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Larvicidal activity of Xenorhabdus and Photorhabdus bacteria against Aedes aegypti and Aedes albopictus

Objective: To evaluate the efficacy of symbiotic bacteria, Xenorhabdus indica, Xenorhabdus stockiae, Photorhabdus luminescens subsp. akhurstii and Photorhabdus luminescens subsp. hainanensis as a larvicide against Aedes aegypti and Aedes albopictus. Methods: Larvae (L3-L4) of Aedes aegypti and Aedes albopictus were given 2 mL of a suspension 107-108 CFU/mL of each symbiotic bacterium. Distilled water and Escherichia coli ATCC® 25922 were used as the control. The mortality rate of the larval mosquitoes was observed at 24, 48, 72 and 96 h. The experiment was performed in triplicates. Results: The larvae of both Aedes species started to die at 24 h exposure. Aedes aegypti showed the highest mortality rate (87%-99%), 96 h after exposure to Xenorhabdus stockiae (bNBP22.2_TH). The mortality rate of Aedes albopictus was between 82% and 96% at 96 h after exposure to Xenorhabdus indica (bKK26.2_TH). Low effectiveness of distilled water and Escherichia coli ATCC® 25922 were observed in both Aedes larvae, with a mortality rate of 2% to 12%. Conclusions: The study confirms the oral toxicity of Xenorhabdus and Photorhabdus bacteria against Aedes spp. Xenorhabdus stockiae and Xenorhabdus indica may be an alternative agent for control Aedes spp. This is basic information for further study on the mechanism of action on Aedes larvae or application to control mosquito larvae in the community.

mechanical, chemical, genetic and biological controls [5]. Elimination of breeding sites of Aedes is a simple method and low cost to reduce the number of mosquitoes. Chemical controls (organochlorides, DDT; organophosphates, OP; pyrethroids) are the first method using in mosquito control. However, repeated use of these insecticides leads to development of insecticidal resistant mosquitoes and toxic to human. Aedes have been reported to be resistant to DDT in worldwide. In addition, mosquitoes in several countries in Asia have been developed to resist pyrethroid [6]. Genetic control of Aedes (the sterile insect technique; rearing of insects carrying a dominant lethal allele) is a species specific method and most are in the laboratory conditions [7,8]. The genetic control methods need more consideration in cost, natural condition and environmental risk assessment [5].
Control of larval mosquitoes is of low cost and can scope the certain source. Therefore, biological control of larval stage of Aedes is considered to be a potential measure to reduce number of mosquitoes leading to prevention and control of viral infection.
However, Aedes spp. can develop moderate resistant to Bacillus thuringiensis subsp. israelensis (B. thuringiensis subsp. israelensis) [23]. Other bacteria commonly used for control of insects are Xenorhabdus and Photorhabdus which are symbiotically associated with entomopathogenic nematodes. These bacteria have also been reported to have oral lethality to Ae. aegypti larvae [17,24].
Cell suspensions of Xenorhabdus and Photorhabdus and their toxins were lethal to Aedes larvae, and a previous study showed that Photorhabdus insect-related protein from Photorhabdus asymbiotica had strong toxicity to Ae. aegypti and Ae. albopictus [33]. More recently, suspensions of Photorhabdus luminescens (P. luminescens) and Xenorhabdus nematophila (X. nematophila) were shown to kill between 42% and 83% of Ae. aegypti larvae in laboratory conditions [24]. In addition, P. luminescens and X. nematophila suspension mixed with Cry4Ba protein from B. thuringiensis subsp.
israelensis produced a mortality rate up to 87% and 95% of Ae.
aegypti [17]. These results suggest that Xenorhabdus and Photorhabdus spp. may be effective alternative agents for the biological control of mosquitoes. Some 30 species of these bacteria have been reported worldwide [34][35][36][37], but few species of these symbiotic bacteria have been tested to determine their efficacy in killing mosquito larvae.
Xenorhabdus stockiae (X. stockiae) and Photorhabdus luminescens subsp. akhurstii (P. luminescens subsp. akhurstii), the majority species found in Thailand, and Xenorhabdus indica (X. indica), and Photorhabdus luminescens subsp. hainanensis (P. luminescens subsp. hainanensis), also found in Thailand [38] suggested that these may be biological agents for controlling mosquito larvae, but the insecticidal or larvicidal activity of these symbiotic bacteria have never been tested against Aedes larvae. During the survey of entomopathogenic nematodes and symbiotic bacteria in northeast of Thailand, we identified several isolates of these symbiotic bacteria including X.
akhurstii and P. luminescens subsp. hainanensis isolated from entomopathogenic nematodes in Thailand against Ae. aegypti and Ae. albopictus larvae.

