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

The regulation of digestive enzyme release in the two-spotted field cricket Gryllus bimaculatus (de Geer): effects of endogenous and environmental factors Kumulative Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth vorgelegt von Sandy Weidlich Bayreuth, Mai 2013 Die vorliegende Arbeit wurde am Lehrstuhl für Tierökologie I der Universität Bayreuth unter der Leitung von Prof. Dr. Klaus H. Hoffmann und Prof. Dr. Joseph Woodring im Zeitraum von April 2009 bis Mai 2013 angefertigt. Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) Dissertation eingereicht am: 14.05.2013 Zulassung durch die Prüfungskommission: 22.05.2013 Wissenschaftliches Kolloquium: 30.10.2013 Amtierender Dekan: Prof. Dr. Rhett Kempe Prüfungsausschuss: Prof. Dr. Klaus H. Hoffmann (Erstgutachter) Prof. Dr. Konrad Dettner (Zweitgutachter) Prof. Ph.D. Harold Drake (Vorsitz) Prof. Dr. Hartmut Frank Prof. Dr. Christian Laforsch Contents I Contents 1 Introduction .................................................................................................... 1 1.1 The morphology of the digestive tract ..................................................... 1 1.2 Digestive enzymes.................................................................................. 2 1.2.1 Proteases ..................................................................................... 3 1.2.2 Carbohydrases ............................................................................. 4 1.2.2.1 Amylases ........................................................................ 4 1.2.2.2 Cellulases ....................................................................... 5 1.2.2.3 Chitinases ....................................................................... 6 1.2.3 Lipases ......................................................................................... 7 1.3 Secretory processes for digestive enzymes ............................................ 8 1.4 Regulation mechanisms controlling digestive enzyme secretion in insects .......................................................................................................... 9 1.4.1 Allatostatins .................................................................................10 1.4.2 Sulfakinins ...................................................................................11 1.5 Enzyme inhibitors ..................................................................................13 1.5.1 Plant protease inhibitors...............................................................13 1.5.2 Endogenous protease inhibitors ...................................................13 1.6 Research gaps ......................................................................................15 2 Synopsis ........................................................................................................17 2.1 Enzyme assays, kinetic parameters and sample preparation .................17 2.1.1 Enzyme activity assays ................................................................17 2.1.2 Kinetic parameters .......................................................................17 2.1.3 Enzyme activity in different samples types ...................................18 2.2 The effect of endogenous factors ...........................................................21 2.2.1 Age-dependent enzyme release ..................................................21 2.2.2 Neuropeptides .............................................................................23 2.2.2.1 Allatostatin type A ..........................................................23 Contents II 2.2.2.2 Sulfakinins .....................................................................31 2.2.3 Calcium ions ................................................................................33 2.2.4 Trypsin activation and autolysis ...................................................34 2.2.5 Endogenous protease inhibitors ...................................................34 2.3 The effect of environmental factors ........................................................36 2.3.1 Temperature ................................................................................36 2.3.1.1 Incubation temperature ..................................................36 2.3.1.2 Rearing temperature ......................................................37 2.3.2 Light-dark cycle ............................................................................38 2.3.3 Food and nutrients .......................................................................40 2.3.3.1 Feeding and starvation ..................................................40 2.3.3.2 Nutrients ........................................................................41 2.3.3.3 Plant protease inhibitors ................................................42 Summary ...........................................................................................................43 Zusammenfassung ...........................................................................................45 Literature ...........................................................................................................47 Publications ......................................................................................................65 (1) Environmental control of trypsin secretion in the midgut of the two-spotted field cricket, Gryllus bimaculatus. ..........................................................67 (2) Regulation of amylase, cellulase and chitinase secretion in the digestive tract of the two-spotted field cricket, Gryllus bimaculatus. .....................68 (3) The secretion of digestive lipase in the midgut of Gryllus bimaculatus: regulation by endogenous and environmental factors. ..........................69 (4) Activation and autolysis of trypsin in the midgut of the Mediterranean field cricket, Gryllus bimaculatus. .................................................................91 Acknowledgments .......................................................................................... 106 Declaration ...................................................................................................... 107 Figures and Tables III List of figures Figure 1: The digestive tract of Gryllus bimaculatus. .................................................. 2 Figure 2: Enzymatic cleavage of a polypeptide chain by aminopeptidase (EC 3.4.11) and trypsin (3.4.21.4). ................................................................................. 3 Figure 3: Enzymatic hydrolysis of amylose (C6H12O6)n by amylases. .......................... 4 Figure 4: Schematic view of the enzymatic hydrolysis of cellulose. ............................ 6 Figure 5: Enzymatic degradation of chitin (C8H13NO5)n by chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.52). .................................................. 7 Figure 6: Stepwise hydrolysis of triacylglycerol to glycerol and carboxylic acids. ........ 7 Figure 7: Models for secretory processes of insect digestive enzymes: exocytic secretion (A), apocrine secretion (B), microapocrine secretion with budding vesicles (C) and with pinched-off vesicles (D). ............................................ 8 Figure 8: Regulation mechanism for digestive enzyme release: small digestive products (prandial) bind to receptors of endocrine cells on the midgut side to free paraneurohormones, which stimulate the enzyme release of zymogene cells (paracrine mechanism). ..................................................................... 10 Figure 9: Nucleotide sequence of the allatostatin (A) and sulfakinin (B) precursor cDNA of Gryllus bimaculatus and the deduced amino acid sequences of the preprohormone polypeptides. .................................................................... 12 Figure 10: Experimental overview: determination of the effects of endogenous (organism) and exogenous (environment) factors controlling the regulation of enzyme secretion in G. bimaculatus. ......................................................... 16 Figure 11: Activity of (A) carbohydrases amylase and cellulase, (B) proteases trypsin and aminopeptidase and (C) lipase in samples of tissue incubation medium (incubate), tissue homogenate (tissue) and luminal content (lumen) of 2-dayold female and male G. bimaculatus. ........................................................ 20 Figure 12: Age-dependent activity of (A) carbohydrases, (B) proteases and (C) lipase in the caecal secretion medium (30 min incubation at 37°C) of male (□) and female (●) last instarlarvaeandadults of G. bimaculatus. ........................... 22 Figure 13: In vitro effect of AST-5 on the amount of protein [µg BSA/mg tissue] released from (A) female and (B) male caecal tissue to incubation medium. ........... 23 Figures and Tables IV Figure 14: In vitro effects of AST-5 on (A) carbohydrases, (B) proteases and (C) lipase secretion from caecal tissue of 2-day-old adult G. bimaculatus. ................ 24 Figure 15: Weight gain of 2-day-old G. bimaculatus last instar larvae and adults, injected with either 2 µg AST-A dsRNA (in 10 µl Ringer) or Ringer only at the day of the preceding moult. .................................................................................. 25 Figure 16: Weight gain of 2-day-old adult G. bimaculatus females, injected with AST-A dsRNA (0-6 µg in 10 µl Ringer) at the day of imaginal moult. .................... 25 Figure 17: Activity of (A) trypsin, (B) aminopeptidase, (C) lipase and (D) amylase in the caecal lumen content of 2-day-old G. bimaculatus adults and last instar larvae injected with either 2 µg AST-A dsRNA (in 10 µl Ringer) (grey) or Ringer only (white) at preceding moult. ..................................................... 26 Figure 18: Activity of (A) trypsin, (B) aminopeptidase, (C) lipase and (D) amylase in the caecal tissue homogenate of 2-day-old G. bimaculatus adults and last instar larvae injected with either 2 µg AST-A dsRNA (in 10 µl Ringer) (grey) or Ringer only (white) at preceding moult. ..................................................... 27 Figure 19: Activity of (A) trypsin, (B) aminopeptidase, (C) lipase and (D) amylase in the caecal tissue incubation medium of 2-day-old G. bimaculatus adults and last instar larvae injected with either 2 µg AST-A dsRNA (in 10 µl Ringer) (grey) or Ringer only (white) at preceding moult. ................................................. 28 Figure 20: The effect of various concentrations of AST-A dsRNA on the protein concentration in (A) tissue incubate, (B) tissue homogenate, and (C) luminal content of 2-day-old adult G. bimaculatus females. ................................... 29 Figure 21: The effect of various concentrations of AST-A dsRNA on (A) amylase, (B) aminopeptidase, (C) trypsin, and (D) lipase activity in tissue incubation medium, tissue homogenate and lumen content of 2-day-old adult G. bimaculatus females. ............................................................................ 30 Figure 22: Protein concentration in samples of tissue incubation medium, tissue homogenate and lumen content from (A) female and (B) male 2-day-old adult G. bimaculatus, injected with either 10 µl Ringer (control) or 2 µg SK dsRNA in 10 µl Ringer at the day of imaginal moult. .............................................. 31 Figure 23: Effect of Ringer and SK dsRNA injection on enzyme secretion of (A) carbohydrases, (B) proteases and (C) lipase of 2-day-old adult female and male G. bimaculatus. ................................................................................. 32 Figures and Tables V Figure 24: In vitro effect of calcium ions (2 mM) on amylase, cellulase, chitinase, and trypsin secretion from caecal epithelium of 2-day-old adult female G. bimaculatus. ......................................................................................... 33 Figure 25: Trypsin activity in mixtures of exogenous bovine trypsin and heated and unheated samples of (A) tissue incubation medium, (B) tissue homogenate and (C) lumen content of diet-fed and starved 2-day-old adultG. bimaculatusfemales. ..................................................................... 35 Figure 26: The effect of incubation temperature (25°C = white, 35°C = grey) on (A) trypsin, (B) aminopeptidase, (C) lipase, and (D) amylase secretion from caecal tissue of 2-day-old adult G. bimaculatus. ........................................ 37 Figure 27: Food uptake and digestive enzyme secretion in the course of a day in 2-dayold penultimate larvae, last instar larvae and adult G. bimaculatus females. .................................................................................................... 39 Figure 28: In vitro effects of nutrients on soluble protein concentration in the incubation medium of caecal tissue from 2-day-old adult female G. bimaculatus. ...... 41 List of tables Table 1: Temperature and pH optima, Km and Vmax values of digestive enzymes from midgut of G. bimaculatus. ............................................................................ 