In the present study, the estimation of protein was performed, followed by their electrophoretic profiling. The total protein contents were measured in total and plasma fraction of larval hemolymph of H. armigera infected with S. abbasi-X. Indica separately. Analysis of variance (two-way) showed that total protein contents in total hemolymph challenged with S. abbasi was affected by time (df = 6; F = 14.584; p = 0.00) and not by treatment (df = 1; F = 1.388; p = 0.241). Effect of time and treatment interaction was significant (df = 5; F = 17.784; p = 0.00). While applying one-way ANOVA, the mean protein contents varied significantly between the infected and control group of H. armigera larvae (df = 12; F = 14.825; p = 0.00) (Fig. 1a). Insignificant changes were recorded in the control group and the amount of protein was almost steady with very minute fluctuations from 129.23 to 129.62 mg/ml. The increment in protein contents was reported over 3 h of infection. This significant increment was 12% as compared to 0 h from 129.49 to 144.81 mg/ml (p < 0.05). With the significant increments of 24.69 and 28.27% protein contents at 6 h and 9 h, respectively, an increment in protein profile was detected in the total protein amount reaching the peak amount of 165.77 mg/ml. From 9 h PIP onwards, a dramatic decline was recorded continuously up to 24 h with 116.92 mg/ml, 109.55 mg/ml, and 97.50 mg/ml at 12, 18, and 24 h, respectively. In a similar way, soluble protein content fluctuated significantly in the plasma fraction of hemolymph (Fig. 1b).
The protein contents in plasma fraction measured lower than the total hemolymph. Two way ANOVA established a significant effect of time (df = 6; F = 15.346; p = 0.00) and nematode infection (df = 1; F = 6.003; p = 0.016) indicating the main role of soluble proteins in combat with the parasitic infection. With the increment in the time, the nematode-bacterium significantly affected the plasma protein contents (df = 5; F = 18.580; p = 0.00) of H. armigera larvae. One-way ANOVA displayed a significant result, when comparing the protein contents in control and infected larvae (df = 6; F = 15.957; p = 0.00). A 26.26% increment (110.26 to 139.30 mg/ml) in protein content was found in the plasma fraction after 3 h of infection, reaching 47.59%) within 9 h (163.01 mg/ml) of infection. From this point onwards, the protein content decreased up to 96.67 in 12 h, 86.48 in 18 h, and 77.12 mg/ml in 24 h after larval infection with nematodes. A similar pattern was observed in total protein contents in the whole hemolymph. Results obtained from previous hemocytic studies revealed that hemocytes of H. armigera played an important role during the nematode-bacterial infection (Istkhar and Chaubey 2018). Increment observed in protein contents by spectrophotometric study, just after 3 h, indicated the activation of H. armigera immune system responses by detecting nematode IJs which may be considered an early defense mechanism. The superior pathogenicity of Steinernema species perceived with the entry of the IJs in the insect host’s body confirmed by the elevated profile of protein in the hemolymph samples, which was almost steady in the control group up to 9 h.
During the analysis of separation of plasma protein contents through SDS-PAGE, lanes were loaded alternatively by plasma fractions of control and infected larval hemolymph plasma of H. armigera. The electrophoretic profile revealed the 8 major bands of proteins in hemolymph plasma with molecular weights of ~ 173, ~ 96, ~ 68, ~ 59, ~ 52, ~ 48, ~ 34, and ~ 17 kDa (Fig. 2). In nematode-bacterium challenged larvae an additional band of ~ 46 kDa was observed at 9 and 12 h of PIPs. This band vanished at 18 h of infection and a new band appeared at 24 h of infection with molecular weight of ~ 28 kDa. When the plasma fractions were analyzed in non-denatured conditions, there were 5 major bands (Rf values 0.09, 0.15, 0.38. 0.53, and 0.57) in the gel and no changes were observed revealed by Native-PAGE electrophoretic profile in control and infected groups of H. armigera (Fig. 3). Electrophoretic study performed on Rhipicephalus microplus (Boophilus) infected with Heterorhabditis indica showed no differences in SDS-PAGE profile of hemolymph proteins (Patrícia Silva Golo et al. 2016). In our study, the electrophoretic patterns of infected and control hemolymph was almost similar; however, the appearance and disappearance of proteins were observed in denatured electrophoretic profile at 9 h, 12 h, and 24 h in infected hemolymph. This can be explained as after entry of nematode-bacterium complex within the body of H. armigera, the host produces some defensive factors to combat with invading pathogens. The proteins are secreted during the initial phase of