Insect rearingThe O. strigicollis colony was provided by the Miaoli District Agricultural Research and Extension Station, Miaoli County, Taiwan. This stock colony was maintained on an alternative prey, eggs of C. cautella, and cultured in plastic containers (37.5 × 23.5 × 15 cm) with a ventilation hole covered with nylon gauze. Soybean (Glycine max L.) seedlings were supplied to provide moisture, shelter, and oviposition substrate for O. strigicollis. The colony was kept at 25 ± 3 °C, 60 ± 15% RH, and a photoperiod of 12:12 h (L:D). To avoid the potential negative effect of long-term inbreeding, more than 100 individuals of O. strigicollis were collected from the field annually and first reared separately for more than two generations. After insectary health inspection, the new cohort was mixed with the lab-reared colony to prevent contamination by parasitoids or entomopathogens.In an artificial diet feeding group, newly hatched neonates were randomly selected and individually fed with a piece of disposable sponge (0.25 × 0.25 × 0.25 cm) soaked with 0.02 ml of fresh diet solution in a Petri dish (9 cm in diameter). Before soaking, the sponge was sterilized with 70% ethanol and then rinsed with autoclaved distilled water. The individual was moved to a sterilized Petri dish containing a new soybean seedling daily when the artificial diet was replaced. The artificial diet used in this study was based on the combined diet 1 described by Hung et al.3 The nymph diet contained 40% fresh egg yolk (Lulon, Changhua, Taiwan), 10% yeast extract (Difco, Becton Dickinson, Rutherford, NJ, USA), 5% beef extract (Difco, Becton Dickinson), 5% sucrose (Sigma-Aldrich, St. Louis, MO, USA), 5% honey (Honey world farm, Taichung, Taiwan), and distilled water. The adult diet consisted of 30% fresh egg yolk, 5% yeast extract, 5% beef extract, 3% sucrose, 20% honey, 10% milk powder (Red Cow, E-mart Marketing Co., Taipei, Taiwan), and distilled water. To prepare the diets, the yeast extract and water were autoclaved before adding the other ingredients, and no preservatives or antibiotics were included. The artificial diet was then stored at − 20 °C until needed.DNA extraction and 16S rRNA gene amplicon sequencingBefore DNA extraction, twenty O. strigicollis females from each feeding group (the artificial diet and moth eggs) were subjected to surface-sterilized with 75% ethanol then rinsed with deionized water three times. Total genomic DNA extraction was performed using DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Extracted DNA samples were quantified using QIAquick PCR Purification Kit (Qiagen), and their integrity was verified by agarose electrophoresis. The V3–V4 region of the bacterial 16S rRNA gene was amplified using primers 314 F with specific barcode and 805R (Supplementary Table S2)47. Each sample was subjected to PCR in 30 µL of the reaction mixture, including 0.5 µL of KAPA HiFi DNA Polymerase, 0.75 µL KAPA dNTP Mix, 0.5 µL KAPA HiFi Fidelity Buffer, 0.75 µL of forward and reverse primers (10 µM), and 10 ng template DNA (the reagents and mix listed above were provided by Kapa Biosystems, Woburn, MA, USA). The PCR conditions were as follows: initial denaturation at 95 °C for 3 min followed by 5 cycles of 98 °C (20 s), 57.5 °C (20 s), and 72 °C (20 s), and a post-PCR incubation at 72 °C for 3 min. Samples containing bright main bands ~ 500 bp were retained for further experiments. The PCR products were pooled and then purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). The sequencing library was generated using the Celero DNA-Seq System (Nugen, San Carlos, CA, USA), with an index code added. Finally, the library was sequenced on the Illumina MiSeq System (Tri-I Biotech Inc., Taipei, Taiwan) and 2 × 301 bp paired-end read segments were generated.Computational analyses of 16S rRNA sequencing dataData obtained from independent sequencing were analyzed separately using QIAGEN CLC Genomics Workbench (v 10.1.1, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Samples were marked as follows: Moth egg group (ME): O. strigicollis fed with eggs of C. cautella; Artificial diet group (AD): O. strigicollis fed with the artificial diet. Paired-end sequences imported into CLC Genomics Workbench were quality-controlled and combined. If the similarity between sequences was more than 97%, they were assigned to the same operational taxonomic units (OTUs). For the taxonomic classification of OTUs, SILVA (v132), comprehensive database of 16S rRNA sequences for microbial classification, were utilized as 16S rRNA gene databases48. Alpha diversity (the number of observed OTUs, Shannon diversity, Faith’s phylogenetic diversity, and Chao1 diversity), were analyzed using R package “microeco”49. The alpha diversity (within samples) of OTUs was analyzed through the number of observed OTUs and Chao1, Shannon, and Faith’s phylogenetic diversity indices to obtain the species richness and uniformity information in the samples, as well as to identify common and unique OTUs among different samples. Linear discriminant analysis effect size (LEfSe) was used to screen the taxa for significant differences between two groups with LDA scores greater than two. A cladogram