18 Abbreviations VI Abbreviations AST allatostatin AST-A allatostatin type A AT acclimation temperature BapNa Nα-benzoyl-DL-arginine-p-nitroanilide hydrochloride CA corpora allata cDNA complementary desoxyribonucleic acid CMC carboxymethyl cellulose CMCh carboxymethyl-chitin-RBV 5R DNS dinitrosalicylic acid dsRNA double-stranded ribonucleic acid EC enzyme commission EPI endogenous protease inhibitor Fig. figure G. bimaculatus Gryllus bimaculatus GHF glycosyl hydrolase family GlcNAc N-acetyl-D-glucosamine IPM integrated pest management IT incubation temperature JH juvenile hormone Km Michaelis-Menten constant LpNa L-leucine p-nitroanilide hydrochloride Ma maltose mRNA messenger RNA PG peritrophic gel PI protease inhibitor PM peritrophic membrane PMx peritrophic matrix pNA p-nitroaniline pNP p-nitrophenol pNPP p-nitrophenyl palmitate RBV remazol brilliant violet RFU relative fluorescent units RNA ribonucleic acid SBTI soybean trypsin inhibitor SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM standard error of the mean SK sulfakinin Vmax maximum reaction rate Part I Synopsis Introduction 7 chitobiose), which are subsequently digested by exo-splitting β-N-acetylglucosaminidases (EC 3.2.1.52) to the monomer GlcNAc (Kramer and Koga, 1986; Reynolds and Samuels, 1996). Figure 5: Enzymatic degradation of chitin (C8H13NO5)n by chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.52).∙∙∙∙∙∙ cutting site. 1.2.3 Lipases Lipids are an important source of energy and essential for insect development, energy storage and oogenesis. Insects have a dietary requirement for polyunsaturated fatty acids during their post-embryonic phases of development, but food requirement differs between species (Dadd, 1983, 1985; Canavoso et al., 2001). Complete lipid digestion is accomplished by carboxylic ester hydrolases (EC 3.1.1: lipases, esterases, phospholipase A and B) (Figure 6), phosphoric monoester hydrolases (EC 3.1.3: phosphatases) and phosphoric diester hydrolases (EC 3.1.4: phospholipase C and D) (Terra et al., 1996). Thereby, lipases are essential compounds of the fat metabolism and hydrolyse the outer ester links of triacylglycerols from the α-position stepwise to diacylglycerols, monoacylglycerols, glycerol and free fatty acids (Bollade et al., 1970; Hoffman and Downer, 1979; Secundo et al., 2006). Figure 6: Stepwise hydrolysis of triacylglycerol to glycerol and carboxylic acids. Introduction 8 1.3 Secretory processes for digestive enzymes Different mechanisms of synthesis, storage and release of digestive enzymes at the cellular level have been described in various insect species (Cristofoletti et al., 2001; Ferreira et al., 2002; Terra et al., 1996; Weidlich et al., 2012). Digestive enzymes are synthesized in the rough endoplasmatic reticulum, processed in the Golgi complex, packed into secretory vesicles and secreted by the gut endothelium via exocytosis, apocrine or microapocrine processes (Terra and Ferreira, 2012) (Figure 7). In insects, most enzyme release is by exocytosis, and less often by apocrine secretion (Terra and Ferreira, 1994), depending on the midgut region, the enzyme and the species (Graf et al., 1986; Santos et al., 1986; Jordão et al., 1996, 1999; Cristofoletti et al., 2001; Ferreira et al., 2002). During exocytosis enzymes are stored in vesicles, which fuse with the plasma membrane and release their content without any loss of cytoplasm (Figure 7A), whereas apocrine secretion involves a loss of apical cytoplasm following vesicle release, in which the enzymes are stored (Figure 7B). Microapocrine secretion is a common type of apocrine secretion (De Priester, 1971; Heinrich and Zebe, 1973; Nopanitaya and Misch, 1974; Lehane, 1976; Humbert, 1979; Santos et al., 1984; Terra et al., 1988), in which the loss of cytoplasm is minimal and small budding double membrane vesicles (Figure 7C) or pinched-off secretory vesicles are released (Figure 7D). The content of those vesicles is freed by membrane fusion or solubilisation in the midgut lumen (Terra and Ferreira, 2012). Figure 7: Models for secretory processes of insect digestive enzymes: exocytic secretion (A), apocrine secretion (B), microapocrine secretion with budding vesicles (C) and with pinched-off vesicles (D). BSV: budding secretory vesicle, CE: cellular extrusion, DE: digestive enzymes; GC: Golgi complex, M: microvilli; N: nucleus, PSV: pinched-off secretory vesicles, RER: rough endoplasmatic reticulum, SV: secretory vesicle. (adapted from Terra and Ferreira, 2009) Introduction 9 1.4Regulation mechanisms controlling digestive enzyme secretion in insects According to their feeding behaviour insects can be classified in two major groups: continuous and discontinuous feeders. Continuous feeders have a continuous stream of food passing through the intestine, while discontinuous feeders (carnivores, haematophagous) have periods where the gut is filled or empty (Lehane et al., 1996). Therefore, discontinuous feeders need a regulation of enzyme secretion. The midgut is the main site of digestive enzyme release and metabolite absorption in insects (Dow, 1992; Chapman, 1998), whereby the control of digestive enzyme secretion depends on various mechanisms (e.g. hormonal, paracrine, prandial) (Lehane et al., 1995) and requires separate regulation of enzyme synthesis and enzyme secretion (Blakemore et al., 1995). Food consumption plays a fundamental role in the secretion of digestive enzymes, in that not only food intake (Engelmann, 1969; Dadd, 1970; Chapman, 1998) but also the composite of the nutrition regulates secretion mechanism (prandial release mechanism) (Chapman, 1985; Terra, 1990; Lehane et al., 1996; Terra et al., 1996). Thereby, small components of the diet (different nutrients) interact directly with the secretory cells of the midgut and stimulate the secretion of specific digestive enzymes (Lehane et al., 1995). Although some studies already reported the influence of hormones on enzyme secretion in the midgut of insects (Applebaum, 1985; Chapman, 1985), it is still controversial whether the hormone system has a direct influence on digestive enzyme release or rather changes in hormone systems are subsequent due to the fact of treatment (Lehane et al., 1996). The insect midgut epithelium contains large numbers of endocrine cells (Montuenge et al., 1989; Endo et al., 1990; Sehnal and Žitňan, 1996), which likely play a role in intestinal activities (Lehane et al., 1996). These cells have a hemolymph side and a gut lumen side. In this model, nutrient receptors on the lumen side can stimulate the release of paraneurohormones into the hemolymph, which bind to receptors and induce the release of digestive enzymes into the gut lumen (Figure 8). Several neuropeptides including FMRFamide-related peptides, proctolin, insect kinins and allatoregulatory peptides have already been identified in the enteric nervous system and in the endocrine cells of the gut (Reichwald et al., 1994; Yu et al., 1995; Sehnal and Žitňan, 1996), and were shown to affect food uptake, gut motility (Wei et al., 2000; Predel et al., 2001; Aguilar et al., 2004; Meyering-Vos and Müller, 2007a; Meyering-Vos and Woodring, 2008; Audsley and Weaver, 2009) and the release of digestive enzymes in particular (Fusé et al., 1999; Harshini et al., 2002a,b; Aguilar et al., 2003; Hill and Orchard, 2005; Sakai et al., 2006; Audsley and Weaver, 2009; Woodring et al., 2009; Lwalaba et al., 2010a). Introduction 10 Figure 8: Regulation mechanism for digestive enzyme release: small digestive products bind to receptors of endocrine cells on the midgut side to free paraneurohormones, which stimulate the enzyme release of zymogene cells (paracrine mechanism). A1: absorption of soluble compounds, A2: absorption of small digestive products via endocytosis, AC: absorptive cell, DE: digestive enzymes (green), DP: digestion products, EC: endocrine cell, GC: Golgi complex, M: microvilli, N: nucleus, PNH: paraneurohormones, R: receptor, RER: rough endoplasmatic reticulum, sDP: small digestive products, SV: secretory vesicles, ZC: zymogen cell. (modified from Terra and Ferreira, 2009) 1.4.1 Allatostatins Allatoregulating peptides are divided in two groups based on their stimulatory (allatotropins) or inhibitory (allatostatins) effect on juvenile hormone biosynthesis in the corpora allata (CA) (Hoffmann et al., 1999; Gäde, 2002). Allatostatins belong to a well-documented group of neurohormones, which has been identified in a large number of insect species (Stay, 2000), and can be classified in three subgroups according to their sequence homology: allatostatin A (AST-A, FGLamides), allatostatin B (AST-B, W(X6)Wamides) and allatostatin C (AST-C, PISCF-OH) (Stay, 2000; Meyering-Vos et al., 2001; Hoffmann, 2003). The Allatostatin type A peptide is characterized by a common C-terminus sequence Tyr/PheXaa-Phe-Gly-Leu-Ile/Val-amid (Stay et al., 1991) and was first identified from Diploptera punctata (Woodhead et al., 1989; Donly et al., 1993) and other cockroach species (Ding et al., 1995; Bellés et al., 1999, Bendena et al., 1999). Due to their pleiotropic function, AST-A peptides are expressed in different tissues (Stay, 2000). In G. bimaculatus the AST-A gene is Introduction 11 strongly expressed in the brain, the suboesophageal ganglion and the caeca of the digestive tract (Meyering-Vos and Hoffmann, 2003). The prohormone precursor encodes for 14 putative Gryllus-AST-A peptides which are interspaced by acidic spacers (Meyering-Vos et al., 2001) (Figure 9A). An inhibitory effect of AST-A peptides on JH biosynthesis was demonstrated for cockroaches, termites, crickets and some beetles (Stay and Tobe, 2007; Abdel-latief and Hoffmann, 2010). But AST-A peptides have also myoinhibiting effects on different parts of the insect gut or on the oviduct (Gäde and Hoffmann, 2005). Moreover, they inhibit the production and release of vitellogenin from the fat body of cockroaches (Martín et al., 1996, 1998) and affect the secretion of digestive enzymes (Fusé et al, 1999; Aguilar et al., 2003; Sakai et al., 2006; Digali et al., 2010). 1.4.2 Sulfakinins Sulfakinins (SK) are another family of neuropeptides with myotropic function, which were primarily isolated from the cockroach Leucophaea maderae (Nachman et al., 1986a). Insect SKs show structural homology to the peptides gastrin and cholecystokinin, which are involved in the regulation of food uptake in vertebrates (Nachman et al., 1986b). SKs are characterized by a highly conserved C-terminal hexapeptide sequence DY(SO3H)GHMRF-NH2and a sulphated tyrosine residue (Audsley and Weaver, 2009).SKs were isolated from several different insect species (Veenstra, 1989; Schoofs et al., 1990; Nichols et al., 1988, 1992; Fonagy et al., 1992; Duve et al., 1995; East et al., 1997; Maestro et al., 2001; Meyering-Vos and Müller, 2007b) and were shown to affect food uptake and enzyme release into the digestive tract (Nachman et al., 1997; Wei et al., 2000; Maestro et al., 2001; Harshini et al., 2002b; Schoofs and Nachman, 2006; Downer et al.,2007; Meyering-Vos and Müller, 2007a; Meyering-Vos and Woodring, 2008). In G. bimaculatus the SK prohormone precursor encodes two SK peptides (Figure 9B), which both show a strong expression only in the brain (Meyering-Vos and Müller, 2007b). Introduction 12 Figure 9: Nucleotide sequence of the allatostatin (A) and sulfakinin (B) precursor cDNA of G. bimaculatus and the deduced amino acid sequences of the preprohormone polypeptides. Adapted from (A) Meyering-Vos et al. (2001), (B) Meyering-Vos and Müller (2007b). The cDNA sequence is numbered from the most distal nucleotide identified on the 5' end. The deduced protein sequence is in boldface. Potential cleavage sites are boxed. Neuropeptide sequences are underlined red, those of acidic spacers blue, preceding glycine residues (required for α-amidation) black. * represents the stop codon. Introduction 13 1.5 Enzyme inhibitors 1.5.1 Plant protease inhibitors Over recent years the adaptation mechanisms of insects to their nutrition and abiotic environment have become more important with view of the increasing number of pest insects and their control. Therefore, a lot of studies focused on the investigation of insect digestion and a probable application of natural enzyme inhibitors for pest control. Many plants produce protease inhibitors (PI) as a defence mechanism against feeding damage (Fan and Wu, 2005). PIs inhibit the proteases present in the midgut lumen and crop of insects (Johnston et al., 1993; Telang et al., 2005; Duncan et al., 2006; Brioschi et al., 2007), but also affect the secretion of proteases by the epithelium (Lwalaba et al., 2010b; Weidlich et al., 2012). While PIs were thought to have the potential to protect plants against herbivorous insects (Broadway and Duffey, 1986; Broadway et al., 1986; Hilder et al., 1987; Johnson et al., 1989; Oppert et al., 1993; Orozco-Cardenas et al., 1993; McManus et al., 1994), it was soon apparent that insects have evolved different strategies to deal with PIs in the diet: (a) enzyme hyperproduction (Broadway and Duffey, 1986; Johnston et al., 1993; Broadway, 1995, Hivrale et al., 2011), (b) upand down-regulation of proteases (Jongsma et al., 1995; Cloutier et al., 2000; Zhu-Salzman et al., 2003; Brioschi et al., 2007; Dunse et al., 2010a, b), (c) increasing release of inhibitor-insensitive enzyme isoforms (Jongsma et al., 1995, 1996; Paulillo et al., 2000; Brito et al., 2001; Volpicella et al., 2003; Brioschi et al., 2007; George et al., 2008; Hivrale et al., 2011; de Oliveira et al., 2013) or (d) secretion of PI-degrading proteases (Jongsma et al., 1996; Michaud, 1997; Girard et al., 1998). The Kunitz type trypsin inhibitor from soybean (SBTI) is a small protein (~25 kDa) which interacts with trypsin-like proteases by forming an irreversible complex with a very low dissociation constant and, therefore, blocking the active site of the enzyme (Kunitz, 1948). Furthermore, SBTI is quite resistant to higher temperatures by changing conformation (Kunitz, 1948). The inhibitory effect of SBTI against midgut proteases was demonstrated for different insects in vitro and in vivo (Applebaum et al., 1963; Miller et al., 1974; Christeller et al., 1990; Johnston et al., 1993; Oppert et al., 2005; Lwalaba et al., 2010b; Weidlich et al., 2012). 