research article next generation sequencing identifies five...

Research ArticleNext Generation Sequencing Identifies Five Major Classes ofPotentially Therapeutic Enzymes Secreted by Lucilia sericataMedical Maggots

Zdenjk Franta1 Heiko Vogel2 Ruumldiger Lehmann1 Oliver Rupp3

Alexander Goesmann3 and Andreas Vilcinskas14

1Department of Bioresources Fraunhofer Institute for Molecular Biology and Applied EcologyWinchesterstraszlige 2 35394 Giessen Germany2Department of Entomology Max Planck Institute for Chemical Ecology Hans-Knoll-Straszlige 8 07745 Jena Germany3Justus-Liebig-University of Giessen Bioinformatics and System Biology Heinrich-Buff-Ring 58 35392 Giessen Germany4Justus-Liebig-University of Giessen Institute for Insect Biotechnology Heinrich-Buff-Ring 26-32 35392 Giessen Germany

Correspondence should be addressed to Andreas Vilcinskas andreasvilcinskasagraruni-giessende

Received 29 January 2016 Accepted 7 March 2016

Academic Editor Yudong Cai

Copyright copy 2016 Zdenek Franta et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Lucilia sericata larvae are used as an alternative treatment for recalcitrant and chronic wounds Their excretionssecretionscontain molecules that facilitate tissue debridement disinfect or accelerate wound healing and have therefore been recognizedas a potential source of novel therapeutic compounds Among the substances present in excretionssecretions various peptidaseactivities promoting the wound healing processes have been detected but the peptidases responsible for these activities remainmostly unidentified To explore these enzymes we applied next generation sequencing to analyze the transcriptomes of differentmaggot tissues (salivary glands gut and crop) associated with the production of excretionssecretions andor with digestion as wellas the rest of the larval body As a result we obtainedmore than 1238 million paired-end reads which were assembled de novo usingTrinity and Oases assemblers yielding 41421 contigs with an N50 contig length of 222 kb and a total length of 6779Mb BLASTpanalysis against the MEROPS database identified 1729 contigs in 577 clusters encoding five peptidase classes (serine cysteineaspartic threonine and metallopeptidases) which were assigned to 26 clans 48 families and 185 peptidase species The individualenzymes were differentially expressed among maggot tissues and included peptidase activities related to the therapeutic effects ofmaggot excretionssecretions

1 Introduction

The maggots of certain flies have been used as traditionalmedicines for centuries [1] but modern maggot debridementtherapy (MDT) was established approximately 100 years agoMDT was then widely used for the treatment of chronicwounds until the mid-1940s since when the technique hasbeen supplanted by antibiotics and improved wound care[2] MDT using exclusively Lucilia sericata maggots hasrecently undergone a renaissance and medical maggots arenow approved as an alternative approach for the treatment ofmany types of chronic and necrotic wounds including dia-betic ulcers [3ndash5] postsurgical wounds [6] and burns [7 8]

Maggots applied to hard to heal wounds debride the necrotictissue disinfect the wound and stimulate the healing process[9] The beneficial effect of MDT cannot be attributed to thesingle molecule but rather to the synergistic action of variousbioactive substances including large variety of proteolyticenzymes which are present in maggots excretionssecretionsproducts (MEP) [10]

Debridement the removal of necrotic tissue and woundslough is a well-documented effect ofMDT [11ndash13]Themag-gots perform physical debridement with their mandibles butchemical debridement with enzymes is the most importantcomponent They do so by releasing their digestive enzymesinto the wound which liquefy necrotic and infected tissues

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 8285428 27 pageshttpdxdoiorg10115520168285428

2 BioMed Research International

before it is consumed back Chambers et al identified threeclasses of proteolytic enzymes (aspartic serine and metal-lopeptidases) from MEP and proposed that mainly serinepeptidases are responsible for the superficial debridementactivity of maggots [14] Only two such peptidases (serinepeptidases) have been identified and characterized thus farChymotrypsin 1 was identified from MEP and producedin the recombinant form [15] Recombinant enzyme wasshown to degrade the eschar from venous leg ulcers in vitro[15] and to be unaffected by two endogenous inhibitors1205721-antichymotrypsin and 1205721-antitrypsin from wound eschar[16] We recently produced and characterized Jonah-likechymotrypsin which digested three specific extracellularmatrix proteins (laminin fibronectin and collagen IV) invitro and proposed its function in wound debridement[17]

The natural habitat of L sericata larvae is rotting organicmatter such as cadavers and excrement but this ecologicalniche also favors many microorganisms so the larvae musthave adequate defenses against infectionThemaggots there-fore protect themselves by producing many antimicrobialsubstances [18ndash22] and by digesting microbes which arethus eliminated in the larval gut [23 24] InterestinglyMEPs also show activity against relevant human pathogensincluding antibiotic-resistant bacterial strains [25ndash27] andbiofilms [28ndash31] Recently two molecules with antibiofilmactivity have been identified from MEP Affinity purifiedDNase disrupted Pseudomonas aeruginosa biofilm [32] andrecombinant chymotrypsin I was active against Staphylococ-cus epidermidis and S aureus biofilms [33]

Surprisingly medical maggots also directly promotewound healing [10] MEPs stimulate fibroblast migration[34 35] and proliferation [36] and increase angiogenesis[37 38] MEPs also influence the activation of the humancomplement system [39] reduce proinflammatory responses[40ndash43] and induce fibrinolysis [44] Recently we discov-ered that MEPs contain peptidases that influence bloodcoagulation as part of the wound healing process [45] andthis activity was attributed to Jonah-like serine peptidaseRecombinant enzyme was shown to reduce the clotting timeof human plasma by substituting for the intrinsic clottingfactors kallikrein factor XI and factor XII respectively [17]

However although severalmolecules have been identifiedfromMEP it is still recognized as a largely unexplored sourceof compounds with therapeutic potential The future studiesshall focus on identification isolation andor production ofeffector molecules and testing of their therapeutic potentialHere we analyzed the transcriptome of different larvaltissues to systematically identify MEP peptidases It is notclear whether MEP components are exclusively producedby salivary glands or also by other tissues so we dissectedthree individual tissues (gut crop and salivary glands) as wellas the remaining larval biomass to generate tissue-specificsequence data The extracted mRNA was sequenced usingthe Illumina HiSeq2000 Genome Analyzer platform andpaired-end read technology After preprocessing 123856654paired reads remained in the panel of libraries These wereprocessed further to yield a final assembly of 41421 contigs in17479 clusters resulting in the identification of 1729 contigs

in 577 clusters encoding five different functional classes ofproteolytic enzymes

2 Materials and Methods

21 Preparation of Biological Material First-instar L sericatamaggots were obtained from BioMonde GmbH (BarsbuttelGermany) and were cultured under sterile conditions onColumbia agar plates with ldquosheep blood +rdquo (Oxoid Deutsch-land GmbH Wesel Germany) at 28∘C for 48 h in the darkThe larvae were cleaned and then infected with a mixtureof Pseudomonas aeruginosa (DSM 50071) and Staphylococcusaureus (DSM 2569) as previously described [21] The larvalgut salivary glands and crop were dissected under a binoc-ular microscope 8 h after infection Dissected tissues and theremaining larval body for Illumina sequencing were frozenin liquid nitrogen and stored at ndash80∘C Samples for qRT-PCRanalysis were processed immediately as described below

22 RNA Isolation and Illumina Sequencing Total RNAwas extracted from individual tissues and the rest of thelarval body using the innuPREP RNA Mini Isolation Kit(Analytik Jena Jena Germany) following the manufacturerrsquosinstructions Additional RNA purification quantificationand quality control were carried out as previously described[46] An additional Turbo DNase treatment (Thermo FisherScientific Waltham MA USA) was applied before thesecond purification step to eliminate contaminating DNAThe DNase was removed and the RNA purified using theRNeasy MinElute Clean up Kit (Qiagen Hilden Germany)following the manufacturerrsquos protocol RNA was eluted in20120583L Ambion RNA Storage Solution (Thermo Fisher Scien-tific) and poly(A)+ mRNA was prepared using the AmbionMicroPoly(A) Purist Kit according to the manufacturerrsquosinstructions (Thermo Fisher Scientific) The integrity andquantity of the mRNA was confirmed using an Agilent 2100Bioanalyser and RNA Nano chips (Agilent TechnologiesSanta Clara CA USA)

Transcriptome sequencingwas carried out on an IlluminaHiSeq2000 Genome Analyzer platform using paired-end(2 times 100 bp) read technology for the larval tissues withRNA fragmented to an average length of 150 nucleotidesSequencing was carried out by Eurofins MWG Operon(Ebersberg Germany) and resulted in totals of 29 33 26 and34 million reads for the rest of body gut crop and salivaryglands respectively

23 Read Preprocessing The quality of the reads was checkedusing the FastQC toolkit [47] Trimmomatic [48] wasused to clip adapters and trim low-quality regions (param-eter ILLUMINACLIPTruSeq3-SE-223010 SLIDINGWIN-DOW520 HEADCROP15 MINLEN50) The reads fromall libraries were then pooled for assembly and digitallynormalized to achieve 100-fold coverage using the Trinity ldquoinsilico normalizationrdquo tool [49]

24 Assembly The reads from all libraries were assembledde novo in two steps One assembly was computed using

BioMed Research International 3

the Trinity assembler [49] followed by 28 individualVelvetOases assemblies [50] with k-mer parameters rangingfrom 21 to 75 In the second step the resulting transcriptsequences were combined and high-quality sequences wereextracted using the EvidentialGene pipeline [51] Potentialisoforms were detected by clustering the protein sequencesfrom the EvidentialGene pipeline using CD-HIT [52] with90 identity

25 Quality Assessment The CEGMAmethod [53] was usedto assess the completeness of the transcriptomeThedetectionstep of theCEGMApipelinewas replaced by aBLASTp search[54] against the CEGMA EuKaryotic Orthologous Groups(KOG) sequences because protein sequences had alreadybeen identified in the previous stepThe completeness of indi-vidual sequences was estimated by computing the ldquoorthologhit ratiordquo [55] against the D melanogaster protein sequences

26 Functional Annotation and Peptidase IdentificationPutative transposable elements were identified using Trans-posonPSI [56] Furthermore all sequences with HMMER 30[57] hits against Pfam domains [58] as previously described[59] were marked as potential transposable elements Allsequences in clusters with at least one putative transpos-able element were annotated as transposable elements Allsequences were uploaded to the SAMS web server [60] andautomatically annotated using BLAST [54] andHMMER [57]searches against different databases Next all the peptidaseswere identified using the ECnumbers [61] from the automaticannotation of the transcriptome data and further classifiedusing MEROPS database [62]

27 Mapping and Digital Gene Expression Analysis Digitalgene expression analysis was carried out by using QSeqSoftware (DNAStar Inc) to remap the Illumina reads ontothe reference backbone and then counting the sequences toestimate expression levels For read mapping we used thefollowing parameters n-mer length = 40 read assignmentquality options required at least 40 bases (the amount ofmappable sequence as a criterion for inclusion) and atleast 90 of bases matching (minimum similarity fractiondefining the degree of preciseness) within each read to beassigned to a specific contig maximum number of hits fora read (reads matching a greater number of distinct placesthan this number are excluded) = 10 n-mer repeat set-tings were automatically determined and other settings werenot changed Biases in the sequence datasets and differenttranscript sizes were corrected using the RPKM algorithm(reads per kilobase of transcript permillionmapped reads) toobtain correct estimates for relative expression levels For theselected protease groups gene expression (log2 transformedRPKM values) was visualized as heat maps using customscripts and matplotlib [63] to generate a 2D plotting libraryusing the Jet Colormap [64]

28 Quantitative Reverse Transcription Real-Time PCRReaction (qRT-PCR) A subset of differentially expressed

Table 1 Illumina sequencing data representing different L sericatatissues

Reads BasesBody 29536054 438GbCrop 26402527 396GbSalivary glands 34206636 509GbGut 33711437 509Gb

peptidases from each peptidase clan was validated by qRT-PCR Total RNA isolation cDNA synthesis primer designand qRT-PCR experiments were performed as describedpreviously [65] Datawere analyzed in Rest 2009 (httpwwwgene-quantificationderest-2009html) using the ΔΔCqmethod [66] Relative mRNA values of individual genes wereadjusted to the sample with the highest value and normalizedusing the 60S acidic ribosomal protein P0 (RPLPO) and 40Sribosomal protein S3 (RPS3) genes

3 Results and Discussion

31 Preprocessing and Assembly of Sequence Reads Afterpreprocessing 123856654 paired reads remained in thefour tissue-specific libraries (Table 1) The ldquoin silico normal-izationrdquo of the pooled reads reduced the total number to16236645 The normalized reads were assembled using theTrinity assembler and 28 individual VelvetOases assembliesproducing a total of 1794145 contigs (Additional file 1 seeSupplementary Material available online at httpdxdoiorg10115520168285428) Filtering and clustering of the contigsusing the EvidentialGene pipeline and CD-HIT produced afinal set of 41421 contigs in 17479 clustersThe final assemblycontained 41421 contigs covering a total of 6779Mb a meancontig length of 164 kb and a N50 contig length of 222 kb

32 Assembly Quality Control We found that 80ndash89 of thenonnormalized reads could be mapped to the final assembly(8461 body 8030 crop 8892 gland and 8795 gut)CEGMA identified 235 of the 248 (9476) core genes withan ortholog ratio of 279 We then mapped 26950Drosophilamelanogaster protein sequences to the final assembly and9961 (3696) could be alignedwith amean ortholog hit ratioof 071 The mean ortholog number (number of contigs map-ping to the same D melanogaster protein sequence) was 35

33 Annotation TransposonPSI and HMMER searchesidentified 470 putative transposable elements in 288 clustersThe automatic functional annotation pipeline involvingBLASTp searches against different databases revealed 17864(4312) ldquohigh confidencerdquo annotations and 15155 (3658)ldquohypothetical proteinsrdquo Gene ontology (GO) analysis wasused to explore the functional characteristics of all contigsand assign them to three independent categories biologicalprocesses molecular function and cellular components(Figure 1) In addition a BLASTp search against theMEROPS database v912 [67] identified 1729 contigs in 577clusters as peptidases The identified peptidases represented


Biol

ogic

al p

roce

ssC

ellu

lar c

ompo

nent

Mol

ecul

ar fu

nctio

n

48763430

3192822041479284257

658530524424390

1603919107533

2799813

355305194

272084411

0 2000 4000 6000Binding

Catalytic activityTransporter activity

Structural molecule activityElectron carrier activity

Enzyme regulator activity

Receptor activityMolecular transducer activity

Cell partOrganelle

MembraneMembrane part

Macromolecular complexExtracellular region

Organelle partVirion part

Extracellular matrixCell junction

Extracellular matrix partMembrane-enclosed lumen

Extracellular region part

Metabolic processCellular process

Biological regulationLocalization

Single-organism processResponse to stimulus

Multicellular organismal processImmune system process

Reproductive processDevelopmental process

SignalingGrowth

Number of ldquohigh confidencerdquo annotations

Protein binding transcription factor activity

Nucleic acid binding transcription factor activity

Figure 1 Gene ontology analysis of the L sericata transcriptome All identified contigs (41421) were categorized using three GO terms(a) biological process (b) cellular component (c) molecular function

sim4 of the total number of contigs This result correlateswith data from other organisms where peptidases representmore than 2 of all genes [68]

34 Peptidases Peptidases are proteolytic enzymes thathydrolyze peptide bonds and they are found ubiquitously inall biological systems from viruses to vertebrates Based onthe key amino acid residues responsible for proteolytic activ-ity six different peptidase classes are recognized (asparticcysteine serine glutamic threonine and metallopeptidases)as well as further unclassified peptidases [69] From the 1729L sericata contigs (in 577 clusters) identified as peptidases1655 contigs (in 557 clusters) were assigned to one of five pep-tidase classes (aspartic cysteine serine threonine and met-allopeptidases) whereas 74 contigs (in 20 clusters) remainedunassigned As summarized in Figure 2 serine peptidaseswere the most prominent class (837 contigs in 270 clusters)followed by metallopeptidases (565 contigs in 202 clusters)cysteine peptidases (145 contigs in 45 clusters) threoninepeptidases (51 contigs in 25 clusters) and aspartic peptidases(57 contigs in 15 clusters) The MEROPS database wasused to subdivide the identified enzymes further into clans(peptidases with evolutionarily conserved tertiary structuresorders of catalytic residues and common sequence motifsaround the catalytic site) families (peptidases with similar

amino acid sequences) and species (peptidases with similarproperties and a unique MEROPS identity) [70 71] Accord-ingly we identified 26 clans containing 48 families and 185peptidase species (Table 2) We found that almost half of theidentified clusters represented serine peptidases in clan PAand family S1

GO analysis was then carried out to assign functionalcategories to each of the identified peptidase clustersWewereable to assign 534 of 577 clusters to three different categoriesbiological process (345 clusters) molecular function (533clusters) and cellular component (70 clusters) (Figure 3) Wefound that most of the peptidases (310 clusters) are involvedin the biological process (level 3) category of ldquoprimarymetabolic processrdquo (Figure 3(a)) The molecular function(level 3) of most peptidases was either catalytic activity (201)or hydrolase activity (253) (Figure 3(b)) as expected given themolecular role of peptidases Interestingly only 70 clusterswere assigned a cellular component function (Figure 3(c))

