Gut immunity in Lepidopteran insects

Gut immunity in Lepidopteran insects

Kai Wu a ,Bing Yang a ,Wuren Huang b ,Leonard Dobens c ,Hongsheng Song d ,**,Erjun Ling a ,*

a

Key Laboratory of Insect Developmental and Evolutionary Biology,Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200032,China b

Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou,Hainan 571101,China c

School of Biological Sciences,University of Missouri-Kansas City,5007Rockhill Road,Kansas City,MO 64110,USA d

College of Life Sciences,Shanghai University,Shanghai 200444,China

a r t i c l e i n f o

Article history:

Received 14September 2015Received in revised form 6February 2016

Accepted 6February 2016Available online xxx Keywords:Lepidoptera Gut

Bacteria

Microsporidia Virus

Immunity

a b s t r a c t

Lepidopteran insects constitute one of the largest fractions of animals on earth,but are considered pests in their relationship with man.Key to the success of this order of insects is its ability to digest food and absorb nutrition,which takes place in the midgut.Because environmental microorganisms can easily enter Lepidopteran guts during feeding,the innate immune response guards against pathogenic bacteria,virus and microsporidia that can be devoured with food.Gut immune responses are complicated by both resident gut microbiota and the surrounding peritrophic membrane and are distinct from immune re-sponses in the body cavity,which depend on the function of the fat body and hemocytes.Due to their relevance to agricultural production,studies of Lepidopteran insect midgut and immunity are receiving more attention,and here we summarize gut structures and functions,and discuss how these confer immunity against different microorganisms.It is expected that increased knowledge of Lepidopteran gut immunity may be utilized for pest biological control in the future.

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1.Introduction

On earth,there are approximately 160,000species of Lepidop-teran insects,some of which act as agricultural pests,while others,such as the silkworm (Bombyx mori ),can be considered economi-cally important.Microorganisms introduced into the body cavities through wounds or infection induce hemocyte-involved

phagocytosis,encapsulation and nodule formation (cellular im-munity)and/or the expression of humoral proteins involved pro-tection (humoral immunity)(Hoffmann,2003;Lemaitre and Hoffmann,2007;Liu et al.,2013a;Strand,2008).In contrast,pathogenic microorganisms transferred into the gut with food activate gut immunity to protect insects (Dillon and Dillon,2004;Engel and Moran,2013;Lemaitre and Miguel-Aliaga,2013).Thus innate immunity is critical to protect insects from infection and to guarantee normal development (Hoffmann,2003;Lemaitre and Hoffmann,2007;Liu et al.,2013a;Strand,2008).Genomes of B.mori (Xia et al.,2004),Plutella xylostella (You et al.,2013)and Danaus plexippus (Zhan et al.,2011)have been decoded,and the detailed genetics of these Lepidopteran insects have proven helpful to understand their development,growth,immunity and prolifer-ation.Here we review Lepidopteran gut structure,function and their respective relationships to gut immunity.Understanding the fundamental molecular mechanisms of insect immunity will have important impacts on pest control (Hakim et al.,2010).

Abbreviations:GNBP,gram negative bacteria binding protein;20E,20-Hydroxyecdysone;CPV,cytoplasmic polyhedrosis virus;Bt,Bacillus thuringiensis ;CF,Cytophyaga/Flavobacteria ;ROS,reactive oxygen species;Imd,immune de ?-ciency;PO,phenoloxidase;DUOX,dual oxidase;NPV,nucleopolyhedrovirus;Bb,Bacillus bombysepticus ;DNV,densovirus;ODV,occlusion derived virus;BV,budded virus;JcDNV,Junonia coenia densovirus;BmDNV-1,B.mori densovirus type 1;DXV,Drosophila X virus;IE1,immediate early-1;BmSP-2,B.mori serine protease-2;BmtryP, B.mori alkaline trypsin protein;ESI,electrospray ionization;AcMNPV,Autographa californica multicapsid nucleopolyhedrovirus;CBD,chitin binding do-mains;PMP,peritrophic membrane proteins;TnGV,Trichoplusia ni granulosis virus;XcGV,Xestia c-nigrum granulovirus;ChiA,chitinase A;PPO,prophenoloxidase;AMP,antibacterial peptides.*Corresponding author.**Corresponding author.

E-mail addresses:hssong@https://www.360docs.net/doc/394394490.html, (H.Song),erjunling@https://www.360docs.net/doc/394394490.html, ,ejling@https://www.360docs.net/doc/394394490.html, (E.

Ling).

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2.Lepidopteran gut

2.1.Structure of Lepidopteran gut

The Lepidopteran larval gut can be divided into three parts: foregut,midgut and hindgut(Chapman,1998)and the morphol-ogies of these three parts in a silkworm B.mori larva is shown in Fig.1A.The foregut,consisting of the pharynx esophagus,crop, proventriculus and cardiac valve,mixes ingested foods with?uids secreted from foregut tissue and salivary glands(Fig.1B).The midgut serves as the main place where food is digested and absorbed(Engel and Moran,2013;Lehane and Billingsley,1996; Lemaitre and Miguel-Aliaga,2013).The peritrophic membrane lines the midgut,which includes three cell types,the columnar cells,goblet cells and stem cells(Lehane,1997)(Fig.1C).The hindgut can be divided into the anterior intestine,composed of ileum and colon,and the posterior rectum(Chapman,1998).In the anterior intestine,the epidermal cells are large,and a layer of thick intima covers epidermal cells facing gut content(Fig.1D).In the posterior rectum of Lepidoptera,including the silkworm and tenebrionid beetles,there are many Malpighian tubules in the (Fig.1E),which enter the rectum and are buried beneath the membrane and muscular sheath.The Malpighian tubules perform the function of water and salt(mainly KCl)resorption from food residues before?nal secretion as feces(Klowden,2013).

