Effect of entomopathogenic nematode, Steinernema feltiae, on survival and plasma phenoloxidase activity of Helicoverpa armigera (Hb) (Lepidoptera: Noctuidae) in laboratory conditions

Lethal effect of S. feltiae against H. armigera

Insects

Bioassay

Late prepupae of H. armigera which stopped their feeding were used in bioassay experiment. The experiment was conducted in cylindrical plastic boxes (3-cm diameter and 5-cm height) which were filled with 15-g autoclaved sandy soil. Based on preliminary experiments, range of the appropriate nematode concentration which causes approximately 20 to 80% mortality was determined. Then, different nematode concentrations (i.e., 10, 20, 45, 95, and 200 infective juveniles (IJs) per larva) were added on to the surface of the wetted soil. In brief, 0.5 ml of distilled water was added to the soil; then, the nematode concentration was added in 1-ml distilled water. One prepupa of H. armigera was placed on the soil, and the boxes were covered with ventilated lids to avoid desiccation. Control boxes received 1.5-ml distilled water without nematode. Each nematode concentration and control was replicated three times, and insect mortality was recorded after 96 h. The boxes were incubated at 26 ± 2 °C, 50 ± 5% RH and 16:8 (L: D) photoperiod.

Plasma penoloxidase activity assay

Infective juveniles (20–25 IJs in 20 μl of Ringer’s solution) of S. feltiae were injected into each of 50 larvae of H. armigera. The control groups consisted of 50 insects without injection and larvae injected with 20 μl of Ringer’s solution. Prior to bleeding, insects were disinfected for 2 min in 70% ethanol and then placed on ice for 2–5 min. Five larvae were bled once at designated time intervals (0 min–48 h pi). Hemolymph samples were centrifuged at 4 °C and 12,000g for 10 min to pellet cell debris. Aliquots (5 μl) of hemolymph supernatant were added to reaction buffer (195 μl 10 mM l-DOPA and 600 μl 10 mM Tris-HCl) and dopachrome formation followed spectrophotometrically (at an optical density (OD) wave length of 490 nm) (Biochrom WPA Biowave S2100 Diode Array Spectrophotometer) in samples incubated at 35 °C for 5 min in a water bath (Ebrahimi et al. 2014b). There were three replicates for each sample. The experiment was repeated twice.

Statistical analysis

LC₂₀, LC₅₀, and LC₈₀ values were obtained by Probit analysis using SAS software (SAS Institute 2004). Analysis of variance was done, and the means were evaluated by Duncan’s multiple-range test (SAS Institute 2004). Lethal experimental data was transformed into square root of (x + 1) where needed, before analysis.

Lethal effect

Pathogenicity of different isolations and strains of EPNs against H. armigera were documented before (Glazer and Navon 1990; Glazer 1997; Navon et al. 2002; Kalia et al. 2014). The result of lethal experiment showed high susceptibility of H. armigera prepupae to S. feltiae which is agreed with the previous studies (Glazer and Navon 1990; Glazer 1997). Glazer and Navon (1990) reported a LC₅₀ value equal to 54 IJs per insects for S. feltiae larvae. The present results with prepupae show either a greater virulence by the Iranian strain or increased pupal susceptibility, which lacking larval mobility and grooming behaviors, would be more easily infected by the IJs.

Plasma phenoloxidase activity

Variance analysis of plasma PO activity data showed significant difference between nematode injected and both control groups (F value = 628.36; df = 2; P < 0.01). Plasma PO activity was 0.184 ± 0.002 (OD/min) for nematode-injected insects while it was 0.119 ± 0.001 (OD/min) and 0.105 ± 0.00 (OD/min) for non-injected and Ringerʼs solution-injected control groups, respectively. All three insect groups showed significantly different PO activity from each other.

A few studies investigated the infection of EPNs on PO activity of host insects (Brivio et al. 2002; Walter et al. 2008; Ebrahimi et al. 2014a, 2014b; Kramarz et al. 2016). PO is the key component of insect humeral immune system. PO activation occurs upon different pathogens such as entomopathogenic nematode invasion in insect hemolymph. However, Brivio et al. (2002) reported S. feltiae inhibited PO activity of G. mellonella until 40 min pi compared to control larvae. They showed that the juveniles of S. feltiae induced the speedy suppression of phenoloxidase tend to avoid host humoral encapsulation. In overall, lepidopterans are susceptible to EPNs and G. mellonella is used as laboratory susceptible host for them. The results of PO activity assay demonstrated higher levels of PO unit in nematode-injected insect compared to both control groups (Table 2), which suggested H. armigera PO activation by nematode infection. Releasing the symbiotic bacteria and increasing the bacterial cells in hemolymph of H. armigera during the experimental time interval were reasonable explanation for increasing trend of PO activity in nematode-injected insects. While it is possible the nematode alone activated the enzyme, a GroEL-like toxin from Xenorhabdus budapestensis which activates PO in G. mellonella larvae. Yang et al. (2012) implies in H. armigera, X. nematophilus complex to activate the enzyme.