Studies on immunomodulatory effect of endophytic fungus Alternaria alternata on Spodoptera litura Harminder Preet Kaur a , Bahaderjeet Singh b , Abhinay Thakur a , Amarjeet Kaur b , Sanehdeep Kaur a, ⁎ a Department of Zoology, Guru Nanak Dev University, Amritsar 143005, India b Department of Microbiology, Guru Nanak Dev University, Amritsar 143005, India abstract article info Article history: Received 4 August 2014 Revised 23 November 2014 Accepted 25 November 2014 Available online 6 December 2014 Keywords: Immune response Total hemocyte count Differential hemocyte count Phenoloxidase and lysozyme activities Secondary metabolites of entomopathogenic fungi have been known to suppress the immune response of vari- ous insects. Hence, we have investigated the effect of endophytic fungus, Alternaria alternata on cellular and hu- moral immune response of Spodoptera litura (Fabricius). Significant drop in total hemocyte count has been observed in the larvae fed on diet supplemented with ethyl acetate extract of A. alternata as compared to those fed on untreated or control diet devoid of ethyl acetate extract. Similarly differential hemocyte count of the larvae feeding on amended diet differed significantly from control. As compared to control significantly higher percent- age of hemocytes exhibited abnormalities like aggregation, change in shape, extensive vacuolization and necro- sis. The cytotoxic effect showed a dose dependent response which increased with time. A reduction in phenoloxidase and lysozyme activities was also observed. The data suggest that the secondary metabolites pro- duced by A. alternata disabled several immune mechanisms of the insect. These studies would help to identify the insect defenses that could be manipulated to accelerate host death due to fungal endophytes. © 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved. Introduction Insects live in different environments where they are exposed to var- ious potential invaders such as pathogens, parasites and parasitoids. However, their success to colonize every niche on earth has been attrib- uted to their ability to neutralize pathogen invasions (Dunn, 1986; Lowenberger, 2001; Silva et al., 2002). Insects have a highly efficient im- mune system that is able to withstand challenges from majority of micro- organisms present in the different habitats where they live. The innate immune system of insects is subdivided into — humoral and cellular de- fenses (Strand, 2008). Humoral response includes the synthesis of broad spectrum of antimicrobial proteins and phenoloxidase (PO) as well as the production of reactive intermediates of oxygen and nitrogen (Ashida and Brey, 1998; Bulet et al., 1999; Lavine and Strand, 2002; Cerenius and Soderhall, 2004). PO is the most important humoral component of insect immune response and has been believed to be involved in wound healing and sclerotization of the cuticle and recognition and melaniza- tion of foreign particles (Ashida and Yamazaki, 1990; Sugumaran, 1998). It is present in an inactive form, prophenoloxidase (Pro PO) and is released by hemocytes upon activation by biological activators. PO generated quinones may serve as toxic metabolites that might be harmful to the intruders (Ashida and Yamazaki, 1990). Lysozyme is one of the antibacterial peptides that is produced by hemocytes and fat body of insects and plays a significant role in immune response. In contrast, cellular defense refers to hemocyte mediated responses like phagocytosis, nodulation and encapsulation (Strand and Petch, 1995; Schmidt et al., 2001). There is considerable overlap between humoral and cellular defenses. Many humoral factors affect hemocyte functions and hemocytes are an important source of many humoral molecules (Lavine and Strand, 2002). Cellular response in the insect immune system has been shown to be an important barrier to the infection process (Hoffmann, 1995, 2003). Hemocyte types and their specific responses during insect–pathogen in- teraction are good indicators of insect defense reactions (Da Silva et al., 2000; Gillespie et al., 2000). Several classes of hemocytes have been morphologically and functionally characterized, mainly in Diptera, Lep- idoptera and Coleoptera (Lavine and Strand, 2002; Giulianini et al., 2003; Costa et al., 2005; Giglio et al., 2008). The most common types of hemocytes reported in the literature are prohemocytes, granulocytes, plasmatocytes, adipohemocytes, and oenocytoids. In larval stage of Lep- idoptera, granulocytes and plasmatocytes are the only hemocyte types capable of adhering to foreign surfaces and together usually comprise more than 50% of the hemocytes in circulation (Ratcliffe, 1993; Strand and Petch, 1995). Multifunctional role of hemocytes such as phagocyto- sis, encapsulation, nodule formation, cell agglutination, detoxification as well as synthesis and secretion of the humoral immunity factors may render them more sensitive than the other cells towards internal and external factors. The hemocytes are found to show changes in their number and configuration under different stresses which finally affect Journal of Asia-Pacific Entomology 18 (2015) 67–75 ⁎ Corresponding author. Tel.: +91 183 2258802 09; fax: +91 183 2258819, 2258820. E-mail address: sanehsaini@gmail.com (S. Kaur). http://dx.doi.org/10.1016/j.aspen.2014.11.004 1226-8615/© 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape