Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 93-106, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-112 Citation: H.P. Shambhavi, P. Mak- wana, B. Surendranath, K.M. Pon- nuvel, R.K. Mishra, A.N.R. Pradeep (2020) Phagocytic events, associated lipid peroxidation and peroxidase activ- ity in hemocytes of silkworm Bombyx mori induced by microsporidian infec- tion. Caryologia 73(1): 93-106. doi: 10.13128/caryologia-112 Received: January 9, 2019 Accepted: February 22, 2020 Published: May 8, 2020 Copyright: © 2020 H.P. Shambhavi, P. Makwana, B. Surendranath, K.M. Pon- nuvel, R.K. Mishra, A.N.R. Pradeep. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Phagocytic events, associated lipid peroxidation and peroxidase activity in hemocytes of silkworm Bombyx mori induced by microsporidian infection Hungund P. Shambhavi1, Pooja Makwana2, Basavaraju Surendranath3, Kangayam M Ponnuvel1, Rakesh K Mishra1, Appukuttan Nair R Pradeep1,* 1 Proteomics Division, Seribiotech Research Laboratory, CSB-Kodathi Campus, Carmel- aram P.O., Bangalore-560035, Karnataka, India 2 Central Sericultural Research and Training Institute, Berhampore, India 3 Central Tasar Research and Training Institute, Ranchi, India *Corresponding author. E-mail: arpradeepnair@gmail.com Abstract. Microbial infections induced humoral and cell- mediated immune events in hemocytes. After infection by the microsporidian Nosema bombycis in the com- mercially important silkworm, Bombyx mori, hemocytes exhibited deformed nucleus and degranulation of structural granules by exocytosis. Granulocytes showed signs of phagocytosis included formation of microvilli, pseudopodia, engulfment of spores, phagosome formation and membrane porosity. Association of membrane disintegra- tion with infection – induced lipid peroxidation (LPO) was revealed by testing level of malondialdehyde, a byproduct of LPO. LPO activity enhanced significantly (P < 0.0002) throughout infection with peak activity in later stages of infection from day 11 accompanied by hemocyte plasma membrane disintegration. Partial increase in LPO activity coupled with increased peroxidase activity recorded in early and mid stag- es of infection. In later stages, peroxidase activity decreased however LPO increased accompanied by phagocytosis events. In hemocytes, phagocytic events are initiated by activation of genes encoding recognition proteins, aggregation factors and immune- associated proteins. ß-GRP expression was down regulated after the infection whereas CTL-11 enhanced expression on day 10. Humoral lectin enhanced expression on day 6 whereas apolipophorin showed 2.59 fold increase on day 10 after infection. Gene encoding cytoskeletal protein, ß- Actin showed stable enhanced expression throughout infection showing positive correlation (R2 = 0.65) with age after infection. Phagocyto- sis- associated gene Eater from Drosophila showed enhanced heterologous expression. Altogether phagocytic events induced by microsporidian infection are accompanied by increased LPO, decreased peroxidase activity and modulated gene activity in hemo- cytes of B. mori. Keywords. Phagocytosis, lipid peroxidation, peroxidase activity, Bombyx mori, micro- sporidian infection, hemocytes. 94 Hungund P. Shambhavi et al. INTRODUCTION Insects have developed functionally active immune system for survival in the widespread habitat. Insect immune system comprises humoral and cellular respons- es as well as phenol oxidase cascade culminates in mela- nisation. Innate immunity components included activa- tion of different pathways such as Toll, IMD and JAK- STAT pathways effecting in production of antimicrobial proteins (Hoffmann 2003; Govind 2008). Cell- medi- ated responses, facilitated instantaneously by hemocytes against pathogens (Barillas-Mury et al. 2000) involved nodulation, encapsulation and phagocytosis depending on size of the pathogen/ parasite (Rosales 2011). Phago- cytic response of a cell is evolutionarily conserved from protozoan to mammals (Faurschou and Borregaard 2003; Nazario-Toole and Wu 2017) which involved recognition, binding and ingestion of parasites (Rosales 2011; Kwon et al. 2014). Phagocytic receptors evoke different signalling pathways such as Draper activated Draper/Src/Syk/CED-6 pathway (Fullard et al. 2009) whereas TEP (thioester con- taining protein) activated CSD6 pathway (Blandin et al. 2004). Activation of signalling leads to cytoskeletal rear- rangement, insertion of new membrane for pseudopodia and microvilli formation for parasitic engulfment (Bajno et al. 2000). In Drosophila, plasmatocytes and granulo- cytes act as major phagocytic cells against bacteria (Cas- tillo et al. 2006; Lamprou et al. 2007) whereas in lepidop- terans granulocytes are modified to become phagocytic cells (Ling and Yu 2006; Rebeiro and Brehelin 2006). Though commercially important silkworm, Bombyx mori (Lepidoptera) is domesticated and reared under protected conditions, worms are exposed to pathogenic attack causing major losses to silk industry. In B. mori, infection by the obligate intracellular microsporidian parasite Nosema bombycis causes devastating disease, pebrine. N. bombycis infects either through transovarian transmission or through secondary contamination by feeding contaminated mulberry leaves (Hukuhara 2017). On reaching midgut, spores germinate and inject sporo- plasm into midgut epithelial cells (Franzen 2005) where it multiplies and spread to other host tissues including haemolymph and hemocytes. Though