	53.18	56,487.2	316

Intestinal Bacterial Alpha and Beta Diversity

The alpha diversity index indicated differences in gut microbiota flora between Wild and Domestic Dhh groups. According to Observed OTUs and Chao1 index, the Wild group is enriched with significantly greater numbers of gut microbiota species compared to the domestic group (Figure. 3A, B). Pielou_e index illustrated the Wild group slightly more even than the domestic group, but no significantly (Figure. 3C). The Simpson index showed that the Wild group had a higher value (Figure. 3D). The indexes above consistently showed that the diversity of microbial communities in the Wild group was the highest.

Analysis of similarity (ANOSIM) at the ASV level based on Bray-Curtis dissimilarity explored an overall significant difference among wild and domestic Dhh groups (R = 0.2463, p = 0.002). PCA analysis and PCOA analysis was further to visualize the results. PCA results showed two major microbial clusters for wild and domestic Dhh respectively (Figure. 3E). Although no clear segregations were observed between the two Dhh groups, the wild sample did express extremely high variability in which the majority of its data points were distant to the domestic cluster. PCoA (P = 0.002) results showed two distinguished microbial clusters (Figure. 3F). The two samples did express a distinct separation, exhibiting substantial discrepancies between Wild and Domestic Dhh fecal microbiota.

Characteristics comparison of the gut microbiota between wild and domestic Dhh

The wild Dhh fecal microbiotas’ majority of abundance was occupied by three phyla: Firmicutes, with a relative abundance (RA) of 24.56–91.36% (mean RA: 62.02%); Proteobacteria, with a RA of 4.28–58.57% (mean RA: 33.71%); and Bacteroidetes, with a RA of 0.47–6.39% (meanRA:1.78%) (Figure. 4A). In the studied wild Dhh, Erysipelotrichaceae was the predominant family within the phylum Firmicutes, with Ruminococcaceae being the second-most dominant family belonging to Firmicutes. These two families are among the top 5 most abundant families in the wild and the overall sample (Figure. 4B). Other phyla observed in low abundance include Fusobacteriota (mean RA: 0.18%) and Actinobacteriota (mean RA: 1.02%), in which Fusobacteriota was almost exclusively seen in wild Dhh fecal samples. In the domestic Dhh samples, the most dominant phylum was Proteobacteria, with a RA of 28.38–89.80% (mean RA: 67.08%), followed by Firmicutes, with a RA of 2.56–67.07% (mean RA: 22.00%); and Actinobacteriota, with a RA of 0.25–10.97% (mean RA: 3.89%). Although at low abundances, multiple trace phyla expressed by domestic Dhh microbiota are almost exclusive to domestic samples, including Methylomirabilota (mean RA: 0.03%) and Desulfobacterota (mean RA: 0.21%) (Figure. 4A)

The RA of Firmicutes was higher in wild Dhh compared to that of domestic Dhh (Wilcoxon test, P < 0.01), whilst the RA of Proteobacteria in wild Dhh was lower compared to that of domestic Dhh (Wilcoxon test, P < 0.05). Bacteroidetes RA showed no significant difference between the two Dhh groups (Wilcoxon test, P = 0.87). Burkholderiaceae, belonging to the phylum Proteobacteria, was the predominant family in both wild and domestic samples and did express significant differences between its abundance in the two Dhh groups. Its RA for the combined sample reached an impressive 30.71% on average, with a mean RA of 41.02% and 19.54% for the domestic and wild samples, respectively. Another family belonging to the Proteobacteria phylum, Rhodocyclaceae, is among the top 5 most abundant bacterial families for the overall & separate samples (Figure. 4B)

Marked differences in identified genera were also observed between wild and domestic Dhh. Ralstonia was the predominant genus in the combined sample and both individual Dhh groups (mean RA: 67.08%). However, there were still stark differences in terms of RA of Ralstonia between the wild and domestic samples, with Ralstonia's RA being significantly higher in domestic Dhh compared to wild Dhh (40.63% vs. 19.17% respectively). The second overall most abundant genus was Turicibacter (mean RA: 9.99%); its mean RA was only 2.75% in domestic Dhh compared to the 17.84% in the wild sample. The abundance of other genera, such as Tyzzerella, Methyloversatilis, and (Candidatus) Soleaferrea, also demonstrated a significant difference in wild Dhh compared to domestic Dhh (Wilcoxon test, P < 0.05). These three genera, combined with Ralstonia and Turicibacter, constitute the five dominant genera in terms of relative abundance in the overall sample (Figure. 4C).