1.5.2 Endogenous protease inhibitors Endogenous serine protease inhibitors (EPI) are widely found in all tissues of all animals. The serpins, a very large class of proteases, are mostly intracellular, and undergo a unique change in shape when they inhibit target proteases (Huntington et al., 2000). They regulate such processes as coagulation, inflammation, and immunity. However, some insect inhibitors involved with immune responses (coagulation, activation of phenoloxidases) belong to the classical Kunitz and Kazal type inhibitors (Kanost, 1999). They bind to the active site of the Introduction 14 proteases and block access (Kunitz, 1948). Protease inhibitors of the Kazal type are found in the salivary gland and saliva of Nauphoeta cinerea, which are particularly effective against the bacterial protease subtilisin. This suggests a defensive mechanism against the masses of bacteria typically found in the cockroach crop, some of which are probably pathogenic (Taranushenko et al., 2009). Inhibitory peptides in the midgut lumen are less well studied, though trypsin and subtilisin inhibitors are described from whole midgut preparations of the cockroach N. cinerea (Elpidina et al., 2001) and in five additional cockroach species (Vinokurov et al., 2007). Inhibitors from the cockroach gut act on endogenous digestive proteinases and may represent a new mechanism of digestion regulation. Introduction 15 1.5 Research gaps Although, a lot of studies focused on the digestion in insects, there is little knowledge on the factors controlling enzyme secretion (Lehane et al., 1996; Blakemore et al., 1995; Woodring et al., 2009; Lwalaba et al., 2010a). Therefore, the present dissertation focuses on the determination of the effect of different endogenous (organismic) and exogenous (environmental) factors on the regulation of digestive enzyme secretion into the midgut of the two-spotted field cricket G. bimaculatus (Figure 10). In particular, the following experiments were carried out:  Sample type: Enzyme activity in different sample types (luminal content, tissue incubation medium, tissue homogenate)  Age-dependent enzyme release: Changes in enzyme release of female and male G. bimaculatus from last larval instar to adult stadium  Neuropeptides: The effect of cricket allatostatin type A and sulfakinin on the release of digestive enzymes using in vitro incubation and RNA interference  Calcium ions: Dependency of enzyme release on the presence or absence of calcium ions in tissue incubation medium  Trypsin zymogen: The presence and activation of a putative trypsin precursor, and the autolysis of Gryllus-trypsin  Endogenous protease inhibitors: The presence of endogenous protease inhibitors in the midgut of adult crickets  Temperature: The effect of incubation and rearing temperature on enzyme release with respect to a putative temperature acclimation  Light-dark cycle: Food uptake and enzyme release during photoand scotophase within 24 h in penultimate instar, last larval instar and adult stadium  Food: The effect of feeding and starvation, and the influence of various nutrients (in vitro and in vivo studies) on enzyme secretion  Plant protease inhibitors: Dose-dependent inhibition of trypsin secretion and activity caused by feeding protease inhibitor (SBTI) enriched diets in adult G. bimaculatus Introduction 16 Figure 10: Experimental overview: determination of the effects of endogenous (organism) and exogenous (environment) factors controlling the regulation of enzyme secretion in G. bimaculatus. Note: not all experiments were carried out for all enzymes. For details, please check the corresponding articles. temperature effectof incubation temperature (in vitro) effectof rearing temperature: acclimation experiments fromdifferent developmental stages light-darkcycle theenzymesecretionandfooduptake over24h food & nutrients feeding& starvation cellulose enriched diets effectof nutrientsin incubation medium SBTI enriched diets refeeding experiments In vivo In vitro age enzymeactivation (zymogen activation, calciumions) sex endogenous enzyme inhibitors ENVIRONMENT ORGANISM developmentalstage THE REGULATION OF ENZYME SECRETION neuropeptides Synopsis 23 23 2.2.2 Neuropeptides 2.2.2.1 Allatostatin type A The allatostatin type A peptide and its gene, which is strongly expressed in brain, suboesophageal ganglion and caeca of G. bimaculatus (Meyering-Vos and Hoffmann, 2003), were analysed with regards to their influence on digestive enzyme release using in vitro and in vivo studies. AST-5 (DRLYSFGK-NH2) (Bachem, Germany) was used for in vitro incubation of caecal tissue following enzyme activity assays and quantification of soluble proteins. RNA interference was used to analyse the gene function of allatostatin by gene silencing in vivo, a method already employed for allatostatin peptides in this species by Meyering-Vos et al. (2006). In vitro studies The influence of AST-5 in vitro on enzyme release of amylase, trypsin, and aminopeptidase has already been reported by Woodring et al. (2009), where amylase and trypsin release increased during incubation with 10-8 to 10-5 M AST-5. The data of the current study did not fully confirm these results. In this study, the in vitro effect of AST-5 on enzyme release from caeca was tested by addition of appropriate dilution of 10-3 M stock solution of AST-5 (in 20 % acetonitrile) in LGR. The incubation medium was tested for enzyme activity and concentration of soluble proteins. There was a trend of decreasing release of soluble proteins in response to higher concentration of AST-5 (Figure 13). Figure 13: In vitro effect of AST-5 on the amount of protein [µg BSA/mg tissue] released from (A) female and (B) male caecal tissue to incubation medium. Mean + SEM. n = 10. Statistics: Kruskal-Wallis test. P > 0.05. Although there were no statistically significant differences, the secretion of amylase and cellulase (Figure 14A), aminopeptidase and trypsin (Figure 14B), and lipase (Figure 14C) in both male and female G. bimaculatus showed a continues trend of increased enzyme release in response to higher concentrations of AST-5 (10-6 to 10-5 M) in incubation medium. AST-5 concentration [M] µg BSA/mg tissue 0 2 4 6 8 10 12 14 AST-5 concentration [M] µg BSA/mg tissue 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 digestion protein digestion carbohydrate digestion amylase AST-5 concentration [M] µmol maltose/30min/µg BSA 0.0 0.1 0.2 0.3 cellulase AST-5 concentration [M] RFU/30min/µg BSA 0 1 2 3 4 male female male female 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 aminopeptidase AST-5 concentration [M] nmol pNA/min/µg BSA 0 1 2 3 trypsin AST-5 concentration [M] nmol pNA/min/µg BSA 0.0 0.1 0.2 0.3 male female male female 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 concentration [M] nmol pNP/30min/µg BSA 0.00 0.25 0.50 0.75 1.00 male female 0 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6 10-5 Figure 14: In vitro effects of AST-5 on (A) carbohydrases, (B) proteases and (C) lipase secretion from caecal tissue of 2-day-old adult G. bimaculatus males (white) and females (grey). Mean + SEM. n = 10. Statistics: Kruskal-Wallis test. P > 0.05; no significant differences. Synopsis 25 25 In vivo studies - RNA interference In vivo effects after injection of dsRNA targeted against AST-A into newly moulted crickets (last instar larvae and adults), following the degradation of the specific mRNAs, were studied by measuring the body weight gain, and the activity of amylase, aminopeptidase, trypsin and lipase in caecal lumen content, tissue homogenate and tissue incubation medium two days after dsRNA injections. The AST-A dsRNA was generated as previously described by Meyering-Vos et al. (2006). 2 to 6 µg AST-A dsRNA in 10 µl Gryllus Ringer were injected once with a 100 µl Hamilton syringe between the third and the fourth abdominal segment. Control crickets were injected with Gryllus Ringer only. The body weight gain of crickets injected with AST-A dsRNA did not differ from those injected with Ringer solution (Figure 16 & Figure 15).This corresponds to the results of Meyering-Vos et al. (2006), where differences in body weight gain following AST-A dsRNA injection were only observed in older adults. Figure 15: Weight gain of 2-day-old G. bimaculatus last instar larvae and adults, injected with either 2 µg AST-A dsRNA (in 10µl Ringer) or Ringer only at the day of the preceding moult. The body weight on the day of injection was set 100%. Mean ± SEM, n = 16-20. Statistics: students t-test, ns = not significant. Figure 16: Weight gain of 2-day-old adult G. bimaculatus females, injected with AST-A dsRNA (0-6 µg in 10 µl Ringer) at the day of imaginal moult. The body weight on the day of injection was set to 100%. Mean ± SEM., n = 9–10. Statistics: ANOVA, P > 0.05 = no significant differences. last instar adult last instar adult body weight gain [%] 0 20 40 60 Ringer dsRNA female male ns ns ns ns concentration dsRNA [µg] 0 µg 2 µg 4 µg 6 µg body weight gain [%] 0 10 20 30 40 50 60 Synopsis 26 26 Newly moulted male and female G. bimaculatus adults and last instar larvae were injected with either 2 µg AST-A dsRNA or Ringer. Two days later, the enzyme activity of trypsin, aminopeptidase, lipase, and amylase was determined in the luminal content (Figure 17), tissue homogenate (Figure 18), and tissue incubation medium (Figure 19). There was no clear trend in enzyme activity of the luminal content for either last instar larvae or adult crickets following AST-A gene knockdown (Figure 17). The enzyme activity in the caecal lumen is an estimate of the amount of enzymes that have accumulated at a given age and time. Therefore, the experimental time of two days may have been too short to detect significant changes in enzyme activity in the lumen content after injection of 2 µg AST-A dsRNA. Figure 17: Activity of (A) trypsin, (B) aminopeptidase, (C) lipase and (D) amylase in the caecal lumen content of 2-day-old G. bimaculatus adults and last instar larvae injected with either 2 µg AST-A dsRNA (in 10 µl Ringer) (grey) or Ringer only (white) at preceding moult. Mean + SEM. n = 9-10. Statistics: Mann-Whitney U-test or student’s t-test. ns = not significant, * = P < 0.05, # = 0.05 < P < 0.1. lipase last instar adult last instar adult lipase activity [nmol pNP/30min/10µl lumen content] 0 200 400 600 800 1000 1200 1400 amylase last instar adult last instar adult amylase activity [µmol Ma/30min/10µl lumen content] 0 5 10 15 20 25 C D aminopeptidase last instar adult last instar adult aminopeptidase activity [nmol pNA/min/10µl lumen content] 0 50 100 150 200 250 trypsin last instar adult last instar adult trypsin activity [nmol pNA/min/10µl lumen content] 0 50 100 150 200 female male female male A B female male female male ns ns ns * ns ns # ns ns ns #ns ns ns ns * Synopsis 27 27 However, there was a significant reduction of protease (Figure 18A,B), lipase (Figure 18C) and amylase activity (Figure 18D) in tissue homogenate of male and female crickets (with few exceptions) following AST-A gene knockdown. The enzyme activity in the tissue homogenate represents the amount of enzymes stored in tissue cells and, therefore, represents an indicator of enzyme synthesis rate. Thus, gene silencing of allatostatin type A reduced the synthesis of digestive enzymes in the midgut of G. bimaculatus. Figure 18: Activity of (A) trypsin, (B) aminopeptidase, (C) lipase and (D) amylase in the caecal tissue homogenate of 2-day-old G. bimaculatus adults and last instar larvae injected with either 2 µg AST-A dsRNA (in 10 µl Ringer) (grey) or Ringer only (white) at preceding moult. Mean + SEM. n = 9-10. Statistics: Mann-Whitney U-test or student’s t-test. ns = not significant, * = P < 0.05, ** = P< 0.01, *** = P < 0.001. ns ns aminopeptidase trypsin A B lipase amylase C D female male female male female male last instar adult last instar adult amylase activity [µmol Ma/30min/mg tissue] 0.0 0.1 0.2 0.3 0.4 0.5 last instar adult last instar adult aminopeptidase activity [nmol pNA/min/mg tissue] 0 5 10 15 20 25 30 female male last instar adult last instar adult lipase activity [nmol pNP/30min/mg tissue] 0 5 10 15 20 25 30 last instar adult last instar adult trypsin activity [nmol pNA/min/mg tissue] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ns ns ns ns ** *** ** * * * *** ** *** *** Synopsis 28 28 Although, gene silencing of allatostatin A resulted in a reduced synthesis rate of digestive enzymes in the caecal tissue, there was a trend of increasing protease (Figure 19A,B) and lipase (Figure 19C) release from the caecal tissue into the incubation medium, especially in female last instar larvae. Figure 19: Activity of (A) trypsin, (B) aminopeptidase, (C) lipase and (D) amylase in the caecal tissue incubation medium of 2-day-old G. bimaculatus adults and last instar larvae injected with either 2 µg AST-A dsRNA (in 10 µl Ringer) (grey) or Ringer only (white) at preceding moult. Mean + SEM. n = 9-10. Statistics: Mann-Whitney U-test or student’s t-test. ns = not significant, * = P < 0.05, ** = P< 0.01, *** = P < 0.001. In general, larval and adult females of G. bimaculatus seem to be more