Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

The roles of mucus-forming mucins, peritrophins and peritrophins with mucin domains in the insect midgut.

Most insects have a gut lined with a peritrophic membrane (PM) consisting of chitin and proteins, mainly peritrophins that have chitin-binding domains. The PM is proposed to originate from mucus-forming mucins (Mf-mucins), which acquired a chitin-binding domain that interlocked with chitin, replacing mucus in function. We evaluated the expression of Mf-mucins and peritrophins by RNA-sequencing (RNA-seq) throughout the midgut of four distantly related insects. Mf-mucins were identified as proteins with high o-glycosylation and a series of uninterrupted Pro/Thr/Ser residues. The results demonstrate that the mucus layer is widespread in insects, and suggest that insect Mf-mucins are derived from those found in other animals by the loss of the cysteine knot and von Willebrand domains. The data also support a role of Mf-mucins in protecting the middle midgut of Musca domestica against acidic buffers. Mf-mucins may also produce a jelly-like material associated with the PM that immobilizes digestive enzymes in Spodoptera frugiperda. Peritrophins with a domain similar to Mf-mucins may be close to the ancestor of peritrophins. Expression data of peritrophins and chitin synthase genes throughout the midgut of M. domestica, S. frugiperda and Tenebrio molitor indicated that peritrophins were incorporated along the PM, according to their preferential sites of formation. Finally, the data support the view that mucus has functions distinct from the PM.

Gelsolin role in microapocrine secretion.

A role of gelsolin in opening the way along the microvilli for secretory vesicles during microapocrine secretion is proposed here. Data obtained with different techniques showed that many digestive enzymes are released by microapocrine secretion in insects. Proteins that might be involved in the machinery of microapocrine secretion were selected from our transcriptomes and literature searches. The proteins were annexin, Complex actin-related proteins 2 and 3 (ARP 2/3) cofilin, fimbrin, gelsolin 1, gelsolin 2, moesin, myosin 1, myosin 6, protein disulphide isomerase 1 (PDI 1), PDI 2 and profilin. The cDNAs coding for annexin, fimbrin, gelsolin 1, myosin 1, PDI 1 and PDI 2 were cloned and their sequences deposited in GenBank. Only gelsolin 1 and myosin 1 are expressed exclusively in the midgut (semiquantitative reverse transcriptase PCR). As myosin 1 may have a structural role in microvilli, gelsolin 1 is the best guess to be involved in the secretory machinery. A truncated recombinant gelsolin 1 was used to generate antibodies with which it was shown labelling inside and around midgut cell microvilli shown in an electron microscope, reinforcing a microvillar role for gelsolin 1. Suppression of gelsolin 1 synthesis by RNA interference prevents secretory vesicles from advancing inside the microvilli, in agreement with its putative role in severing the actin filaments to free the way for the vesicles.

Insect midgut carboxypeptidases with emphasis on S10 hemipteran and M14 lepidopteran carboxypeptidases.

We compared the whole complement of midgut carboxypeptidases from 10 insects pertaining to five orders based on transcriptomes obtained by deep sequencing and biochemical data. Most of the carboxypeptidases were metallocarboxypeptidases from family M14, with carboxypeptidase A (CPA) predominating over carboxypeptidase B (CPB). They were found in all of the insects studied except for the hemipterans and a bruchid beetle. M14 carboxypeptidases were expressed only in the midgut of Spodoptera frugiperda (Lepidoptera). The most expressed CPA from this insect (SfCPA) was cloned, sequenced and expressed as a recombinant enzyme. This enzyme was used to generate antibodies used to demonstrate that SfCPA is secreted by an exocytic route. Serine carboxypeptidases from family S10 were found in all of the insects studied here. In S. frugiperda, they are expressed in all tissues besides the midgut, in accordance with their presumed lysosomal role. In the hemipteran Dysdercus peruvianus, S10 carboxypeptidases are expressed only in midgut, suggesting that they are digestive enzymes. This was confirmed by enzyme assays of midgut contents. Furthermore, the substrate specificity of D. peruvianus S10 carboxypeptidases are predicted to be one CPC (preferring hydrophobic residues) and one CPD (preferring basic residues), thus able to hydrolyse the peptides formed by their digestive cathepsin D and cathepsin L, respectively. The role of S10 carboxypeptidases in bruchid beetles are suggested to be the same as in hemipterans.