35 Aspartic Peptidases Aspartic peptidases contain anaspartic acid residue at the active site [72] An asparticpeptidase activity was previously identified in maggot MEPsusing class-specific inhibitors [14] and the correspondinggene was shown to be strongly upregulated in L sericatalarvae following an immune challenge [18] We identified 57


Table 2 General overview of L sericata peptidases

Class Clans Families Species Unassigned peptidases Nonpeptidase homologsAspartic peptidase 2 3 7 1 mdashCysteine peptidase 6 11 23 8 1Metallopeptidase 9 20 53 12 9Threonine peptidase 1 2 16 mdash mdashSerine peptidase 8 12 86 2 1Total 26 48 185 23 11

47

35

8

43 3

Serine peptidases (270)Metallopeptidases (202)Cysteine peptidases (45)

Threonine peptidases (25)Aspartic peptidases (15)Unidentifiedunassigned (20)

Figure 2 Proportional representation of each peptidase class inthe L sericata transcriptome Among 577 clusters identified aspeptidases 557 clusters were assigned to 5 peptidase classes (serinepeptidases (270) metallopeptidases (202) cysteine peptidases (45)threonine peptidases (25) and aspartic peptidases (15)) and 20clusters remained unassigned

contigs in 15 clusters representing aspartic peptidases andthese were further assigned to two clans three families andseven peptidase species (Table 3) with different tissue-specificexpression profiles (Additional file 2)

351 Family A1 Preprocathepsin D-like peptidases are thelargest group of aspartic peptidases The majority of clustersincluded a signal peptide propeptide and mature enzymecontaining all of the conserved catalytic and substrate-binding residues found in human lysosomal cathepsinD [69]With the exception of cluster LST LS5572 and two incom-plete clusters (LST LS009595 and LST LS016491) all clusterslacked the polyproline loop (DxPxPx(GA)P) (Figure 4)Theabsence of this loop is a characteristic of pepsin and diges-tive cathepsin D peptidases in the Brachyceran infraorder

Muscomorpha and may be associated with the extracellularrole of these enzymes [73] The aspartic peptidase gene pre-viously identified in challenged L sericata larvae [GenBankFG360526] is homologous to cluster LST LS005916 whichwas predominantly expressed in the larval gut Asparticpeptidases can kill bacteria in vitro in an acidic medium[74] and may also kill bacteria in the Musca domesticalarval midgut (Espinoza-Fuentes Terra 1987) Based on itslocalization in the gut and induction by an immune chal-lenge we propose a similar role for this L sericata asparticpeptidase However heat map analysis (Additional file 2)revealed that themajority of A1 family aspartic peptidases arepredominantly expressed in the larval gut suggesting a rolein digestion andor the elimination of bacteria

352 Family A22 This family of intramembrane peptidasescomprises two subfamilies The A22a subfamily is typified bypresenilin an enzyme that plays central role in intramem-brane proteolysis [75] and the pathogenesis of Alzheimerrsquosdisease [76] The A22b subfamily is typified by impas 1 pep-tidase which is responsible for the degradation of liberatedsignal peptides and may play an essential role in the devel-opment of D melanogaster larvae [77] We identified fourclusters assigned to three peptidase species encoding mem-bers of the A22b subfamily and two of them (LST LS005714and LST LS015224) were strongly upregulated in the sali-vary glands (Additional file 2) as previously reported in Dmelanogaster [77] We therefore propose a similar functionfor the L sericata peptidase in larval development

353 Family A28 Only one contig in one cluster was iden-tified assigned to family A28 A homologous skin asparticpeptidase (SASPase) was recently identified in human skin[78] although its biological role remains unclear

36 Cysteine Peptidases Cysteine peptidases are ubiquitousand mediate diverse biological processes including immuneresponses [79] extracellular matrix remodeling [80] anddevelopment and apoptosis [81]The deregulation of cysteinepeptidases is associated with human diseases such as cancerand atherosclerosis [82] There are 65 cysteine peptidasefamilies listed in MEROPS database v912 [62] 56 of whichare assigned to 10 annotated clans whereas 9 families remainunassigned Our L sericata transcriptome contained 145contigs in 45 clusters belonging to 6 clans 11 families and 23peptidase species (Table 4)


290203145

8986

174

029058 058 058

Metabolic process (10)Nitrogen compound metabolic process (7)Catabolic process (5)Primary metabolic process (310)Cellular process (6)Multicellular organismal process (1)Single-organism process (2)Localization (2)Biological regulation (2)

(a) Biological process

3771

019

4747

0191426

019

Catalytic activity (201)Transferase activity (1)Hydrolase activity (253)Lyase activity (1)Binding (76)Enzyme regulator activity (1)

(b) Molecular function

429

5286

571571

1571

1571

Extracellular region (3)Membrane (37)Extracellular matrix (4)Macromolecular complex (4)Membrane part (11)Cell part (11)

(c) Cellular component

Figure 3 Gene ontology analysis of L sericata peptidases All identified peptidase clusters (577 in total) were categorized using three GOterms (a) biological process (b)molecular function and (c) cellular component Analysis of (a) biological process and (b)molecular functionwas performed at level three whereas analysis of (c) cellular component was performed at level two

361 Family C1 The biggest family of cysteine peptidasesin the L sericata transcriptome is clan CA family C1and its members were moderately abundant in all tissues(Additional file 3) Of 16 clusters 12 were assigned to 7

peptide species including several with known roles Theseinclude insect 2629 kDA peptidase which plays a rolein immunity [83 84] vitellogenic cathepsin B whichdegrades vitellogenin [85] and bleomycin hydrolase


Table 3 List of aspartic peptidases in the L sericata transcriptome database

Clan Family Peptidase type Peptidase species MEROPS ID Number of clusters

AA A1 Pepsin A

CAD2 peptidase (M domestica) A01092 5CAD3 peptidase (M domestica) A01093 2CG10104 gp (D melanogaster) A01A64 2

CG17134 protein (D melanogaster) A01A78 1

AD A22 Presenilin 1impas 1 peptidase A22003 2impas 2 peptidase A22005 1

Psn peptidase (Drosophila-type) A22014 1AA A28 DNA-damage inducible protein 1 Family A28 unassigned peptidases A28UPW 1

Table 4 List of cysteine peptidases in the L sericata transcriptome database


CA C1 Papain

Insect 2629 kDa peptidase C01067 2Vitellogenic cathepsin B C01068 1Bleomycin hydrolase (animal) C01084 2Papain homologue (Rattus-type) C01107 3Cathepsin B-like cysteine peptidase (Raphanus sativus) C01144 1CG12163 protein (D melanogaster) C01A27 2CG4847 protein (D melanogaster) C01A28 1Nonpeptidase homologues C01UNA 3Subfamily C1A unassigned peptidases C01UPA 1

CA C2 Calpain 2 Calpain-15 C02010 1Calpain A C0214 1

CA C12 Ubiquitinyl hydrolase-L1Ubiquitinyl hydrolase-L5 C12005 1CG8445 protein (D melanogaster) C12A09 1Family C12 unassigned peptidases C12UPW 1

CA C54 Autophagin-1 CG4428 protein (D melanogaster) C54A04 1Family C54 unassigned peptidases C54UPW 2

CA C65 Otubain-1 Otubain-1 C65001 1

CD C13 Legumain Glycosylphosphatidylinositolprotein transamidase C13005 2Family C13 unassigned peptidases C13UPW 2

CD C14 Caspase-1

Caspase (insect 1) C14015 2Caspase (insect 2) C14016 1Caspase DRONC- (D melanogaster-) type peptidase C14019 2Caspase DECAY C14022 1Caspase STRICA (Drosophila sp) C14023 1Caspase Dredd C14040 1

CE C48 Ulp1 peptidase Ulp1 peptidase (Drosophila-type) C48024 1Family C48 unassigned peptidases C48UPW 1

CF C15 Pyroglutamyl-peptidase Family C15 unassigned peptidases C15UPW 1

PC C26 Gamma-glutamyl hydrolase CG32155 protein (D melanogaster) C26A22 2Family C26 unassigned peptidases C26UPW 1

PD C46 Hedgehog protein Hedgehog protein C46001 1Family C46 unassigned peptidases C46UPW 1

whose natural function remains unknown although it caninactivate bleomycin [69] Heat map analysis (Additionalfile 4) revealed only three papain homologs present mostlyin the larval gut (LST LS004517 LST LS006643 andLST LS006644) and two of them (LST LS004517 andLST LS006644) are identical to previously identified partial

sequences of L sericata cysteine peptidases [GenBankFG360492 FG360504] that are upregulated in response toan immune challenge [18] Although papain-type cysteinepeptidases were previously shown to play important digestiverole in ticks [86] hemipterans [87] and beetles [88] theirdigestive role in dipteran species remains unclear [89] Based


lowastlowast lowast lowastlowast lowast lowast lowast lowast lowast lowast lowastlowastlowastlowastlowast lowastlowastlowastLST_LS005916 A I G A T F N Y D Y Y T Y T V D C S S I D S L P A L T L N I G G T T F T I E A S D Y I L Q - - - - S E G V C S S A F E N I G T D F - - - - - - - W I L G D I - F I G R Y Y S I F D L A N N R V G F A T A V LST_LS005431 A I G A T F N Y D Y Y T Y T V D C S S I D S L P A L T L N I G G T T F T I E A S D Y I L Q - - - - S E G V C S S A F E N A G T D F - - - - - - - W I L G D I - F I G R - - - - - - - - - - - - - - - - - - LST_LS005915 A I G A T F N Y D Y Y T Y T V D C S S I D S L P A L T L N I G G T T F T I E A S D Y I L Q - - - - S E G V C S S A F E N I G T D F - - - - - - - W I L G D I - F I G R Y Y S I F D L A N N R V G F A T A V LST_LS005911 A I G A T F N E T T Y E F M L D C S T L D S L P D V N F H I G D G I Y T L E P S D Y V L Q - - - - A D D Q C A T A F E D A G M N I - - - - - - - W I L G D V - F I G K Y Y T T F D L K H K R V G F A R A I LST_LS005551 E I G G M Y N S E D G N Y Y V D C S A I D S L P V V N F V I G G R N F S L E P S A Y I V N - - - - L E G S C M S S F T T M G T D F - - - - - - - W I L G D V - F I G D V W L K M L W C L L S I I C S P K V LST_LS005572 A I G G T P M I G G - Q Y I V S C D S I P N L P V I K F V L G G K T F E L E G K D Y I L R V A Q M G K T I C L S G F - - M G I E I P P P N G P L W I L G D V F F I G K Y Y T E F D M A N D R V G F A V A K

Figure 4 Partial amino acid sequence alignment of A1 peptidases found in the L sericata transcriptome Amino acid sequences of A1aspartic peptidases were aligned using MAFFT [199] Only one cluster (LST LS005572) contained a polyproline loop (underlined) Asterisk(lowast) indicates positions which have a single fully conserved residue Colon () indicates conservation between groups of strongly similarproperties scoring gt 05 in the Gonnet PAM 250 matrix Period () indicates conservation between groups of weakly similar propertiesscoring =lt 05 in the Gonnet PAM 250 matrix

on the strict localization in the L sericata gut and inductionby an immune challenge we speculate that these enzymes areprobably required for the elimination of bacteria in the larvalgut [23] rather than the digestion of food but additionalexperiments are needed to confirm their specific function

362 Family C13 The C13 family of cysteine peptidasescomprises two types of enzymes The first is the asparaginylendopeptidases which were originally found in legumes [90]and later in schistosomes [91] mammals [92] and recentlyalso arthropods [93] These are acidic lysosomal enzymesthat favor asparagine at the P1 position [94] and whose rolesinclude antigen presentation [95] enzyme transactivation[96] and blood meal digestion [97] The second is the glyco-sylphosphatidylinositol (GPI)protein transamidases whichare required for the removal of C-terminal peptides andthe attachment of GPI anchors [98] We identified twoclusters encodingGPIprotein transamidases and two clustersremained unidentified

363 Family C14 Caspases are intracellular endopeptidasesthat are highly specific for the cleavage of aspartyl bondsWith the exception of caspase 1 which is responsible forthe production of interleukin-1120573 in monocytes [69] mostcaspases regulate apoptosis by taking part in a proteasecascade [99]TheDmelanogaster genome encodes seven cas-pases Dronc (Drosophila Nedd2-like caspase) Dredd (deathrelated ced-3Nedd2like) and Strica (serinethreonine richcaspase) possess long N-terminal domains and function asupstream or initiator enzymes whereas Drice (DrosophilaICE) Dcp-1 (death caspase-1) and Decay (death executionercaspase related to ApopainYama) are downstream or effectorcaspases [100 101] Damm(death-associatedmolecule relatedto Mch2) caspase shares the features of both groups but itsbiological role is not fully understood [69] The L sericatatranscriptome database contained eight clusters in sevenpeptidase species representing caspases with different tissue-specific expression profiles (Additional file 4) Phylogeneticanalysis (Figure 5) revealed one L sericata homolog eachfor the effector caspases Dcp-1 and Decay two homologs forDrice one homolog each for the initiator caspases Dreddand Strica and two homologs for Dronc We did not find asequence representing the D melanogaster Damm caspase

364 Other Cysteine Peptidase Families Several cysteinepeptidase families were more or less equally distributed

among the L sericata tissues we tested and these are probablyrequired for essential cellular functions The C2 familyof calcium-dependent peptidases (calpains) is formed ofubiquitous intracellular neutral peptidases associated withdiverse biological functions ranging from signal transductionto apoptosis [102] Ubiquitinyl hydrolases (family C12) areintracellular enzymes that remove ubiquitin from ubiquitiny-lated proteins and peptides [69] Members of family C15 areubiquitous intracellular peptidases that remove pyrogluta-mate from the N-terminus of peptides and hydrolyze biolog-ically active peptides such as neurotensin and gonadotropin[103] Gamma-glutamyl hydrolases (family C26) are pri-marily lysosomal enzymes which are widely distributed innature and probably required for the turnover of cellularfolates [69] Hedgehog proteins (family C46) are self-splicingtwo-domain signaling proteins originally discovered in Dmelanogaster [104]They are found in most metazoan speciesand play multiple roles in pattern formation during develop-ment [105] Members of family C54 which was first discov-ered in the budding yeast Saccharomyces cerevisiae are neces-sary for autophagy [106] Otubains (family C65) are isopepti-dases involved in the removal of ubiquitin frompolyubiquitin[107] These enzymes share no homology to other deubiqui-tinylating enzymes but belong to the ovarian tumor family(OTU) and possess a cysteine peptidase domain [108]

37 Metallopeptidases The metallopeptidases are a ubiqui-tous and highly diverse group of enzymes containing bothendopeptidases and exopeptidases MEROPS database v912[62] lists more than 15 clans and 71 families involved indiverse biological processes such as digestion wound heal-ing reproduction and host-pathogen interactions Althoughthese enzymes vary widely at the sequence structural andeven functional levels all members require a metal ion forcatalytic activity [69] More than 30 of all clusters in ourL sericata transcriptome database (202 clusters) were foundto encode metallopeptidases which were further assignedto 9 clans 20 families and 53 peptidase species (Table 5)The metallopeptidases are therefore the second largest groupof peptidases in the L sericata transcriptome and the mostdiverse in terms of the number of families Although thevariability and abundance of metallopeptidases in L sericataindicate their importance their roles are not well understoodand few studies have addressed specific biological activitiesA metallopeptidase with exopeptidase characteristics anda pH optimum of 8 was detected in L sericata MEPs


LST LS002862StricaA0A0A1XQW9 BACCUags9

ags12ags10

ags13T1P9A0 MUSDO

LST LS005021LST LS006099

T1PAQ7 MUSDODronc

A0A0A1X1L7 BACCUagL2

E0D2V3 BOMMOD6X3B3 TRICALST LS003797T1PK82 MUSDOA0A0A1XM78 BACCU

DreddagL1D6WJH5 TRICAA9ZMY8 BOMMO

D6WD86 TRICAA0A0A1XL94 BACCU

ags8Damm

E9JEH0 BOMMOLST LS017291LST LS000019

B5AK94 MUSDODriceA0A0A1X8B8 BACCUags7A0A0A1WTD1 BACCULST LS006067

T1PN44 MUSDODcp-1

Q8I9V7 BOMMOD6WFK4 TRICALST LS006780T1PFC3 MUSDO

DecayA0A0A1XRT4 BACCU

ags4ags3

ags5ags11ags6ags14

ags2ags1

D6WIV9 TRICAQ2HZ05 BOMMOD6WD84 TRICA

026026

032001001

002007

041021

026025

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038024024

028027

033035037041

043044

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012018

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014014

016023

022022

002000

022020

018019

035035

038

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019

012015

003

001

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001003

003001

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001

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001004

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002006

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01

Figure 5 Phylogenetic analysis of L sericata caspases The phylogenetic analysis of eight identified caspases in the transcriptome of Lsericata For the analysis the sequences were compared with Bombyx mori (BOMMO) UniProtKB [E0D2V3 E9JEH0 E0D2V3 E9JEH0Q2HZ05 Q8I9V7] Tribolium castaneum (TRICA) UniProtKB [D6WD84 D6WD86 D6WFK4 D6WIV9 D6WJH5 D6X3B3] Muscadomestica (MUSDO) UniProtKB [T1P9A0 T1PAQ7 T1PFC3 T1PK82 T1PN44 B5AK94] Bactrocera cucurbitae (BACCU) UniProtKB[A0A0A1WTD1 A0A0A1X1L7 A0A0A1X8B8 A0A0A1XL94 A0A0A1XM78 A0A0A1XQW9 A0A0A1XRT4] D melanogaster (StricaUniProtKB [Q7KHK9] Dronc UniProtKB [Q9XYF4] Dredd UniProtKB [Q8IRY7] Damm UniProtKB [O44252] Drice UniProtKB[O01382] Dcp-1 UniProtKB [O02002] Decay UniProtKB [Q9VET9]) and Anopheles gambiae (agL1 agL2 ags1ndashags14) UniProtKB[Q5TMK1 Q7Q2Q0 Q5TMS0 Q7PZJ9 Q7PZK0 Q7PV92 Q7Q802 Q7Q803 Q7Q4X6 Q7PWK2 Q7QGM9 Q7PSJ2 Q7Q801 Q7QGN0Q7QGM8 Q7QKT2] For the alignment MAFFT [199] was used


