Investigating the pathogenicity of surface sterilised entomopathogenic nematode Oscheius sp TEL-

3.4 Pathogenicity of surface-sterilised nematodes

Oscheius sp. TEL-2014 that had been surface sterilized for at varying time periods, 0min

(control, treated with sterile distilled water), 30min, 60min, 90min, 120min, 150min and 180 min in 0.1% sodium hypochlorite solution resulted in 100% mortality of G. mellonella larvae. 100% mortality of the larvae was also achieved with the control treatment where the nematodes were not surface sterilised. However, as the length of surface sterilisation increased, the percentage of larvae in which IJs were recovered gradually dropped (Table 2). The remaining cadaver where dissected all were found to have IJs. Even if surface sterilised IJs had the ability to penetrate into the G. mellonella, nematodes performance declined as the exposure time in 0.1% NaClO increased.

Time (Days) 0 1 2 3 4 5 6 7 8 la rv a mo rta lit y % -20 0 20 40 60 80 100 120

Time (Days) vs 0 min Time (Days) vs 30 min Time (Days) vs 60 min Time (Days) vs 90 min Time (Days) vs 120 min Time (Days) vs 150 min Time (Days) vs 180 min

Fig. 4 Galleria mellonella percentage mortality caused by Oscheius sp. TEL-2014 treated with 0.1% sodium hypochlorite solution for different periods of time.

This Oscheius sp. TEL-2014 was able to invade Galleria mellonella larvae, release the bacteria into the larvae’s gut and cause 20% mortality within 24 hours when surface- sterilised for 30-60 minutes and 100% larvae mortality is observed within 72 hours. However, as the length of surface-sterilisation time increases (90-180) minutes, the 10-20%

sterilised for 90-120 minutes and by day 7 for 150-180 minutes. These results show that the virulence of the bacteria decreases with an incline in the amount of time the nematodes are exposed to 0.1% sodium hypochlorite solution. In a study conducted in 2011, Oscheius

carolinensis IJs taken from cadavers surface-sterilised for 1 minute using 1% NaClO and

used to infect Galleria mellonella larvae and in their results, 100% insect mortality was reported (Torres-Barragan et al, 2011). These results prove bacteria involved in the mortality of the insects are found inside the nematodes not on the surface of the nematodes. This implies that the hypothesis stated in the investigation is rejected based on the results obtained.

Table 2

Time to host death, time to nematode emergence after host death, and numbers of adults and juveniles emerged from host after inoculation with Oscheius sp. TEL-2014 nematodes (100 per host larva). The IJs emerged from one dead larvae and time for IJs emergence values represent the mean +/- standard error.

Treatment (length of

nematode surface sterilisation in minutes)

IJs Emerged from dead larva

Percentage (%) of larvae in which IJs were recovered Time for IJ emergence (in days) 0 850+/-76.7 100 2+/-0.05 30 720 +/-54.1 100 2+/-0.049 60 680 +/-28.3 100 4+/-0.197 90 590 +/-24.4 70 4+/-0.2 120 225+/-15.5 40 5+/-0.32 150 65+/-10.2 30 6+/-0.34 180 50+/-8.12 10 7+/-0.35

Our findings are not supported by some of the previous studies done on Oscheius

Carolinensis which had relations with four bacteria.

In our study, surface sterilised nematodes performed well in causing mortality of G.

mellonella insect larvae while in previous studies surface sterilised O. carolinensis nematodes

The combination of P. rettgeri with S. marcescens caused mortality of insect larvae however it was reported that further studies need to be conducted in order to confirm and describe the relationship and role of between O. carolinensis and S. marcescens (Torres-Barragan et al, 2011)

Fig. 5 Infection symptoms recorded for each treatment after 24 hours of inoculating G.

mellonella larvae surface-sterilised Oscheius sp. TEL-2014. A=0min, B=30min, C=60min,

D=90min, E=120min, F=150min and 180min. It is evident that as the length of surface sterilisation time increases, the larva displays less infection symptoms.

3.5 Pathogenicity of Infective juveniles which are not surface sterilised and

identification of associated bacterial isolates using Sanger sequencing

and Illumina 16S rDNA-based metagenome sequencing

Table 3

Sanger sequencing results which are also support the Illumina 16S rDNA-based sequencing Bacterial isolate Species identified

1 Citrobacter freundii

2 Serratia species

3 Uncultured Klebsiella

Fig. 6 Sunburst Classification Chart. This sunburst chart shows the relative abundance of the classification results within each taxonomic level. The bacterial species identified using Illumina 16S rDNA-based metagenome analysis were obtained from infective juveniles not surface sterilised.

Surface sterilised nematodes were able to infect and cause mortality of the insect larvae which indicates that the Serratia species isolated from the gut of the nematode was pathogenic.

Fig. 7 Mortality observed when 10 insect larvae per bacterial isolate were inoculated with bacterial isolate no. 1, which was identified as Citrobacter freundii, isolate no. 2 Serratia sp and isolate no. 3 Uncultured Klebsiella. Mortality was monitored over 24 hours.

No death or any signs of infection were observed in insect larvae inoculated with Citrobacter

freundii and Uncultured Klebsiella after 24 hours, while all 10 larvae died within 24 hours of

infection by the Serratia species.

Fig. 8 the evolutionary history of several species of Serratia and other selected sequences was centred on the analysis of 16S rDNA ITS region inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The bootstrap consensus tree inferred from 1000 replications and tree is drawn to scale, with branch lengths measured in the number of substitutions per site (next to the branches).

Fig. 9 the evolutionary history of several species of Serratia sequences only was centred on the analysis of 16S rDNA ITS region inferred by using the Maximum Likelihood method based on the Tamura-Nei model.

EF635247 Serratia sp. DM1 16S AB453291 Serratia sp. HI10

HM136580 Serratia marcescens strain SA1 KM252937.1 Serratia marcescens strain SW2-9-3 KP318498.1 Serratia marcescens strain DUCC3751 Serratia sp. HX-3-3

Uncultured Serratia sp. XYE 107R KP711410 Serratia sp. TEL

KF793930.1 Serratia marcescens strain MCB KM242583 Serratia sp. INBio 4516E

KP325089 Serratia sp. CH-B17

KP004440 Serratia entomophila strain CT8

Photorhabdus sp. Caborca 0.010 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.059 0.000 0.000 0.000 0.073 0.009 0.005 0.011 0.000 0.01 Serratia sp. HX-3-3

Uncultured Serratia sp. XYE 107R

KP318498.1 Serratia marcescens strain DUCC3751 AB453291 Serratia sp. HI10

EF635247 Serratia sp. DM1 16S

KM252937.1 Serratia marcescens strain SW2-9-3 HM136580 Serratia marcescens strain SA1

KP711410 Serratia sp. TEL KM242583 Serratia sp. INBio 4516E

KP325089 Serratia sp. CH-B17

KP004440 Serratia entomophila strain CT8

KF793930.1 Serratia marcescens strain MCB

0.010 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.057 0.000 0.000 0.000 0.005 0.036 0.010 0.01

3.7 Bacterial growth curve

In document Genome analysis of an entomopathogenic nematode belonging to the genus Oscheius and its insect pathogenic bacterial endosymbiont (Page 71-78)