Identification of entomopathogenic nematodes and symbiotic bacteria from Nam Nao National Park in Thailand and larvicidal activity of symbiotic bacteria against Aedes aegypti and Aedes albopictus

Entomopathogenic nematodes (EPNs) that are symbiotically associated with Xenorhabdus and Photorhabdus bacteria can kill target insects via direct infection and toxin action. There are limited reports identifying such organisms in the National Park of Thailand. Therefore, the objectives of this study were to identify EPNs and symbiotic bacteria from Nam Nao National Park, Phetchabun Province, Thailand and to evaluate the larvicidal activity of bacteria against Aedes aegypti and Ae. albopictus. A total of 12 EPN isolates belonging to Steinernema and Heterorhabditis were obtained form 940 soil samples between February 2014 and July 2016. EPNs were molecularly identified as S. websteri (10 isolates) and H. baujardi (2 isolates). Symbiotic bacteria were isolated from EPNs and molecularly identified as P. luminescens subsp. akhurstii (13 isolates), X. stockiae (11 isolates), X. vietnamensis (2 isolates) and X. japonica (1 isolate). For the bioassay, bacterial suspensions were evaluated for toxicity against third to early fourth instar larvae of Aedes spp. The larvae of both Aedes species were orally susceptible to symbiotic bacteria. The highest larval mortality of Ae. aegypti was 99% after exposure to X. stockiae (bNN112.3_TH) at 96 h, and the highest mortality of Ae. albopictus was 98% after exposure to P. luminescens subsp. akhurstii (bNN121.4_TH) at 96 h. In contrast to the control groups (Escherichia coli and distilled water), the mortality rate of both mosquito larvae ranged between 0 and 7% at 72 h. Here, we report the first observation of X. vietnamensis in Thailand. Additionally, we report the first observation of P. luminescens subsp. akhurstii associated with H. baujardi in Thailand. X. stockiae has potential to be a biocontrol agent for mosquitoes. This investigation provides a survey of the basic diversity of EPNs and symbiotic bacteria in the National Park of Thailand, and it is a bacterial resource for further studies of bioactive compounds.

Introduction subsp. namnaonensis, was isolated from H. baujardi from the Nam Nao district in Phetchabun province [36]. This suggests that EPNs and their symbionts may be diverse in Phetchabun province, a mountainous region of central Thailand. Therefore, other areas in Phetchabun province should be explored for EPNs and their symbiotic bacteria. Nam Nao National Park includes the mountainous forests of Phetchabun and Chaiyaphum provinces in Thailand. The flora consists mainly of dry dipterocarps, mixed deciduous, hill evergreens, vast bamboo groves, pine forests and some grassland areas. Despite the interesting location for the surveying of EPNs and their symbiotic bacteria, there is only one report of these organisms in the National Park (Mae Wong) of Thailand. This area is abundant with insect hosts and might lead to the recovery of several species of EPNs and symbiotic bacteria. In addition, four organisms were newly recorded in Mae Wong National Park, namely H. zealandica, S. kushidai, X. japonica and P. temparata subsp. temperata. Therefore, the objectives of this study were to isolate and identify EPNs and their associated Xenorhabdus and Photorhabdus from Nam Nao National Park of Thailand and to determine the larvicidal activity of these bacteria against Ae. aegypti and Ae. albopictus.

Collection of soil samples
The methods for soil collection in Nam Nao National Park of Phetchabun province were approved and permitted by the Department of National Park, Wildlife and Plant Conservation, Thailand (Permission number 0907.4/8245). Overall, 940 soil samples were collected from 188 soil sites in Nam Nao National Park between February 2014 and July 2016. At each site, five soil samples (300-500 g) were collected using a hand shovel. The soil collection process was performed according to Thanwisai et al. [15]. The latitude and altitude of each soil site were determined using a GPS navigator (Garmin nüvi 1250, Taiwan). The moisture and pH of each soil sample were recorded using the soil pH and moisture meter Takemura DM15 (Takemura Electronic Works, Chiba, Japan). The soil temperature of each sample was also measured using a soil survey instrument (Yancheng Kecheng Optoelectronic Technology, Jiangsu, China). Soil texture was also noted. All soil samples were transported in ambient temperature to the Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok province, Thailand for the isolation of EPNs. Soil parameters (soil temperature, pH and moisture) of samples with and without EPNs were compared statistically using the Mann-Whitney test in the SPSS Program, Version 17 (P < 0.05).