Sequencing of Spodoptera frugiperda midgut trehalases and demonstration of secretion of soluble trehalase by midgut columnar cells.

Both soluble (SfTre1) and membrane-bound (SfTre2) trehalases occur along the midgut of Spodoptera frugiperda larvae. Released SfTre2 was purified as a 67 kDa protein. Its K(m) (1.6 mM) and thermal stability (half life 10 min at 62 degrees C) are different from the previously isolated soluble trehalase (K(m)= 0.47 mM; 100% stable at 62 degrees C). Two cDNAs coding for S. frugiperda trehalases have been cloned using primers based on consensus sequences of trehalases and having as templates a cDNA library prepared from total polyA-containing RNA extracted from midguts. One cDNA codes for a trehalase that has a predicted transmembrane sequence and was defined as SfTre2. The other, after being cloned and expressed, results in a recombinant trehalase with a K(m) value and thermal stability like those of native soluble trehalase. This enzyme was defined as SfTre1 and, after it was used to generate antibodies, it was immunolocalized at the secretory vesicles and at the glycocalyx of columnar cells. Escherichia coli trehalase 3D structure and sequence alignment with SfTre1 support a proposal regarding the residue modulating the pKa value of the proton donor.

The Aedes aegypti larval transcriptome: a comparative perspective with emphasis on trypsins and the domain structure of peritrophins.

The genome sequence of Aedes aegypti was recently reported. A significant amount of Expressed Sequence Tags (ESTs) were sequenced to aid in the gene prediction process. In the present work we describe an integrated analysis of the genomic and EST data, focusing on genes with preferential expression in larvae (LG), adults (AG) and in both stages (SG). A total of 913 genes (5.4% of the transcript complement) are LG, including ion transporters and cuticle proteins that are important for ion homeostasis and defense. From a starting set of 245 genes encoding the trypsin domain, we identified 66 putative LG, AG, and SG trypsins by manual curation. Phylogenetic analyses showed that AG trypsins are divergent from their larval counterparts (LG), grouping with blood-induced trypsins from Anopheles gambiae and Simulium vittatum. These results support the hypothesis that blood-feeding arose only once, in the ancestral Culicomorpha. Peritrophins are proteins that interlock chitin fibrils to form the peritrophic membrane (PM) that compartmentalizes the food in the midgut. These proteins are recognized by having chitin-binding domains with 6 conserved Cys and may also present mucin-like domains (regions expected to be highly O-glycosylated). PM may be formed by a ring of cells (type 2, seen in Ae. aegypti larvae and Drosophila melanogaster) or by most midgut cells (type 1, found in Ae. aegypti adult and Tribolium castaneum). LG and D. melanogaster peritrophins have more complex domain structures than AG and T. castaneum peritrophins. Furthermore, mucin-like domains of peritrophins from T. castaneum (feeding on rough food) are lengthier than those of adult Ae. aegypti (blood-feeding). This suggests, for the first time, that type 1 and type 2 PM may have variable molecular architectures determined by different peritrophins and/or ancillary proteins, which may be partly modulated by diet.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae.

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (mu = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (mu = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotriozide (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (ì = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (ì = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Subsite substrate specificity of midgut insect chymotrypsins.

Insect chymotrypsins are distinctively sensitive to plant protein inhibitors, suggesting that they differ in subsite architecture and hence in substrate specificities. Purified digestive chymotrypsins from insects of three different orders were assayed with internally quenched fluorescent oligopeptides with three different amino acids at P1 (Tyr, Phe, and Leu) and 13 amino acid replacements in positions P1', P2, and P3. The binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T)++, determined from k(cat)/K(m) values) were calculated. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results showed that except for S1, the other subsites (S2, S3, and S1') vary among chymotrypsins. This result contrasts with insect trypsin data that revealed a trend along evolution, putatively associated with resistance to plant inhibitors. In spite of those differences, the data suggested that in lepidopteran chymotrypsins S2 and S1' bind the substrate ground state, whereas only S1' binds the transition state, supporting aspects of the present accepted mechanism of catalysis.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae.