pathogens spread through eggs to newly hatched larvae, symptoms or presence of spores could not be identified in initial stag- es of infection and spores could be identified only after six days of infection (Ma et al. 2013). In B. mori, infection by N. bombycis suppressed host responses, inhibited melanization events and down reg- ulated immune genes in midgut (Pan et al. 2013; Ma et al. 2013). However hemocyte- mediated cellular respons- es against microsporidian infection is not known. In this investigation, cellular, biochemical and molecu- lar immune responses of hemocytes in B. mori were revealed showing induction of LPO activity and per- oxidase activity in association with phagocytosis events against N. bombycis infection. MATERIALS AND METHODS Infection and sample collection B. mori larvae were reared on mulberry (Morus sp) leaves under standard rearing conditions of 25 ± 2oC, 70% relative humidity and natural photoregime (13L : 11D). Initially larvae were grown till third instar and separated after third moulting to fourth instar. Day 0 fourth instar larvae were collected and exposed to experimental infection by feeding spores of N. bombycis (standard strain: NIK-1s_mys) smeared on mulberry leaf with a dose of 1 x 106 spores / larva that induced 50% mortality within the lethal time LT50 (Rao et al. 2004). Non-infected B. mori larvae of the same age group were used as control and reared separately. In order to analyse hemocyte responses after N. bombycis infection, haemolymph samples were collect- ed from control and infected larvae on day 0, 2, 6, 8, 9, 10, 11 and 12 after the infection. Haemolymph was col- lected by piercing first abdominal proleg using a steri- lized needle. Haemolymph of pupae were collected from newly formed pupa (14 days after infection) by punctur- ing leg impressions on ventral side. The hemocytes were separated by centrifugation at 880 g for 10 min at 4oC. The hemocyte pellets were washed with anticoagulant solution (0.098M NaOH, 0.186M NaCl, 0.017M EDTA, 0.041M Citric acid, pH 4.5 adjusted using NaCl) twice and stored at -80oC for protein analysis. For total RNA extraction, hemocyte pellets were stored in RNA stabili- zation reagent RNA later (Qiagen). Light microscopy and transmission electron microscopy (TEM) Hemocytes of infected and non-infected control lar- vae were observed under inverted tissue culture micro- scope (Leica) and the hemocytes and spores were count- ed using hemocytometer. In order to carryout TEM analysis, hemocyte samples were processed essentially as described earlier (Pradeep et al. 2013). Briefly, hemocytes were fixed in 3 % glutaraldehyde upto 24 h before fixa- tion in 1% osmium tetroxide. The samples were dehydrat- ed through alcohol series, brought to acetone and then stained with 2 % uranyl acetate. Using an embedding kit 95Phagocytosis and lipid peroxidation in hemocytes of B. mori induced by microsporidian infection (Araldite Embed- 812) hemocyte samples were embedded in araldite for 48h. Ultrathin sections (100nm) were cut using Ultramicrotome (Leica –EM UC6) and placed on a copper grid. The ultrathin sections were stained with uranyl acetate and lead citrate and observed under TEM. Ultrastructural variations in the hemocytes (n = 50 each) were observed at 60 kV in a Tecnai G2 transmission elec- tron microscope attached with Megaview Soft Imaging System and photographed (at the TEM facility in Nation- al Institute for Mental Health and Neuro Sciences (NIM- HANS), Bangalore, India). Lipid peroxidation In order to examine lipid peroxidation in the hemo- cytes of B. mori larva infected with N. bombycis, hemo- cyte samples were collected at different time points after infection along with sample from same aged non- infected control. The assay was performed using EZAs- sayTM TBARS lipid peroxidation estimation kit following manufacturer’s (HiMedia) protocol. Membrane lipids are destructed by phagocytes to form lipid peroxides (Mylonas and  Kouretas 1999) which in turn break- down to form a by-product malondialdehyde (MDA) which is measured by absorbance. The lipid peroxidation was estimated as malondialdehyde (MDA) equivalents (Hodges et al. 1999) in a 96 well microtiter plate using absorbance reading at 532 nm in a microtiter plate read- er (Multiskan spectrum, Thermo). From 1- 10 µM MDA, a standard curve was obtained by using a slope of stand- ard curve (y = 0.00457+0.00416x) and MDA concentra- tion in the sample was calculated (Yagi 1998; Fatima et al. 2011). Ascorbate peroxidase activity In order to test whether peroxidase activity var- ied in hemocytes after the infection, ascorbate peroxi- dase (PRX) activity was determined as described earlier (Nakano and Asada 1981). Hemocyte pellet was collected from haemolymph at different time points after the infec- tion and then lyzed in Insect cell lysis buffer and quan- tified the protein (Lowry et al. 1951). Briefly, PRX assay was performed using 750 µl 100 mM phosphate buffer (pH- 7.0), 150 µl of 5.0 mM ascorbate and 300 µl 0.5 mM of H2O2. The reaction was carried out in 1.5 ml Eppen- dorf tubes using 300 µl of hemocyte lysate sample con- taining 100 µg protein. The reaction mixture was trans- ferred to 96 well plate. The rate of decrease in absorbance was read at 290 nm at every one minute interval for ten minutes duration. The enzymatic activity was calculated using a molar extinction coefficient of 2.8 mM-1 cm-1 and the result was expressed as micromoles per minute per milligram of protein (Nakano and