Identification of entomopathogenic nematodes
A baiting technique [37] using Galleria mellonella larvae followed by a White trap [38] was performed to isolate EPNs from soil samples. The infective juvenile stages (IJs) of EPNs emerged from the G. mellonella cadaver and moved to water. The IJs were collected in a culture flask and cleaned by a sedimentation technique, along with several changes of sterile distilled water. The clean IJs were kept in aerated water and stored at 13˚C in a refrigerator. To increase the number and confirm the entomopathogenicity of EPNs, a new G. mellonella larva was infected with IJs.
The genomic DNA of EPNs was extracted from the IJs as described previously [15]. The genomic DNA was dissolved in 20 μl of sterile distilled water and stored at -20˚C for future use.
A PCR-based analysis and DNA sequencing were conducted to identify Steinernama and Heterorhabditis species. The primers used for amplifying Steinernama 28S rDNA were 539_F (5'GGATTTCCTTAGTAACTGCGAGTG-3') and 535_R (5'-TAGTCTTTCGCCCCTATACCC TT-3') [39]. The PCR conditions consisted of pre-denaturation at 95˚C for 1 min, followed by 35 cycles of denaturation at 94˚C for 1 min, annealing at 55˚C for 30 s, extension at 72˚C for 45 s, and a final extension at 72˚C for 7 min. The size of amplification fragment was 870 bp.

Isolation and identification of symbiotic bacteria
Symbiotic bacteria were isolated from the hemolymph of dead G. mellonella larvae, which had been infected with the IJs of EPNs. The dead larvae of G. mellonella were surface-sterilized by dipping into absolute ethanol for 1 min and placed in a sterile petri dish to dry. Sterile forceps were used to open the 3rd segment from the head of G. mellonella. A sterile loop was used to touch hemolymph and streaked on nutrient agar supplemented with bromothymol blue and triphenyl-2,3,5-tetrazolium chloride (NBTA). Colonies of Xenorhabdus and Photorhabdus were blue and green on NBTA after four days of incubation in the dark at room temperature. A single colony of each isolate of symbiotic bacteria was inoculated in 3 ml of Luria-Bertani (LB) broth and incubated with shaking at 180 rpm overnight (approximately 18-24 h). The genomic DNA of symbiotic bacteria was extracted from bacterial pellets using the Blood/Cell DNA Mini Kit (Geneaid Biotech Ltd., New Taipei, Taiwan). The genomic DNA of symbiotic bacteria was stored at -20˚C for further use in PCR.
PCR-based analysis and recA gene sequencing was performed to identify Xenorhabdus and Photorhabdus species. Primers used were recA_F (5'-GCTATTGATGAAAATAAACA-3') and recA_R (5'-RATTTTRTCWCCRTTRTAGCT-3') to obtain an 890 pb amplicon [41]. The PCR conditions for Xenorhabdus and Photorhabdus were based on a previous report by Thanwisai el at. (2012) [15]. A total of 30 μl of the PCR reaction contained 3 μl of 10x buffer (1x), 4. PCR was conducted in a thermal cycler (Applied Biosystems, Pittsburgh, PA, USA). The thermal profile was as previously described [15]. PCR products were visualized on a ethidium bromide-stained 1.2% agarose gel and purified using a PCR clean-up Gel Extraction Kit (Macherey-nagel, Düren, Germany). The DNA sequencing for both directions was analyzed by Macrogen Inc. service in Korea.

Sequencing and phylogenetic analysis
To identify species of EPNs (Steinernema and Heterorhabditis) and symbiotic bacteria (Xenorhabdus and Photorhabdus), comparison of the partially edited nucleotide sequences (recA, ITS and 28S rDNA) was performed using the BLASTN program from NCBI. A cut-off of ! 97% identity was considered for the same species. The nucleotide sequences were aligned using ClustalW, and a phylogenetic tree was constructed using the maximum likelihood method with the MEGA Version 7.0 program [42].
The eggs of Ae. aegypti and Ae. albopictus (laboratory strain) on a filter paper 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, Nonthaburi Province, Thailand. Aedes eggs were placed into distilled water to allow hatching of first instar larvae, which were fed with minced food pets. The late third and early fourth instar larvae were used in the bioassays.
Escherichia coli ATCC 1 25922 and distilled water were used as controls for the bioassays. Escherichia coli ATCC 1 25922 was inoculated in the LB broth and incubated at 37˚C for 24 h, while each isolate of Xenorhabdus and Photorhabdus was inoculated on 5YS medium (5% (w/v) yeast extract, 0.5% (w/v) NaCl, 0.05% (w/v) NH 4 H 2 PO 4 , 0.05% (w/v) K 2 HPO 4 and 0.02% (w/v) MgSO 4 .7H 2 O) and incubated at room temperature for 48 h. All broth cultures of bacteria were centrifuged at 10,000 x g for 10 min. The supernatant was discarded and the pellets were suspended in sterile distilled water to an OD 600 of 0.1 with a spectrophotometer (Metertech SP-880, Taiwan).
Suspensions containing Xenorhabdus and Photorhabdus was evaluated for toxicity against the larvae (third late to early fourth instars) of Ae. aegypti and Ae. albopictus. Distilled water and a suspension of E. coli ATCC 1 25922 were used as controls. In each bioassay, 30 larvae of each mosquito species were placed in three wells of a 24-well plate (10 larvae/well). Subsequently, 2 ml (10 7 -10 8 CFU/ml) from each bacterial suspension was added to each well. This concentration of symbiotic bacteria showed the enhancement of oral toxicity against Ae. aegypti [10]. The larvae were fed with minced pet food during the experiment. The mortality rate of larvae was monitored after exposure to bacterial suspensions at 24, 48, 72 and 96 h. Each bioassay was tested in three replicates on different dates. The larvae were considered dead when no movement was detected when teased with a fine sterile toothpick. Larval mortality of Ae. aegypti and Ae. albopictus after exposure to symbiotic bacteria was analyzed by survival analysis (Kaplan-Meier Estimate) of SPSS Version 17 (P < 0.05).

