Insect Digestion Research Articles

Probing the conformation and dynamics of allatostatin neuropeptides: A structural model for functional differences

Allatostatins are a family of related neuropeptides that play an important role in development, reproduction, and digestion in insects. The cockroach Diploptera punctata has 13 allatostatin neuropeptides, with pleiotropic functions, two of which are: inhibition of juvenile hormone (JH) production and inhibition of gut muscle contraction. In this study, the conformation and dynamics of D. punctata allatostatin 5 (Dippu-AST 5) and allatostatin 8 (Dippu-AST 8) are investigated by CD, NMR, and molecular dynamics simulations. These peptides contain eight and nine residues, respectively, and the identical six-residue C-terminal motif. Yet Dippu-AST 5 and Dippu-AST 8 affect juvenile hormone production and hindgut contraction with different potencies. Dippu-AST 5 is one of the most potent inhibitors of juvenile hormone production and one of the least potent inhibitors of gut contraction, whereas Dippu-AST 8 has the opposite potencies with respect to these tissues. From the NMR structure, it is clear that Dippu-AST 5 has a 3 10 helix involving three of its residues and a “γ” turn at the end of its C-terminal motif. In contrast Dippu-AST 8 has an open “π” turn among five of its central residues. In addition, the orientation preferences within the membrane of the two peptides were simulated. Our simulation results show that the C-terminal segment of Dippu-AST 5 orients in the membrane surface with an average angle of 17.5°, whereas Dippu-AST 8 orients with an average angle of 5.1°. Taken together, from the structures and orientation preferences of these peptides within the membrane, it appears that these peptides may interact with the receptor very differently.

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides. A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.

Carnivorous pitcher plant uses free radicals in the digestion of prey

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.

Microhabitat preferences of six Drosera (Droseraceae) from Tibagi river basin, Paraná state, Brazil

We studied the microhabitats and the climatic requirements of six Drosera taxa, which occur in the subtropical grasslands of the Tibagi river basin. They are annual,or most communly, perennial herbs known as carnivorous plants, whose leaves are transformed into traps for the capture and digestion of small insects as an adaptation for supplying nutricional deficiencies. They usually occur in distrophic substrata. In order to contribute to conservation plans for the species of the genus, threatened by the conversion of the natural grasslands into pastures, we tried to define their preferential habitats and climatic requirements. We verified that the variables of microhabitat and altitude presented significant correlation at 5% probability level, while the variables in annual average relative humidity, annual average temperature and type of substratum types did not show significant correlation.

Insect digestion: Potential applications in insect management

AbstractThe papers assembled in this special issue were originally presented by researchers gathered at an informal symposium entitled “Insect Digestion: Potential Applications in Insect Management” at the National Meeting of the Entomological Society of America in Fort Lauderdale, Florida in November 2002. Although symposium participants and attendees clearly came from a diversity of backgrounds, they shared a common goal: to bring together their collective knowledge of insect digestive physiology and promote discussions of control methods that target insect digestion. The manuscripts published in this issue reflect the breadth of that knowledge. They include a structural and functional study of a peptide hormone that modulates digestion in mosquitoes, investigation of responses of Colorado potato beetle to transgenically expressed proteinase inhibitors, demonstration of the importance of phosphorous as a nutritive element of caterpillars, and phylogenetic analysis of insect trypsins and inhibitor resistance. The papers reinforce the notion that approaches to the study of insect digestion are as diverse as the insects we study, and it is our hope that this endeavor will promote new ideas for effective pest management tools.The organizers would like to thank the Entomological Society of America for hosting the informal symposium, and to all who presented and attended. We are especially grateful to the Executive Editor of Archives of Insect Biochemistry and Physiology, David Stanley, and his editorial staff for making publication of this special issue possible.

MALARIA IN EXPERIMENTAL BIOLOGY

![Figure][1] The hard statistics about malaria are far from comforting. Killing over one million victims every year and infecting another 300 million, the disease continues to have a catastrophic effect in many developing countries, striking the most vulnerable members of society: pregnant

Diverse enzymatic specificities of digestive proteases, 'intestains', enable Colorado potato beetle larvae to counteract the potato defence mechanism.

In response to insect attack, high levels of proteinase inhibitors are synthesised in potato leaves. This can cause inefficient protein digestion in insects, leading to reduced growth, delayed development and lower fecundity. It has been suggested that Colorado potato beetle overcomes this defence mechanism by inducing the production of a set of cysteine proteases that are resistant to potato proteinase inhibitors. Experiments with gut extracts showed that these proteases have unusual inhibition profiles as they are not inhibited by most of the cystatins but are strongly inhibited by thyropins. In this study we have isolated three cysteine proteases from adapted guts of Colorado potato beetle larvae, named intestains 1, 2 and 3, the first cysteine proteases known to be involved in extracellular protein digestion. The N-terminal sequences suggest their classification into the papain family. Intestains differ in substrate specificities and inhibitory profiles. Their substrate specificities suggest that intestains 1 and 2 are general digestive enzymes, while intestain 3 has a more specific function. The inhibitory profile of intestain 1 is similar to that of proteases of the papain family. However, the Ki values for the interaction of intestain 2 with the same set of inhibitors are several hundred fold higher, which would enable the enzyme to circumvent the potato defence mechanism characterised by high concentrations of protease inhibitors in attacked potato leaves. A further, different strategy of the Colorado potato beetle to avoid potato defence is exhibited by intestain 3, which is able to cleave off the N-terminus of model cystatin and thus inactivate the inhibitor. These results suggest that the Colorado potato beetle combines different strategies to counteract plant defence mechanisms.

Digestion of nectar and insects by Palestine sunbirds.

In nectarivorous birds, specialization for feeding on nectar has led to a simple gut structure with high sugar digestive efficiencies and rapid gut passage rates. These features of the digestive system may make digestion of more complex, protein-rich food sources, such as pollen or insects, less efficient. In this light, we hypothesized that sugar metabolizability in nectarivorous Palestine sunbirds (Nectarinia osea) would be high, whereas nitrogen metabolizability would be lower than typically found for birds. We measured glucose and fructose apparent metabolizabilities (*MCs) and transit times (TTs) in eight Palestine sunbirds offered either a 10% or a 50% mixed sugar diet. *MC for glucose (99.9%+/-0.1%) was significantly greater than for fructose (99.6%+/-0.4%; ANOVA; P<0.001). TT for the 10% sugar diet (26.3+/-10.1 min) was significantly shorter than for the 50% sugar diet (47.0+/-7.8 min). We measured nitrogen true metabolizability (MC) and TT in Palestine sunbirds offered a daily fruit fly intake of either 40 or 200 flies. Nitrogen MC was not significantly different between diets, and average MC for both diets was 58.5%+/-8.5% (n=8). TT was not significantly different when birds ate 10 flies (50.1+/-13.6 min) than when they ate 50 flies (48.5+/-16.5 min). The high sugar *MC and relatively rapid TT of nectar in Palestine sunbirds are similar to those found for other nectarivorous species. Transit times of insect material are longer that those found in small insectivorous species. However, MCs of insect material are lower. Thus, even though sunbirds consume easily digestible soft-bodied insects, they are less efficient at extracting protein than nonnectarivores.

What physiological processes permit insects to eat Eucalyptus leaves?

AbstractMany species of insects eat Eucalyptus foliage despite its relatively low nutritional value and the many plant secondary metabolites (PSMs) present, for example, terpenes, phenols and formylated phloroglucinols (FPGs). Formylated phloroglucinols are a new class of PSMs that act as antifeedants for possums and koalas. What physiological processes are present that permit insects to eat eucalypt foliage and how do PSMs influence insect feeding or digestion? Some trees seem to be repeatedly infested with eucalypt‐feeding insects, possibly as a result of previous chemosensory cues remaining from parental selection of a plant. Avoidance or storage of PSMs permit jarrah leafminers (Perthida glyphopa) and sawflies (Perga sp.) to consume eucalypt foliage without dealing with the majority of these compounds. Some PSMs can be metabolized by polysubstrate membrane oxidases as found in caterpillars or sawflies that feed on eucalypts. High midgut pH may be advantageous for nutrient extraction and PSM metabolism, and midgut pH ranges between 8.5 and 8.9 for caterpillars of Hyalarcta huebneri. Plant secondary metabolites may not be absorbed as a result of the combined presence of the peritrophic matrix and endogenous surfactants. Excretion of PSMs can be as metabolites or intact compounds. Both putative metabolites and sideroxylonal‐A, an FPG, are present in the faeces of larvae of the case moth, H. huebneri. The presence of sideroxylonal‐A in the food had an effect on the presence of 5‐hydroxytryptamine (5HT) in the central nervous system of caterpillars, as larvae fed leaves with a high concentration of sideroxylonal‐A had relatively more 5HT in the brain and central nervous system ganglia than larvae fed leaves containing a low concentration. Further work is necessary to clarify how PSMs are handled by eucalypt‐feeding insects and what effect FPGs have on feeding and digestion.

A tubular network associated with the brush-border surface of the Aedes aegypti midgut: implications for pathogen transmission by mosquitoes.

The mosquito Aedes aegypti is capable of transmitting a variety of pathogens to man and to other vertebrates. The midgut of this insect has been well-studied both as the tissue where the first contact occurs between ingested pathogens and the insect host, and as a model system for blood meal digestion in blood-sucking insects. To understand better the nature of the midgut surface encountered by parasites or viruses, we used scanning electron microscopy to identify the most prominent structures and cell morphologies on the luminal midgut surface. The luminal side of the midgut is a complex and layered set of structures. The microvilli that are found on most, but not all, cells are covered by a network of fine strands that we have termed the microvilli-associated network (MN). The MN strands are membranous, as shown by a membrane bilayer visible in cross sections of MN strands at high magnification in transmission electron micrographs. The MN is found in blood-fed as well as unfed mosquitoes and is not affected by chitinase treatment, suggesting that it is not related to the chitinous peritrophic membrane that is formed only after blood feeding. The cells in the midgut epithelium have two distinct morphologies: the predominant cell type is densely covered with microvilli, while cells with fewer microvilli are found interspersed throughout the midgut. We used lectins to probe for the presence of carbohydrates on the midgut surface. A large number of lectins bind to the luminal midgut surface, suggesting that a variety of sugar linkages are present on the structures visualized by electron microscopy. Some of these lectins partially block attachment of malaria ookinetes to the midgut surface in vitro. Thus, the mosquito midgut epithelium, like the lining of mammalian intestines, is complex, composed of a variety of cell types and extensively covered with surface carbohydrate that may play a role in pathogen attachment.

Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes.

Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In Nepenthes, insect-derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in Nepenthes pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of Nepenthes. Expression of the ammonium transporter gene NaAMT1 was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene, NaAAP1, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by NaAAP1 may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene NaNTR1 was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem.

Changes in the Digestive Tract and Feeding Behavior of Anuran Amphibians during Metamorphosis

During metamorphic climax, anuran amphibians undergo dramatic changes as they pass from aquatic to terrestrial life. In particular, the digestive tract is completely modified. The pharyngeal filtering apparatus disappears. Striking transformations take place in the gastric region. The tadpole's glandular sheath regresses and ceases protruding from the gut wall. At the same time, a large permanent stomach is formed to replace the sheath, gastric glands develop, and the muscle layer thickens. The intestine also changes greatly. An overall shortening and rearrangement of its coils occurs. The histological changes that affect the intestine are largely confined to the epithelium. Degeneration occurs in the larval epithelium, while intense proliferation of stem cells results in the development of a new epithelium that is folded. During metamorphosis, the endocrine cells of the digestive tract are also redistributed. Such changes are associated with modifications in feeding behavior. Most tadpoles are microphagous and feed on particles suspended in the water, which they retain on the surface of the gill filters and other buccopharyngeal structures. Anuran tadpoles usually stop feeding at climax because the filtering apparatus degenerates. Moreover, at climax the larval intestinal epithelium is too altered to absorb anything, and the new intestinal epithelium has not yet developed. Juveniles and adults are predators (carnivores or insect eaters). The acquisition of binocular vision permits efficient perception of prey in a terrestrial environment. The new feeding behavior is also associated with the appearance of new enzymes. The secretion of proteolytic enzymes at the pancreas (trypsin) and stomach (pepsin) levels prepares the animals to digest meat. Chitinases are secreted by the stomach as soon as it differentiates and allow the digestion of chitinous insects. Moreover, the new intestinal mucosa is well equipped for the absorption of protein hydrolysis residues. Control of the digestive secretions and feeding behavior in developing anurans is also discussed in the present article.