Table 5 List of metallopeptidases in the L sericata transcriptome database


MA M1 Aminopeptidase N

ERAP2 aminopeptidase M01024 5Slamdance (D melanogaster) M01A02 2

CG5845 protein (D melanogaster) M01A05 1CG11956 protein (D melanogaster) M01A09 6Dgri protein (D melanogaster) M01A10 2

CG8774 protein (D melanogaster) M01A11 3CG8773 protein (D melanogaster) M01A12 1

AT4g33090 gp (Arabidopsis thaliana) M01A25 6Family M1 nonpeptidase homologues M01UNW 3Family M1 unassigned peptidases M01UPW 4

MA M2Angiotensin-converting enzymepeptidase unit 1

Peptidyl-dipeptidase Ance M02003 16Family M2 nonpeptidase homologues M02UNW 6Family M2 unassigned peptidases M02UPW 4

MA M3 Thimetoligopeptidase

Subfamily M3A nonpeptidase homologues M03UNA 1Subfamily M3A unassigned peptidases M03UPA 1

MA M8 Leishmanolysin Family M8 unassigned peptidases M08UPW 2

MA M10 Matrixmetallopeptidase-1

Dm1 matrix metallopeptidase M10031 2Dm2-matrix metallopeptidase M10036 2

MA M12 Astacin

Mammalian-type tolloid-like 2 protein M12018 1ADM-4 peptidase (Caenorhabditis elegans) M12329 1

ADAMTS-A (D melanogaster) M12A03 1CG4096 protein (D melanogaster) M12A04 2CG4096 protein (D melanogaster) M12A05 1CG6696 protein (D melanogaster) M12A08 1CG15254 protein (D melanogaster) M12A09 1

Subfamily M12A unassigned peptidases M12UPA 10Subfamily M12B unassigned peptidases M12UPB 8

MA M13 Neprilysin

Zmp1 peptidase (Mycobacterium-type) M13009 2Nep2 peptidase (insect) M13012 2

Neprilysin-4 (D melanogaster) M13014 2CG3775 protein (D melanogaster) M13A01 2CG14526 protein (D melanogaster) M13A04 2CG14528 protein (D melanogaster) M13A06 1CG9507 protein (D melanogaster) M13A11 2CG5894 protein (D melanogaster) M13A15 2

Family M13 nonpeptidase homologues M13UNW 6Family M13 unassigned peptidases M13UPW 13

MA M41 FtsH peptidaseParaplegin M41006 1

YME1L1 gp (Homo sapiens) and similar M41026 1Family M41 nonpeptidase homologues M41UNW 1

MA M48 Ste24 peptidase CG9001 protein (D melanogaster) M48A06 2

MA M49 Dipeptidyl-peptidaseIII CG7415 gp (D melanogaster) M49A01 2


Table 5 Continued


MC M14 Carboxypeptidase A1

CG8560 protein (D melanogaster) M14A04 2CG2915 protein (D melanogaster) M14A05 6CG17633 protein (D melanogaster) M14A08 1CG14820 protein (D melanogaster) M14A12 2CG8562 protein (D melanogaster) M14A15 1CG18417 protein (D melanogaster) M14A16 1CG3097 protein (D melanogaster) M14A19 1

Silver protein domain 2 (D melanogaster) M14A20 2CG11428 (D melanogaster) M14A21 2

Subfamily M14A nonpeptidase homologues M14UNA 1Subfamily M14A unassigned peptidases M14UPA 11Subfamily M14B unassigned peptidases M14UPB 3

ME M16 Pitrilysin

Mitochondrial processing peptidase beta-subunit M16003 1Eupitrilysin M16009 1

Mername-AA224 nonpeptidase homologue M16975 2CG2025 protein (D melanogaster) M16A06 3

C02G61 gp (C elegans) M16A08 1LOC133083 gp (H sapiens) M16P01 2

MF M17 Leucineaminopeptidase 3

Leucyl aminopeptidase-1 (Caenorhabditis-type) M17006 2Leucine aminopeptidase (Fasciola-type) M17011 2

MG M24 Methionylaminopeptidase 1

Xaa-Pro dipeptidase (eukaryote) M24007 2Methionyl aminopeptidase 1 (eukaryote) M24017 1

Subfamily M24B nonpeptidase homologues M24UNB 1Family M24 nonpeptidase homologues M24UNW 1Subfamily M24A unassigned peptidases M24UPA 2Subfamily M24B unassigned peptidases M24UPB 2

MH M20 Glutamatecarboxypeptidase Peptidase T M20003 1

M28 Aminopeptidase S Aminopeptidase ES-62 (Acanthocheilonema viteae) M28A15 1Family M28 unassigned peptidases M28UPW 6

MJ M19 Membranedipeptidase Family M19 nonpeptidase homologues M19UNW 1

MM M50 Site 2 peptidase dS2P peptidase (D melanogaster) M50007 1MP M67 RPN11 peptidase At1g71230 (A thaliana) M67A02 1UNA M79 RCE1 peptidase RCE1 peptidase (H sapiens-type) M79002 1

using FITC-casein as a substrate [14] and a partial sequenceencoding a clanMC familyM14metallopeptidase [GenBankFG360509] was identified among the upregulated genes inimmune challenged larvae [18]

371 Family M1 Family M1 is one of 16 peptidase familiesfound in the genomes of all forms of cellular life [109]This family mostly comprises membrane-bound or cytosolicexopeptidases that remove theN-terminus of their substratesHowever the specificity of the S1 subsite varies considerablywhich allows this family to be involved in many differentbiological processes [110] Insect M1 peptidases are mainlyexpressed in the gut where they play important intermediate

roles in protein digestion [111] as well as host-pathogeninteractions Membrane-bound aminopeptidases in the gutare receptors for Bacillus thuringiensis toxins in several insectspecies [112ndash114] Aminopeptidases have also been detectedin other insect tissues such as the fat body [115] salivaryglands [116] and Malpighian tubes [116] Although theirinteractionswithB thuringiensis toxins have been confirmedtheir endogenous role is unclear [116] Aminopeptidase N inthe hemocoel plays an important role in the postembryonicdevelopment of the pest moth Achaea janata [117] Weidentified 33 clusters encoding 8 peptidase species (Table 5)and 6 of them are predominantly expressed in the larval gut(Additional file 4)


372 FamilyM2 FamilyM2 contains angiotensin convertingenzyme (ACE) the dipeptidyl peptidase that removes dipep-tides from the C-terminus of angiotensin ACEwas originallyidentified in mammals where it regulates vascular home-ostasis [69] The first insect ACE was found in M domestica[118] and several ACE paralogs have been identified in everyinsect genome investigated thus far [119] Insect ACE cleavespeptides with roles in development [119 120] reproduction[121] and immunity [122 123] Recently ACE was shownto be involved in aphid-plant interactions by modulatingthe feeding behavior and survival of aphids on plants [124]Six ACE paralogs were identified in the D melanogastergenome but only Ance and Acer are active enzymes [125]These enzymes have distinct tissue localization and substrateprofiles but their exact role is unclear Ance is expressedmostly in the gut and around the reproductive organs thussuggesting a role processing peptides in gut muscle cells[126] and during spermatogenesis [121] In contrast Acerwas exclusively found in developing heart cells [127] Weidentified 26 clusters belonging to the M2 family 16 ofwhichwere assigned to theAnce peptidyl-dipeptidase species(Table 5) and were predominantly expressed in the larval gut(Additional file 4) Based on this localization we speculatethat L sericataAnce plays a similar role to itsDmelanogasterortholog Interestingly D melanogaster Ance was shown tohydrolyze the two important bioactive peptides angiotensinI and bradykinin [119] which are the major substrates ofmammalian ACE It would be interesting to see whether Lsericata Ance can also cleave these substrates which wouldsuggest a potential endogenous role in hormonal signaling

373 Family M3 The L sericata transcriptome was shownto contain mitochondrial intermediate peptidase and thimetoligopeptidase which were expressed similarly in all thetissues we sampled (Additional file 4) Both enzymes areintracellular endopeptidases Mitochondrial intermediatepeptidase processes mitochondrial protein precursors duringtheir import into the mitochondria [128] whereas thimetoligopeptidase degrades small peptides (5ndash53 residues) withbroad specificity and plays an important role in antigenpresentation [129]

374 Family M8 Two L sericata clusters belong to fam-ily M8 which is typified by leishmanolysin an importantvirulence factor found in leishmania parasites [130] Leish-manolysin is a membrane-bound peptidase which degradesextracellular matrix proteins thus enabling parasite migra-tion [131] Furthermore a D melanogaster M8 metallopep-tidase was found to be involved in cell migration duringembryogenesis and coordinated mitotic progression [132]

375 Family M10 Family M10 comprises secreted matrixmetalloendopeptidases (MMPs) that are synthesized as inac-tive proenzymes and become functional in the extracellularenvironment MMPs are strongly conserved and have beenidentified in plants [133] cnidarians [134] nematodes [135]insects (Vilcinskas and Wedde 2002) and humans [136]

As their name indicates MMPs play important roles in extra-cellular matrix remodeling and turnover Aberrant MMPactivity is associatedwithmany forms of cancermaking themmedically relevant [137] Most MMPs are oncogenic that ishigher activity promotes cancer but some (including MMP3and MMP8) have the opposite effect [138] It is difficultto determine their precise individual roles because thereare 24 human MMPs with overlapping expression profilesand activities but insects could be used as a simplifiedmodel to probe their functions in more detail Only twoD melanogaster MMPs have been described [139 140] aswell as three from the red flour beetle Tribolium castaneum[141] and one from the greater wax moth Galleria mellonella[142] All insect MMPs play important physiological rolesand some also promote tumor progression suggesting theyhave similar functions to their human counterparts [143] Weidentified only four clusters representing L sericata MMPswhich were assigned to two peptidase species (Table 5)These enzymes were generally expressed at low levels butwere slightly upregulated in the larval gut (Additional file4) The role of these enzymes remains unknown and furtherstudies are required to clarify their physiological functionsand whether L sericataMMPs contribute to the degradationof extracellular matrix proteins in human wounds

376 Family M12 Family M12 comprises two subfamiliesnamely subfamily M12a which is typified by astacin andsubfamily M12b which is typified by adamalysin Astacinis an endopeptidase originally identified in the crayfishAstacus astacus which is probably involved in digestion [69]Hundreds of astacins have been identified in many differentspecies but no examples have yet been identified in plantsand fungi [144] In addition to digestion astacins may alsoplay roles in embryogenesis and extracellular protein remod-eling [145] Adamalysins are membrane-bound proteins withdisintegrin andmetallopeptidase domainsThey have a broadsubstrate range and are therefore involved inmany importantphysiological processes such as protein shedding devel-opment and spermatogenesis [146] Adamalysins are alsoknown to facilitate cell signaling and have been implicatedin carcinogenesis making them medically relevant [147] Weidentified 13 L sericata clusters representing subfamily M12aand another 13 clusters representing subfamily M12b Onlyone cluster (LST LS007850) was mainly expressed in thelarval gut indicating a potential role in digestion whereas theothers showed diverse tissue-specific expression profiles andtheir roles remain unclear

377 Family M13 Neprilysin and endothelin convertingenzyme (ECE) are the two best characterized members ofmetallopeptidase family M13 in mammals Neprilysin isinvolved in biological processes such as reproduction and themodulation of neuronal activity and blood pressure whereasECEs are responsible for the final step in the synthesis ofendothelins which are potent vasoconstrictors [69] Insectfamily M13 metallopeptidases are membrane-bound pepti-dases with a broad substrate range and tissue distribution[125] The precise biological roles of these enzymes in insects


are still unclear but they are associated with immunityto bacteria fungi and protozoa [122 148] metamorphosis[149] reproduction [150] and neuropeptide metabolism[151] We identified 34 clusters coding for M13 peptidasesin L sericata and they were predominantly expressed inthe larval body following the removal of the gut crop andsalivary glands (Additional file 4) Among 34 clusters 15 werefurther assigned to 8 peptidase families whereas 13 remainedunassigned and 6 represent nonpeptidase homologs (Table 5)

378 Family M14 Most family M14 enzymes are car-boxypeptidases that remove a single amino acid residuefrom the C-terminus of polypeptides Carboxypeptidases arerequired for digestion and are widely distributed amonginsects [152] but they also process bioactive peptides(carboxypeptidase E) and hydrolyze bacterial cell walls(120574-glutamyl-(L)-meso-diaminopimelate peptidase I) [69]Recently a partial L sericata sequence encoding an M14metallopeptidase was found to be upregulated by an immunechallenge [18] We identified 33 clusters representing M14family metallopeptidases that were differentially expressedamong the L sericata tissues we tested (Additional file4) These clusters were assigned to 9 peptidase specieswhereas 14 remained unassigned and one was shown torepresent a nonpeptidase homolog (Table 5) The previouslyidentified M14 peptidase [GenBank FG360509] was foundto be homologous to cluster LST LS004029 which is stronglyexpressed in the gutThe localization of this enzyme in the gutand its induction in response to an immune challenge suggestthat it contributes to the elimination of ingested bacteria aspreviously described for L sericata larvae [23]

379 Family M16 FamilyM16 can be divided into three sub-families M16A comprising oligopeptidases such as insulysinnardilysin andpitrilysinM16Bwhich includesmitochondrialprocessing peptidase and M16C which includes eupitrilysin[69] We identified four M16A clusters and two peptidasespecies with pitrilysin-like characteristics Pitrilysin is anendopeptidase originally found in Escherichia coli which ishomologous to human insulin-degrading enzyme [153] Wealso identified five M16B clusters and three peptidase speciessimilar to mitochondrial processing peptidase which cleavesthe N-terminal signals of mitochondrial proteins duringtheir import from the cytosol [69] We also identified oneM16C cluster representing one peptidase species similar toeupitrilysin

3710 Family M17 Leucyl aminopeptidase (LAP) is a cocat-alytic peptidase that is it requires two metal ions foractivity with diverse biological roles [154] We identified fourclusters and two peptidase species similar to LAP with thestrongest expression in gut tissues (Additional file 4) LAPswere previously identified in the digestive organs of blood-feeding parasites including ticks [155] schistosomes [156]and Plasmodium spp [157] and were found to be involved inthe final stage of hemoglobin digestion Because hemoglobincould also represent part of the L sericata diet we can

speculate that L sericata LAPs similarly are required forhemoglobin digestion

3711 Families M19 and M50 Families M19 and M50 eachcomprise strictlymembrane-bound enzymesThe familyM19dipeptidases degrade extracellular glutathione or inactivatedleukotriene D4 whereas the family M50 enzymes regulategene expression by processing different transcription fac-tors [69] We identified one cluster coding for a familyM19 nonpeptidase homolog and one representing a familyM50 S2P peptidase The latter is a strongly hydrophobicpeptidase found on the endoplasmic reticulum membraneD melanogaster S2P (ds2p) is required to cleave the sterolregulatory element binding protein (SREBP) and thus helpsto regulate lipid biosynthesis [158]

3712 Families M20 and M28 Families M20 and M28 com-prise divergent cocatalytic exopeptidases Family M20 con-tains only carboxypeptidases whereas family M28 includesboth carboxypeptidases and aminopeptidases We identifiedone cluster in family M20 which was tentatively identifiedas peptidase T and one cluster tentatively identified as ahomolog of D melanogaster putative protein CG10062 Sixunassigned clusters were also identified in family M28

3713 Family M24 Members of family M24 are mostlyintracellular cocatalytic exopeptidases characterized by theso-called pita-bread fold [159] which have been found inevery genome sequence published thus far [109] They areinvolved in many fundamental biological processes includ-ing the removal of N-terminal methionine residues fromnascent polypeptides (methionyl aminopeptidase) intracel-lular protein turnover and collagen metabolism (Xaa-Prodipeptidase) They are also involved in angiogenesis andtheir specific inhibitors are therefore sought as potential anti-cancer drugs [160] We identified nine clusters representingmethionyl aminopeptidases and one Xaa-Pro dipeptidaseClusters LST LS003866 LST LS017277 and LST LS003028were predominantly expressed in the larval gut whereas theotherM24 familymetallopeptidases were expressed at similarlevels in all the tissues we investigated (Additional file 4)

3714 Families M48 andM79 Themembers of families M48and M79 are membrane-bound metallopeptidases involvedin the release of tripeptides from Saccharomyces cerevisiae120572 mating factor [161] and the Ras oncoprotein [162] tofacilitate membrane attachment Both families are medicallyrelevant because of their ability to regulate the function ofRas which is involved inmany forms of cancerWe identifiedtwo clusters in one peptidase species coding for M48 familyand one cluster in one peptidase species coding for M79 Allclusterswere expressed at similar levels in all dissected tissues