was drawn to show the distribution of these taxa at different taxonomic levels by R package “microeco”49.Isolation and identification of bacteriaBacteria were identified from the 5-day to 7-day old female O. strigicollis fed on moth eggs following the below isolation and culture procedure, all conducted were carried out in biosafety cabinet using an aseptic technique. Five female O. strigicollis were surface sterilized with 75% ethanol, and placed to a sterile tube, following by rinsing three times in distilled water prior to homogenization with a plastic pestle in 200 µL double distilled water. Ten µL of the homogenate was spotted on NB/LB plates, and the plates were incubated at 28 °C for 24 h (Supplementary Table S4). Single colonies were chosen and inoculated into the liquid culture mentioned in Supplementary Table S4 to establish pure cultures, and the culture tubes were incubated at 28 °C for 16 h. The pH of all liquid media and plates was adjusted to pH 6.5 before inoculation. To preserve the bacteria, liquid cultures were cryopreserved by adding a 20% (V/V) glycerol solution and storing them at − 80 °C.To identify isolated bacteria, a 1 mL overnight culture was transferred into a sterile 1.75 mL tube and DNA was extracted using QIAamp DNA Microbiome Kit (Qiagen) according to the manufacturer’s instructions. The 16 s rRNA was amplified with universal primers 27F and 1492R Supplementary Table S2)50. Each PCR sample had a total volume of 50 μL, contained 25 μL 2 × PCR supermix (TOOLS, Taiwan), 10 μM of primer pair, and 10 ng template DNA. The PCR amplification reaction was conducted according to described by Wu et al.51. The amplified 16S rDNA gene fragments were then cloned into a pGEM-T easy vector (Promega, USA) and transformed into competent DH5α cells according to the manufacturer’s protocol. Blue/white selection was carried out and the plasmids were isolated for bidirectional sequencing. The obtained sequences were subjected to BLAST analysis using the GenBank database established by the US National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) to allow taxonomic identification by similarity.The 16S rDNA phylogenetic analysis of the OS1 isolate was performed for 22 nucleotide sequences, which included the sequence of the OS1 obtained in this study, as well as 10 sequences belonging to the genus Pantoea, 5 sequences belonging to the genus Kluyvera, and 5 sequences belonging to the genus Erwinia. These sequences were obtained from BLAST results in the GenBank database to perform a comparison with our sequences. In addition, the sequence of Escherichia coli (X80725.1), which was used as an outgroup, was included. Multiple sequence alignment was performed using the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) program, and phylogenetic trees were constructed using MEGA11 software52. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Bootstrapping was performed for 1000 replicates.In vivo assaysAbundance of Pantoea in O. strigicollis after supplementationTo assess the abundance of Pantoea in O. strigicollis after continually supplementation of the OS1, newly emerged females fed from moth eggs were individually placed in Petri dishes individually and fed the artificial diet supplemented with the OS1 in sucrose solution (108 bacteria per mL, Sigma-Aldrich). Prior to supplementation, the OS1 was cultured in LB media at 28 °C for 16 h, pelleted (by centrifugation for 1 min at 8000 rpm), and resuspended in 5% sucrose solution. Females fed on the artificial diet supplemented only with sucrose solution, devoid of OS1, were collected to form the control group. On day 1, 3, and 5, ten insects from each group were sampled to assess bacterial abundance using quantitative PCR (qPCR). Before DNA extraction, each female was surface sterilized with 75% ethanol and washed in sterilized deionized water three times. Total genomic DNA extraction was performed using a DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s instructions. Two primer pairs, 27F/ 355R, and Pantoea_F/Pantoea_R, were used to determine the abundance of total bacteria and Pantoea spp. in O. strigicollis (Supplementary Table S2). The abundance of Pantoea spp. was determined using the same thermal cycle conditions as described in Huang et al.53. but the annealing step was modified (60 °C for 30 s) for the total bacterial abundance assay. Using standard curves from the amplification of the cloned target sequence in a pGEM-T easy vector (Promega), we calculated the absolute DNA copy number for the reaction template and then adjusted it based on the dilution to calculate the total DNA copy number for each sample. Each qPCR experiment was performed using three independent biological replicates with three technical replicates. Biological replicates address natural variation among organisms, while technical replicates address variability in the experimental process.Life table parameters evaluationNewly hatched neonates were randomly collected within 12 h of emergence. Three treatment groups were designed: (1) Nym group: neonates fed on the artificial diet supplemented with the OS1 sucrose solution at their nymphal stage, while adults were fed only the