sensitive to physiological effects on digestive enzymes caused by injections of AST-A dsRNA, than males. In the following experiment the effects of different concentrations of AST-A dsRNA injections on the digestive enzyme activities of adult females were analysed. In addition the concentration of soluble proteins was determined for each sample. aminopeptidase trypsin A B lipase amylase C D female male last instar adult last instar adult amylase activity [µmol Ma/30min/mg tissue] 0.0 0.1 0.2 0.3 0.4 0.5 female male last instar adult last instar adult lipase activity [nmol pNP/30min/mg tissue] 0 2 4 6 8 last instar adult last instar adult aminopeptidase activity [nmol pNA/min/mg tissue] 0.0 0.5 1.0 1.5 2.0 female male last instar adult last instar adult trypsin activity [nmol pNA/min/mg tissue] 0.0 0.5 1.0 1.5 2.0 female male ns ns ns ns ns ns ns ns nsns ns ns ** ** * *** Synopsis 29 29 The amount of total soluble protein in samples of the luminal content (Figure 20C) decreased at higher amounts of AST-A dsRNA, but not in samples of tissue incubation medium or tissue homogenates (Figure 20A,B). Figure 20: The effect of various concentrations of AST-A dsRNA on the protein concentration in (A) tissue incubate, (B) tissue homogenate, and (C) luminal content of 2-day-old adult G. bimaculatus females. The AST-A dsRNA (0-6 µg in 10 µl Ringer) was injected into crickets at day of imaginal moult. Mean ± SEM. n = 9-10. Statistics: ANOVA and post hoc Bonferroni t-test. * indicates significant differences to control (0 µg dsRNA). The activities of amylase, aminopeptidase, trypsin, and lipase were analysed in luminal content, tissue homogenate and tissue incubation medium (secretion) (Figure 21). Injection of AST-A dsRNA did not show a dose-dependent effect on enzyme activity neither in tissue homogenate nor in tissue incubation medium, for all tested enzymes. However, higher concentration of 6 µg AST-A dsRNA resulted in significant higher amylase activity in the lumen content. Similar trends of increasing enzyme activity in the lumen content were observed for aminopeptidase and trypsin, respectively (Figure 21B,C). C concentration dsRNA 0µg 2µg 4µg 6µg µg BSA/10µl lumen content 0 100 200 300 400 * * A concentration dsRNA 0µg 2µg 4µg 6µg µg BSA/mg tissue 0 2 4 6 8 10 12 B concentration dsRNA 0µg 2µg 4µg 6µg µg BSA/mg tissue 0 2 4 6 8 10 12 Synopsis 30 30 Figure 21: The effect of various concentrations of AST-A dsRNA on (A) amylase, (B) aminopeptidase, (C) trypsin, and (D) lipase activity in tissue incubation medium, tissue homogenate and lumen content of 2-day-old adult G. bimaculatus females. The AST-A dsRNA (0-6 µg in 10 µl Ringer) was injected at day of imaginal moult. Mean + SEM. n = 9-10. Statistics: Kruskal-Wallis test + post hoc Dunn’s method. * indicates significant differences to control (0 µg dsRNA). tissue homogenate concentration dsRNA 0µg 2µg 4µg 6µg µmol maltose/30min/µg BSA 0.00 0.02 0.04 0.06 0.08 tissue homogenate concentration dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.00 0.05 0.10 0.15 0.20 0.25 0.30 tissue homogenate concentration dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.00 0.05 0.10 0.15 0.20 tissue homogenate concentration dsRNA 0µg 2µg 4µg 6µg nmol pNP/30min/µg BSA 0.0 0.2 0.4 0.6 tissue incubation medium concentration dsRNA 0µg 2µg 4µg 6µg µmol maltose/30min/µg BSA 0.00 0.05 0.10 0.15 0.20 0.25 0.30 tissue incubation medium concentration dsRNA 0µg 2µg 4µg 6µg nmol pNP/30min/µg BSA 0.0 0.2 0.4 0.6 0.8 1.0 tissue incubation medium concentration dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 tissue incubation medium concentration 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 content concentration dsRNA 0µg 2µg 4µg 6µg µmol maltose/30min/µg BSA 0.00 0.05 0.10 0.15 0.20 lumen content concentration dsRNA 0µg 2µg 4µg 6µg nmol pNA/min/µg BSA 0.0 0.5 1.0 1.5 2.0 2.5 lumen content concentration 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 content concentration dsRNA 0µg 2µg 4µg 6µg nmol pNP/30min/µg BSA 0 1 2 3 4 amylase aminopeptidase lipase trypsin A B C D * Synopsis 31 31 2.2.2.2 Sulfakinins Previous studies using RNAi suggested that SK peptides affect satiety in G. bimaculatus by reducing food uptake (Meyering-Vos and Müller, 2007a), as was also reported for cockroaches and locusts (Wei et al., 2000; Maestro et al., 2001). A stimulating effect of sulfakinins on digestive enzyme release has already been demonstrated for beetles and moths (Nachman et al., 1997; Harshini et al., 2002b), but preliminary RNAi studies in G. bimaculatus showed no effect (Meyering-Vos and Müller, 2007a). Therefore, RNAi experiments regarding gene silencing of sulfakinin were repeatedly done, and subsequent physiological effects were analysed in more detail. The SK dsRNA was generated as previously described by MeyeringVos and Müller (2007a). Male and female crickets were injected with either 2 µg dsRNA in 10 µl Ringer solution or 10 µl Ringer solution (control) with a 100 µl Hamilton syringe between the third and the fourth abdominal segment on the day of imaginal moult. Crickets were dissected two days after injection and all samples were analysed for soluble protein concentration and enzyme activities of amylase, cellulase, aminopeptidase, trypsin, and lipase. tissue homogenate Ringer dsRNA µg BSA/mg tissue 0 10 20 30 40 tissue incubation medium Ringer dsRNA µg BSA/mg tissue 0 2 4 6 8 10 12 14 lumen content Ringer dsRNA µg BSA/mg tissue 0 100 200 300 400 lumen content Ringer dsRNA µg BSA/10µl lumen content 0 100 200 300 400 tissue incubation medium Ringer dsRNA µg BSA/mg tissue 0 2 4 6 8 10 12 14 tissue homogenate Ringer dsRNA µg BSA/mg tissue 0 10 20 30 40 A B #* ns ns ns ns Figure 22: Protein concentration in samples of tissue incubation medium, tissue homogenate and lumen content from (A) female and (B) male 2-day-old adult G. bimaculatus, injected with either 10 µl Ringer (control) or 2 µg SK dsRNA in 10 µl Ringer at the day of imaginal moult. Mean ± SEM. n = 9-10. Statistics: student’s t-test or Mann-Whitney U-test. * = P < 0.05, # = 0.05 < P< 0.1, ns = P> 0.1. Synopsis 32 32 Injection of SK dsRNA induced a significant decrease in the protein concentration of lumen samples of females (Figure 22A). A similar trend was observed for the female tissue incubate. SK dsRNA injections had no effect on the protein concentrations in samples of male crickets (Figure 22B). Gene silencing of sulfakinin resulted in higher amylase and cellulase release in female crickets (Figure 23A), but not in males. Therefore, SK peptides seem to have an inhibitory effect on carbohydrate digestion, at least in female G. bimaculatus. amylase female male µmol maltose/30min/µg BSA 0.0 0.1 0.2 0.3 Ringer dsRNA A cellulase female male RFU/30min/µg BSA 0 20 40 80 120 BC ns ns ** # ns aminopeptidase female male nmol pNA/min/µg BSA 0 1 2 3 4 5 trypsin female male nmol pNA/min/µg BSA 0.0 0.1 0.2 0.3 0.4 0.5 0.6 lipase female male nmol pNP/30min/µg BSA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 # # ns ns carbohydrate digestion protein digestion lipid digestion Figure 23: Effect of Ringer and SK dsRNA injection on enzyme secretion of (A) carbohydrases, (B) proteases and (C) lipase of 2-day-old adult female and male G. bimaculatus. 2 µg SK dsRNA in 10 µl Ringer or Ringer alone (control) was injected to crickets at the day of imaginal moult. Mean ± SEM. n = 9-10. Statistics: t-test or Mann-Whitney U-test. * = P < 0.05, # = 0.05 < P <0.1, ns = not significant. Furthermore, there was a trend of decreasing protease (Figure 23B) and lipase (Figure 23C) release in male crickets, which may indicate a stimulatory effect of SK peptides on protein and lipid digestion. Injections of SK dsRNA did not affect enzyme activities in lumen content and tissue homogenate (not shown). Synopsis 39 39 Figure 27: Food uptake and digestive enzyme secretion in the course of a day in 2-day-old penultimate larvae, last instar larvae and adult G. bimaculatus females. Scotophase was from 22:00-6:00 CEST (grey). Mean ± SEM. n= 9-10. 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 amylase activity [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 activity [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 aminopeptidase activity [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 trypsin activity [nmol pNA/min/mg caeca] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 time of day 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 lipase activity [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 crop weight [mg] 0 50 100 150 penultimate instar last instar adult carbohydrases proteases lipase food uptake Synopsis 40 40 2.3.3 Food and nutrients Feeding is the most important exogenous factor affecting enzyme secretion, which correlates with both the quality and the quantity of food. The importance of feeding on the secretion of digestive enzymes in G. bimaculatus was analysed in detail in publications 1 - 4. The effect of food availability on enzyme secretion was determined in either 'feeding and starvation' or 'refeeding' experiments, while the importance of food quality was investigated by feeding different diets, and by in vitro incubation of caecal tissue in LGR in the presence of various nutrients. 2.3.3.1 Feeding and starvation In order to test the effect of feeding and starvation on digestive enzyme release, crickets were placed individually into boxes shortly after imaginal moult to prevent cannibalism. They were provided with either a fresh cube of standard agar-diet or no food at all. Two days later the activities of amylase, cellulase, trypsin and lipase was determined in samples of tissue incubation medium, tissue homogenate and luminal content. For the refeeding experiment newly moulted crickets were isolated and not fed for 5 days. Afterwards, they were provided with the agar-standard diet and the release of amylase and chitinase was determined every hour. The enzyme release and enzyme activity in midgut lumen for amylase (Fig. 4, publication 2), cellulase (Fig. 3, publication 2), trypsin (Fig. 6, publication 1), and lipase (Fig. 2, publication 3), strongly increased in the presence of food compared to starvation. Enzyme activities in tissue homogenate were also significantly higher in fed crickets compared to starved ones. In refeeding experiments, amylase showed no response to the food uptake within 5 h (Fig. 5A, publication 3). However, 24 h later there was a significant increase of secretion indicating a strong decrease of amylase synthesis in the caecal tissue during starvation (Fig. 5B, publication 2). At least 6 h are required by caecal tissue to respond to the presence of food. In contrast, chitinase secretion was slightly increased shortly after food uptake (~ 10% in 2 h), but generally remained at low level (Fig. 6, publication 2). In conclusion, feeding has a significant positive influence on digestive enzyme activity and stimulates not only the secretion of digestive enzymes, but also the synthesis rate in the caecal tissue. Synopsis 41 41 2.3.3.2 Nutrients The influence of different nutrients on digestive enzyme release has already been reported for trypsin, aminopeptidase and amylase (Woodring et al., 2009; Digali et al., 2010). Therefore, the effects of nutrients were analysed only for lipase (publication 3) and cellulase (publication 2). maltose concentration [mg/ml] 0 1 2 4 µg BSA/mg tissue 0 2 4 6 8glucose concentration [mg/ml] 0 1 2 4 µg BSA/mg tissue 0 2 4 6 8 cellubiose concentration [mg/ml] 0 1 2 4 µg BSA/mg tissue 0 2 4 6 8oleic acid concentration [mM] 0.00 0.05 0.10 µg BSA/mg tissue 0 2 4 6 8 A C B D ** * Figure 28: In vitro effects of nutrients on soluble protein concentration in the incubation medium of caecal tissue from 2-day-old adult female G. bimaculatus. Caecal tissues were incubated for 30 min at 37°C in LGR containing (A) maltose, (B) glucose, (C) cellubiose and (D) oleic acid. Mean ± SEM. n = 10. Statistics: Kruskal-Wallis test and post hoc Dunn’s test with multiple comparisons versus control group (concentration = 0). * indicates significant differences to control group. Additions of higher concentrations of cellubiose (2-4 mg/ml) and oleic acid (0.1 mM) to tissue incubation medium resulted in increased protein concentration and, therefore, in higher enzyme release from the caecal tissue (Figure 28C,D). There was no significant effect of maltose or glucose on protein concentrations (Figure 28A,B). Synopsis 42 42 In vitro incubation of caecal tissue with oleic acid enriched LGR led to a significant higher lipase release from caecal endothelium (Fig 3, publication 3). Glucose and maltose had no effect on lipase activity (data not shown). In contrast to the stimulating effect of cellubiose on protein release from caecal tissue (Figure 28C), cellulase activity was much less in tissue incubation medium with increased concentration of cellubiose (Fig. 8A, publication 2). In vivo studies feeding cellulose-enriched diets (40, 70 or 100%) also resulted in decreased cellulase activity in luminal content and tissue incubation medium (Fig. 7, publication 2), and in reduced body weight (Fig. 9A, publication 2). However, the food uptake of the animals increased following feeding of 40-70% cellulose-enriched diets, which may indicate an attempt to compensate less energy uptake (Fig. 9B, publication 2). 