3715 Family M49 Dipeptidyl-aminopeptidase III (DPP-3)is an exopeptidase that may be involved in the metabolismof angiotensin peptide and encephalin in mammals [163]Insect orthologs of DPP-3 were purified from the foregutmembrane of the cockroach Blaberus craniifer [164] and


Table 6 List of threonine peptidases in the L sericata transcriptome database


PB T1 Archaean proteasome beta component

Constitutive proteasome catalytic subunit 1 T01010 2Proteasome subunit alpha 6 T01971 2Proteasome subunit alpha 2 T01972 1Proteasome subunit alpha 4 T01973 2Proteasome subunit alpha 7 T01974 2Proteasome subunit alpha 5 T01975 2Proteasome subunit alpha 1 T01976 1Proteasome subunit alpha 3 T01977 2Proteasome subunit beta 2 T01984 1Proteasome subunit beta 1 T01986 1Proteasome subunit beta 4 T01987 2

Proteasome subunit beta2 (D melanogaster) T01A02 1Beta 5 subunit (D melanogaster) T01A06 1

Mername-AA231 pseudogene (H sapiens) T01P02 1

PB T2 Glycosylasparaginase precursor Isoaspartyl dipeptidase (threonine type) T02002 3Taspase-1 T02004 1

from adult D melanogaster [165] Purified DPP-3 hydrolyzedan insect neuropeptide (proctocolin) suggesting a role inneuropeptide signaling activity We identified two clustersand one peptidase species related to DPP-3 expressed atsimilar levels in all the L sericata tissues we tested

3716 Family M67 Family M67 metallopeptidases areresponsible for the removal of ubiquitin from ubiquitinylatedproteins prior to their degradation in the proteasome Weidentified one cluster and one peptidase species representingfamily M67 expressed at similar levels in all the L sericatatissues we tested

38Threonine Peptidases Threonine peptidases were discov-ered in 1995 in archaean proteasomes [166] They are N-terminal nucleophile peptidases belonging to clan PB and canbe divided into three families namely T1 T2 and T3 TheT1 family comprises peptidases of the proteasome and relatedcompound peptidasesThe proteasome plays a central role inintracellular protein turnover and is a complex supramolec-ular complex with many subunits [167] The T2 family com-prises the aspartyl glucosylaminases which are necessary forthe degradation of asparagine-linked glycoproteins [168]TheT3 family comprises the 120574-glutamyltransferases which play akey role in glutathionemetabolism [69] Among 25 L sericataclusters identified as threonine peptidases 21 clusters and 14peptidase species represented family T1 whereas 4 clustersand 2 peptidase species represented family T2 (Table 6) All25 clusters were expressed in all the L sericata tissues weinvestigated and the expression levels were universally low(Additional file 5)

39 Serine Peptidases Serine peptidases require a serineresidue for their catalytic activity and represent one of themost abundant and functionally diverse groups of enzymesSerine peptidases are involved in a broad range of biologicalprocesses including digestion development immunity andblood coagulation [169] MEROPS database v912 [62] lists 45

families in 15 clans as well as further 7 unassigned familiesWe found that serine peptidases are the largest group ofpeptidases in the L sericata transcriptome We identifiedmore than 800 contigs in 270 clusters which were assignedto 8 clans 12 families and 86 peptidase species (Table 7)These clusters showed a number of distinct tissue-specificexpression profiles (Additional file 6)

391 Family S1 Clan PA family S1 comprises endopeptidasescontaining the catalytic triad His-Asp-Ser and this wasthe largest peptidase family we found in the L sericatatranscriptome Most S1 peptidases possess an N-terminalsignal peptide and are synthesized as propeptides that mustbe cleaved to generate the active form S1 peptidases areusually soluble secreted enzymes but membrane-bound andinactive homologs have also been described [69] Many S1peptidases have been identified in insects where their rolesinclude digestion [111] immunity [170] wound responses[171] and development [172] S1 serine peptidases from L ser-icatamaggots have been associated with several of the benefi-cial effects ofMEPs including blood coagulation [45] biofilmeradication [33] and wound debridement [15] Althoughserine peptidases play an important role in MDT only asmall number of complete and partial L sericata sequencesrepresenting these enzymes have been published thus far

We detected 230 clusters representing S1 peptidasesof subfamily S1A which is typified by chymotrypsin andtrypsin We assigned 216 clusters to 62 peptidase specieswhereas 14 clusters represented nonpeptidase homologs(Table 7) Interestingly only 21 of the 62 species have alreadybeen provisionally identified and associated with specificfunctions whereas the remaining 41 putative peptidases havenot been characterized Among the identified peptidaseswe detected 39 clusters in 7 peptidase species encodingfor trypsin-like peptidases (S01110 S01116 S01117 S01130S01A83 S01A87 and S01A91) These were mainly expressedin the larval gut (Additional file 6) and probably function asdigestive enzymes as reported for other insect species [111]


Table 7 List of serine peptidases in the L sericata transcriptome database

Clan Family Peptidase type Peptidase species MEROPS ID Number ofclusters

PA S1 Chymotrypsin A

Ovochymase domain 2 (X laevis) S01024 3Trypsin alpha (insect) (D melanogaster) S01110 23Trypsin zeta (insect) (D melanogaster) S01116 7Trypsin eta (insect) (D melanogaster) S01117 3Trypsin (mosquito type) (Anopheles gambiae) S01130 2Chymotrypsin m-type 2 (insect) (Lucilia cuprina) S01168 7Easter peptidase (D melanogaster) S01201 6Melanization peptidase-2 (D melanogaster) S01203 2CG4386 peptidase (Sarcophaga peregrina) S01316 2Prophenoloxidase-activating peptidase (Pacifastacusleniusculus) S01413 2

Persephone peptidase (D melanogaster) S01421 1Tequila peptidase (D melanogaster) S01461 2DmHtrA2-type peptidase (M domestica) S01476 2Proteolytic lectin (Glossina-type) (Glossina fuscipesfuscipes) S01493 3

Grass peptidase (Insecta) (D melanogaster) S01502 3TmSPE peptidase (Tenebrio-type) (Tenebrio molitor) S01507 2CLIP-domain prophenoloxidase activating factor(Cotesia rubecula) S01960 8

Testis-specific protein 50 (H sapiens) S01993 1CG34409 protein (D melanogaster) S01A23 1CG11670 protein (D melanogaster) S01A31 1CG14780 protein (D melanogaster) S01A35 1CG8952 protein (D melanogaster) S01A36 2CG8170 protein (D melanogaster) S01A37 1CG2105 (D melanogaster) S01A51 2Sp212 protein (D melanogaster) S01A52 1CG9733 protein (D melanogaster) S01A61 2CG7829 protein (D melanogaster) S01A62 1CG5909 protein (D melanogaster) S01A64 1CG6048 protein (D melanogaster) S01A77 3CG6041 protein (D melanogaster) S01A78 4Trypsin beta (D melanogaster) S01A83 1CG17571 protein (D melanogaster) S01A85 9Trypsin lambda (D melanogaster) S01A87 2CG9676 protein (D melanogaster) S01A89 7Try29F protein (D melanogaster) S01A91 1CG17477 protein (D melanogaster) S01A92 1CG5246 protein (D melanogaster) S01A94 7CG17475 protein (D melanogaster) S01A96 1CG5233 protein (D melanogaster) S01A97 2Jonah 65Aiv (D melanogaster) S01B05 10CG7542 protein (D melanogaster) S01B07 3CG10477 protein (D melanogaster) S01B12 19CG6457 protein (D melanogaster) S01B13 2CG6483 protein (D melanogaster) S01B14 1CG10472 protein (D melanogaster) S01B16 2CG11529 protein (D melanogaster) S01B22 1


Table 7 Continued

ClanFamily Peptidase type Peptidase species MEROPS ID Number ofclusters

IP21814 protein (D melanogaster) S01B25 2Spirit protein (D melanogaster) S01B27 4CG3700 protein (D melanogaster) S01B30 2CG1299 protein (D melanogaster) S01B33 12CG34350 protein (D melanogaster) S01B35 1CG4914 protein (D melanogaster) S01B41 1CG11836 protein (D melanogaster) S01B43 1CG16821 protein (D melanogaster) S01B44 1CG32808 protein (D melanogaster) S01B47 1CG16749 protein (D melanogaster) S01B48 5CG8299 protein (D melanogaster) S01B50 2IP10861 protein (D melanogaster) S01B61 1CG16996 protein (D melanogaster) S01B64 2CG7142 protein (D melanogaster) S01B65 1CG11911 protein (D melanogaster) S01B68 8CG10663-PC isoform C (D melanogaster) S01B77 4Subfamily S1A non-peptidase homologues S01UNA 14

SB S8 Subtilisin Carlsberg

Furin-1 (arthropod-type) S08048 1Furin-2 (insect) S08049 1Site-1 peptidase S08063 1KPC2-type peptidase S08109 2Subfamily S8A unassigned peptidases S08UPA 2

SC S9 Prolyl oligopeptidase

Omega peptidase (D melanogaster) S09069 2CG11034 protein (D melanogaster) S09A64 2CG11319 (D melanogaster) S09A65 4Xaa-Pro dipeptidylpeptidase S09A73 1

SC S10 Carboxypeptidase Y Vitellogenic carboxypeptidase-like protein S10003 2CG32483 protein (D melanogaster) S10A52 2

SC S28 Lysosomal Pro-Xaa carboxypeptidase Lysosomal Pro-Xaa carboxypeptidase S28001 1CG9953 protein (D melanogaster) S28A10 2

SF S26 Signal peptidase ISignal peptidase (invertebrate) S26023 1CG11110 protein (D melanogaster) S26A09 1Subfamily S26A unassigned peptidases S26UPA 1

SJ S16 Lon-A peptidase Lon-A peptidase S16001 2PIM1 peptidase S16002 1

SK S14 Peptidase Clp Peptidase Clp (type 3) S14003 1

SK S41 C-terminal processing peptidase-1 C-terminal processing peptidase-1 S41001 1C-terminal processing peptidase-2 S41004 2

SK S49 Signal peptide peptidase A BSn5 05605 gp (Bacillus subtilis) S48A08 1

ST S54 Rhomboid-1Rhomboid-1 (Diptera) S54001 1Rhomboid-4 (insect) S54012 2Cg8972 protein S54A12 1

PC S51 Dipeptidase E Alpha-aspartyl dipeptidase (eukaryote) S51002 2

We provisionally identified two chymotrypsin-like peptidasespecies namely chymotrypsin m-type 2 (S01168) and Jonah65Aiv (S01B05) both of which were strongly expressed inthe gut (Additional file 6) as discussed in more detail belowWe also identified 8 peptidase species (S01203 S01413S01421 S01493 S01502 S01507 S01960 and S01B27) withdifferent tissue-specific expression profiles (Additional file 6)representing immunity-related peptidases Melanization

peptidase (S01203) prophenoloxidase-activating peptidase(S01413) and CLIP-domain prophenoloxidase-activatingfactor (S01960) are involved in the regulation of invertebrateinnate defenses [170] Proteolytic lectin (S01493) was firstidentified inGlossina spp where it regulates interactions withtrypanosome parasites [173] TmSPE peptidase (S01507)[174] Persephone (S01421) [175] Grass (S01502) and Spirit(S01B27) [176] facilitate the activation of Toll pathway


signaling which triggers the synthesis of antimicrobialpeptides in response to fungi and Gram-negative bacteria[177] We also identified ovochymase (S01024) whichwas discovered in Xenopus laevis eggs and may play arole in fertilization or early development [178] the Easterpeptidase (S01201) required for dorsoventral patterningin D melanogaster embryos [179] the Tequila peptidase(S01461) that mediates long term memory formation inD melanogaster [180] the proapoptotic DmHtrA2-typemitochondrial peptidase (S01476) [181] and testis-specificprotein 50 (S01993) which is necessary for spermatogenesisin mammals and is upregulated in breast cancer [182]

All the previously identified L sericata serine pep-tidase genes were also identified in the transcriptomedataset (Table 8) Interestingly only four of these previouslydescribed genes could be assigned to peptidase species witha known function whereas most were identified based onhomology to putative proteins in D melanogaster (Table 8)We also found that although many of the enzymes weredetected in MEPs the corresponding mRNA was predomi-nantly expressed in the larval gut (Additional file 6)The samephenomenon was confirmed for the Jonah-like peptidasewhere high expression level of Jonah mRNA was observedin the gut but the native enzyme was only detected in MEPs[17] These results indicate that peptides in MEPs are notexclusively produced by the salivary glands but rather acombination of the salivary glands gut and crop Althoughfurther studies are required to confirm this hypothesiswe suggest that regurgitation andor vomiting in dipteranspecies [183] contributes to the production of beneficial MEPmolecules in L sericata larvae

Cluster LST LS005873 was tentatively identified as chy-motrypsin m-type 2 (S01168) and this is identical to thepreviously described L sericata chymotrypsin I [GenBankCAS92770] A recombinant form of this enzyme was shownto degrade wound eschar ex vivo [15] and to degrademicrobial surface components recognizing adhesive matrixmolecules from the slough [184] As shown in Additionalfile 6 we identified seven further clusters representingthe same peptidase species (S01168) Another recombinantserine peptidase known as sericase [GenBank AAA17384]was shown to enhance fibrinolysis [44] Sericase was foundto be identical to L sericata trypsin-like serine peptidasewhich was proposed to facilitate wound debridement [185]We identified three clusters (LST LS007476 LST LS007613and LST LS010750) homologous to sericase which repre-sent one peptidase species provisionally identified as Dmelanogaster putative protein CG7542 (S01B07) Moreoverthe most prominent cluster (LST LS007476) was also foundto be homologous to a previously identified serine peptidase[GenBank FG360529]which is induced at the transcriptionallevel following an immune challenge [18] Our data indicatethat sericase is probably involved in several MEP func-tions including fibrinolysis debridement and other immuneresponses

Debrilase [GenBank AJN88395] is a serine peptidaseknown to play a role in L sericataMDT Debrilase is homol-ogous to cluster LST LS015273 which along with anothereight clusters represents one peptidase species provisionally

identified as D melanogaster putative protein CG17571(S01A85) All the clusters share the same tissue-specificexpression profile with strong upregulation in the gut (Addi-tional file 6) Recently L sericataMEPswere shown to reducethe clotting time of human plasma and this phenomenonwas attributed to a serine peptidase activity [45] Recom-binant Jonah-like chymotrypsin was confirmed to reducethe clotting time of human plasma and to degrade certainextracellular matrix proteins [17] We found 10 clustersrepresenting one peptidase species tentatively identified asJonah 65Aiv (S01B05) These clusters were predominantlyexpressed in the gut (Additional file 6) Interestingly clusterLST LS015269 was found to be homologous to a L sericataserine peptidase [GenBank FG360505] that is upregulated inimmune challenged larvae thus indicating a role in immunity[18]

392 Family S8 Family S8 comprises two subfamilies ofenzymes Subfamily S8a is typified by the endopeptidase sub-tilisin originally identified in Bacillus subtilis [69] as well astripeptidyl-peptidase II an exopeptidase involved in generalintracellular protein turnover [69] Subfamily S8b is typifiedby kexin (whose mammalian homolog is known as furin)which processes numerous proteins ranging from growthfactors and chemokines to extracellularmatrix proteins [186]and is therefore associated with diseases such as Alzheimerrsquosdisease atherosclerosis and cancer [187] We identified threeclusters of L sericata subtilisin-like enzymes two clusterssimilar to tripeptidyl-peptidase II and two clusters as furin-like enzymes (Table 7)

393 Family S9 The family S9 prolyl oligopeptidases areintracellular enzymes that strictly cleave substrates con-taining proline residues and they are thought to pro-cess neuropeptides in humans [188] Interestingly a prolyloligopeptidase was recently identified in the human parasiteSchistosoma mansoni Although the enzyme is not secretedby the parasite it cleaves the human vasoregulatory pep-tides bradykinin and angiotensin I in vitro thus potentiallymodulating or dysregulating homeostasis in its host [189]We identified 9 clusters and 4 peptidase species represent-ing L sericata prolyl oligopeptidases The clusters showeddifferent tissue-specific expression profiles but three of them(LST LS016452 LST LS001966 and LST LS016700) werepredominantly expressed in the gut andor the salivary glands(Additional file 6) This specific distribution in L sericatatissues associated with the production of MEPs indicatesa potential role in wound homeostasis but more detailedexperiments are required to confirm this hypothesis

394 Family S10 Family S10 comprises lysosomal car-boxypeptidases with predominantly regulatory functionsalthough hemipteran S10 peptidases were recently shownto be involved in the digestion of food [152] Among fourfamily S10 clusters identified inL sericata two (LSTLS003337and LST LS015778) were strongly upregulated in the gutwhereas the others (LST LS004162 and LST LS016840) wereexpressed at similar levels in the L sericata tissues we


Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin

Fibrinolysisdebridem

ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07


Table 9 Genes and qRT-PCR primers evaluated in this study

ClanFamily MEROPS ID Peptidase sp Cluster Primer sequences (51015840- 31015840) 119871 (bp)a 119864 ()b 1198772c

AA A1 A01092 CAD2 peptidase(M domestica) LST LS005916 51015840-GCTGCCAAGCTATTGCTGAT-31015840 75 102 0997

51015840-CGGCTTGAATGTTTTCGTATT-31015840

CA C1 C01A27 CG12163 protein(D melanogaster) LST LS006048 51015840- TTTCACCGGTAATCGCAAAT -31015840 66 105 0997

