Changes in protein profile and encapsulation avoiding responses of entomopathogenic nematode in the American bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)
- 108 Downloads
The American bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is a highly destructive agriculture pest of worldwide importance. The aim of the present study was to infect H. armigera larvae with entomopathogenic nematode juveniles for hematological study of proteins and encapsulation responses to evaluate using this nematode for the management of this pest. Total protein estimation and the electrophoretic profiling carried out in the hemolymph showed a high pathogenicity of Steinernema abbasi to H. armigera. The control group survived and succeeded to develop to adults, while the infected ones died within 24 h. An increase in the protein contents in the total and plasma hemolymph was observed just after 3 h of infection with an increase at 6 h and 9 h as symptoms of early defence of the insect. SDS-PAGE profile also showed an evolvement of a protein band of 46 kDa. No self-association or aggregation and binding of other proteins were found in the hemolymph as revealed by Native-PAGE. The encapsulation avoidance rate of nematode juvenile gave good results with (> 33%) in 2 IJs/larva to 5% in 20 IJs/larva doses at24 h post infection. Loss of hemolymph proteins continued for more than 24 h with a very low recognition of nematode rate in hemolymph, followed by the death of larvae within 48 h, which proved the high pathogenicity of S. abbasi and suppression of host immune system of H. armigera.
KeywordsHelicoverpa armigera Entomopathogenic nematode Steinernema abbasi Protein estimation Encapsulation
Insect immunology is a rapidly growing discipline with exciting and innovative application, especially in control of agricultural pests by understanding the host parasite interactions such as entomopathogenic nematodes (EPNs). When insect pests of agricultural crops are targeted by microorganisms, they rely on innate immunity for quick response comprises cellular and humoral activity.
The American bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a polyphagous worldwide distributed pest infests more than 181 plant species belonging to 45 families (Singh 2005). Defensive strategies of H. armigera are poorly understood, and this insect has developed a strong resistance against several chemical pesticides (Bass et al. 2015). Studying the immune system of pests helps better understanding of their defenses against invading parasites (EPNs), which are nowadays been used in insect pest management. In this regard, less information is available on immunological defenses in hemolymph of H. armigera creating its management difficult. Results of pathogenicity of EPNs against H. armigera larvae provided a good picture and better management in a comparatively environmentally safer way (Istkhar and Chaubey 2017).
The EPNs, steinernematids and heterorhabditids have become a new tool as they have a wide variety of host range, high virulence, less killing time, host finding ability, fast acting generations, and safe to vertebrates and non-target invertebrates (Lacey and Georgis 2012). Steinernematids are naturally associated with symbiotic bacteria of the genus Xenorhabdus (Gammaproteobacteria: Enterobacteriaceae). Researchers showed that not only the nematodes, but also the bacterial symbionts are highly pathogenic to many insect pests once they reached the hemocoel of the insect (Forst and Nealson 1996). During their complex life cycle in a common insect host, bacteria carried by the nematodes in their gut are regurgitated in the hemocoel of insect, where they multiply along with nematodes and produced several toxins, leading to the death of insect. Hemolymph plays a vital role for insects as the place where they compete with invading microorganisms. Production of antimicrobial peptides (AMPs), activation of melanization, phagocytosis, and encapsulation are the main events against EPN infection in the insect hemolymph (Castillo et al. 2011). When the nematode enters in the hemocoel of insect, soluble proteins in hemolymph and proteins released by hemocytes and hematopoietic organs participate in their encapsulation (Lemaitre and Hoffmann 2007). To avoid the targeting, nematode and/or nematode-bacterial complex also attempt to evade the poorly understood immune response of insect immune system (Brivio et al. 2010). Little information is available about the nematode infection and hematological changes in H. armigera.
The present study was planned to study the hematological responses of H. armigera larvae infected with 3rd stage infective juvenile (IJs) of Steinernema abbasi for protein and nematode encapsulation.
Materials and methods
Eggs of H. armigera were obtained from the National Bureau of Agriculturally Important Resources (NBAIR), ICAR, Bangalore, India (National Accession No. NBAII-MP-NOC-01), and the culture was maintained in the laboratory. Larvae were fed on a semisynthetic diet (Nagarkatti and Prakash 1974), while the adults were fed on 10% honey solution containing vitamin E for better egg laying. A culture of the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), was also maintained in the laboratory for in vivo culturing of the EPNs.
In vivo nematode culture
Steinernema abbasi (Rhabditida: Steinerneamtidae) was found to be the most dominating and pathogenic species during the survey conducted in studied area and showed a strong pathogenicity to H. armigera (Istkhar and Chaubey 2017). Isolate CS2 of S. abbasi originally isolated from the soil of District Meerut (U.P.), India, was used to infect the larvae of H. armigera for hematological study. Population of S. abbasi was maintained in vivo on larvae of G. mellonella, which considered as the universal host of EPNs. Freshly emerged 3rd stage infective juvenile (IJs) were collected from White trap (Stock and Goodrich-Blair 2012) and stored in BOD at 15 ± 1 °C and used to infect the larvae of H. armigera for experimentation within a week of emergence.
