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Regulation of digestive enzyme levels in insects
Comp. Biochem. Physiol. Vol. 110B, No. 2, pp. 285-289, 1995
~
Elsevier Science Ltd Printed in Great Britain 0305-0491/95 $9.50 + 0.00
Pergamon
0305-0491(94)00157-X
MINI REVIEW Regulation of digestive enzyme levels in insects M. J. Lehane, D. Blakemore, S. Williams and M. R. Moffatt School of Biological Sciences, University of Wales, Bangor LL57 2UW, Gwynedd, U.K. Because of confusion over the use of the term secretagogue we propose that the direct interaction of an element of the meal with digestive enzyme-producing cells, resulting in increased rates of enzyme synthesis or secretion, should be referred to as a prandial mechanism. Most studies suggest that paracrine and/or prandial mechanisms are the main factors controlling digestive enzyme synthesis and secretion in insects. Distinguishing between the two mechanisms is a significant challenge. In many insects, soluble proteins are potent stimulants of proteinase synthesis and secretion probably through the prandial]paracrine pathways. The details of the mechanisms involved are unknown. Hormones (other than paracrine) probably play a role in modulating the levels of digestive enzymes rather than in their absolute control. Feeding can effect the control of digestive enzyme synthesis at either the transcriptional or translational level. Key words: Insects; Midgut; Digestion; Digestive enzymes; Trypsin; Regulation; Secretagogue; Prandial mechanism; Hormone; Transcription; Translation.
Comp. Biochem. Physiol. llOB, 285-289, 1995.
digesters usually have a midgut in which the epithelium is little differentiated along its length. In the second group, digestion is commonly organized in a production line with food storage, digestion and absorption occurring at different positions along the length of the gut. The midgut epithelium in continuous digesters is often morphologically, as well as functionally, differentiated along its length. During the digestive cycle, there are considerable changes in the levels of midgut digestive enzymes, particularly in batch digesters. This strongly suggests that digestive enzyme synthesis and secretion are regulated during the digestive cycle. It is possible that control over secretion and synthesis may be separate events particularly in cells undertaking regulated secretion, but these aspects are only beginning to be investigated (Blakemore et al., 1995). Given this diversity in digestive organization in insects and the evolCorrespondence to: M. J. Lehane° School of Biological utionary age of the class, it is surprising that the Sciences, University of Wales, Bangor LL57 2UW, emerging picture for the control of digestive Gwynedd, U.K. Received 13 February 1994;revised 12 July 1994;accepted enzyme levels is showing consistency across the 18 July 1994. class. Before discussing what is known about
Insect midgut cells synthesizing and secreting digestive enzymes can be divided into two types. Constitutively secreting cells do not accumulate secretory products, synthesized enzymes being released immediately following their synthesis. Regulated secretory cells accumulate secretory material which is rapidly released in response to an appropriate signal. Constitutive secretion appears to be the rule in most insects with regulated secretion being reported in relatively few to date. Insects can also be divided into two groups dependent on the organization of their digestive system. One group, characterized by adult mosquitoes, undertakes batch digestion in which a single discrete blood meal is digested as a bolus with digestion proceeding over the entire surface of the meal simultaneously. Batch
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these control mechanisms we need to clarify a mechanisms merely have a modulating effect confusion which has arisen in the terminology on enzyme levels as trypsin levels were still used in this field. sufficient for normal digestion of the meal in The term "secretagogue" is widely used in the operated mosquitoes. A more pronounced effect vertebrate literature to refer to any substance can be seen in mosquitoes with mature or nearly which elicits secretion, e.g. a hormone. Some mature eggs when the insect is incapable of insect physiologists use the term in this sense. digesting a blood meal (Detinova, 1962). The However, others have followed the lead of effects seen in these experiments may involve the Shambaugh (l 954) and Gooding (1975) who use decapeptide termed "trypsin modulating oostthe term to refer to elements of the meal directly atic factor" (TMOF) isolated from the ovaries stimulating secretory cells. Because of this con- of Aedes aegypti (Borovsky et al., 1990, 1993). fusion over the use of the term secretagogue, we When 0.5 pl of TMOF or its analogs are inpropose that increased rates of enzyme synthesis jected into mosquitoes, biting midges, flies and or secretion caused when an element of the meal fleas at concentrations up to 7/tM, trypsin directly interacts with digestive enzyme-produc- production was reduced by up to 90%. Whether ing cells should be referred to as a prandial TMOF exerts its effect directly on digestive mechanism. enzyme secreting cells or indirectly by, for Four categories of control mechanism regu- example, affecting general protein metabolism, lating digestive enzyme levels in insects have remains to be seen. TMOF has no effect on been suggested--nervous, hormonal, paracrine protein-stimulated secretion from in vitro prepand prandial. Direct nervous control of diges- arations of opaque zone cells of S. calcitrans tive enzyme synthesis has been largely dis- (Millett and Lehane, unpublished). counted