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

Apichat Vitta, Punnawat Thimpoo, Wipanee Meesil, Thatcha Yimthin, Chamaiporn Fukruksa, Raxsina Polseela,2, Bandid Mangkit, Sarunporn Tandhavanant, Aunchalee Thanwisai,2

1Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand

2Centre of Excellence in Medical Biotechnology, Naresuan University, Phitsanulok, Thailand

3Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

4Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok, Thailand

1. Introduction

Aedesmosquitoes are the main vectors of West Nile, chikungunya,and dengue viruses[1,2]. Recently the zika virus, with devastating effects, particularly for pregnant women, was proven to be transmitted to humans byAedes[3].Aedes aegypti(Ae. aegypti) andAedes albopictus(Ae. albopictus) are the main vectors of the dengue virus, causing dengue fever which has affected over 390 million people living in more than 100 countries[1,4]. At present, there are no specific treatments or vaccines for these viruses, and the best approach to prevent infection is avoidance of mosquito bites[3].Therefore, control adult and larvalAedesis an important measure to prevent the viral infection to human. Control methods for adult and larvalAedesspp. have been categorized as environmental,mechanical, chemical, genetic and biological controls[5]. Elimination of breeding sites ofAedesis 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.Aedeshave 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 ofAedes(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 ofAedesis considered to be a potential measure to reduce number of mosquitoes leading to prevention and control of viral infection.

Biological control forAedesspp. using protozoa[9], copepods[10-12], plant extracts[13-15], fungi[16], bacteria and their toxins[17-20] are promoted as being ecologically friendly, which is important for human life.Bacillus thuringiensis(B. thuringiensis),entomopathogenic bacteria have potential for biological control ofAedesspp.[20,21]. This bacterium shows rapid killing of the mosquito larvae and has no cross-resistant with chemical insecticides[22].However,Aedesspp. can develop moderate resistant toBacillus thuringiensissubsp.israelensis(B. thuringiensissubsp.israelensis)[23]. Other bacteria commonly used for control of insects areXenorhabdusandPhotorhabduswhich are symbiotically associated with entomopathogenic nematodes. These bacteria have also been reported to have oral lethality toAe. aegyptilarvae[17,24].

XenorhabdusandPhotorhabdusare symbiotically associated with entomopathogenic nematodes which are Gram negative bacteria with the rod shape and peritichous flagella of the family Enterobacteriaceae. These bacteria produce several bioactive compounds with cytotoxic, antifungal, antibacterial, antiparasitic and insecticidal activities[25-31]. Isopropylstilbene and ethylstilbene produced byPhotorhabdus, and xenorhabdin and xenematide produced byXenorhabdus, have also shown insecticidal activity[32].Cell suspensions ofXenorhabdusandPhotorhabdusand their toxins were lethal toAedeslarvae, and a previous study showed thatPhotorhabdusinsect-related protein fromPhotorhabdus asymbioticahad strong toxicity toAe. aegyptiandAe. albopictus[33]. More recently, suspensions ofPhotorhabdus luminescens(P. luminescens)andXenorhabdus nematophila(X. nematophila) were shown to kill between 42% and 83% ofAe. aegyptilarvae in laboratory conditions[24]. In addition,P. luminescensandX. nematophilasuspension mixed with Cry4Ba protein fromB. thuringiensissubsp.israelensisproduced a mortality rate up to 87% and 95% ofAe.aegypti[17]. These results suggest thatXenorhabdusandPhotorhabdusspp. may be effective alternative agents for the biological control of mosquitoes. Some 30 species of these bacteria have been reported worldwide[34-37], but few species of these symbiotic bacteria have been tested to determine their efficacy in killing mosquito larvae.Xenorhabdus stockiae(X. stockiae) andPhotorhabdus luminescenssubsp.akhurstii(P. luminescenssubsp.akhurstii), the majority species found in Thailand, andXenorhabdus indica(X. indica), andPhotorhabdus luminescenssubsp.hainanensis(P. luminescenssubsp.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 againstAedeslarvae. During the survey of entomopathogenic nematodes and symbiotic bacteria in northeast of Thailand, we identified several isolates of these symbiotic bacteria includingX.stockiae,X. indica,P. luminescenssubsp.akhurstiiandP. luminescenssubsp.hainanensis. Therefore, the objective of this study was to evaluate the effect ofX. stockiae,X. indica,P. luminescenssubsp.akhurstiiandP. luminescenssubsp.hainanensisisolated from entomopathogenic nematodes in Thailand againstAe. aegyptiandAe.albopictuslarvae.