Lepidopteran larval midguts including other insects secrete many enzymes required for food digestion(Engel and Moran,2013; Lehane and Billingsley,1996;Lemaitre and Miguel-Aliaga,2013).In the midgut of Helicoverpa armigera,enzymes that can digest proteins,carbohydrates and lipids were identi?ed through prote-omics analysis(Pauchet et al.,2008).Similar approaches were used to show that midguts produce key immunity proteins required for protection,including(1)Immune-related Hdd13,(2)Cyclophilin A and(3)Cyclophilin in B.mori(Zhang et al.,2011),and(4)Gram negative bacteria binding protein(GNBP),which is involved in recognition of Gram negative and positive bacteria and fungi (Pauchet et al.,2008).

2.2.Goblet cell

Lepidopteran goblet cells can be distinguished from other midgut cells by their large chalice-shaped central cavity(Billingsley and Lehane,1996;Moffett and Koch,1992b)(Fig.1C),which forms from an apical membrane invagination(Moffett and Koch,1992b). This class of cells distinguishes Lepidopteran gut morphology from other insects and may confer protection from pathogenic microbes these pests ingest.Goblet cells have very small nuclei located near to the basal membrane(Fig.1C)and a valve facing the lumen,which is narrow and interdigitated through microvilli in the end(Moffett and Koch,1992b).According to cell culture analysis in vitro,goblet cells are thought to differentiate from a source of stem cells (Tettamanti and Casartelli,2010).

So far we know very little on the function of goblet cell.The interior surface of goblet structure is covered with microvilli rich in mitochondria(Moffett and Koch,1992b)and vacuolar Ht-ATPase (Gomes et al.,2013;Levy et al.,2004)and functions to transport Ktfrom the hemolymph across the goblet cavity into the midgut lumen through the goblet valve(Moffett and Koch,1992a,b).

In

Fig.1.Structure of Bombyx mori larval gut.(A)Morphology of a gut dissected from a silkworm larva on day2of5th larval stage.The foregut(FG),midgut(MG)and hindgut(HG) are indicated.The inset shows the structure of foregut in detail and the white dotted line delineates the foregut from the midgut(MG).The foregut has an esophagus(es)and crop, which contains ingested mulberry leaf fragments.For comparison,the hindgut is divided into two parts:hindgut one(HG1)and hindgut two(HG2).The midgut is notably larger than the foregut and hindgut.(B)A longitudinal section showing the corresponding structure of the foregut and part of midgut after being stained by haematoxylin and eosin.In the crop of the foregut and midgut,mulberry leaf(ML)fragments can be observed.Proventriculus(PR)that connects the foregut and midgut is visible after tissue sectioning.Peritrophic matrix,the non-cellular structure(Lehane,1997),is invisible since it cannot be stained by haematoxylin and eosin.The arrowhead points to stomodeal valve(sv).(C)Structure of larval midgut.There are columnar cells(C)and goblet cells(G).The arrow indicates the basal lamina.Stem cells of midgut are very few on the5th larval stage.(D)Morphology of the hindgut one(HG1).This part of hindgut is composed of many large epidermal cells(EC)with thick intima(arrow-indicated)facing the feces(f).The outside is a lay of muscle(M) tissues.(E)Morphology of the hindgut two(HG2).There are many Malpighian tubules(MT)between the epidermal cells(EC)and the basal lamina(arrow-indicated).In(B e E),the tissue sections were stained using haematoxylin and eosin(Xu et al.,2012).

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Anticarsia gemmatalis,the observation of excreted cellular compo-nents accumulated on the microvilli of the goblet cavity suggests a secretion function for this type of cell(Gomes et al.,2013).Thus goblet cells may regulate the ionic exchange between the gut and hemolymph and serve as a depot to transfer cell debris and metabolize gut constitutents(Gomes et al.,2013).Immuno-staining shows that the enzymes ecdysone oxidase and3-dehydroecdyson-3a-reductase accumulate in the goblet cavities of B.mori(Sun et al., 2012).Because these enzymes share an important role in the metabolism of the insect molting hormone20-Hydroxyecdysone (20E)(Riddiford,2012;Riddiford et al.,2000)and their expres-sion correlates with the ecdysteroid titer(Sun et al.,2012),it is interesting to consider whether goblet cells in Lepidopteran insect are involved in regulating20E levels in the midgut.

In the mammalian small intestine,there are four types of cells: goblet cells,absorptive cells,paneth cells,and enteroendocrine cells(Crosnier et al.,2006).Absorptive cells are very similar in appearance to Lepidopteran columnar cells,with both cell types exhibiting many microvilli on the side facing gut lumen(Crosnier et al.,2006).In addition,mammalian goblet cells closely resemble Lepidopteran goblet cells,with a large cavity inside the cell.Mucus secreted by mammalian goblet cells forms a liquid layer on the internal surface to lubricate and protect small intestinal cells from physical abrasion by foods and invading bacteria(Cone,2009)and extensive studies reveal that mucus is composed of mucin,a type of glycoprotein(Kim and Ho,2010;Rogers,2003),and key immunity-related proteins such as trefoil peptides,resistin like molecule b and Fc-g-binding protein(Herbert et al.,2009;Johansson et al.,2009; Taupin and Podolsky,2003).