on the grounds that innervation Many studies have shown that the ingestion appears limited to motor innervation of the of some nutrients, but not others, will stimulate midgut musculature (Day and Powning, 1949; synthesis of new digestive enzymes (Champlain Garcia and Garcia, 1977; Zitnan et al., 1993). and Fisk, 1956; Hori et al., 1982; Engelmann, Designing experiments to successfully separate 1969; Engelmann and Wilkens, 1969; Garcia the other three potential control mecihanisms is and Garcia, 1977; Muraleedharan and Prabhu, proving a difficult challenge and the picture is 1979; Houseman et al., 1988; Felix et al., 1991; Stiles et al., 1991). Stimulation has been shown still very far from complete. Many investigations of the role of hormones to be independent of meal size, greatly diminishin the control of midgut enzyme synthesis and ing the likelihood of stretch receptor involvesecretion have produced inconclusive results. ment (Briegel and Lea, 1975), so these specific While it is possible hormonal influence over responses to particular nutrients are likely to be digestive enzyme synthesis and secretion may be operating through hormonal mechanisms linked direct, it is difficult to separate direct from to foregut receptors or to paracrine or prandial indirect influences. These may be general in mechanisms in the midgut itself. Work in which nature; thus it would be surprising, indeed, if these stimulating nutrients are supplied to the hormones influencing general protein metab- insect by enema or in which the nutrients are olism did not affect digestive enzyme levels. used on in vitro preparations of midgut suggest However, researchers should be aware of more that control is probably operating through subtle influences; for example, Engelmann prandial and/or paracrine mechanisms. Thus (1969) suggested hormones may affect peristaltic the in vitro preparation of Blakemore et al. movements of the alimentary canal, altering (1995) in which responses to protein stimulants food passage rates. This, in turn, may influence are shown to be dose-dependent, demonstrate digestive enzyme levels through the pran- prandial and/or paracrine control of secretion in dial/paracrine and other mechanisms discussed the midgut of S. calcitrans. Similarly, the findbelow. Similarly, neurosecretory cell ablation ings of Briegel and Lea (1975, 1979) that inmay affect food intake by as much as 50% creased trypsin levels were incurred in Aedes (Engelmann and Wilkens, 1969; Muraleedharan aegypti following feeding by enema, suggest that and Prabhu, 1979) so again achieving an indi- prandial and/or paracrine mechanisms are at work. Graf and Briegel (1989) took this work rect control over digestive enzyme levels. Possibly the best body of evidence for hor- further, showing that trypsin secretion is depenmonal involvement in the control of digestive dent on meal size; they suggested that part of enzyme levels comes from the mosquitoes. this prandial and/or paracrine response in Briegel and Lea (1979) showed the halving of mosquitoes may be mechanical or osmotic stress midgut trypsin activity in Aedes aegypti follow- on the epithelium. Separating prandial from paracrine mechaning ovariectomy or MNC ablation and that this could be reversed by reimplantation of an isms is a serious experimental challenge because ovary. In these experiments, the underlying of the composition of the insect gut. The insect
Regulation of insect digestive enzymes midgut epithelium contains a significant proportion of putative endocrine cells; these have been demonstrated universally in all developmental stages of insect species examined. These cells contain epitopes recognized by antibodies raised against vertebrate neuropeptides (Endo et al., 1990; Sehnal and Zitnan, 1990; Zitnan et al., 1993). The cells are ultrastructurally similar to mammalian gut endocrine cells and quantitative changes in the levels of putative "peptide hormones" in the cells have been recorded in response to feeding (Brown et al., 1986; Jenkins et al., 1989). It seems highly probable these cells have a role in the control of insect intestinal activity as they do in vertebrates (Fujita et al., 1988). However, the difficulty of designing experiments to separate prandial from paracrine events means that, as yet, there is no clear, direct evidence for their involvement although there is some weak evidence for paracrine control. Sreekumar and Prabhu (1988) claimed raised proteolytic and amylase activity after incubating midgut preparations of Oryctes rhinocerus in vitro with midgut epithelial, extract. They concluded that digestive enzyme release into the gut lumen was probably due to the action of a hormone present in the midgut epithelium, however, the protein content of their homogenates may be playing a role in stimulation (Blakemore et al., 1995) and the presentation of data in that paper prevents re-evaluation of their results. Rounds (1968) claimed raised midgut proteolytic activity in Periplaneta americana by injection of midgut tissue extracts into the haemocoel but, again, data presentation makes it difficult to judge these claims. Many studies have looked at the function of proteins in the regulation of digestive enzyme levels. In general, soluble, but not insoluble, proteins have been shown to play an important role. Thus Felix et al. (1991) showed that insoluble proteins are ineffective in raising enzyme levels in Aedes aegypti while Briegel and Lea (1975) showed that egg albumin and blood plasma caused raised levels of proteolytic activity in the same mosquito. Our in vitro assay for digestive enzyme secretion in S. calcitrans has shown that regulated secretion of digestive enzymes is stimulated by a wide range of proteins but is not stimulated by poly-L amino acid sequences, amino acids or by small peptides (Blakemore et al., 1995). The fact that so many proteins can stimulate secretion and synthesis begs the question of the nature of a mechanism capable of recognizing so many disparate molecules. In continuous digesters such as S. calcitrans, trypsin and other digestive enzymes are always present in the gut lumen. Thus, due to the nature of enzymatic