nematode growth more abundantly (Simões et al. 2000) as observed in the present study, where the amount of proteins in hemolymph increased significantly during initial hours of infection. The increment reported in hemocytes during initial hours of infection in the previous study (Istkhar and Chaubey 2018) acclaimed the increment in protein contents in present study and showed the secretion of some major proteins of hemolymph and some unknown proteins by hemocytes, which may help in initial protection and defend host to parasitic infection. This statement was supported by the presence of extra band at 9 h of infection. The disappearance of proteins bands occurred at 9 and 12 h as well as in the total protein contents were explained as the action of hydrolysing of the host proteins. These results agree with the Schmidt and Platzer (1980), who reported protein degradation, when Culex pipiens (L.) was infected by Romanomermis culicivorax. They suggested the production of some proteases from the nematodes leads to the degradation of hemolymph proteins of C. pipiens. Wee et al. (2000) suggested the production of proteases by bacterial cells, followed by the breakdown of insect’s protein and serving as nutritional resources for nematode-bacterium development. In another study, the reduction in total protein contents of hemolymph was observed in the hemolymph of Schistocerca gregaria (Forsskål) during the course of infection with EPF, Mertarhizium anisopliae var acridium (Gillespie et al. 2000). Other workers supported the study, where the productions of proteases and subsequent reduction in proteins by Steinernema carpocapsae and bacteria were reported (Toubarro et al. 2013). While studying the electrophoretic band pattern in native-PAGE for the agglutination and/or association of proteins during infection, no changes were observed and similar pattern banding was found in the control and the infected groups.
For encapsulation study, the first batch of nematode-infected larvae was dissected after 24 h only and not after 48 h as infected larvae were found dead after 48 h. During the time of dissection, the adhering of hemocytes was observed around the encapsulated nematode. In the first batch, 30, 20, 30, and 10% of larvae injected with 2, 5, 10, and 20 IJs of nematode were found having encapsulated IJs within their hemocoel. No strong melanization was noticed over encapsulated IJs. Mortality was observed in the larvae and 10, 30, and 50% mortality rates were found in H. armigera larvae infected with 5, 10, and 20 IJs/larva, respectively, at 24 h (Fig. 4a). No mortality was observed in the larvae injected with 2 IJs/larva. In a second batch of larvae injected with nematode, all nematodes were found dead in dissected larvae at 48 h in 10 and 20 IJs/larva doses, whereas in 2 and 5 IJs/larva doses, 70 and 80% after 72 h and one of the larvae attained pupation, but could not achieved emergence (Fig. 4b). The percentage of encapsulation of IJs was calculated in the larvae found positive for encapsulation, when injected with different doses of IJs. The proportion of encapsulated nematodes declined with increasing dose from 66.67% for 2 IJs/larva to 5% for 20 IJs/larva. For 5 and 10 IJs/larva, the encapsulation was 30 and 16.67%, respectively.
However, EPNs were found effective against the insect pests; the insect has also evolved defensive mechanisms against these nematode invaders (Feldhaar and Gross 2008). The innate immune responses included hemocytes, which participated in phagocytosis, nodulation, and encapsulation, and played a significant role and provide a protection to the insect hosts (Schmidt et al. 2001). The increments in hemocyte number in hemolymph of H. armigera infected with S. abbasi have already been observed in previous study (Istkhar and Chaubey 2018). The encapsulation responses of different hosts varied according to host species and nematode species. In Manduca sexta (L.), H. bacteriophora was recognized by (> 99 %) value, while S. glaseri showed only (28%) recognition (Li et al. 2009). The encapsulation study of S. feltiae and H. bacteriophora, against the prepupae of Leptinotarsa decemlineata (Say) showed a more frequent encapsulation of S. feltiae than for H. bacteriophora (Ebrahimi et al. 2011). On the other hand, in the rose sawfly Arge ochropus (Gmelin), the cellular responses were weaker to S. carpocapsae than in H. bacteriophora (Sheykhnejad et al. 2014). In another study, 6% encapsulation of S. feltiae was reported, while it was 24% in H. bacteriophora in Agriotes lineatus (L.) (Rahatkhah et al. 2015). Obtained results showed that the S. abbasi isolate CS2 had the strong capability to avoid encapsulation responses in larval H. armigera leading to the death of larvae within a very short time.