The low excretory rates of secreted digestive enzymes, such as trypsins, in insect species with peritrophic membranes led to the hypothesis of ectoperitrophic countercurrent water fluxes causing enzyme recycling. The midgut water flux model of Tenebrio molitor (T. molitor) is revisited and supported by in vivo experiments. Sequences from proteins putatively involved in water transport were retrieved from the T. molitor transcriptome by Blast and analyzed using bioinformatics tools. Gene expression of selected proteins was determined in three midgut sections (anterior, AM; middle, MM; posterior, PM) by RNA-seq, and transporter proteins were verified in microvillar-membrane-enriched midgut samples by proteomics. Genes encoding three cation chloride cotransporters (CCC) and four aquaporins were expressed in the midgut. TmNaCCC2, TmPrip, and TmEglp1 showed higher expression in the front half, while TmKCC, TmNKCC1, TmDrip, and TmEglp2 were more highly expressed in the back half. However, only TmNaCCC2 was found by proteomics. Midgut water fluxes were quantified by feeding T. molitor larvae with nonabsorbable dye and measuring its concentration along the midgut. The results suggest water absorption in AM and secretion in MM and PM, potentially caused by TmNaCCC2 and TmPrip in AM, and TmKCC and TmDrip in PM, whereas MM serves as a transition region. Larvae fed on furosemide, an NKCC and KCC inhibitor, showed altered midgut water fluxes, resulting in higher trypsin excretion into the hindgut, thus reinforcing the hypothesis of a countercurrent water flux generated by CCCs powering enzyme recycling in insect midguts.

Peptidases constitute at least 2% of genes in living organisms and participate in nearly all physiological processes across life forms. Conversely, peptidase inhibitors are essential for regulating proteolytic activity and have been widely applied. Combining high-throughput sequencing of novel peptidase inhibitor sources with molecular modeling and drug design currently represents an efficient strategy for developing new molecules. Venomous spiders harbor a wide array of peptidase inhibitors in both their venom and digestive system. However, biochemical and transcriptomic investigations of non-venomous spiders (Uloboridae) remain recent and scarce. Here, transcriptomic and biochemical analyses of the Uloboridae spider Zosis geniculata's digestive midgut diverticula (MD) revealed that this species exhibited a digestive enzyme profile similar to that of other spiders. Furthermore, the MD transcriptome identified 19 peptidase inhibitors belonging to six inhibitor families. Serine peptidase inhibitors were the most abundant and diverse, while metallopeptidases represented the main proteolytic enzymes, suggesting that these inhibitors may have evolved to counteract prey-derived peptidases. Inhibitory assays using trypsin from potential insect prey confirmed this activity. The diversity and abundance of these molecules highlight Uloboridae spiders as promising novel sources of proteolytic inhibitors.

Opiliones (Arachnida) comprises approximately 7,000 species. Due to their lack of venom glands, their defense is mainly based on the secretion of scent glands, which also play roles in communication and antimicrobial activity. This odoriferous secretion has a diverse composition according to harvestmen species but frequently contains benzoquinones. Studies of benzoquinone synthesis intermediates suggest a pathway based on lipid metabolism from diet. This study provides the first transcriptomic and enzymatic analysis of the midgut of Mischonyx squalidus to understand the acquisition of nutrients. The enzymatic analysis tested 11 digestive enzymes and found high lipase activity and moderate and low activity for peptidases and carbohydrases, respectively. Transcriptome sequencing yielded 19,658 unigenes, predominating enzymes and binding proteins closely associated with Xiphosura and Scorpiones proteins; a third of which are hydrolases. A multigene lipase family is expressed. Cathepsin L peptidases were prominently abundant, indicating their relevance for protein digestion. Additionally, toxin-like proteins and lipid metabolism-related enzymes, such as phospholipase A2 and NPC cholesterol transporters, were represented, indicating a sophisticated digestive and metabolic system for lipids. These findings suggest a link between lipid digestive metabolism and defensive and communicative molecule synthesis, underscoring Opiliones’ unique evolutionary adaptations.