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cleavage, partial digestion products with structural similarities will be produced irrespective of the nature of the original protein substrate. If one of these similar, partial digestion products was recognized by a receptor, it would neatly explain how so many disparate proteins, some unlikely to be ever encountered by the living insect, can be identified by the gut epithelium. With this in mind, it is interesting to note that a 5 kDa trypsin digestion product of bovine serum albumin is a potent promoter of trypsin and carboxypeptidase B in the tsetse fly (Gooding, 1977). One of the weaknesses of this hypothesis is that it cannot explain control in batch digesters, such as the mosquitoes, because luminal trypsin is not present in the unfed insect. In vertebrates, cholecystokinin (CCK) stimulates secretion from the pancreas and gall bladder. CCK is released from I cells in the proximal intestine in response to ingestion of food. The mechanisms controlling CCK release are not well understood but, in the rat, intact proteins are the only nutrients effecting release (Liddle et al., 1986; Sharara et al., 1993). Proteins do not directly affect 1 cells; rather, they compete for luminal trypsin allowing the build up in the lumen of two trypsin-sensitive molecules, monitor peptide and CCK intestinal releasing factor. Elevated levels of these peptides stimulate I cells to release CCK. Such a model would be consistent with gut conditions in many insects but cannot be applied to batch digesters, such as mosquitoes, where trypsin is not present in the unfed insect. Also, it is difficult to reconcile the results of Blakemore et al. (1995) with such an indirect control mechanism given the nature of their in vitro preparation. However, with the vertebrate model in mind, it is interesting to note that Gooding (1977) found that the fraction of bovine serum displaying the highest trypsin inhibition capacity also stimulated trypsin and carboxypeptidase B production to the greatest extent. Protein content of the digestive region of the midgut and extracellular proteinase levels (but not other digestive enzymes; e.g. Billingsley and Hecker, 1991; Houseman et al., 1985) correlate strongly in several insects. Perhaps the simplest explanation for this phenomenon was suggested by Lehane (1977). He proposed that midgut protein concentration above a certain level caused increased synthesis and secretion through a prandial/paracrine mechanism and that protein levels in the absorptive region of the midgut then determined the quantities of amino acids available for the synthesis of new proteinases. Another possibility is that the control may be regulated through a monitor peptide-like mechanism. In this model lumenal levels of a protease-sensitive monitor peptide-like material
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w o u l d be d e t e r m i n e d by the levels o f p r o t e i n in the digestive p a r t o f the m i d g u t (see above). Changes in the level o f this m o n i t o r i n g m a t e r i a l would, in turn, regulate the levels o f p r o t e o l y t i c enzyme in the gut t h r o u g h a paracrine pathway. C o r r e l a t i o n s between levels o f extracellular p r o t e o l y t i c enzymes have also been d e m o n strated on several occasions. F o r example, in S. c a l c i t r a n s trypsin, c h y m o t r y p s i n a n d carb o x y p e t i d a s e A a n d B activities all c o r r e l a t e d d u r i n g the digestive cycle (Schneider et al., 1987). These a u t h o r s suggested that this might be explained neatly if all extracellular proteinases were under the c o n t r o l o f the same " o p e r o n " but the o b s e r v a t i o n s o f G o o d i n g (1977) that the relationship only holds true after certain types o f p r o t e i n meal suggests that the controlling m e c h a n i s m s are likely to be m o r e complex. The c o n t r o l exerted by p r o t e i n s can result in regulation o f digestive enzyme synthesis at either the t r a n s c r i p t i o n a l or t r a n s l a t i o n a l level. F o r example, the early a n d late trypsins p r o d u c e d in the m o s q u i t o A n . g a m b i a e are regulated at the t r a n s c r i p t i o n a l level (Miiller et al., 1993) a l t h o u g h A n t r y p l is detectable in unfed insects a n d so a p p e a r s to be less tightly regulated than A n t r y p 2 . In S. calcitrans, increased synthetic rate i m m e d i a t e l y following the b l o o d meal a p p e a r s to be regulated at the t r a n s l a t i o n a l level ( M o f f a t t et al., 1995). Clearly, there is considerable scope for further physiological investigation o f the mechanisms controlling digestive enzyme synthesis and secretion in insects. In the short term, the develo p m e n t o f suitable in vivo a n d in vitro assays a n d their use in c o m b i n a t i o n with peptides purified from the p u t a t i v e m i d g u t e n d o c r i n e cells m a y well yield useful results. R a p i d a d v a n c e s at the m o l e c u l a r level are also likely because o f the strong interest being shown in the o r g a n i z a t i o n a n d regulation o f trypsin genes in the m i d g u t epithelial cells o f b l o o d - s u c k i n g insects.
References BillingsleyP. F. and Hecker H. (1991) Blood digestion in the mosquito, Anopheles stephensi Liston (Diptera: Culicidae), activity and distribution of trypsin, aminopeptidase and alpha-glucosidase in the midgut. J. Med. Ent. 78, 865-871. Blakemore D., Lehane M. J. and Williams S. (1993) Cyclic AMP can promote the secretion of digestive enzymes in Stomoxys calcitrans. Insect Biochem. molec. Biol. 23, 331-335. Blakemore D., Williams S. and Lehane M. J. (1995) Protein stimulation of trypsin secretion from the opaque zone midgut cells of Stomoxys calcitrans. Comp. Biochem, Physiol. IlOB, 301-307. Borovsky D., Carlson D. A., Griffin P. R., Shabanowitz J. and Hunt D. F. 0990) Mosquito oostatic factor: a novel
decapeptide modulating trypsin-like enzyme biosynthesis in the midgut. FASEB J. 4, 3015 3020. Borovsky D., Carlson D. A., Griffin P. R., Shabanowitz J. and Hunt D. F. (1993) Mass-spectrometry and characterisation of Aedes aegypti trypsin modulating oostatic factor (TMOF) and its analogs. Insect Biochem. molec Biol. 23, 703-712. Briegel H. and Lea A. O. (1975) Relationship between protein and proteolytic activity in the midgut of mosquitoes. J. Insect Physiol. 21, 1597-1604. Briegel H. and Lea A. O. (1979) Influence of the endocrine system on tryptic activity in female Aedes aegypti. J. Insect PhysioL 25, 227-230. Brown M. R., Crim J. W. and Lea A. O. (1986) FMRFamide- and pancreatic polypeptide-like immunoreactivity of endocrine cells in the midgut of a mosquito. Tissue Cell 18, 419~425. Champlain R. A. and Fisk F. W. (1956) The digestive enzymes of the stablefly Stomoxys calcitrans (L.) Ohio J. Sci. 56, 52 56. Day M. F. and Powning R. F. (1949) A study of the processes of digestion in certain insects. Aust. J. Sci. Res. B. 2, 175 215. Detinova T. S. (1962) Age-grouping methods in Diptera of medical importance. W.H.O. Monogr. 47, 216. Geneva. Endo Y., lwanga T. and Fumita T. (1990) Gut endocrine cells of invertebrates. In Progress in Comparative Endocrinology (Edited by Epple A., Scanes C. G. and Stetson M. H.), pp. 499 503. Wiley-Liss, New York. Engelmann F. (1969) Food-stimulated synthesis of intestinal proteolytic enzymes in the cockroach Leucophaea maderae. J. Insect Physiol. 15, 217-235. Engelmann F. and Wilkens J. L. (1969) Synthesis of digestive enzyme in the fleshfly Sarcophaga bullata stimulated by food. Nature 222, 798. Felix C. R., Betschart B., Billingsley P. F. and Freyvogel T. A. (1991) Post-feeding induction of trypsin in the midgut of Aedes aegypti L. (Diptera: Culicidae) is separable into two cellular phases. Insect Biochem. 21, 197 203. Fujita T., Kanno T. and Kobayashi S. (1988) The Paraneuron, p. 367. Springer, Heidelberg. Garcia E. de S. and Garcia M. L. M. (1977) Control of protease secretion in the intestine of fifth instar larvae of Rhodnius prolixus. J. Insect Physiol. 23, 247 251. Gooding R. H. (1975) Digestive enzymes and their control in haematophagous arthropods. Acta Tropiea 32, 96 111. Gooding R. H. (1977) Digestive processes of haematophagous insects. XII. Secretion of trypsin and carboxypeptidase B by Glossina morsitans morsitans Westwood (Diptera: Glossinidae). Can J. ZooL 55, 215-222. Graf R. and Briegel H. (1989) The synthetic pathway of trypsin in the mosquito Aedes aegypti L. (Diptera: Culicidae) and in vitro stimulation in isolated midguts. Insect Biochem. 19, 129 137. Hori K., Araki S. and Kuramochi K. (1982) Trypsin-like enzyme and aminopeptidase in the midguts of the adult horn fly, Hematobia irritans and of the stable fly, Stomoxys calcitrans: change of activity in relation to blood ingestion and age. Ent. exp. Appl. 31, 421~,27. Houseman J. G., Aresta F., Mark W. A. and Morrison P. E. (1988) Effect of fractionated blood components on trypsin activity in the stable fly Stomo.~:ys calcitrans (L.) (Diptera: Muscidae). Can. J. Zool. 66, 1188-1190. Houseman J. G., Downe A. E. R. and Morrison P. E. (1985) Similarities in digestive proteinase production in Rhodnius prolixus (Hemiptera: Reduviidae) and Stomoxys calcitrans (Diptera: Muscidae). Insect Biochem. 15, 471474. Jenkins A. C., Brown M. R. and Crim J. W. (1989) FMRF-amide immunoreactivity and the midgut of the corn earworm (Heliothis ~ea). J. exp. Zool. 252, 71-78.
Regulation of insect digestive enzymes Lehane M. J. (1977) A hypothesis of the mechanism controlling proteolytic digestive enzyme production levels in Stomoxys calcitrans. J. Insect Physiol. 23, 713-715. Liddle R. A., Green G. M., Conrad C. K. and Williams J. A. (1986) Proteins but not amino acids, carbohydrates, or fats stimulate cholecystokinin secretion in the rat. Am. J. Physiol. 251, G243-G248. Moffatt M., Blakemore D. and Lehane M. J. (1995) Studies on the synthesis and secretion of trypsin in the midgut of Stomoxys calcitrans. Comp. Bioehem. Physiol. I10B, 291-300. Miiller H. M., Crampton J. M., della Torre A., Sinden R. and Crisanti A. (1993) Members of a trypsin gene family in Anopheles gambiae are induced in the gut by blood meal. EMBO J. 12, 2891-2900. Muraleedharan D. and Prabhu V. K. K. (1979) Role of the median neurosecretory cells in secretion of protease and inverase in the red cotton bug, Dysdereus cingulatus. J. Insect Physiol. 25, 237-240. Rounds H. D. (1968) Diurnal variation in the effectiveness of extracts of cockroach midgut in the release of intestinal proteinase activity. Comp. Biochem. Physiol. 25, 1125-1128.
Schneider F., Houseman J. G. and Morrison P. E. (1987)
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Activity cycles and the regulation of digestive proteases in the posterior midgut of Stomoxys calcitrans (L.) (Diptera: Muscidae). Insect Biochem. 17, 859-862. Sehnal F. and Zitnan D. (1990) Endocrines of insect gut. In Progress in Comparative Endocrinology (Edited by Epple A., Scanes C. G. and Stetson M. H.), pp. 510-515. Wiley-Liss, New York. Shambaugh G. F. (1954) Protease stimulation by foods in adult Aedes aegypti Linn. Ohio J. Sci. 54, 151-160. Sharara A. I., Bouras E. P., Misukonis M. A, and Liddle R. A. (1993) Evidence for indirect dietary regulation of cholecystokinin release in rats. Am. J. Physiol. 265, GI07-G112. Sreekumar S. and Prabhu V. K. K. (1988) Probable endocrine role of midgut tissue in stimulation of digestive enzyme secretion in Oryctes rhinoceros (Coleoptera: Scarabidae). Proc. Indian Acad. Sci. 97, 73-78. Stiles J. K., Wallbanks K. R. and Molyneux D. H. (1991) The use of casein substrate gels for determining trypsinlike activity in the midgut of Glossina palpalis spp. J. Insect Physiol. 37, 247. Zitnan D., Sauman I. and Sehnal F. (1993) Peptidergic innervation and endocrine cells of insect midgut. Arch. Insect Biochem. Physiol. 22, 113-132.
~
Elsevier Science Ltd Printed in Great Britain 0305-0491/95 $9.50 + 0.00
Pergamon
0305-0491(94)00157-X
MINI REVIEW Regulation of digestive enzyme levels in insects M. J. Lehane, D. Blakemore, S. Williams and M. R. Moffatt School of Biological Sciences, University of Wales, Bangor LL57 2UW, Gwynedd, U.K. Because of confusion over the use of the term secretagogue we propose that the direct interaction of an element of the meal with digestive enzyme-producing cells, resulting in increased rates of enzyme synthesis or secretion, should be referred to as a prandial mechanism. Most studies suggest that paracrine and/or prandial mechanisms are the main factors controlling digestive enzyme synthesis and secretion in insects. Distinguishing between the two mechanisms is a significant challenge. In many insects, soluble proteins are potent stimulants of proteinase synthesis and secretion probably through the prandial]paracrine pathways. The details of the mechanisms involved are unknown. Hormones (other than paracrine) probably play a role in modulating the levels of digestive enzymes rather than in their absolute control. Feeding can effect the control of digestive enzyme synthesis at either the transcriptional or translational level. Key words: Insects; Midgut; Digestion; Digestive enzymes; Trypsin; Regulation; Secretagogue; Prandial mechanism; Hormone; Transcription; Translation.