The Linear discriminant analysis effect size (LEfSe) analysis further identified numerous taxonomic clades that is demonstrating statistically significant differences between wild and domestic Dhh (Figure. 5A). The logarithmic discriminant analysis (LDA) results showed that the genus level biomarkers in the gut microbiota of domestic Dhh group were Ralstonia, and Methyloversatilis, while the enriched genera of wild Dhh group were Turicibacter, Gandidatus_Soleaferrea, Tyzzerella, and two unclassified genera of Firmicutes and Ruminococcaceae respectively (Figure. 5B).

Network analysis of wild and domestic Dhh gut microbiota

Interaction network analysis of the bacterial microbiome in each group were used to select the key species and to unveil the relationship of microbiome in the community (Fig. 6). We found 48 nodes and 109 edges in Dhh_W and 51 nodes and 69 edges in Dhh_D. Compared to the Dhh_D, the edges, average triangles were presented much higher, which indicated that a more complex network was observed in Dhh_W. Also, average weighted degree was much higher in Dhh_W, which probably indicates that the nodes in network of Dhh_W were more important than that of Dhh_D. More network topological attributes were present in Fig. 6C. Also, different dominant genera were observed in three Dhh groups individually (estimated by weighted degree). In Dhh_D, Tyzzerella, Ralstonia, (Candidatus) Soleaferrea, Turicibacter and Vibrio greatly contributed to the interaction complexity. In Dhh_W, Tyzzerella, (Candidatus) Soleaferrea, Pseudomonas, Christensenellaceae_R-7_group and Ralstonia played an important role in the complexity of community.

Predicted functions of wild and domestic Dhh gut microbiota

The microbiota functions were predicted using PICRUSt2 based on the detected 16S rRNA gene amplicon sequences. The anticipated pathways and functions showed significant differences in microbiota functionalities between wild and domestic Dhh. KEGG level 2 and level 3 pathways (Fig. 7), such as “Nucleotide metabolism”, “replication and repair pathways”, “environmental adaptation”, “starch and sucrose metabolism”, “ribosome biogenesis pathway”, “sporulation”, and “bacterial toxin synthesis” pathways were significantly more enriched (Student's t-test, P < 0.01) in wild Dhh, whereas “glycan biosynthesis and metabolism pathway”, “lipid metabolism pathway”, “metabolism of other amino acids”,“xenobiotics5 biodegradation and metabolism”, “energy metabolism”, “TCA cycle”, “glyoxylate and dicarboxylate metabolism”, and “oxidative phosphorylation pathways” were enriched significantly greater in domestic Dhh group.

Microbiota differences of larval Dhh of different sizes

All the 4 indexes used previously for the alpha diversity of Wild vs. Domestic Dhh group, demonstrated no significance in terms of gut microbe OTUs between different Dorcus size groups (Figure S1) (Table S1). Analysis of similarity (ANOSIM) at the ASV level based on Bray-Curtis dissimilarity explored no significant difference among Dhh_S, Dhh_M and Dhh_L. The PCA (P = 0.416) and PCoA (p = 0.342) analysis were used to visualize the results, which coincides with the anosim analysis result (Figure S2). The bacterial community possessed similar structure among Dhh_S, Dhh_M and Dhh_L, which indicated the size of Dhh make no significant difference in the gut bacterial microbiota.

At phylum level, the overall top 5 most dominant phyla remain to be Proteobacteria, Firmicutes, Actinobacteriota, Bacteroidetes, and Cyanobacteria. The column chart depicted the significant differences in relative abundance of top 5 phyla in larval Dhh of different sizes (Figure S3A). The depicted disparities of relative genera abundance were also less pronounced compared to the wild vs. domestic analysis, with only 9 of the 718 tested genera revealing statistically significant differences for relative abundance between the three Dhh groups. Of the 9 genera, only Turicibacter was among the top 5 most dominant genera in the overall sample (Figure S3B).