Asada 1981). Hydrogen peroxide activity In order to test variation in hydrogen peroxide (H2O2) level after infection, assay was performed using hemocyte extract in phosphate buffered saline (PBS) buffer (pH 7.1) as described earlier (Velikova et al. 2000; Pooja et al. 2017). The absorbance was measured at 390 nm in the microplate reader and H2O2 content was cal- culated based on a standard curve. Total RNA extraction and cDNA synthesis Total RNA was extracted from hemocytes collected from control and N. bombycis- infected B. mori larva. Genomic DNA contamination was removed from total RNA by incubation with RNAse free – DNAse I (Taka- ra). Complementary DNA (cDNA) was synthesized from 1 µg total RNA using oligo d(T) primer and M-MuLV (Moloney Murine Leukemia virus) reverse transcriptase using cDNA synthesis kit as per manufacturer’s pro- tocol (Primescript; Takara). RT-PCR was performed to analyse semiquantitative expression on 0, 2, 6, 8 and 10 days after infection using gene- specific primers (Table 1) which was validated by qPCR. Quantitative PCR (qPCR) qPCR was performed using DyNaMo SYBR GREEN qPCR Master Mix (Thermo - Finzyme) with 0.3X Rox as passive reference dye, on Agilent Stratagene Mx3005P qPCR system. A 25 μl reaction mixture contained 2.5 μl cDNA template, one pmol each of forward and reverse primers and 12.5 μl SYBR Green qPCR master mix (1X) containing 4 mM MgCl2. The thermal program was set as 95°C for 15 min, followed by 40 cycles of 95°C for 30 seconds and at primer- specific annealing tempera- ture (Tm) for a minute (Table 1).The PCR products were resolved by 1.5 % agarose gel electrophoresis and con- firmed target-specific amplification. For housekeeping gene, ribosomal protein gene was used and fold change in expression relative to calibrator was calculated. Cloning and sequencing From B. mori genome phagocytosis- associated genes TEP-1 and Eater are not reported. In order to con- 96 Hungund P. Shambhavi et al. Ta bl e 1. K ey to th e ge ne s an d en co di ng p ro te in s as so ci at ed w ith p ha go cy to si s in du ce d in h em oc yt es o f B . m or i l ar va a fte r in fe ct io n w ith th e m ic ro sp or id ia n N . b om by ci s. G en e na m e N uc le ot id e A cc es si on N o. Pr im er (L eft ( L) a nd r ig ht ( R )) Tm (° C ) Pr ot ei n A cc es si on N o. Fu nc tio ns R ef er en ce T EP - 1 A Y 43 37 51 5’ G T G G C TA A G C C C A G T T T C A G 3’ L 5’A G T C A C T C G G A A G C A C T C G T 3’ R 59 .4 A B G -0 03 44 13 H em oc yt e m ed ia te d co m pl em en t l ik e pr ot ei n, Pl as m od iu m b er gh ei b in di ng a nd m ed ia te s ki lli ng Bl an di n et a l, 20 04 Ea te r N M _1 43 27 6 5’A TA G C C G C T G C T G A T G A C T C 3’ 5’ T C T T C G A T C C G G C A A A A C 3’ 56 .5 Q 9V B 78 Tr an sm em br an e pr ot ei n, s ca ve ng er r ec ep to r, ba ct er ia l r ec og ni tio n an d ph ag oc yt os is . K oc ks e t a l, 20 05 ; C hu ng e t a l., 2 01 1 βG R P- 2 N M _0 01 04 39 85 5’A A T G A C A C T G T T C G C G T T C C 3’ 5’ T C G C A C T C T C G T C T T T G T T G 3’ 57 .3 H 9J Q 04 R ec og ni tio n of β - 1, 3 g lu ca n fr om fu ng i a nd m ed ia te c el lu la r re sp on se s. K im e t a l, 20 00 X ia ng -J un R ao e t a l 2 01 4 βG R P- 4 N M _0 01 16 61 42 5’A C C T T G T C G A A T C C A G A G G C 3’ 5’ C G G G T C TA T T G T T G A A G C C G 3’ 59 .4 Q 9N L8 9 R ec og ni tio n of β - 1, 3 g lu ca n fr om fu ng i a nd m ed ia te c el lu la r re sp on se s. K im e t a l, 20 00 X ia ng -J un R ao e t a l 2 01 4 C T L- 1 1 B G IB M G A 00 66 23 5’ T C T G G T C G G T C G G C T G TA TA 3’ 5’ G A G C T G C T C C G C TA T G A A C T 3’ 59 .4 D 2X 2F 7 A ct s as o ps on in a nd in cr ea se p ha go cy to si s. Pe nd la nd e t a l, 19 88 C T L- 1 7 N M _0 01 13 08 99 5’A C G T C C T G C A TA C C G A A A A G 3’ 5’ G C C T C G T C TA A C G A T T C A G G 3’ 58 .3 Q 06 FJ 6 A ct s as o ps on in a nd in cr ea se p ha go cy to si s. Pe nd la nd e t a l, 19 88 A po lip op h- or in I /I I A B 64 06 23 5’ T G G C G G A TA A A T G C T C G T T G 3’ 5’ T C T T C T T C C G C G C A A A T C T G 3’ 57 .3 G 1U IS 8 A ct a s pa tt er n re co gn iti on p ro te in a nd b in ds to fu ng al β - 1, 3 g lu ca n, a id in p ha go cy to si s. W hi tt en e t a l. 20 04 ; B ar ab as a nd C yt ry ńs ka , 2 01 3 H um or al le ct in N P_ 00 11 04 81 7. 1 5’ G G C G G TA C A A C G T TA A G G A G 3’ 5’A A C G A G C A C C G A C A C A A G TA 3’ 58 .3 P9 80 92 A dh es iv e pr ot ei n an d re la te s to h em os ta si s or en ca ps ul at io n of fo re ig n su bs ta nc es fo r se lf- de fe ns e. K ot an i e t a l. 19 95 Be ta A ct in A 4 N M _0 01 12 62 55 5’A T C C T G C G T C T G G A C T TA G C 3’ 5’A A G A C T T C T C G A G G G A G C T G 3’ 59 .4 P8 41 83 C yt os ke le ta l p ro te in , h el ps in fo rm at io n of ce llu la r pr oj ec tio ns . M ay e t a l. 20 01 ; M ay a nd M ac he sk 20 01 Ri bo so m al p ro te in N P_ 00 10 37 25 9. 