2. Materials and methods

2.1. Bacterial isolates

XenorhabdusandPhotorhabduswere isolated from entomopathogenic nematodes collected from soil samples from northeast of Thailand. These bacteria were previously identified by the sequencing of a partial region of therecAgene. To identifyXenorhabdusandPhotorhabdusinto species level, BLASTN analysis of the 588 bprecAgene was performed with cut-off at 97% identity. Two species ofXenorhabduswere identified asX.stockiaeisolate bNBP22.2_TH (Accession No. KY809323) andX. indicaisolate bKK26.2_TH (Accession No. KY809302). Two subspecies ofPhotorhabduswere identified asP. luminescenssubsp.akhurstiiisolate bMSK25.5_TH (Accession No. KY809375) andP.luminescenssubsp.hainanensisisolate bKK17.1_TH (Accession No.KY809363). These four entomopathogenic bacteria were used in bioassays.

2.2. Preparation of bacterial cell suspension

XenorhabdusandPhotorhabdusin LB broth with 20% glycerol were kept at -80 ℃ 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%K2HPO4(w/v), 0.05% NH2H2PO4(w/v), and 0.02% MgSO4·7H2O(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®25922 that is used as the negative control was cultured on tryptone soy agar. The culturing process for theE. coliATCC®25922 was performed similarly to the preparation of theXenorhabdusandPhotorhabdusbacteria.

To prepare bacterial cell suspension, the overnight cultures ofXenorhabdus,PhotorhabdusandE. coliATCC®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 OD600nm by spectrophotometer. These bacterial suspensions were ready for using in bioassays.

2.3. Mosquito strains

Ae. aegyptiandAe. albopictuseggs were purchased from the Taxonomy and Reference Museum of the Department of Medical Sciences at the National Institute of Health of Thailand, Ministry of Public Health, Thailand. The filter papers containing the dried eggs of eachAedesspecies were placed in separate plastic containers containing dechlorinated water to allow theAedeslarvae to hatch.Larvae at the late third and early fourth instar were then selected out and feed with minced pet food.

2.4. Bioassay

Four different isolates of symbiotic bacteria (X. stockiaebNBP22.2_TH,X. indicabKK26.2_TH,P. luminescenssubsp.akhurstiibMSK25.5_TH andP. luminescenssubsp.hainanensisbKK17.1_TH)were tested as a larvicide againstAe. aegyptiandAe. albopictus. The efficacy ofXenorhabdusandPhotorhabdussuspensions against late third to fourth early instar larvae of bothAe. aegyptiandAe. albopictuswas evaluated under laboratory conditions. In each bioassay, ten larvae were placed in 100 μL of water in a well in a 24-well plate(COSTAR®, USA). Two mL of each bacterial suspension (107-108CFU/mL) was added to the well. Distilled water and suspension ofE. coliATCC®25922 were used as the negative control. The bioassay was designed to test two groups, the ‘fed group’ which wasAedeslarvae fed with minced pet food during exposure to bacterial suspension and the ‘unfed group’ which was not fed during the experiment. All bioassays were conducted in triplicate on different dates. The mortality of theAedeslarvae was monitored at 24, 48, 72 and 96 h exposure to the bacterial suspensions. The dead larvae were determined when no movement was detected when teasing with fine sterile toothpick.

2.5. Data analysis

Mortality ofAedeslarvae 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 theAedeslarvae from both the fed and unfed groups was statistically analyzed by Mann-Whitney test.

3. Result

BothAe. aegyptiandAe. albopictus(late 3rd to early 4th instars larvae) were susceptible to all isolates ofXenorhabdusandPhotorhabdusbacteria. The mortality of the larvae began to die at 24 h after exposure to the bacterial suspension. In the fed group, a cell suspension ofX. stockiae(bNBP22.2_TH) demonstrated the highest toxicity toAe. aegyptilarvae (99% mortality) at 72 h after exposure.In the unfed group,X. stockiae(bNBP22.2_TH) showed the highest pathogenic effect onAe. aegyptilarvae, with 87% mortality at 96 h after exposure. Significant mortality among all bacterial isolates and negative controls (distilled water andE. coliATCC®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 byAe. aegyptiwas not significantly different among the four bacterial isolates.

Table 2 shows the mortality rate ofAe. albopictuslarvae after exposure to cell suspension ofXenorhabdusandPhotorhabdus.X.indica(bKK26.2_TH) was highest toxic toAe. albopictusat 96 h in both fed (82%) and unfed (96%) condition. This bacterial isolate seemed to be fast pathogens toAe. albopictushaving kill 84% of 24 h. Mortality rate at each time among bacterial isolates and controls was significantly different in both fed and unfed conditions.

Mortality rate ofAe. aegyptiat each time between fed and unfed groups was not significant different. Significant mortality between fed and unfed groups ofAe. albopictuslarvae after exposure toX. indica(bKK26.2_TH) andP. luminescenssubsp.hainanensis(bKK17.1_TH) was observed at 24 h.

