The regulation of digestive enzyme release in the two-spotted field cricket Gryllus bimaculatus (de Geer): effects of endogenous and environmental factors

The egula ion o diges i e enzyme elease in he wo-spo ed ield c icke G yllus bimacula us (de Gee ): e ec s o endogenous and en i onmen al ac o s Kumula i e Disse a ion zu E langung des Dok o g ades de Na u wissenscha en (D . e . na .) de Fakul ä ü Biologie, Chemie und Geowissenscha en de Uni e si ä Bay eu h o geleg on Sandy Weidlich Bay eu h, Mai 2013 Die o liegende A bei wu de am Leh s uhl ü Tie ökologie I de Uni e si ä Bay eu h un e de Lei ung on P o . D . Klaus H. Ho mann und P o . D . Joseph Wood ing im Zei aum on Ap il 2009 bis Mai 2013 ange e ig . Volls ändige Abd uck de on de Fakul ä ü Biologie, Chemie und Geowissenscha en de Uni e si ä Bay eu h genehmig en Disse a ion zu E langung des akademischen G ades eines Dok o s de Na u wissenscha en (D . e . na .) Disse a ion einge eich am: 14.05.2013 Zulassung du ch die P ü ungskommission: 22.05.2013 Wissenscha liches Kolloquium: 30.10.2013 Am ie ende Dekan: P o . D . Rhe Kempe P ü ungsausschuss: P o . D . Klaus H. Ho mann (E s gu ach e ) P o . D . Kon ad De ne (Zwei gu ach e ) P o . Ph.D. Ha old D ake (Vo si z) P o . D . Ha mu F ank P o . D . Ch is ian La o sch Con en s I Con en s 1 In oduc ion .................................................................................................... 1 1.1 The mo phology o he diges i e ac ..................................................... 1 1.2 Diges i e enzymes.................................................................................. 2 1.2.1 P o eases ..................................................................................... 3 1.2.2 Ca bohyd ases ............................................................................. 4 1.2.2.1 Amylases ........................................................................ 4 1.2.2.2 Cellulases ....................................................................... 5 1.2.2.3 Chi inases ....................................................................... 6 1.2.3 Lipases ......................................................................................... 7 1.3 Sec e o y p ocesses o diges i e enzymes ............................................ 8 1.4 Regula ion mechanisms con olling diges i e enzyme sec e ion in insec s .......................................................................................................... 9 1.4.1 Alla os a ins .................................................................................10 1.4.2 Sul akinins ...................................................................................11 1.5 Enzyme inhibi o s ..................................................................................13 1.5.1 Plan p o ease inhibi o s...............................................................13 1.5.2 Endogenous p o ease inhibi o s ...................................................13 1.6 Resea ch gaps ......................................................................................15 2 Synopsis ........................................................................................................17 2.1 Enzyme assays, kine ic pa ame e s and sample p epa a ion .................17 2.1.1 Enzyme ac i i y assays ................................................................17 2.1.2 Kine ic pa ame e s .......................................................................17 2.1.3 Enzyme ac i i y in di e en samples ypes ...................................18 2.2 The e ec o endogenous ac o s ...........................................................21 2.2.1 Age-dependen enzyme elease ..................................................21 2.2.2 Neu opep ides .............................................................................23 2.2.2.1 Alla os a in ype A ..........................................................23 Con en s II 2.2.2.2 Sul akinins .....................................................................31 2.2.3 Calcium ions ................................................................................33 2.2.4 T ypsin ac i a ion and au olysis ...................................................34 2.2.5 Endogenous p o ease inhibi o s ...................................................34 2.3 The e ec o en i onmen al ac o s ........................................................36 2.3.1 Tempe a u e ................................................................................36 2.3.1.1 Incuba ion empe a u e ..................................................36 2.3.1.2 Rea ing empe a u e ......................................................37 2.3.2 Ligh -da k cycle ............................................................................38 2.3.3 Food and nu ien s .......................................................................40 2.3.3.1 Feeding and s a a ion ..................................................40 2.3.3.2 Nu ien s ........................................................................41 2.3.3.3 Plan p o ease inhibi o s ................................................42 Summa y ...........................................................................................................43 Zusammen assung ...........................................................................................45 Li e a u e ...........................................................................................................47 Publica ions ......................................................................................................65 (1) En i onmen al con ol o ypsin sec e ion in he midgu o he wo-spo ed ield c icke , G yllus bimacula us. ..........................................................67 (2) Regula ion o amylase, cellulase and chi inase sec e ion in he diges i e ac o he wo-spo ed ield c icke , G yllus bimacula us. .....................68 (3) The sec e ion o diges i e lipase in he midgu o G yllus bimacula us: egula ion by endogenous and en i onmen al ac o s. ..........................69 (4) Ac i a ion and au olysis o ypsin in he midgu o he Medi e anean ield c icke , G yllus bimacula us. .................................................................91 Acknowledgmen s .......................................................................................... 106 Decla a ion ...................................................................................................... 107 Figu es and Tables III Lis o igu es Figu e 1: The diges i e ac o G yllus bimacula us. .................................................. 2 Figu e 2: Enzyma ic clea age o a polypep ide chain by aminopep idase (EC 3.4.11) and ypsin (3.4.21.4). ................................................................................. 3 Figu e 3: Enzyma ic hyd olysis o amylose (C6H12O6)n by amylases. .......................... 4 Figu e 4: Schema ic iew o he enzyma ic hyd olysis o cellulose. ............................ 6 Figu e 5: Enzyma ic deg ada ion o chi in (C8H13NO5)n by chi inase (EC 3.2.1.14) and β-N-ace ylglucosaminidase (EC 3.2.1.52). .................................................. 7 Figu e 6: S epwise hyd olysis o iacylglyce ol o glyce ol and ca boxylic acids. ........ 7 Figu e 7: Models o sec e o y p ocesses o insec diges i e enzymes: exocy ic sec e ion (A), apoc ine sec e ion (B), mic oapoc ine sec e ion wi h budding esicles (C) and wi h pinched-o esicles (D). ............................................ 8 Figu e 8: Regula ion mechanism o diges i e enzyme elease: small diges i e p oduc s (p andial) bind o ecep o s o endoc ine cells on he midgu side o ee pa aneu oho mones, which s imula e he enzyme elease o zymogene cells (pa ac ine mechanism). ..................................................................... 10 Figu e 9: Nucleo ide sequence o he alla os a in (A) and sul akinin (B) p ecu so cDNA o G yllus bimacula us and he deduced amino acid sequences o he p ep oho mone polypep ides. .................................................................... 12 Figu e 10: Expe imen al o e iew: de e mina ion o he e ec s o endogenous (o ganism) and exogenous (en i onmen ) ac o s con olling he egula ion o enzyme sec e ion in G. bimacula us. ......................................................... 16 Figu e 11: Ac i i y o (A) ca bohyd ases amylase and cellulase, (B) p o eases ypsin and aminopep idase and (C) lipase in samples o issue incuba ion medium (incuba e), issue homogena e ( issue) and luminal con en (lumen) o 2-day- old emale and male G. bimacula us. ........................................................ 20 Figu e 12: Age-dependen ac i i y o (A) ca bohyd ases, (B) p o eases and (C) lipase in he caecal sec e ion medium (30 min incuba ion a 37°C) o male (□) and emale (●) las ins a la aeandadul s o G. bimacula us. ........................... 22 Figu e 13: In i o e ec o AST-5 on he amoun o p o ein [µg BSA/mg issue] eleased om (A) emale and (B) male caecal issue o incuba ion medium. ........... 23 Figu es and Tables IV Figu e 14: In i o e ec s o AST-5 on (A) ca bohyd ases, (B) p o eases and (C) lipase sec e ion om caecal issue o 2-day-old adul G. bimacula us. ................ 24 Figu e 15: Weigh gain o 2-day-old G. bimacula us las ins a la ae and adul s, injec ed wi h ei he 2 µg AST-A dsRNA (in 10 µl Ringe ) o Ringe only a he day o he p eceding moul . .................................................................................. 25 Figu e 16: Weigh gain o 2-day-old adul G. bimacula us emales, injec ed wi h AST-A dsRNA (0-6 µg in 10 µl Ringe ) a he day o imaginal moul . .................... 25 Figu e 17: Ac i i y o (A) ypsin, (B) aminopep idase, (C) lipase and (D) amylase in he caecal lumen con en o 2-day-old G. bimacula us adul s and las ins a la ae injec ed wi h ei he 2 µg AST-A dsRNA (in 10 µl Ringe ) (g ey) o Ringe only (whi e) a p eceding moul . ..................................................... 26 Figu e 18: Ac i i y o (A) ypsin, (B) aminopep idase, (C) lipase and (D) amylase in he caecal issue homogena e o 2-day-old G. bimacula us adul s and las ins a la ae injec ed wi h ei he 2 µg AST-A dsRNA (in 10 µl Ringe ) (g ey) o Ringe only (whi e) a p eceding moul . ..................................................... 27 Figu e 19: Ac i i y o (A) ypsin, (B) aminopep idase, (C) lipase and (D) amylase in he caecal issue incuba ion medium o 2-day-old G. bimacula us adul s and las ins a la ae injec ed wi h ei he 2 µg AST-A dsRNA (in 10 µl Ringe ) (g ey) o Ringe only (whi e) a p eceding moul . ................................................. 28 Figu e 20: The e ec o a ious concen a ions o AST-A dsRNA on he p o ein concen a ion in (A) issue incuba e, (B) issue homogena e, and (C) luminal con en o 2-day-old adul G. bimacula us emales. ................................... 29 Figu e 21: The e ec o a ious concen a ions o AST-A dsRNA on (A) amylase, (B) aminopep idase, (C) ypsin, and (D) lipase ac i i y in issue incuba ion medium, issue homogena e and lumen con en o 2-day-old adul G. bimacula us emales. ............................................................................ 30 Figu e 22: P o ein concen a ion in samples o issue incuba ion medium, issue homogena e and lumen con en om (A) emale and (B) male 2-day-old adul G. bimacula us, injec ed wi h ei he 10 µl Ringe (con ol) o 2 µg SK dsRNA in 10 µl Ringe a he day o imaginal moul . .............................................. 31 Figu e 23: E ec o Ringe and SK dsRNA injec ion on enzyme sec e ion o (A) ca bohyd ases, (B) p o eases and (C) lipase o 2-day-old adul emale and male G. bimacula us. ................................................................................. 32 Figu es and Tables V Figu e 24: In i o e ec o calcium ions (2 mM) on amylase, cellulase, chi inase, and ypsin sec e ion om caecal epi helium o 2-day-old adul emale G. bimacula us. ......................................................................................... 33 Figu e 25: T ypsin ac i i y in mix u es o exogenous bo ine ypsin and hea ed and unhea ed samples o (A) issue incuba ion medium, (B) issue homogena e and (C) lumen con en o die - ed and s a ed 2-day-old adul G. bimacula us emales. ..................................................................... 35 Figu e 26: The e ec o incuba ion empe a u e (25°C = whi e, 35°C = g ey) on (A) ypsin, (B) aminopep idase, (C) lipase, and (D) amylase sec e ion om caecal issue o 2-day-old adul G. bimacula us. ........................................ 37 Figu e 27: Food up ake and diges i e enzyme sec e ion in he cou se o a day in 2-day- old penul ima e la ae, las ins a la ae and adul G. bimacula us emales. .................................................................................................... 39 Figu e 28: In i o e ec s o nu ien s on soluble p o ein concen a ion in he incuba ion medium o caecal issue om 2-day-old adul emale G. bimacula us. ...... 41 Lis o ables Table 1: Tempe a u e and pH op ima, Km and Vmax alues o diges i e enzymes om midgu o G. bimacula us. ............................................................................ 