Comp. Biochem. Physiol. llOB, 285-289, 1995.
digesters usually have a midgut in which the epithelium is little differentiated along its length. In the second group, digestion is commonly organized in a production line with food storage, digestion and absorption occurring at different positions along the length of the gut. The midgut epithelium in continuous digesters is often morphologically, as well as functionally, differentiated along its length. During the digestive cycle, there are considerable changes in the levels of midgut digestive enzymes, particularly in batch digesters. This strongly suggests that digestive enzyme synthesis and secretion are regulated during the digestive cycle. It is possible that control over secretion and synthesis may be separate events particularly in cells undertaking regulated secretion, but these aspects are only beginning to be investigated (Blakemore et al., 1995). Given this diversity in digestive organization in insects and the evolCorrespondence to: M. J. Lehane° School of Biological utionary age of the class, it is surprising that the Sciences, University of Wales, Bangor LL57 2UW, emerging picture for the control of digestive Gwynedd, U.K. Received 13 February 1994;revised 12 July 1994;accepted enzyme levels is showing consistency across the 18 July 1994. class. Before discussing what is known about
Insect midgut cells synthesizing and secreting digestive enzymes can be divided into two types. Constitutively secreting cells do not accumulate secretory products, synthesized enzymes being released immediately following their synthesis. Regulated secretory cells accumulate secretory material which is rapidly released in response to an appropriate signal. Constitutive secretion appears to be the rule in most insects with regulated secretion being reported in relatively few to date. Insects can also be divided into two groups dependent on the organization of their digestive system. One group, characterized by adult mosquitoes, undertakes batch digestion in which a single discrete blood meal is digested as a bolus with digestion proceeding over the entire surface of the meal simultaneously. Batch
285
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these control mechanisms we need to clarify a mechanisms merely have a modulating effect confusion which has arisen in the terminology on enzyme levels as trypsin levels were still used in this field. sufficient for normal digestion of the meal in The term "secretagogue" is widely used in the operated mosquitoes. A more pronounced effect vertebrate literature to refer to any substance can be seen in mosquitoes with mature or nearly which elicits secretion, e.g. a hormone. Some mature eggs when the insect is incapable of insect physiologists use the term in this sense. digesting a blood meal (Detinova, 1962). The However, others have followed the lead of effects seen in these experiments may involve the Shambaugh (l 954) and Gooding (1975) who use decapeptide termed "trypsin modulating oostthe term to refer to elements of the meal directly atic factor" (TMOF) isolated from the ovaries stimulating secretory cells. Because of this con- of Aedes aegypti (Borovsky et al., 1990, 1993). fusion over the use of the term secretagogue, we When 0.5 pl of TMOF or its analogs are inpropose that increased rates of enzyme synthesis jected into mosquitoes, biting midges, flies and or secretion caused when an element of the meal fleas at concentrations up to 7/tM, trypsin directly interacts with digestive enzyme-produc- production was reduced by up to 90%. Whether ing cells should be referred to as a prandial TMOF exerts its effect directly on digestive mechanism. enzyme secreting cells or indirectly by, for Four categories of control mechanism regu- example, affecting general protein metabolism, lating digestive enzyme levels in insects have remains to be seen. TMOF has no effect on been suggested--nervous, hormonal, paracrine protein-stimulated secretion from in vitro prepand prandial. Direct nervous control of diges- arations of opaque zone cells of S. calcitrans tive enzyme synthesis has been largely dis- (Millett and Lehane, unpublished). counted on the grounds that innervation Many studies have shown that the ingestion appears limited to motor innervation of the of some nutrients, but not others, will stimulate midgut musculature (Day and Powning, 1949; synthesis of new digestive enzymes (Champlain Garcia and Garcia, 1977; Zitnan et al., 1993). and Fisk, 1956; Hori et al., 1982; Engelmann, Designing experiments to successfully separate 1969; Engelmann and Wilkens, 1969; Garcia the other three potential control mecihanisms is and Garcia, 1977; Muraleedharan and Prabhu, proving a difficult challenge and the picture is 1979; Houseman et al., 1988; Felix et al., 1991; Stiles et al., 1991). Stimulation has been shown still very far from complete. Many investigations of the role of hormones to be independent of meal size, greatly diminishin the control of midgut enzyme synthesis and ing the likelihood of stretch receptor involvesecretion have produced inconclusive results. ment (Briegel and Lea, 1975), so these specific While it is possible hormonal influence over responses to particular nutrients are likely to be digestive enzyme synthesis and secretion may be operating through hormonal mechanisms linked direct, it is difficult to separate direct from to foregut receptors or to paracrine or prandial indirect influences. These may be general in mechanisms in the midgut itself. Work in which nature; thus it would be surprising, indeed, if these stimulating nutrients are supplied to the hormones influencing general protein metab- insect by enema or in which the nutrients are olism did not affect digestive enzyme levels. used on in vitro preparations of midgut suggest However, researchers should be aware of more that control is probably operating through subtle influences; for example, Engelmann prandial and/or paracrine mechanisms. Thus (1969) suggested hormones may affect peristaltic the in vitro preparation of Blakemore et al. movements of the alimentary canal, altering (1995) in which responses to protein stimulants food passage rates. This, in turn, may influence are shown to be dose-dependent, demonstrate digestive enzyme levels through the pran- prandial and/or paracrine control of secretion in dial/paracrine and other mechanisms discussed the midgut of S. calcitrans. Similarly, the findbelow. Similarly, neurosecretory cell ablation ings of Briegel and Lea (1975, 1979) that inmay affect food intake by as much as 50% creased trypsin levels were incurred in Aedes (Engelmann and Wilkens, 1969; Muraleedharan aegypti following feeding by enema, suggest that and Prabhu, 1979) so again achieving an indi- prandial and/or paracrine mechanisms are at work. Graf and Briegel (1989) took this work rect control over digestive enzyme levels. Possibly the best body of evidence for hor- further, showing that trypsin secretion is depenmonal involvement in the control of digestive dent on meal size; they suggested that part of enzyme levels comes from the mosquitoes. this prandial and/or paracrine response in Briegel and Lea (1979) showed the halving of mosquitoes may be mechanical or osmotic stress midgut trypsin activity in Aedes aegypti follow- on the epithelium. Separating prandial from paracrine mechaning ovariectomy or MNC ablation and that this could be reversed by reimplantation of an isms is a serious experimental challenge because ovary. In these experiments, the underlying of the composition of the insect gut. The insect
Regulation of insect digestive enzymes midgut epithelium contains a significant proportion of putative endocrine cells; these have been demonstrated universally in all developmental stages of insect species examined. These cells contain epitopes recognized by antibodies raised against vertebrate neuropeptides (Endo et al., 1990; Sehnal and Zitnan, 1990; Zitnan et al., 1993). The cells are ultrastructurally similar to mammalian gut endocrine cells and quantitative changes in the levels of putative "peptide hormones" in the cells have been recorded in response to feeding (Brown et al., 1986; Jenkins et al., 1989). It seems highly probable these cells have a role in the control of insect intestinal activity as they do in vertebrates (Fujita et al., 1988). However, the difficulty of designing experiments to separate prandial from paracrine events means that, as yet, there is no clear, direct evidence for their involvement although there is some weak evidence for paracrine control. Sreekumar and Prabhu (1988) claimed raised proteolytic and amylase activity after incubating midgut preparations of Oryctes rhinocerus in vitro with midgut epithelial, extract. They concluded that digestive enzyme release into the gut lumen was probably due to the action of a hormone present in the midgut epithelium, however, the protein content of their homogenates may be playing a role in stimulation (Blakemore et al., 1995) and the presentation of data in that paper prevents re-evaluation of their results. Rounds (1968) claimed raised midgut proteolytic activity in Periplaneta americana by injection of midgut tissue extracts into the haemocoel but, again, data presentation makes it difficult to judge these claims. Many studies have looked at the function of proteins in the regulation of digestive enzyme levels. In general, soluble, but not insoluble, proteins have been shown to play an important role. Thus Felix et al. (1991) showed that insoluble proteins are ineffective in raising enzyme levels in Aedes aegypti while Briegel and Lea (1975) showed that egg albumin and blood plasma caused raised levels of proteolytic activity in the same mosquito. Our in vitro assay for digestive enzyme secretion in S. calcitrans has shown that regulated secretion of digestive enzymes is stimulated by a wide range of proteins but is not stimulated by poly-L amino acid sequences, amino acids or by small peptides (Blakemore et al., 1995). The fact that so many proteins can stimulate secretion and synthesis begs the question of the nature of a mechanism capable of recognizing so many disparate molecules. In continuous digesters such as S. calcitrans, trypsin and other digestive enzymes are always present in the gut lumen. Thus, due to the nature of enzymatic