1 5’ T G G A G C G C C T TA C A A A C T C T 3’ 5’ G C C A G A T T G C T T G G T T G A C T 3’ 57 .3 Q 5U A N 9 H ou se - ke ep in g ge ne Lu e t a l 2 01 3 97Phagocytosis and lipid peroxidation in hemocytes of B. mori induced by microsporidian infection firm heterologous expression of TEP-1 and Eater, mRNA sequence of TEP-1 of Aedes aegypti (Acc No.AY432915.1) and Eater of Drosophila melanogaster (Acc. No. HM165182) were collected from NCBI database and prim- ers were designed (Primer 3 program) and used for qPCR (Table 1). The amplicons were resolved on 1.2 % agarose gel containing the fluorescent dye ethidium bromide. The Eater gene amplified at expected product size. In order to confirm the sequence identity Eater amplicon from RT- PCR was purified using a PCR purification kit (QIAquick, Qiagen) and cloned into a vector pJET 1.2/blunt using CloneJet PCR cloning kit (Thermo Scientific) as per man- ufacturer’s protocol. The cloned product was transformed into JM 109 competent cells. Through colony PCR, plas- mids were confirmed and the plasmid DNA was isolated using QIAprep spin miniprep kit (Qiagen). Presence of the fragment was confirmed by PCR and sequenced using Sanger method at a facility (Applied Biosystems) available at Eurofins Genomics India Pvt. Ltd., Bangalore, India. The nucleotide sequence was used to perform BLASTN 2.8.1+ (NCBI) search against non-redundant database. The nucleotide sequence was then translated to protein sequence using Expasy – Translate tool (https:// web.expasy.org/translate/). The 3’5’ Frame 2 of the trans- lated sequence was used for analysis by BLASTP 2.8.1+ (NCBI) and searched against non-redundant GenBank CDS translations, PDB, SWISSPROT, PIR and PRF. Statistical analyses The data were presented as Mean ± SD. Significance of difference between means was evaluated by Students’ t- test or single factor ANOVA. Correlation between var- iables was analysed by linear regression (y = a + bx). Quantitative gene expression was performed using cDNA synthesized from total RNA of hemocytes of control and infected B. mori larva, relative to the cali- brator using Mx3500P Real Time PCR system (Agilent). Average threshold cycle (Ct) value of transcript expres- sion was calculated from triplicates using ΔΔ Ct method (Livak and Schmittagen 2001) and normalized by house- keeping gene encoding ribosomal protein. Comparative Ct values of the genes were standardized by Ct values for the house keeping gene encoding ribosomal protein. Ct values were standardized with average control value, providing ΔCt value which is standardized to make the average control value ‘1’ (the ΔΔ Ct values) (Gerardo et al. 2010). Fold change in gene expression was calculated with reference to the calibrator, which in turn presented down regulated relative quantities as negative values. The data (mean ± SD) denoted is the gene expression induced by infection after eliminating the changes in control. RESULTS Organismal variations Day 0 fourth instar larvae were experimentally infected by feeding spores of N. bombycis smeared on mulberry leaf with LD50 dose of 1 x 106 spores / larva. After the infection, changes were not observed in lar- val behaviour and activity till day 2. Larval death was recorded from four days after infection. Moulting of infected larvae to fifth instar delayed by 24 h in compar- ison to control. Infected larvae were smaller in size and showed reduced growth from day 8. Infected larvae ini- tiated cocoon spinning at 24 h after control larvae spun silk. Cocoons of infected larvae were smaller and flimsy with lower silk content. Infected larvae were black and showed melanization under cuticle. Infected pupae were smaller in size and acutely melanized (Fig 1 A-C). N. bombycis spores were absent in non- infected control larval haemolymph and tissue samples. In N. bombycis- infected larvae (n = 30 each), spores were not found till day 6. On day 6, spore count was 0.1 x 105 / ml haemolymph. On day 9 the count was 0.95 x 105 spores/ ml showing significant (P< 0.001) increase in compari- son to day 6. Spore count then enhanced significantly (P < 0.001) to 4.4 x 105 spores / ml on day 10 and to 5.6 x 105 spores / ml on day 11 after infection showing the increase in a sigmoid fashion. Within 11 days, average of 60% larval mortality was recorded after the infection. Cellular variations in hemocytes Total hemocyte count of control and N. bomby- cis- infected larvae did not vary significantly on day 0 of infection, however significant (P < 0.005; ANOVA) decrease observed in later stages (8 to 11 days) of infec- tion. In control, total hemocyte count on day 0 fourth instar larvae was 14.4 x 105 cells/ml, which did not vary significantly (P < 0.1), on day 0 after infection. The count increased to 16.55 x 105 cells/ ml on day 6 how- ever hemocyte count significantly (P< 0.002) increased to 17.95 x 105 cells/ml after infection. In control, hemo- cyte count significantly (P < 0.001) increased on day 8 to 37.05 x 105 cells/ ml whereas after infection it decreased to 32.35 x 105 cells/ ml. On day 9 the count was 49.15 x 105 cells / ml in control and 45.05 x 105 cells / ml after infection. In control on day 10 and 11, mean hemocyte count was 50.4 x 105 cells/ ml in comparison to 42.1x 105 cells/ ml after infection. Under light microscope, four types of hemocytes viz., granulocytes, plasmatocytes, spherulocytes and oenocytes were observed in addition to the precursor 98 Hungund P. Shambhavi et al. prohemocytes. In control, hemocytes appeared intact with clear cytoplasm and less granules whereas after infection, cy toplasm becomes granulated on day 6. Many cells ruptured and degranulated from day 8 after infection (Fig.1 D - E). In order to examine subcellular variations induced by N. bombycis infection, infected and control hemo- cytes were examined under TEM on day 6, 8 and 11 after infection. In hemocytes of control day 6 larvae, cytoplasm was clear. RER and mitochondria with well developed cristae distributed in cytoplasm (Fig. 2 A- C). In plasmatocytes, nuclei were round or ovoid and in granulocytes, smooth or