18 Abb e ia ions VI Abb e ia ions AST alla os a in AST-A alla os a in ype A AT acclima ion empe a u e BapNa Nα-benzoyl-DL-a ginine-p-ni oanilide hyd ochlo ide CA co po a alla a cDNA complemen a y desoxy ibonucleic acid CMC ca boxyme hyl cellulose CMCh ca boxyme hyl-chi in-RBV 5R DNS dini osalicylic acid dsRNA double-s anded ibonucleic acid EC enzyme commission EPI endogenous p o ease inhibi o Fig. igu e G. bimacula us G yllus bimacula us GHF glycosyl hyd olase amily GlcNAc N-ace yl-D-glucosamine IPM in eg a ed pes managemen IT incuba ion empe a u e JH ju enile ho mone Km Michaelis-Men en cons an LpNa L-leucine p-ni oanilide hyd ochlo ide Ma mal ose mRNA messenge RNA PG pe i ophic gel PI p o ease inhibi o PM pe i ophic memb ane PMx pe i ophic ma ix pNA p-ni oaniline pNP p-ni ophenol pNPP p-ni ophenyl palmi a e RBV emazol b illian iole RFU ela i e luo escen uni s RNA ibonucleic acid SBTI soybean ypsin inhibi o SDS-PAGE sodium dodecyl sul a e polyac ylamide gel elec opho esis SEM s anda d e o o he mean SK sul akinin Vmax maximum eac ion a e Pa I Synopsis In oduc ion 7 chi obiose), which a e subsequen ly diges ed by exo-spli ing β-N-ace ylglucosaminidases (EC 3.2.1.52) o he monome GlcNAc (K ame and Koga, 1986; Reynolds and Samuels, 1996). Figu e 5: Enzyma ic deg ada ion o chi in (C8H13NO5)n by chi inase (EC 3.2.1.14) and β-N-ace ylglucosaminidase (EC 3.2.1.52).∙∙∙∙∙∙ cu ing si e. 1.2.3 Lipases Lipids a e an impo an sou ce o ene gy and essen ial o insec de elopmen , ene gy s o age and oogenesis. Insec s ha e a die a y equi emen o polyunsa u a ed a y acids du ing hei pos -emb yonic phases o de elopmen , bu ood equi emen di e s be ween species (Dadd, 1983, 1985; Cana oso e al., 2001). Comple e lipid diges ion is accomplished by ca boxylic es e hyd olases (EC 3.1.1: lipases, es e ases, phospholipase A and B) (Figu e 6), phospho ic monoes e hyd olases (EC 3.1.3: phospha ases) and phospho ic dies e hyd olases (EC 3.1.4: phospholipase C and D) (Te a e al., 1996). The eby, lipases a e essen ial compounds o he a me abolism and hyd olyse he ou e es e links o iacylglyce ols om he α-posi ion s epwise o diacylglyce ols, monoacylglyce ols, glyce ol and ee a y acids (Bollade e al., 1970; Ho man and Downe , 1979; Secundo e al., 2006). Figu e 6: S epwise hyd olysis o iacylglyce ol o glyce ol and ca boxylic acids. In oduc ion 8 1.3 Sec e o y p ocesses o diges i e enzymes Di e en mechanisms o syn hesis, s o age and elease o diges i e enzymes a he cellula le el ha e been desc ibed in a ious insec species (C is o ole i e al., 2001; Fe ei a e al., 2002; Te a e al., 1996; Weidlich e al., 2012). Diges i e enzymes a e syn hesized in he ough endoplasma ic e iculum, p ocessed in he Golgi complex, packed in o sec e o y esicles and sec e ed by he gu endo helium ia exocy osis, apoc ine o mic oapoc ine p ocesses (Te a and Fe ei a, 2012) (Figu e 7). In insec s, mos enzyme elease is by exocy osis, and less o en by apoc ine sec e ion (Te a and Fe ei a, 1994), depending on he midgu egion, he enzyme and he species (G a e al., 1986; San os e al., 1986; Jo dão e al., 1996, 1999; C is o ole i e al., 2001; Fe ei a e al., 2002). Du ing exocy osis enzymes a e s o ed in esicles, which use wi h he plasma memb ane and elease hei con en wi hou any loss o cy oplasm (Figu e 7A), whe eas apoc ine sec e ion in ol es a loss o apical cy oplasm ollowing esicle elease, in which he enzymes a e s o ed (Figu e 7B). Mic oapoc ine sec e ion is a common ype o apoc ine sec e ion (De P ies e , 1971; Hein ich and Zebe, 1973; Nopani aya and Misch, 1974; Lehane, 1976; Humbe , 1979; San os e al., 1984; Te a e al., 1988), in which he loss o cy oplasm is minimal and small budding double memb ane esicles (Figu e 7C) o pinched-o sec e o y esicles a e eleased (Figu e 7D). The con en o hose esicles is eed by memb ane usion o solubilisa ion in he midgu lumen (Te a and Fe ei a, 2012). Figu e 7: Models o sec e o y p ocesses o insec diges i e enzymes: exocy ic sec e ion (A), apoc ine sec e ion (B), mic oapoc ine sec e ion wi h budding esicles (C) and wi h pinched-o esicles (D). BSV: budding sec e o y esicle, CE: cellula ex usion, DE: diges i e enzymes; GC: Golgi complex, M: mic o illi; N: nucleus, PSV: pinched-o sec e o y esicles, RER: ough endoplasma ic e iculum, SV: sec e o y esicle. (adap ed om Te a and Fe ei a, 2009) In oduc ion 9 1.4Regula ion mechanisms con olling diges i e enzyme sec e ion in insec s Acco ding o hei eeding beha iou insec s can be classi ied in wo majo g oups: con inuous and discon inuous eede s. Con inuous eede s ha e a con inuous s eam o ood passing h ough he in es ine, while discon inuous eede s (ca ni o es, haema ophagous) ha e pe iods whe e he gu is illed o emp y (Lehane e al., 1996). The e o e, discon inuous eede s need a egula ion o enzyme sec e ion. The midgu is he main si e o diges i e enzyme elease and me aboli e abso p ion in insec s (Dow, 1992; Chapman, 1998), whe eby he con ol o diges i e enzyme sec e ion depends on a ious mechanisms (e.g. ho monal, pa ac ine, p andial) (Lehane e al., 1995) and equi es sepa a e egula ion o enzyme syn hesis and enzyme sec e ion (Blakemo e e al., 1995). Food consump ion plays a undamen al ole in he sec e ion o diges i e enzymes, in ha no only ood in ake (Engelmann, 1969; Dadd, 1970; Chapman, 1998) bu also he composi e o he nu i ion egula es sec e ion mechanism (p andial elease mechanism) (Chapman, 1985; Te a, 1990; Lehane e al., 1996; Te a e al., 1996). The eby, small componen s o he die (di e en nu ien s) in e ac di ec ly wi h he sec e o y cells o he midgu and s imula e he sec e ion o speci ic diges i e enzymes (Lehane e al., 1995). Al hough some s udies al eady epo ed he in luence o ho mones on enzyme sec e ion in he midgu o insec s (Applebaum, 1985; Chapman, 1985), i is s ill con o e sial whe he he ho mone sys em has a di ec in luence on diges i e enzyme elease o a he changes in ho mone sys ems a e subsequen due o he ac o ea men (Lehane e al., 1996). The insec midgu epi helium con ains la ge numbe s o endoc ine cells (Mon uenge e al., 1989; Endo e al., 1990; Sehnal and Ži ňan, 1996), which likely play a ole in in es inal ac i i ies (Lehane e al., 1996). These cells ha e a hemolymph side and a gu lumen side. In his model, nu ien ecep o s on he lumen side can s imula e he elease o pa aneu oho mones in o he hemolymph, which bind o ecep o s and induce he elease o diges i e enzymes in o he gu lumen (Figu e 8). Se e al neu opep ides including FMRFamide- ela ed pep ides, p oc olin, insec kinins and alla o egula o y pep ides ha e al eady been iden i ied in he en e ic ne ous sys em and in he endoc ine cells o he gu (Reichwald e al., 1994; Yu e al., 1995; Sehnal and Ži ňan, 1996), and we e shown o a ec ood up ake, gu mo ili y (Wei e al., 2000; P edel e al., 2001; Aguila e al., 2004; Meye ing-Vos and Mülle , 2007a; Meye ing-Vos and Wood ing, 2008; Audsley and Wea e , 2009) and he elease o diges i e enzymes in pa icula (Fusé e al., 1999; Ha shini e al., 2002a,b; Aguila e al., 2003; Hill and O cha d, 2005; Sakai e al., 2006; Audsley and Wea e , 2009; Wood ing e al., 2009; Lwalaba e al., 2010a). In oduc ion 10 Figu e 8: Regula ion mechanism o diges i e enzyme elease: small diges i e p oduc s bind o ecep o s o endoc ine cells on he midgu side o ee pa aneu oho mones, which s imula e he enzyme elease o zymogene cells (pa ac ine mechanism). A1: abso p ion o soluble compounds, A2: abso p ion o small diges i e p oduc s ia endocy osis, AC: abso p i e cell, DE: diges i e enzymes (g een), DP: diges ion p oduc s, EC: endoc ine cell, GC: Golgi complex, M: mic o illi, N: nucleus, PNH: pa aneu oho mones, R: ecep o , RER: ough endoplasma ic e iculum, sDP: small diges i e p oduc s, SV: sec e o y esicles, ZC: zymogen cell. (modi ied om Te a and Fe ei a, 2009) 1.4.1 Alla os a ins Alla o egula ing pep ides a e di ided in wo g oups based on hei s imula o y (alla o opins) o inhibi o y (alla os a ins) e ec on ju enile ho mone biosyn hesis in he co po a alla a (CA) (Ho mann e al., 1999; Gäde, 2002). Alla os a ins belong o a well-documen ed g oup o neu oho mones, which has been iden i ied in a la ge numbe o insec species (S ay, 2000), and can be classi ied in h ee subg oups acco ding o hei sequence homology: alla os a in A (AST-A, FGLamides), alla os a in B (AST-B, W(X6)Wamides) and alla os a in C (AST-C, PISCF-OH) (S ay, 2000; Meye ing-Vos e al., 2001; Ho mann, 2003). The Alla os a in ype A pep ide is cha ac e ized by a common C- e minus sequence Ty /Phe- Xaa-Phe-Gly-Leu-Ile/Val-amid (S ay e al., 1991) and was i s iden i ied om Diplop e a punc a a (Woodhead e al., 1989; Donly e al., 1993) and o he cock oach species (Ding e al., 1995; Bellés e al., 1999, Bendena e al., 1999). Due o hei pleio opic unc ion, AST-A pep ides a e exp essed in di e en issues (S ay, 2000). In G. bimacula us he AST-A gene is In oduc ion 11 s ongly exp essed in he b ain, he suboesophageal ganglion and he caeca o he diges i e ac (Meye ing-Vos and Ho mann, 2003). The p oho mone p ecu so encodes o 14 pu a i e G yllus-AST-A pep ides which a e in e spaced by acidic space s (Meye ing-Vos e al., 2001) (Figu e 9A). An inhibi o y e ec o AST-A pep ides on JH biosyn hesis was demons a ed o cock oaches, e mi es, c icke s and some bee les (S ay and Tobe, 2007; Abdel-la ie and Ho mann, 2010). Bu AST-A pep ides ha e also myoinhibi ing e ec s on di e en pa s o he insec gu o on he o iduc (Gäde and Ho mann, 2005). Mo eo e , hey inhibi he p oduc ion and elease o i ellogenin om he a body o cock oaches (Ma ín e al., 1996, 1998) and a ec he sec e ion o diges i e enzymes (Fusé e al, 1999; Aguila e al., 2003; Sakai e al., 2006; Digali e al., 2010). 1.4.2 Sul akinins Sul akinins (SK) a e ano he amily o neu opep ides wi h myo opic unc ion, which we e p ima ily isola ed om he cock oach Leucophaea made ae (Nachman e al., 1986a). Insec SKs show s uc u al homology o he pep ides gas in and cholecys okinin, which a e in ol ed in he egula ion o ood up ake in e eb a es (Nachman e al., 1986b). SKs a e cha ac e ized by a highly conse ed C- e minal hexapep ide sequence DY(SO3H)GHMRF-NH2and a sulpha ed y osine esidue (Audsley and Wea e , 2009).SKs we e isola ed om se e al di e en insec species (Veens a, 1989; Schoo s e al., 1990; Nichols e al., 1988, 1992; Fonagy e al., 1992; Du e e al., 1995; Eas e al., 1997; Maes o e al., 2001; Meye ing-Vos and Mülle , 2007b) and we e shown o a ec ood up ake and enzyme elease in o he diges i e ac (Nachman e al., 1997; Wei e al., 2000; Maes o e al., 2001; Ha shini e al., 2002b; Schoo s and Nachman, 2006; Downe e al.,2007; Meye ing-Vos and Mülle , 2007a; Meye ing-Vos and Wood ing, 2008). In G. bimacula us he SK p oho mone p ecu so encodes wo SK pep ides (Figu e 9B), which bo h show a s ong exp ession only in he b ain (Meye ing-Vos and Mülle , 2007b). In oduc ion 12 Figu e 9: Nucleo ide sequence o he alla os a in (A) and sul akinin (B) p ecu so cDNA o G. bimacula us and he deduced amino acid sequences o he p ep oho mone polypep ides. Adap ed om (A) Meye ing-Vos e al. (2001), (B) Meye ing-Vos and Mülle (2007b). The cDNA sequence is numbe ed om he mos dis al nucleo ide iden i ied on he 5' end. The deduced p o ein sequence is in bold ace. Po en ial clea age si es a e boxed. Neu opep ide sequences a e unde lined ed, hose o acidic space s blue, p eceding glycine esidues ( equi ed o α-amida ion) black. * ep esen s he s op codon. In oduc ion 13 1.5 Enzyme inhibi o s 1.5.1 Plan p o ease inhibi o s O e ecen yea s he adap a ion mechanisms o insec s o hei nu i ion and abio ic en i onmen ha e become mo e impo an wi h iew o he inc easing numbe o pes insec s and hei con ol. The e o e, a lo o s udies ocused on he in es iga ion o insec diges ion and a p obable applica ion o na u al enzyme inhibi o s o pes con ol. Many plan s p oduce p o ease inhibi o s (PI) as a de ence mechanism agains eeding damage (Fan and Wu, 2005). PIs inhibi he p o eases p esen in he midgu lumen and c op o insec s (Johns on e al., 1993; Telang e al., 2005; Duncan e al., 2006; B ioschi e al., 2007), bu also a ec he sec e ion o p o eases by he epi helium (Lwalaba e al., 2010b; Weidlich e al., 2012). While PIs we e hough o ha e he po en ial o p o ec plan s agains he bi o ous insec s (B oadway and Du ey, 1986; B oadway e al., 1986; Hilde e al., 1987; Johnson e al., 1989; Oppe e al., 1993; O ozco-Ca denas e al., 1993; McManus e al., 1994), i was soon appa en ha insec s ha e e ol ed di e en s a egies o deal wi h PIs in he die : (a) enzyme hype p oduc ion (B oadway and Du ey, 1986; Johns on e al., 1993; B oadway, 1995, Hi ale e al., 2011), (b) up- and down- egula ion o p o eases (Jongsma e al., 1995; Clou ie e al., 2000; Zhu-Salzman e al., 2003; B ioschi e al., 2007; Dunse e al., 2010a, b), (c) inc easing elease o inhibi o -insensi i e enzyme iso o ms (Jongsma e al., 1995, 1996; Paulillo e al., 2000; B i o e al., 2001; Volpicella e al., 2003; B ioschi e al., 2007; Geo ge e al., 2008; Hi ale e al., 2011; de Oli ei a e al., 2013) o (d) sec e ion o PI-deg ading p o eases (Jongsma e al., 1996; Michaud, 1997; Gi a d e al., 1998). The Kuni z ype ypsin inhibi o om soybean (SBTI) is a small p o ein (~25 kDa) which in e ac s wi h ypsin-like p o eases by o ming an i e e sible complex wi h a e y low dissocia ion cons an and, he e o e, blocking he ac i e si e o he enzyme (Kuni z, 1948). Fu he mo e, SBTI is qui e esis an o highe empe a u es by changing con o ma ion (Kuni z, 1948). The inhibi o y e ec o SBTI agains midgu p o eases was demons a ed o di e en insec s in i o and in i o (Applebaum e al., 1963; Mille e al., 1974; Ch is elle e al., 1990; Johns on e al., 1993; Oppe e al., 2005; Lwalaba e al., 2010b; Weidlich e al., 2012). 