51015840- GCAGCCTTTTGACGATGTTT -31015840

MA M1 M01024 ERAP2 aminopeptidase LST LS004632 51015840- CCATTCCGTCCGAT TCTT -31015840 68 98 099951015840- ATCGGCATACTGGGCAAA -31015840

PB T1 T01976 Proteasome subunitalpha 1 LST LS006843 51015840- TCTTCACGCTCTCAAGACGA -31015840 61 103 0993

51015840- GGTCTTGTGTTTCGCCATCT -31015840

PA S1 S01993 Testis-specific protein50 (H sapiens) LST LS010066 51015840- TATGGGCAGCCCTAACGA -31015840 60 104 0998

51015840- AAAGAACGGAGAGCATCACCT -31015840aLength of ampliconbQuantitative qRT-PCR efficiencycCoefficient of determination

investigatedThe clusters induced in the gut were assigned tothe peptidase species without a known function whereas theother two were annotated as vitellogenic carboxypeptidase-like proteins suggesting a role in vitellogenesis

395 Families S14 and S41 Family S14 comprises cytosolicATP-dependent Clp endopeptidases and their homologs Clppeptidases together with their ATP-binding subunits createan oligomeric complex of 20ndash26 subunits [69] that mediateprotein quality control and regulatory degradation [190]Family S41 comprises endopeptidases that are involved in thedegradation of incorrectly synthesized proteinsThey possessthe catalytic tetrad SerndashHisndashSerndashGlu which is unusual forserine peptidase and neither the position of the active siteresidues nor the residues themselves are conserved [69] Wefound that the L sericata families S14 (one cluster in onepeptidase species) and S41 (three clusters in two peptidasespecies) endopeptidases were similarly expressed in all thetissues we analyzed and are likely to be involved in theregulation of protein synthesis

396 Family S16 The family S16 enzyme Lon is a bacte-rial ATP-dependent endopeptidase containing a peptidasedomain and an ATP-binding domain in a single subunitSimilar enzymes are found in many other organisms [109]where they facilitate the degradation of unfolded proteins[191] We identified three clusters assigned to two peptidasespecies which were present in all L sericata tissues

397 Family S26 Family S26 consists of ubiquitousmembrane-bound enzymes with a catalytic dyad which areinvolved in the cleavage of signal peptides thus facilitating thesecretion of proteins [69]We identified two L sericata familyS26 clusters and two peptidase species representing signalpeptidases and another cluster that remained unassigned(Table 7) Cluster LST LS009731 was strongly expressed inthe salivary glands which are known to secret large amountsof protein thus indicating a role in protein secretion

398 Family S28 Family S28 comprises the lysosomalPro-Xaa carboxypeptidases which are lysosome-specificexopeptidase found solely in eukaryotes featuring an unusualselectivity for the cleavage of ProXaa bonds In humanssuch enzymes inactivate angiotensin II and activate plasmakallikrein [192] We identified three L sericata family S28clusters assigned to two peptidase species that were upreg-ulated in the crop and larval body samples (Additional file 6)The precise role of these enzymes remains unclear althoughthey may contribute to the procoagulation activity of MEPsas recently described [45]

399 Family S49 Only one cluster in one peptidase specieswas assigned to the family S49 Family S49 is the signalpeptidases required for intracellular protein processing andthe regulation of protein export [193]

3910 Family S51 Family S51 is typified by aspartyl dipep-tidase an exopeptidase originally identified in Salmonellatyphimurium [194] that hydrolyzes 120572-aspartyl bonds Thecrystal structure of the S typhimurium aspartyl dipeptidasehas been solved revealing an unusual catalytic triad withSer and His as predicted but Glu instead of Asp [195] Thebiological role for this enzyme is not clear but it seems tobe involved in the production of nutritional amino acids[69] Two clusters belonging to one peptidase species wereidentified in L sericata transcriptome

3911 Family S54 Family S54 is typified by Rhomboid-1 anintramembrane enzyme identified in D melanogaster [196]that plays an important role in embryonic development bycleaving the Spitz protein and thus activating the epidermalgrowth factor receptor [197] We identified three clusters inthree peptidase species whichwere expressed in allL sericatatissues

310 qRT-PCR Verification of Gene Expression To exper-imentally verify the results from digital gene expression


LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100

Body Gland Crop Gut Body Gland Crop Gut


Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)

Figure 6 Verification of digital gene expression analysis by quantitative RT-PCR (a) The individual clusters encoding for aspartic(LST LS005916) cysteine (LST LS006048) metallo (LST LS004632) threonine (LST LS006843) and serine (LST LS010066) peptidases aredepicted on the left Shown are log

2

-transformed RPKM values (blue resembles lower-expressed genes while red represents highly expressedgenes) (b)ThemRNA expression profiles of five enzymatic genes in various larval tissues were determined by qRT-PCR and normalized withthe 60S acidic ribosomal protein P0 (RPLPO) and 40S ribosomal protein S3 (RPS3) genes The expressions of individual genes were related tothe maximum mRNA level of each gene set as 100

analysis we performed qRT-PCR analysis of one peptidasegene from each peptidase clan (aspartic cysteine metallothreonine and serine) These genes (Table 9) code for pepti-dases with various physiological function and show differenttissue expression profile All the tested genes show the similarexpression profiles as acquired by digital gene expressionanalysis (Figure 6)

4 Conclusion

The purpose of this study was to provide an overview of thedistribution of proteolytic enzymes in L sericata focusing onthe tissue-specific expression profiles and potential functionsas the basis for further more detailed studies of individualpeptidasesWe identified 577 clusters representing five classes

of proteolytic enzymes (aspartic cysteine threonine serineand metallopeptidases) which were further assigned into 26clans 48 families and 185 peptidase species with diversetissue-specific patterns of distribution We identified all pre-viously described therapeutic peptidases and found thatmostof them were most highly expressed in the larval gut thusindicating that the larval gut contributes to the production ofbeneficial enzymes found in theMEPs Although themajorityof the enzymes we identified were serine peptidases mostof them were novel putative peptidases whose function isunclear but whose specific tissue-specific expression profilesindicate an important role in MEP activity Several peptideswith the most intriguing expression profiles have been pre-pared as synthetic genes allowing the functional analysis ofthe corresponding recombinant peptides


Additional Points

The dataset supporting the results of this paper is availablein the (EBI Short Read Archive [SRA]) repository under theaccession number PRJEB7567 The complete study can beaccessed directly at httpwwwebiacukenadataviewPRJ-EB7567

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors acknowledge funding by the Hessen State Min-istry of Higher Education Research and the Arts includinga generous grant for the LOEWE research center ldquoInsectBiotechnology and Bioresourcesrdquo Zdenek Franta was sup-ported by an Alexander von Humboldt Research Fellowshipfor Postdoctoral ResearchersThe authors would like to thankAndre Baumann for his help with qRT-PCR analysis Theauthors thank Richard M Twyman for editing of the paper

References

[1] J C T Church ldquoThe traditional use of maggots in woundhealing and the development of larva therapy (biosurgery) inmodern medicinerdquo Journal of Alternative and ComplementaryMedicine vol 2 no 4 pp 525ndash527 1996

[2] R A Sherman M J R Hall and S Thomas ldquoMedicinalmaggots an ancient remedy for some contemporary afflictionsrdquoAnnual Review of Entomology vol 45 pp 55ndash81 2000

[3] J Edwards and S Stapley ldquoDebridement of diabetic foot ulcersrdquoCochrane Database of Systematic Reviews no 1 Article IDCD003556 2010

[4] M L Marineau M T Herrington KM Swenor and L J EronldquoMaggot debridement therapy in the treatment of complexdiabetic woundsrdquoHawaiiMedical Journal vol 70 no 6 pp 121ndash124 2011

[5] C Wilasrusmee M Marjareonrungrung S Eamkong et alldquoMaggot therapy for chronic ulcer a retrospective cohort andameta-analysisrdquoAsian Journal of Surgery vol 37 no 3 pp 138ndash147 2014

[6] J-H Hwang H N Modi S-W Suh J-Y Hong J-H Yangand J-H Park ldquoMaggot debridement therapy for postsurgicalwound infection in scoliosis a case series in five patientsrdquo Spinevol 36 no 4 pp 313ndash319 2011

[7] N Akhtar S Abdel-Rehim J Rodrigues and P BrooksldquoThe use of larvae therapy to debride full thickness burns inthe anaesthetically unfit patient the Nottingham experiencerdquoBurns vol 37 no 6 pp e44ndashe49 2011

[8] N Namias J E Varela R P Varas O Quintana and C GWard ldquoBiodebridement a case report of maggot therapy forlimb salvage after fourth-degree burnsrdquo Journal of Burn Careand Rehabilitation vol 21 no 3 pp 254ndash257 2000

[9] R A Sherman ldquoMechanisms of maggot-induced wound heal-ing what do we know and where do we go from hererdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 592419 13 pages 2014

[10] G Cazander D I Pritchard Y Nigam W Jung and PH Nibbering ldquoMultiple actions of Lucilia sericata larvae inhard-to-heal wounds larval secretions contain molecules thataccelerate wound healing reduce chronic inflammation andinhibit bacterial infectionrdquo BioEssays vol 35 no 12 pp 1083ndash1092 2013

[11] K Y Mumcuoglu A Ingber L Gilead et al ldquoMaggot therapyfor the treatment of intractable woundsrdquo International Journalof Dermatology vol 38 no 8 pp 623ndash627 1999

[12] R A Sherman ldquoMaggot versus conservative debridementtherapy for the treatment of pressure ulcersrdquoWound Repair andRegeneration vol 10 no 4 pp 208ndash214 2002

[13] P Steenvoorde C E Jacobi L Van Doorn and J OskamldquoMaggot debridement therapy of infected ulcers patient andwound factors influencing outcomemdasha study on 101 patientswith 117 woundsrdquo Annals of the Royal College of Surgeons ofEngland vol 89 no 6 pp 596ndash602 2007

[14] L Chambers S Woodrow A P Brown et al ldquoDegradationof extracellular matrix components by defined proteinasesfrom the greenbottle larva Lucilia sericata used for the clinicaldebridement of non-healing woundsrdquo British Journal of Derma-tology vol 148 no 1 pp 14ndash23 2003

[15] G Telford A P Brown R A M Seabra et al ldquoDegradationof eschar from venous leg ulcers using a recombinant chy-motrypsin fromLucilia sericatardquoBritish Journal ofDermatologyvol 163 no 3 pp 523ndash531 2010

[16] G Telford A P Brown A Kind J S C English and DI Pritchard ldquoMaggot chymotrypsin I from Lucilia sericata isresistant to endogenous wound protease inhibitorsrdquo BritishJournal of Dermatology vol 164 no 1 pp 192ndash196 2011

[17] A Poppel M Kahl A Baumann et al ldquoA Jonah-like chy-motrypsin from the therapeutic maggot Lucilia sericata playsa role in wound debridement and coagulationrdquo Insect Biochem-istry and Molecular Biology vol 70 pp 138ndash147 2016

[18] B Altincicek and A Vilcinskas ldquoSeptic injury-inducible genesin medicinal maggots of the green blow fly Lucilia sericatardquoInsect Molecular Biology vol 18 no 1 pp 119ndash125 2009

[19] L Huberman N Gollop K Y Mumcuoglu et al ldquoAntibacterialsubstances of low molecular weight isolated from the blowflyLucilia sericatardquoMedical and Veterinary Entomology vol 21 no2 pp 127ndash131 2007

[20] L Huberman N Gollop K Y Mumcuoglu C Block and RGalun ldquoAntibacterial properties of whole body extracts andhaemolymph of Lucilia sericata maggotsrdquo Journal of WoundCare vol 16 no 3 pp 123ndash127 2007

[21] A-K Poppel H Vogel J Wiesner and A Vilcinskas ldquoAntimi-crobial peptides expressed in medicinal maggots of the blowfly Lucilia sericata show combinatorial activity against bacteriardquoAntimicrobial Agents andChemotherapy vol 59 no 5 pp 2508ndash2514 2015

[22] A-K Poppel A Koch K-H Kogel et al ldquoLucimycin anantifungal peptide from the therapeutic maggot of the commongreen bottle fly Lucilia sericatardquo Biological Chemistry vol 395no 6 pp 649ndash656 2014

[23] K Y Mumcuoglu J Miller M Mumcuoglu M Friger and MTarshis ldquoDestruction of bacteria in the digestive tract of themaggot of Lucilia sericata (Diptera Calliphoridae)rdquo Journal ofMedical Entomology vol 38 no 2 pp 161ndash166 2001

[24] I Valachova P Takac and J Majtan ldquoMidgut lysozymes ofLucilia sericata-new antimicrobials involved inmaggot debride-ment therapyrdquo Insect Molecular Biology vol 23 no 6 pp 779ndash787 2014


[25] A Bexfield YNigam SThomas andNA Ratcliffe ldquoDetectionand partial characterisation of two antibacterial factors from theexcretionssecretions of the medicinal maggot Lucilia sericataand their activity against methicillin-resistant Staphylococcusaureus (MRSA)rdquoMicrobes and Infection vol 6 no 14 pp 1297ndash1304 2004

[26] A S Andersen D Sandvang K M Schnorr et al ldquoA novelapproach to the antimicrobial activity of maggot debridementtherapyrdquo Journal of Antimicrobial Chemotherapy vol 65 no 8pp 1646ndash1654 2010

[27] A Bexfield A E Bond E C Roberts et al ldquoThe antibacterialactivity against MRSA strains and other bacteria of a lt500 Dafraction from maggot excretionssecretions of Lucilia sericata(Diptera Calliphoridae)rdquoMicrobes and Infection vol 10 no 4pp 325ndash333 2008

[28] M J A van der Plas G N Jukema S-W Wai et al ldquoMaggotexcretionssecretions are differentially effective against biofilmsof Staphylococcus aureus and Pseudomonas aeruginosardquo Journalof Antimicrobial Chemotherapy vol 61 no 1 pp 117ndash122 2008

[29] L G Harris A Bexfield Y Nigam H Rohde N A Rat-cliffe and D Mack ldquoDisruption of Staphylococcus epider-midis biofilms by medicinal maggot Lucilia sericata excre-tionssecretionsrdquo International Journal of Artificial Organs vol32 no 9 pp 555ndash564 2009

[30] G Cazander M C van de Veerdonk C M J EVandenbroucke-Grauls M W J Schreurs and G N JukemaldquoMaggot excretions inhibit biofilm formation on biomaterialsrdquoClinical Orthopaedics and Related Research vol 468 no 10 pp2789ndash2796 2010

[31] J Bohova J Majtan V Majtan and P Takac ldquoSelectiveantibiofilm effects of Lucilia sericata larvae secretionsexcre-tions against wound pathogensrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2014 Article ID 857360 9pages 2014

[32] A Brown A Horobin D G Blount et al ldquoBlow flyLucilia sericata nuclease digests DNA associated with woundslougheschar and with Pseudomonas aeruginosa biofilmrdquoMedical and Veterinary Entomology vol 26 no 4 pp 432ndash4392012

[33] L G Harris Y Nigam J Sawyer D Mack and D IPritchard ldquoLucilia sericata chymotrypsin disrupts protein adh-esin-mediated staphylococcal biofilm formationrdquo Applied andEnvironmental Microbiology vol 79 no 4 pp 1393ndash1395 2013

[34] A J Horobin K M Shakesheff S Woodrow C Robinson andD I Pritchard ldquoMaggots and wound healing an investigationof the effects of secretions from Lucilia sericata larvae uponinteractions between human dermal fibroblasts and extracellu-larmatrix componentsrdquoBritish Journal of Dermatology vol 148no 5 pp 923ndash933 2003

[35] A J Horobin KM Shakesheff andD I Pritchard ldquoPromotionof human dermal fibroblast migration matrix remodelling andmodification of fibroblast morphology within a novel 3Dmodelby Lucilia sericata larval secretionsrdquo Journal of InvestigativeDermatology vol 126 no 6 pp 1410ndash1418 2006

[36] P E Prete ldquoGrowth effects of Phaenicia sericata larval extractson fibroblasts mechanism for wound healing by maggot ther-apyrdquo Life Sciences vol 60 no 8 pp 505ndash510 1997

[37] Z Zhang S Wang Y Diao J Zhang and D Lv ldquoFatty acidextracts from Lucilia sericata larvae promote murine cutaneouswound healing by angiogenic activityrdquo Lipids in Health andDisease vol 9 article 24 2010

[38] A Bexfield A E Bond C Morgan et al ldquoAmino acid deriva-tives from Lucilia sericata excretionssecretions may contributeto the beneficial effects of maggot therapy via increased angio-genesisrdquo British Journal of Dermatology vol 162 no 3 pp 554ndash562 2010

[39] G Cazander M W J Schreurs L Renwarin C DorresteijnD Hamann and G N Jukema ldquoMaggot excretions affect thehuman complement systemrdquo Wound Repair and Regenerationvol 20 no 6 pp 879ndash886 2012

[40] J Pecivova T Macickova P Takac M Kovacsova DCupanıkova and M Kozanek ldquoEffect of the extract fromsalivary glands of Lucilia sericata on human neutrophilsrdquoNeuroendocrinology Letters vol 29 no 5 pp 794ndash797 2008

[41] M J A van der Plas A M van der Does M Baldry et alldquoMaggot excretionssecretions inhibit multiple neutrophil pro-inflammatory responsesrdquo Microbes and Infection vol 9 no 4pp 507ndash514 2007