branched. Chromatin was uniformly spread in nucleoplasm. Granulocytes showed presence of few granules and plasmatocytes with few electron dense particles (EDP). Plasma membrane was smooth and with pinocytic vesicles showing active mem- brane transport. On day 6 after infection, several packs of structured granules appeared in granulocytes. Large- sized vacuoles occupied major cytoplasmic area. Mitochondria increased in number. Many granulocytes featured highly irregu- lar branched nucleus with condensed chromatin. Plasma membrane showed few cellular projections (Fig. 3 A). On day 8 after infection, granulocytes showed plas- ma membrane with cytoplasmic extensions, lysosomes and phagosomes with engulfed spores (Fig. 3 B). More granulocytes were with pseudopodia and microvilli (Fig. 3 C). Phagosomes enclosing spores were observed and were associated with lysosomes (Fig. 3 B). On day 11 after infection, hemocytes showed porous plasma membrane that lost integrity. In few cells, cell membrane completely degenerated. Degranulation by exocytosis observed in close vicinity of spores (Fig. 4 A-B). Many phagocytic granulocytes were observed with cy toplasmic extensions, developed pseudopo- dia, whirled sporoplasm, engulfed mature spores and ghost spores (Fig. 3 D - F). Rough endoplasmic reticu- lum (RER) and several mitochondria were observed in Figure 1. Organismal effects of microsporidian infection on B. mori larva: In comparison to control larva (A), infected larvae showed retarded growth and both larvae and pupae melanised (B – C). Control larval hemocytes illustrate clear cytoplasm and less gran- ules (D) whereas infected larval hemocytes were with granulated cytoplasm and few showed degranulation (E). Figure 2. Transmission electron microscopy (TEM) of hemocytes of control fifth instar larvae of B. mori (A) showed smooth plasma membrane, clear cytoplasm and cells with few granules and elec- tron dense particles (EDP), nucleus (N) with oval or branched nuclear membrane and evenly distributed chromatin, several mito- chondria (M) with developed cristae (B) and rough endoplasmic reticulum (RER) (C). Figure 3. TEM observations on hemocytes of fifth instar larvae of B. mori showing subcellular variations after infection with N. bom- bycis: Granulocytes turn phagocytic on day 6 (A-B) showing highly deformed nucleus with condensed chromatin (CC), cellular pro- jections (CP) and few vacuoles (V) in cytoplasm. On day 10 well differentiated pseudopodia (C; black arrow heads), multivesicular body (MV) and phagosomes (P) in association with lysosomes (L) were found. On day 11, hemocytes (D - F) with dense cytoplas- mic contents, invaginated nucleus (NI) with condensed chromatin, packets of structured granules (SG), engulfed mature spores (S; white arrows), ghost spores (GS) and whirled sporoplasm (WS) in vacuoles found. Cytoplasm was with RER, mitochondria (M), and vacuoles with cellular remnants (E). 99Phagocytosis and lipid peroxidation in hemocytes of B. mori induced by microsporidian infection dense cytoplasm. Nucleus demorphed, highly intended and showed deep invagination (Fig. 3D). Number of lys- osomes increased and located adjacent to spores or fused with vacuoles formed phagosomes. Lipid peroxidation In order to examine involvement of lipid peroxida- tion (LPO) in inducing membrane permeability, LPO was assayed by measuring malondialdehyde (MDA) which is a by-product of lipid peroxidation. After N. bombycis infection, MDA levels significantly (P < 0.012; ANOVA) increased from day 2 to day 14 with signifi- cantly (P < 0.000004) larger increase from day 11 indi- cating increased LPO activity after infection (Fig. 5 A). The increase in LPO activity showed positive correla- tion with increase in age after infection (R2 = 0.58) as well as number of spores increased exponentially (y = 0.0003e0.5572x; R² = 0.80) with increasing age. In order to verify relation between infection level and LPO increase, correlation analysis was performed which showed sig- nificant linear correlation (R2= 0.65) between increase in spore number and LPO activity. Ascorbate peroxidase activity In order to examine change in activity of ascorbate peroxidase (PRX) with increase in infection, PRX activ- ity was measured in hemocytes using ascorbic acid as substrate. PRX activity significantly (P < 0.025; ANOVA) enhanced from day 2 to 10 after infection with peak activ- ity on day 10 (Fig. 5 B) whereas control hemocytes showed PRX activity at basic level and increased activity on day 12, during spinning duration. Relation between increase in LPO activity (MDA level) with changes in PRX activity in infected hemocytes was analyzed by correlation- regres- sion which showed positive linear correlation though with low correlation coefficient value (R² = 0.294) during initial ten days of infection. Notably, from day 11 after infection PRX activity significantly decreased and LPO increased. Figure 4. TEM observations on hemocytes of fifth instar larvae of B. mori showing subcellular variations after infection with N. bom- bycis. Hemocytes (A) showed degranulation by exocytosis (arrow) to the microsporidian infection locale; (B) Plasmatocyte (PC) and granulocytes (GC) showed membrane disintegration (arrows) after infection with N. bombycis. S- spore; GS- ghost spore; SG- struc- tured granules; N- nucleus. Figure 5 A-B. Lipid peroxidation in hemocytes of B. mori larva induced after infection by N. bombycis indicated by the variation in malondialdehyde which is a bye-product of lipid peroxidation (A): Lipid peroxidation was at significantly higher level from ini- tial stages of infection and at peak level from day 11 onwards. (B): Variation in ascorbate peroxidase (PRX) activity in hemocytes of B. mori larva induced after infection by N. bombycis: PRX activity was significantly higher after infection with peak activity on day 2 and 10 followed by decline from day 11 onwards. 