1.5.2 Endogenous p o ease inhibi o s Endogenous se ine p o ease inhibi o s (EPI) a e widely ound in all issues o all animals. The se pins, a e y la ge class o p o eases, a e mos ly in acellula , and unde go a unique change in shape when hey inhibi a ge p o eases (Hun ing on e al., 2000). They egula e such p ocesses as coagula ion, in lamma ion, and immuni y. Howe e , some insec inhibi o s in ol ed wi h immune esponses (coagula ion, ac i a ion o phenoloxidases) belong o he classical Kuni z and Kazal ype inhibi o s (Kanos , 1999). They bind o he ac i e si e o he In oduc ion 14 p o eases and block access (Kuni z, 1948). P o ease inhibi o s o he Kazal ype a e ound in he sali a y gland and sali a o Nauphoe a cine ea, which a e pa icula ly e ec i e agains he bac e ial p o ease sub ilisin. This sugges s a de ensi e mechanism agains he masses o bac e ia ypically ound in he cock oach c op, some o which a e p obably pa hogenic (Ta anushenko e al., 2009). Inhibi o y pep ides in he midgu lumen a e less well s udied, hough ypsin and sub ilisin inhibi o s a e desc ibed om whole midgu p epa a ions o he cock oach N. cine ea (Elpidina e al., 2001) and in i e addi ional cock oach species (Vinoku o e al., 2007). Inhibi o s om he cock oach gu ac on endogenous diges i e p o einases and may ep esen a new mechanism o diges ion egula ion. In oduc ion 15 1.5 Resea ch gaps Al hough, a lo o s udies ocused on he diges ion in insec s, he e is li le knowledge on he ac o s con olling enzyme sec e ion (Lehane e al., 1996; Blakemo e e al., 1995; Wood ing e al., 2009; Lwalaba e al., 2010a). The e o e, he p esen disse a ion ocuses on he de e mina ion o he e ec o di e en endogenous (o ganismic) and exogenous (en i onmen al) ac o s on he egula ion o diges i e enzyme sec e ion in o he midgu o he wo-spo ed ield c icke G. bimacula us (Figu e 10). In pa icula , he ollowing expe imen s we e ca ied ou :  Sample ype: Enzyme ac i i y in di e en sample ypes (luminal con en , issue incuba ion medium, issue homogena e)  Age-dependen enzyme elease: Changes in enzyme elease o emale and male G. bimacula us om las la al ins a o adul s adium  Neu opep ides: The e ec o c icke alla os a in ype A and sul akinin on he elease o diges i e enzymes using in i o incuba ion and RNA in e e ence  Calcium ions: Dependency o enzyme elease on he p esence o absence o calcium ions in issue incuba ion medium  T ypsin zymogen: The p esence and ac i a ion o a pu a i e ypsin p ecu so , and he au olysis o G yllus- ypsin  Endogenous p o ease inhibi o s: The p esence o endogenous p o ease inhibi o s in he midgu o adul c icke s  Tempe a u e: The e ec o incuba ion and ea ing empe a u e on enzyme elease wi h espec o a pu a i e empe a u e acclima ion  Ligh -da k cycle: Food up ake and enzyme elease du ing pho o- and sco ophase wi hin 24 h in penul ima e ins a , las la al ins a and adul s adium  Food: The e ec o eeding and s a a ion, and he in luence o a ious nu ien s (in i o and in i o s udies) on enzyme sec e ion  Plan p o ease inhibi o s: Dose-dependen inhibi ion o ypsin sec e ion and ac i i y caused by eeding p o ease inhibi o (SBTI) en iched die s in adul G. bimacula us In oduc ion 16 Figu e 10: Expe imen al o e iew: de e mina ion o he e ec s o endogenous (o ganism) and exogenous (en i onmen ) ac o s con olling he egula ion o enzyme sec e ion in G. bimacula us. No e: no all expe imen s we e ca ied ou o all enzymes. Fo de ails, please check he co esponding a icles. empe a u e e ec o incuba ion empe a u e (in i o) e ec o ea ing empe a u e: acclima ion expe imen s omdi e en de elopmen al s ages ligh -da kcycle heenzymesec e ionand oodup ake o e 24h ood & nu ien s eeding& s a a ion cellulose en iched die s e ec o nu ien sin incuba ion medium SBTI en iched die s e eeding expe imen s In i o In i o age enzymeac i a ion (zymogen ac i a ion, calciumions) sex endogenous enzyme inhibi o s ENVIRONMENT ORGANISM de elopmen als age THE REGULATION OF ENZYME SECRETION neu opep ides Synopsis 23 23 2.2.2 Neu opep ides 2.2.2.1 Alla os a in ype A The alla os a in ype A pep ide and i s gene, which is s ongly exp essed in b ain, suboesophageal ganglion and caeca o G. bimacula us (Meye ing-Vos and Ho mann, 2003), we e analysed wi h ega ds o hei in luence on diges i e enzyme elease using in i o and in i o s udies. AST-5 (DRLYSFGK-NH2) (Bachem, Ge many) was used o in i o incuba ion o caecal issue ollowing enzyme ac i i y assays and quan i ica ion o soluble p o eins. RNA in e e ence was used o analyse he gene unc ion o alla os a in by gene silencing in i o, a me hod al eady employed o alla os a in pep ides in his species by Meye ing-Vos e al. (2006). In i o s udies The in luence o AST-5 in i o on enzyme elease o amylase, ypsin, and aminopep idase has al eady been epo ed by Wood ing e al. (2009), whe e amylase and ypsin elease inc eased du ing incuba ion wi h 10-8 o 10-5 M AST-5. The da a o he cu en s udy did no ully con i m hese esul s. In his s udy, he in i o e ec o AST-5 on enzyme elease om caeca was es ed by addi ion o app op ia e dilu ion o 10-3 M s ock solu ion o AST-5 (in 20 % ace oni ile) in LGR. The incuba ion medium was es ed o enzyme ac i i y and concen a ion o soluble p o eins. The e was a end o dec easing elease o soluble p o eins in esponse o highe concen a ion o AST-5 (Figu e 13). Figu e 13: In i o e ec o AST-5 on he amoun o p o ein [µg BSA/mg issue] eleased om (A) emale and (B) male caecal issue o incuba ion medium. Mean + SEM. n = 10. S a is ics: K uskal-Wallis es . P > 0.05. Al hough he e we e no s a is ically signi ican di e ences, he sec e ion o amylase and cellulase (Figu e 14A), aminopep idase and ypsin (Figu e 14B), and lipase (Figu e 14C) in bo h male and emale G. bimacula us showed a con inues end o inc eased enzyme elease in esponse o highe concen a ions o AST-5 (10-6 o 10-5 M) in incuba ion medium. AST-5 concen a ion [M] µg BSA/mg issue 0 2 4 6 8 10 12 14 AST-5 concen a ion [M] µg BSA/mg issue 0 2 4 6 8 10 12 14 A B 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 Synopsis 24 24 A B Clipid diges ion p o ein diges ion ca bohyd a e diges ion amylase AST-5 concen a ion [M] µmol mal ose/30min/µg BSA 0.0 0.1 0.2 0.3 cellulase AST-5 concen a ion [M] RFU/30min/µg BSA 0 1 2 3 4 male emale male emale 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 aminopep idase AST-5 concen a ion [M] nmol pNA/min/µg BSA 0 1 2 3 ypsin AST-5 concen a ion [M] nmol pNA/min/µg BSA 0.0 0.1 0.2 0.3 male emale male emale 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 lipase AST-5 concen a ion [M] nmol pNP/30min/µg BSA 0.00 0.25 0.50 0.75 1.00 male emale 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 Figu e 14: In i o e ec s o AST-5 on (A) ca bohyd ases, (B) p o eases and (C) lipase sec e ion om caecal issue o 2-day-old adul G. bimacula us males (whi e) and emales (g ey). Mean + SEM. n = 10. S a is ics: K uskal-Wallis es . P > 0.05; no signi ican di e ences. Synopsis 25 25 In i o s udies - RNA in e e ence In i o e ec s a e injec ion o dsRNA a ge ed agains AST-A in o newly moul ed c icke s (las ins a la ae and adul s), ollowing he deg ada ion o he speci ic mRNAs, we e s udied by measu ing he body weigh gain, and he ac i i y o amylase, aminopep idase, ypsin and lipase in caecal lumen con en , issue homogena e and issue incuba ion medium wo days a e dsRNA injec ions. The AST-A dsRNA was gene a ed as p e iously desc ibed by Meye ing-Vos e al. (2006). 2 o 6 µg AST-A dsRNA in 10 µl G yllus Ringe we e injec ed once wi h a 100 µl Hamil on sy inge be ween he hi d and he ou h abdominal segmen . Con ol c icke s we e injec ed wi h G yllus Ringe only. The body weigh gain o c icke s injec ed wi h AST-A dsRNA did no di e om hose injec ed wi h Ringe solu ion (Figu e 16 & Figu e 15).This co esponds o he esul s o Meye ing-Vos e al. (2006), whe e di e ences in body weigh gain ollowing AST-A dsRNA injec ion we e only obse ed in olde adul s. Figu e 15: Weigh gain o 2-day-old G. bimacula us las ins a la ae and adul s, injec ed wi h ei he 2 µg AST-A dsRNA (in 10µl Ringe ) o Ringe only a he day o he p eceding moul . The body weigh on he day o injec ion was se 100%. Mean ± SEM, n = 16-20. S a is ics: s uden s - es , ns = no signi ican . Figu e 16: Weigh gain o 2-day-old adul G. bimacula us emales, injec ed wi h AST-A dsRNA (0-6 µg in 10 µl Ringe ) a he day o imaginal moul . The body weigh on he day o injec ion was se o 100%. Mean ± SEM., n = 9–10. S a is ics: ANOVA, P > 0.05 = no signi ican di e ences. las ins a adul las ins a adul body weigh gain [%] 0 20 40 60 Ringe dsRNA emale male ns ns ns ns concen a ion dsRNA [µg] 0 µg 2 µg 4 µg 6 µg body weigh gain [%] 0 10 20 30 40 50 60 Synopsis 26 26 Newly moul ed male and emale G. bimacula us adul s and las ins a la ae we e injec ed wi h ei he 2 µg AST-A dsRNA o Ringe . Two days la e , he enzyme ac i i y o ypsin, aminopep idase, lipase, and amylase was de e mined in he luminal con en (Figu e 17), issue homogena e (Figu e 18), and issue incuba ion medium (Figu e 19). The e was no clea end in enzyme ac i i y o he luminal con en o ei he las ins a la ae o adul c icke s ollowing AST-A gene knockdown (Figu e 17). The enzyme ac i i y in he caecal lumen is an es ima e o he amoun o enzymes ha ha e accumula ed a a gi en age and ime. The e o e, he expe imen al ime o wo days may ha e been oo sho o de ec signi ican changes in enzyme ac i i y in he lumen con en a e injec ion o 2 µg AST-A dsRNA. Figu e 17: Ac i i y o (A) ypsin, (B) aminopep idase, (C) lipase and (D) amylase in he caecal lumen con en o 2-day-old G. bimacula us adul s and las ins a la ae injec ed wi h ei he 2 µg AST-A dsRNA (in 10 µl Ringe ) (g ey) o Ringe only (whi e) a p eceding moul . Mean + SEM. n = 9-10. S a is ics: Mann-Whi ney U- es o s uden ’s - es . ns = no signi ican , * = P < 0.05, # = 0.05 < P < 0.1. lipase las ins a adul las ins a adul lipase ac i i y [nmol pNP/30min/10µl lumen con en ] 0 200 400 600 800 1000 1200 1400 amylase las ins a adul las ins a adul amylase ac i i y [µmol Ma/30min/10µl lumen con en ] 0 5 10 15 20 25 C D aminopep idase las ins a adul las ins a adul aminopep idase ac i i y [nmol pNA/min/10µl lumen con en ] 0 50 100 150 200 250 ypsin las ins a adul las ins a adul ypsin ac i i y [nmol pNA/min/10µl lumen con en ] 0 50 100 150 200 emale male emale male A B emale male emale male ns ns ns * ns ns # ns ns ns #ns ns ns ns * Synopsis 27 27 Howe e , he e was a signi ican educ ion o p o ease (Figu e 18A,B), lipase (Figu e 18C) and amylase ac i i y (Figu e 18D) in issue homogena e o male and emale c icke s (wi h ew excep ions) ollowing AST-A gene knockdown. The enzyme ac i i y in he issue homogena e ep esen s he amoun o enzymes s o ed in issue cells and, he e o e, ep esen s an indica o o enzyme syn hesis a e. Thus, gene silencing o alla os a in ype A educed he syn hesis o diges i e enzymes in he midgu o G. bimacula us. Figu e 18: Ac i i y o (A) ypsin, (B) aminopep idase, (C) lipase and (D) amylase in he caecal issue homogena e o 2-day-old G. bimacula us adul s and las ins a la ae injec ed wi h ei he 2 µg AST-A dsRNA (in 10 µl Ringe ) (g ey) o Ringe only (whi e) a p eceding moul . Mean + SEM. n = 9-10. S a is ics: Mann-Whi ney U- es o s uden ’s - es . ns = no signi ican , * = P < 0.05, ** = P< 0.01, *** = P < 0.001. ns ns aminopep idase ypsin A B lipase amylase C D emale male emale male emale male las ins a adul las ins a adul amylase ac i i y [µmol Ma/30min/mg issue] 0.0 0.1 0.2 0.3 0.4 0.5 las ins a adul las ins a adul aminopep idase ac i i y [nmol pNA/min/mg issue] 0 5 10 15 20 25 30 emale male las ins a adul las ins a adul lipase ac i i y [nmol pNP/30min/mg issue] 0 5 10 15 20 25 30 las ins a adul las ins a adul ypsin ac i i y [nmol pNA/min/mg issue] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ns ns ns ns ** *** ** * * * *** ** *** *** Synopsis 28 28 Al hough, gene silencing o alla os a in A esul ed in a educed syn hesis a e o diges i e enzymes in he caecal issue, he e was a end o inc easing p o ease (Figu e 19A,B) and lipase (Figu e 19C) elease om he caecal issue in o he incuba ion medium, especially in emale las ins a la ae. Figu e 19: Ac i i y o (A) ypsin, (B) aminopep idase, (C) lipase and (D) amylase in he caecal issue incuba ion medium o 2-day-old G. bimacula us adul s and las ins a la ae injec ed wi h ei he 2 µg AST-A dsRNA (in 10 µl Ringe ) (g ey) o Ringe only (whi e) a p eceding moul . Mean + SEM. n = 9-10. S a is ics: Mann-Whi ney U- es o s uden ’s - es . ns = no signi ican , * = P < 0.05, ** = P< 0.01, *** = P < 0.001. In gene al, la al and adul emales o G. bimacula us seem o be mo e sensi i e o physiological e ec s on diges i e enzymes caused by injec ions o AST-A dsRNA, han males. In he ollowing expe