2.3.3.3 Plant protease inhibitors Crickets are omnivorous and may encounter plant material containing proteinase inhibitors, such as SBTI. Therefore, the adaptation of G. bimaculatus to SBTI enriched diet was studied in publication 1 in detail. SBTI in the diet reduced trypsin activity in the lumen content and tissue incubation medium in a dose-dependent manner (Tables 1 and 2, publication 1). To investigate a putative adaptation of trypsin secretion to SBTI, newly moulted female last instar larvae were fed 0.1% and 0.4% SBTI enriched diets until day 2 after imaginal moult. G. bimaculatus seems to deal with lower concentrations of SBTI (0.1-0.2%) by hyperproduction of trypsin within the first 72 h, but are unable to adapt to higher concentrations (0.4%). Furthermore, larvae fed 0.4% SBTI enriched diet showed a reduced growth compared to crickets fed diet without SBTI (Fig. 9A, publication 1). Because plants are not the sole food source, G. bimaculatus is not under selective pressure to evolve a specific adaptation mechanism to protease inhibitors. Summary 43 43 Summary Insects are the most abundant animal species on earth with a huge economical and ecological impact. In spite of intensive research in the field of integrated pest management there are still a lot of questions concerning the adaptation mechanism of insects to their environment. As the digestive tract displays a putative target for effective pest management, this study worked on the effects of endogenous and environmental factors on digestive enzyme release in the omnivorous cricket, Gryllus bimaculatus. The age-dependent enzyme release of carbohydrases, proteases and lipase correlates with the daily feeding rate of the crickets and peaked between days 2 to 4 in last instar larvae as well as in adult crickets. In contrast, the secretion of chitinase was affected by the moulting cycle of the insects reaching maximum activity at the day of moult. Therefore, chitinase plays only a minor role in food digestion. The cellulase activity in the midgut of G. bimaculatus resulted from an endogenous cellulase and was not caused by bacteria or eukaryotic endosymbionts in the digestive tract. The endoprotease trypsin was stored in the caecal tissue as an inactive precursor, and is secreted to the lumen by exocytosis. Following activation Gryllus-trypsin (~24 kDa) is protected from proteolytic degradation, but there is no endogenous protease inhibitor in the midgut. Gene knockdown by RNA interference was used to analyse the endogenous regulation of digestive enzyme release by the neuropeptides allatostatin A and sulfakinin, which had already been shown to affect feeding in G. bimaculatus. Functional analysis of the AST-A gene was investigated for last instar larvae and adult crickets, whereby female crickets seemed to be more sensitive to this method. The gene suppression of AST-A resulted in a decreased synthesis of amylase, trypsin, aminopeptidase and lipase in the caecal tissue, but enzyme release varied between sexes and developmental stages. The knockdown of SK expression led to an increase of amylase and cellulase secretion in female crickets, and to a reduction of protease and lipase release in males. As food plays a fundamental role in digestive enzyme release, both quality and quantity of nutrition are ample factors. There was always a higher digestive enzyme activity in fed crickets compared to starved ones. Furthermore, starvation resulted in a decrease of enzyme synthesis in the caecal tissue. In general, nutrients in the incubation medium led to a stimulation of digestive enzyme secretion, but in the case of cellulase the presence of both cellubiose in the incubation medium and cellulose in the diet caused a strong decline in cellulase release. Addition of the plant protease inhibitor SBTI to the diet caused a dose-dependent inhibition of protease activity in the caeca, whereby minor concentrations of SBTI were compensated by enzyme hyperproduction. Summary 44 44 In addition to the food uptake, the daily light-dark cycle seems to affect digestive enzyme release. Crickets started to feed at the beginning of the scotophase, which led to an increase of protease and lipase secretion in larvae and adults. The secretion of carbohydrases was highest during the photophase. This means that enzyme release is not solely affected by the time of food uptake. Temperature is one of the most important environmental factors, but seems to play only a minor role in the release of digestive enzymes. All tested enzymes showed a broad optimal temperature range (30°C - 40°C), but there was no difference in the release of amylase or lipase after tissue incubation at 25°C or 35°C. In contrast, trypsin and aminopeptidase showed a higher secretion after incubation at 35°C compared to 25°C. Furthermore, insect rearing at 22°C and 32°C during various developmental stages resulted in a positive acclimation of trypsin secretion to rearing temperature. Zusammenfassung 45 45 Zusammenfassung Insekten stellen mit Abstand die größte und vielfältigste Tiergruppe auf Erden dar und üben einen großen ökologischen wie auch ökonomischen Einfluss aus. Trotz intensiver Forschung im Bereich der integrierten Schädlingsbekämpfung sind bislang noch viele Fragen über die Adaptionsmechanismen von Insekten gegenüber ihrer Umwelt ungeklärt. Da der Verdauungstrakt von Insekten ein potentielles Angriffsziel für effektive Schädlingsbekämpfung darstellt, wurde innerhalb dieser Studie die Freisetzung von Verdauungsenzymen bei der omnivoren Grille, Gryllus bimaculatus, in Abhängigkeit von endogenen und Umweltfaktoren (Temperatur, Licht-Dunkel Rhythmus, Futter) analysiert. Die Freisetzung von Carbohydrasen, Proteasen und Lipasen in den Caeca von adulten und larvalen G. bimaculatus korreliert stark mit der täglichen Futteraufnahme. Dabei wurde eine maximale Enzymsekretion zwischen Tag 2 und 4 des letzten Larvenstadiums bzw. des Adultstadiums gefunden. Die Freisetzung von Chitinase wird hingegen maßgeblich vom Zeitpunkt der Häutung beeinflusst, so dass Chitinase bei der Nahrungsverwertung eher eine untergeordnete Rolle spielt. Darüber hinaus wird vermutet, dass die gemessene Cellulaseaktivität in den Caeca auf eine endogene Cellulase zurückzuführen ist, da aufgrund der Probenaufarbeitung mögliche eukaryotischen Endosymbionten oder Bakterien aus dem Darm entfernt wurden. Nähere Untersuchungen an der Protease Trypsin zeigten, dass das Enzym in Form einer Vorstufe (Zymogen) im Darmgewebe gespeichert wird. Die Freisetzung erfolgt über Exocytose aus den zymogenen Zellen des Mitteldarmes in das Darmlumen. Nach Aktivierung der Vorstufe wies das Gryllus-Trypsin ein Molekulargewicht von ~24 kDa auf und war gegen proteolytischen Abbau im Lumen geschützt. Eine Regulation der Proteaseaktivität im Mitteldarm durch endogene Protease-Inhibitoren konnte nicht gefunden werden. Mithilfe der RNA-Interferenz Methode (Gen-Knockdown) wurde die endogene Steuerung der Enzymsekretion im Mitteldarm durch die Neuropeptide Allatostatin A und Sulfakinin näher untersucht. Beide Peptide beeinflussen nachweislich die Futteraufnahme der Tiere. Die Funktionsanalyse des Allatostatin A Gens auf physiologischer Ebene wurde an Larven und adulten Grillen durchgeführt, wobei Weibchen auf die Methode sensibler zu reagieren scheinen. Gensuppression von AST-A führte meist zu einer reduzierten Syntheserate von Amylase, Trypsin, Aminopeptidase und Lipase in den Caeca-Geweben. Dabei schwankte die Höhe der Freisetzung zwischen den untersuchten Stadien und Geschlechtern. Der Knockdown der Sulfakininexpression führte hingegen zu einer deutlichen Erhöhung der Amylaseund Cellulasesekretion bei Weibchen, während bei Männchen ein Trend zu reduzierter Proteaseund Lipasefreisetzung ersichtlich war. Zusammenfassung 46 46 Sowohl die Quantität als auch die Qualität der Nahrung haben einen maßgeblichen Einfluss auf die Freisetzung aller untersuchten Enzyme. Gefütterte Tiere wiesen stets eine höhere Aktivität und eine stärkere Freisetzung der Verdauungsenzyme auf als gehungerte Grillen. Längere Hungerphasenführten zu einer Reduktion der Enzymaktivität im Darm und zu einer deutlich verminderten Syntheserate in den Caeca-Geweben. Obwohl die Anwesenheit von Nährstoffen im Inkubationsmedium eher stimulierend auf die Freisetzung der Enzyme wirkte, konnte bei Cellulase eine starke Reduktion der Sekretion bei Anwesenheit von Cellubiose im Inkubationsmedium oder Cellulose in der Nahrung festgestellt werden. Zusatz des pflanzlichen Protease-Inhibitors SBTI zur Nahrung führte zu einer konzentrationsabhängigen Reduktion der Aktivität und Freisetzung von Trypsin in den Caeca, wobei die Grillen geringere Konzentrationen an SBTI durch Enzym-Hyperproduktion kompensieren konnten. Neben der Futteraufnahme wurde die Freisetzung der Verdauungsenzyme durch den TagNacht-Rhythmus der Tiere beeinflusst. Das Einsetzen der Futteraufnahme zu Beginn der Dunkelphase erklärt die erhöhte Proteaseund Lipasefreisetzung, jedoch konnte in adulten wie larvalen G. bimaculatus ein Anstieg der Freisetzung von Carbohydrasen während der Photophase verzeichnet werden. Die Regulation der Enzymfreisetzung wird folglich nicht allein durch den Zeitpunkt der Futteraufnahme bestimmt. Temperatur als Umweltfaktor spielt bei der Freisetzung von Verdauungsenzymen in G. bimaculatus eine eher untergeordnete Rolle. Obwohl alle untersuchten Enzyme Temperaturoptima im Bereich von 30°C - 40°C aufwiesen, zeigten Lipase und Amylase keinen Unterschied in der Freisetzung nach Gewebeinkubationen bei 25°C oder 35°C. Die Sekretion von Proteasen hingegen war bei 35°C Inkubationstemperaturdeutlich erhöht. 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Publications 70 Submitted to: 1 Physiological Entomology 2 March 2013 3 4 5 The secretion of digestive lipase in the midgut of 6 Gryllus bimaculatus: regulation by endogenous and environmental 7 factors. 8 9 Authors: Sandy Weidlich1, Klaus H. Hoffmann1 and Joseph Woodring2 10 Institution: 1 Department of Animal Ecology I, University of Bayreuth, 95440 Bayreuth, Germany 11 2 Department of Animal Ecology II, University of Bayreuth, 95440 Bayreuth, Germany 12 Correspondence to: Sandy Weidlich 13 Address: Department of Animal Ecology I, University of Bayreuth, 95440 Bayreuth, Germany 14 E-mail: [email protected] 15 Publications 71 Abstract. Lipase release in Gryllus bimaculatus depends on various endogenous (age, sex, 16 developmental stage) and exogenous (light-dark cycle, food quality, temperature) factors. Whereas 17 lipase secretion was very similar in both sexes of last instar larvae, lipase release peaked on day 5 after 18 ecdysis in adult females and on day 7 in adult males; increased feeding resulted in increased lipase 19 release. In last instar larvae and adults feeding and lipase release showed a circadian rhythm and 20 increased from 4:00 to 24:00 CEST, but not in penultimate larvae. Lipase activity in the luminal contents 21 and in caecal secretion was higher in fed crickets than in those fed a non-nutritive cellulose diet or 22 starved. Increasing concentration of fatty acids in the caecal incubation medium led to increasing lipase 23 release. The lipase release from caeca incubated at either 25°C or 35°C showed little difference. Crickets 24 acclimated at 32°C showed higher lipase release than those acclimated at 22°C, indicating no 25 temperature acclimation. Lipase secretion increased with a longer exposure time (adult stage, last larval 26 stage, last two larval instars) when the crickets were acclimated at 32°C, but not when acclimated at 27 22°C. There was no difference in total food uptake in crickets maintained at 22°C or 32°C, but at 22°C 28 development was slowed and, therefore, there was a longer time in which the daily food uptake was 29 reduced. 30 Key words: lipase, digestive enzyme, cricket, temperature, food uptake, photoperiod 31 Publications 72 Introduction 32 The two-spotted field cricket, Gryllus bimaculatus, is an omnivorous insect adapted to consumption of 33 plant, fungi and insect material. The secretion of digestive enzymes requires control mechanisms to cope 34 with the variable quality and quantity of food found in nature. A regulation of enzyme secretion and 35 synthesis is therefore essential (Blakemore et al., 1995). The midgut is the main site of digestive enzyme 36 release and metabolite absorption in insects (Dow, 1992; Chapman, 1998), whereby the regulation of 37 digestive enzyme secretion is subject to various mechanisms (Lehane et al., 1995). The midgut 38 epithelium of insects consists of a single cell layer with numerous interspersed endocrine cells 39 (Montuenge et al., 1989; Endo et al., 1990; Sehnal & Zitnan, 1996), which are likely to play a role in 40 intestinal activities (Lehane et al., 1996). 41 Numerous studies already demonstrated the action of various neuropeptides on digestive enzyme release 42 in insects (Fusé et al., 1999; Harshini et al., 2002a, b; Aguilar et al., 2003; Hill & Orchard, 2005; Sakai 43 et al., 2006; Woodring et al., 2009; Lwalaba et al., 2010). The release of neuropeptides is induced by 44 nutrients in the lumen, and these peptides act as parahormones inducing the release of digestive enzymes 45 from nearby zymogen cells in the endothelium (Lehane et al., 1996). Therefore, the secretion of 46 digestive enzymes is strongly correlated to the food intake of an insect (Engelmann, 1969; Dadd, 1970; 47 Chapman, 1985; Terra, 1990; Lehane et al., 1996; Terra et al., 1996). 