**Background**
Lactic acid bacteria (LAB) are well-known beneficial microorganisms for humans and animals, but their characteristics and functions in insects remain poorly understood. The bean bug, Riptortus pedestris (Hemiptera: Alydidae), is a significant pest of leguminous crops in Korea, Japan, and China, causing severe agricultural losses. While previous research has focused on the specific symbiotic relationship between R. pedestris and Burkholderia (Caballeronia) bacteria, which improve host growth and confer pesticide resistance, the presence and role of LAB in this insect had not been investigated. This study aimed to isolate, identify, and characterize LAB from the gut of R. pedestris and evaluate their probiotic potential and effects on insect host fitness.
**Methods**
Adult R. pedestris were collected from soybean fields in Jinju, South Korea in 2018. Guts from 20 surface-sterilized adults were homogenized, serially diluted, and plated on de Man, Rogosa, and Sharpe (MRS) agar incubated at 28°C for 3 days in a CO₂ incubator. Three bacterial isolates (B103–B105) were selected based on colony morphology (Gram-positive, oxidase-negative, round, white colonies). Identification was performed using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonics microflex LT) and 16S rRNA gene sequencing (primers 27mF/1492mR). Survival assays were conducted at alkaline pH (8 and 9), in simulated human gastric juice (pH 2.2 and 2.5 with 1% pepsin), and in bile salt solutions (0.1% and 0.5% oxgall). Glucose utilization, pH changes, and organic acid production (lactic acid, acetic acid, propionate) were measured using a D-glucose assay kit, pH meter, and HPLC, respectively. For in vivo colonization, spontaneous rifampicin-resistant LAB strains were fed to second-instar nymphs at ~10⁸ CFU/mL; gut colonization was assessed at 21 days post-inoculation by colony counting on rifampicin-containing MRS agar. Survival rates were monitored daily for 20 days; adult body weight (dry, after acetone immersion and 70°C drying) and body length (head to abdomen tip) were measured at 21 days. Natural LAB infection frequency was surveyed in wild adults collected from 18 regions of Gyeongsangnam-do, South Korea during 2021–2022.
**Key Results**
MALDI-TOF analysis identified B103 and B104 as Lactococcus lactis (scores 2.236 and 2.256) and B105 as Enterococcus faecalis (score 2.331). 16S rRNA sequencing confirmed 100% similarity to reference strains. All three strains survived at pH 8 for 24 h; L. lactis B103 and E. faecalis B105 also survived at pH 9, though L. lactis B104 decreased sharply to 2.63 log₁₀ CFU/mL after 24 h at pH 9. In simulated gastric juice at pH 2.2, E. faecalis B105 showed the highest survival; L. lactis B104 decreased by 4.26 log₁₀ CFU/mL at 120 min, and B103 decreased by 4.78 log₁₀ CFU/mL. All strains maintained high survival at pH 2.5 and in 0.1% and 0.5% bile salt over 24 h. E. faecalis B105 consumed 65% of glucose over 72 h and reduced pH to 4.4, while L. lactis B103 and B104 consumed 43% and 50% of glucose, reaching pH 5.0 and 4.8, respectively. Organic acid analysis showed E. faecalis B105 produced the highest levels of acetate, lactate, and propionate; L. lactis B104 produced intermediate levels; and B103 produced low lactate and propionate with no detectable acetate. Gut colonization densities at 21 days post-inoculation were 1.21 × 10⁷ CFU/gut for L. lactis B103, 5.27 × 10⁵ CFU/gut for L. lactis B104, and 3.24 × 10⁶ CFU/gut for E. faecalis B105. Survival rates were 78.9% for B103, 60.5% for B104, 55% for B105, and 47.3% for the DWA control. No significant differences in body weight or length were observed between LAB-treated and control insects. Natural LAB infection frequency in wild populations was 89% (16/18), with L. lactis and E. faecalis detected across 16 regions.
**Clinical Implications**
This study demonstrates that insect-derived LAB from R. pedestris possess key probiotic traits, including survival under gastrointestinal conditions (acidic pH, bile salts) and the ability to colonize the insect gut at high densities. The significant improvement in host survival (up to 78.9% with L. lactis B103 vs. 47.3% control) suggests these LAB could be developed as probiotics for beneficial insect rearing in agriculture. The lack of host-specificity and dependence on habitat environment for LAB–insect symbiosis indicates these strains may be broadly applicable. However, the absence of growth-promoting effects and the potential pathogenicity of E. faecalis warrant caution. These findings provide fundamental insights into insect–LAB symbiosis and propose a novel concept for pest management through probiotic-based approaches.