imen he e ec s o di e en concen a ions o AST-A dsRNA injec ions on he diges i e enzyme ac i i ies o adul emales we e analysed. In addi ion he concen a ion o soluble p o eins was de e mined o each sample. aminopep idase ypsin A B lipase amylase C D emale male las ins a adul las ins a adul amylase ac i i y [µmol Ma/30min/mg issue] 0.0 0.1 0.2 0.3 0.4 0.5 emale male las ins a adul las ins a adul lipase ac i i y [nmol pNP/30min/mg issue] 0 2 4 6 8 las ins a adul las ins a adul aminopep idase ac i i y [nmol pNA/min/mg issue] 0.0 0.5 1.0 1.5 2.0 emale male las ins a adul las ins a adul ypsin ac i i y [nmol pNA/min/mg issue] 0.0 0.5 1.0 1.5 2.0 emale male ns ns ns ns ns ns ns ns nsns ns ns ** ** * *** Synopsis 29 29 The amoun o o al soluble p o ein in samples o he luminal con en (Figu e 20C) dec eased a highe amoun s o AST-A dsRNA, bu no in samples o issue incuba ion medium o issue homogena es (Figu e 20A,B). Figu e 20: The e ec o a ious concen a ions o AST-A dsRNA on he p o ein concen a ion in (A) issue incuba e, (B) issue homogena e, and (C) luminal con en o 2-day-old adul G. bimacula us emales. The AST-A dsRNA (0-6 µg in 10 µl Ringe ) was injec ed in o c icke s a day o imaginal moul . Mean ± SEM. n = 9-10. S a is ics: ANOVA and pos hoc Bon e oni - es . * indica es signi ican di e ences o con ol (0 µg dsRNA). The ac i i ies o amylase, aminopep idase, ypsin, and lipase we e analysed in luminal con en , issue homogena e and issue incuba ion medium (sec e ion) (Figu e 21). Injec ion o AST-A dsRNA did no show a dose-dependen e ec on enzyme ac i i y nei he in issue homogena e no in issue incuba ion medium, o all es ed enzymes. Howe e , highe concen a ion o 6 µg AST-A dsRNA esul ed in signi ican highe amylase ac i i y in he lumen con en . Simila ends o inc easing enzyme ac i i y in he lumen con en we e obse ed o aminopep idase and ypsin, espec i ely (Figu e 21B,C). C concen a ion dsRNA 0µg 2µg 4µg 6µg µg BSA/10µl lumen con en 0 100 200 300 400 * * A concen a ion dsRNA 0µg 2µg 4µg 6µg µg BSA/mg issue 0 2 4 6 8 10 12 B concen a ion dsRNA 0µg 2µg 4µg 6µg µg BSA/mg issue 0 2 4 6 8 10 12 Synopsis 30 30 Figu e 21: The e ec o a ious concen a ions o AST-A dsRNA on (A) amylase, (B) aminopep idase, (C) ypsin, and (D) lipase ac i i y in issue incuba ion medium, issue homogena e and lumen con en o 2-day-old adul G. bimacula us emales. The AST-A dsRNA (0-6 µg in 10 µl Ringe ) was injec ed a day o imaginal moul . Mean + SEM. n = 9-10. S a is ics: K uskal-Wallis es + pos hoc Dunn’s me hod. * indica es signi ican di e ences o con ol (0 µg dsRNA). issue homogena e concen a ion dsRNA 0µg 2µg 4µg 6µg µmol mal ose/30min/µg BSA 0.00 0.02 0.04 0.06 0.08 issue homogena e concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.00 0.05 0.10 0.15 0.20 0.25 0.30 issue homogena e concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.00 0.05 0.10 0.15 0.20 issue homogena e concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNP/30min/µg BSA 0.0 0.2 0.4 0.6 issue incuba ion medium concen a ion dsRNA 0µg 2µg 4µg 6µg µmol mal ose/30min/µg BSA 0.00 0.05 0.10 0.15 0.20 0.25 0.30 issue incuba ion medium concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNP/30min/µg BSA 0.0 0.2 0.4 0.6 0.8 1.0 issue incuba ion medium concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 issue incuba ion medium concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.00 0.05 0.10 0.15 0.20 0.25 0.30 lumen con en concen a ion dsRNA 0µg 2µg 4µg 6µg µmol mal ose/30min/µg BSA 0.00 0.05 0.10 0.15 0.20 lumen con en concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.0 0.5 1.0 1.5 2.0 2.5 lumen con en concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 lumen con en concen a ion dsRNA 0µg 2µg 4µg 6µg nmol pNP/30min/µg BSA 0 1 2 3 4 amylase aminopep idase lipase ypsin A B C D * Synopsis 31 31 2.2.2.2 Sul akinins P e ious s udies using RNAi sugges ed ha SK pep ides a ec sa ie y in G. bimacula us by educing ood up ake (Meye ing-Vos and Mülle , 2007a), as was also epo ed o cock oaches and locus s (Wei e al., 2000; Maes o e al., 2001). A s imula ing e ec o sul akinins on diges i e enzyme elease has al eady been demons a ed o bee les and mo hs (Nachman e al., 1997; Ha shini e al., 2002b), bu p elimina y RNAi s udies in G. bimacula us showed no e ec (Meye ing-Vos and Mülle , 2007a). The e o e, RNAi expe imen s ega ding gene silencing o sul akinin we e epea edly done, and subsequen physiological e ec s we e analysed in mo e de ail. The SK dsRNA was gene a ed as p e iously desc ibed by Meye ing- Vos and Mülle (2007a). Male and emale c icke s we e injec ed wi h ei he 2 µg dsRNA in 10 µl Ringe solu ion o 10 µl Ringe solu ion (con ol) wi h a 100 µl Hamil on sy inge be ween he hi d and he ou h abdominal segmen on he day o imaginal moul . C icke s we e dissec ed wo days a e injec ion and all samples we e analysed o soluble p o ein concen a ion and enzyme ac i i ies o amylase, cellulase, aminopep idase, ypsin, and lipase. issue homogena e Ringe dsRNA µg BSA/mg issue 0 10 20 30 40 issue incuba ion medium Ringe dsRNA µg BSA/mg issue 0 2 4 6 8 10 12 14 lumen con en Ringe dsRNA µg BSA/mg issue 0 100 200 300 400 lumen con en Ringe dsRNA µg BSA/10µl lumen con en 0 100 200 300 400 issue incuba ion medium Ringe dsRNA µg BSA/mg issue 0 2 4 6 8 10 12 14 issue homogena e Ringe dsRNA µg BSA/mg issue 0 10 20 30 40 A B #* ns ns ns ns Figu e 22: P o ein concen a ion in samples o issue incuba ion medium, issue homogena e and lumen con en om (A) emale and (B) male 2-day-old adul G. bimacula us, injec ed wi h ei he 10 µl Ringe (con ol) o 2 µg SK dsRNA in 10 µl Ringe a he day o imaginal moul . Mean ± SEM. n = 9-10. S a is ics: s uden ’s - es o Mann-Whi ney U- es . * = P < 0.05, # = 0.05 < P< 0.1, ns = P> 0.1. Synopsis 32 32 Injec ion o SK dsRNA induced a signi ican dec ease in he p o ein concen a ion o lumen samples o emales (Figu e 22A). A simila end was obse ed o he emale issue incuba e. SK dsRNA injec ions had no e ec on he p o ein concen a ions in samples o male c icke s (Figu e 22B). Gene silencing o sul akinin esul ed in highe amylase and cellulase elease in emale c icke s (Figu e 23A), bu no in males. The e o e, SK pep ides seem o ha e an inhibi o y e ec on ca bohyd a e diges ion, a leas in emale G. bimacula us. amylase emale male µmol mal ose/30min/µg BSA 0.0 0.1 0.2 0.3 Ringe dsRNA A cellulase emale male RFU/30min/µg BSA 0 20 40 80 120 BC ns ns ** # ns aminopep idase emale male nmol pNA/min/µg BSA 0 1 2 3 4 5 ypsin emale male nmol pNA/min/µg BSA 0.0 0.1 0.2 0.3 0.4 0.5 0.6 lipase emale male nmol pNP/30min/µg BSA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 # # ns ns ca bohyd a e diges ion p o ein diges ion lipid diges ion Figu e 23: E ec o Ringe and SK dsRNA injec ion on enzyme sec e ion o (A) ca bohyd ases, (B) p o eases and (C) lipase o 2-day-old adul emale and male G. bimacula us. 2 µg SK dsRNA in 10 µl Ringe o Ringe alone (con ol) was injec ed o c icke s a he day o imaginal moul . Mean ± SEM. n = 9-10. S a is ics: - es o Mann-Whi ney U- es . * = P < 0.05, # = 0.05 < P <0.1, ns = no signi ican . Fu he mo e, he e was a end o dec easing p o ease (Figu e 23B) and lipase (Figu e 23C) elease in male c icke s, which may indica e a s imula o y e ec o SK pep ides on p o ein and lipid diges ion. Injec ions o SK dsRNA did no a ec enzyme ac i i ies in lumen con en and issue homogena e (no shown). Synopsis 39 39 Figu e 27: Food up ake and diges i e enzyme sec e ion in he cou se o a day in 2-day-old penul ima e la ae, las ins a la ae and adul G. bimacula us emales. Sco ophase was om 22:00-6:00 CEST (g ey). Mean ± SEM. n= 9-10. 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 amylase ac i i y [µmol Ma/30min/mg caeca] 0.5 1.0 1.5 2.0 2.5 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 cellulase ac i i y [RFU/30min/mg caeca] 0 50 100 150 200 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 aminopep idase ac i i y [nmol pNA/min/mg caeca] 5 10 15 20 25 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 ypsin ac i i y [nmol pNA/min/mg caeca] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ime o day 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 lipase ac i i y [nmol pNP/30min/mg caeca] 0 2 4 6 8 10 12 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 c op weigh [mg] 0 50 100 150 penul ima e ins a las ins a adul ca bohyd ases p o eases lipase ood up ake Synopsis 40 40 2.3.3 Food and nu ien s Feeding is he mos impo an exogenous ac o a ec ing enzyme sec e ion, which co ela es wi h bo h he quali y and he quan i y o ood. The impo ance o eeding on he sec e ion o diges i e enzymes in G. bimacula us was analysed in de ail in publica ions 1 - 4. The e ec o ood a ailabili y on enzyme sec e ion was de e mined in ei he ' eeding and s a a ion' o ' e eeding' expe imen s, while he impo ance o ood quali y was in es iga ed by eeding di e en die s, and by in i o incuba ion o caecal issue in LGR in he p esence o a ious nu ien s. 2.3.3.1 Feeding and s a a ion In o de o es he e ec o eeding and s a a ion on diges i e enzyme elease, c icke s we e placed indi idually in o boxes sho ly a e imaginal moul o p e en cannibalism. They we e p o ided wi h ei he a esh cube o s anda d aga -die o no ood a all. Two days la e he ac i i ies o amylase, cellulase, ypsin and lipase was de e mined in samples o issue incuba ion medium, issue homogena e and luminal con en . Fo he e eeding expe imen newly moul ed c icke s we e isola ed and no ed o 5 days. A e wa ds, hey we e p o ided wi h he aga -s anda d die and he elease o amylase and chi inase was de e mined e e y hou . The enzyme elease and enzyme ac i i y in midgu lumen o amylase (Fig. 4, publica ion 2), cellulase (Fig. 3, publica ion 2), ypsin (Fig. 6, publica ion 1), and lipase (Fig. 2, publica ion 3), s ongly inc eased in he p esence o ood compa ed o s a a ion. Enzyme ac i i ies in issue homogena e we e also signi ican ly highe in ed c icke s compa ed o s a ed ones. In e eeding expe imen s, amylase showed no esponse o he ood up ake wi hin 5 h (Fig. 5A, publica ion 3). Howe e , 24 h la e he e was a signi ican inc ease o sec e ion indica ing a s ong dec ease o amylase syn hesis in he caecal issue du ing s a a ion (Fig. 5B, publica ion 2). A leas 6 h a e equi ed by caecal issue o espond o he p esence o ood. In con as , chi inase sec e ion was sligh ly inc eased sho ly a e ood up ake (~ 10% in 2 h), bu gene ally emained a low le el (Fig. 6, publica ion 2). In conclusion, eeding has a signi ican posi i e in luence on diges i e enzyme ac i i y and s imula es no only he sec e ion o diges i e enzymes, bu also he syn hesis a e in he caecal issue. Synopsis 41 41 2.3.3.2 Nu ien s The in luence o di e en nu ien s on diges i e enzyme elease has al eady been epo ed o ypsin, aminopep idase and amylase (Wood ing e al., 2009; Digali e al., 2010). The e o e, he e ec s o nu ien s we e analysed only o lipase (publica ion 3) and cellulase (publica ion 2). mal ose concen a ion [mg/ml] 0 1 2 4 µg BSA/mg issue 0 2 4 6 8glucose concen a ion [mg/ml] 0 1 2 4 µg BSA/mg issue 0 2 4 6 8 cellubiose concen a ion [mg/ml] 0 1 2 4 µg BSA/mg issue 0 2 4 6 8oleic acid concen a ion [mM] 0.00 0.05 0.10 µg BSA/mg issue 0 2 4 6 8 A C B D ** * Figu e 28: In i o e ec s o nu ien s on soluble p o ein concen a ion in he incuba ion medium o caecal issue om 2-day-old adul emale G. bimacula us. Caecal issues we e incuba ed o 30 min a 37°C in LGR con aining (A) mal ose, (B) glucose, (C) cellubiose and (D) oleic acid. Mean ± SEM. n = 10. S a is ics: K uskal-Wallis es and pos hoc Dunn’s es wi h mul iple compa isons e sus con ol g oup (concen a ion = 0). * indica es signi ican di e ences o con ol g oup. Addi ions o highe concen a ions o cellubiose (2-4 mg/ml) and oleic acid (0.1 mM) o issue incuba ion medium esul ed in inc eased p o ein concen a ion and, he e o e, in highe enzyme elease om he caecal issue (Figu e 28C,D). The e was no signi ican e ec o mal ose o glucose on p o ein concen a ions (Figu e 28A,B). Synopsis 42 42 In i o incuba ion o caecal issue wi h oleic acid en iched LGR led o a signi ican highe lipase elease om caecal endo helium (Fig 3, publica ion 3). Glucose and mal ose had no e ec on lipase ac i i y (da a no shown). In con as o he s imula ing e ec o cellubiose on p o ein elease om caecal issue (Figu e 28C), cellulase ac i i y was much less in issue incuba ion medium wi h inc eased concen a ion o cellubiose (Fig. 8A, publica ion 2). In i o s udies eeding cellulose-en iched die s (40, 70 o 100%) also esul ed in dec eased cellulase ac i i y in luminal con en and issue incuba ion medium (Fig. 7, publica ion 2), and in educed body weigh (Fig. 9A, publica ion 2). Howe e , he ood up ake o he animals inc eased ollowing eeding o 40-70% cellulose-en iched die s, which may indica e an a emp o compensa e less ene gy up ake (Fig. 9B, publica ion 2). 