Previous studies suggest that some insects require dietary arginine because they cannot synthesize this amino acid through the urea cycle. To determine whether this finding applies to all insects and what its metabolic implications are, we analysed the conservation of 20 genes involved in arginine biosynthesis and metabolism in the genomes of 150 species from 11 taxonomic orders. Our results showed that no insect can synthesize arginine via the urea cycle, as ornithine carbamoyltransferase is absent from all genomes analysed. While we found losses in other genes encoding urea cycle enzymes, nitric oxide synthase (NOS) was conserved across orders. However, the citrulline produced by NOS cannot be converted back to arginine in several insects due to the loss of argininosuccinate synthase and argininosuccinate lyase genes. Despite the inability to synthesize arginine, all insects (except some Hemiptera) can degrade it to ornithine and urea, as the arginase (ARG) gene is conserved across the orders analysed. For some Hemiptera that have lost ARG, we investigated how these insects produce or metabolize ornithine. Our results show that the genes for converting ornithine to glutamate, proline and putrescine are conserved across orders. However, while all insects have enzymes to synthesize putrescine and spermidine, some lack the ability to produce spermine due to the absence of the spermine synthase gene. Taken together, our results show that the loss of the urea cycle has led to significant changes in the pathways by which insects metabolize and recover arginine, which is particularly important for the diversification of hemipterans.

Hemiptera Order comprises insect species adapted to different diets regarding water and nutrient content and availability, thus suggesting different combinations of proteins to ensure their absorption. To find out whether hemipterans use the same or distinct set of proteins and whether these differences are related to the phylogeny or the diet, RNAseq analyses were conducted in gut sections of three hemipterans, M. fimbriolata, D. peruvianus, and R. prolixus, with remarkable distinct diet. Since only a few of the selected proteins were functionally characterized, the coded putative proteins were manually curated by bioinformatics to infer their physiological function. The results suggest a relationship between gene expression patterns and water and nutrient dietary content and availability. In contrast, putative gene expansions and deletions are related to phylogeny, corresponding to evolutionary adaptations of ancestral forms to feed on xylem, cotton seeds, and blood, resulting in more resemblances between D. peruvianus and R. prolixus than M. fimbriolata. M. fimbriolata absorbs water through aquaporins Drip and Prip in the filtration chamber by passive diffusion, with a higher contribution of water-selective Drip. D. peruvianus water absorption involves Drip and Prip, but Prip contribution appears to be higher, and they probably cooperate with water-ion cotransporters in the posterior midgut. R. prolixus absorbs water in the anterior midgut involving a sodium transporter and a putative water-urea Prip. Sugars, amino acids, and lipids might be absorbed along the midgut in the three species, with a higher contribution of the posterior midgut for amino acid and lipid absorption in M. fimbriolata and D. peruvianus and the middle midgut in R. prolixus.

A model of protein digestion and peptide and amino acid absorption along the midgut of Musca domestica larvae was proposed and supported by RNA-Seq analyses, protein bioinformatics, microvillar-membrane-enriched midgut proteomics, and enzymatic activities. Peptidase genes are highly expressed in the posterior midgut (PM), whereas those for cathepsins have expression limited to the middle midgut (MM). MM has the lowest levels of gene expression of almost all peptidases but has high expression of genes for membrane-bound serine endopeptidases. The anterior midgut (AM) has intermediate expression values of serine endopeptidase and aminopeptidase (AP) genes and low expression of carboxypeptidases (CPs). Gene expression and peptidase activities were usually consistent for putative intracellular and membrane-bound enzymes. However, secreted peptidase gene expression and activities have divergent values, especially in the PM, which may be due to the countercurrent water flux causing enzyme recycling, thus decreasing their excretion. Data suggest that Trys and APs act in the AM. In the acidic MM, lysozymes kill microorganisms found in the diet, releasing proteins digested by cathepsins D, which may also digest Trys coming from the AM. Finally, highly active serine endopeptidases, CPs, dipeptidases, and APs complete protein digestion in PM. Absorption of peptides and amino acids coupled to protons may occur along the midgut, especially in PM, as occurs for facilitated amino acid transport. Absorption with sodium ions is probably restricted to AM and PM. Our findings provide valuable insights into the protein digestion and amino acid absorption mechanism in M. domestica larvae.