Very minimal predicted pathways and functions were shown to have different levels of enrichment between the three Dorcus size groups. The "signaling molecules and interaction" pathway (level 2) was shown to be distinguished enriched among the three groups (Figure S4A), while the "ribosome biogenesis" function (level 3) is only enriched at inferior levels in the "Small" group and the "cyanoamino acid metabolism" (level 3) is only greater enriched in the "Large" group (Figure S4B). The "Wnt signaling pathway" was also enriched at statistically significant levels; although significant, this pathway's depicted deficient proportion was deemed unfit for further inspection (Figure S4C).

Drastic transformations in gut microbial community structures were found between wild and domestic Dhh larvae, suggesting that the host environment serves a crucial role in shaping intestinal bacterial structure. However, there are also observable similarities between each group's presented major taxonomy contributions as well. For instance, although Firmicutes were expressed with higher RA and Proteobacteria expressed at a lower RA in wild Dhh compared to the domestic group, the two phyla combined comprised more than 85% of the bacterial species in the domestic group and more than 95% in the wild group. The statistics reflect the significance of these two phyla in forming larval Dhh gut microbiota; furthermore, the discrepancy in RA of the two major phyla among the two Dhh groups demonstrates the environmental and ecological impact on the Firmicutes-to-Proteobacteria ratio and overall microbiota compositional balance.

Overall, although existing at different abundances, both Wild and Domestic Dhh group exhibited significant gut microbiota communities associated with xenobiotic degradation and metabolism. Out of the top 5 genera, the ones responsible for metabolism and plant material breakdown (Turicibacter, Tyzzerella, Soleaferrea) were more significantly enriched in the Wild group; genera with xenobiotic degradation abilities (Ralsonia, Methyloversatilis) were more abundant in the Domestic group, completely aligning with trends identified by PICRUSt2 results. Xenobiotics- degrading microbes’ elevated presence is potentially the direct effect of the artificial exacerbation of Dhh larvae’s habitat ecology.

The top5 most dominant families in the overall sample are Burkholderiaceae, Erysipelotrichaceae, Ruminococcaceae, Lachnospiraceae, and Rhodocyclaceae. Three belong to Firmicutes and two to Proteobacteria, parallel to the obtained phylum-level statistics. Ralstonia was responsible for the abundance of Burkholderiaceae, Turicibacter for Erysipelotrichaceae, Tyzzerella for Lachnospiraceae, and Methyloversatilis for Rhodocyclaceae. However, we discovered that the results for Rhodocyclaceae and Erysipelotrichaceae’s abundance were erroneously produced due to the data analysis algorithm's ignorance of certain contemporary modifications in bacterial taxonomy. Specifically, Turicibacter was reclassified into the new family of Turicibacteraceae in 2020. Methyloversatilis was categorized into the new family of Sterolibacteriaceae in 2017, both of which were not updated in the analysis program's data bank [19, 20]. Nonetheless, this taxonomic error will not affect the later discussions as previously stated, the functional and compositional disparities of gut microbiota were principally attributed to major genera, and genus-level data presents no taxonomical issues. Network analysis indicated that a more complex and stable bacterial community was observed in Dhh_W, which coincided with our anticipation. It’s probably the diverse food intake that leads to the need of complex intestinal bacterial community and its potential function.

The family Ruminococcaceae was distinctive as its abundance was not due to individual genera and contained no exceptionally expressed genera within, and numerous species in the Ruminococcaceae family presumably share certain beneficial features favorable for Dhh metabolism. This hypothesis is supported by general Ruminococcaceae known capacity for structural polysaccharides breakdown [21, 22] and further testified by the strong positive association between Ruminococcaceae and tetrapod herbivory [21]. Although we cannot conclude that specific function of Ruminococcaceae in Dhh gut microbiota will be identical to that of other tetrapod herbivores due to physiological differences, current knowledge about biochemical potentials of Ruminococcaceae does imply high probabilities of this bacterial family conducting similar roles in Dhh gut microbiota.