2.3.3.3 Plan p o ease inhibi o s C icke s a e omni o ous and may encoun e plan ma e ial con aining p o einase inhibi o s, such as SBTI. The e o e, he adap a ion o G. bimacula us o SBTI en iched die was s udied in publica ion 1 in de ail. SBTI in he die educed ypsin ac i i y in he lumen con en and issue incuba ion medium in a dose-dependen manne (Tables 1 and 2, publica ion 1). To in es iga e a pu a i e adap a ion o ypsin sec e ion o SBTI, newly moul ed emale las ins a la ae we e ed 0.1% and 0.4% SBTI en iched die s un il day 2 a e imaginal moul . G. bimacula us seems o deal wi h lowe concen a ions o SBTI (0.1-0.2%) by hype p oduc ion o ypsin wi hin he i s 72 h, bu a e unable o adap o highe concen a ions (0.4%). Fu he mo e, la ae ed 0.4% SBTI en iched die showed a educed g ow h compa ed o c icke s ed die wi hou SBTI (Fig. 9A, publica ion 1). Because plan s a e no he sole ood sou ce, G. bimacula us is no unde selec i e p essu e o e ol e a speci ic adap a ion mechanism o p o ease inhibi o s. Summa y 43 43 Summa y Insec s a e he mos abundan animal species on ea h wi h a huge economical and ecological impac . In spi e o in ensi e esea ch in he ield o in eg a ed pes managemen he e a e s ill a lo o ques ions conce ning he adap a ion mechanism o insec s o hei en i onmen . As he diges i e ac displays a pu a i e a ge o e ec i e pes managemen , his s udy wo ked on he e ec s o endogenous and en i onmen al ac o s on diges i e enzyme elease in he omni o ous c icke , G yllus bimacula us. The age-dependen enzyme elease o ca bohyd ases, p o eases and lipase co ela es wi h he daily eeding a e o he c icke s and peaked be ween days 2 o 4 in las ins a la ae as well as in adul c icke s. In con as , he sec e ion o chi inase was a ec ed by he moul ing cycle o he insec s eaching maximum ac i i y a he day o moul . The e o e, chi inase plays only a mino ole in ood diges ion. The cellulase ac i i y in he midgu o G. bimacula us esul ed om an endogenous cellulase and was no caused by bac e ia o euka yo ic endosymbion s in he diges i e ac . The endop o ease ypsin was s o ed in he caecal issue as an inac i e p ecu so , and is sec e ed o he lumen by exocy osis. Following ac i a ion G yllus- ypsin (~24 kDa) is p o ec ed om p o eoly ic deg ada ion, bu he e is no endogenous p o ease inhibi o in he midgu . Gene knockdown by RNA in e e ence was used o analyse he endogenous egula ion o diges i e enzyme elease by he neu opep ides alla os a in A and sul akinin, which had al eady been shown o a ec eeding in G. bimacula us. Func ional analysis o he AST-A gene was in es iga ed o las ins a la ae and adul c icke s, whe eby emale c icke s seemed o be mo e sensi i e o his me hod. The gene supp ession o AST-A esul ed in a dec eased syn hesis o amylase, ypsin, aminopep idase and lipase in he caecal issue, bu enzyme elease a ied be ween sexes and de elopmen al s ages. The knockdown o SK exp ession led o an inc ease o amylase and cellulase sec e ion in emale c icke s, and o a educ ion o p o ease and lipase elease in males. As ood plays a undamen al ole in diges i e enzyme elease, bo h quali y and quan i y o nu i ion a e ample ac o s. The e was always a highe diges i e enzyme ac i i y in ed c icke s compa ed o s a ed ones. Fu he mo e, s a a ion esul ed in a dec ease o enzyme syn hesis in he caecal issue. In gene al, nu ien s in he incuba ion medium led o a s imula ion o diges i e enzyme sec e ion, bu in he case o cellulase he p esence o bo h cellubiose in he incuba ion medium and cellulose in he die caused a s ong decline in cellulase elease. Addi ion o he plan p o ease inhibi o SBTI o he die caused a dose-dependen inhibi ion o p o ease ac i i y in he caeca, whe eby mino concen a ions o SBTI we e compensa ed by enzyme hype p oduc ion. Summa y 44 44 In addi ion o he ood up ake, he daily ligh -da k cycle seems o a ec diges i e enzyme elease. C icke s s a ed o eed a he beginning o he sco ophase, which led o an inc ease o p o ease and lipase sec e ion in la ae and adul s. The sec e ion o ca bohyd ases was highes du ing he pho ophase. This means ha enzyme elease is no solely a ec ed by he ime o ood up ake. Tempe a u e is one o he mos impo an en i onmen al ac o s, bu seems o play only a mino ole in he elease o diges i e enzymes. All es ed enzymes showed a b oad op imal empe a u e ange (30°C - 40°C), bu he e was no di e ence in he elease o amylase o lipase a e issue incuba ion a 25°C o 35°C. In con as , ypsin and aminopep idase showed a highe sec e ion a e incuba ion a 35°C compa ed o 25°C. Fu he mo e, insec ea ing a 22°C and 32°C du ing a ious de elopmen al s ages esul ed in a posi i e acclima ion o ypsin sec e ion o ea ing empe a u e. Zusammen assung 45 45 Zusammen assung Insek en s ellen mi Abs and die g öß e und iel äl igs e Tie g uppe au E den da und üben einen g oßen ökologischen wie auch ökonomischen Ein luss aus. T o z in ensi e Fo schung im Be eich de in eg ie en Schädlingsbekämp ung sind bislang noch iele F agen übe die Adap ionsmechanismen on Insek en gegenübe ih e Umwel ungeklä . Da de Ve dauungs ak on Insek en ein po en ielles Ang i sziel ü e ek i e Schädlingsbekämp ung da s ell , wu de inne halb diese S udie die F eise zung on Ve dauungsenzymen bei de omni o en G ille, G yllus bimacula us, in Abhängigkei on endogenen und Umwel ak o en (Tempe a u , Lich -Dunkel Rhy hmus, Fu e ) analysie . Die F eise zung on Ca bohyd asen, P o easen und Lipasen in den Caeca on adul en und la alen G. bimacula us ko elie s a k mi de äglichen Fu e au nahme. Dabei wu de eine maximale Enzymsek e ion zwischen Tag 2 und 4 des le z en La ens adiums bzw. des Adul s adiums ge unden. Die F eise zung on Chi inase wi d hingegen maßgeblich om Zei punk de Häu ung beein luss , so dass Chi inase bei de Nah ungs e we ung ehe eine un e geo dne e Rolle spiel . Da übe hinaus wi d e mu e , dass die gemessene Cellulaseak i i ä in den Caeca au eine endogene Cellulase zu ückzu üh en is , da au g und de P obenau a bei ung mögliche euka yo ischen Endosymbion en ode Bak e ien aus dem Da m en e n wu den. Nähe e Un e suchungen an de P o ease T ypsin zeig en, dass das Enzym in Fo m eine Vo s u e (Zymogen) im Da mgewebe gespeiche wi d. Die F eise zung e olg übe Exocy ose aus den zymogenen Zellen des Mi elda mes in das Da mlumen. Nach Ak i ie ung de Vo s u e wies das G yllus-T ypsin ein Molekula gewich on ~24 kDa au und wa gegen p o eoly ischen Abbau im Lumen geschü z . Eine Regula ion de P o easeak i i ä im Mi elda m du ch endogene P o ease-Inhibi o en konn e nich ge unden we den. Mi hil e de RNA-In e e enz Me hode (Gen-Knockdown) wu de die endogene S eue ung de Enzymsek e ion im Mi elda m du ch die Neu opep ide Alla os a in A und Sul akinin nähe un e such . Beide Pep ide beein lussen nachweislich die Fu e au nahme de Tie e. Die Funk ionsanalyse des Alla os a in A Gens au physiologische Ebene wu de an La en und adul en G illen du chge üh , wobei Weibchen au die Me hode sensible zu eagie en scheinen. Gensupp ession on AST-A üh e meis zu eine eduzie en Syn hese a e on Amylase, T ypsin, Aminopep idase und Lipase in den Caeca-Geweben. Dabei schwank e die Höhe de F eise zung zwischen den un e such en S adien und Geschlech e n. De Knockdown de Sul akininexp ession üh e hingegen zu eine deu lichen E höhung de Amylase- und Cellulasesek e ion bei Weibchen, wäh end bei Männchen ein T end zu eduzie e P o ease- und Lipase eise zung e sich lich wa . Zusammen assung 46 46 Sowohl die Quan i ä als auch die Quali ä de Nah ung haben einen maßgeblichen Ein luss au die F eise zung alle un e such en Enzyme. Ge ü e e Tie e wiesen s e s eine höhe e Ak i i ä und eine s ä ke e F eise zung de Ve dauungsenzyme au als gehunge e G illen. Länge e Hunge phasen üh en zu eine Reduk ion de Enzymak i i ä im Da m und zu eine deu lich e minde en Syn hese a e in den Caeca-Geweben. Obwohl die Anwesenhei on Näh s o en im Inkuba ionsmedium ehe s imulie end au die F eise zung de Enzyme wi k e, konn e bei Cellulase eine s a ke Reduk ion de Sek e ion bei Anwesenhei on Cellubiose im Inkuba ionsmedium ode Cellulose in de Nah ung es ges ell we den. Zusa z des p lanzlichen P o ease-Inhibi o s SBTI zu Nah ung üh e zu eine konzen a ionsabhängigen Reduk ion de Ak i i ä und F eise zung on T ypsin in den Caeca, wobei die G illen ge inge e Konzen a ionen an SBTI du ch Enzym-Hype p oduk ion kompensie en konn en. Neben de Fu e au nahme wu de die F eise zung de Ve dauungsenzyme du ch den Tag- Nach -Rhy hmus de Tie e beein luss . Das Einse zen de Fu e au nahme zu Beginn de Dunkelphase e klä die e höh e P o ease- und Lipase eise zung, jedoch konn e in adul en wie la alen G. bimacula us ein Ans ieg de F eise zung on Ca bohyd asen wäh end de Pho ophase e zeichne we den. Die Regula ion de Enzym eise zung wi d olglich nich allein du ch den Zei punk de Fu e au nahme bes imm . Tempe a u als Umwel ak o spiel bei de F eise zung on Ve dauungsenzymen in G. bimacula us eine ehe un e geo dne e Rolle. Obwohl alle un e such en Enzyme Tempe a u op ima im Be eich on 30°C - 40°C au wiesen, zeig en Lipase und Amylase keinen Un e schied in de F eise zung nach Gewebeinkuba ionen bei 25°C ode 35°C. Die Sek e ion on P o easen hingegen wa bei 35°C Inkuba ions empe a u deu lich e höh . Bei länge e Zuch de Tie e bei 22°C und 32°C konn e nu ü T ypsin eine Anpassung de Sek e ions a e an die Hal ungs empe a u in Fo m eine posi i en Akklima isa ion ge unden we den. Li e a u e 47 47 Li e a u e Abdel-la ie M., Ho mann K.H., 2010. 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Wood ing J., Die sch S., Lwalaba D., Ho mann K.H., Meye ing-Vos M., 2009. Con ol o he elease o diges i e enzymes in he caeca o he c icke G yllus bimacula us. Physiological En omology 34: 144–151. Wood ing J., Ho mann K.H., Lo enz M.W., 2007. Ac i i y, elease and low o diges i e enzymes in he c icke , G yllus bimacula us. Physiological En omology 32: 56–63. Yokoe Y., Yasumasu I., 1964. Dis ibu ion o cellulose in in e eb a es. Compa a i e Biochemis y and Physiology 13: 323–338. Yu C.G., S ay B., Ding Q., Tobe S.S., 1995. Immunochemical iden i ica ion and exp ession o alla os a ins in he gu o Diplop e a punc a a. Jou nal o Insec Physiology 41: 1035–1043. Zhu-Salzman K., Koiwa H., Salzman R.A., Shade R.E., Ahn J.-E., 2003. Cowpea b uchid Callosob uchus macula us uses a h ee-componen s a egy o o e come a plan de ensi e cys eine p o ease inhibi o . Insec Molecula Biology 12: 135–145. Zibaee A., Bandani A.R., Ka il M., Ramzi S., 2008. Cha ac e iza ion o α-amylase in he midgu and he sali a y glands o ice s iped s em bo e , Chilo supp essalis Walke . (Lepidop e a: Py alidae).Jou nal o Asia-Paci ic En omology 11: 201–205. Publica ions 70 Submi ed o: 1 Physiological En omology 2 Ma ch 2013 3 4 5 The sec e ion o diges i e lipase in he midgu o 6 G yllus bimacula us: egula ion by endogenous and en i onmen al 7 ac o s. 8 9 Au ho s: Sandy Weidlich1, Klaus H. Ho mann1 and Joseph Wood ing2 10 Ins i u ion: 1 Depa men o Animal Ecology I, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many 11 2 Depa men o Animal Ecology II, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many 12 Co espondence o: Sandy Weidlich 13 Add ess: Depa men o Animal Ecology I, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many 14 E-mail: sandy.weidlich@uni-bay eu h.de 15 Publica ions 71 Abs ac . Lipase elease in G yllus bimacula us depends on a ious endogenous (age, sex, 16 de elopmen al s age) and exogenous (ligh -da k cycle, ood quali y, empe a u e) ac o s. Whe eas 17 lipase sec e ion was e y simila in bo h sexes o las ins a la ae, lipase elease peaked on day 5 a e 18 ecdysis in adul emales and on day 7 in adul males; inc eased eeding esul ed in inc eased lipase 19 elease. In las ins a la ae and adul s eeding and lipase elease showed a ci cadian hy hm and 20 inc eased om 4:00 o 24:00 CEST, bu no in penul ima e la ae. Lipase ac i i y in he luminal con en s 21 and in caecal sec e ion was highe in ed c icke s han in hose ed a non-nu i i e cellulose die o 22 s a ed. Inc easing concen a ion o a y acids in he caecal incuba ion medium led o inc easing lipase 23 elease. The lipase elease om caeca incuba ed a ei he 25°C o 35°C showed li le di e ence. C icke s 24 acclima ed a 32°C showed highe lipase elease han hose acclima ed a 22°C, indica ing no 25 empe a u e acclima ion. Lipase sec e ion inc eased wi h a longe exposu e ime (adul s age, las la al 26 s age, las wo la al ins a s) when he c icke s we e acclima ed a 32°C, bu no when acclima ed a 27 22°C. The e was no di e ence in o al ood up ake in c icke s main ained a 22°C o 32°C, bu a 22°C 28 de elopmen was slowed and, he e o e, he e was a longe ime in which he daily ood up ake was 29 educed. 30 Key wo ds: lipase, diges i e enzyme, c icke , empe a u e, ood up ake, pho ope iod 31 Publica ions 72 In oduc ion 32 The wo-spo ed ield c icke , G yllus bimacula us, is an omni o ous insec adap ed o consump ion o 33 plan , ungi and insec ma e ial. The sec e ion o diges i e enzymes equi es con ol mechanisms o cope 34 wi h he a iable quali y and quan i y o ood ound in na u e. A egula ion o enzyme sec e ion and 35 syn hesis is he e o e essen ial (Blakemo e e al., 1995). The midgu is he main si e o diges i e enzyme 36 elease and me aboli e abso p ion in insec s (Dow, 1992; Chapman, 1998), whe eby he egula ion o 37 diges i e enzyme sec e ion is subjec o a ious mechanisms (Lehane e al., 1995). The midgu 38 epi helium o insec s consis s o a single cell laye wi h nume ous in e spe sed endoc ine cells 39 (Mon uenge e al., 1989; Endo e al., 1990; Sehnal & Zi nan, 1996), which a e likely o play a ole in 40 in es inal ac i i ies (Lehane e al., 1996). 