100 Hungund P. Shambhavi et al. Hydrogen peroxide assay In order to quantify reactive oxygen species level in hemocyte- phagocytes, level of hydrogen peroxide (H2O2) in the hemocytes was tested at 0, 2, 6, 8 and 10 days after the infection using hemocyte lysate extracted in PBS. In hemocytes of N. bombycis- infected larvae, H2O2 levels did not show significant variation from con- trol (data not provided). Expression of phagocytosis- associated genes Humoral immune responses of hemocytes are ini- tiated with recognition of pathogen followed by signal- ling and effector action. Expression of genes encoding recognition proteins β- glucan recognition proteins (BGRP2 and BGRP4), opsonins C- type lectin (CTL11 and CTL 17), hemocy te aggregation factor humoral lectin, phagocytosis enhancer apolipophorin, cytoskel- etal protein β- Actin, hemocyte mediated complement like protein that bind and kill Plasmodium berghei in Aedes aegypti Thioester containing protein (TEP-1) and bacterial phagocytosis associated Eater from Drosophi- la melanogaster was analysed by RT- PCR (Table 1) and qPCR (Fig. 6). RT-PCR profile and qPCR revealed down regula- tion of βGRP2 and BGRP4 expression after N. bom- bycis infection. CTL genes showed low level of expres- sion in earlier days of N. bombycis infection whereas 0.334 fold increase in expression was noticed on 10th day after infection (Fig. 6 B). Humoral lectin enhanced relative expression on day 6 (0.79 fold) after infection followed by down regulation (-1.97 fold) on day 10. After infection by N. bombycis, apolipophorin showed increase in expression by 1.96 fold on 6th day and by 2.59 fold on 10t h day. Expression of β- actin showed stable increase from early to late stages of infection with strong positive correlation (R 2 = 0.65) with age after infection (Fig. 6 C). In the dipterans A. aegypti and D. melanogaster, TEP1 (Blandin et al. 2004) and Eater (Kocks et al. 2005; Juneja and  Lazzaro 2010) respectively are closely asso- ciated with phagocytosis however these genes are not reported from B. mori. Primers derived from TEP of A. aegypti and Eater of Drosophila was used for amplifica- tion with template cDNA from hemocytes of B. mori after infection with N. bombycis. In this study TEP did not show expression in hemocytes of B. mori whereas Eater showed enhanced relative expression on day 2 and 6 after infection followed by significant (P < 0.005) decrease (Fig. 6 D). Figure 6 A-D. Modulation in expression of immune genes in hemocytes of B. mori larva after infection with N. bombycis: (A) RT-PCR profile of gene expression of different genes including cytoskeletal protein gene β- Actin and the house keeping gene encoding ribosomal protein resolved from hemocytes collected from control and infected larvae at 2, 6, 8 and 10 days after infec- tion. M- Massruler DNA marker (Thermo). (B) qPCR showed relative expression of different immune genes in hemocytes at 6 and 10 h after infection. (C) β- Actin, the cytoskeletal protein gene showed gradual increase in expression represented by den- sitometric units. Increase in expression was correlated with age after infection shown by allometric line. The linear regression formula and correlation coefficient are inserted. (D) Relative expression pattern of the phagocytosis associated gene Eater like after standardization with expression of the house keeping gene, ribosomal protein and after elimination of control value. Figure 7. Alignment of nucleotide sequence (A) of amplification product from heterologous expression profile of Eater gene of Dros- ophila (query) with nucleotide sequence (subject) of the most simi- lar gene, B. mori uncharacterized transcript variant X2 (Accession No. XM_004928120.3) revealed by NCBI-BLASTn analysis, showed 98 % similarity. (B) Protein sequence of the uncharacterized tran- script variant (subject) is aligned with translated sequence of the Eater like gene (query) showing 97% similarity. 101Phagocytosis and lipid peroxidation in hemocytes of B. mori induced by microsporidian infection Sequence analysis In order to confirm presence of Eater like gene in B. mori, amplification products were ligated, cloned and sequenced. The nucleotide sequence was analysed by NCBI-BLAST. Eater like sequence showed 98 % simi- larity with B. mori uncharacterized transcript variant X2 (Accession No. XM_004928120.3) with an expect value 7e-110. The translated sequence of Eater like (3’5’ frame 2) showed 97 % similarity with translated amino acid sequence of Bm uncharacterized protein (BmUCP) BmUCP LOC101736235 isoform X2 (H9JFY7_BOMMO of Uniprot; BGIBMGA008434 of B. mori) revealing existence of Eater like sequence in B. mori genome (Fig. 7). This sequence showed orthology in the lepidopterans Heliconi- us melpomene and Danus plexippus with unknown func- tion (EggNOG 4.5.1). However complete sequencing of the gene has to be performed for gene structure confirmation. DISCUSSION In the initial stages of microsporidian infection in B. mori, spores were not detected microscopically for six days after infection. In the mid and later stages, expo- nential