Experimental design for protein estimation
For studying the changes in protein quantity and protein profiling through electrophoresis in hemolymph of H. armigera, the hemolymph was collected at different post infection periods (PIP) after infection with 100 IJs of S. abbasi (isolate CS2) in six-well Petri plates (Himedia) lined with a double layer of Whatman filter Paper (No. 1) by maintaining the adequate moisture. Larvae were then surface sterilized and 50 μl of hemolymph from each larva was drawn directly in pre-chilled 1.5 ml centrifuge tubes containing 450 μl of phosphate buffer saline (PBS, pH 7.2) by cutting their proleg at 0, 3, 6, 9, 12, 18, and 24 h post infection period (PIP). Control group received 450 μl of distilled water and used for hour to hour protein comparison. Control and treated group were placed in BOD at 27°±2 °C. For each hour interval, 10 out of 20 infected larvae were randomly selected for hemolymph collection, and the whole experiment was repeated twice. Hemolymph of larvae in the control group was also collected in a similar way. Hemolymph was collected singly from each larva, and mean values of 10 replicates were taken as mean quantity of protein in milligram/milliliter of hemolymph.
For the assessment of combined effect of soluble proteins and hemocytic protein (proteins secreted by hemocytes) changes due to nematode infection in hemolymph and soluble proteins only, the hemolymph samples were tested as total hemolymph and plasma fractions, respectively. Total protein contents in plasma fraction of hemolymph samples, collected from the infected and control larvae of H. armigera in PBS solution (pH 7.2), were estimated according to Bradford’s method (Bradford 1976). Two-hundred microliters of the hemolymph-PBS mixture mentioned above was retained as a whole hemolymph, and the rest was centrifuge at 10,000 g for 20 min at 4 °C to separate out plasma contents. Protein was estimated as total protein in hemolymph and in plasma fraction in terms of soluble proteins. Both samples were used for protein estimation and were expressed in milligram/milliliter of hemolymph. Dilutions of bovine serum albumin (BSA) were used to prepare a standard curve for estimation of protein.
Electrophoresis of hemolymph samples
Reducing electrophoresis of cell-free hemolymph was performed using 12% resolving and 4% stacking polyacrylamide gel (SDS-PAGE) (Laemmli 1970) on Genei electrophoretic unit for 1-D gel electrophoresis using power supply set at 15 mA for 10 min and at 30 mA for 120 min. A broad range (10–250 kDa) multicolour ladder (Thermo ScientificTM) was run along with hemolymph samples for estimating and comparing changes in protein samples. Lanes of gel were loaded with 5-μl plasma sample side by side collected from parasitized and unparasitized larvae of H. armigera, respectively. After electrophoresis, the gels were stained, using coomasie brilliant blue R250 stain. The analyses of gels were performed through My Image Analysing Software Version 2.0 (Thermo Scientific). For non-reducing electrophoresis, Native-PAGE was performed for all the samples to study self-association or aggregation and the binding of other proteins if any.
Experimental design for encapsulation responses
Descriptive analysis was performed for quantified protein at each hour interval and presented as mean ± standard error of mean (SEM) of 10 replicates randomly selected from infected with EPNs and control larvae of H. armigera. Two-way ANOVA was performed to find out the effect of time and treatment on total protein contents. One-way ANOVA was executed to compare protein differences in infected and control larvae. Fisher’s least significance difference (Fisher’s LSD) was used as post hoc test for comparison within infected and control at each hour interval.
Results and discussion
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.
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.
This study revealed that the presence of nematode within the hemolymph significantly decreased the amount of protein contents. The low encapsulation avoiding response led to a high number of nematode in hemolymph and subsequently reduced the amount of defensive proteins. More number of IJs in the hemolymph produced more toxins, which weaken the insect’s immune system because of the quick death of the host larvae. The results showed that S. abbasi isolate CS2 had a high pathogenicity merit and provided an insight to use it as a better alternative of biological control agent in future insect pest management programs.
Authors greatly acknowledge the Department of Zoology, Chaudhary Charan Singh University, Meerut, India, for providing infrastructure.
Istkhar and AKC conceived and designed the study and critically revised the manuscript. Istkhar performed the experiments and analyzed the data. Both authors have approved the final manuscript.
Authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for funding through EMR project (37(1550)/12-EMR-II).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
- Gillespie JP, Burnettand C, Charnley AK (2000) The immune response of the desert locust Schistocerca gregaria during mycosis of the fungus, Metarhizium anisopliae. J Physiol 46:429–437Google Scholar
- Nagarkatti S, Prakash A (1974) Rearing of Heliothis armigera (Hubn) on artificial diet. Technical Bulletin of Commonwealth Institute of Commonwealth Institute of Biological Control, Bangalore, pp 17, pp 169–173Google Scholar
- Patrícia Silva Golo PS, de Oliveira dos Santos AS, Monteiro CMO, de Souza Perinotto WM, Quinelato S, Camargo MG, de Sá FA, da Costa AI, Martins MF, de Azevedo Prata MC, Bittencourt VREP (2016) Lab-on-a-chip and SDS-PAGE analysis of hemolymph protein profile from Rhipicephalus microplus (Acari: Ixodidae) infected with entomopathogenic nematode and fungus. Parasitol Res. https://doi.org/10.1007/s00436-016-5109-z CrossRefGoogle Scholar
- Schmidt SP, Platzer EG (1980) Hemolymph composition of mosquito larvae infected with a mermithid nematode. J Nematol 10:299–304Google Scholar
- Singh OP (2005) Consumption pattern of insecticide in Helicoverpa armigera management in India. In: Saxena H, Rai AB, Ahmed R, Gupta S (eds) Recent Advances in Helicoverpa armigera Management. Indian Society of Pulses Research and Development, Kanpur, pp 17–24Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.