Both species and abundance of gut bacteria positively influence Fortica okadai behavior as parasites and vectors

Fortica Okadai Rearing and microbiota manipulation

The species used in the study was caught in a pear orchard in Zunyi, a city in southwest China [15], and then cultured and reared in an artificial climate box (BIC-400, Shanghai, China) at 28 ± 1 °C and 70 ± 5% relative humidity in a 14 h:8 h light/dark cycle. Pears that were naturally overripe (28 ± 1 °C) after 3 days were used as culture media.

GF generation P.Okadai Newly emerged adult individuals were acquired by feeding autoclaved pears containing a mixture of three antibiotics, ampicillin (300 μg/mL), streptomycin (300 μg/mL), and tetracycline (90 μg/mL) for 72 h in a biosafety cabinet. [16], Single-bacterium-seeded strains were produced by cultivating GF P.Okadai Autoclaved pears were inoculated with single bacteria isolated from fly intestine using 100 μl bacterial suspension at a density of 1.5 × 10.8 cells/ml for 24 hours in a biosafety cabinet. Each whole gut of GF or single-bacteria-seeded P.Okadai were dissected and authenticated using Luria-Bertani (LB) medium.

DNA extraction and amplification

Eggs, larvae, pupae and adults of both sexes were collected and washed with 75% ethanol for 1 min, 1% sodium hypochlorite for 3 min and sterile water three times to remove surface contaminants. Thirty adults of each sex were dissected under a stereomicroscope (Leica S9i, Germany) to isolate the midgut, frozen in liquid nitrogen and stored at −80°C. [17],

Genomic DNA was extracted from various stages P.Okadai and naturally overripe pears after 3 days (28 ± 1 °C) using the TiGuide S96 bead-based fecal genomic DNA extraction kit (Tiangen, DP812, China). DNA concentration was measured using a multimode reader (Synergy HTX, BioTek Instruments, China). The V3–V4 hypervariable region of the 16S ribosomal RNA (rRNA) gene (primers 338F: 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′) was amplified by PCR. [18], PCR was performed in a 10 μl reaction mixture containing 10 ng genomic DNA, 0.2 μl KOD FX Neo (Toyobo, Japan), 5 μl KOD FX Neo buffer, 2 μl deoxyribonucleoside triphosphate (dNTP) (2 mM), 0.3 . μl of primers 338F (10 mM) and 806R (10 mM), and sufficient distilled water (dH).2o) To maintain a final volume of up to 10 μl under the following conditions: an initial denaturation step of 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 30 sec, 50 °C for 30 min. Annealing at Celsius. , and elongation at 72 °C for 40 sec, followed by a final elongation of 7 min at 72 °C. DNA integrity was checked by 1.8% (w/v) agarose gel electrophoresis.

Library creation and indexing

Target region PCR products (10 μl) were purified by adding VAHTS™ DNA Clean beads at a 1:1 ratio, and then barcoded indexing and Illumina adapters were added by Solexa PCR (20 μl reaction volume), to a 5 μl pool. Was used. PCR products, 2.5 μl of each index (forward and reverse), and 10 μl of 2× Q5 High-Fidelity Master Mix (NEB, USA) [19], PCR conditions were 98 °C for 30 sec, followed by 10 cycles of 98 °C for 10 sec, 65 °C for 30 sec, and 72 °C for 30 sec and a final extension of 5 min at 72 °C. It was Charan. , Agarose gel electrophoresis was performed on 1.8% (w/v) agarose gel, and the results were quantified by the ImageJ program. Each sample was mixed by aspirating 150 ng, and the mixed samples were purified before gel cutting using the Cycle Pure kit and recovered by the Monarch DNA kit. The final gene library was assessed for concentration and quality on a Qsep400 (BiOptic, Taipei, Taiwan) and then sequenced on an Illumina NovaSeq 6000. Construction and sequencing of the 16S rRNA gene library was accomplished by Biomarker Technologies Corporation (Beijing, China).

Processing of sequence data and bioinformatics analysis

Using Trimmomatic v0.33 software, the raw reads obtained from sequencing were filtered. Paired-end FastQ files removed using VSEARCH v2.8.1 [20], Then, we used UCHIME v4.2 software to identify and remove chimeric sequences to obtain final effective reads. Sequences were removed from inclusion according to the following criteria: average mass of bases was less than 20, length of sequences was less than 350 base pairs (bp), sequences had primer mismatches, reads were of low quality, and sequences Could not connect. Clustering of reads was performed at 97.0% similarity level to obtain operational taxonomic units (OTUs), and OTUs were taxonomically annotated based on the SILVA taxonomic database.