5439821 DNA encoding peptide hormone that inhibits digestion in insects : Borovsky Dov; Carlson David A Vero Beach, FL, United States assigned to University of Florida Research Foundation; The United States of America as represented by the United States Department of Agriculture

5439821 DNA encoding peptide hormone that inhibits digestion in insects : Borovsky Dov; Carlson David A Vero Beach, FL, United States assigned to University of Florida Research Foundation; The United States of America as represented by the United States Department of Agriculture

The Black Fly Simulium vittatum Trypsin Gene: Characterization of the 5′-Upstream Region and Induction by the Blood Meal

The events leading to blood digestion in hematophagous insects are nor well understood at the molecular level. Here we report on the characterization of a trypsin gene from the black fly Simulium vittatum. Southern blot analysis indicated that the S. vittatum genome contains at least two trypsin genes. Primer extension experiments identified two closely spaced transcription initiation sites, 3 bp apart. A genomic clone containing the trypsin gene was restriction mapped and the nucleotide sequence of the 5′-upstream region was determined. Several sequence elements were identified, including a consensus TATA box 35 nucleotides upstream from the transcription initiation sire, a consensus arthropod initiator site at position −7, and two conserved sequence elements, 5′GGATTAA (position −77) and 5′TGTTTCCT (position −148). The latter two elements are present at comparable distances from the transcription initiation sites in black fly and mosquito trypsin genes. Significant amounts of trypsin mRNA were detected in guts of sugar-fed flies. Upon blood feeding, trypsin mRNA levels gradually increased to reach twice the initial abundance at 8 hr after blood ingestion. In contrast to other hematophagous insects, the induced mRNA level persists for a prolonged period of time (at least 48 hr) after blood ingestion. The results of this study provide information that may be useful for the expression of transgenes in insects of medical importance.

Carboxy-Terminal Truncation of Oryzacysatin II by Oryzacytatin-Insensitive Insect Digestive Proteinases

The biochemical interactions between digestive proteinases of the Coleoptera pest black vine weevil (Otiorynchus sulcatus) and two plant cysteine proteinase inhibitors, oryzacystatin I (OCI) and oryzacystatin II (OCII), were assessed using gelatin-polyacrylamide gel electrophoresis, OCI-affinity chromatography, and recombinant forms of the two plant inhibitors. The insect proteinases were resolved in gelatin-containing polyacrylamide gels as five major bands, only three of them being totally or partially inactivated by OCI and OCII. The maximal inhibitory effect of both OCs at pH 5.0 was estimated at 40% and the inhibition was stable with time despite the presence of OC-insensitive proteases, indicating the stability of the OCI and OCII effects. After removing OC-sensitive proteinases from the insect crude extract by OCI-affinity chromatography, the effects of the insect cystatin-insensitive proteases on the structural integrity of the free OCs were analyzed. While OCI remained stable, OCII was subjected to limited proteolysis leading to its gradual transformation into a ∼10.5-kDa unstable intermediate, OCIIi. As shown by the degradation pattern of a glutathione S-transferase (GST)/OCII fusion protein, the appearence of OCII resulted from the C-terminal truncation of OCII. Either free or linked to GST, OCIIi was as active against papain and human cathepsin H as OCII, and the initial specificities of the inhibitor for these two cysteine proteinases were conserved after cleavage. Although these observations indicate the high conformational stability of OCII near its active (inhibitory) site, they also suggest a general conformational destabilization of this inhibitor following its initial cleavage, subsequently leading to its complete hydrolysis. This apparent susceptibility of OCII to proteolytic cleavage by the insect proteinases could have major implications when planning the use of this plant cystatin for insect pest control.

Cellulose digestion in termites and cockroaches: What role do symbionts play?