A great deal of evidence shows that mammalian goblet cells have an important role in intestinal innate immunity defense(Kim and Ho,2010)and due to their similar morphology to mammalian goblet cells,it is quite natural to consider whether the Lepidopteran goblet cells have an immunity function as well.When H.armigera larvae were infected by cytoplasmic polyhedrosis virus(CPV), goblet cells degenerated and some microvillus-like cytoplasmic projections were left(Marzban et al.,2013).If Bacillus thuringiensis (Bt)toxin was fed to Lepidopteran larvae,goblet cells were specif-ically affected(Baines et al.,1997).From these limited studies,it appears that Lepidopteran goblet cells are affected directly or indirectly by pathogen infection.

2.3.Peritrophic membrane and protection

In most insects,there is an acellular structure called peritrophic membrane on the inner surface of midgut(Lehane,1997;Tellam, 1996;Umut et al.,2010).Lepidopteran peritrophic membranes are secreted by midgut epithelial cells(the cardia)and are composed of chitin and glycoproteins cross-linked to form the net-like membrane(Umut et al.,2010)with a thickness of about 0.5e1.0m m(Hegedus et al.,2009;Lehane,1997).The membrane functions as a semi-permeable barrier for transporting nutrients, water and minerals and protecting the midgut cells from both mechanic abrasion by foods and infection by microorganisms devoured with foods(Lehane,1997;Tellam,1996;Umut et al., 2010).Proteins associated with the peritrophic membrane (collectively referred to as peritrophic matrix)commonly include chitin binding domains(CBD)(Umut et al.,2010).More focused studies have identi?ed305peritrophic membrane proteins in B.mori(Zhong et al.,2012),while in H.armigera,41different pro-teins were identi?ed(Campbell et al.,2008).A genome-wide analysis and RNAseq assays of M.sexta midguts identi?ed the expression of17peritrophic membrane proteins(PMP)(Tetreau et al.,2015),and in Tribolium castaneum,RNAi knockdown of candidate peritrophic matrix proteins identi?ed genes essential for insect survival(Agrawal et al.,2014).

Evidence indicates that the peritrophic membrane protects in-sects from damage by bacteria toxin.In Lepidopteran larvae,peri-trophic membranes bind to Bt toxin Cry1A(Hayakawa et al.,2004; Rodrigo-Simon et al.,2006)to delay the contacting of Bt to the midgut cells,suggesting that the peritrophic membrane plays a key role in preventing bacterial toxins from damaging the gut.In Drosophila,the importance of Drosocrystallin(Dcy)protein to adult peritrophic membrane formation was demonstrated in loss-of-function mutants that exhibited thinned peritrophic membrane with increased permeability(Kuraishi et al.,2011).dcy mutant midgut cells had increased susceptibility to damage by both pore forming toxins secreted by Pseudomonas entomophila,and to oral infection(Kuraishi et al.,2011).

Peritrophic membranes of Lepidopterans also serve as a natural barrier to virus.In most Lepidopteran insects,the pore sizes of peritrophic membrane are approximately21e29nm(Barbehenn and Martin,1995)while the pore sizes of Calliphora eryth-rocephala peritrophic membrane are about3e4nm(Zimmermann and Mehlan,1976).Contrasted with the20e300nm sizes of most viruses(Arnone and Walling,2007),peritrophic membranes effectively inhibit viral penetration and infection.Nevertheless, viruses can break through this innate barrier by altering peritrophic membrane permeability or destroying its structure and three ex-amples can be cited:(1)the metalloprotease enhancin produced by TnGV(Trichoplusia ni granulosis virus)likely affects the perme-ability of peritrophic membranes(Peng et al.,1999);(2)enhancin produced by Mamestra con?gurata NPV selectively degraded the peritrophic membrane proteins(Toprak et al.,2012);and(3) enhancin from XcGV(Xestia c-nigrum granulovirus)broke down the peritrophic membrane to accelerate infection of S.Litrue(Guo et al.,2007).Similar to enhancin,the viral protein Chitinase A (ChiA)from AcMNPV disrupted the integrity of peritrophic mem-brane when expressed and fed to B.mori larvae,with a striking 100%mortality associated with the appearance of perforations on the peritrophic membrane(Rao et al.,2004),indicating that this viral protein serves to disrupt the gut barrier to facilitate infection.

The thickness of peritrophic membrane may affect virus infec-tion.The peritrophic membrane is more fragile in A.gemmatalis larvae susceptible to A.gemmatalis multicapsid nucleopolyhe-drovirus(AgMNPV)infection compared to membrane in resistant strains,due in part to lower chitin levels and more abundant non-solubilized vesicular materials(Levy et al.,2011).Lepidopteran Heliothis virescens https://www.360docs.net/doc/394394490.html,rvae fed on foliage had thicker peritrophic membrane associated with reduced baculoviral ef?cacy compared to those fed on the arti?cial diet(Plymale et al.,2008).Conversely, feeding calco?uor caused mechanical damage to peritrophic membrane,which in turn enhanced the susceptibility of Tricho-plusia ni to NPV(Wang and Granados,2000).

