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. Neu opep ide egula o s o he ju enile ho mone
biosyn hesis (in i o) in he bee le Teneb io moli o (Coleop e a, Teneb ionidae). A chi es
o Insec Biochemis y and Physiology 74: 135–146.
Aguila R., Maes o J.L., Vilaplana L., Chi a C., And eu D., Bellés X., 2004. Iden i ica ion o
leucomyosupp essin in he Ge man cock oach, Bla ella ge manica, as an inhibi o o ood
in ake. Regula o y Pep ides 119: 105–112.
Aguila R., Maes o J.L., Vilaplana L., Pascual N., Piulachs M.D., Bellés X., 2003. Alla os a in
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unc ion. Compa a i e Biochemis y and Physiology 109B:1–62.
Te a W.R., Fe ei a C., Jo dão B.P., Dillon R.J., 1996. Diges i e enzymes. In: Lehane M.J.,
Billingsley P.F., edi o s. Biology o he Insec Midgu . U.K.: Chapman & Hall, p 153–193.
Te a W.R., Espinoza-Fuen es F.P., Ribei o A.F., Fe ei a C., 1988. The la al midgu o he
house ly (Musca domes ica): ul as uc u e, luid luxes and ion sec e ion in ela ion o he
o ganiza ion o diges ion. Jou nal o Insec Physiology 34: 463–472.
Thomas K.K., Na ion J.L., 1984. P o ease, amylase and lipase ac i i ies in he midgu and
hindgu o he c icke , G yllus ubens and mole c icke , Scap e iscus acle us. Compa a i e
Biochemis y and Physiology 79A: 297–304.
Ti a enko E., Ch ispeels M.J., 2000. cDNA cloning, biochemical cha ac e iza ion and inhibi ion
by plan inhibi o s o he α-amylases o he Wes e n co n oo wo m, Diab o ica i gi e a.
Insec Biochemis y and Molecula Biology 30: 979–990.
Tokuda G., Wa anabe H., Ma sumo o T., Noda H., 1997. Cellulose diges ion in he wood-ea ing
highe e mi e, Nasu i e mes akasagoensis (Shi aki): dis ibu ion o cellulases and
p ope ies o endo-be a-1,4-glucanase. Zoological Science 14: 83–93.
T e es D.S., Ma in M.M., 1994. Cellulose diges ion in p imi i e hexapods: e ec o inges ed
an ibio ics on gu mic obial popula ions and gu cellulase le els in he i eb a , The mobia
domes ica (Zygen oma, Lepisma idae). Jou nal o Chemical Ecology 20: 2003–2020.
Valencia-Jiménez A., A boleda J.W., López A ila A., G ossi-de-Sá M.F., 2008. Diges i e
α-amylases om Tecia solani o a la ae (Lepidop e a: Gelechiidae): esponse o pH,
empe a u e and plan amylase inhibi o s. Bulle in o En omological Resea ch 98: 575–579.
Veens a J.A., 1989. Isola ion and s uc u e o wo gas in/CCK-like neu opep ides om he
Ame ican cock oach homologous o he leucosul akinins. Neu opep ides 14: 145–149.
Villalon J.M., Ghosh A., Jacobs-Lo ena M., 2003. The pe i ophic ma ix limi s he a e o
diges ion in adul Anopheles s ephensi and Aedes aegyp i mosqui oes. Jou nal o Insec
Physiology 49: 891–895.
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. Jou nal o Insec
Physiology 53: 794–802.
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Beekwilde J., 2003. P ope ies o pu i ied gu ypsin om Helico e pa zea, adap ed o
p o einase inhibi o s. Eu opean Jou nal o Biochemis y 270: 10–19.
Wa anabe H., Tokuda G., 2010. Celluloly ic sys ems in insec s. Annual Re iew o En omology
53: 609–632.
Wa anabe H., Tokuda G., 2001. Animal cellulases. Cellula and Molecula Li e Sciences 58:
1167–1178.
Wa anabe H., Noda H., Tokuda G., Lo N., 1998. A cellulase gene o e mi e o igin. Na u e 394:
330–331.
Wa anabe H., Nakamu a M., Tokuda G., Yamaoka I., Sc i ene A.M., Noda H., 1997. Si e o
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Wei Y.D., Lee K.S., Gui Z.Z., Yoon H.J., Kim I., Zhang G.Z., Guo X., Sohn H.D., Jin B.R., 2006.
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Na ional Academy o Science o he USA 86: 5997–6001.
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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
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G yllus bimacula us: egula ion by endogenous and en i onmen al
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ac o s.
8
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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

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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
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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).
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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;
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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.
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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
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om ela i ely ew species., and sec e ion om gu issue is no well unde s ood (Wein aub & Tie z,
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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
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G. bimacula us.
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Ma e ials and me hods
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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
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(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).
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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
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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.
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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
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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
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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
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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
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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.
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Gu dissec ion and sample p epa a ion
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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
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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).
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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
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incuba ion, he caecal issue was disca ded and cells we e emo ed by cen i uga ion a low speed
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(2000 g) o 2 min a 4°C.
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To de e mine lipase ac i i y in issue cells, insed caecal issue o indi idual c icke was added o
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150 - 200 µl G yllus Ringe wi h a ew c ys als o N-phenyl hiou ea (PTH) and homogenized wi h an
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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.
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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
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190 µl G yllus-Ringe wi h PTH and cen i uged a 16000 g a 4°C o 2 min.
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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).
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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).
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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.
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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 .
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Resul s
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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
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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
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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
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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
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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
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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
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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).
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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
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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
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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

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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
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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
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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
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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
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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

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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
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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
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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.
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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
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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.

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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
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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
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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