Characterisation of insecticide degrading bacterial endosymbionts of cowpea pod bug Riptortus pedestris (Hemiptera :Alydidae)

Abstract

Cowpea Vigna unguiculata (L), commonly known as vegetable ‘meat', is widely grown in India for its high nutritional value, drought tolerance, and ability to improve soil fertility. Riptortus pedestris (Hemiptera: Alydidae), is a major pest of cowpea, causing significant damage during the post-flowering phase to immature pods and reducing pod quality and yield up to 80 per cent (Prasad et al., 2021). The cowpea pod bug has increasingly shown resistance to commonly used insecticides, largely due to endosymbiotic bacteria it acquires from soil during the second instar. Identifying and characterising endosymbiotic bacteria and assessing their insecticide-degrading potential can aid in developing environmentally sound strategies. Hence, this study, “Characterisation of insecticide-degrading bacterial endosymbionts of cowpea pod bug Riptortus pedestris (Hemiptera: Alydidae)”, conducted at the Department of Agricultural Entomology, College of Agriculture, Vellanikkara (2023–2025), aimed to characterise the culturable endosymbionts of R. pedestris and evaluate their insecticide-degrading potential for targeted pest management. To identify and characterise the endosymbiotic bacteria of R.pedestris, adult pod bugs were collected and dissected under sterile conditions to separate the midgut. Serially diluted gut contents were plated on nutrient agar and Burkholderia cepacia selective agar. Four culturable endosymbiotic bacteria were identified from the M4 region of the pod bug, viz., Burkholderia cepacia, Pantoea dispersa, Macrococcus equipersicus, and Serratia sp. Morphological characterisation showed that B. cepacia produced light pink, smooth, raised, circular colonies with entire margins and Gram-negative rod-shaped cells. P. dispersa formed yellow, smooth, circular colonies with entire margins and slightly raised elevation, appearing as Gram-negative rods. M. equipersicus developed orange, smooth, circular colonies with an umbonate elevation and entire margins, exhibiting Gram -positive cocci. Serratia sp. produced white, smooth, raised, round colonies with entire margins and Gram - negative rod-shaped cells. Sequences were deposited in NCBI with accession numbers for B. cepacia (PX128431), P. dispersa (PX128399), and M. equipersicus (PX128423).

Metagenomic profiling showed that the cowpea pod bug gut microbiome was dominated by few taxa, reflected in a low Shannon index (0.63) and Chao1 richness (37). Burkholderiaceae, particularly the Burkholderia–Caballeronia–Paraburkholderia

cluster, comprised 94.8 per cent of all reads, while other groups such as Bacillaceae, Streptococcaceae, Caulobacteraceae and the genera Bacillus, Pantoea, and Azospirillum occurred only in trace amounts. The insecticide-degrading potential of the culturable endosymbiotic bacteria was assessed using the spectrophotometric method and validated through enumerating bacterial growth and Gas chromatography-flame photometric detector (GC-FPD) analysis. Each bacterial cultures was amended with each insecticide, and B. cepacia showed the highest degradation ability, especially for quinalphos 25% EC followed by malathion 50% EC. OD₆₀₀ for Burkholderia cepacia recorded 1.84 for quinalphos 25% EC and 1.76 for malathion 50%EC, far higher than M. equipersicus (0.38), Serratia sp. (0.21), and P. dispersa (0.38). The corresponding bacterial count reached 1.95 × 10⁷ CFU/mL in quinalphos 25%EC and 1.81 × 10⁷ CFU/mL in malathion 50% EC, compared to the control (1.13 × 10⁷ CFU/mL). GC-FPD analysis further confirmed the degradation potential of B. cepatia, wherein the quantity of quinalphos 25% EC dropped from 10 ppm (0 day) to 3.79 ppm (3rd day) and 0.97 ppm (7th day), whereas the corresponding reduction in control was 9.04 ppm and 5.27 ppm, respectively, at the third and seventh day. Malathion 50% EC declined from an initial quantity of 2 ppm to 0.49 ppm (3 rd day) and became undetectable by day seven, whereas in the control, the reduction was 1.45 ppm (3rd day) and 0.008 ppm (7th day). Agar well diffusion assay was conducted to identify the most effective antimicrobial agents against B. cepacia. Among the treatments, chloramphenicol (25 ppm) showed a clear inhibitory effect against Burkholderia, with a distinct inhibition zone of 7.625 mm, followed by lemongrass essential oil (1.375 mm). Sequential application of chloramphenicol (25 ppm) followed by malathion 50% EC (2mL/L) resulted in 97.5 per cent mortality, while their combined application resulted in 92.5 per cent mortality on the third day. Additionally, egg laying was inhibited in both treatments. However, malathion alone caused 65 per cent mortality only. Beauveria bassiana (1×10⁸ cells/mL), neem oil nanoemulsion (500 ppm), and lemongrass essential oil (1000 ppm) exhibited marginal lethality. Burkholderia symbiosis markedly enhanced the biological fitness of R. pedestris. To study the fitness offered by Burkholderia, two groups, i.e. symbiont-colonised (Sym) and non-colonised (Apo) pod bugs, were reared. The morphometric analysis revealed that symbiotic adults were larger, with a mean body length of 15.20 ± 0.061 mm (males) and 16.6 ± 0.042 mm (females) while for apo symbiotic, it is 12.3 ± 0.064 mm (males) and 13.4 ± 0.054 mm (females). Adult longevity was higher in sym. insects (33.05 ± 0.76 days) as against 22.2 ± 0.57 days in apo symbiotic. The fecundity was higher to the tu ne of 65.28 ± 1.93 eggs in symbiotic, whereas it was 34.57 ± 1.45 in apo symbiotic females. In conclusion, this study established the symbiosis of B. cepacia with R. pedestris, which significantly enhanced the biological fitness and insecticide degradation in the host. Symbiont-targeted pest management offers an eco-friendly approach against R. pedestris. Most effective antimicrobials against B. cepacia need to be identified for the sustainable management of R.pedestris in cowpea.