In summary,the acellular peritrophic membranes are important to prevent physical abrasion to the midgut cells and to permit nutrition and water to pass through the gut freely.Due to the small pore sizes,the Lepidopteran peritrophic membranes exclude most bacteria and virus,and effectively serves as the?rst line to defend the gut from invasive microorganisms.

3.Microbiota in the Lepidopteran gut

Midguts of Lepidopteran insects harbor large amount of microbiota consisting of both non-pathogenic and pathogenic bacteria devoured with food(Engel and Moran,2013).These commensal and symbiotic bacteria serve many important host functions,including decomposition of toxins and insecticides,de-fense against parasites and other pathogens,transfer of signals among individuals,and the production of nutrients to supplement

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poor diet and help digestion(Dillon and Dillon,2004;Engel and Moran,2013).The power of Drosophila genetics has been used to reveal an elegant immunity system that can effectively distinguish commensal and symbiotic bacteria from pathogenic ones(Bae et al., 2010;Ha et al.,2009;Ryu et al.,2010).

Gene sequencing approaches can identify species-speci?c insect midgut microbiota mainly through16S rRNA(Woo et al.,2000). Bacteria isolated from the midguts of Spodoptera litura(Fab.)larvae were distinguished based on16S rRNA gene sequencing and include the microbiota Microbacterium arborescens(SL6),Entero-coccus casseli?avus(SL10)and Enterobacter cloacae(SL11)(Thakur et al.,2015).Midgut microbiota vary among species of Lepidop-tera so that in the oriental armyworm(Mythimna separata),bacteria such as Enterobacteriales,Cyanobacteria,Firmicute s,Actinobacteria, Gracilicutes and Proteobacteria were identi?ed via analysis of a16S rDNA clone library(He et al.,2013)while in Spodopter littoralis and H.armigera larva,midgut microbiota include Enterococci,Lactoba-cilli and Clostridia(Tang et al.,2012).Midgut microbiota vary by strain,too,with two insecticide(chlorpyrifos and?pronil)resistant lines of P.xylostella(L.)larvae having more Lactobacillales,Pseudo-monadales and Xanthomonadales in the midgut than susceptible lines(Xia et al.,2013).

Feeding can change the make-up of the Lepidopteran gut bac-teria community(Hammer et al.,2014;Liang et al.,2014;Sittenfeld et al.,2002).In B.mori fed on mulberry leaves,the midgut micro-biota are mainly Bacillus and Arcobacter(Liang et al.,2014).How-ever when fed on lettuce leaves in a bioregenerative life support system,Acinetobacter and Bacteroides predominate(Liang et al., 2014).The butter?y Heliconius erato has markedly distinct midgut microbiota among the leaf chewing,nectar-and pollen-feeding adults(Hammer et al.,2014).Although the reason for these dif-ferences is unclear,plant secondary metabolites that accompany food have been demonstrated to alter the midgut microbiota.For example,when Lepidopteran Acentria ephemerella larvae were fed on Myriophyllum spicatum,a freshwater angiosperm containing a signi?cant amount of tannins,levels of both Gram positive bacteria and Cytophyaga/Flavobacteria(CF)were reduced(Walenciak et al., 2002).Microbiota identi?ed in larvae living in the?eld are very different from those lines cultured in the laboratory(Belda et al., 2011;Hammer et al.,2014;Mason and Raffa,2014)and when larvae derived from the?eld and laboratory were fed under the same conditions,their microbiota became similar(Mason and Raffa,2014;Tang et al.,2012).

Lepidopteran midgut microbiota confer host protection in some circumstances.In some Lepidopteran insects,depletion of midgut microbiota increases the susceptibility of hosts to pathogenic infection.Photorhabdus temperata is associated with nematodes of the Heterorhabditidae family as symbiotic bacteria(Carneiro et al., 2008).However,after oral infection of P.temperata in Lepidop-teran Diatraea saccharalis larvae,90%of gut microbiota disappeared within2days(Carneiro et al.,2008)and the resulting midgut bacteria showed increase infectivity of the nematode symbiont. Lepidopteran gut microbiota are also crucial to decrease the dam-age caused by bacteria toxin.Puri?ed Bt toxin was pathogenic to the aseptically reared larvae(Raymond et al.,2009),however when bacteria Enterobacter sp.Mn2was fed to the germ-free P.xylostella larvae,reduced Bt toxicity was observed(Raymond et al.,2009). This result suggests that midgut microbiota protect Lepidopteran larvae from pathogen attack and consistent with this,levels of Lactobacillales in the midguts of P.xylostella(L.)were increased when larvae were treated with insecticides(Xia et al.,2013). Accordingly,enhanced immune activity increases the midgut microbiota load(Hernandez-Martinez et al.,2010)so that when Spodoptera exigua larvae with high immunity were speci?cally selected,they were found to have greater bacteria loads in the midguts along with an enhanced tolerance to Bt(Hernandez-Martinez et al.,2010),a tolerance that could be genetically trans-ferred to offspring.All these works demonstrate that the gut microbiota can indeed protect Lepidopteran insects from pathogen infection and toxin damage.