increase in spore count was recorded. In insects host responses in hemocytes initiated with activation of cell surface receptors and signal transduction (Lamprou et al 2007; Tsakas and Marmaras 2010). Hemocytes rec- ognize pathogens entered in larval body with assistance from recognition proteins. The proteins that recognize Nosema sporoplasm / spores have not been identified in B. mori though microbial recognition proteins such as peptidoglycan recognition proteins (PGRPs) have role in host responses of honey bees against infection by N. ceranae through Toll / IMD pathways (Li et al 2017). β- GRP (β-1,3-glucan recognition proteins) are recognition proteins in Toll/ Dif pathway (Gobert et al 2003) and are associated with detection of bacterial endotoxin in Dros- ophila (Kim et al. 2000) and phenol oxidase activation in B. mori (Yoshida et al. 1986) indicating multiple role associated with immune reactions. Following recogni- tion, hemocytes initiated cellular immune events such as cell aggregation, nodulation, cytokine release, melaniza- tion and encapsulation depend on size of the pathogen / parasite (Lavine and Strand 2002). Similarly, hemocytes initiate phagocytosis against bacteria and fungi particu- larly against those with size less than five microns (Pech and Strand 1996; Scapigliati and Mazzini 2009). Notably, N. bombycis spores infecting B. mori larva are of 2.6 to 3.8 microns (breadth x length) (Rao et al. 2007) which could be phagocytosed by hemocytes though mechanism of parasite destruction is not clearly known. Phago- cytic uptake of KOH treated- or cold storaged Nosema spores is found in insect cell line (Cai et al. 2012) how- ever phagocytosis of live Nosema spores by larval hemo- cytes in vivo had not been reported in B. mori. After N. bombycis infection in B. mori larva, TEM observation showed symptoms of phagocytosis in granulocytes such as formation of pseudopodia and appearance of phago- somes with lysosomal bags. Spores and ghost spores were observed within phagosomes of hemocytes indica- tive of lysosomal activity on spores. Phagosomes enclos- ing spore / meront were found in granulocytes of B. mori as noticed in A. aegypti after infection by Plasmo- dium gallinaceum (Hillyer et al. 2003) and in the coleop- teran Rhynchophorus ferrugineus infected with the yeast Saccharomyces cerevisae (Manachini et al. 2011). In the coleopteran flower chafers Protaetia brevitarsis seulensis, development of autophagic vacuoles observed in asso- ciation with phagocytosis by granulocytes indicative of autophagic elimination of pathogens (Kwon et al. 2014). After N. bombycis infection, granulocytes showed cyto- plasmic projections, pseudopodia, microvilli, membrane porosity and disruption as characteristics of phagocytic cells (Castillo et al. 2006; Williams 2007). Moreover granulocytes showed degranulation by exocytosis in spore ‘locales’ indicating active transportation of struc- tural granules to the plasma membrane and degranula- tion in the site of infection by the spores as observed in mouse models (Dias et al. 2018). Hemocytes with extended pseudopodia, cy toplasmic projections and phagosomes were observed in Anopheles quadrimacula- tus infected with nematode, Romanomermis culicivorax (Shamseldean et al. 2006), Culex quinquefasciatus infect- ed by Wuchereria bancrofti (Brayner et al. 2007) and in plasmatocytes of the tick Rhipicephalus sanguineus infected with Leishmania infantum (Feitosa et al. 2015). Stable increase in expression of the cytoskeletal protein gene β- actin with age was noticed in hemocytes of B. mori after N. bombycis infection indicated continuous requirement of actin to redistribute the cytoskeletal pro- tein during formation of pseudopodia and microvilli after N. bombycis infection and to meet rearrangement of cytoskeletal proteins for engulfment (Moore et al. 1992; Kwon et al. 2014). Variation in actin protein con- tent and its critical role was reported in association with formation of pseudopodia in other models also (May and Machesky 2001; Baranov et al. 2016). LPO and peroxidase activity associated with phagocytosis Infection with N. bombycis increased malondialde- hyde production in hemocytes revealed increased lipid 102 Hungund P. Shambhavi et al. peroxidation (LPO). Lipoprotein structure of the mem- brane is disrupted by LPO, which caused membrane porosity and disintegration in association with phago- cytosis. LPO activity significantly increased with age in early and mid stages of infection however it was higher in later stages of infection. Moreover spore count is increased exponentially after six days of infection and lipid peroxidation increased simultaneously showing correlated increase. During lipid peroxidation, carbon- carbon double bonds present in the polyunsaturated fatty acids of plasma membrane are attacked (Yin et al. 2011; Wong-ekkabut et al. 2007) which is initiated with oxidation of few lipid molecules and subsequently con- tinued as a chain reaction leading to disintegration of cell membrane (Mylonas and Kouretas 1999; Ayala et al. 2014). Though infection induced oxidative stress caused lipid peroxidation (Milei et al. 2007; Pooja et al. 2017), increment in reactive oxygen species (H2O2) was not observed in hemocytes of B. mori larva after N. bombycis infection indicating a direct effect of LPO on hemocyte membrane integrity probably through accumulated toxic products (Clark et al 1987). Similar direct impact of lipid peroxidation on tissue damage was