[42] M J A van der Plas M Baldry J T van Dissel G N Jukemaand P H Nibbering ldquoMaggot secretions suppress pro-inflam-matory responses of human monocytes through elevation ofcyclic AMPrdquo Diabetologia vol 52 no 9 pp 1962ndash1970 2009

[43] M J A van der Plas J T van Dissel and P H NibberingldquoMaggot secretions skewmonocyte-macrophage differentiationaway from a pro-inflammatory to a pro-angiogenic typerdquo PLoSONE vol 4 no 11 Article ID e0008071 2009

[44] M J A van der Plas A S Andersen S Nazir et al ldquoAnovel serine protease secreted by medicinal maggots enhancesplasminogen activator-induced fibrinolysisrdquo PLoS ONE vol 9no 3 Article ID e92096 2014

[45] M Kahl A Gokcen S Fischer et al ldquoMaggot excretionproducts from the blowfly Lucilia sericata contain contactphaseintrinsic pathway-like proteases with procoagulant func-tionsrdquoThrombosis and Haemostasis vol 114 no 2 pp 277ndash2882015

[46] M de la Paz Celorio-Mancera C W Wheat H Vogel LSoderlind N Janz and S Nylin ldquoMechanisms of macroevolu-tion polyphagous plasticity in butterfly larvae revealed byRNA-SeqrdquoMolecular Ecology vol 22 no 19 pp 4884ndash4895 2013

[47] Babraham Bioinformatics ldquoFastQCmdasha quality control tool forhigh throughput sequence datardquo httpwwwbioinformaticsbabrahamacukprojectsfastqc

[48] AM BolgerM Lohse and B Usadel ldquoTrimmomatic a flexibletrimmer for Illumina sequence datardquoBioinformatics vol 30 no15 pp 2114ndash2120 2014

[49] M G Grabherr B J Haas M Yassour et al ldquoFull-lengthtranscriptome assembly fromRNA-Seq datawithout a referencegenomerdquoNature Biotechnology vol 29 no 7 pp 644ndash652 2011

[50] M H Schulz D R Zerbino M Vingron and E Birney ldquoOasesrobust de novo RNA-seq assembly across the dynamic range ofexpression levelsrdquo Bioinformatics vol 28 no 8 pp 1086ndash10922012

[51] D Gilbert ldquoEvidentialGene tr2aacds mRNA TranscriptAssembly Softwarerdquo 2013 httparthropodseugenesorgEvid-entialGeneaboutEvidentialGene trassembly pipehtml

[52] W Li and A Godzik ldquoCd-hit a fast program for clusteringand comparing large sets of protein or nucleotide sequencesrdquoBioinformatics vol 22 no 13 pp 1658ndash1659 2006

[53] G Parra K Bradnam and I Korf ldquoCEGMA a pipelineto accurately annotate core genes in eukaryotic genomesrdquoBioinformatics vol 23 no 9 pp 1061ndash1067 2007


[54] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[55] S T OrsquoNeil and S J Emrich ldquoAssessing De Novo transcriptomeassembly metrics for consistency and utilityrdquo BMC Genomicsvol 14 no 1 article 465 2013

[56] ldquoTransposonPSI An Application of PSI-Blast to Mine (Retro-)Transposon ORF Homologiesrdquo httptransposonpsisourcefo-rgenet

[57] S R Eddy ldquoA new generation of homology search tools basedon probabilistic inferencerdquo Genome Informatics vol 23 no 1pp 205ndash211 2009

[58] R D Finn J Mistry J Tate et al ldquoThe Pfam protein familiesdatabaserdquo Nucleic Acids Research vol 38 no 1 Article IDgkp985 pp D211ndashD222 2009

[59] E M Zdobnov M Campillos E D Harrington D Torrentsand P Bork ldquoProtein coding potential of retroviruses and othertransposable elements in vertebrate genomesrdquo Nucleic AcidsResearch vol 33 no 3 pp 946ndash954 2005

[60] O Rupp J Becker K Brinkrolf et al ldquoConstruction of a publicCHO cell line transcript database using versatile bioinformaticsanalysis pipelinesrdquo PLoS ONE vol 9 no 1 Article ID e855682014

[61] ldquoEnzymeNomenclaturerdquo httpwwwchemqmulacukiubmbenzyme

[62] N D Rawlings M Waller A J Barrett and A BatemanldquoMEROPS the database of proteolytic enzymes their substratesand inhibitorsrdquoNucleic Acids Research vol 42 no 1 pp D503ndashD509 2014

[63] J D Hunter ldquoMatplotlib a 2D graphics environmentrdquo Comput-ing in Science and Engineering vol 9 no 3 Article ID 4160265pp 99ndash104 2007

[64] Matplotlib ldquoColor example code colormaps referencepyrdquohttpmatplotliborgexamplescolorcolormaps referencehtml

[65] A Baumann R Lehmann A Beckert A Vilcinskas and ZFranta ldquoSelection and evaluation of tissue specific referencegenes in Lucilia sericata during an immune challengerdquo PLoSONE vol 10 no 8 Article ID e0135093 2015

[66] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquoNucleic Acids Research vol 29 no9 article e45 2001

[67] ldquoMEROPS the peptidase databaserdquo httpmeropssangeracuk

[68] N D Rawlings and A J Barrett ldquoMEROPS the peptidasedatabaserdquoNucleic Acids Research vol 27 no 1 pp 325ndash331 1999

[69] N D Rawlings andG S Salvesen EdsHandbook of ProteolyticEnzymes Elsevier San Diego Calif USA 2013

[70] A J Barrett and N D Rawlings ldquorsquoSpeciesrsquo of peptidasesrdquoBiological Chemistry vol 388 no 11 pp 1151ndash1157 2007

[71] A J Barrett N D Rawlings and E A OrsquoBrien ldquoThe MEROPSdatabase as a protease information systemrdquo Journal of StructuralBiology vol 134 no 2-3 pp 95ndash102 2001

[72] P B Szecsi ldquoThe aspartic proteasesrdquo Scandinavian Journal ofClinical and Laboratory Investigation Supplement vol 210 pp5ndash22 1992

[73] M H P Padilha A C Pimentel A F Ribeiro and W R TerraldquoSequence and function of lysosomal and digestive cathepsinD-like proteinases ofMusca domesticamidgutrdquo Insect Biochem-istry and Molecular Biology vol 39 no 11 pp 782ndash791 2009

[74] H Zhu C AHart D Sales andN B Roberts ldquoBacterial killingin gastric juicemdasheffect of pH and pepsin on Escherichia coli andHelicobacter pylorirdquo Journal of Medical Microbiology vol 55 no9 pp 1265ndash1270 2006

[75] W Xia and M S Wolfe ldquoIntramembrane proteolysis by prese-nilin and presenilin-like proteasesrdquo Journal of Cell Science vol116 no 14 pp 2839ndash2844 2003

[76] A L Brunkan and A M Goate ldquoPresenilin function and 120574-secretase activityrdquo Journal of Neurochemistry vol 93 no 4 pp769ndash792 2005

[77] D J Casso S Tanda B Biehs B Martoglio and T B KornbergldquoDrosophila signal peptide peptidase is an essential protease forlarval developmentrdquo Genetics vol 170 no 1 pp 139ndash148 2005

[78] D Bernard B Mehul A Thomas-Collignon C Delattre MDonovan and R Schmidt ldquoIdentification and characterizationof a novel retroviral-like aspartic protease specifically expressedin human epidermisrdquo Journal of Investigative Dermatology vol125 no 2 pp 278ndash287 2005

[79] A-M Lennon-Dumenil A H Bakker P Wolf-Bryant H LPloegh and C Lagaudriere-Gesbert ldquoA closer look at proteoly-sis and MHC-class-II-restricted antigen presentationrdquo CurrentOpinion in Immunology vol 14 no 1 pp 15ndash21 2002

[80] M Fonovic and B Turk ldquoCysteine cathepsins and extracellularmatrix degradationrdquo Biochimica et Biophysica Acta vol 1840no 8 pp 2560ndash2570 2014

[81] H Wu X Che Q Zheng et al ldquoCaspases a molecularswitch node in the crosstalk between autophagy and apoptosisrdquoInternational Journal of Biological Sciences vol 10 no 9 pp1072ndash1083 2014

[82] O Vasiljeva T Reinheckel C Peters D Turk V Turk and BTurk ldquoEmerging roles of cysteine cathepsins in disease and theirpotential as drug targetsrdquo Current Pharmaceutical Design vol13 no 4 pp 387ndash403 2007

[83] H Saito S Kurata and S Natori ldquoPurification and charac-terization of a hemocyte proteinase of Sarcophaga possiblyparticipating in elimination of foreign substancesrdquo EuropeanJournal of Biochemistry vol 209 no 3 pp 939ndash944 1992

[84] C Serbielle S Moreau F Veillard et al ldquoIdentification ofparasite-responsive cysteine proteases in Manduca sextardquo Bio-logical Chemistry vol 390 no 5-6 pp 493ndash502 2009

[85] W-L Cho S-M Tsao A R Hays et al ldquoMosquito cathepsinB-like protease involved in embryonic degradation of vitellinis produced as a latent extraovarian precursorrdquo The Journal ofBiological Chemistry vol 274 no 19 pp 13311ndash13321 1999

[86] D Sojka Z Franta M Horn C R Caffrey M Mares and PKopacek ldquoNew insights into the machinery of blood digestionby ticksrdquoTrends in Parasitology vol 29 no 6 pp 276ndash285 2013

[87] C FialhoMdoW R Terra N RMoreira J C Zanuncio and JE Serrao ldquoUltrastructure and immunolocalization of digestiveenzymes in the midgut of Podisus nigrispinus(HeteropteraPentatomidae)rdquo Arthropod Structure and Development vol 42no 4 pp 277ndash285 2013

[88] A G Martynov E N Elpidina L Perkin and B OppertldquoFunctional analysis of C1 family cysteine peptidases in thelarval gut of Tenebrio molitor and Tribolium castaneumrdquo BMCGenomics vol 16 no 1 article 75 2015

[89] W R Terra and C Ferreira ldquoBiochemistry and molecular biol-ogy of digestionrdquo in Insect Molecular Biology and BiochemistryL I Gilbert Ed Elsevier San Diego Calif USA 2012

[90] Y Abe K Shirane H Yokosawa et al ldquoAsparaginyl endopepti-dase of jack bean seeds Purification characterization and high


utility in protein sequence analysisrdquo The Journal of BiologicalChemistry vol 268 no 5 pp 3525ndash3529 1993

[91] C R CaffreyMAMathieuAMGaffney et al ldquoIdentificationof a cDNA encoding an active asparaginyl endopeptidase ofSchistosoma mansoni and its expression in Pichia pastorisrdquoFEBS Letters vol 466 no 2-3 pp 244ndash248 2000

[92] J-M Chen P M Dando N D Rawlings et al ldquoCloningisolation and characterization of mammalian legumain anasparaginyl endopeptidaserdquoThe Journal of Biological Chemistryvol 272 no 12 pp 8090ndash8098 1997

[93] D Sojka O Hajdusek J Dvorak et al ldquoIrAE-an asparaginylendopeptidase (legumain) in the gut of the hard tick Ixodesricinusrdquo International Journal for Parasitology vol 37 no 7 pp713ndash724 2007

[94] J-M Chen M Fortunato and A J Barrett ldquoActivation ofhuman prolegumain by cleavage at a C-terminal asparagineresiduerdquoBiochemical Journal vol 352 part 2 pp 327ndash334 2000

[95] C Watts S P Matthews D Mazzeo B Manoury and C XMoss ldquoAsparaginyl endopeptidase case history of a class IIMHC compartment proteaserdquo Immunological Reviews vol 207pp 218ndash228 2005

[96] M Sajid J H McKerrow E Hansell et al ldquoFunctional expres-sion and characterization of Schistosoma mansoni cathepsin Band its trans-activation by an endogenous asparaginyl endopep-tidaserdquo Molecular and Biochemical Parasitology vol 131 no 1pp 65ndash75 2003

[97] M Horn M Nussbaumerova M Sanda et al ldquoHemoglobindigestion in blood-feeding ticks mapping a multipeptidasepathway by functional proteomicsrdquo Chemistry and Biology vol16 no 10 pp 1053ndash1063 2009

[98] M JMcConville andAKMenon ldquoRecent developments in thecell biology and biochemistry of glycosylphosphatidylinositollipids (review)rdquo Molecular Membrane Biology vol 17 no 1 pp1ndash16 2000

[99] W C Earnshaw L M Martins and S H Kaufmann ldquoMam-malian caspases structure activation substrates and functionsduring apoptosisrdquo Annual Review of Biochemistry vol 68 pp383ndash424 1999

[100] D Xu S E Woodfield T V Lee Y Fan C Antonio andA Bergmann ldquoGenetic control of programmed cell death(apoptosis) in Drosophilardquo Fly vol 3 no 1 pp 78ndash90 2009

[101] S Kumar and J Doumanis ldquoThe fly caspasesrdquo Cell Death andDifferentiation vol 7 no 11 pp 1039ndash1044 2000

[102] H Sorimachi S Hata and Y Ono ldquoExpanding members androles of the calpain superfamily and their genetically modifiedanimalsrdquo Experimental Animals vol 59 no 5 pp 549ndash5662010

[103] P M Cummins and B OrsquoConnor ldquoPyroglutamyl peptidase anoverview of the three known enzymatic formsrdquo Biochimica etBiophysica ActamdashProtein Structure and Molecular Enzymologyvol 1429 no 1 pp 1ndash17 1998

[104] C Nusslein-Volhard and E Wieschaus ldquoMutations affectingsegment number and polarity in Drosophilardquo Nature vol 287no 5785 pp 795ndash801 1980

[105] T R Burglin ldquoThe Hedgehog protein familyrdquo Genome Biologyvol 9 no 11 p 241 2008

[106] T Lang E Schaeffeler D Bernreuther M BredschneiderD H Wolf and M Thumm ldquoAut2p and Aut7p two novelmicrotubule-associated proteins are essential for delivery ofautophagic vesicles to the vacuolerdquo The EMBO Journal vol 17no 13 pp 3597ndash3607 1998

[107] M Y Balakirev S O Tcherniuk M Jaquinod and JChroboczek ldquoOtubains a new family of cysteine proteases inthe ubiquitin pathwayrdquo EMBOReports vol 4 no 5 pp 517ndash5222003

[108] K S Makarova L Aravind and E V Koonin ldquoA novelsuperfamily of predicted cysteine proteases from eukaryotesviruses and Chlamydia pneumoniaerdquo Trends in BiochemicalSciences vol 25 no 2 pp 50ndash52 2000

[109] M J Page and E Di Cera ldquoEvolution of peptidase diversityrdquoJournal of Biological Chemistry vol 283 no 44 pp 30010ndash30014 2008

[110] S Dalal D R T Ragheb F D Schubot and M KlembaldquoA naturally variable residue in the S1 subsite of M1 familyaminopeptidases modulates catalytic properties and promotesfunctional specializationrdquo The Journal of Biological Chemistryvol 288 no 36 pp 26004ndash26012 2013

[111] W R Terra and C Ferreira ldquoInsect digestive enzymes prop-erties compartmentalization and functionrdquo Comparative Bio-chemistry and Physiology Part B Biochemistry and vol 109 no1 pp 1ndash62 1994

[112] P Denolf K Hendrickx J Van Damme et al ldquoCloning andcharacterization of Manduca sexta and Plutella xylostellamidgut aminopeptidase N enzymes related to Bacillusthuringiensis toxin-binding proteinsrdquo European Journal ofBiochemistry vol 248 no 3 pp 748ndash761 1997

[113] R Rajagopal N Agrawal A Selvapandiyan S Sivakumar SAhmad and R K Bhatnagar ldquoRecombinantly expressed isoen-zymic aminopeptidases from Helicoverpa armigera (Americancotton bollworm) midgut display differential interaction withclosely related Bacillus thuringiensis insecticidal proteinsrdquo Bio-chemical Journal vol 370 no 3 pp 971ndash978 2003

[114] A Aroonkesorn K Pootanakit G Katzenmeier andC Angsuthanasombat ldquoTwo specific membrane-boundaminopeptidase N isoforms from Aedes aegypti larvae serveas functional receptors for the Bacillus thuringiensis Cry4Batoxin implicating counterpart specificityrdquo Biochemical andBiophysical Research Communications vol 461 no 2 pp300ndash306 2015

[115] M Budatha G Meur and A Dutta-Gupta ldquoA novel aminopep-tidase in the fat body of the moth Achaea janata as a receptorfor Bacillus thuringiensis Cry toxins and its comparison withmidgut aminopeptidaserdquo Biochemical Journal vol 405 no 2pp 287ndash297 2007

[116] T J Ningshen R K Chaitanya P P Hari P S Vimala Devi andA Dutta-Gupta ldquoCharacterization and regulation of Bacillusthuringiensis Cry toxin binding aminopeptidases N (APNs)from non-gut visceral tissues Malpighian tubule and salivarygland comparison with midgut-specific APN in the mothAchaea janatardquo Comparative Biochemistry and Physiology BBiochemistry and Molecular Biology vol 166 no 3-4 pp 194ndash202 2013

[117] T J Ningshen P Aparoy V R Ventaku and A Dutta-GuptaldquoFunctional interpretation of a non-gut hemocoelic tissueaminopeptidase n (APN) in a lepidopteran insect pest Achaeajanatardquo PLoS ONE vol 8 no 11 Article ID e79468 2013

