Survival and Infectivity of Entomopathogenic Nematodes Formulated in Sodium Alginate Beads

Infective juveniles of the nematodes Steinernema carpocapsae (Sc), Heterorhabditis bacteriophora (Hb), and Steinernema glaseri (Sg) collected within a week after their emergence from the host cadavers were used. The nematodes had been reared in last instar larvae of the greater wax moth, Galleria mellonella, at 23 ± 3°C and stored at 12°C.

Two capture methods of IJs were used, a modified White trap (WT) and a WT filled with a layer of plaster of Paris (PP) instead of the filter paper on small petri dish (Stock and Goodrich-Blair, 2014). The PP was used for the pre-conditioning of IJs upon emergence from infected hosts. A total of 30 Galleria mellonella larvae were inoculated with Sc or Hb and 60 with Sg IJs in petri dishes with a filter paper disc (90 mm diameter, Whatman No. 1). The inoculation rate was 100 IJs per insect. Three days after inoculation, the infected hosts were divided between the two IJs capture treatments and incubated at 23 ± 3°C. A few drops of bidistilled water were occasionally applied on the plaster to prevent desiccation of the host.

Infected cadavers placed in WT or PP were checked daily to inspect the emergence of IJs. Due to the faster movement of juveniles in PP, they were collected from the surrounding water three days after the onset of the massive emergence. The IJs from the water in the WTs were collected 7 d after the emergence. Then, the collected juveniles were separately concentrated in bidistilled water suspension at the rate of 25,000 IJs/ml into partially closed 250 ml tissue culture flasks and stored at 12°C for a week.

The ABs were made following the ionic gelation method (Vemmer and Patel, 2013). 20 ml of IJs aqueous suspension was deposited in 20 ml of a 1% solution of sodium alginate, 36% glycerol, and 0.05% green vegetable dye for 24 h at 12°C. Final composition was 0.5% of sodium alginate, 18% glycerol, and 0.025% green vegetable dye. Droplets of 83.33 µl of the alginate nematode suspension containing approximately 1,000 IJs were deposited in a 2% CaCl₂ solution using a 10 ml Mohr pipette with an exit hole of 3 mm in diameter, the drops remained in the solution for 20 min. A total of 470 ABs were recovered by treatment with a sieve, rinsed three times with bidistilled water to remove residues from the CaCl₂ solution and deposited in a 500 ml beaker which was covered with Parafilm® tape.

Each AB was individually immersed for 2 s in a sodium alginate solution containing 0.5% of sodium alginate, 18% glycerol, and 0.025% green vegetable dye, to be deposited afterwards in the CaCl₂ solution for 20 min. The ABs were recovered with a sieve, rinsed three times with bidistilled water, and deposited in a 500 ml beaker which was covered with Parafilm® tape.

The beakers containing the ABs were stored at room temperature (23 ± 3°C) for 50 d. Monitoring of storage conditions was done with a temperature and relative humidity data logger (Control Company®, model 4040, accuracy of ±1°C and ±5% RH).

Every two days, a dissected AB was placed in a petri dish (60 mm diameter) and rehydrated with 7 ml of bidistilled water for a period of 24 h to soften them and facilitate the release of IJs (Kaya and Nelsen, 1985). Then, a 1 ml sample containing approximately 143 IJs was taken, divided in ten 100 µl-subsamples and the alive and dead juveniles were counted using a stereoscopic microscope at 30×; the living ones had mobility by themselves or moved when stimulated with a dissecting needle, while dead IJs had shriveled surface or gas bubbles inside body (Peters, 2016). The survival rate (SR) was calculated as the proportion of alive juveniles over the total juveniles (alive and dead).

Infectivity was evaluated every 7 d according to the mortality of Galleria mellonella (Ricci et al., 1996). A filter paper disc (15 mm diameter, Whatman no. 413) was placed in each cell of the 24-well plate, where 100 μl of bidistilled water and a dissected AB were placed afterwards. The plates were maintained in an incubator at 25 ± 2°C for 24 h for reactivation of the EPNs. Subsequently, the Galleria mellonella larvae were individually placed in each well plate, covered with Parafilm® tape, and returned to the incubator. Insect mortality was checked 72 h later. To verify the mortality of Galleria mellonella larvae by EPNs, each host with signs of infection was placed in a petri dish and incubated at 25 ± 2°C until the IJs emerged.

The experiment was performed by duplication and each bioassay was repeated five times. The results of the duplicate tests were combined for the final analysis. Survival analysis was performed using Kaplan-Meier plots and the Log-Rank test (P ⩽ 0.001). The analysis of variance (ANOVA on ranks) and the SNK test (P < 0.05) were performed between the species of EPNs, pre-conditioning, coating of Abs and % infectivity on Galleria mellonella. All analyzes were performed using SigmaPlot® 12 software (Systat Software, Inc., San Jose, CA, USA).

The survival rates per week of EPNs in ABs are presented in Table 1, which shows that Sc had the best viability, maintaining a SR value of 58.8% for 28 d, followed by Hb with a SR of 51.4% at 21 d and Sg with the shortest survival time with 7 d and 15% of SR. The differences between the survival curves by nematode species were highly significant (χ ² = 2,507, Log-Rank < 0.001).