While in general Lepidopteran midgut microbiota are commensal to hosts,a commensal-to-pathogen switch is observed under some conditions.While Enterococcus faecalis is not associ-ated with infection in the midguts of M.sexta,injection into larval hemocoels rapidly leads to sepsis(Mason et al.,2011).Oral feeding of Bt toxin with E.faecalis enhanced larval mortality rate with E.faecalis detected in the hemolymph(Mason et al.,2011).In contrast,midgut sourced Gram-negative Enterobacter sp.NAB3 enhanced the susceptibility of several Lepidopteran larvae to B.thuringiensis toxin(Broderick et al.,2006,2009).Very interest-ingly,when the commensal bacteria E.cloacae were coated on caster leaves and fed to S.litura,E.cloacae dominated the popula-tion of the midgut bacteria in the resulting dead larvae(Thakur et al.,2015),suggesting that Lepidopteran midgut commensal bacteria may become pathogenic under some conditions.All together,the Lepidopteran midgut microbiota vary from species to species and between diets and symbiotic and commensal bacteria offer protection of the Lepidoptera.

4.Lepidopteran gut immunity against pathogenic bacteria, microsporidia and virus

The insect midgut makes?rst contact with pathogens including Gram positive and negative bacteria,fungi,virus and parasites.To support this?rst line of immunity defense,insects utilize anti-bacterial peptides and reactive oxygen species(ROS)to clean out gut pathogens and protect commensal microbiota(Bae et al.,2010; Ha et al.,2009;Ryu et al.,2010).Most bacteria enter the hemocoel through the wounds,while parasites and fungi can penetrate the integument and further infect in the hemocoel(Clarkson and Charnley,1996;Libersat et al.,2009).In contrast,viruses invade the hemocoel mainly via the gut(Tanada and Hess,1976;Tanada and Leutenegger,1970).Insects defend against Gram positive bac-teria and fungi using the Toll pathway,and utilize the immune de?ciency(Imd)pathway to target Gram negative bacteria (Hoffmann,2003;Lemaitre and Hoffmann,2007).To supplement these immune responses,melanization induced by activated phe-noloxidase(PO)physically isolates the invading pathogens(Ashida and Brey,1998;Lu et al.,2014).Viruses are targeted through RNAi, apoptosis and autophagy(Clem,2005;Nakamoto et al.,2012)and the JNK,JAK/STAT and p38pathways are also crucial to the insect innate immunity(Chen et al.,2010;Hoffmann,2003;Tanaka et al., 2008).The diverse immune responses insects use to actively defend against pathogens in gut are summarized below.

4.1.Bacteria

Lepidoptera mount immunity responses against both patho-genic bacteria and their toxins as evidenced by increases in expression of key immunity genes.When B.mori larvae were fed food carrying Gram positive(Staphylococcus aureus)and negative (Escherichia coli)bacteria,transcription of ceropin A,gloverin and lysozyme genes were up-regulated(Wu et al.,2010b).Glovorin2 and Glovorin3were up-regulated in the silkworm midguts when Pseudomonas aeruginosa were fed to the silkworm larvae(Zhang et al.,2015).Toll-9expression is up-regulated after oral feeding with different microorganisms,revealing the close relationship between Toll pathway signaling and midgut immunity(Wu et al., 2010a).Very interestingly,when LPS was fed,expression of Toll 9-1was down-regulated(Liu et al.,2013b).When B.mori larvae

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were orally infected by P.aeruginosa and S.aureus separately,PGRP-C1,Serine protease precursor gene,and the30kD protease A pre-cursor gene were up-regulated in the larval midgut(Zhu and Lu, 2013).In gypsy moth Lymantria dispar,when larvae were orally infected by B.thuringiensis Kurstaki,several genes associated with digestion,development,binding proteins and immunity were broadly affected in the midguts(Sparks et al.,2013).The ingestion of Bt induced the up-regulation of antibacterial peptides and ly-sozymes in S.exigua larval midguts(Crava et al.,2015)and in the absence of any pathogen,sublethal doses of Bt toxin(CrylCa and Vip3Aa)induced a similar immunity response(Crava et al.,2015). Surprisingly,injection of soluble peptidoglycan into the silkworm larvae slightly up-regulated transcription of Cecropin A and B in the midgut(Yamano et al.,1994),indicating that in addition to their toxins,pathogen proteins can induce immune responses in the midguts of Lepidoptera.

In addition to anti-bacterial peptides,evidence from Drosophila indicates that ROS is an important means to defend against path-ogens(Ha et al.,2009;Kuraishi et al.,2013).In midguts of the fruit ?y,ROS is produced by the membrane associated dual oxidase (DUOX)under the control of p38pathway(Bae et al.,2010;Ha et al., 2009;Ryu et al.,2010).In B.mori,the expression of DUOX was up-regulated in larval midguts orally fed with E.coli and nucleopoly-hedrovirus(NPV)(Hu et al.,2013).Conversely,bacterial prolifera-tion in the larval midguts was increased when the DUOX gene was knocked out(Hu et al.,2013).In addition to DUOX,in B.mori, BmPrx5was found to degrade increasing levels of H2O2,to protect larvae from oxidative damage associated with pathogenic infection in the hemocoel and midguts(Zhang and Lu,2015).Therefore,ROS serves as an important immunity factor for Lepidopteran larvae to defend against bacteria.