[118] N S Lamango and R E Isaac ldquoIdentification and propertiesof a peptidyl dipeptidase in the housefly Musca domesticathat resembles mammalian angiotensin-converting enzymerdquoBiochemical Journal vol 299 no 3 pp 651ndash657 1994

[119] R E Isaac N S Lamango U Ekbote et al ldquoAngiotensin-converting enzyme as a target for the development of novel


insect growth regulatorsrdquo Peptides vol 28 no 1 pp 153ndash1622007

[120] E Lemeire B Vanholme T Van Leeuwen J Van Camp andG Smagghe ldquoAngiotensin-converting enzyme in Spodopteralittoralis molecular characterization expression and activityprofile during developmentrdquo Insect Biochemistry and MolecularBiology vol 38 no 2 pp 166ndash175 2008

[121] R E Isaac U Ekbote D Coates and A D Shirras ldquoInsectangiotensin-converting enzyme A processing enzyme withbroad substrate specificity and a role in reproductionrdquo Annalsof the New York Academy of Sciences vol 897 pp 342ndash347 1999

[122] R Aguilar A E Jedlicka M Mintz V Mahairaki A LScott and G Dimopoulos ldquoGlobal gene expression analysisof Anopheles gambiae responses to microbial challengerdquo InsectBiochemistry and Molecular Biology vol 35 no 7 pp 709ndash7192005

[123] NMacours KHens C Francis ADe Loof andRHuybrechtsldquoMolecular evidence for the expression of angiotensin convert-ing enzyme in hemocytes of Locusta migratoria stimulationby bacterial lipopolysaccharide challengerdquo Journal of InsectPhysiology vol 49 no 8 pp 739ndash746 2003

[124] W Wang L Luo H Lu S Chen L Kang and F CuildquoAngiotensin-converting enzymes modulate aphid-plant inter-actionsrdquo Scientific Reports vol 5 article 8885 2015

[125] N Macours and K Hens ldquoZinc-metalloproteases in insectsACE and ECErdquo Insect Biochemistry and Molecular Biology vol34 no 6 pp 501ndash510 2004

[126] M J Cornell T A Williams N S Lamango et al ldquoCloningand expression of an evolutionary conserved single-domainangiotensin converting enzyme fromDrosophila melanogasterrdquoThe Journal of Biological Chemistry vol 270 no 23 pp 13613ndash13619 1995

[127] C Harrison and K R Acharya ldquoACE for allmdasha molecularperspectiverdquo Journal of Cell Communication and Signaling vol8 no 3 pp 195ndash210 2014

[128] D M Gordon A Dancis and D Pain ldquoMechanisms ofmitochondrial protein importrdquo Essays in Biochemistry vol 36pp 61ndash73 2000

[129] I A York A X Y Mo K Lemerise et al ldquoThe cytosolicendopeptidase thimet oligopeptidase destroys antigenic pep-tides and limits the extent ofMHC class I antigen presentationrdquoImmunity vol 18 no 3 pp 429ndash440 2003

[130] C Yao J E Donelson and M E Wilson ldquoThe major surfaceprotease (MSP or GP63) of Leishmania sp Biosynthesis regu-lation of expression and functionrdquo Molecular and BiochemicalParasitology vol 132 no 1 pp 1ndash16 2003

[131] B S McGwire K-P Chang and D M Engman ldquoMigrationthrough the extracellular matrix by the parasitic protozoanLeishmania is enhanced by surface metalloprotease gp63rdquoInfection and Immunity vol 71 no 2 pp 1008ndash1010 2003

[132] BMcHugh S A Krause B Yu et al ldquoInvadolysin a novel con-served metalloprotease links mitotic structural rearrangementswith cell migrationrdquo Journal of Cell Biology vol 167 no 4 pp673ndash686 2004

[133] J M Maidment D Moore G P Murphy G Murphy and I MClark ldquoMatrixmetalloproteinase homologues fromArabidopsisthaliana Expression and activityrdquo The Journal of BiologicalChemistry vol 274 no 49 pp 34706ndash34710 1999

[134] A A Leontovich J Zhang K-I Shimokawa H Nagase andMP Sarras Jr ldquoA novel hydra matrix metalloproteinase (HMMP)functions in extracellular matrix degradation morphogenesis

and the maintenance of differentiated cells in the foot processrdquoDevelopment vol 127 no 4 pp 907ndash920 2000

[135] B Altincicek M Fischer M Fischer et al ldquoRole of matrix met-alloproteinase ZMP-2 in pathogen resistance and developmentin Caenorhabditis elegansrdquo Developmental and ComparativeImmunology vol 34 no 11 pp 1160ndash1169 2010

[136] R Visse and H Nagase ldquoMatrix metalloproteinases and tis-sue inhibitors of metalloproteinases structure function andbiochemistryrdquo Circulation Research vol 92 no 8 pp 827ndash8392003

[137] H Hua M Li T Luo Y Yin and Y Jiang ldquoMatrix metallopro-teinases in tumorigenesis an evolving paradigmrdquo Cellular andMolecular Life Sciences vol 68 no 23 pp 3853ndash3868 2011

[138] C Lopez-Otın and L M Matrisian ldquoEmerging roles of pro-teases in tumour suppressionrdquo Nature Reviews Cancer vol 7no 10 pp 800ndash808 2007

[139] E Llano A M Pendas P Aza-Blanc T B Kornberg andC Lopez-Otin ldquoDm1-MMP a matrix metalloproteinase fromDrosophila with a potential role in extracellular matrix remod-eling during neural developmentrdquo The Journal of BiologicalChemistry vol 275 no 46 pp 35978ndash35985 2000

[140] E Llano G Adam A M Pendas et al ldquoStructural and enzy-matic characterization of DrosophilaDm2-MMP a membrane-bound matrix metalloproteinase with tissue-specific expres-sionrdquo Journal of Biological Chemistry vol 277 no 26 pp 23321ndash23329 2002

[141] E Knorr H Schmidtberg A Vilcinskas and B AltincicekldquoMMPs regulate both development and immunity in the Tri-boliummodel insectrdquo PLoS ONE vol 4 no 3 Article ID e47512009

[142] B Altincicek andAVilcinskas ldquoIdentification of a lepidopteranmatrixmetalloproteinase with dual roles inmetamorphosis andinnate immunityrdquo Developmental and Comparative Immunol-ogy vol 32 no 4 pp 400ndash409 2008

[143] A Page-McCaw ldquoRemodeling the model organism matrixmetalloproteinase functions in invertebratesrdquo Seminars in Celland Developmental Biology vol 19 no 1 pp 14ndash23 2008

[144] F X Gomis-Ruth ldquoStructural aspects of the metzincin clan ofmetalloendopeptidasesrdquoMolecular Biotechnology vol 24 no 2pp 157ndash202 2003

[145] J S Bond and R J Beynon ldquoThe astacin family of metalloen-dopeptidasesrdquo Protein Science vol 4 no 7 pp 1247ndash1261 1995

[146] D F Seals and S A Courtneidge ldquoThe ADAMs family of met-alloproteases multidomain proteins with multiple functionsrdquoGenes and Development vol 17 no 1 pp 7ndash30 2003

[147] M L Moss and J W Bartsch ldquoTherapeutic benefits fromtargeting of ADAM family membersrdquo Biochemistry vol 43 no23 pp 7227ndash7235 2004

[148] Y Zhu T J Johnson A A Myers and M R Kanost ldquoIden-tification by subtractive suppression hybridization of bacteria-induced genes expressed in Manduca sexta fat bodyrdquo InsectBiochemistry and Molecular Biology vol 33 no 5 pp 541ndash5592003

[149] C L Wilson A D Shirras and R E Isaac ldquoExtracellular pep-tidases of imaginal discs of Drosophila melanogasterrdquo Peptidesvol 23 no 11 pp 2007ndash2014 2002

[150] J L Sitnik C Francis K Hens R Huybrechts M F Wolfnerand P Callaerts ldquoNeprilysins an evolutionarily conserved fam-ily ofmetalloproteases that play important roles in reproductionin Drosophilardquo Genetics vol 196 no 3 pp 781ndash797 2014


[151] R E IsaacND Bland andAD Shirras ldquoNeuropeptidases andthemetabolic inactivation of insect neuropeptidesrdquoGeneral andComparative Endocrinology vol 162 no 1 pp 8ndash17 2009

[152] C Ferreira K G O Rebola C Cardoso I Bragatto A FRibeiro and W R Terra ldquoInsect midgut carboxypeptidaseswith emphasis on S10 hemipteran and M14 lepidopteran car-boxypeptidasesrdquo Insect Molecular Biology vol 24 no 2 pp222ndash239 2015

[153] J A Affholter V A Fried and R A Roth ldquoHuman insulin-degrading enzyme shares structural and functional homologieswith E coli protease IIIrdquo Science vol 242 no 4884 pp 1415ndash1418 1988

[154] MMatsui J H Fowler and L LWalling ldquoLeucine aminopepti-dases diversity in structure and functionrdquo Biological Chemistryvol 387 no 12 pp 1535ndash1544 2006

[155] T Hatta N Tsuji T Miyoshi M A Alim M K Islamand K Fujisaki ldquoLeucine aminopeptidase in the ixodid tickHaemaphysalis longicornis endogenous expression profiles inmidgutrdquo Journal of Veterinary Medical Science vol 71 no 5 pp589ndash594 2009

[156] E McCarthy C Stack S M Donnelly et al ldquoLeucineaminopeptidase of the human blood flukes Schistosoma man-soni and Schistosoma japonicumrdquo International Journal forParasitology vol 34 no 6 pp 703ndash714 2004

[157] J-Y Lee S-M Song J-W Seok et al ldquoM17 leucine aminopep-tidase of the human malaria parasite Plasmodium vivaxrdquoMolecular and Biochemical Parasitology vol 170 no 1 pp 45ndash48 2010

[158] A C Seegmiller I Dobrosotskaya J L Goldstein Y K HoM S Brown and R B Rawson ldquoThe SREBP pathway inDrosophila regulation by palmitate not sterolsrdquoDevelopmentalCell vol 2 no 2 pp 229ndash238 2002

[159] J F Bazan L H Weaver S L Roderick R Huber and B WMatthews ldquoSequence and structure comparison suggest thatmethionine aminopeptidase prolidase aminopeptidase P andcreatinase share a common foldrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 91 no7 pp 2473ndash2477 1994

[160] E C Griffith Z Su B E Turk et al ldquoMethionine aminopepti-dase (type 2) is the common target for angiogenesis inhibitorsAGM-1470 and ovalicinrdquo Chemistry and Biology vol 4 no 6pp 461ndash471 1997

[161] K Fujimura-Kamada F J Nouvet and S Michaelis ldquoA novelmembrane-associated metalloprotease Ste24p is required forthe first step of NH2-terminal processing of the yeast a-factorprecursorrdquo Journal of Cell Biology vol 136 no 2 pp 271ndash2851997

[162] V L Boyartchuk M N Ashby and J Rine ldquoModulation ofRas and a-factor function by carboxyl-terminal proteolysisrdquoScience vol 275 no 5307 pp 1796ndash1800 1997

[163] M Smyth and G OrsquoCuinn ldquoDipeptidyl aminopeptidase III ofguinea-pig brain specificity for short oligopeptide sequencesrdquoJournal of Neurochemistry vol 63 no 4 pp 1439ndash1445 1994

[164] C Mazzocco K M Fukasawa A-A Raymond and J PuirouxldquoPurification partial sequencing and characterization of aninsect membrane dipeptidyl aminopeptidase that degrades theinsect neuropeptide proctolinrdquo European Journal of Biochem-istry vol 268 no 18 pp 4940ndash4949 2001

[165] C Mazzocco J Gillibert-Duplantier V Neaud et al ldquoIden-tification and characterization of two dipeptidyl-peptidase IIIisoforms in Drosophila melanogasterrdquo FEBS Journal vol 273no 5 pp 1056ndash1064 2006

[166] E Seemuller A Lupas D Stock J Lowe R Huber and WBaumeister ldquoProteasome from Thermoplasma acidophilum athreonine proteaserdquo Science vol 268 no 5210 pp 579ndash5821995

[167] M H Glickman and A Ciechanover ldquoThe ubiquitin-proteasome proteolytic pathway destruction for the sake ofconstructionrdquo Physiological Reviews vol 82 no 2 pp 373ndash4282002

[168] I Mononen K J Fisher V Kaartinen and N N AronsonJr ldquoAspartylglycosaminuria protein chemistry and molecularbiology of the most common lysosomal storage disorder ofglycoprotein degradationrdquoThe FASEB Journal vol 7 no 13 pp1247ndash1256 1993

[169] M J Page and E Di Cera ldquoSerine peptidases classificationstructure and functionrdquo Cellular and Molecular Life Sciencesvol 65 no 7-8 pp 1220ndash1236 2008

[170] L Cerenius andK Soderhall ldquoTheprophenoloxidase-activatingsystem in invertebratesrdquo Immunological Reviews vol 198 pp116ndash126 2004

[171] R A Patterson M T Juarez A Hermann R Sasik G Hardi-man and W McGinnis ldquoSerine proteolytic pathway activationreveals an expanded ensemble of wound response genes inDrosophilardquo PLoS ONE vol 8 no 4 Article ID e61773 2013

[172] Z Zou D L Lopez M R Kanost J D Evans and HJiang ldquoComparative analysis of serine protease-related genesin the honey bee genome possible involvement in embryonicdevelopment and innate immunityrdquo Insect Molecular Biologyvol 15 no 5 pp 603ndash614 2006

[173] D N Amin S G Kamita G M Muluvi J Machuka B DHammock and E O Osir ldquoGlossina proteolytic lectin does notrequire a carbohydrate moiety for enzymatic or trypanosome-transforming activitiesrdquo Journal of Medical Entomology vol 43no 2 pp 301ndash308 2006

[174] I-H Jang N Chosa S-H Kim et al ldquoA Spatzle-processingenzyme required for toll signaling activation in drosophilainnate immunityrdquo Developmental Cell vol 10 no 1 pp 45ndash552006

[175] P Ligoxygakis N Pelte J A Hoffmann and J-M ReichhartldquoActivation ofDrosophila toll during fungal infection by a bloodserine proteaserdquo Science vol 297 no 5578 pp 114ndash116 2002

[176] Z Kambris S Brun I-H Jang et al ldquoDrosophila immunity alarge-scale in vivo RNAi screen identifies five serine proteasesrequired for toll Activationrdquo Current Biology vol 16 no 8 pp808ndash813 2006

[177] B Lemaitre and J Hoffmann ldquoThe host defense of Drosophilamelanogasterrdquo Annual Review of Immunology vol 25 pp 697ndash743 2007

[178] L L Lindsay J C Yang and J L Hedrick ldquoOvochymase aXenopus laevis egg extracellular protease is translated as part ofan unusual polyproteaserdquo Proceedings of the National Academyof Sciences of the United States of America vol 96 no 20 pp11253ndash11258 1999

[179] S Misra P Hecht R Maeda and K V Anderson ldquoPositive andnegative regulation of Easter a member of the serine proteasefamily that controls dorsal-ventral patterning in the Drosophilaembryordquo Development vol 125 no 7 pp 1261ndash1267 1998

[180] G Didelot F Molinari P Tchenio et al ldquoTequila a neu-rotrypsin ortholog regulates long-term memory formation inDrosophilardquo Science vol 313 no 5788 pp 851ndash853 2006

[181] T Igaki Y Suzuki N Tokushige H Aonuma R Takahashiand M Miura ldquoEvolution of mitochondrial cell death pathway


proapoptotic role of HtrA2Omi in Drosophilardquo Biochemicaland Biophysical Research Communications vol 356 no 4 pp993ndash997 2007

[182] J Shan L Yuan Q Xiao et al ldquoTSP50 a possible proteasein human testes is activated in breast cancer epithelial cellsrdquoCancer Research vol 62 no 1 pp 290ndash294 2002

[183] J G Stoffolano Jr and A T Haselton ldquoThe adult dipteran cropa unique and overlooked organrdquo Annual Review of Entomologyvol 58 pp 205ndash225 2013

[184] D I Pritchard and A P Brown ldquoDegradation of MSCRAMMtarget macromolecules in VLU slough by Lucilia sericatachymotrypsin 1 (ISP) persists in the presence of tissue gelatinaseactivityrdquo InternationalWound Journal vol 12 no 4 pp 414ndash4212015

[185] D I Pritchard V Cerovsky Y Nigam et al ldquoTIME manage-ment by medicinal larvaerdquo International Wound Journal 2015

[186] G Thomas ldquoFurin at the cutting edge from protein trafficto embryogenesis and diseaserdquo Nature Reviews Molecular CellBiology vol 3 no 10 pp 753ndash766 2002

[187] F Couture A Kwiatkowska Y L Dory and R Day ldquoThera-peutic uses of furin and its inhibitors a patent reviewrdquo ExpertOpinion onTherapeutic Patents vol 25 no 4 pp 379ndash396 2015

[188] J A Garcıa-Horsman P T Mannisto and J I VenalainenldquoOn the role of prolyl oligopeptidase in health and diseaserdquoNeuropeptides vol 41 no 1 pp 1ndash24 2007

[189] P Fajtova S Stefanic M Hradilek et al ldquoProlyl oligopeptidasefrom the blood fluke Schistosoma mansoni from functionalanalysis to anti-schistosomal inhibitorsrdquo PLoS Neglected Trop-ical Diseases vol 9 no 6 Article ID e0003827 2015