reported in liver of the fish Pimephales promelas infested by liver trematode Ornithodiplostomum  sp (Stumbo et al. 2012). Accumula- tion of LPO toxic products could suppress the phagocyt- ic action of hemocytes which defend parasite survival. A possibility for less H2O2 content observed in the infected hemocytes might be due to relatively shorter half life induced by its reactivity with biomolecules (Lennicke et al. 2015). In order to protect the cells from peroxidation, enzymatic antioxidant peroxidases are activated (Brige- lius-Flohe and Maiorino 2013; Jablonska et al. 2015). Ascorbate peroxidase removed lipid peroxides in the lep- idopteran Helicoverpa zea (Mathews et al. 1997) through ascorbate recycling system (Summers and Felton 1993; Krishnan and Kodrik 2006; Lukasik et al. 2009). After N. bombycis infection, in hemocytes of B. mori, per- oxidase activity was significantly higher in early and mid- stages of infection which regulated LPO activity to comparatively lower level. In the later stages of infection, peroxidase activity significantly reduced, in contrast, lipid peroxidation increased significantly indicating neg- ative interaction between lipid peroxidation and ascor- bate peroxidase activity, corroborating with the negative relation noticed between LPO and peorxidase activity in humans under diseased conditions (McCay et al. 1976; Motghare et al. 2001). The peroxidase regulation of LPO in association with phagocytic events is unknown in invertebrates after parasitic infection. Modulation in gene expression after infection In order to enhance immune reactions after N. bom- bycis infection, genes encoding proteins associated with humoral and cellular immune response were activated before eliciting the host responses (Brown and Gordon 2003; Manachini et al. 2011). Notably β-GRP genes did not show significant variability in expression after infec- tion showing an ambiguity in its role in immune reac- tions against N. bombycis infection. On the other hand CTL genes showed upregulated expression on day 6 after infection and its role was suggested to be in spore recognition and signal trasnsduction (Ma et al. 2013). CTL-11 and 17 implicated in opsonising blastospores of the fungus, Beauveria bassiana to make fungal spores susceptible to phagocy tosis (Pendland et al. 1988). Notably, taxonomic position of N. bombycis is shifted from protozoan to fungus (Han and Weiss 2017) based on molecular phylogeny. CTL proteins activated dur- ing infection by other fungus could be effective during infection by N. bombycis probably due to activation of similar immune mechanisms against different species of fungi. Gene encoding hemocyte adhesive factor humoral lectin (Kotani et al. 1995) and the phagocytosis enhanc- er Apolipoprotein III (Whitten et al. 2004) enhanced expression after infection. Apolipophorin III together with I/ II help in pattern recognition as well as enhances phagocytic action of hemocytes in insects (Barabas and Cytryńska 2013; Whitten et al. 2004). In the dipterans Aedes and Drosophila, Thioester containing protein (TEP1) (Blandin et al. 2004) and Eat- er (Kocks et al. 2005) respectively are associated with phagocytosis however these genes have not reported from B. mori. Though TEP1 did not show expression, Eater like showed heterologous expression in hemocytes of B. mori larva on day 2 and 6 after infection. Both nucleotide sequence and translated amino acid sequence of Eater like amplicon showed 98% similarity with that of an uncharacterized transcript variant from B. mori indicating activation of Eater like gene in B. mori in association with hemocy te- mediated phagocy tosis against N. bombycis. In Drosophila, Eater is a transmem- brane protein involved in binding and internalization of bacteria in the phagosomes (Stuart et al. 2005; Kocks et al. 2005; Chung and Kocks 2011) however role of Eater like protein in B. mori immune responses is unknown. N. bombycis infection induced subcellular variations in hemocytes of B. mori including demorphed nucleus, activation of phagocytosis including formation of pseu- dopodia, microvilli, porous plasma membrane and for- mation of phagosomes. In addition, lipid peroxidation was increased in hemocytes with increase in age after 103Phagocytosis and lipid peroxidation in hemocytes of B. mori induced by microsporidian infection infection. Simultaneous increase in peroxidase reduced the LPO activity. In the later stage, peroxidase activ- ity reduced and LPO activity increased showing nega- tive relation. Moreover infection by N. bombycis induced modulation of phagocytosis- associated genes where heterologous expression of Eater also observed indicat- ing activation of phagocytic events and associated events against N. bombycis infection in B. mori larva which are potential novel targets for developing new control meas- ures. The protein- based targets could be used to devel- op antibody- based mechanisms for early detection of microsporidian infection. ACKNOWLEDGEMENTS The authors thank anonymous reviewers for valu- able suggestions, Central Silk Board, Bangalore for the facilities and Department of Biotechnology (Government of India), New Delhi for financial support in the form of a research project to ARP (BT/PR6355/PBD/19/236/2012 dated 08/01/2013). SPH and PM were supported by research fellowships from the project. CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES Ayala A, Munoz MF, Arguelles S. 2014. 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