In Lepidoptera,ingested microorganisms can also induce im-munity responses in hemocoel.When Galleria mellonella larvae were fed with non-pathogenic and pathogenic bacteria,the activity of lysozyme and PO in hemolymph increased and some antibac-terial proteins were also induced(Freitak et al.,2014).Further work indicates that the ingested bacteria can translocate from midgut into hemocoel,and in females these bacteria deposit with laid eggs to transmit offspring immunity(Freitak et al.,2014).When B.mori larvae were fed with Bacillus bombysepticus(Bb),host cellular and humoral immunities were triggered(Huang et al.,2009).Mean-while,several basal metabolic pathways were regulated and the genes of juvenile hormone synthesis and metabolism pathway were increased(Huang et al.,2009).Thus,immunity responses are not limited to the midgut after oral infection,and both hemocoel immunity and metabolism are also altered.

4.2.Microsporidia

Microsporidia are a group of fungi-like pathogens(V a vra and Larsson,1999)with a broad spectrum of hosts including humans (Didier et al.,2000).During the process of infection,each micro-sporidia cell extrudes a specialized polar tube to inject its spore content into the target cell(Xu and Weiss,2005).Microsporidia can spread by vertical transmission,exempli?ed by the midgut path-ogens Endoreticulatus schubergi and Nosema lymantriae in L.dispar larvae(Goertz and Hoch,2008),and can overwinter in the dead bodies of larvae as seen for the microsporidia Vairimorpha disparis which targets fat bodies(Goertz and Hoch,2008).

Most insect microsporidia are intestinal pathogens.Nosema bombycis is an important pathogen to B.mori,and it can also infect another Lepidopteran insect P.xylostella(Kermani et al.,2013). Histological observations demonstrated that Nosema infect the P.xylostella midgut epithelial cells resulting in marked vacuoliza-tion of the cytoplasm(Kermani et al.,2013).When B.mori larvae were orally infected by N.bombycis,the subtilism-like serine pro-tease NbSLP1was transcribed in the larval midgut(Dang et al., 2013).NbSLP1is localized at the two poles of spore and is likely involved in the polar tube extrusion process.Separate studies showed that oral infection of B.mori larvae with N.bombycis led to activation of the Toll and JAK/STAT pathways in the midguts,which probably induced the up-regulation of antimicrobial peptides to resist the invading microsporidia(Ma et al.,2013).Very interest-ingly,silkworm hemolymph melanization was also inhibited following microsporidia infection.While clearly an important pathogen to Lepidoptera,we still know very little about the ability of microsporidia to elicit an immunity response,and further work is warranted.

4.3.Virus

As a family of important pathogens,the interaction of virus and Lepidopteran insect has been extensively studied.Viruses that can infect Lepidopteran insects include Cytoplasmic polyhedrosis virus (CPV),Nucleopolyhedrovirus(NPV)and Densovirus(DNV).CPV virions are occluded by viral polyhedrin proteins to form the polyhedra(inclusion bodies)within the host midgut cell cytoplasm after infecting the Lepidopteran midguts(Ince et al.,2007).Lepi-dopteran NPV replicates in two forms,the occlusion derived virus (ODV)formed in the midgut cells and the budded virus(BV)pro-duced and released to induce systemic infection after midgut cell infection(Hou et al.,2013).These two viral sub-types can be distinguished by both their envelope proteins and their respective protein posttranslational modi?cations.During the progress of infection,viruses?rst break through the peritrophic membrane (Bao et al.,2013)and bind Lepidopteran midgut proteins to infect speci?c cell types(Bao et al.,2013).After infection,many protective antiviral proteins including small RNA are induced in the midgut to block the viral DNA replication and protect Lepidopteran insects (Asser-Kaiser et al.,2011).

Each virus spreads differently in Lepidoptera.In B.mor i larvae, the spreading of B.mori nucleopolyhedrovirus(BmNPV)infection from the midgut to hemocoel occurs via the tracheae(Rahman and Gopinathan,2004).Junonia coenia densovirus(JcDNV)is internal-ized via endocytosis by absorptive cells of Spodoptera frugiperda larvae and crosses the midguts through transcytosis(Wang et al., 2013).B.mori densovirus type1(BmDNV-1)proliferates in the nuclei of columnar cells(Ito et al.,2013)and kills larvae four days post oral infection,resulting in phenotypic changes in the midguts, changes that suggest the importance of virus-host interactions during infection.

In Drosophila,RNAi is a potent pathway to defend against virus (Sabin et al.,2010)and the activation of Toll pathway inhibited Drosophila X virus(DXV)(Zambon et al.,2005).In addition,auto-phagy and JAK/STAT signaling are involved in the antiviral process (Sabin et al.,2010).In B.mori larvae,Bmlipase-1,puri?ed from midgut juice,has strong antiviral activity against BmNPV(Ponnuvel et al.,2003),and midgut overexpression under the control of either midgut-speci?c promoter P2or baculoviral immediate early-1(IE1) promoter conferred signi?cant antiviral activity(Jiang et al.,2012, 2013).In the midgut, B.mori serine protease-2(BmSP-2) (Nakazawa et al.,2004)and alkaline trypsin protein(BmtryP)also showed strong antiviral activity against BmNPV(Ponnuvel et al., 2012),while plasma proteins including Hemolin,a Lepidopteran immunoglobulin protein produced by hemocytes and fat bodies upon viral injection,demonstrated antiviral activity in Chinese oak silkworm,Cecropia moth and M.sexta(Terenius,2008;Terenius et al.,2009).Hemolymph genes serpin-2,RFP,lipase,sp-2and NOX also exhibit anti-viral activity in B.mori(Chen et al.,2014). Taken together,these works indicate that Lepidoptera circulate key

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proteins in the midgut juice and hemolymph that serve as potent antiviral factors.