Venn diagrams were used to show the number of common, unique features between samples from groups with bacterial abundance percentage greater than 0.1% ( The community composition of each sample was determined at each level (phylum, genus) and mapped using R tools ( Alpha diversity was calculated using species-level annotation information statistics, and the Wilcoxon test was used to analyze variability between groups. P <0.05 indicates a significant difference. The number of sequences versus the number of species was used to construct a rarefaction curve. UPGMA (Unweighted Pair-Group Method with Arithmetic Means) analysis was based on the Bray-Curtis distance between samples. The results of principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) analysis were plotted separately using the online website, and one-to-one test for variance (PERMANOVA). Permutational multivariate analysis was used. Was beta diversity significantly different between samples from different groups P <0.05 indicates a significant difference. Analysis of variance (ANOVA), a nonparametric statistical method (P<0.05), was used to test the average values ​​of multiple samples at the genus level to determine the significance of differences in the average values ​​of samples at different developmental stages.

Isolation, identification and culture of bacteria P.OkadaiCourage

Under a postural dissector (S9i, Leica, Wetzlar, Germany), three males and three females P.OkadaiThey were fed normally, and the intestine was dissected using the above method. Phosphate-buffered saline (PBS) solution (0.5 ml) was added to the centrifuge tube, and the mixture was centrifuged. Then, 10, 102103104and 105 Dilutions were obtained, and 50 μl of the diluted solution was coated on a blood plate (90 mm) and cultured for 24 h, 48 h, and 72 h (37 °C, 5% CO).2 Atmosphere). According to the growth characteristics of the colonies on the plates, different forms of bacteria were isolated, cultured and purified twice through zoning line.

Bacterial genome extraction was performed using the Bacterial Genomic DNA Kit (ZP301, Zoman Biotechnology, Beijing, China). PCR was performed using common 16S primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCCTTGTTACGACTT-3′). [21], followed by sequencing (Tsingke, Chongqing, China). The PCR consisted of 12.5 μl of K5 HiFi 2× PCR Master Mix (ZT211), 1 μl of template DNA, and 1 μl of primer in a total volume of 25 μl. Cycling conditions were 95 °C for 5 min; 30 cycles of 94 °C for 30 sec, 56 °C for 30 sec, and 72 °C for 60 sec; and final extension for 5 min at 72 °C. The PCR products were sequenced by an ABI 3730xl DNA analyzer (Fuzhou, China) with both strands using the 27F-1492R primer. Sequences of different bacterial isolates were analyzed using EZBioCloud ( [22] To select the most identical 16S ribosomal DNA (rDNA) sequences.

applied experimental design

four species of gut bacteria, Lactiplantibacillus argentoratensis, Lisinibacillus fusiformis, Leuconostoc CitrumAnd Levilactobacillus brevis Cultivation was carried out on de Man-Rogosa-Sharp (MRS) medium (Solarbio, Beijing, China) in 12 ml culture tubes (natural, dual cap, sterile) under the following conditions: 20 μl bacterial suspension at a density of 1.5 × 10.8 Colony forming units (CFU)/ml were added to 2.5 ml medium and incubated at 37 °C and 180 r/min for 24 h, 48 h and 72 h. The same method was used to culture other bacteria, Acetobacter tropicalis But with a custom medium consisting of 10 g of glucose, 10 g of yeast powder, and 1000 ml of deionized water, which was autoclaved for 20 min, and then 40 ml of anhydrous ethanol was added.

To evaluate the gut bacterial species attracted to the host, media containing individual bacterial species cultured for different periods of time were subjected to behavioral experiments with a wild-type four-armed attraction apparatus. P.Okadai , The most attractive medium was then selected for further validation with GF. P.Okadai To distinguish between wild type and GF strains. Furthermore, to explore whether gut bacterial abundance also plays a role in influencing host behavior, single-bacteria-seed P.OkadaiBehavioral experiments were conducted using the same method using the most attractive medium.

Additionally, to test the attractiveness of the bacterial fermentation liquid and autoclaved supernatant, the most attractive bacterial cultures were subsequently transferred to 2 ml tubes and labeled as bacterial fermentation liquid, and then, the remaining bacterial cultures was centrifuged at 10,000 rpm for 20 min. , Subsequently, 2 ml of the resulting supernatant was autoclaved at 121 °C for 20 min and denoted as autoclaved supernatant. MRS medium and live bacteria were used as controls. Finally, to evaluate the potential application of the most attractive bacterial culture, 3-day-old naturally overripe pears were used as reference.

All behavioral experiments were conducted using a four-armed olfactometer or Y-tube bioassay. Fortica OkadaiSubjects were fasted (only water was provided) for 12 hours before the experiment began and results were recorded after an additional 12 hours. 30 were used in each experiment P.OkadaiSamples with 10 replicates to ensure statistical robustness.

Statistical analysis

Data were tested for normal distribution and subsequently analyzed for multiple (one-way ANOVA, least significant difference) [LSD] Post hoc test) or two-group (students). Tea-Examination, P<0.05) treatment using GraphPad Prism 9. These differences were considered significant P<0.05 level.

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