As the most abundant genus in Dhh_W and Dhh_D, the soil-borne bacterial genus Ralstonia was widely regarded as a plant pathogen, causing bacterial wilt in plants [23]. Ralstonia includes mostly plant pathogenic bacteria capable of growing on plant oils/fatty acids, and they express genes such as aceA1 and aceA2 that code for enzymes involved in the glyoxylate cycle6 [24]. This indicates that Ralstonia potentially provides Dhh with metabolites derived from previously undegradable plant lipids and aid in Dhh cells aerobic respiration. Ralstonia could utilize an array of aromatic compounds and heavy metals for energy for detoxification [25]. These compounds are primarily xenobiotic and hazardous to organism health, thus, Ralstonia species are valuable gut microbes to form symbiotic relationships with, especially in larval Dhh, whose nutrition sources often experience contamination during decay. PICRUSt2 reinforced our hypothesized function of Ralstonia in larval Dhh gut microbiota. The functions of "lipid metabolism," "xenobiotics biodegradation and metabolism," "TCA (tricarboxylic acid) cycle," and "glyoxylate metabolism" were all significantly enriched in the domestic group, the group of higher Ralstonia RA. These results indicate us that the microorganisms being regarded as plant pathogen probably play a vital role in insects by improving metabolism and detoxification. Furthermore, since the rearing of domestic larvae occurred in human infrastructure, the domestic group is indeed expected to be exposed to greater levels of xenobiotics, validating PICRUSt2 data and Ralstonia's predicted functions.

The genera (Candidatus) Soleaferrea and Tyzzerella are denoted to contribute to plant-matter breakdown. Both were identified as core microbiota genera of Macrotermes falciger (a termite species), a species sharing similar diets with Dhh larvae (xylophagous & mycophagous) [26]. Furthermore, Tyzzerella was significantly enriched in the gut microbiota of crayfish, which enjoy feeding on fresh or decayed aquatic plant material.

The microbiota differences between the Dorcus size groups (L, M, S) are of less significance, as could be seen from abundance analysis and PICRUSt2 results. One actual difference between the Dhh groups gut microbiota is the Turicibacter RA, which depicts a descending tendency as larvae size decreases. This suggests that larger Dhh larvae growth was potentially attributed to reinforced metabolic capabilities and internal hormone regulation provided by Turicibacter. The "large" group was the most enriched, the "medium" group came second, and the "small" group was the least enriched. Overall, the patterns demonstrated by Dhh larvae of different sizes contrasted with the former research results, as size and microbiota composition/diversity were demonstrated to be associative in the stag beetle Odontolabis fallaciosa [27].

This present study results could be beneficial for improving the current artificial rearing methods of Dhh by suggesting advantageous microbial communities for larval growth, potentially leading to modified feeding procedures that cultivate more aesthetic and healthier commercial Dhh. More importantly, the obtained data are beneficial for developing future environmental protection and conservation methods. One hypothesized technique to be considered is the release of artificially bred Dhh larvae or adults with modified microbiota profiles imitating wild individuals into designated areas to facilitate material decomposition and nutrient cycling in any required habitat, such as the insect-based agri-food waste valorization [18]. Modifying the gut microbiome reduces the released specimens' repulsive responses to a feral environment and lowers any risks of introducing foreign gut microbes into the wild population. This could alleviate nutrient cycling issues in the specified environment with minimal contamination on gut microbiome and overall biochemical diversity of the local species.

As this study is one of the very few research focusing on gut microbiota of the Dorcus hopei and specifically the Dhh subspecies, these results provided additional insights into the area of Lucanidae gut microbiota and outlined several new research orientations to be conducted for further exploration or validation of information the regarding gut microbiota of Docus hopei phpei.

The present study utilized 16S rDNA sequencing to compare the fecal bacteria of wild and domestic Dhh larvae, and between larvae of different sizes. The relative abundance of fecal microbiota showed a notable difference between wild and domestic Dhh. Overall, the domestic Dhh individuals contained more gut microbial taxa with xenobiotic degrading functions, such as genera Ralstonia and Methyloversatilis, while the wild sample possesses gut microbiota compositions more appropriate for energy metabolism and potential growth, such as Turicibacter and Tyzzerella. On the other hand, Dhh larvae of different Dorcus size groups exhibited significantly fewer disparities in their gut microbiota composition, suggesting that habitat-ecology factors, rather than individual growth potential, are responsible for the assemblage of detected distinguished microbiota patterns. These results provided preliminary insights into the gut microbiota of Dorcus hopei phpei and its most common subspecies, increased our current understanding of Lucanidae microbiota composition, and indicated future research orientations for exploring insect gut microbiome. Furthermore, the significant enrichment of xenobiotics-degrading gut microbiota functionalities in Dhh larvae, especially in the Wild group, demonstrates the inherent environmental presence of factors pernicious to the survival of Dhh larvae. This calls for conservational actions aimed at alleviating the influence of artificial infrastructure and contaminants on local ecology and biodiversity.