Isolation of entomopathogenic nematodes from soil samples
Out of the 188 soil sites from Nam Nao National Park, Phetchabun Province, Thailand, 25 tested positive for EPNs (Fig 1). Of the 940 soil samples, 27 (2.87%) were found positive for Steinernema or Heterorhabditis. This yielded 13 isolates of Heterorhabditis and 14 isolates of Steinernema. Most EPNs were isolated from loam (96.30%), with the soil pH ranging between 4.8 and 7.0, soil temperature ranging between 19˚C and 30˚C and soil moisture ranging between 1.0 and 8.0 (Table 1). One isolate of the EPNs was isolated from sandy loam and none of the EPNs were isolated from soil with a clay texture.

Identification and phylogeny of entomopathogenic nematodes
To identify Heterorhabditis and Steinernema species, PCR-based analysis and sequencing of the ITS and 28S rDNA regions were performed together with a BLASTN search of the edited sequences. Two isolates of Heterorhabditis (accession no. MG209260 and MG209261) were identified as Heterorhabditis baujardi with 99% identity after BLASTN search using 542 nucleotides   (Fig 2) and 10 isolates of the genus Steinernema grouped in S. websteri (accession no. AY841762.1) (Fig 3).

Isolation, identification and phylogeny of Xenorhabdus and Photorhabdus
Based on colony morphology on NBTA agar, 27 isolates of symbiotic bacteria were identified as Photorhabdus (13 isolates

Larvicidal activity of symbiotic bacteria against Aedes larvae
The symbiotic bacteria used for the bioassay were selected based on their locations in different groups on the phylogenetic tree. Both, the Xenorhabdus and Photorhabdus isolates in the present study were assumed to be pathogenic when ingested by Ae. aegypti and Ae. albopictus larvae. Fig 6 and (Fig 6 and Table 3). Moreover, Ae. aegypti and Ae. albopictus larvae exhibited a low susceptibility to X. japonica (bNN165.4_TH) and X. vietnamensis (bNN167.2_TH and bNN167.3_TH) as indicated by the 22-61% mortality after exposure for 96 h.