41 Nume ous s udies al eady demons a ed he ac ion o a ious neu opep ides on diges i e enzyme elease 42 in insec s (Fusé e al., 1999; Ha shini e al., 2002a, b; Aguila e al., 2003; Hill & O cha d, 2005; Sakai 43 e al., 2006; Wood ing e al., 2009; Lwalaba e al., 2010). The elease o neu opep ides is induced by 44 nu ien s in he lumen, and hese pep ides ac as pa aho mones inducing he elease o diges i e enzymes 45 om nea by zymogen cells in he endo helium (Lehane e al., 1996). The e o e, he sec e ion o 46 diges i e enzymes is s ongly co ela ed o he ood in ake o an insec (Engelmann, 1969; Dadd, 1970; 47 Chapman, 1985; Te a, 1990; Lehane e al., 1996; Te a e al., 1996). 48 Lipids a e u ilized o ene gy s o age ( a body) and o oogenesis in all insec s, and in some insec s 49 (seed eede s) lipids a e an impo an sou ce o ene gy. Fo mos insec s, howe e , including c icke s, 50 e y li le die a y lipid is equi ed o g ow h and de elopmen (Pa on, 1967; Chippendale, 1971; 51 Wood ing e al., 1979), bu almos all insec s ha e a speci ic die a y equi emen o s e ols and 52 polyunsa u a ed a y acids (Cana oso e al., 2001). Many insec s can ob ain he essen ial polyunsa u ed 53 a y acids by diges ion o phospholipids ia sec e ion o phospholipase A2 om he midgu endo helium 54 (Rana & S anley, 1999). C icke s ha e a e y lipid ich a body (o e 50% iglyce ides) (Lo enz & 55 Gäde, 2009), bu hese lipids a e p ima ily de i ed om inges ed ca bohyd a es. C icke s, howe e do 56 syn hesize and elease signi ican amoun s o lipases in o he midgu (Teo & Wood ing, 1988; Wood ing 57 e al., 2009), meaning ha hey can use nu ien lipids ( iglyce ides) o ene gy s o es, bu hey a e no 58 equi ed. 59 Comple e lipid diges ion is accomplished by ca boxylic es e hyd olases (EC 3.1.1; lipases, es e ases, 60 phospholipase A and B), phospho ic monoes e hyd olases (EC 3.1.3; phospha ases) and phospho ic 61 dies e hyd olases (EC 3.1.4; phospholipase C and D) (Te a e al., 1996). The eby, lipases (EC 3.1.1.3) 62 a e essen ial compounds o he a me abolism and hyd olyse he ou e es e links o iacylglyce ols 63 om he α-posi ion s epwise o diacylglyce ols, monoacylglyce ols, glyce ol and ee a y acids 64 (Bollade e al., 1970; Ho man & Downe , 1979; Secundo e al., 2006). 65 Lipid me abolic ac i i ies in he issues o insec s a e well cha ac e ized (Cana oso e al., 2001; A ese 66 e al., 2001; Van de Ho s & Ryan, 2012), bu o da e he e a e only a ew epo s on diges i e lipases 67 Publica ions 73 om ela i ely ew species., and sec e ion om gu issue is no well unde s ood (Wein aub & Tie z, 68 1973; Male & S o ey, 1981; M dako ić e al., 2008; Ho ne e al., 2009; Wood ing e al., 2009; Lwalaba 69 e al., 2010; Ch is elle e al., 2010, 2011; Zibaee, 2012; Zibaee & Fazeli-Dinan, 2012 ). The cu en 70 s udy ocuses on he e ec o ex insic ac o s ( empe a u e, ligh -da k cycle, ood consump ion) and 71 in insic ac o s (age, de elopmen al s age, sex) on he elease o diges i e lipase in he midgu o 72 G. bimacula us. 73 Publica ions 74 Ma e ials and me hods 74 Rea ing me hod and eeding 75 The Medi e anean ield c icke G. bimacula us de Gee (Ensi e a, G yllidae) was aised unde a long- 76 day egime (LD 16:8 pho ocycle, ligh om 6 a.m. o 10 p.m. CEST) a 27°. Newly eme ged c icke s 77 we e isola ed wi hin 1 h a e he imaginal moul and we e designa ed 0-day-old c icke s. C icke s 78 ecei ed a mixed die (c icke chow) consis ing o g ound abbi , a and ca ood in a a io o 4:2:1 79 (w/w), all om Al omin Lage, Ge many. The o al nu ien alue o he c icke chow was 40 % 80 ca bohyd a es, 25 % p o ein, and 6 % lipids (Lo enz & Anand, 2004). 81 The in i o e ec o eeding and s a a ion on enzyme elease was es ed by p o iding s anda d aga 82 die (40 g c icke chow + 3.6 g aga + 160 ml H2O) and a non-nu ien cellulose die (120 g cellulose 83 powde + 12 g aga pe li e H2O) o by s a a ion (access o wa e , bu no ood a all). A e imaginal 84 moul c icke s we e placed indi idually in o boxes and p o ided a esh cube o an aga -die o no ood 85 a all. 86 To in es iga e he in luence o empe a u e acclima ion on lipase elease c icke s we e di ided in o wo 87 g oups, one main ained a 22°C he o he a 32°C (acclima ion empe a u e, AT). Each o hese wo 88 g oups was di ided in o h ee g oups, which we e se o AT om (a) day 0 o he penul ima e ins a , (b) 89 day 0 o he las ins a , and om (c) day 0 o he adul li e. Hence he c icke s we e exposed o he wo 90 AT o a sho , in e media e o long pe iod o ime. Du ing his ime c icke s we e kep indi idually in 91 boxes (10x10x6 cm). Fo each o he o al o six g oups he caeca we e emo ed on day 2 o he adul 92 s age, di ided in hal and each hal incuba ed in low glucose Ringe a ei he 25°C o 35°C incuba ion 93 empe a u e (IT) o 30 min. The esul s a e gi en pe mg caeca o compensa e o he di e en size o 94 each hal . In addi ion, ood up ake was de e mined o c icke s acclima ed a bo h 22°C and 32°C om 95 he beginning o las la al s age. 96 Gu dissec ion and sample p epa a ion 97 The c icke s we e en ally cu open om he las abdominal segmen o he neck. The caeca we e 98 emo ed, cu open and insed 3- imes wi h G yllus Ringe (138 mM NaCl, 5 mM KCl, 2 mM 99 CaCl2∙2 H2O, 4 mM Hepes, pH 7.2). Con ac ion o he muscles o he opened caeca leads o a cup- 100 shaped s uc u e wi h he lumen side ou e mos and he hemolymph side inne mos , which was 101 designa ed a la -shee gu p epa a ion (Blakemo e e al., 1995). 102 To de e mine he lipase elease (sec e ion), he opened and insed caecal issue o indi idual c icke s 103 we e incuba ed in low glucose Ringe (LGR) (10 mg glucose / 100 ml G yllus Ringe ) o 30 min a 104 37°C wi hou shaking. The ai - illed achea kep he caeca a he su ace o he medium. Following 105 incuba ion, he caecal issue was disca ded and cells we e emo ed by cen i uga ion a low speed 106 (2000 g) o 2 min a 4°C. 107 Publica ions 75 To de e mine lipase ac i i y in issue cells, insed caecal issue o indi idual c icke was added o 108 150 - 200 µl G yllus Ringe wi h a ew c ys als o N-phenyl hiou ea (PTH) and homogenized wi h an 109 ul asonica o a he lowes se ing o ew seconds (Soni ie 250, B anson). Tissue homogena e (TH) 110 was cen i uged a 16000 g o 10 min a 4°C and he supe na an was used o he enzyme assay. 111 To es he lipase ac i i y in caecal lumen con en , 10 µl aliquo s o luminal con en s we e mixed wi h 112 190 µl G yllus-Ringe wi h PTH and cen i uged a 16000 g a 4°C o 2 min. 113 Enzyme assay 114 Lipase ac i i y was measu ed using he subs a e p-ni ophenyl palmi a (pNPP) (Winkle & S uckman, 115 1979). 15 mg pNPP was dissol ed in 5 ml 2-p opanol by hea ing o abou 50°C un il clea (8 mM pNPP 116 s ock solu ion). The s ock solu ion was dilu ed wi h 50 mM T is-HCl bu e , pH 8 + 0.1 % T i on X100 117 and gen ly mixed o hea ed un il a clea suspension esul ed (0.4 mM wo king solu ion). The T i on X 118 p e en ed he o ma ion o a u bid suspension (Gup a e al., 2002). 119 190 µl o a eshly p epa ed 0.4 mM wo king solu ion pNPP was added o 10 µl sample and he change 120 in abso bance a 410 nm o e 30 min a 25°C was measu ed in 96 well mic opla es using a mic opla e 121 eade (Syne gy HT, BioTek). 122 The op imal empe a u e and pH o lipase was measu ed using pNPP as subs a e. The op imal 123 empe a u e o he lipase (a pH 8.2) was e y b oad (30 - 40°C) wi h a sligh peak a ound 37°C, and 124 he op imal pH (a 25°C) was abou 8.5. 125 In i o e ec o nu ien s on enzyme elease 126 The e ec o nu ien s on he in i o elease o lipase om caecal epi helium was es ed by he addi ion 127 o glucose (1 - 4 mg nu ien /ml LGR) and oleic acid (0.05 - 0.1 mM) o he incuba ion medium (LGR) 128 con aining eshly insed caeca o one c icke . Lipase ac i i y in he incuba ion medium wi hou any 129 added nu ien s (con ol) was se o 100 % o compa ison o media wi h nu ien s. 130 S a is ical analyses 131 The SigmaPlo 11.0 p og am (Sys a So wa e GmbH) was used o e alua e he da a. All da a we e 132 s a is ically es ed o homogenei y o a iance (Le ene’s es ) and no mal dis ibu ion (Shapi o-Wilk 133 es ). Pai ed - es was used o linked da a o he e ec o incuba ion empe a u e on lipase elease. 134 Independen da a we e e alua ed using ei he ANOVA o K uskal-Wallis es and indi idual pos hoc 135 analysis. Fo s a is ical analysis da a o empe a u e e ec (Fig.7) and ligh -da k cycle (Fig. 5A) on 136 enzyme elease we e no malized by log10 ans o ma ion, da a o ood up ake p esen ed as c op weigh 137 (Fig. 5B) we e no malized by squa e oo ans o ma ion. The s a is ical signi icance is designa ed in 138 he g aphs and ex . 139 Publica ions 76 Resul s 140 Lipase ac i i y was measu ed in he luminal con en , issue homogena e and sec e ion o wo day old 141 ed G. bimacula us. In emales lipase ac i i y was nea ly equal in he luminal con en and issue 142 homogena e, bu was signi ican ly highe han ha sec e ed o e 30 min (Fig. 1A). In male c icke s 143 lipase ac i i y showed a simila end wi h highe enzyme ac i i y in luminal con en (Fig. 1B). 144 Bo h, a non nu ien die (cellulose die ) as well as s a a ion caused a signi ican ly lowe lipase ac i i y 145 in he luminal con en and dec eased lipase sec e ion om caecal epi helium. In s a ed and cellulose 146 ed c icke s lipase ac i i y in luminal con en was abou 90 % less han in ed ones (Fig. 2A), bu lipase 147 elease was educed by only abou 50 % (Fig. 2B). The in i o e ec o nu ien s on lipase elease was 148 es ed by incuba ion o caecal issue in low glucose Ringe wi h he addi ion o ei he glucose o oleic 149 acid. The e was a dose dependen inc ease o lipase elease in esponse o oleic acid (0.05 - 0.1mM) 150 (Fig. 3B), bu glucose showed no e ec (Fig. 3A). 151 Feeding beha iou in insec s is co ela ed o he quali y and a ailabili y o ood, bu also ela ed o 152 on ogenesis. The age-dependen lipase elease in G. bimacula us (Fig. 4) showed inc easing ac i i y 153 om day 0 o 5 o he las ins a la ae. The e was no di e ence in lipase sec e ion be ween male and 154 emale la ae wi h a maximum ac i i y o 65-78 nmol pNP/30min a day 5, and he lipase ac i i y 155 dec eased om day 5 o 8 (5.9-6.4 nmol pNP/30min). A e he imaginal moul lipase elease in emale 156 c icke s inc eased apidly om 8 o 100 nmol pNP/30min wi hin i e days, whe eas lipase ac i i y in 157 males emained low (~30 nmol pNP/30min). F om day 6 lipase sec e ion in males inc eased eaching 158 maximum a day 7, whe eas lipase elease in emales declined. In gene al howe e , he e was a highe 159 lipase elease in adul emales han in males. 160 Lipase sec e ion om caecal epi helium (Fig. 5A) and ood up ake (c op weigh ) (Fig. 5B) was ollowed 161 o e a pe iod o one day (24 h) in he las wo la al s ages o emales and in he adul s. C icke s we e 162 ea ed a 27°C unde a long-day egime wi h a pho ophase om 6:00 o 22:00 CEST (see ma e ials and 163 me hods). The e was a signi ican e ec o s age and ime on lipase elease. Adul c icke s and las ins a 164 la ae showed inc easing lipase sec e ion om 4:00 o 24:00 CEST, eaching a maximum (116 - 136 165 nmol pNP/30min) in he la e pho ophase o ea ly sco ophase (22:00-24:00 CEST) (Fig. 5A). In 166 penul ima e la ae he e was no clea end o inc easing lipase ac i i y o e 24 h. The c op weigh o 167 c icke s was de e mined a he ime o dissec ion (Fig. 5B). The e was a signi ican e ec o ime and 168 s age on he c op weigh o he c icke s wi h a signi ican in e ac ion, which indica es di e ences in 169 eeding beha iou ( ime o ood up ake) wi hin indi idual de elopmen al s ages. The c op weigh o las 170 ins a la ae and adul c icke s was highes a 24:00 CEST, wi h a highe ood up ake o he beginning 171 o da kness (Fig. 5B). In con as , he c op o penul ima e la ae was illed a he end o he sco ophase 172 a 4:00 CEST. Howe e , he e was no co ela ion be ween c op weigh and lipase elease o any 173 de elopmen al s age (Spea man ank o de : p > 0.05) (da a no shown). 174 Publica ions 77 Tempe a u e is one o he mos impo an en i onmen al ac o s ha di ec ly in luence he me abolic 175 a e, g ow h, ood consump ion and enzyme sec e ion in insec s. A an incuba ion empe a u e o ei he 176 25°C o 35°C he e was no e ec on lipase elease om caecal issue o c icke s acclima ed a 22°C 177 (Fig. 6A) o 32°C (Fig. 6B) om he ime o he imaginal moul o hose acclima ed since he moul o 178 las la al ins a . Bu he e we e sligh bu signi ican di e ences in emales ea ed since he moul o 179 he penul ima e ins a . Lipase elease inc eased a 35°C IT when ea ed a 22°C, bu dec eased a 35°C 180 when ea ed a 32°C (Fig. 6A,B). Howe e , lipase elease was always highe in c icke s ea ed a 32°C 181 han hose ea ed a 22°C (Fig. 7). Fu he mo e, he e was a signi ican e ec o exposu e ime o he 182 wo acclima ion empe a u es on lipase elease and a s ong in e ac ion o ea ing empe a u e and 183 exposu e ime. Lipase elease inc eased he longe c icke s we e acclima ed a 32°C, bu no when 184 acclima ed a 22°C (Fig. 6). 