Abstract 1. 1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts. 2. 2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase. 3. 3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO2 in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs. 4. 4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers. 5. 5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose. 6. 6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.

Comparative studies on the distribution of quinoidal chalcone pigments in the extracts from insect wastes and intact tissues of dyer's saffron florets

Distribution of quinoidal chalcone pigments in aqueous extracts from insect wastes and intact tissues of dyer's saffron (C. tinctorius) florets was compared by applying HPLC techniques. The analytical data indicated that the polyoxychalcone constituents are not seriously changed even after exposed to the insect digestion. The findings endorse the possibility that the spoiled florets can be used safely as raw materials for preparation of red and/or yellowCarthamus dyes.

The evolution of cellulose digestion in insects

Despite the abundance and diversity of species that include living or dead plant tissue in their diets, the ability to digest cellulose is rare in insects and is restricted to a small number of orders and families. In this paper it is argued that cellulolytic capacity is uncommon in insects simply because it is a trait that is rarely advantageous to possess. Although there is a growing body of evidence for the occurrence of symbiont-independent cellulose digestion in cockroaches and in higher termites from the subfamily Nasutitermitinae, cellulose digestion in insects is usually mediated by microorganisms. It is proposed that non-cellulolytic omnivorous scavengers and detritivores may be preadapted to evolve symbiont-mediated cellulolytic mechanisms because of the prevalence of mutualistic associations between such species and the microorganisms that normally reside in their hindguts. A scenario is proposed for the evolution of symbiont-mediated cellulolytic capacity in roaches and lower termites. Finally, it is suggested that biochemical studies of insect cellulases might provide crucial insights that would greatly advance our understanding of the evolution of cellulose digestion in insects.

Comparative aspects of plant cell wall digestion in insects

Although many phytophagous and wood-eating invertebrates form their own cellulases, there is an overwhelming variety of symbioses between plant- and wood-utilising insects and microorganisms. In one type of symbiosis (endosymbiosis), insects (rhinoceros beetle, cockroach, lower termites) host cellulolytic protozoa and/or bacteria in the hindgut. In a number of insect taxa (higher termites, cerambycid beetles) cellulose digestion is aided by the ingestion of fungal tissues for the delivery of certain components of the cellulase complex needed to complement the host enzymes for action on crystalline forms of cellulose. While the bulk of the plant lignin is not significantly degraded under the conditions characteristic of gut contents of herbivores, some groups of insects are thought to be able to digest a part of this non-carbohydrate polymer.

Phylogenetic considerations of insect digestion: Disaccharidases and the spatial organization of digestion in the Tenebrio molitor larvae

Amylase, cellobiase, cellulase, trehalase and trypsin are found in major amounts in the midgut lumen, whereas aminopeptidase (which is membrane bound) occurs mainly in the midgut tissue of Tenebrio molitor larvae. Cellulase and a minor aminopeptidase seem to be derived from fungi contaminating the wheat bran used as food. The data suggest that the majority of carbohydrate digestion should take place in the lumen of anterior midgut, whereas protein digestion should occur partly in the lumen and partly at the cell surface of the posterior midgut. The finding that less than 5% of the total amylase, cellobiase, maltase and trypsin are excreted, after a time identical to the passage time of the food bolus, leads to the proposal that there exists an endo-ectoperitrophic circulation of enzymes by which these enzymes are recovered from the undigested food before it is excreted. There is only one molecular species of cellobiase (pH optimum 5.3, K m 1.1 mM, pI 3.7, M r 75,000), maltase (pH optimum 5.3, K m 3.7 mM, pI 3.6, M r 60,000) and trehalase (pH optimum 5.0, K m 0.40 mM, pI 4.0, M r 60,000) in T. molitor larval midguts as judged by electrophoretic, isoelectric focusing and density-gradient centrifugation data. The data led to the proposal that dimer and oligomer hydrolases are small in Coleoptera ancestors and large in Diptera and Lepidoptera ancestors. For this, the majority of T. molitor larval digestion takes place inside the peritrophic membrane, whereas in Rhynchosciara americana (Diptera) and Erinnyis ello (Lepidoptera) larvae initial digestion occurs inside and intermediate and final digestion outside the peritrophic membrane.