Discussion
Here, we report a low recovery of EPNs (2.87%) from Nam Nao National Park of Thailand. Our findings revealed two genera of EPNs, which consisted of 10 isolates of Steinernema and two isolates of Heterorhabditis. Most EPNs were isolated from loam, which is consistent with the study by Thanwisai et al. (2012) [15]. In addition, EPNs were not found in clay in the present study.
Soil parameters including temperature, pH, and moisture are important for EPN survival and infectivity. In the present study, the soil moisture was 1.0-3.0% (average 1.8%) for the EPN-positive samples and 1.0-8.0% (average 2.0%) for the EPN-negative samples. This indicated that EPNs, especially S. websteri and H. indica (the most common species in Thailand), could live in a restricted range of moisture contents. In addition, soil moisture contents between 4.0-8.0% may not suitable for the survival of EPNs because none were recovered from this range of moisture contents. Although the difference in soil moisture contents found in the present study between the EPN-positive and EPN-negative samples was not significantly great (P-value = 0.972), soil moisture is an important factor that supports the ability of EPNs to infect hosts and its survival in different environments [43,44]. High and low moisture content could affect the movement of EPNs to find a new host; thus, this soil parameter might affect survival rates. The appropriate moisture content for survival is 8-18% for H. indica, 6-20% for S. themophilum and 8.0-25.0% for S. glaseri [45]. This indicates that each species of EPN responds to different levels of moisture. Soil temperatures between EPN-positive samples (20-25˚C) and EPN-negative samples (19-30˚C) in the present study was not significantly great (P-value = 0.055). In general, temperatures under 0˚C and above 40˚C would lead to EPN death. Temperatures between 10 and 15˚C could limit the movement of EPNs, while temperatures between 30 and 40˚C could result in low infectivity of the EPNs [46]. In the present study, the soil pH between EPN-positive samples (6.2-7.0) and EPN negative samples (4.8-7.0) was not significantly great (P-value = 0.165). Soil pH is one of the factors that is important for the infectivity and survival of EPNs. For Steinernema, infection and survival rates showed a low level of decrease when the soil pH was between 4 and 8. Rapid decreases in EPN infectivity and survival rates were observed with pH = 10 [47].
Herein, we isolated and identified two isolates of H. baujardi and 10 isolates of S. websteri. The most common species found in Nam Nao National Park was S. websteri, which has previously been isolated from soil samples collected from the roadside areas of several provinces of Thailand [15,31,32]. This EPN was also found in Mae Wong National Park, Kampheang Phet province and central Thailand [33]. This confirmed that S. websteri was abundant in natural and cultivated fields across the country. H. baujardi, a less common species that was found in the present study, is also recovered in a low number of isolates across the country [16]. In the early studies of EPNs in Thailand, Tangchitsomkid and Sontirat reported the first isolation of the Steinernema and Heterorhabditis genera from soil samples from Kanchanaburi, Kalasin, Maha Sarakham, Khon Kaen, Nong Khai, Phichit and Sa Kaeo provinces in Thailand [48]. Steinernema saimkayi, a novel species, has been isolated from a soil sample collected from the land used to cultivate tamarind in Phetchabun province [28]. Steinernema minutum, a second novel species reported in our country, was recovered from soil samples in Chumphon province, which is in south Thailand [29]. Our finding is consistent with previous studies that the   most common species of EPNs in Nam Nao National Park was S. websteri. However, when comparing our findings with a previous study in Mae Wong National Park, we did not isolate H. zealandica and S. kushidai, which may be due to the low distribution of these two EPNs in the National Park.
Of the symbiotic bacteria, all 13 isolates of Photorhabdus were molecularly identified as P. luminescens subsp. akhurstii. Xenorhabdus species (14 isolates) were molecularly identified as X. stockiae (11 isolates), X. vietnamensis (2 isolates) and X. japonica (1 isolate). In the present study, we found X. stockiae was associated with S. websteri, which was consistent with the previous research in Thailand [15,33]. However, S. websteri was also symbiotically associated with X. nematophila [49]. Two isolates of X. vietnamensis were first identified in Nam Nao National Park of Thailand. Unfortunately, the EPN host X. vietnamensis was only identified to the genus level in the present study. A previous study in Vietnam reported X. vietnamensis was associated with S. sangi [50,51]. The finding of one isolate of X. japocina in Nam Nao National Park is similar to the finding of Muangpat, who first recorded X. japonica associated with S. kushidai in Mae Wong National Park of Thailand [33]. For the genus Photorhabdus, P. luminescens subsp. akhurstii was associated with H. baujardi. Elsewhere, P. luminescens subsp. hainanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. laumondii were associated with H. indica [15]. The relationship found in this study between P. luminescens subsp. akhurstii and H. baujardi is a new observation in Thailand.