[190] S Gottesman S Wickner and M R Maurizi ldquoProtein qualitycontrol triage by chaperones and proteasesrdquo Genes and Devel-opment vol 11 no 7 pp 815ndash823 1997

[191] S G Roudiak A Seth N Knipfer and T E Shrader ldquoThe Lonprotease from Mycobacterium smegmatis molecular cloningsequence analysis functional expression and enzymatic char-acterizationrdquo Biochemistry vol 37 no 1 pp 377ndash386 1998

[192] Z Shariat-Madar F Mahdi and A H Schmaier ldquoRecombinantprolylcarboxypeptidase activates plasma prekallikreinrdquo Bloodvol 103 no 12 pp 4554ndash4561 2004

[193] P Novak and I K Dev ldquoDegradation of a signal peptide byprotease IV and oligopeptidase Ardquo Journal of Bacteriology vol170 no 11 pp 5067ndash5075 1988

[194] T H Carter and C G Miller ldquoAspartate-specific peptidasesin Salmonella typhimurium mutants deficient in peptidase ErdquoJournal of Bacteriology vol 159 no 2 pp 453ndash459 1984

[195] K Hakansson A H-J Wang and C G Miller ldquoThe structureof aspartyl dipeptidase reveals a unique fold with a Ser-His-Glucatalytic triadrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 26 pp 14097ndash141022000

[196] S Urban J R Lee and M Freeman ldquoDrosophila rhomboid-1defines a family of putative intramembrane serine proteasesrdquoCell vol 107 no 2 pp 173ndash182 2001

[197] S Urban J R Lee and M Freeman ldquoA family of rhomboidintramembrane proteases activates all Drosophila membrane-tethered EGF ligandsrdquo The EMBO Journal vol 21 no 16 pp4277ndash4286 2002

[198] I Valachova T Majtan P Takac and J Majtan ldquoIdentificationand characterisation of different proteases in Lucilia sericatamedicinal maggots involved in maggot debridement therapyrdquoJournal of Applied Biomedicine vol 12 no 3 pp 171ndash177 2014

[199] K Katoh and D M Standley ldquoMAFFT multiple sequencealignment software version 7 improvements in performanceand usabilityrdquo Molecular Biology and Evolution vol 30 no 4pp 772ndash780 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


before it is consumed back Chambers et al identified threeclasses of proteolytic enzymes (aspartic serine and metal-lopeptidases) from MEP and proposed that mainly serinepeptidases are responsible for the superficial debridementactivity of maggots [14] Only two such peptidases (serinepeptidases) have been identified and characterized thus farChymotrypsin 1 was identified from MEP and producedin the recombinant form [15] Recombinant enzyme wasshown to degrade the eschar from venous leg ulcers in vitro[15] and to be unaffected by two endogenous inhibitors1205721-antichymotrypsin and 1205721-antitrypsin from wound eschar[16] We recently produced and characterized Jonah-likechymotrypsin which digested three specific extracellularmatrix proteins (laminin fibronectin and collagen IV) invitro and proposed its function in wound debridement[17]

The natural habitat of L sericata larvae is rotting organicmatter such as cadavers and excrement but this ecologicalniche also favors many microorganisms so the larvae musthave adequate defenses against infectionThemaggots there-fore protect themselves by producing many antimicrobialsubstances [18ndash22] and by digesting microbes which arethus eliminated in the larval gut [23 24] InterestinglyMEPs also show activity against relevant human pathogensincluding antibiotic-resistant bacterial strains [25ndash27] andbiofilms [28ndash31] Recently two molecules with antibiofilmactivity have been identified from MEP Affinity purifiedDNase disrupted Pseudomonas aeruginosa biofilm [32] andrecombinant chymotrypsin I was active against Staphylococ-cus epidermidis and S aureus biofilms [33]

Surprisingly medical maggots also directly promotewound healing [10] MEPs stimulate fibroblast migration[34 35] and proliferation [36] and increase angiogenesis[37 38] MEPs also influence the activation of the humancomplement system [39] reduce proinflammatory responses[40ndash43] and induce fibrinolysis [44] Recently we discov-ered that MEPs contain peptidases that influence bloodcoagulation as part of the wound healing process [45] andthis activity was attributed to Jonah-like serine peptidaseRecombinant enzyme was shown to reduce the clotting timeof human plasma by substituting for the intrinsic clottingfactors kallikrein factor XI and factor XII respectively [17]

However although severalmolecules have been identifiedfromMEP it is still recognized as a largely unexplored sourceof compounds with therapeutic potential The future studiesshall focus on identification isolation andor production ofeffector molecules and testing of their therapeutic potentialHere we analyzed the transcriptome of different larvaltissues to systematically identify MEP peptidases It is notclear whether MEP components are exclusively producedby salivary glands or also by other tissues so we dissectedthree individual tissues (gut crop and salivary glands) as wellas the remaining larval biomass to generate tissue-specificsequence data The extracted mRNA was sequenced usingthe Illumina HiSeq2000 Genome Analyzer platform andpaired-end read technology After preprocessing 123856654paired reads remained in the panel of libraries These wereprocessed further to yield a final assembly of 41421 contigs in17479 clusters resulting in the identification of 1729 contigs

in 577 clusters encoding five different functional classes ofproteolytic enzymes

2 Materials and Methods

21 Preparation of Biological Material First-instar L sericatamaggots were obtained from BioMonde GmbH (BarsbuttelGermany) and were cultured under sterile conditions onColumbia agar plates with ldquosheep blood +rdquo (Oxoid Deutsch-land GmbH Wesel Germany) at 28∘C for 48 h in the darkThe larvae were cleaned and then infected with a mixtureof Pseudomonas aeruginosa (DSM 50071) and Staphylococcusaureus (DSM 2569) as previously described [21] The larvalgut salivary glands and crop were dissected under a binoc-ular microscope 8 h after infection Dissected tissues and theremaining larval body for Illumina sequencing were frozenin liquid nitrogen and stored at ndash80∘C Samples for qRT-PCRanalysis were processed immediately as described below

22 RNA Isolation and Illumina Sequencing Total RNAwas extracted from individual tissues and the rest of thelarval body using the innuPREP RNA Mini Isolation Kit(Analytik Jena Jena Germany) following the manufacturerrsquosinstructions Additional RNA purification quantificationand quality control were carried out as previously described[46] An additional Turbo DNase treatment (Thermo FisherScientific Waltham MA USA) was applied before thesecond purification step to eliminate contaminating DNAThe DNase was removed and the RNA purified using theRNeasy MinElute Clean up Kit (Qiagen Hilden Germany)following the manufacturerrsquos protocol RNA was eluted in20120583L Ambion RNA Storage Solution (Thermo Fisher Scien-tific) and poly(A)+ mRNA was prepared using the AmbionMicroPoly(A) Purist Kit according to the manufacturerrsquosinstructions (Thermo Fisher Scientific) The integrity andquantity of the mRNA was confirmed using an Agilent 2100Bioanalyser and RNA Nano chips (Agilent TechnologiesSanta Clara CA USA)

Transcriptome sequencingwas carried out on an IlluminaHiSeq2000 Genome Analyzer platform using paired-end(2 times 100 bp) read technology for the larval tissues withRNA fragmented to an average length of 150 nucleotidesSequencing was carried out by Eurofins MWG Operon(Ebersberg Germany) and resulted in totals of 29 33 26 and34 million reads for the rest of body gut crop and salivaryglands respectively

23 Read Preprocessing The quality of the reads was checkedusing the FastQC toolkit [47] Trimmomatic [48] wasused to clip adapters and trim low-quality regions (param-eter ILLUMINACLIPTruSeq3-SE-223010 SLIDINGWIN-DOW520 HEADCROP15 MINLEN50) The reads fromall libraries were then pooled for assembly and digitallynormalized to achieve 100-fold coverage using the Trinity ldquoinsilico normalizationrdquo tool [49]

24 Assembly The reads from all libraries were assembledde novo in two steps One assembly was computed using















Biol

ogic

al p

roce

ssC

ellu

lar c

ompo

nent

Mol

ecul

ar fu

nctio

n

48763430

3192822041479284257

658530524424390

1603919107533

2799813

355305194

272084411

0 2000 4000 6000Binding





Cell partOrganelle












SignalingGrowth













47

35

8

43 3











290203145

8986

174

029058 058 058



3771

019

4747

0191426

019



429

5286

571571

1571

1571









AA A1 Pepsin A







CA C1 Papain







CD C14 Caspase-1



















ags12ags10

ags13T1P9A0 MUSDO


T1PAQ7 MUSDODronc





ags8Damm



T1PN44 MUSDODcp-1



ags4ags3

ags5ags11ags6ags14

ags2ags1


026026

032001001

002007

041021

026025

031030

033037

038024024

028027

033035037041

043044

045037

000000

006011

012018

017012014

018019019

014014

016023

022022

002000

022020

018019

035035

038

006003

019

012015

003

001

004005

001003

003001

003

001

004

001

005

002

001004

002

001

001

001

000

007004

001007

001

005

001

003

002

002

010

002006

006

001

021

002

004

001

001

010

002

001

001

001

001

01

















MA M12 Astacin




MA M13 Neprilysin









Table 5 Continued






ME M16 Pitrilysin



























































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Biol

ogic

al p

roce

ssC

ellu

lar c

ompo

nent

Mol

ecul

ar fu

nctio

n

48763430

3192822041479284257

658530524424390

1603919107533

2799813

355305194

272084411

0 2000 4000 6000Binding





Cell partOrganelle












SignalingGrowth













47

35

8

43 3











290203145

8986

174

029058 058 058



3771

019

4747

0191426

019



429

5286

571571

1571

1571









AA A1 Pepsin A







CA C1 Papain







CD C14 Caspase-1



















ags12ags10

ags13T1P9A0 MUSDO


T1PAQ7 MUSDODronc





ags8Damm



T1PN44 MUSDODcp-1



ags4ags3

ags5ags11ags6ags14

ags2ags1


026026

032001001

002007

041021

026025

031030

033037

038024024

028027

033035037041

043044

045037

000000

006011

012018

017012014

018019019

014014

016023

022022

002000

022020

018019

035035

038

006003

019

012015

003

001

004005

001003

003001

003

001

004

001

005

002

001004

002

001

001

001

000

007004

001007

001

005

001

003

002

002

010

002006

006

001

021

002

004

001

001

010

002

001

001

001

001

01

















MA M12 Astacin




MA M13 Neprilysin









Table 5 Continued






ME M16 Pitrilysin



























































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




47

35

8

43 3











290203145

8986

174

029058 058 058



3771

019

4747

0191426

019



429

5286

571571

1571

1571









AA A1 Pepsin A







CA C1 Papain







CD C14 Caspase-1



















ags12ags10

ags13T1P9A0 MUSDO


T1PAQ7 MUSDODronc





ags8Damm



T1PN44 MUSDODcp-1



ags4ags3

ags5ags11ags6ags14

ags2ags1


026026

032001001

002007

041021

026025

031030

033037

038024024

028027

033035037041

043044

045037

000000

006011

012018

017012014

018019019

014014

016023

022022

002000

022020

018019

035035

038

006003

019

012015

003

001

004005

001003

003001

003

001

004

001

005

002

001004

002

001

001

001

000

007004

001007

001

005

001

003

002

002

010

002006

006

001

021

002

004

001

001

010

002

001

001

001

001

01

















MA M12 Astacin




MA M13 Neprilysin









Table 5 Continued






ME M16 Pitrilysin



























































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


290203145

8986

174

029058 058 058



3771

019

4747

0191426

019



429

5286

571571

1571

1571









AA A1 Pepsin A







CA C1 Papain







CD C14 Caspase-1



















ags12ags10

ags13T1P9A0 MUSDO


T1PAQ7 MUSDODronc





ags8Damm



T1PN44 MUSDODcp-1



ags4ags3

ags5ags11ags6ags14

ags2ags1


026026

032001001

002007

041021

026025

031030

033037

038024024

028027

033035037041

043044

045037

000000

006011

012018

017012014

018019019

014014

016023

022022

002000

022020

018019

035035

038

006003

019

012015

003

001

004005

001003

003001

003

001

004

001

005

002

001004

002

001

001

001

000

007004

001007

001

005

001

003

002

002

010

002006

006

001

021

002

004

001

001

010

002

001

001

001

001

01

















MA M12 Astacin




MA M13 Neprilysin









Table 5 Continued






ME M16 Pitrilysin



























































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




AA A1 Pepsin A







CA C1 Papain







CD C14 Caspase-1



















ags12ags10

ags13T1P9A0 MUSDO


T1PAQ7 MUSDODronc





ags8Damm



T1PN44 MUSDODcp-1



ags4ags3

ags5ags11ags6ags14

ags2ags1


026026

032001001

002007

041021

026025

031030

033037

038024024

028027

033035037041

043044

045037

000000

006011

012018

017012014

018019019

014014

016023

022022

002000

022020

018019

035035

038

006003

019

012015

003

001

004005

001003

003001

003

001

004

001

005

002

001004

002

001

001

001

000

007004

001007

001

005

001

003

002

002

010

002006

006

001

021

002

004

001

001

010

002

001

001

001

001

01

















MA M12 Astacin




MA M13 Neprilysin









Table 5 Continued






ME M16 Pitrilysin



























































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












ags12ags10

ags13T1P9A0 MUSDO


T1PAQ7 MUSDODronc





ags8Damm



T1PN44 MUSDODcp-1



ags4ags3

ags5ags11ags6ags14

ags2ags1


026026

032001001

002007

041021

026025

031030

033037

038024024

028027

033035037041

043044

045037

000000

006011

012018

017012014

018019019

014014

016023

022022

002000

022020

018019

035035

038

006003

019

012015

003

001

004005

001003

003001

003

001

004

001

005

002

001004

002

001

001

001

000

007004

001007

001

005

001

003

002

002

010

002006

006

001

021

002

004

001

001

010

002

001

001

001

001

01

















MA M12 Astacin




MA M13 Neprilysin









Table 5 Continued






ME M16 Pitrilysin



























































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















MA M12 Astacin




MA M13 Neprilysin









Table 5 Continued






ME M16 Pitrilysin



























































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 5 Continued






ME M16 Pitrilysin



























































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































Table 7 Continued




























Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Table8Com

paris

onof

cluste

rswith

previouslyidentifi

edLseric

ataS1

peptidases

GenBa

nkID

Nam

eProp

osed

functio

nRe

ference

Cluster

Peptidases

pecies

MER

OPS

ID

CAS92770

Chym

otrypsin

IWou

nddebridem

ent

[15]

LSTLS

005873

Chym

otrypsin

m-ty

pe2(in

sect)(L

cuprina)

S01168

KT158461

Jonah-lik

echymotrypsin

Plasma

clotting

debrid

ement

[17]

LSTLS

007060

Jonah65Aiv(D

mela

nogaste

r)S01B

05

AAA17384

Seric

asetrypsin


ent

[44]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07[185]

CG7542

protein(D

mela

nogaste

r)S01B

07

AEX

33291

Putativ

esalivarytrypsin

Not

describ

ed[19

8]

LSTLS

016882

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005035

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005470

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

0164

40CL

IP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005552

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

LSTLS

005028

CLIP-dom

ainprop

heno

loxidase

activ

atingfactor

(Crub

ecula)

S019

60

AEX

33294

Putativ

esalivarytrypsin

Not

describ

ed[19

8]LS

TLS

003737

IP10861p

rotein

(Dm

elanogaste

r)S01B

61

AEX

33290

Putativ

echymotrypsin

Not

describ

ed[19

8]LS

TLS

0160

62CG

10477protein(D

mela

nogaste

r)S01B

12LS

TLS

007621

CG10477protein(D

mela

nogaste

r)S01B

12AJ

N88395

Debrilase

Debrid

ement

Patent

US8623810

LSTLS

015273

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360474

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007407

CG119

11protein(D

mela

nogaste

r)S01B

68LS

TLS

015021

CG119

11protein(D

mela

nogaste

r)S01B

68

FG360505

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015269

Jonah65Aiv(D

mela

nogaste

r)S01B

05

FG360512

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008125

Subfam

ilyS1Ano

n-peptidase

homologues

S01U

NA

FG360521

Upregulated

upon

immun

echalleng

e[18]

LSTLS

008080

CG9676

protein(D

mela

nogaste

r)S01A

89

FG360523

Upregulated

upon

immun

echalleng

e[18]

LSTLS

003034

Trypsin

alph

a(insect)(D

mela

nogaste

r)S01110

FG360527

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007843

CG17571p

rotein

(Dm

elanogaste

r)S01A

85

FG360528

Upregulated

upon

immun

echalleng

e[18]

LSTLS

015518

CG16749protein(D

mela

nogaste

r)S01B

48LS

TLS

007441

CG16749protein(D

mela

nogaste

r)S01B

48

FG360529

Upregulated

upon

immun

echalleng

e[18]

LSTLS

007476

CG7542

protein(D

mela

nogaste

r)S01B

07























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























LST_LS005916 129630

LST_LS006048

LST_LS004632

LST_LS006843

LST_LS010066

Body

Gla

nd

Crop

Gut

RPKM

minus3

minus6

minus9

(a)

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS005916

1

10

100Re

lativ

e exp

ress

ion

()

LST_LS006048

1

10

100



Body Gland Crop Gut

Relat

ive e

xpre

ssio

n(

)

LST_LS004632

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS006843

1

10

100

Relat

ive e

xpre

ssio

n (

)

LST_LS010066

(b)


2



4 Conclusion




Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Additional Points


Competing Interests


Acknowledgments


References

























































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article next generation sequencing identifies five...

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