Diversity of entomopathogenic nematodes and symbiotic bacteria in Thailand and their application for the control of Aedes larvae

Abstract

Aedes mosquitoes, especially Aedes aegypti and Aedes albopictus are important vectors for several arboviruses such as the dengue virus. The chemical control of Aedes spp., which is usually implemented, affects both humans and the environment. Moreover, Ae. aegypti resistance to chemical insecticides has been reported worldwide. To tackle this, entomopathogenic nematodes (EPNs) and their symbiotic bacteria (Photorhabdus and Xenorhabdus) may be an alternative bio-control agent that can overcome such issues. Thus, this study aims to isolate, identify, and analyze the phylogeny of EPNs and their symbiotic bacteria in Thailand and evaluate their efficacy in controlling the Aedes larvae. From 12 provinces in Thailand, soil samples were randomly collected, with 118 out of 1,100 them being positive for EPNs (10.73% prevalence) in genera Steinernema (4.46%) and Heterorhabditis (6.27%). Then, molecular discrimination of these two genera was performed based on the sequencing and phylogenetic analysis of the 28S rDNA and internal transcribed spacer regions (ITS). The most abundant species of EPN were Heterorhabditis indica, with minor species of Heterorhabditis sp. SGmg3, H. baujardi, S. surkhetense, S. kushidai, S. siamkayai, Steinernema sp. YNd80, Steinernema sp. YNc215, S. guangdongense, and S. huense. Symbiotic bacteria were isolated from the EPNs and identified based on the colony morphology as Photorhabdus (69 isolates) and Xenorhabdus (49 isolates). The molecular identification of symbiotic bacteria with recA sequencing indicated that most were P. luminescens subsp. akhurstii and X. stockiae with minor prevalence of P. luminescens subsp. hainanensis, P. asymbiotica subsp. australis, X. indica, X. griffiniae, X. japonica, X. thuongxuanensis, and X. eapokensis. The larvicidal activity of five selected EPN isolates were tested against Ae. aegypti. Ten larvae of Ae. aegypti were incubated with different concentrations (80, 160, 320, and 640 IJs/larva) of the infective juveniles of EPN in 24-well and 6-well plates for 4 days. The mortality rates of the larvae were observed daily. Steinernema surkhetense (ePYO8.5_TH) showed the potential to kill Ae. aegypti larvae, with the highest mortality rate of 92 ± 9.37% and 89 ± 9.91% after it was treated with 640 IJs/larva in a 24-well plate and 1600 IJs/larva in a 6-well plate, respectively. The larvicidal bioassays of symbiotic bacteria were also tested with Ae. aegypti and Ae. albopictus larvae. The results suggested that a whole-cell suspension of X. griffiniae (bMSN3.3_TH) had the highest efficiency in eradicating Ae. aegypti and A. albopictus, with 90 ± 3.71% and 81 ± 2.13% mortality, respectively, after 96 h exposure. In contrast, 1% of ethyl acetate extracted from X. indica (bSNK8.5_TH) showed reduced mortality for Ae. aegypti of only 50 ± 3.66% after 96 h exposure. In conclusion, this study revealed an abundant distribution of EPNs and their symbiotic bacteria across Thailand, and S. surkhetense (ePYO8.5_TH), X. griffiniae (bMSN3.3_TH) and X. indica (bSNK8.5_TH) may be used as a biocontrol agent against Aedes larvae.