185 C icke s acclima ed a 22°C, compa ed o hose acclima ed a 32°C om he beginning o he las ins a 186 o penul ima e ins a showed e a ded (slowe ) p og ess in de elopmen he longe hey we e exposed 187 o he lowe empe a u e (Table 1). In e es ingly he o al amoun o consumed ood h oughou he las 188 ins a did no di e be ween c icke s acclima ed a 22°C o 32°C (Fig. 8), bu he daily amoun o ood 189 up ake a 22°C was g ea ly educed (Fig. 9). A 22°C ood up ake inc eased o e he i s h ee days o 190 80 - 100 mg ood/body weigh [g] and s ayed he same un il day 13 o las ins a . The ea e , eeding 191 g adually dec eased un il he inal moul (Fig. 9A). Food up ake o c icke s ea ed a 32°C on he o he 192 hand inc eased quickly om day 0 o 2 wi h maximum o 400 mg ood/g body weigh , and dec eased 193 apidly om day 3 o 6 p io o he inal moul (Fig. 9B). A e he imaginal moul he daily ood up ake 194 wi hin he i s h ee days was h ee imes highe a 32°C (~300 mg ood/ g body weigh ) compa ed o 195 22°C (~100 mg/g body weigh ), and also he o al amoun o consumed ood h ough adul li e was 196 signi ican highe a 32°C (Fig. 8). 197 198 Publica ions 78 Discussion 199 Tempe a u e is one o he mos impo an abio ic ac o s, ha in luence insec de elopmen ( ime, 200 numbe o s ages, g ow h a e) and biology ( ood consump ion, me abolic a e, e ili y, locomo ion, 201 ep oduc ion, diges ion), and he e o e i s li e his o y and pheno ypic plas ici y (Chown & Te blanche, 202 2007). Insec s a e ec o he mic and he a e unc ions o mos ac i i ies a e ela ed o ambien empe a u e 203 (Ho mann, 1974; Me kel, 1977, Beh ens e al., 1983; Hade speck & Ho mann, 1991; Boo h & Kiddell, 204 2007; Lachennich e al., 2010). Inc easing ea ing empe a u e om 22°C o 32°C led o he expec ed 205 dec ease in he du a ion o he las ins a and penul ima e ins a o G. bimacula us. All epo ed diges i e 206 enzymes ha e in i o empe a u e op ima, howe e , hese op ima o en do no co espond o he ambien 207 empe a u e (Te a e al., 1996). Acco ding o Wood ing e al. (2009) he empe a u e op imum o 208 lipase in G. bimacula us is abou 37°C a an op imal pH 8.0. Incuba ion empe a u e o caecal issue 209 (25°C o 35°C) has no e ec on enzyme elease in 2-day adul s a e sho - e m acclima ion (since he 210 beginning o las ins a o since adul ecdysis), bu he e was a sligh di e ence wi h longe acclima ion 211 (since he beginning o he penul ima e la al ins a ), which may indica e a elease o di e en isozymes 212 wi h highe ac i i y a highe empe a u es. 213 An acclima ion o he a e o diges i e enzyme sec e ion has sca cely been in es iga ed in insec s 214 (Weidlich e al., 2012). The highe acclima ion empe a u e o 32°C led o a signi ican inc ease in lipase 215 elease o adul c icke s o e di e en exposu e imes compa ed o hose ea ed a 22°C. This indica es 216 no acclima ion o a a e unc ion in he classical sense, as de ined by P osse (1991). The ypsin 217 sec e ion in G. bimacula us on he o he hand showed a highe a e o sec e ion a e acclima ion o 218 22°C han when acclima ed a 32°C (Weidlich e al., 2012), which is he classical posi i e empe a u e 219 acclima ion pa e n. Tempe a u e has a di ec s imula o y e ec on he amoun o lipase eleased and a 220 highe empe a u e also appea s o s imula e lipase syn hesis, in ha mo e lipase is sec e ed a a highe 221 incuba ion empe a u e (35°C compa ed o 25°C). Mo eo e , lipase syn hesis is s ongly in luenced by 222 exposu e ime a di e en acclima ion empe a u es. The longe c icke s we e ea ed a 32°C, he mo e 223 lipase was syn hesized, s o ed in endo helial cells and subsequen ly sec e ed. 224 In insec s ood consump ion is co ela ed o he sex, age, de elopmen al s age, ea ing empe a u e and 225 ligh -da k cycle, and hese ac o s ( ia ood consump ion) also in luence he elease o diges i e 226 enzymes. The sec e ion o lipase in he caeca o G. bimacula us is simila o ha o ypsin, amylase and 227 cellulase (Weidlich e al., 2012, 2013), and is di ec ly ela ed o ood up ake (Wood ing & Lo enz, 228 2007). Ele a ed lipase elease in emale c icke s migh be associa ed wi h a g ea e need o lipids o 229 egg p oduc ion (Espig & Ho mann, 1985). In bo h sexes las ins a la ae showed a apid decline o 230 lipase elease om day 6 o 8 associa ed wi h he p epa a ion o he inal moul in ha he gu is emp ied 231 and much less o no ood is consumed (Anand & Lo enz, 2008). 232 Diges i e enzyme elease in G. bimacula us depends on bo h quan i y and quali y o he die . Lipase 233 sec e ion and lipase ac i i y in he luminal con en s in s a ed and non-nu ien (cellulose) ed c icke s 234 Publica ions 79 was g ea ly educed compa ed o die - ed c icke s. Speci ic nu ien s in he die s imula e he elease o 235 lipase. Oleic acid, o example, when added o he incuba ion medium o caeca leads o a signi ican 236 inc ease o lipase sec e ion, indica ing a p andial elease mechanism, simila o ha epo ed o ypsin 237 and amylase (Wood ing e al., 2009). Rana & S anley (1999) al eady epo ed a s imula o y e ec o 238 he p esence o phospholipids on he sec e ion o phospholipase A2 in he midgu o Manduca sex a. 239 In e es ingly, glucose in he incuba ion medium has no e ec on lipase elease. 240 The e was a signi ican e ec o pho ope iod and de elopmen al s age on lipase sec e ion in 241 G. bimacula us. Feeding and lipase sec e ion was highes a he beginning o he sco ophase (24:00 242 CEST) in he las ins a and in he adul s age, bu o unknown easons no in he penul ima e ins a . 243 Las ins a la ae and adul c icke s a e basically noc u nal, in ha locomo o y ac i i ies ake place in 244 he sco ophase (Nowosielski & Pa on, 1963; Nielsen & D eisig, 1970; Lohe , 1972; Tanaka e al., 1999; 245 Lo enz, 2007). The inc ease o ood up ake and lipase sec e ion was shown o esul om an inc ease 246 o locomo o y ac i i y in Ache a domes icus (Wood ing & Cli o d, 1986). A simila e ec was also 247 epo ed o ypsin sec e ion in G. bimacula us (Weidlich e al., 2012). Bo h sexes o las ins a 248 G. bimacula us show an age-dependen cyclic pa e n o ac i i y wi h maxima du ing ea ly o mid 249 sco ophase and minima du ing ea ly pho ophase. A e he imaginal moul he c icke s show a 250 con inuing cyclic o ac i i y un il day 6 o adul li e (Faßold e al., 2010). 251 In insec s he pa s in e ce eb alis is in ol ed in he ci cadian egula ion o ac i i y le els (Ma sui e al., 252 2009). S udies on Pe iplane a ame icana showed inc easing locomo ion, ood consump ion, as well as 253 amylase and p o ease ac i i y in he da k phase (Ma sui e al., 2009). Fu he mo e diges i e enzyme 254 elease in he insec midgu unde lies he in luence o neu opep ides (Lehane e al., 1995; Fusé e al., 255 1999; Ha shini e al., 2002a, b; Aguila e al., 2003; Hill & O cha d, 2005; Sakai e al., 2006; Wood ing 256 e al., 2009; Lwalaba e al., 2010). 257 In conclusion, lipase sec e ion in G. bimacula us is s ongly in luenced by endogenous and 258 en i onmen al ac o s. Highe empe a u es associa ed wi h longe exposu e o di e en acclima ion 259 empe a u es lead o an inc ease o lipase syn hesis in caecal issue and, he e o e, o an inc eased 260 sec e ion. Lipase sec e ion is co ela ed o sex, age, de elopmen al s age, and ci cadian ac i i y hy hm 261 o he c icke s, and all hese ac o s in luence eeding beha iou . In gene al inc eased ood in ake esul s 262 in inc eased lipase elease. 263 Acknowledgemen s 264 We hank Juliane Hus e and Sonja Mülle o p o ing pa o samples on age-dependen enzyme 265 elease and in i o s udies o nu ien s. We also hank Ma ion P eiß o echnical assis ance. 266 Publica ions 86 s anda d cellulose s a ed s anda d cellulose s a ed lipase ac i i y [nmol pNP/30min] 0 20 40 60 80 100 A B a bb a bb die die die die 439 Fig. 3: In i o e ec o glucose (A) and oleic acid (B) on lipase elease om incuba ed caeca (37°C, 440 30 min) o 2-day-old adul G. bimacula us emales. Lipase ac i i y o con ols (0 mg/ml glucose, 441 0 mM oleic acid) was se 100 %. Mean ± SEM. n = 10. S a is ics: K uskal-Wallis es (A: H = 442 3.524, d = 3, p > 0.05; B: H = 19.424, d = 2, p < 0.001) and pos hoc Tukey es . Di e en 443 le e s indica e signi ican di e ences. 444 age [d] 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 9 10 lipase ac i i y [nmol pNP/30min] 0 20 40 60 80 100 120 las ins a la ae adul s adium 445 Fig. 4: Age-dependen ac i i y o lipase sec e ion (30 min incuba ion a 37°C) o male (○) and emale 446 (●) las ins a la ae and adul s o G. bimacula us. Mean ± SEM. n = 10. 447 Publica ions 87 a b a,b a,b a,b a,b a,b aa a,b a,b ba,b a,b aa,b a,b b a,c b,c a c,d d,a b,c,d aa,b bbbb aa,b a c cb,c ime o day 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 c op weigh [mg] 0 50 100 150 B ime o day 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 lipase ac i i y [nmol pNP/30min] 0 50 100 150 200 Apenul ima e la ae penul ima e la ae las ins a la ae las ins a la ae adul s adium adul s adium 448 Fig. 5: Lipase elease (A) and c op weigh (B) o 2-day-old penul ima e la ae, las ins a la ae and 449 adul emales o G. bimacula us o e a 24 h pe iod. Sco ophase was om 22:00 o 6:00 CEST 450 (g ey). Mean ± SEM. n = 9-10. 451 S a is ics: (A) wo-way ANOVA: s age (F2,178 = 20.09; p < 0.001); ime (F5,178 = 11.64; p < 452 0.001); in e ac ion s age* ime (F10,178 = 4.76, p < 0.001). (B) wo-way ANOVA: s age (F2,178 = 453 89.68; p < 0.001); ime (F5,178 = 6.62; p < 0.001); in e ac ion s age* ime (F10,178 = 2.18; p = 454 0.022). Pos hoc compa ison (Tukey es ) o ac o ime wi hin indi idual s ages. Di e en 455 le e s indica e signi ican di e ence. 456 Publica ions 88 ALI PI ALI PI lipase ac i i y [nmol pNP/30min/mg caeca] 0 2 4 6 incuba ion a 25°C incuba ion a 35°C A B ns ns ns ns * ** 457 Fig. 6: The e ec o incuba ion empe a u e on lipase sec e ion om caecal epi helium o 2-day-old 458 adul emale G. bimacula us acclima ed a (A) 22°C and (B) 32°C o h ee di e en exposu e 459 imes. A = om day 0 o adul s adium, LI = om day 0 o las ins a , PI = om day 0 o 460 penul ima e ins a . Mean ± SEM. n = 20-40. S a is ics: pai ed - es . ns = no signi ican , * = p 461 < 0.05, ** = p < 0.01. 462 exposu e ime ALI PI lipase ac i i y [nmol pNP/30min/mg caea] 0 1 2 3 4 5 6 22°C 32°C 463 Fig. 7: Lipase sec e ion om caecal issue o emale G. bimacula us acclima ed a 22°C and 464 32°C o h ee di e en exposu e imes (see Fig. 6). Mean ± SEM. n = 19–36. 465 S a is ics: wo-way ANOVA: empe a u e (F1,139 = 236.71, p < 0.001); exposu e ime (F2,139 = 466 24.5, p < 0.001); in e ac ion empe a u e*exposu e ime (F2,139 = 19.54, p < 0.001). 467 Publica ions 89 las ins a la ae adul ood up ake [mg] 0 1000 2000 3000 22°C 32°C ns *** 468 Fig. 8: A e age ood up ake [mg] o emale G. bimacula us in las la al ins a and adul s adium. Mean 469 SEM. n = 5-10. S a is ics: las ins a la ae ( - es : = -0.863, d = 13, p > 0.05), adul ( - es : 470 = -9.543, d = 13, p < 0.001),. ns = p > 0.05; *** = p < 0.001. 471 age [d] 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 3 4 5 6 7 8 9 ood up ake [mg] pe body weigh c icke [g] 0 50 100 150 200 age [d] 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8 9 ood up ake [mg] pe body weigh c icke [g] 0 100 200 300 400 500 600 adul s age las ins a la ae adul s agelas ins a la ae A B 472 Fig. 9: Age-dependen ood up ake o emale G. bimacula us h ough las la al ins a and adul 473 s adium acclima ed a (A) 22°C and (B) 32°C. Mean ± SEM. n = 5-10. 474 Publica ions 90 Tables Table 1: Du a ion o he las wo la al ins a s [days] o emale G. bimacula us acclima ed a 22°C and 32°C and he o al exposu e ime a acclima ion empe a u e un il dissec ion. Mean ± SEM. n = 19-36. since penul ima e ins a since las ins a 22°C 32°C 22°C 32°C penul ima e ins a 15.26 ± 0.20 5.00 ± 0.00 - - las ins a 18.16 ± 0.23 6.20 ± 0.09 16.37 ± 0.21 6.56 ± 0.13 o al exposu e ime 33.75 ± 1.71 13.20 ± 0.09 17.55 ± 0.84 8.36 ± 0.08 Publica ions 91 Publica ion 4 Weidlich S., Ho mann K.H. and Wood ing J. Ac i a ion and au olysis o ypsin in he midgu o he Medi e anean ield c icke , G yllus bimacula us. will be submi ed sho ly Publica ions 92 Ac i a ion and au olysis o ypsin in he midgu o he Medi e anean ield c icke , G yllus bimacula us. Au ho s: Sandy Weidlich1, Klaus H. Ho mann1 and Joseph Wood ing2 Ins i u ion: 1 Depa men o Animal Ecology I, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many 2 Depa men o Animal Ecology II, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many Abb e ia ed i le: Ac i a ion and au olysis o G yllus ypsin Co espondence o: Sandy Weidlich, Depa men o Animal Ecology I, Uni e si y o Bay eu h, 95440 Bay eu h, Ge many Email: sandy.weidlich@uni-bay eu h.de Publica ions 93 Abs ac In G. bimacula us he cells o he midgu epi helium syn hesize ypsin p ecu so (TP), which is s o ed in cy oplasmic esicles. TP o insed ypsin- ee caecal issue was ee om esicles by soni ica ion. The comple e sel -ac i a ion o TP in i o equi es abou 60 min. The maximum sel -ac i a ion (measu ed by hyd olysis o BApNA) was 4- imes highe in homogena es om ed c icke s compa ed o s a ed c icke s, indica ing a posi i e in luence o eeding on TP syn hesis in he epi helial cells. Nei he he addi ion o G yllus- ypsin ( om lumen con en s) no bo ine ypsin (Sigma), when added o he issue homogena e, accele a ed he maximum ac i i y. The p esence o calcium ions in he incuba ion medium esul ed in inc eased sec e ion o TP om caecal endo helium, indica ing an exocy osis mechanism o elease. The ypsin ac i i y o incuba ed lumen con en e ained i s ac i i y o e a pe iod o a leas 4 h, whe eas a bo ine ypsin solu ion