Preparation of bacterial cell suspension
Xenorhabdus and Photorhabdus in LB broth with 20% glycerol were kept at -80 曟 in our laboratory. Each bacterial isolate was grown on NBTA agar for 4 d and incubated at room temperature. To prepare a starter, a single colony was sub-cultured into 5 mL of 5YS medium containing 5% yeast extract (w/v), 0.5% NaCl (w/v), 0.05% K 2 HPO 4 (w/v), 0.05% NH 2 H 2 PO 4 (w/v), and 0.02% MgSO 4 ·7H 2 O (w/v). The tube was then incubated in the dark for 24 h with shaking at 160 rpm. One mL of the starter was transferred into a 50 mL tube containing 39 mL of 5YS medium. The tubes were then incubated in the dark for 24 h with shaking at 160 rpm.
Escherichia coli (E. coli) ATCC 襅 25922 that is used as the negative control was cultured on tryptone soy agar. The culturing process for the E. coli ATCC 襅 25922 was performed similarly to the preparation of the Xenorhabdus and Photorhabdus bacteria.
To prepare bacterial cell suspension, the overnight cultures of Xenorhabdus, Photorhabdus and E. coli ATCC 襅 25922 were then centrifuged at 10 000 rpm at room temperature for 20 min. The supernatants were discharged. The bacterial pellets were resuspended with sterile distilled water. The turbidity of bacterial suspension was adjusted to 1.0 with sterile distilled water at OD 600 nm by spectrophotometer. These bacterial suspensions were ready for using in bioassays.

Mosquito strains
Ae. aegypti and Ae. albopictus eggs were purchased from the Larvae at the late third and early fourth instar were then selected out and feed with minced pet food.

Data analysis
Mortality of Aedes larvae after exposure to the bacteria suspension with the comparison with the control groups was analyzed by Kruskal-Wallis test using SPSS version 17.0. P-value < 0.05 was considered as significant differences. The mortality of the Aedes larvae from both the fed and unfed groups was statistically analyzed by Mann-Whitney test.

Result
Both Ae. aegypti and Ae. albopictus (late 3rd to early 4th instars larvae) were susceptible to all isolates of Xenorhabdus and Photorhabdus bacteria. The mortality of the larvae began to die at 24 h after exposure to the bacterial suspension. In the fed group, a cell suspension of X. stockiae (bNBP22.2_TH) demonstrated the highest toxicity to Ae. aegypti larvae (99% mortality) at 72 h after exposure.
In the unfed group, X. stockiae (bNBP22.2_TH) showed the highest pathogenic effect on Ae. aegypti larvae, with 87% mortality at 96 h after exposure. Significant mortality among all bacterial isolates and negative controls (distilled water and E. coli ATCC 襅 25922) was observed at each time in the unfed group, although at a low rate of mortality (Table 1). However, the mortality rate of both the fed and unfed groups by Ae. aegypti was not significantly different among the four bacterial isolates.

Discussion
In the present study, we demonstrate the alternative bacterial agent for control Aedes spp., a main vector for important virus infection in man. Both Aedes spp. are susceptible to X. stockiae (bNBP22.2_ TH) X. indica (bKK26.2_TH) P. luminescens subsp. akhurstii (bMSK25.5_TH) and P. luminescens subsp. hainanensis (bKK17.1_ TH). It seems that the symbiotic bacteria of genus Xenorhabdus and Photorhabdus cause superior mortality of Aedes. X. stockiae, a symbiotic bacterium that is found to be associated with Steinernema websteri, have been used for acaricidal and antibacterial activity [39,40].

X. indica produces several bioactive compounds including taxlllaids
A-G which has weakly effect on Plasmodium falciparum [41]. In addition, metalloprotease purified from X. indica showed insecticidal activity against Helicoverpa armigera [42]. P. luminescens subsp.
Ae. aegypti and Ae. albopictus, both serious transmitting vectors of West Nile, chikungunya, dengue and zika viruses to humans, are globally distributed [1,4]. Although several control methods against these vectors have been attempted to stop the transmission of viral infections, the numbers of human case has not declined, especially dengue infection [44]. Biological controls of the vectors are an alternative measure to reduce human-mosquito contact. Our study demonstrated larvicidal activity of X. stockiae (bNBP22.2_TH), X.
To support this scenario, Photorhabdus insect-related protein from Photorhabdus asymbiotica showed strong toxicity to Ae. aegypti and Ae. albopictus [33]. In addition, a suspension of Photorhabdus luminescens subsp. laumondii TT01 DSM15139 and X. nematophila ATCC 襅 19061 showed orally lethality to Ae. aegypti larvae in laboratory conditions [24]. P. luminescens and X. nematophila suspension mixed with Cry4Ba protein from B. thuringiensis subsp.
In our study, we confirmed the oral toxicity of Xenorhabdus and Photorhabdus against Ae. aegypti and Ae. albopictus. However, it remains unknown as to the mechanism of killing effect of these bacteria on Aedes spp.
Xenorhabdus and Photorhabdus have orally toxicity to Aedes spp., but mortality rates vary. It is possible that the different pathogenicity from each bacterial species or isolates produces different amounts and kinds of bioactive compounds. Phurealipid derivatives, the inhibitor of juvenile hormone epoxide hydrolase in insects, were produced by different isolates of P. luminescens subsp. akhurstii [45,46].
In addition, the virulence of Xenorhabdus and Photorhabdus varied among insect species is related to foraging behavior [47]. This suggests that the virulent factors of Xenorhabdus and Photorhabdus require further study for more deeply understanding.
We demonstrate the potential of entomopathogenic bacteria, X.
stockiae, X. indica, P. luminescens subsp. akhurstii and P. luminescens   larval and adult mosquitoes, and their mechanisms of killing mosquitoes, are suggested.

Conflict of interest statement
We declare that we have no conflict of interest.