48 Lipids are utilized for energy storage (fat body) and for oogenesis in all insects, and in some insects 49 (seed feeders) lipids are an important source of energy. For most insects, however, including crickets, 50 very little dietary lipid is required for growth and development(Patton, 1967; Chippendale, 1971; 51 Woodring et al., 1979), but almost all insects have a specific dietary requirement for sterols and 52 polyunsaturated fatty acids (Canavoso et al., 2001). Many insects can obtain the essential polyunsatured 53 fatty acids by digestion of phospholipids via secretion of phospholipase A2 from the midgut endothelium 54 (Rana & Stanley, 1999). Crickets have a very lipid rich fat body (over 50% triglycerides) (Lorenz & 55 Gäde, 2009), but these lipids are primarily derived from ingested carbohydrates. Crickets, however do 56 synthesize and release significant amounts of lipases into the midgut (Teo & Woodring, 1988; Woodring 57 et al., 2009), meaning that they can use nutrient lipids (triglycerides) for energy stores, but they are not 58 required. 59 Complete lipid digestion is accomplished by carboxylic ester hydrolases (EC 3.1.1; lipases, esterases, 60 phospholipase A and B), phosphoric monoester hydrolases (EC 3.1.3; phosphatases) and phosphoric 61 diester hydrolases (EC 3.1.4; phospholipase C and D) (Terra et al., 1996). Thereby, lipases (EC 3.1.1.3) 62 are essential compounds of the fat metabolism and hydrolyse the outer ester links of triacylglycerols 63 from the α-position stepwise to diacylglycerols, monoacylglycerols, glycerol and free fatty acids 64 (Bollade et al., 1970; Hoffman & Downer, 1979; Secundo et al., 2006). 65 Lipid metabolic activities in the tissues of insects are well characterized (Canavoso et al., 2001; Arrese 66 et al., 2001; Van der Horst & Ryan, 2012), but to date there are only a few reports on digestive lipases 67 Publications 73 from relatively few species., and secretion from gut tissue is not well understood (Weintraub & Tietz, 68 1973; Male & Storey, 1981; Mrdaković et al., 2008; Horne et al., 2009; Woodring et al., 2009; Lwalaba 69 et al., 2010; Christeller et al., 2010, 2011; Zibaee, 2012; Zibaee & Fazeli-Dinan, 2012 ). The current 70 study focuses on the effect of extrinsic factors (temperature, light-dark cycle, food consumption) and 71 intrinsic factors (age, developmental stage, sex) on the release of digestive lipase in the midgut of 72 G. bimaculatus. 73 Publications 74 Materials and methods 74 Rearing method and feeding 75 The Mediterranean field cricket G. bimaculatus de Geer (Ensifera, Gryllidae) was raised under a long76 day regime (LD 16:8 photocycle, light from 6 a.m. to 10 p.m. CEST) at 27°. Newly emerged crickets 77 were isolated within 1 h after the imaginal moult and were designated 0-day-old crickets. Crickets 78 received a mixed diet (cricket chow) consisting of ground rabbit, rat and cat food in a ratio of 4:2:1 79 (w/w), all from Altromin Lage, Germany. The total nutrient value of the cricket chow was 40 % 80 carbohydrates, 25 % protein, and 6 % lipids (Lorenz & Anand, 2004). 81 The in vivo effect of feeding and starvation on enzyme release was tested by providing standard agar 82 diet (40 g cricket chow + 3.6 g agar + 160 ml H2O) and a non-nutrient cellulose diet (120 g cellulose 83 powder + 12 g agar per litre H2O) or by starvation (access to water, but no food at all). After imaginal 84 moult crickets were placed individually into boxes and provided a fresh cube of an agar-diet or no food 85 at all. 86 To investigate the influence of temperature acclimation on lipase release crickets were divided into two 87 groups, one maintained at 22°C the other at 32°C (acclimation temperature, AT). Each of these two 88 groups was divided into three groups, which were set to AT from (a) day 0 of the penultimate instar, (b) 89 day 0 of the last instar, and from (c) day 0 of the adult life. Hence the crickets were exposed to the two 90 AT for a short, intermediate or long period of time. During this time crickets were kept individually in 91 boxes (10x10x6 cm). For each of the total of six groups the caeca were removed on day 2 of the adult 92 stage, divided in half and each half incubated in low glucose Ringer at either 25°C or 35°C incubation 93 temperature (IT) for 30 min. The results are given per mg caeca to compensate for the different size of 94 each half. In addition, food uptake was determined for crickets acclimated at both 22°C and 32°C from 95 the beginning of last larval stage. 96 Gut dissection and sample preparation 97 The crickets were ventrally cut open from the last abdominal segment to the neck. The caeca were 98 removed, cut open and rinsed 3-times with Gryllus Ringer (138 mM NaCl, 5 mM KCl, 2 mM 99 CaCl2∙2 H2O, 4 mM Hepes, pH 7.2). Contraction of the muscles of the opened caeca leads to a cup100 shaped structure with the lumen side outermost and the hemolymph side innermost, which was 101 designated a flat-sheet gut preparation (Blakemore et al., 1995). 102 To determine the lipase release (secretion), the opened and rinsed caecal tissue of individual crickets 103 were incubated in low glucose Ringer (LGR) (10 mg glucose / 100 ml Gryllus Ringer) for 30 min at 104 37°C without shaking. The air-filled trachea kept the caeca at the surface of the medium. Following 105 incubation, the caecal tissue was discarded and cells were removed by centrifugation at low speed 106 (2000 g) for 2 min at 4°C. 107 Publications 75 To determine lipase activity in tissue cells, rinsed caecal tissue of individual cricket was added to 108 150 - 200 µl Gryllus Ringer with a few crystals of N-phenylthiourea (PTH) and homogenized with an 109 ultrasonicator at the lowest setting for few seconds (Sonifier 250, Branson). Tissue homogenate (TH) 110 was centrifuged at 16000 g for 10 min at 4°C and the supernatant was used for the enzyme assay. 111 To test the lipase activity in caecal lumen content, 10 µl aliquots of luminal contents were mixed with 112 190 µl Gryllus-Ringer with PTH and centrifuged at 16000 g at 4°C for 2 min. 113 Enzyme assay 114 Lipase activity was measured using the substrate p-nitrophenyl palmitat (pNPP) (Winkler & Stuckman, 115 1979). 15 mg pNPP was dissolved in 5 ml 2-propanol by heating to about 50°C until clear (8 mM pNPP 116 stock solution). The stock solution was diluted with 50 mM Tris-HCl buffer, pH 8 + 0.1 % Triton X100 117 and gently mixed or heated until a clear suspension resulted (0.4 mM working solution). The Triton X 118 prevented the formation of a turbid suspension (Gupta et al., 2002). 119 190 µl of a freshly prepared 0.4 mM working solution pNPP was added to 10 µl sample and the change 120 in absorbance at 410 nm over 30 min at 25°C was measured in 96 well microplates using a microplate 121 reader (Synergy HT, BioTek). 122 The optimal temperature and pH of lipase was measured using pNPP as substrate. The optimal 123 temperature of the lipase (at pH 8.2) was very broad (30 - 40°C) with a slight peak around 37°C, and 124 the optimal pH (at 25°C) was about 8.5. 125 In vitro effect of nutrients on enzyme release 126 The effect of nutrients on the in vitro release of lipase from caecal epithelium was tested by the addition 127 of glucose (1 - 4 mg nutrient/ml LGR) and oleic acid (0.05 - 0.1 mM) to the incubation medium (LGR) 128 containing freshly rinsed caeca of one cricket. Lipase activity in the incubation medium without any 129 added nutrients (control) was set to 100 % for comparison to media with nutrients. 130 Statistical analyses 131 The SigmaPlot 11.0 program (Systat Software GmbH) was used to evaluate the data. All data were 132 statistically tested for homogeneity of variance (Levene’s test) and normal distribution (Shapiro-Wilk 133 test). Paired t-test was used for linked data of the effect of incubation temperature on lipase release. 134 Independent data were evaluated using either ANOVA or Kruskal-Wallis test and individual post hoc 135 analysis. For statistical analysis data of temperature effect (Fig.7) and light-dark cycle (Fig. 5A) on 136 enzyme release were normalized by log10 transformation, data of food uptake presented as crop weight 137 (Fig. 5B) were normalized by square root transformation. The statistical significance is designated in 138 the graphs and text. 139 Publications 76 Results 140 Lipase activity was measured in the luminal content, tissue homogenate and secretion of two day old 141 fed G. bimaculatus. In females lipase activity was nearly equal in the luminal content and tissue 142 homogenate, but was significantly higher than that secreted over 30 min (Fig. 1A). In male crickets 143 lipase activity showed a similar trend with higher enzyme activity in luminal content (Fig. 1B). 144 Both, a non nutrient diet (cellulose diet) as well as starvation caused a significantly lower lipase activity 145 in the luminal content and decreased lipase secretion from caecal epithelium. In starved and cellulose 146 fed crickets lipase activity in luminal content was about 90 % less than in fed ones (Fig. 2A), but lipase 147 release was reduced by only about 50 % (Fig. 2B). The in vitro effect of nutrients on lipase release was 148 tested by incubation of caecal tissue in low glucose Ringer with the addition of either glucose or oleic 149 acid. There was a dose dependent increase of lipase release in response to oleic acid (0.05 - 0.1mM) 150 (Fig. 3B), but glucose showed no effect (Fig. 3A). 151 Feeding behaviour in insects is correlated to the quality and availability of food, but also related to 152 ontogenesis. The age-dependent lipase release in G. bimaculatus (Fig. 4) showed increasing activity 153 from day 0 to 5 of the last instar larvae. There was no difference in lipase secretion between male and 154 female larvae with a maximum activity of 65-78 nmol pNP/30min at day 5, and the lipase activity 155 decreased from day 5 to 8 (5.9-6.4 nmol pNP/30min). After the imaginal moult lipase release in female 156 crickets increased rapidly from 8 to 100 nmol pNP/30min within five days, whereas lipase activity in 157 males remained low (~30 nmol pNP/30min). From day 6 lipase secretion in males increased reaching 158 maximum at day 7, whereas lipase release in females declined. In general however, there was a higher 159 lipase release in adult females than in males. 160 Lipase secretion from caecal epithelium (Fig. 5A) and food uptake (crop weight) (Fig. 5B) was followed 161 over a period of one day (24 h) in the last two larval stages of females and in the adults. Crickets were 162 reared at 27°C under a long-day regime with a photophase from 6:00 to 22:00 CEST (see materials and 163 methods). There was a significant effect of stage and time on lipase release. Adult crickets and last instar 164 larvae showed increasing lipase secretion from 4:00 to 24:00 CEST, reaching a maximum (116 - 136 165 nmol pNP/30min) in the late photophase to early scotophase (22:00-24:00 CEST) (Fig. 5A). In 166 penultimate larvae there was no clear trend of increasing lipase activity over 24 h. The crop weight of 167 crickets was determined at the time of dissection (Fig. 5B). There was a significant effect of time and 168 stage on the crop weight of the crickets with a significant interaction, which indicates differences in 169 feeding behaviour (time of food uptake) within individual developmental stages. The crop weight of last 170 instar larvae and adult crickets was highest at 24:00 CEST, with a higher food uptake to the beginning 171 of darkness (Fig. 5B). In contrast, the crop of penultimate larvae was filled at the end of the scotophase 172 at 4:00 CEST. However, there was no correlation between crop weight and lipase release of any 173 developmental stage (Spearman rank order: p > 0.05) (data not shown). 174 Publications 77 Temperature is one of the most important environmental factors that directly influence the metabolic 175 rate, growth, food consumption and enzyme secretion in insects. At an incubation temperature of either 176 25°C or 35°C there was no effect on lipase release from caecal tissue of crickets acclimated at 22°C 177 (Fig. 6A) or 32°C (Fig. 6B) from the time of the imaginal moult or those acclimated since the moult to 178 last larval instar. But there were slight but significant differences in females reared since the moult to 179 the penultimate instar. Lipase release increased at 35°C IT when reared at 22°C, but decreased at 35°C 180 when reared at 32°C (Fig. 6A,B). However, lipase release was always higher in crickets reared at 32°C 181 than those reared at 22°C (Fig. 7). Furthermore, there was a significant effect of exposure time to the 182 two acclimation temperatures on lipase release and a strong interaction of rearing temperature and 183 exposure time. Lipase release increased the longer crickets were acclimated at 32°C, but not when 184 acclimated at 22°C (Fig. 6). 185 Crickets acclimated at 22°C, compared to those acclimated at 32°C from the beginning of the last instar 186 or penultimate instar showed retarded (slower) progress in development the longer they were exposed 187 to the lower temperature (Table 1). Interestingly the total amount of consumed food throughout the last 188 instar did not differ between crickets acclimated at 22°C or 32°C (Fig. 8), but the daily amount of food 189 uptake at 22°C was greatly reduced (Fig. 9). At 22°C food uptake increased over the first three days to 190 80 - 100 mg food/body weight [g] and stayed the same until day 13 of last instar. Thereafter, feeding 191 gradually decreased until the final moult (Fig. 9A). Food uptake of crickets reared at 32°C on the other 192 hand increased quickly from day 0 to 2 with maximum of 400 mg food/g body weight, and decreased 193 rapidly from day 3 to 6 prior to the final moult (Fig. 9B). After the imaginal moult the daily food uptake 194 within the first three days was three times higher at 32°C (~300 mg food/ g body weight) compared to 195 22°C (~100 mg/g body weight), and also the total amount of consumed food through adult life was 196 significant higher at 32°C (Fig. 8). 