los 80% o i s ac i i y in 30 min. I is sugges ed ha insec ypsin in he diges i e ac is p o ec ed om au olysis by he p esence o pep ides, a mechanism long known in mammalian sys ems, Key wo ds: diges i e enzyme, zymogen, ypsin, p o ease, c icke , au olysis, ac i a ion Publica ions 94 In oduc ion P o eases a e enzymes wi h a wide ange o physiological oles, including he diges ion o die a y p o eins, he emo al o damaged issues, and ha e highly specialized oles in a ious ac i a ion o inhibi o y cascades (Kanos and Clem, 2012). Thus p o eases a e equi ed in a wide ange o i al p ocesses such as diges ion, g ow h, e iliza ion, immunological eac ions, wound healing and cell dea h (Lazu e, 2002). Se ine p o eases, especially ypsins, occu in he diges i e ac o almos all animals, and he e is an ex ensi e li e a u e on he occu ence, dis ibu ion, sec e ion, cha ac e is ics, and e ec s o in insic o ex insic ac o s egula ing he sec e ion (Applebaum, 1985; Da is e al., 1985; Chapman, 1989; Mo a and Lehane, 1990; G a e al., 1991; Ramos e al., 1993; Te a and Fe ei a, 1994; Te a e al., 1996a,b; C is o ole i e al., 2001; Wood ing e al., 2007, 2009; Weidlich e al., 2012). Inspi e o his, he e a e h ee a he simple ques ions conce ning insec s ypsins ha emain inadequa ely answe ed. Fi s , wha is he sec e o y mechanism o ypsin in insec s? Two, is ypsin sec e ed as a p ecu so molecule, ha mus be ac i a ed o is i sec e ed in an ac i e o m? Thi d, how s able is ypsin once sec e ed and ac i a ed? Mos p o eoly ic enzymes a e indeed syn hesized as inac i e p ecu so s (deAlbuque que e al., 2001; Khan and James, 1998; Lazu e, 2002), which also enables a spa ial and empo al egula ion o enzyme ac i i y. T ypsin p ecu so s (TP) o e eb a es a e ac i a ed by hyd olysis o sho polypep ide chains be ween he amino acids isoleucine and lysine o a ginine, and by changing con o ma ion o ac i e subs a e binding si es (Eh mann and Clausen, 2004; Pas e nak e al., 1999; Walsh, 1970).T ypsin and TP we e ea ly a ge s o p o ein sequencing s udies in e eb a es, bu TP has also been desc ibed in some insec species (Da is e al., 1985; Mo a and Lehane, 1990; G a e al., 1991; Ramos e al., 1993). T ypsin iso o ms o a ious leng hs p esen in he midgu o Locus a mig a o ia p obably indica es he p esence o p ecu so p o eins (Lam e al., 2000). In Musca domes ica memb ane-bound and newly syn hesized TPs a e s o ed in esicles o he endo helium, which la e on use wi h he plasma memb ane eleasing hei con en in o he lumen (Lemos and Te a, 1992; Te a and Fe ei a, 1994; Jo dão e al., 1996). The egula ion o p o ease ac i i y includes mic oen i onmen al ac o s (pH, ions), gene egula ion, syn hesis o speci ic inhibi o s, subs a e inhibi ion, o cascade egula ion (Lazu e, 2002). Se e al endogenous se ine p o ease inhibi o s ha e al eady been iden i ied in a ious cock oach species (Elpidina e al., 2001a,b; Engelmann and Ge ae s, 1980; Vinoku o e al., 2007; Zhuzhiko , 1997), bu such endogenous ypsin inhibi o s we e no ound in G. bimacula us (Weidlich e al., 2012). Fu he mo e, au olysis is an impo an ac o in p o ease egula ion oo. In his s udy he p esence o a pu a i e ypsin p ecu so in G. bimacula us and i s ac i a ion was in es iga ed, and he in luence o eeding on he syn hesis a e was de e mined. Finally, he au olysis o na i e G yllus- ypsin was compa ed o ha o bo ine ypsin. Publica ions 95 Ma e ials and me hod Rea ing me hods The Medi e anean ield c icke , G. bimacula us de Gee (Ensi e a, G yllidae), was aised unde a long- day egime (LD 16:8 h pho ocycle) a 27°C. Newly-eme ged emales we e isola ed wi hin one hou a e he imaginal moul (be o e hey s a ed o eed) and we e designa ed 0-day old. C icke s ecei ed a mixed die (c icke chow) consis ing o g ound abbi , a and ca ood in a a io o 4:2:1 (w/w), all om Al omin Lage, Ge many. The o al nu ien alue o he chow was 40% ca bohyd a e, 25% p o ein, and 6% lipids (Lo enz and Anand, 2004). The op imal g ow h (maximal weigh gain and sho es ime) o G. bimacula us ed a die con aining om 10 o 50% casein was achie ed wi h a 30% casein die (Me kel, 1977). To compa e he ac i a ion o ypsin p ecu so in ed and s a ed G. bimacula us, c icke s we e placed indi idually in o boxes and p o ided a cube o an aga -die (40 g c icke chow + 3.6 g aga + 160 ml wa e ) o no ood a all (access o wa e ). Gu dissec ion and sample p epa a ion The c icke s we e en ally cu open om he las abdominal segmen o he neck. The caeca we e emo ed, cu open and insed 3- imes wi h G yllus Ringe (138 mM NaCl, 5 mM KCl, 2 mM CaCl2∙2 H2O, 4 mM Hepes, pH 7.2) (GR). This assu ed ha pe haps mo e han 95% o he diges i e enzymes be ween he in ica e olds o he epi helial issue we e emo ed. A spon aneous con ac ion o he ex e nal muscles o he caeca led o he o ma ion o an open, cup shaped s uc u e wi h he lumen side ou e mos and he hemolymph side inne mos ( e med a la -shee gu p epa a ion; Blakemo e e al., 1995). The e o e, bo h sides o he caecal epi helium we e equally exposed o he medium du ing incuba ion. The caeca a e ichly supplied wi h la ge achea (Wood ing and Lo enz, 2007) and hese emain connec ed, so ha he p epa a ion loa s on he su ace o he incuba ion medium. The ole o calcium ions The insed caecal issue o single c icke s was di ided in hal . One hal was incuba ed in low glucose Ringe (10 mg glucose/100 ml G yllus Ringe )(LGR) ei he wi h o wi hou calcium ions a 37°C o 30 min. A e wa ds, he issue was disca ded, and he supe na an (incuba e) was cen i uged a 2,000 g o 2 min and subsequen ly assayed o ypsin ac i i y. Ac i a ion o ypsin p ecu so F esh insed caeca we e ans e ed o 600 µl 50 mM phospha e bu e (pH 7.2) con aining ew c ys als o N-phenol hiou ea ( o inhibi phenoloxidase) and we e homogenised a he lowes se ing o ew seconds wi h ul asonica o (B anson Soni ie 250). The sample was cen i uged a 11,000g o 10 min, he pelle disca ded and he supe na an was ozen a -20°C. The homogena e con ained li le o no ypsin, bu sonica ion and eezing dis up ed all esicles and he eby eleased TP. A e wa ds, he Publica ions 102 Sipos T, Me kel JR. 1970. An e ec o calcium ions on he ac i i y, hea s abili y, and s uc u e o ypsin. Biochemis y 9:2766-2775. Spiess C, Beil A., Eh mann M. 1999. A empe a u e-dependen swi ch om chape one o p o ease in a widely conse ed hea shock p o ein. Cell 97:339-347. Te a WR, Fe ei a C, Bake JE. 1996a. Compa men aliza ion o diges ion. In: Lehane MJ and Billingsley PF, edi o s. Biology o he insec midgu . London: Chapman & Hall. p 206-235. Te a WR, Fe ei a C, Jo dão BP, Dillon RJ. 1996b. Diges i e enzymes. In: Lehane MJ and Billingsley PF, edi o s. Biology o he insec midgu . London: Chapman & Hall. p 153-194. Te a WR, Fe ei a C. 1994. Insec diges i e enzymes: P ope ies, compa men aliza ion and unc ion. Comp Biochem Physiol B Biochem Mol Biol 109:1-62. Vajda T, Ga ai A. 1981. Compa ison o he e ec o calcium (II) and manganese (II) ions on ypsin au olysis. J Ino g Biochem 15:307-315. Vinoku o K, Ta anushenko Y, K ishnan N, Sehnal F. 2007. P o einase, amylase, and p o einase- inhibi o ac i i ies in he gu o six cock oach species. J Insec Physiol 53:794-802. Walsh KA. 1970. T ypsinogens and ypsins o a ious species. Me hods Enzymol 19:41-63. Weidlich S, Hus e J, Ho mann KH, Wood ing J. 2012. En i onmen al con ol o ypsin sec e ion in he midgu o he wo-spo ed ield c icke , G yllus bimacula us. J Insec Physiol 58:1477-1484. Wood ing J, Die sch S, Lwalaba D, Ho mann KH, Meye ing-Vos M. 2009. Con ol o he elease o diges i e enzymes in he caeca o he c icke G yllus bimacula us. Physiol En omol 34:144–151. Wood ing J, Ho mann KH, Lo enz MW. 2007. Ac i i y, elease and low o diges i e enzymes in he c icke G yllus bimacula us. Physiol En omol 31:1-8. Wood ing J, Lo enz MW. 2007. Feeding, nu ien low and unc ional gu mo phology in he c icke G yllus bimacula us. J Mo phol 268:815-825. Zhuzhiko DP. 1997. Inhibi o o se ine p o einases in in es ine o cock oach Nauphoe a cine ea. J E ol Biochem Physiol 33:524-528. Publica ions 103 Figu es Fig. 1 In i o e ec o calcium ions on ypsin sec e ion om caecal epi helium o 2-day-old G. bimacula us adul s. Mean ± SEM. n = 10-15. S a is ics: pai ed - es : P emale = 0.001 (**), Pmale = 0.051 (#) Fig. 2: Sel -ac i a ion o ypsin p ecu so om caecal issue in s a ed (A) and ed (B) 2-day-old emale G. bimacula us. Tissue homogena e was incuba ed a 37°C o 4 h and aliquo s o 50 µl we e measu ed o ypsin ac i i y e e y 30 min. MW ± SEM. n = 20. S a is ics: K uskal-Wallis es (P < 0.001) and pos hoc Dunn’s me hod. Di e en le e s indica e signi ican di e ences. emale male ypsin ac i i y [nmol pNA/min/mg issue] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 LGR wi hou calcium LGR wi h calcium ** # ime [min] 030 60 90 120 150 180 210 240 ypsin ac i i y 0 50 100 150 200 030 60 90 120 150 180 210 240 [nmol p-ni oaniline/min] 0 100 200 300 400 500 600 B A a a,b bcc c b,cb,c c a a,b a,b b b b bbb Publica ions 104 Fig. 3: Ac i a ion o ypsin p ecu so in caecal issue homogena e o s a ed (A)and ed (B) 2-day- old adul G. bimacula us emales o e 2 h a 37°C by addi ion o G yllus- ypsin om lumen con en . Aliquo s o 100 µl we e aken o measu e ypsin ac i i y e e y 30 min. MW ± SEM. n = 30. S a is ics: (A) Repea ed measu emen ANOVA (P < 0.001) and pos hoc Tukey- es , (B) F iedman-Tes (P < 0.001) and pos hoc Tukey- es . Di e en le e s indica e signi ican di e ences. Fig. 4: Ac i a ion o ypsin p ecu so in 2-day-old adul s a ed (A)and ed (B) emale G. bimacula us induced by addi ion o 20 µl bo ine ypsin (1 µg/µl) du ing incuba ion a 37°C o 2 h. Aliquo s o 100 µl we e measu ed o ypsin ac i i y e e y 30 min. MW ± SEM. n = 15. S a is ics: F iedman-Tes (P < 0.001) and pos hoc Wilcoxon-Tes + Bon e oni co ec ion. Di e en le e s indica e signi ican di e ences. ime [min] 030 60 90 120 ypsin ac i i y [nmol p-ni oaniline/min] 0 100 200 300 400 030 60 90 120 aabbb a b cc c AB 030 60 90 120 ypsin ac i i y [nmol p-ni oaniline/min] 0 100 200 300 400 ime [min] 030 60 90 120 AB abc c d a bcbb Publica ions 105 Fig. 5: Ac i i y o G yllus- ypsin, bo ine ypsin and a mix u e o G yllus- and bo ine ypsin du ing 4 h incuba ion a 37°C. Aliquo s o 50 µl we e measu ed o ypsin ac i i y e e y 30 min. MW ± SEM. n = 10-20. ime [min] 030 60 90 120 150 180 210 240 ypsin ac i i y [%] 0 50 100 150 200 bo ine ypsin G yllus ypsin G yllus ypsin + bo ine ypsin Acknowledgmen s 106 Acknowledgemen s I wan o hank all colleagues and pe sons who con ibu ed o his wo k. Fi s I hank P o . D . Klaus H. Ho mann who enabled and suppo ed his p ojec , p o ided guidance and ad ice whene e needed. P o . D . Joseph Wood ing became a ue men o and iend o me. He posi i ely in luenced my wo k and encou aged me a all imes. I was a pleasu e o wo k wi h him side by side and I am e y g a e ul o his suppo , guidance and ad ices du ing my wo k and he w i ing o manusc ip s. He is an inspi a ion o e e y scien is o enjoy wo k and ne e lose sigh o you aims. Ca mela He mann has been he hea and he soul o he Depa men and ook ca e o o ganiza ion, logis ics and helped wi h all ques ions and p oblems. Special hanks go o Ma ion P eiß who became indispensable assis ance because o he dedica ion and pa ience. I am g a e ul o he suppo and echnical assis ance o M.Sc. Sonja Mülle , M.Sc. Alexande Meye , U sula Wilczek and Do o hea Wiesne . I also hank all my colleagues who c ea ed a e y iendly wo king en i onmen and helped wi h scien i ic discussions and eaching: D . F anziska Wende, PD D . Ma ina Meye ing-Vos, S e anie Schapp (Depa men o Animal Ecology I), D . S e an Küchle , D . Sieg ied Kehl (Depa men o Animal Ecology II), P o . D . Heike Feldhaa , D . Oli e O i and D . Simon T agus (Depa men o Animal Ecology I - AG Popula ion Ecology). Fu he mo e I wan o hank my bachelo s uden s and s uden assis an s Jö n He e , Ma io Schwa z, Sand a Wal he , Bas ian Schaue and especially Juliane Hus e o hei help and con ibu o wo k. Since e hanks a e gi en o all p o esso s and collabo a o s o he di e en depa men s o biology and chemis y who suppo ed my scien i ic and eaching wo k. I hank he boa d o p o esso s o he Eu opean PhD Ne wo k o Insec Science and Bio echnology who ga e me he oppo uni y o p esen and discuss my wo k in a e y iendly and kind a mosphe e and o ex end my ne wo k o new young scien is s. Abo e all, I hank my amily and iends o hei pe sonal suppo and hei con ibu ion o become Bay eu h a new home. Decla a ion 107 Decla a ion Hie mi e siche e ich, Sandy Weidlich, die o liegende A bei selbs s ändig e ass und keine ande en als die on mi angegebenen Quellen und Hil smi el benu z zu haben. Da übe hinaus e siche e ich, dass ich diese ode eine gleicha ige Disse a ion nich ande wei ig e such habe einzu eichen und mich keine gleicha igen Dok o p ü ung, mi ode ohne E olg, an eine ande en Hochschule un e zogen habe. I, Sandy Weidlich, decla e ha his hesis he eby submi ed o he Doc o deg ee a he Uni e si y o Bay eu h is my own wo k and has no been p e iously submi ed by me a any ano he Uni e si y o any o he deg ee. The wo k is o iginal excep whe e indica ed by special e e ence in he ex and has no been p esen ed o any o he Uni e si y o examina ion. Bay eu h, Mai 2013 __________________ Sandy Weidlich