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)expresses p35to inhibit host cell apoptosis(Clem,2005) and mutations in p35reduced AcMNPV infection activity to S.fru-giperda larvae(Clem and Miller,1994).Over-expression of apoptosis inhibitor Cp-iap in the mutant AcMNPV p35strain effectively rescued the infection activity,to almost the wild type levels(Clem and Miller,1994).These data support the notion that cell apoptosis is an important Lepidopteran host defense and sug-gests that some virus may speci?cally inhibit cell apoptosis to accelerate the speed of infection.

With the genome sequence resolved in some important Lepi-dopteran insects(Xia et al.,2004;You et al.,2013;Zhan et al.,2011), transcriptome and proteomics approaches have given important insights into immunity responses to midgut virus infection.Next generation sequencing of B.mori(Daizo)midguts following oral infection with cytoplasmic polyhedrosis virus(BmCPV)identi?ed 167up-regulated and141down-regulated genes,and among these were known immunity genes against BmCPV(Kolliopoulou et al., 2015).Among the genes up-regulated were the core RNAi genes Ago-2and Dcr-2,suggesting that the RNAi machinery probably responds to virus infection(Kolliopoulou et al.,2015).In an earlier study using BmCPV to orally infect the4008silkworm strain,there were649up-regulated and103down-regulated genes detected (Gao et al.,2014).Among a subset tested,the Calreticulin FK506-binding protein was concluded to induce calcium dependent apoptosis as a cellular antiviral response(Gao et al.,2014). Sequencing of small RNA libraries constructed from CPV-infected B.mori midguts identi?ed approximately58miRNAs showing dif-ferential expression between infected and normal midguts(Wu et al.,2013).The role of these miRNA and their targets in medi-ating antiviral immunity in B.mori deserves further work.Elec-trospray ionization(ESI)mass spectrometry assay of B.mori larval guts following oral BmNPV viral infection identi?ed many novel secreted proteins including HSP70,lipase-1and chlorophyllide A binding protein precursor(Hu et al.,2015).From these pioneering transcriptome and proteomics studies,a complex picture of the Lepidopteran gut immunity responses designed to defend against viral infection has emerged.

Extensive work to identify strain-speci?c differences in viral susceptibility and resistance in Lepidoptera has the potential to shed light on the mechanism of antiviral immunity.B.mori den-sovirus(BmDNV)causes lethality due to replication in the midgut columnar cells(Ito et al.,2013).In a B.mori virus resistant strain, positional cloning was used to identify a6-kb deletion in the ORF of the transmembrane protein,nsd-2(Ito et al.,2008).To demonstrate that nsd-2is a receptor for BmDNV-2,over-expression of the wild type nsd-2ORF in the deletion resistant strain made the transgenic individuals susceptible to BmDNV-2again(Ito et al.,2008).To broaden the understanding to the mechanism of strain-speci?c differences in viral effects,transcriptome and proteomics compar-isons have been used to identify key genes involved in antiviral activity.Up-regulation of heat shock70-kDa protein cognate,cy-tochrome P450,vacuolar ATP synthase subunit B,arginine kinase, vacuolar ATP synthase subunit D and glutathione S-transferase sigma occurred in the midguts of the silkworm resistant strains Qiufeng and the near isogenic line BC6,suggesting the potential immunity activity of both these genes against BmDNV(Chen et al., 2012).In another study,when the B.mori strains KN(resistant)and 306(susceptible)were simultaneously orally infected with BmNPV and subjected to subtractive hybridization(Bao et al.,2009),62 differentially expressed genes were identi?ed,and among the genes associated with midgut immunity against BmNPV infection in the KN strain were gloverin-3,gloverin-4,lebocin,serpin-5,arylphorin,promoting protein,cathepsin B and actin A3(Bao et al.,2009).Four resistant strains of B.mori(P50,A35,A40,A53) exhibited higher expression of antiviral proteins like lipase-1,Nox and serine protease-2compared to susceptible ones(Cheng et al., 2014).With new,powerful techniques to measure global tran-scriptome changes between susceptibile and resistant strains, important genes involved in antiviral infection can be identi?ed and their functions assayed directly.

4.4.Gut immunity on molting and metamorphosis stages

While Lepidopteran larva have active immunity against patho-gens in the midguts during feeding stage,the importance of im-mune activity during molting and metamorphosis stages when no foods are ingested is unclear.Studies in Lepidopteran and other species of insects indicate that immunity proteins expressed during the molting and wandering stages likely defend eclosing insects against potential infection from the gut microbiota due to signi?-cant remodeling of gut structures(Kim et al.,2014;Xu et al.,2012). Consistent with this,in Anopheles punctipennis(Say),Culex pipiens (L.)and Aedes aegypti(L.),microbiota in the midgut decreased signi?cantly from fourth instar to the?nal larval defecation(Moll et al.,2001).Levels of many immunity proteins increase in the Lepidopteran midguts,suggesting that these proteins probably serve to clean the microbiota during the wandering stage in prep-aration for metamorphosis.In B.mori,the immunity proteins lysozyme,b GRP2and TAK1were speci?cally expressed in the midguts during the wandering stage(Xu et al.,2012).In Riptortus-Burkholderia,the number of bene?cial symbionts decreased from the pre-molt to the inter-molt stages(Kim et al.,2014),which was explained in part by molting stage expression of immunity proteins such as C-type lysozyme and pyrrhocoricin-like antimicrobial peptide(Kim et al.,2014).In the midguts of mosquitoes,molting ?uids,probably ingested through the true mouth during the pro-cess of adult emergence contain many immunity proteins(Zhang et al.,2014),and are thought to be bactericidal during meta-morphosis(Moll et al.,2001).These limited studies suggest that the regulation of immunity and the role of immune defenses during molting and metamorphosis stages deserve further study.