Food may be acquired by biting with chewing parts, sucking by pierce-sucking mouthparts, or by ingesting a pre-digested or pre-dispersed meal. Digestion is a stepwise process by which the molecules of food are hydrolyzed into components able to be absorbed. The first step of digestion is the initial digestion, when food polymers result in oligomers, followed by the second step, intermediary digestion, corresponding to the conversion of oligomers into dimers and, finally, the third step, final digestion, in which the dimers are cleaved into monomers that are absorbed. The gut morphology varies among insect taxa from the basal plan formed by a capacious crop followed by a midgut with anteriorly placed ceca, ending in a hindgut. The midgut has inside an anatomical chitin-protein film, the peritrophic membrane (PM), that separates two luminal compartments: endoperitrophic (inside PM) and ectoperitrophic (outside PM) spaces. In polyneopterans and lower holometabolans, the enzymes of initial and intermediary digestion move freely inside the midgut, whereas in higher holometabolans, only the enzymes of initial digestion traverse PM into the endoperitrophic space. The enzymes of intermediary digestion are retained in the ectoperitrophic space because they are larger than the PM pores (7–9 nm dia). There are midgut countercurrent fluxes caused by the secretion of fluid in a posterior region and its absorption in an anterior region. These countercurrent fluxes decrease the loss of enzymes by excretion as part of the enhancement of digestive efficiency caused by the compartmentalization of digestion. The sites of water and nutrient absorption are identified with the use of a non-absorbable dye and nutrients. The concentration of dye indicates water removal, its dilution indicates water secretion, whereas a change in the ratio of nutrient and dye indicates nutrient absorption.

The knowledge of diets and their chemical components demanding digestion is a prerequisite to understanding the evolution of insect midguts. The clade Insecta is a hyper-diverse group, and this diversity extends to an impressive number of different feeding habits. Therefore, we can only summarize some of the most conspicuous and representative types of diets used by insects in their natural environments. The diversity of chemical compounds found in these diets is also staggering. This makes insects excellent models for investigating the comparative physiology of digestion among animals. The following diets will be discussed: detritus, plant exudates, other plant material, animal flesh, blood, fungi, lichens, bacteria, and nectar. In this chapter, we will discuss some of the major types and chemical compositions of diets used by insects. Our focus will be on chemical constituents that require some special type of digestion or that offer some challenge that leads to adaptations in the insect digestive system.

Insect digestion is regulated mainly by peptides that affect food intake, gut motility, digestive enzyme release, and nutrient absorption. The regulatory peptides are produced by the central nervous system, the stomatogastric nervous system, and midgut endocrine cells. There are peptides with stimulatory (allatotropin, proctolin, sulfakinins, tachyinins, kinins, etc.) or inhibitory (allatostatin, myoinhibitory peptides, myosuppressins, etc.) effects. In this chapter, we will review the chemical identity, site of production, and function of those peptides that differ somewhat among different insects.

