Research
Identification and functional analysis of insect immune genes of silkmoths
Diverse organisms like insects and mammals are now known to share many of molecular, cellular and developmental processes. A prominent example is the evolutionary conservation of innate immune system amongst the vertebrates and invertebrates. Insects rely solely on innate immune system, albeit recent studies on dipteran insect Drosophila melanogaster indicated the existence of systems with memory and adaptive ability. Due to the absence of adaptive immune system, insects provide an ideal model to study innate immune mechanisms of higher organisms. ‘Innate immunity’ referring to the first line of defense against microbial infections, comprises of humoral and cellular immune responses. Humoral immune responses include synthesis of antimicrobial peptides, and activation of the prophenoloxidase cascade leading to melanization; while cellular immune responses involve hemocytes in phagocytosis of microbes, nodule formation, and encapsulation of large pathogens.
An understanding of insect immune mechanisms offers strategies to control spread of insect vector borne diseases in animals. In addition, an efficient management of insect pests of agricultural crops and disease resistance of beneficial insects can also be achieved via a thorough knowledge of its immune system. D. melanogaster has been widely investigated to study insect immune responses towards diverse pathogens like bacteria, fungi, parasites and viruses. However, lepidopteran immune mechanisms remain less understood. Our lab is interested in identification and functional analysis of insect immune genes from silkmoths.
An expressed sequence tag (EST) library from bacteria challenged fat body of tasar silkmoth A. mylitta has generated a repertoire of information on both known as well as putative immune genes. The immune transcriptome consisted of known immune gene categories like antimicrobial proteins (attacins, cecropins, lysozymes, gloverins), recognition proteins (PGRPs, lectins, hemolins, GNBPs and mucins), serine proteases and protease inhibitors (Figure 1).
Figure 1: Distribution of immune-related transcripts in Antheraea mylitta fat body transcriptome. The figure in parentheses indicates the number of isoforms identified in that particular gene family.
Additionally, we found a number of genes with unknown function that could be implicated in control and execution of the immune response. Among the class of antimicrobial proteins, we found two lysozyme-like proteins (BLLP1 and ALLP1) that lacked one or both of catalytic residues. These proteins have not been reported in lepidopterans before and are homologous to dipteran lysozyme-like proteins of unknown function. The functional analysis of these proteins revealed that their antibacterial mechanism depended on peptidoglycan binding unlike peptidoglycan hydrolysis or membrane permeabilization as observed with classical lysozymes. This study thus led to the identification and further characterization of several antimicrobial genes.
The immune response in insects is dynamic and involves differential response towards different pathogens leading to expression of different effector genes during infection. Recognition of non-self from self is achieved by pattern recognition, mediated by a set of pattern recognition receptors (PRRs). These PRRs specifically recognize and bind to the pathogen-associated molecular patterns (PAMPs) (such as lipopolysaccharide, lipoteichoic acid and peptidoglycan from bacteria, and beta-1,3-glucan from fungi) present on the surface of microorganisms. Recognition of PAMPs by different PRRs will trigger a variety of humoral and cellular immune responses. Our study on the functional analysis of a putative defense related gene DFP-1 (designated as Noduler) from A. mylitta immune transcriptome indicated a role for this protein in nodulation response of insects. Noduler specifically bound lipopolysaccharide, lipotechoic acid and ?-1, 3 glucan components of microbial cell walls and also insect hemocytes. RNA-interference mediated knock-down of Noduler resulted in significant reduction in the number of nodules and level of phenoloxidase but showed increase in bacterial load in larval hemolymph. Thus, Noduler seemed to play a role in early clearance of bacteria by forming nodules of hemocytes and bacterial complexes (Figure 2). These results drew our attention to understand the logistic modules operating in the binding of Noduler to bacterial components and the hemocytes during nodulation response.
Our research is now focused on understanding the molecular mechanisms underlying the recognition of pathogens, as well as the signaling pathways implicated in immune response.
Figure 2: The suggested role for Noduler in the formation of nodules.
The results emanated from the studies point towards a vast repertoire of molecules involved in a variety of immune functions in insects. As more and more whole genome sequence data and expressed sequence tags data become available from diverse insect sources many more novel molecules implicated in immune function will emerge to be added to the already existing amazing diversity of immune molecules in insects that has contributed to the evolutionary success of insects as the most diverse and abundant taxon in the animal kingdom.
Projects being pursued
- Genetics of immunity in silkmoths
- Elucidation of immune pathways in silkmoths
- Identification of novel immune response genes in insects
Working group
- V V Satyavathi
- Asha Minz
- Akanksha Kakkar
Past Phd students
- Nirotpal Mrinal
Publications
- Mrinal, N., Tomar, A. and Nagaraju, J. (2011) Role of sequence encoded .B DNA geomtery in gene regulation by Dorsal. Nucleic Acids Research.
- Mrinal N and Nagaraju J (2010) Dynamic repositioning of dorsal to two different kB motifs controls its autoregulation during immune response in Drosophila. Journal of Biological Chemistry. 285:24206-24216.
- Mrinal N, Nagaraju J (2008) Intron loss is associated with gain of function in the evolution of gloverin family of antibacterial genes in Bombyx mori. Journal of Biological Chemistry 283: 23376-23387.
- Gandhe A S, John S H, Nagaraju J (2007) Noduler, a novel immune upregulated protein mediates nodulation response in insects. Journal of Immunology 179: 6943-6951.
- Gandhe AS, Janardhan G, Nagaraju J (2007) Immune upregulation of novel antibacterial proteins from silkmoths (Lepidoptera) that resemble lysozymes but lack muramidase activity. Insect Biochemistry and Molecular Biology 37: 655-666.
- Gandhe AS, Arunkumar KP, John SH, Nagaraju J (2006) Analysis of bacteria-challenged wild silkmoth, Antheraea mylitta (lepidoptera) transcriptome reveals potential immune genes. BMC Genomics 7: 184.
- Khurad AM, Kanginakudru S, Qureshi SO, Rathod MK, Rai MM, Nagaraju J (2006) A new Bombyx mori larval ovarian cell line highly susceptible to nucleopolyhedrovirus. Journal of Invertebrate Pathology 92: 59-65.
- Johny S, Kanginakudru S, Muralirangan MC, Nagaraju J (2006) Morphological and molecular characterization of a new microsporidian (Protozoa: Microsporidia) isolated from Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Parasitology 132: 803-814.
- Khurad AM, Mahulikar A, Rathod MK, Rai MM, Kanginakudru S, Nagaraju J (2004) Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L. Journal of Invertebrate Pathology 87: 8-15.
- Jain D, Nair DT, Swaminathan GJ, Abraham EG, Nagaraju J, Salunke DM (2001) Structure of the induced antibacterial protein from tasar silkworm, Antheraea mylitta. Implications to molecular evolution. Journal of Biological Chemistry 276: 41377-41382.
- Abraham EG, Nagaraju J, Salunke D, Gupta HM, Datta RK (1995) Purification and partial characterization of an induced antibacterial protein in the silkworm, Bombyx mori. Journal of Invertebrate Pathology 65: 17-24.