Sample Collection and Rearing Condition

The individuals from the wild were collected in decayed timber located in Hangzhou, Zhejiang Province, China. Out of the 12 wild larvae, four specimens were from Yang jia pai Village (30° 14' 57.0768'' N, 120° 3' 54.3384'' E), four from Yuewang Temple (30°09'07.0"N, 120°13'46.9"E), three from Wenbi Mountain (30°12'34.6"N, 120°05'36.6"E), and one from Baoshi Mount (30°15'40.0"N 120°08'38.9"E). They were excavated out of their initial shelter, rotten logs in broad-leaved forests shaded area, from the range of altitude between 200–600 meters. with proper instruments, no larvae were harmed in the process, and each one health condition was examined before being accepted as one sample individual. Each individual was assigned a number; their physical data (head capsule diameter) and feces samples were obtained.

Domestic larvae were raised in captivity. The larvae were given either fermented wood flakes or bottled macrofungi, instead of rotten timber presence in the wild group. All the domestic larvae were reared in thermal control accurately restricted between 22–24°C for their life cycle. The fungus bottles and wood flakes’ humidity remained in the good range between 55 ± 5% constantly. The fungus bottle and fermented wood flake (compressed in a bottle) were changed periodically about every 3 months to prevent food decayness fluctuation. Larvae were kept in a dark environment and the container remained a viable ventilation, and any other human disturbance was prohibited to eliminate confounding variables. Likewise, each individual was assigned a number, and their physical data & feces samples were obtained.

Body data collection and Grouping

The head capsule diameter was measured for each larva. The larvae were held up, and the horizontal diameter of the head capsule was measured using a caliper. The results were recorded and used for grouping. The utilization of head capsule size data instead of other features as growth-potential indicators have been justified in preceding texts. The body weight of each larva was measured using a balance. Body weight was seen as the standard of fat accumulation, which is directly associated with material processing and nutrient absorption. In the process, the larvae body surface feces and wood dust were cleaned before measuring to prevent deviation.

Two grouping plans were used in this study. The purchased and foraged individuals were isolated into "Domestic" and "Wild" Dhh groups (Dhh_D and Dhh_W), each including 11 and 12 Dhh sample individuals, respectively. The same experimental individuals were assigned into groups "Large," "Medium," and "Small" Dhh groups (Dhh_L, Dhh_M, Dhh_S). Each corresponds to a head capsule size of equal/over 11mm, less than 11mm & equal/over 9 mm, and less than 9mm), each including 6, 10, and 6 Dhh sample individuals respectively. Analyses concerning the two grouping systems were separately done.

Fecal sample collection method

The development stages overview of Dorcus hopei hopei was shown in Fig. 1. We collected samples while the individuals are in the stage of third instar larvae. Wild individuals were collected and installed in a container filled with the wood dust processed from the original sheltering rotten timber. Domestic individuals were taken out of their feeding medium and installed in containers. larvae were brought to the laboratory under safety and were left undisturbed for a 3-day acclimation period, in which they were monitored for any potential repulsive behaviors or health condition changes. Throughout the transportation and acclimation process, the living temperature of the larvae was controlled at 24°C. After three days, the larvae were gathered for feces collection. The larvae's abdomen and anal region were disinfected by wiping them with 75% alcohol-soaked gauze. The feces were then naturally excreted by the larvae individually into a disinfected test tube. Sterile forceps and scoopulas were used to remove 1 gram of feces sample from each test tube and place it on a sterile foil, measuring on an analytical balance to ensure it is the precise amount. After reaffirmation, feces samples were transferred into iLongsee Stool Storage Kit (Longsee Biomedical, China), kept in -80℃ condition.

PCR amplification and 16S rDNA sequencing

DNA from different samples was extracted using the E.Z.N.A. ® Stool DNA Kit (D4015, Omega, Inc., USA) according to the manufacturer's instructions. Nuclear-free water was used for blank. Total DNA was eluted in 50 µL of Elution buffer and stored at -80°C until amplification in the PCR by LC-Bio Technology Co., Ltd, Hangzhou, Zhejiang Province, China.

The V3-V4 hypervariable region of the 16S rRNA gene was amplified using indicated primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHV GGGTATCTAATCC-3') (Logue et al., 2015). PCR amplifications were performed in 25 µL of reaction mixture including 25 ng of template DNA, 12.5 µL of PCR Premix, 2.5 µL of each primer, and PCR-grade water. The PCR conditions go as follows: 98°C for 30 seconds; 32 cycles of 98°C for 10 seconds, 54°C for 30 seconds, and 72°C for 45 seconds; and then finally 72°C for 10 minutes. PCR products were verified with agarose gel electrophoresis. Throughout the DNA extraction process, high-purity water will be used to exclude possible false-positive PCR results. The PCR products were purified by AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by Qubit (Invitrogen, USA). Amplicon pools were prepared for sequencing; the amplicon library size and quantity were assessed on Agilent 2100 Bioanalyzer (Agilent, USA) and with the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. The libraries were sequenced on the NovaSeq PE250 platform.

Data analysis

Samples were sequenced on an Illumina NovaSeq platform according to the manufacturer's recommendations provided by LC-Bio. Paired-end reads were assigned to samples based on their individual barcoding and trimmed by cutting off the primer sequence. Paired-end reads were merged using FLASH. Raw reads filtering was performed under specific filtering conditions to obtain precise tags according to fqtrim (v0.94). Chimeric sequences were filtered using Vsearch (v2.3.4). After dereplication, feature tables and sequences were obtained using DADA2. Alpha diversity and beta diversity were calculated using QIIME2, then according to SILVA (release 132) classifier, feature abundance was normalized using the relative abundance of each sample. The graphs for beta diversity were drawn by R package. BLAST was used for sequence alignment, and the feature sequences were annotated via SILVA database. Additional statistical analyses were manipulated utilizing R package (v3.5.2).

Alpha diversity analyses were calculated using the vegan package and plotted using the ggplot2 package. Principal Coordinates Analysis (PCoA) [28] was calculated using ade4 package and Principal Components Analysis (PCA) [29] was calculated using stat package. Analysis of similarities (ANOSIM), a non-parametric statistical test, used to test the difference between Dhh groups, was calculated using vegan package [30]. Log-linear discriminant analysis effect size algorithm (LEfSe) analysis was performed to identify potential taxonomic groups (LDA scores > 4.0, p < 0.05) that can be regarded as biomarkers for different Dhh groups [31] A bacterial interaction network was performed using the Fastspar [32] with a Sparcc correlation coefficient (|correlation r)|>0.2, and p < 0.05), and the network map was visualized using Gephi 0.9.4. Prediction of the Microbial functional was undertaken using the PICRUSt2 by searching data on the 16S rDNA gene sequencing in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (www.genome.jp/). Other diagrams were visualized using the R package (v3.5.2). Wilcoxon rank-sum test, Kruskal-Wallis H test, or student's t-test was employed to determine statistical significance (P < 0.05 is statistically significant).

Supplementary Information

The online version contains supplementary material available at

Acknowledgements

Not applicable.

Authors’ Contributions

Lu.Y., Chu S., and Chen.H. contributions to the conception and design of the work. Lu.Y., Chu S. and Shi. Z. wrote the main manuscript text. Lu.Y., Chu S., and You R. prepared all the figures. Chen.H. revised the manuscript. All authors have read and approved the final manuscript.

Funding

This research was funded by Zhejiang Provincial Natural Science Foundation of China (Grant No. LY22H280011); Zhejiang University Student Science and Technology Innovation Activity Plan (Grant No. 2023R06039) and Science Foundation of Zhejiang Sci-Tech University (Grant No. 21042252-Y).

Data Availability

All data are provided in the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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No competing interests reported.

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published 18 Jan, 2024

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Marked variations in diversity and functions of gut microbiota between wild and domestic stag bettle Dorcus hopei hopei

Status:

Journal Publication

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Abstract

Figures

Background

Results

Sequencing Information

Intestinal Bacterial Alpha and Beta Diversity

Characteristics comparison of the gut microbiota between wild and domestic Dhh

Network analysis of wild and domestic Dhh gut microbiota

Predicted functions of wild and domestic Dhh gut microbiota

Microbiota differences of larval Dhh of different sizes

Discussion

Conclusions

Materials and Methods

Sample Collection and Rearing Condition

Body data collection and Grouping

Fecal sample collection method

PCR amplification and 16S rDNA sequencing

Data analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1