197 198 Publications 78 Discussion 199 Temperature is one of the most important abiotic factors, that influence insect development (time, 200 number of stages, growth rate) and biology (food consumption, metabolic rate, fertility, locomotion, 201 reproduction, digestion), and therefore its life history and phenotypic plasticity (Chown & Terblanche, 202 2007). Insects are ectothermic and the rate functions of most activities are related to ambient temperature 203 (Hoffmann, 1974; Merkel, 1977, Behrens et al., 1983; Haderspeck & Hoffmann, 1991; Booth & Kiddell, 204 2007; Lachennicht et al., 2010). Increasing rearing temperature from 22°C to 32°C led to the expected 205 decrease in the duration of the last instar and penultimate instar of G. bimaculatus. All reported digestive 206 enzymes have in vitro temperature optima, however, these optima often do not correspond to the ambient 207 temperature (Terra et al., 1996). According to Woodring et al. (2009) the temperature optimum for 208 lipase in G. bimaculatus is about 37°C at an optimal pH 8.0. Incubation temperature of caecal tissue 209 (25°C or 35°C) has no effect on enzyme release in 2-day adults after short-term acclimation (since the 210 beginning of last instar or since adult ecdysis), but there was a slight difference with longer acclimation 211 (since the beginning of the penultimate larval instar), which may indicate a release of different isozymes 212 with higher activity at higher temperatures. 213 An acclimation of the rate of digestive enzyme secretion has scarcely been investigated in insects 214 (Weidlich et al., 2012). The higher acclimation temperature of 32°C led to a significant increase in lipase 215 release of adult crickets over different exposure times compared to those reared at 22°C. This indicates 216 no acclimation of a rate function in the classical sense, as defined by Prosser (1991). The trypsin 217 secretion in G. bimaculatus on the other hand showed a higher rate of secretion after acclimation to 218 22°C than when acclimated at 32°C (Weidlich et al., 2012), which is the classical positive temperature 219 acclimation pattern. Temperature has a direct stimulatory effect on the amount of lipase released and a 220 higher temperature also appears to stimulate lipase synthesis, in that more lipase is secreted at a higher 221 incubation temperature (35°C compared to 25°C). Moreover, lipase synthesis is strongly influenced by 222 exposure time at different acclimation temperatures. The longer crickets were reared at 32°C, the more 223 lipase was synthesized, stored in endothelial cells and subsequently secreted. 224 In insects food consumption is correlated to the sex, age, developmental stage, rearing temperature and 225 light-dark cycle, and these factors (via food consumption) also influence the release of digestive 226 enzymes. The secretion of lipase in the caeca of G. bimaculatus is similar to that of trypsin, amylase and 227 cellulase (Weidlich et al., 2012, 2013), and is directly related to food uptake (Woodring & Lorenz, 228 2007). Elevated lipase release in female crickets might be associated with a greater need of lipids for 229 egg production (Espig & Hoffmann, 1985). In both sexes last instar larvae showed a rapid decline of 230 lipase release from day 6 to 8 associated with the preparation of the final moult in that the gut is emptied 231 and much less or no food is consumed (Anand & Lorenz, 2008). 232 Digestive enzyme release in G. bimaculatus depends on both quantity and quality of the diet. Lipase 233 secretion and lipase activity in the luminal contents in starved and non-nutrient (cellulose) fed crickets 234 Publications 79 was greatly reduced compared to diet-fed crickets. Specific nutrients in the diet stimulate the release of 235 lipase. Oleic acid, for example, when added to the incubation medium of caeca leads to a significant 236 increase of lipase secretion, indicating a prandial release mechanism, similar to that reported for trypsin 237 and amylase (Woodring et al., 2009). Rana & Stanley (1999) already reported a stimulatory effect of 238 the presence of phospholipids on the secretion of phospholipase A2 in the midgut of Manduca sexta. 239 Interestingly, glucose in the incubation medium has no effect on lipase release. 240 There was a significant effect of photoperiod and developmental stage on lipase secretion in 241 G. bimaculatus. Feeding and lipase secretion was highest at the beginning of the scotophase (24:00 242 CEST) in the last instar and in the adult stage, but for unknown reasons not in the penultimate instar. 243 Last instar larvae and adult crickets are basically nocturnal, in that locomotory activities take place in 244 the scotophase (Nowosielski & Patton, 1963; Nielsen & Dreisig, 1970; Loher, 1972; Tanaka et al., 1999; 245 Lorenz, 2007). The increase of food uptake and lipase secretion was shown to result from an increase 246 of locomotory activity in Acheta domesticus (Woodring & Clifford, 1986). A similar effect was also 247 reported for trypsin secretion in G. bimaculatus (Weidlich et al., 2012). Both sexes of last instar 248 G. bimaculatus show an age-dependent cyclic pattern of activity with maxima during early to mid 249 scotophase and minima during early photophase. After the imaginal moult the crickets show a 250 continuing cyclic of activity until day 6 of adult life (Faßold et al., 2010). 251 In insects the pars intercerebralis is involved in the circadian regulation of activity levels (Matsui et al., 252 2009). Studies on Periplaneta americana showed increasing locomotion, food consumption, as well as 253 amylase and protease activity in the dark phase (Matsui et al., 2009). Furthermore digestive enzyme 254 release in the insect midgut underlies the influence of neuropeptides (Lehane et al., 1995; Fusé et al., 255 1999; Harshini et al., 2002a, b; Aguilar et al., 2003; Hill & Orchard, 2005; Sakai et al., 2006; Woodring 256 et al., 2009; Lwalaba et al., 2010). 257 In conclusion, lipase secretion in G. bimaculatus is strongly influenced by endogenous and 258 environmental factors. Higher temperatures associated with longer exposure to different acclimation 259 temperatures lead to an increase of lipase synthesis in caecal tissue and, therefore, to an increased 260 secretion. Lipase secretion is correlated to sex, age, developmental stage, and circadian activity rhythm 261 of the crickets, and all these factors influence feeding behaviour. In general increased food intake results 262 in increased lipase release. 263 Acknowledgements 264 We thank Juliane Huster and Sonja Müller for proving part of samples on age-dependent enzyme 265 release and in vitro studies of nutrients. We also thank Marion Preiß for technical assistance. 266 Publications 86 standard cellulose starved standard cellulose starved lipase activity [nmol pNP/30min] 0 20 40 60 80 100 A B a bb a bb diet diet diet diet 439 Fig. 3: In vitro effect of glucose (A) and oleic acid (B) on lipase release from incubated caeca (37°C, 440 30 min) of 2-day-old adult G. bimaculatus females. Lipase activity of controls (0 mg/ml glucose, 441 0 mM oleic acid) was set 100 %. Mean ± SEM. n = 10. Statistics: Kruskal-Wallis test (A: H = 442 3.524, df = 3, p > 0.05; B: H = 19.424, df = 2, p < 0.001) and post hoc Tukey test. Different 443 letters indicate significant differences. 444 age [d] 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 9 10 lipase activity [nmol pNP/30min] 0 20 40 60 80 100 120 last instar larvae adult stadium 445 Fig. 4: Age-dependent activity of lipase secretion (30 min incubation at 37°C) of male (○) and female 446 (●) last instar larvae and adults of G. bimaculatus. Mean ± SEM. n = 10. 447 Publications 87 a b a,b a,b a,b a,b a,b aa a,b a,b ba,b a,b aa,b a,b b a,c b,c a c,d d,a b,c,d aa,b bbbb aa,b a c cb,c time of day 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 crop weight [mg] 0 50 100 150 B time of day 4 8 12 16 20 24 4 8 12 16 20 24 4 8 12 16 20 24 lipase activity [nmol pNP/30min] 0 50 100 150 200 Apenultimate larvae penultimate larvae last instar larvae last instar larvae adult stadium adult stadium 448 Fig. 5: Lipase release (A) and crop weight (B) of 2-day-old penultimate larvae, last instar larvae and 449 adult females of G. bimaculatus over a 24 h period. Scotophase was from 22:00 to 6:00 CEST 450 (grey). Mean ± SEM. n = 9-10. 451 Statistics: (A) two-way ANOVA: stage (F2,178 = 20.09; p < 0.001); time (F5,178 = 11.64; p < 452 0.001); interaction stage*time (F10,178 = 4.76, p < 0.001). (B) two-way ANOVA: stage (F2,178 = 453 89.68; p < 0.001); time (F5,178 = 6.62; p < 0.001); interaction stage*time (F10,178 = 2.18; p = 454 0.022). Post hoc comparison (Tukey test) for factor time within individual stages. Different 455 letters indicate significant difference. 456 Publications 88 ALI PI ALI PI lipase activity [nmol pNP/30min/mg caeca] 0 2 4 6 incubation at 25°C incubation at 35°C A B ns ns ns ns * ** 457 Fig. 6: The effect of incubation temperature on lipase secretion from caecal epithelium of 2-day-old 458 adult female G. bimaculatus acclimated at (A) 22°C and (B) 32°C for three different exposure 459 times. A = from day 0 of adult stadium, LI = from day 0 of last instar, PI = from day 0 of 460 penultimate instar. Mean ± SEM. n = 20-40. Statistics: paired t-test. ns = not significant, * = p 461 < 0.05, ** = p < 0.01. 462 exposure time ALI PI lipase activity [nmol pNP/30min/mg caea] 0 1 2 3 4 5 6 22°C 32°C 463 Fig. 7: Lipase secretion from caecal tissue of female G. bimaculatus acclimated at 22°C and 464 32°Cfor three different exposure times (see Fig. 6). Mean ± SEM. n = 19–36. 465 Statistics: two-way ANOVA: temperature (F1,139 = 236.71, p < 0.001); exposure time (F2,139 = 466 24.5, p < 0.001); interaction temperature*exposure time (F2,139 = 19.54, p < 0.001). 467 Publications 89 last instar larvae adult food uptake [mg] 0 1000 2000 3000 22°C 32°C ns *** 468 Fig. 8: Average food uptake [mg] of female G. bimaculatus in last larval instar and adult stadium. Mean 469 SEM. n = 5-10. Statistics: last instar larvae (t-test: t = -0.863, df = 13, p > 0.05), adult (t-test: t 470 = -9.543, df = 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 food uptake [mg] per body weight cricket [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 food uptake [mg] per body weight cricket [g] 0 100 200 300 400 500 600 adult stage last instar larvae adult stagelast instar larvae A B 472 Fig. 9: Age-dependent food uptake of female G. bimaculatus through last larval instar and adult 473 stadium acclimated at (A) 22°C and (B) 32°C. Mean ± SEM. n = 5-10. 474 Publications 90 Tables Table 1: Duration of the last two larval instars [days] of female G. bimaculatus acclimated at 22°C and 32°C and the total exposure time at acclimation temperature until dissection. Mean ± SEM. n = 19-36. since penultimate instar since last instar 22°C 32°C 22°C 32°C penultimate instar 15.26 ± 0.20 5.00 ± 0.00 - - last instar 18.16 ± 0.23 6.20 ± 0.09 16.37 ± 0.21 6.56 ± 0.13 total exposure time 33.75 ± 1.71 13.20 ± 0.09 17.55 ± 0.84 8.36 ± 0.08 Publications 91 Publication 4 Weidlich S., Hoffmann K.H. and Woodring J. Activation and autolysis of trypsin in the midgut of the Mediterranean field cricket, Gryllus bimaculatus. will be submitted shortly Publications 92 Activation and autolysis of trypsin in the midgut of the Mediterranean field cricket, Gryllus bimaculatus. Authors: Sandy Weidlich1, Klaus H. Hoffmann1 and Joseph Woodring2 Institution: 1 Department of Animal Ecology I, University of Bayreuth, 95440 Bayreuth, Germany 2 Department of Animal Ecology II, University of Bayreuth, 95440 Bayreuth, Germany Abbreviated title: Activation and autolysis of Gryllus trypsin Correspondence to: Sandy Weidlich, Department of Animal Ecology I, University of Bayreuth, 95440 Bayreuth, Germany Email: [email protected] Publications 93 Abstract In G. bimaculatus the cells of the midgut epithelium synthesize trypsin precursor (TP), which is stored in cytoplasmic vesicles. TP of rinsed trypsin-free caecal tissue was free from vesicles by sonification. The complete self-activation of TP in vitro requires about 60 min. The maximum self-activation (measured by hydrolysis of BApNA) was 4-times higher in homogenates from fed crickets compared to starved crickets, indicating a positive influence of feeding on TP synthesis in the epithelial cells. Neither the addition of Gryllus-trypsin (from lumen contents) nor bovine trypsin (Sigma), when added to the tissue homogenate, accelerated the maximum activity. The presence of calcium ions in the incubation medium resulted in increased secretion of TP from caecal endothelium, indicating an exocytosis mechanism of release. The trypsin activity of incubated lumen content retained its activity over a period of at least 4 h, whereas a bovine trypsin solution lost 80% of its activity in 30 min. It is suggested that insect trypsin in the digestive tract is protected from autolysis by the presence of peptides, a mechanism long known in mammalian systems, Key words: digestive enzyme, zymogen, trypsin, protease, cricket, autolysis, activation Publications 94 Introduction Proteases are