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cleavage, partial digestion products with structural similarities will be produced irrespective of the nature of the original protein substrate. If one of these similar, partial digestion products was recognized by a receptor, it would neatly explain how so many disparate proteins, some unlikely to be ever encountered by the living insect, can be identified by the gut epithelium. With this in mind, it is interesting to note that a 5 kDa trypsin digestion product of bovine serum albumin is a potent promoter of trypsin and carboxypeptidase B in the tsetse fly (Gooding, 1977). One of the weaknesses of this hypothesis is that it cannot explain control in batch digesters, such as the mosquitoes, because luminal trypsin is not present in the unfed insect. In vertebrates, cholecystokinin (CCK) stimulates secretion from the pancreas and gall bladder. CCK is released from I cells in the proximal intestine in response to ingestion of food. The mechanisms controlling CCK release are not well understood but, in the rat, intact proteins are the only nutrients effecting release (Liddle et al., 1986; Sharara et al., 1993). Proteins do not directly affect 1 cells; rather, they compete for luminal trypsin allowing the build up in the lumen of two trypsin-sensitive molecules, monitor peptide and CCK intestinal releasing factor. Elevated levels of these peptides stimulate I cells to release CCK. Such a model would be consistent with gut conditions in many insects but cannot be applied to batch digesters, such as mosquitoes, where trypsin is not present in the unfed insect. Also, it is difficult to reconcile the results of Blakemore et al. (1995) with such an indirect control mechanism given the nature of their in vitro preparation. However, with the vertebrate model in mind, it is interesting to note that Gooding (1977) found that the fraction of bovine serum displaying the highest trypsin inhibition capacity also stimulated trypsin and carboxypeptidase B production to the greatest extent. Protein content of the digestive region of the midgut and extracellular proteinase levels (but not other digestive enzymes; e.g. Billingsley and Hecker, 1991; Houseman et al., 1985) correlate strongly in several insects. Perhaps the simplest explanation for this phenomenon was suggested by Lehane (1977). He proposed that midgut protein concentration above a certain level caused increased synthesis and secretion through a prandial/paracrine mechanism and that protein levels in the absorptive region of the midgut then determined the quantities of amino acids available for the synthesis of new proteinases. Another possibility is that the control may be regulated through a monitor peptide-like mechanism. In this model lumenal levels of a protease-sensitive monitor peptide-like material
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w o u l d be d e t e r m i n e d by the levels o f p r o t e i n in the digestive p a r t o f the m i d g u t (see above). Changes in the level o f this m o n i t o r i n g m a t e r i a l would, in turn, regulate the levels o f p r o t e o l y t i c enzyme in the gut t h r o u g h a paracrine pathway. C o r r e l a t i o n s between levels o f extracellular p r o t e o l y t i c enzymes have also been d e m o n strated on several occasions. F o r example, in S. c a l c i t r a n s trypsin, c h y m o t r y p s i n a n d carb o x y p e t i d a s e A a n d B activities all c o r r e l a t e d d u r i n g the digestive cycle (Schneider et al., 1987). These a u t h o r s suggested that this might be explained neatly if all extracellular proteinases were under the c o n t r o l o f the same " o p e r o n " but the o b s e r v a t i o n s o f G o o d i n g (1977) that the relationship only holds true after certain types o f p r o t e i n meal suggests that the controlling m e c h a n i s m s are likely to be m o r e complex. The c o n t r o l exerted by p r o t e i n s can result in regulation o f digestive enzyme synthesis at either the t r a n s c r i p t i o n a l or t r a n s l a t i o n a l level. F o r example, the early a n d late trypsins p r o d u c e d in the m o s q u i t o A n . g a m b i a e are regulated at the t r a n s c r i p t i o n a l level (Miiller et al., 1993) a l t h o u g h A n t r y p l is detectable in unfed insects a n d so a p p e a r s to be less tightly regulated than A n t r y p 2 . In S. calcitrans, increased synthetic rate i m m e d i a t e l y following the b l o o d meal a p p e a r s to be regulated at the t r a n s l a t i o n a l level ( M o f f a t t et al., 1995). Clearly, there is considerable scope for further physiological investigation o f the mechanisms controlling digestive enzyme synthesis and secretion in insects. In the short term, the develo p m e n t o f suitable in vivo a n d in vitro assays a n d their use in c o m b i n a t i o n with peptides purified from the p u t a t i v e m i d g u t e n d o c r i n e cells m a y well yield useful results. R a p i d a d v a n c e s at the m o l e c u l a r level are also likely because o f the strong interest being shown in the o r g a n i z a t i o n a n d regulation o f trypsin genes in the m i d g u t epithelial cells o f b l o o d - s u c k i n g insects.
References BillingsleyP. F. and Hecker H. (1991) Blood digestion in the mosquito, Anopheles stephensi Liston (Diptera: Culicidae), activity and distribution of trypsin, aminopeptidase and alpha-glucosidase in the midgut. J. Med. Ent. 78, 865-871. Blakemore D., Lehane M. J. and Williams S. (1993) Cyclic AMP can promote the secretion of digestive enzymes in Stomoxys calcitrans. Insect Biochem. molec. Biol. 23, 331-335. Blakemore D., Williams S. and Lehane M. J. (1995) Protein stimulation of trypsin secretion from the opaque zone midgut cells of Stomoxys calcitrans. Comp. Biochem, Physiol. IlOB, 301-307. Borovsky D., Carlson D. A., Griffin P. R., Shabanowitz J. and Hunt D. F. 0990) Mosquito oostatic factor: a novel
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