The SR of pre-conditioned EPNs in PP and formulated in ABs remained 39.6% and 53.3% in 7 d for Sg and Sc, respectively, while SR of Hb was above 9.8% in 21 d (Table 1). The maximum extension of the survival time and the highest SR were, using Sg, 21 d and 22.5%, respectively. The differences between the survival curves of the treatments with pre-conditioning IJs were statistically significant (χ ² = 3,207, Log-Rank < 0.001).

Amongst the three EPNs formulated in ABs coated with a layer of sodium alginate, Sc was maintained with an SR of 41.6% for 28 d, Hb with 21.7% for 28 d, and Sg with 20% for 7 d (Table 1). The differences between the survival curves of the EPNs in coated ABs were statistically significant (χ ² = 5,086, Log-Rank < 0.001). The SR trends for Sg and Sc are similar to those observed in the corresponding treatments by EPN factor; therefore, no benefit on survival by coating of ABs was observed on EPNs.

The maximum extent of the survival time of Sg and Hb was just 7 d when pre-conditioned IJs were formulated in coated ABs. The SR of Sg decreased rapidly to 40% in the seventh day, whereas SR of Hb was maintained above 92.4% in the seventh day (Table 1). The SR of Sc remained above 67% in 14 d and its survival time was extended to 35 d, albeit with about 10% SR. The differences between the survival curves of the preconditioned EPNs in coated ABs were statistically significant (χ ² = 4,288, Log-Rank < 0.001).

All three treatments within the four factors tested: nematode species (EPN), pre-conditioning (Prec), coating (C), and the Prec-C combination, were statistically significant different, reaching the lower P-values the first two (Table 2). For example, according to ANOVA, where the F-test applies to the means by factor, the infectivity (IN) of the EPNs in sodium ABs against Galleria mellonella was significantly different for each species applied (F = 6.289, P = 0.023) and the difference in the mean IN between Sc and Sg was greater (P < 0.05) than would be expected by chance after allowing for effects of differences in time after formulation. Sc was the most infectious, killing all larvae in a period of 28 d (100%), Hb killed almost all of them in a period of 14 d (~100%), but Sg killed a few larvae in the first 7 d (15%), which lowered its mean IN to 3.0% for a period of 35 d.

The IN of preconditioned EPNs formulated in sodium ABs against Galleria mellonella over time ranged from 55% to 100% (Table 2). The difference in the mean IN among the different EPN species is greater (F = 5.795, P = 0.006) than would be expected by chance after allowing for effects of differences in time after formulation, and the statistical difference was significant only between Sg and Sc nematodes (P < 0.05).

The IN of EPNs formulated in coated ABs on Galleria mellonella was between 35% and 100% and mean IN was significantly different between each specie applied (P < 0.001). Sg was the less infectious and therefore statistically significant different if compared with Sc or Hb (P < 0.001). As it can be seen in Table 2, this trend in statistical difference was maintained during all times after formulation.

The formulation of preconditioned EPNs in coated ABs results in statistically significant difference on mean IN for each species applied on Galleria mellonella larvae (F = 160.548, P < 0.001). The combined treatment Prec-C improves the mean IN of Sc much more than on Sg or Hb (P < 0.05). Table 2 shows that the mean IN of Sc had increased by 45% when compared with Prec and by 20% when compared with C and, the combination Prec-C produced negative results in Sg with respect to Prec only, while Hb-Prec-C had lower IN with respect to Hb-C only.

In Table 1, the mean survival time (MSt) of EPNs in ABs by each factor is shown for a time of 35 dpf. Non pre-conditioned Sc in uncoated ABs had the highest MSt (26.07 ± 0.389 d), but pre-conditioning of IJs before the formulation shows a negative effect on MSt (13.02 ± 0.218 d), and the same trend in MSt was observed using pre-conditioned Hb. The coating of ABs did not show a significant difference on MSt of Sg, Sc or Hb, in relation to ABs uncoated. Only in the case of Sc, the coating of ABs reversed the negative effect of pre-conditioning of IJs, when both factors were combined, increasing MSt from 13.02 ± 0.218 d to 22.06 ± 0.372 d. Sg had the lowest performance formulated in Abs, but pre-conditioning alone increased the MSt significantly from 8.05 ± 0.079 d to 13.21 ± 0.262 d.

The mean infectivity (mean IN) increased significantly with the pre-conditioning of Sg, since it went from 3 ± 2.19% to 29 ± 9.5%, while for Sc a significant decrease occurred from 80 ± 9.18% to 51 ± 11.3% (Table 2). Coating of ABs had its greatest effect on the mean IN of Hb, with a value of 77 ± 9.0%, while the pre-conditioning combined with the ABs coating increase the mean IN of Sc to 96 ± 2.7%. Based on these mean results, it could be recommended doing more research to validate the use both pre-conditioning and coating treatments for the entire formulation process of Sc in ABs.