enzymes with a wide range of physiological roles, including the digestion of dietary proteins, the removal of damaged tissues, and have highly specialized roles in various activation or inhibitory cascades (Kanost and Clem, 2012). Thus proteases are required in a wide range of vital processes such as digestion, growth, fertilization, immunological reactions, wound healing and cell death (Lazure, 2002). Serine proteases, especially trypsins, occur in the digestive tract of almost all animals, and there is an extensive literature on the occurrence, distribution, secretion, characteristics, and effects of intrinsic or extrinsic factors regulating the secretion (Applebaum, 1985; Davis et al., 1985; Chapman, 1989; Moffat and Lehane, 1990; Graf et al., 1991; Ramos et al., 1993; Terra and Ferreira, 1994; Terra et al., 1996a,b; Cristofoletti et al., 2001; Woodring et al., 2007, 2009; Weidlich et al., 2012). Inspite of this, there are three rather simple questions concerning insects trypsins that remain inadequately answered. First, what is the secretory mechanism of trypsin in insects? Two, is trypsin secreted as a precursor molecule, that must be activated or is it secreted in an active form? Third, how stable is trypsin once secreted and activated? Most proteolytic enzymes are indeed synthesized as inactive precursors (deAlbuquerque et al., 2001; Khan and James, 1998; Lazure, 2002), which also enables a spatial and temporal regulation of enzyme activity. Trypsin precursors (TP) of vertebrates are activated by hydrolysis of short polypeptide chains between the amino acids isoleucine and lysine or arginine, and by changing conformation of active substrate binding sites (Ehrmann and Clausen, 2004; Pasternak et al., 1999; Walsh, 1970).Trypsin and TP were early targets of protein sequencing studies in vertebrates, but TP has also been described in some insect species (Davis et al., 1985; Moffatt and Lehane, 1990; Graf et al., 1991; Ramos et al., 1993). Trypsin isoforms of various lengths present in the midgut of Locusta migratoria probably indicates the presence of precursor proteins (Lam et al., 2000). In Musca domestica membrane-bound and newly synthesized TPs are stored in vesicles of the endothelium, which later on fuse with the plasma membrane releasing their content into the lumen (Lemos and Terra, 1992; Terra and Ferreira, 1994; Jordão et al., 1996). The regulation of protease activity includes microenvironmental factors (pH, ions), gene regulation, synthesis of specific inhibitors, substrate inhibition, or cascade regulation (Lazure, 2002). Several endogenous serine protease inhibitors have already been identified in various cockroach species (Elpidina et al., 2001a,b; Engelmann and Geraerts, 1980; Vinokurov et al., 2007; Zhuzhikov, 1997), but such endogenous trypsin inhibitors were not found in G. bimaculatus (Weidlich et al., 2012). Furthermore, autolysis is an important factor in protease regulation too. In this study the presence of a putative trypsin precursor in G. bimaculatus and its activation was investigated, and the influence of feeding on the synthesis rate was determined. Finally, the autolysis of native Gryllus-trypsin was compared to that of bovine trypsin. Publications 95 Materials and method Rearing methods The Mediterranean field cricket, G. bimaculatus de Geer (Ensifera, Gryllidae), was raised under a longday regime (LD 16:8 h photocycle) at 27°C. Newly-emerged females were isolated within one hour after the imaginal moult (before they started to feed) and were designated 0-day old. Crickets received a mixed diet (cricket chow) consisting of ground rabbit, rat and cat food in a ratio of 4:2:1 (w/w), all from Altromin Lage, Germany. The total nutrient value of the chow was 40% carbohydrate, 25% protein, and 6% lipids (Lorenz and Anand, 2004). The optimal growth (maximal weight gain and shortest time) for G. bimaculatus fed a diet containing from 10 to 50% casein was achieved with a 30% casein diet (Merkel, 1977). To compare the activation of trypsin precursor in fed and starved G. bimaculatus, crickets were placed individually into boxes and provided a cube of an agar-diet (40 g cricket chow + 3.6 g agar + 160 ml water) or no food at all (access to water). Gut dissection and sample preparation The crickets were ventrally cut open from the last abdominal segment to the neck. The caeca were removed, cut open and rinsed 3-times with Gryllus Ringer (138 mM NaCl, 5 mM KCl, 2 mM CaCl2∙2 H2O, 4 mM Hepes, pH 7.2) (GR). This assured that perhaps more than 95% of the digestive enzymes between the intricate folds of the epithelial tissue were removed. A spontaneous contraction of the external muscles of the caeca led to the formation of an open, cup shaped structure with the lumen side outermost and the hemolymph side inner most(termed a flat-sheet gut preparation; Blakemore et al., 1995). Therefore, both sides of the caecal epithelium were equally exposed to the medium during incubation. The caeca are richly supplied with large trachea (Woodring and Lorenz, 2007) and these remain connected, so that the preparation floats on the surface of the incubation medium. The role of calcium ions The rinsed caecal tissue of single crickets was divided in half. One half was incubated in low glucose Ringer (10 mg glucose/100 ml Gryllus Ringer)(LGR) either with or without calcium ions at 37°C for 30 min. Afterwards, the tissue was discarded, and the supernatant (incubate) was centrifuged at 2,000 g for 2 min and subsequently assayed for trypsin activity. Activation of trypsin precursor Fresh rinsed caeca were transferred to 600 µl 50 mM phosphate buffer (pH 7.2) containing few crystals of N-phenolthioureat (to inhibit phenoloxidase) and were homogenised at the lowest setting for few seconds with ultrasonicator (Branson Sonifier 250). The sample was centrifuged at 11,000g for 10 min, the pellet discarded and the supernatant was frozen at -20°C. The homogenate contained little or no trypsin, but sonication and freezing disrupted all vesicles and thereby released TP. Afterwards, the Publications 102 Sipos T, Merkel JR. 1970. An effect of calcium ions on the activity, heat stability, and structure of trypsin. Biochemistry 9:2766-2775. Spiess C, Beil A., Ehrmann M. 1999. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97:339-347. Terra WR, Ferreira C, Baker JE. 1996a. Compartmentalization of digestion. In: Lehane MJ and Billingsley PF, editors. Biology of the insect midgut. London: Chapman & Hall. p 206-235. Terra WR, Ferreira C, Jordão BP, Dillon RJ. 1996b. Digestive enzymes. In: Lehane MJ and Billingsley PF, editors. Biology of the insect midgut. London: Chapman & Hall. p 153-194. Terra WR, Ferreira C. 1994. Insect digestive enzymes: Properties, compartmentalization and function. Comp Biochem Physiol B Biochem Mol Biol 109:1-62. Vajda T, Garai A. 1981. Comparison of the effect of calcium (II) and manganese (II) ions on trypsin autolysis. J Inorg Biochem 15:307-315. Vinokurov K, Taranushenko Y, Krishnan N, Sehnal F. 2007. Proteinase, amylase, and proteinaseinhibitor activities in the gut of six cockroach species. J Insect Physiol 53:794-802. Walsh KA. 1970. Trypsinogens and trypsins of various species. Methods Enzymol 19:41-63. Weidlich S, Huster J, Hoffmann KH, Woodring J. 2012. Environmental control of trypsin secretion in the midgut of the two-spotted field cricket, Gryllus bimaculatus. J Insect Physiol 58:1477-1484. Woodring J, Diersch S, Lwalaba D, Hoffmann KH, Meyering-Vos M. 2009. Control of the release of digestive enzymes in the caeca of the cricket Gryllus bimaculatus. Physiol Entomol 34:144–151. Woodring J, Hoffmann KH, Lorenz MW. 2007. Activity, release and flow of digestive enzymes in the cricket Gryllus bimaculatus. Physiol Entomol 31:1-8. Woodring J, Lorenz MW. 2007. Feeding, nutrient flow and functional gut morphology in the cricket Gryllus bimaculatus. J Morphol 268:815-825. Zhuzhikov DP. 1997. Inhibitor of serine proteinases in intestine of cockroach Nauphoeta cinerea. J Evol Biochem Physiol 33:524-528. Publications 103 Figures Fig. 1 In vitro effect of calcium ions on trypsin secretion from caecal epithelium of 2-day-old G. bimaculatus adults. Mean ± SEM. n = 10-15. Statistics: paired t-test: Pfemale = 0.001 (**), Pmale = 0.051 (#) Fig. 2: Self-activation of trypsin precursor from caecal tissue in starved (A) and fed (B) 2-day-old female G. bimaculatus. Tissue homogenate was incubated at 37°C for 4 h and aliquots of 50 µl were measured for trypsin activity every 30 min. MW ± SEM. n = 20. Statistics: Kruskal-Wallis test (P < 0.001) and post hoc Dunn’s method. Different letters indicate significant differences. female male trypsin activity [nmol pNA/min/mg tissue] 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 LGR without calcium LGR with calcium ** # time [min] 030 60 90 120 150 180 210 240 trypsin activity 0 50 100 150 200 030 60 90 120 150 180 210 240 [nmol p-nitroaniline/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 Publications 104 Fig. 3: Activation of trypsin precursor in caecal tissue homogenate of starved (A)and fed (B) 2-dayold adult G. bimaculatus females over 2 h at 37°C by addition of Gryllus-trypsin from lumen content. Aliquots of 100 µl were taken to measure trypsin activity every 30 min. MW ± SEM. n = 30. Statistics: (A) Repeated measurement ANOVA (P < 0.001) and post hoc Tukey-test, (B) Friedman-Test (P < 0.001) and post hoc Tukey-test. Different letters indicate significant differences. Fig. 4: Activation of trypsin precursor in 2-day-old adult starved (A)and fed (B) female G. bimaculatus induced by addition of 20 µl bovine trypsin (1 µg/µl) during incubation at 37°C for 2 h. Aliquots of 100 µl were measured for trypsin activity every 30 min. MW ± SEM. n = 15. Statistics: Friedman-Test (P < 0.001) and post hoc Wilcoxon-Test + Bonferroni correction. Different letters indicate significant differences. time [min] 030 60 90 120 trypsin activity [nmol p-nitroaniline/min] 0 100 200 300 400 030 60 90 120 aabbb a b cc c AB 030 60 90 120 trypsin activity [nmol p-nitroaniline/min] 0 100 200 300 400 time [min] 030 60 90 120 AB abc c d a bcbb Publications 105 Fig. 5: Activity of Gryllus-trypsin, bovine trypsin and a mixture of Gryllusand bovine trypsin during 4 h incubation at 37°C. Aliquots of 50 µl were measured for trypsin activity every 30 min. MW ± SEM. n = 10-20. time [min] 030 60 90 120 150 180 210 240 trypsin activity [%] 0 50 100 150 200 bovine trypsin Gryllus trypsin Gryllus trypsin + bovine trypsin Acknowledgments 106 Acknowledgements I want to thank all colleagues and persons who contributed to this work. First I thank Prof. Dr. Klaus H. Hoffmann who enabled and supported this project, provided guidance and advice whenever needed. Prof. Dr. Joseph Woodring became a true mentor and friend for me. He positively influenced my work and encouraged me at all times. It was a pleasure to work with him side by side and I am very grateful for his support, guidance and advices during my work and the writing of manuscripts. He is an inspiration for every scientist to enjoy work and never lose sight of your aims. Carmela Herrmann has been the heart and the soul of the Department and took care of organization, logistics and helped with all questions and problems. Special thanks go to Marion Preiß who became indispensable assistance because of her dedication and patience. I am grateful for the support and technical assistance of M.Sc. Sonja Müller, M.Sc. Alexander Meyer, Ursula Wilczek and Dorothea Wiesner. I also thank all my colleagues who created a very friendly working environment and helped with scientific discussions and teaching: Dr. Franziska Wende, PD Dr. Martina Meyering-Vos, Stefanie Schapp (Department of Animal Ecology I), Dr. Stefan Küchler, Dr. Siegfried Kehl (Department of Animal Ecology II), Prof. Dr. Heike Feldhaar, Dr. Oliver Otti and Dr. Simon Tragust (Department of Animal Ecology I - AG Population Ecology). Furthermore I want to thank my bachelor students and student assistants Jörn Herfert, Mario Schwartz, Sandra Walther, Bastian Schauer and especially Juliane Huster for their help and contributor work. Sincere thanks are given to all professors and collaborators of the different departments of biology and chemistry who supported my scientific and teaching work. I thank the board of professors of the European PhD Network of Insect Science and Biotechnology who gave me the opportunity to present and discuss my work in a very friendly and kind atmosphere and to extend my network to new young scientists. Above all, I thank my family and friends for their personal support and their contribution to become Bayreuth a new home. Declaration 107 Declaration Hiermit versichere ich, Sandy Weidlich, die vorliegende Arbeit selbstständig verfasst und keine anderen als die von mir angegebenen Quellen und Hilfsmittel benutzt zu haben. Darüber hinaus versichere ich, dass ich diese oder eine gleichartige Dissertation nicht anderweitig versucht habe einzureichen und mich keiner gleichartigen Doktorprüfung, mit oder ohne Erfolg, an einer anderen Hochschule unterzogen habe. I, Sandy Weidlich, declare that this thesis hereby submitted for the Doctor degree at the University of Bayreuth is my own work and has not been previously submitted by me at any another University for any other degree. The work is original except where indicated by special reference in the text and has not been presented to any other University for examination. Bayreuth, Mai 2013 __________________ Sandy Weidlich