We demonstrated the toxicity of Thai Xenorhabdus and Photorhabdus isolates against Ae. aegypti and Ae. albopictus larvae. Xenorhabdus stockiae bNN112.3_TH and P. luminescens subsp. akhurstii bNN121.4 isolated in the present study have potential to be control agents for Ae. aegypti and Ae. albopictus. We suppose that the symbiotic bacteria are pathogenic when ingested by Aedes larvae. In general, the diet of Aedes larvae are dead and living organic material. These are bacteria, fungi and protozoans that grow on container or are suspended in fluid [52]. These bacteria may produce toxins and secondary metabolites that kill the Aedes larvae. To support this scenario, Xenorhabdus spp. might produce insecticidal compounds including toxin complexes (Tcs), which play an important role in the pathogenicity of immunosuppression by inhibiting eicosanoid biosynthesis in the target insect [4,53]. Xenorhabdus nematophila produces at least eight bacterial metabolites that play crucial roles in suppressing the immune responses of target insects [54]. X. stockiae PB09 showed miticidal activity against mushroom E. coli ATCC 1 25922 0 ± 0.00 0 ± 0.00 0 ± 0.00 7 ± 0.00 0 ± 0.00 0 ± 0.00 0 ± 0.00 0 ± 0.00 Distilled water 0 ± 0.00 0 ± 0.00 0 ± 0.00 7 ± 0.00 0 ± 0.00 0 ± 0.00 7 ± 0.00 17 ± 0.00 https://doi.org/10.1371/journal.pone.0195681.t003 mites [55]. Several species of Photorhabdus have been reported to produce several toxins including toxin complexes (Tcs), make caterpillars floppy (Mcf), Photorhabdus insect-related (Pir) proteins and Photorhabdus virulence cassettes (PVC) [56]. The Tcs destroy the epithelial cells of the insect midgut and are similar to the δ-endotoxin from Bacillus thuringiensis (Bt) [5]. The Mcf is active upon injection and disrupts the insect hemocytes by promoting their apoptosis [57]. The Photorhabdus Pir toxin is composed of PirA and PirB, which have been found to be effect against mosquito larvae especially the dengue vectors, Ae. aegypti and Ae. albopictus [8,58]. Xenorhabdus nematophila and P. luminescens showed oral toxicity against Ae. aegypti [9]. Recently, an enhanced mortality rate of Ae. aegypti was demonstrated by mixing the Cry4Ba protein of Bacillus thuringiensis with X. nematophila and P. luminescens [10]. The PVC causes toxicities against a variety of insects such as Manduca sexta and Gallaria mellonella [59]. Moreover, culture fluids from X. nematophila caused a delay in pupation and the emergence of adults. The fluids were lethal to larvae, with a cumulative mortality higher than 90% by day 14 [11]. In addition, cell suspensions of X. ehlersii isolated from S. scarabaei from Mae Hong Son Province in northern Thailand showed a potential efficacy in killing Ae. aegypti with 100% mortality [60]. In contrast, Ae. aegypti and Ae. albopictus larvae had low mortality rates (4-61%) after exposure to X. japonica NN165.4_TH, X. vietnamensis NN167.2_TH and X. vietnamensis NN167.3_TH. This may have been due to their inability to produce very toxic metabolites to Aedes. In addition, different strains of symbiotic bacteria may produce different secondary metabolites. This may give a variable effectiveness in killing the target organisms. Also, the test of the mortality rate of the insects may be related to the variable number of bacterial cells ingested by each larva. It is possible that the symbiotic bacteria are alive in the water and may produce some secondary metabolites in the water. This may be the cause of the larval death. At present, 2.5 billion people in 100 countries are considered living in areas with a risk infection of the Dengue virus [61,62]. It is transmitted by female mosquitoes mainly of the species Ae. aegypti and, to a lesser extent, Ae. albopictus. This mosquito also transmits chikungunya, yellow fever and Zika infection [63,64]. At present, there are no particular treatments or vaccines available to combat infections by these viruses; the only effective method to prevent infection is to avoid mosquito bites [65]. Control measures for Ae. aegypti and Ae. albopictus can be achieved using repellents, by cleaning the water source where larvae breed, or applying synthetic insecticides such as organophosphates (temephos and fenthion). The insect growth regulators (diflubenzuron and methoprene) are generally used for the control of mosquito larvae [66][67][68]. The constant use of such insecticides induces resistance in populations of Aedes species [69]. Furthermore, biological control might be an alternative approach to avoid these effects. The advantage of using Xenorhabdus and Photorhabdus bacteria is that they rapidly kill both Ae. aegypti and Ae. albopictus larvae within 48 h. These bacteria also may be non-toxic to humans. Xenorhabdus and Photorhabdus may thus be effective in all stages of Aedes larvae.

Conclusion
We report a diversity of EPNs and symbiotic bacteria from Nam Nao National Park, Phetchabun Province, Thailand, with the most commonly isolated EPN species being Steinernema websteri. The most commonly isolated species of symbiotic bacteria was X. stockiae. Xenorhabdus vietnamensis is the first recorded member of this species of symbiotic bacteria in Thailand. P. luminescens subsp. akhurstii associated with H. baujardi is a new observation in Thailand. Xenorhabdus stockiae and P. luminescens subsp. akhurstii are potential bio-control agents for Ae. aegypti and Ae. albopictus. Our findings provide information regarding the diversity of EPNs and symbiotic bacteria in Nam Nao National Park, Phetchabun Province, Thailand and provide a natural resource for further studies of bioactive compounds for controlling mosquitoes. Further investigations of the structure and isolation of the metabolic compounds from Xenorhabdus and Photorhabdus are needed to update the basic knowledge of the secondary metabolites found in these symbiotic bacteria.