artificial diet; (2) Nym + Adu group: neonates fed on the artificial diet supplemented with the OS1 sucrose solution at all life stages; (3) control group: neonates fed on the artificial diet supplemented with 5% sucrose solution alone at all life stages. The survival was recorded daily until all individuals of the tested cohorts developed into adults. Once the adults emerged, one male and one female were transferred to a new Petri dish. Stems of soybean seedlings wrapped individually with wet cotton were provided for oviposition, and egg numbers were counted daily under a dissection microscope. The female fecundity was recorded until all individuals died.Gene expression measurement using reverse transcription-quantitative PCR (RT-qPCR)All adults were 7 days old at the time of sampling. Five females were preserved in the RNAprotect Tissue Reagent (Qiagen) at − 20 °C freezer before homogenization for total RNA extraction. Insects were ground using a motorized hand pestle and total RNA was isolated using RNeasy Kit (Qiagen) according to the manufacturer’s instructions. Residual DNA was removed using the RNeasy columns (Qiagen) with silica-membrane technology. The pure RNA samples were then quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at − 80 °C.The iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) was used to reverse transcribe 1 μg of total RNA into cDNA. Next, qPCR was performed to determine the expression of two antibacterial peptides, genes defensin (Def) and diptericin (Dip). Primers for the application of Def, Dip, and β-actin genes were designed using the gene sequences of Rhodnius prolixus (Stål) (Hemiptera: Reduviidae) and A. lucorum from the GenBank database (AY196130.1, EU448993.1, and KU188517.1) and using Primer3 software54 (Supplementary Table S2). β-actin was used as a reference gene for internal control. Each assay included a negative control without a cDNA template. Each qPCR assay was conducted in a 96-well plate, and every 20 μL of reaction solution contained 10 μL of 2 × iQ SYBR Green Supermix (Bio-Rad, Taipei, Taiwan), 2.5 μL of 1.6 μM of each gene-specific primer, and 5 μL of diluted cDNA. After the reaction agents were added, the 96-well plate was placed inside a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) and allowed to react. The reaction conditions as described in Hsu et al.55. For each cycle, the fluorescent reaction signals were detected and collected using Bio-Rad CFX Maestro software (Bio-Rad). The relative gene expression data were analyzed using the 2−ΔΔCt method56. Each RT-qPCR assay involved three independent biological replicates with three technical repetitions.Life table performance and statistical analysesThe raw data of each O. strigicollis were analyzed according to an age-stage, two-sex life table36,57,58 using the TWOSEX-MSChart computer program59. The age-specific survival rate (lx), age-stage fecundity (fx7, where x is age and 7 is the stage of female) and life table parameters (intrinsic rate of increase; finite rate of increase; net reproduction rate; mean generation time) were calculated according to Chi and Liu57 and Chi58. The adult preoviposition period (APOP) is defined as the time from female adult emergence to its initial oviposition. Oviposition days refers to the exact period during which oviposition took place. The intrinsic rate of increase (r) was estimated using the Euler–Lotka formula through the interactive bisection method with the age indexed from 060, which quantifies the rate at which a population grows when there are no limitations on resources. The finite rate of increase (λ) is calculated as:The net reproduction rate (R0) is defined as the average number of off-spring that an individual can produce throughout its entire lifespan. The mean generation time (T) means the time that a population needs to increase to R0-times its size at the stable age-stage distribution. The formulas of these parameters are defined as below:$$\sum\limits_{x = 0}^\infty {{e^{ – r(x + 1)}}} {l_x}{m_x} = 1$$
(2)
$${R_0} = \sum\limits_{x = 0}^\infty {{l_x}{m_x}}$$
(3)
$$T = \frac{{\ln {R_0}}}{r}$$
(4)
where x is age and j is stage. The standard errors of the nymphal survival rate, adult longevity, APOP, oviposition period, fecundity, and population parameters of O. strigicollis fed on the artificial diet supplemented with the OS1, and sucrose solution were calculated using the bootstrap resampling method61,62 (m = 100,000). The statistical test used for comparing these parameters involved evaluating bootstrap percentile confidence intervals, with significance determined at the 5% level61,62. The difference in alpha diversity and bacterial abundance among groups was compared using the Kruskal–Wallis test, with values of p < 0.05 considered statistically significant. For RT-qPCR, Student’s t-test were used to determine the level of significance of differences in all gene expression in relation to the bacteria addition treatment. Differences were considered significant at p < 0.05. These statistical analyses and graphs were performed using R (version 4.3.2).
Impact of diet and bacterial supplementation regimes on Orius strigicollis microbiota and life history performance