5.Prophenoloxidase in the Lepidopteran gut

In insects,prophenoloxidase(PPO)is typically cleaved and activated into PO to induce melanization around pathogens and wounds(Lu et al.,2014).However,in the mosquito adult midguts, the ookinete stage of the malarial parasite Plasmodium is some-times melanized before traversing the midgut basal lamina and entering the hemocoel(Wang and Jacobs-Lorena,2013;Whitten et al.,2006).Likewise,in some Drosophila mutants,melanization occurs inside the hindguts as well(Chen et al.,2010;Seisenbacher et al.,2011).

The source of PO in the insect midgut was unclear(Ashida and Brey,1998).In the mosquito adult midguts,it is concluded that PO likely comes from hemolymph(Christophides et al.,2004;Osta et al.,2004;Whitten et al.,2006).Recent work shows PPOs are secreted into the foregut in Lepidoptera(Shao et al.,2012)where they are activated likely by a chymotrypsin-like enzyme to catalyze plant phenolics into intermediates to detoxify phenolics in the diet (Wu et al.,2015).In the hindgut of Lepidopteran phytophagous insects like B.mori,PPO is secreted to induce melanization of feces, by which to clean fecal microorganisms(Shao et al.,2012).A physical wound made in the Lepidopteran larval midgut by a needle induced melanization around the wound(Huang et al.,2016), however melanization was limited to the encapsulated hemocytes and no cells in the midgut were melanized directly.

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These observations of melanization in Lepidopteran gut likely have broader implications.In Drosophila,larvae mutant for the p38 mitogen-activated protein kinase(MAPK)show immunode?ciency to microbes in?y food resulting in extensive melanization in the hindgut(Chen et al.,2010).Notably,the thickness of circular muscle ?bers was decreased in the hindguts of these p38b mutants(Chen et al.,2010).During chronic stress,the MAPK-activated protein ki-nase MK2associated with p38b can counteract the JNK-involved apoptosis in the enterocytes in Drosophila larvae(Seisenbacher et al.,2011).In MK2mutant larvae fed on diet containing high salt,hindgut tissue damage was increased and this damage was associated with melanization(Seisenbacher et al.,2011).In hon-eybees,the gut microbiota Frischella perrara colonizes a restricted niche in the hindgut to form a melanized scab(Engel et al.,2015), though it is unclear how this melanized scab is formed through hindgut PPO and how the colonized bacteria in the scab escape the toxicity of melanization.While it was unexpected to?nd PPO in the foregut and hindgut of insects(Shao et al.,2012;Wu et al.,2015), the functions of PPO in insect guts deserve further studies in the future.

6.Conclusions and perspectives

As the main site for digestion and nutrition absorption,the gut of Lepidopteran insect is the?rst line of defense against ingested pathogens.In Drosophila,oral pathogenic infections in the midguts induce cell proliferation,differentiation,apoptosis and autophagy to maintain midgut cell homeostasis(Buchon et al.,2010;Lemaitre and Miguel-Aliaga,2013).Comparatively,midgut responses to oral pathogenic infections in Lepidoptera are still poorly understood, and deserve efforts to elucidate their nature in the future.

Midgut microbiota likely offer the host protection but it is poorly understood how they are protected from antibacterial peptides (AMP)and other immunity proteins including increased ROS pro-duced in the midguts to pathogen infections(Engel and Moran, 2013).In Drosophila,the DUOX-dependent ROS and NF-k B depen-dent AMP system offer protection from pathogenic bacteria but do so without damaging the gut commensal microbiota(Bae et al., 2010;Ha et al.,2009;Ryu et al.,2010).Our limited knowledge of gut immunity in Lepidoptera will be improved by transcriptome and proteomic methods,and while it is not convenient to geneti-cally modify Lepidoptera as it is for Drosophila.Furthermore,RNAi is not so effective in all Lepidoptera insects(Terenius et al.,2011). With the improvement of CRISPR/Cas9and targeted genome edit-ing technology(Bolukbasi et al.,2015;Sander and Joung,2014).It offers a chance to genetically dissect pathways regulating Lepi-doptera gut immunity in the future.

Due to their impact on agriculture,it is urgent to better under-stand Lepidopteran gut immunity as a potential target for pest control with a goal of designing more effective and less harmful pesticides.The surprising similarities in morphology of Lepidop-teran goblet cells to mammalian goblet cells may make them a model to understand the general functions of this class of cells. Likewise,it may be possible to improve insect sericulture by creating transgenic silkworms with the enhanced gut immunity to defend against pathogens effectively.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(31472043,31172147,31172151).

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