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation.

Identification of midgut microvillar proteins from Tenebrio molitor and Spodoptera frugiperda by cDNA library screenings with antibodies.

The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera) in evolution. For this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor (Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were sequenced, assembled and searched for similarities with gene/protein databases. The predicted midgut microvillar proteins from T. molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes (aminopeptidase, alpha-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a) proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase, serpin and juvenile hormone epoxide hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypeptidase); (c) peritrophins; (d) proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin, calmodulin) and (f) unidentified proteins. The novel approach is compared with others and microvillar function is discussed in the light of the predicted proteins.

Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica.

Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 angstroms using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P2(1) (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 angstroms, beta = 102.97 degrees) and the orthorhombic space group P2(1)2(1)2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 angstroms), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.

Substrate specificity of insect trypsins and the role of their subsites in catalysis.

Trypsins have high sequence similarity, although the responses of insect trypsins to chemical and natural inhibitors suggest they differ in specificities. Purified digestive trypsins from insects of four different orders were assayed with internally quenched fluorescent oligopeptides with two different amino acids at P1 (Arg/Lys) and 15 amino acid replacements in positions P1', P2', P2, and P3. The binding energy (deltaG(s), calculated from Km values) and the activation energy (deltaG(T)(double dagger), determined from kcat/Km values) were calculated. Dictyoptera, Coleoptera and Diptera trypsins hydrolyze peptides with Arg at P1 at least 3 times more efficiently than peptides with Lys at P1, whereas Lepidoptera trypsins have no preference between Arg and Lys at that position. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results suggested that insect trypsin subsites become progressively more hydrophobic along evolution. Apparently, this is an adaptation to resist plant protein inhibitors, which usually have polar residues at their reactive sites. Results also suggested that, at least in lepidopteran trypsins, S3, S2, S1', and S2' significantly bind the substrate ground state, whereas in the transition state only S1' and S2' do that, supporting aspects of the presently accepted mechanism of trypsin catalysis. Homology modeling showed differences among those trypsins that may account for the varied kinetic properties.

Coevolution of insect trypsins and inhibitors.

Many plant proteinase inhibitors have lysine at the P1 position, presumably to avoid hydrolysis by insect trypsins. Lepidopteran trypsins appear to have adapted to resist proteinase inhibitors through increased inhibitor hydrolysis and decreased binding to inhibitor hydrophilic reactive sites. Lepidopteran digestive trypsins prefer lysine at the P1 position and have substrate binding subsites more hydrophobic than trypsins from insects in other orders. All available sequences of sensitive and inhibitor-insensitive insect trypsins were aligned with porcine trypsin, for which interactions with Kunitz and Bowman-Birk inhibitor are known. After discounting conserved positions and positions not typical of sensitive or insensitive trypsins, the following residues were considered important to insect trypsin-PI interactions (chymotrypsin numbering): 60, 94, 97, 98, 99, 188, 190, 213, 215, 217, 219, 228. These residues support the Neighbor Joining analysis tree branches separating sensitive and insensitive trypsin sequences. Primary sequences interacting with PIs are around the active site, with some forming part of the S1 (188, 217, 219 and 228) or S4 (99, 215) pockets.

Purification, properties and substrate specificity of a digestive trypsin from Periplaneta americana (Dictyoptera) adults.