4. Discussion

In the present study, we demonstrate the alternative bacterial agent for controlAedesspp., a main vector for important virus infection in man. BothAedesspp. are susceptible toX. stockiae(bNBP22.2_TH)X. indica(bKK26.2_TH)P. luminescenssubsp.akhurstii(bMSK25.5_TH) andP. luminescenssubsp.hainanensis(bKK17.1_TH). It seems that the symbiotic bacteria of genusXenorhabdusandPhotorhabduscause superior mortality ofAedes.X. stockiae, asymbiotic bacterium that is found to be associated withSteinernema websteri, have been used for acaricidal and antibacterial activity[39,40].X. indicaproduces several bioactive compounds including taxlllaids A-G which has weakly effect onPlasmodium falciparum[41]. In addition, metalloprotease purified fromX. indicashowed insecticidal activity againstHelicoverpa armigera[42].P. luminescenssubsp.akhurstiiandP. luminescenssubsp.hainanensisshowed less effective againstAedes aegypti[43]. To our knowledge, it is reported for the first time that four symbiotic bacteria [P. luminescenssubsp.akhurstii(bMSK25.5_TH),P. luminescenssubsp.hainanensis(bKK17.1_TH),X. stockiae(bNBP22.2_TH) andX. indica(bKK26.2_TH) in the present study are symbiotic bacteria for oral pathogenicity againstAe. albopictus.

Table 1 Mortality rate of Ae. aegypti larvae after exposure to cell suspension of Xenorhabdus and Photorhabdus in fed and unfed conditions in laboratory.

Table 2 Mortality rate of Ae. albopictus larvae after exposure to cell suspension of Xenorhabdus and Photorhabdus in fed and unfed conditions in laboratory.

Ae. aegyptiandAe. 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 ofX. stockiae(bNBP22.2_TH),X.indica(bKK26.2_TH),P. luminescenssubsp.akhurstii(bMSK25.5_TH) andP. luminescenssubsp.hainanensis(bKK17.1_TH) againstAe. aegyptiandAe. albopictus. Both vectors were susceptible toXenorhabdusandPhotorhabdusbacteria by oral ingestion. This may be due to the bacteria producing insecticidal compounds including isopropylstilbene, ethylstilbene, xenorhabdin and xenematide[32].To support this scenario,Photorhabdusinsect-related protein fromPhotorhabdus asymbioticashowed strong toxicity toAe. aegyptiandAe. albopictus[33]. In addition, a suspension ofPhotorhabdus luminescenssubsp.laumondiiTT01 DSM15139 andX. nematophilaATCC®19061 showed orally lethality toAe. aegyptilarvae in laboratory conditions[24].P. luminescensandX. nematophilasuspension mixed with Cry4Ba protein fromB. thuringiensissubsp.israelensisenhanced the mortality rate ofAe. aegyptiup to 87%and 95%, respectively[17]. Recently,X. nematophilamixed withB. thuringiensissubsp.israelensiswas observed to enhance the toxicity toAe. albopictusandCulex pipiens pallens[18]. In addition,Xenorhabdus ehlersiiisolated fromSteinernema scarabaeishowed good potential efficacy in killingAe. aegyptiwith 100% mortality[43].In our study, we confirmed the oral toxicity ofXenorhabdusandPhotorhabdusagainstAe. aegyptiandAe. albopictus. However, it remains unknown as to the mechanism of killing effect of these bacteria onAedesspp.

XenorhabdusandPhotorhabdushave orally toxicity toAedesspp.,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 ofP. luminescenssubsp.akhurstii[45,46].In addition, the virulence ofXenorhabdusandPhotorhabdusvaried among insect species is related to foraging behavior[47]. This suggests that the virulent factors ofXenorhabdusandPhotorhabdusrequire further study for more deeply understanding.

We demonstrate the potential of entomopathogenic bacteria,X.stockiae,X. indica,P. luminescenssubsp.akhurstiiandP. luminescenssubsp.hainanensis, for the control of arbovirus vectors,Ae. aegyptiandAe. albopictus, by oral ingestion. This study confirms thatXenorhabdusandPhotorhabdushave orally toxicity againstAedeslarvae and provides further information relevant to the biological control of mosquito larvae. Further studies on identification and isolation of purified useful bioactive compounds to control both 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.

Acknowledgements

This study was supported by Higher Education Research Promotion, The Commission on Higher Education, Thailand (Grant No. R2558A008) and Naresuan University (Grant No. R2557B013).We would like to thank Miss Chutima Sarai and Miss Ponsuwan Aeiwong for their assistance with the laboratory technique. Many thanks were extended to Mr. Roy Morien of the Naresuan University Language Centre for his editing assistance and advice on English expression in this document.

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