A digestive trypsin from the American cockroach (Periplaneta americana, Dictyoptera) males was purified by a combination of anionic chromatographies in low and high pressure systems. The yield was 70% with a final specific activity of 2,000 units per mg protein (substrate: benzoyl-Arg-p-nitroanilide, BRpNA). Chemical modification with TLCK (k(obs)=3.3 M(-1) s(-1); stoichiometry 1:1) and PMSF (k(obs)=0.18 M(-1) s(-1); stoichiometry 1:1) confirmed that this peptidase is a trypsin. This enzyme has a molecular weight of 29 kDa (SDS-PAGE), a pI of 6.0 and a pH optimum of 8.9. Kinetic parameters using different colorimetric, fluorimetric and internally-quenched substrates indicated that P. americana trypsin prefers to hydrolyze synthetic substrates containing more than one amino acid residue and with an arginine residue at P1 position and a hydrophobic residue at P2. This enzyme presented a Km of 120 microM for BRpNA and is competitively inhibited by benzamidine (Ki=0.25 microM). Soybean trypsin inhibitor is a tight-binding inhibitor presenting a K(D) of 0.4 nM. Differences in substrate specificity and in the reactivity of the trypsin active site groups can be related to adaptation of insects to different hosts. P. americana trypsin is an excellent model for comparison as a basal group on evolutionary studies of insect trypsins.

Secretion of beta-glycosidase by middle midgut cells and its recycling in the midgut of Tenebrio molitor larvae.

There are four beta-glycosidases (betagly1, betagly2, betagly3, and betagly4) in Tenebrio molitor midgut larvae. betagly1 and betagly2 have identical kinetic properties, and differ in a few amino acid residues. Purified betagly1 was used to raise antibodies in a rabbit. The resulting antiserum recognizes in a Western blot only betagly1 and betagly2 in midgut tissue homogenates and contents. An immunocytochemical study carried out using confocal fluorescence and immunogold techniques showed that betagly1+betagly2 are secreted by exocytosis mainly from the distal part of the second third of T. molitor midguts. This is the first immunocytochemical study of an insect digestive enzyme that does not have polymers as substrates. Enzyme assays with 0.3 mM amygdalin, a condition that detects only betagly1+betagly2, revealed that most of those beta-glycosidases are found in the lumen of anterior and middle midgut. This supports the hypothesis that a countercurrent flux of fluid occurs in T. molitor midgut that is able to carry betagly1 and betagly2 to anterior midgut, in agreement with the enzyme recycling mechanism thought to occur in most insects.

Purification, molecular cloning, and properties of a beta-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae.

Two beta-glycosidases (M(r) 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (betaGly1 and betaGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E379 (nucleophile) and E169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant beta-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellodextrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified beta-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose beta-1,4-glucosidic bond is 21.2 s(-1) x M(-1). The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.

Amino acid residues involved in substrate binding and catalysis in an insect digestive beta-glycosidase.

A beta-glycosidase (M(r) 50000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl beta-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pK(a1)=4.9 and pK(a2)=7.5). Effect of pH on carbodiimide inactivation indicates that the pK(a) 7.5 group is a carboxyl. k(cat) and K(m) values were obtained for different p-nitrophenyl beta-glycosides and K(i) values were determined for a range of alkyl beta-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature beta-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E(187) (proton donor) and E(399) (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.

The origin and functions of the insect peritrophic membrane and peritrophic gel.

There is a a fluid (peritrophic gel) or membranous (peritrophic membrane, PM) film surrounding the food bolus in most insects. The PM is composed of chitin and proteins, of which peritrophins are the most important. It is proposed here that, during evolution, midgut cells initially synthesized chitin and peritrophins derived from mucins by acquiring chitin-binding domains, thus permitting the formation of PM. Since PM compartmentalizes the midgut, new physiological roles were added to those of the ancestral mucus (protection against abrasion and microorganism invasion). These new roles are reviewed in the light of data on PM permeability and on enzyme compartmentalization, fluid fluxes, and ultrastructure of the midgut. The importance of the new roles in relation to those of protection is evaluated from data obtained with insects having disrupted PM. Finally, there is growing evidence suggesting that a peritrophic gel occurs when a highly permeable peritrophic structure is necessary or when chitin-binding molecules or chitinase are present in food.