In this chapter, we cover the new insect control techniques that target the intestinal tract and are not based on chemical insecticides. Crystal (Cry) and Cytolitic (Cyt) protein families from Bacillus thuringiensis (Bt) and related toxins are used worldwide for insect control. Their primary action is to lyse midgut epithelial cells via binding to specific brush border receptors and elicit the formation of pores or trigger a necrotic pathway, which leads to the destruction of the midgut. We discuss in some detail recent findings on their mode of action. The use of proteinaceous inhibitors expressed in transgenic plants directed to enzyme targets, such as endopeptidases, α-amylases, and polygalacturonases, is reviewed. The success of transgenic crop plants expressing Cry toxin genes has paved the way for the development of genetically modified plants expressing double-stranded RNA (dsRNA) and CRISPR/Cas that can suppress the expression of genes coding for selected vital proteins. We discuss the recent developments in dsRNA and CRISPR/Cas technologies for insect pest management focusing on their actions in insect midgut.

Insects are the most diverse living beings and their ancestors moved from the sea and colonized the land long before the chordates. Phylogenetic and fossil data were combined to detail the patterns of insect evolution. Insects able to flex their wings over the back (Neoptera) correspond to most of the insects and evolved along the major lineages: Polyneoptera, Condylognatha, and Holometabola. Polyneoptera includes Dictyoptera – cockroaches and termites that are omnivorous or wood feeders and the carnivorous mantids – and Orthoptera – the omnivorous crickets and grass-feeding grasshoppers. Condylognatha includes Hemiptera which are the only insects able to live entirely on plant sap such as aphids, cicadas, and spittlebugs; and others like bugs adapted to different diets. Holometabola is the most successful lineage with 86% of the insect species, have complete metamorphosis (larva, pupa, and adult) and comprises the major insect orders: Coleoptera (beetles); Hymenoptera (wasps, ants, and bees); Diptera (mosquitoes and flies); and Lepidoptera (butterflies and moths). Insects of these orders explore the most diverse food sources like other insects, stems, leaves and wood, seeds, keratin (like woolen carpets), pollen, nectar, fungi, and vertebrate blood. The major selective pressures affecting insect guts identified were: (a) adaptations to deal with large amounts of dilute fluid food, (b) adaptations to digesting plant and fungal cells as a result of horizontal transfer of genes from microorganisms and recruitment of lysosomal proteins as digestive enzymes, (c) adaptations to avoiding plant inhibitors by gene expansion and new functionalization, (d) and adaptations to avoiding prolonged exposure to natural enemies and to living in short-lived media by reduction of life span permitted by more efficient midguts.

Most insects have plant material in their diets. Plant cell walls are broken by mastication or under the action of plant cell wall-degrading enzymes. Primary plant cell walls, as those of grasses, are composed of cellulose, pectin, and a network of polysaccharides named hemicelluloses. Secondary plant cell walls, as those in wood, are formed by lignocellulose, which are cellulose and hemicelluloses cross-linked by lignin. Insect cellulases are always β-1,4-endoglucanases of family GH9 or GH45. GH9 cellulases are widespread but were lost in dipterans and lepidopterans. Those of GH45 were acquired by beetles of the clade Phytophaga by horizontal transfer from fungi. Despite the existence of endogenous cellulases, in many insects microbiota cellulases also concur. Pectinases are particularly important in hemipterans to facilitate the insertion of their stylets in sap-conducting structures and to beetles that bore plant tissues. Hemicellulases hydrolyze hemicelluloses. They are licheninases, laminarinases, xylanases, and mannanases. Laminarinases are widespread among insects and hydrolyze β-1,3-glucans (laminarins) and some of them, like the one of Tenebrio molitor, also hydrolyze yeast β-1,3-1,6-glucans. Laminarinases are supposed to digest fungal cells in contaminated food (T. molitor) or callose (lepidopterans), which is deposited in response to wounding caused by the larvae and that impairs nutrient availability for the larvae. The degradation of lignin can only be efficiently performed by oxidative depolymerization catalyzed by laccases aided by redox mediators that usually are produced by microbes. Because of that, most insects only attack wood partly digested by microbes. Fungi are nutrients for detritivorous and stored product insects. Digestive chitinases lack chitin-binding domains, so they are efficient in digesting fungi cell walls but are harmless for the peritrophic membrane. Lysozyme catalyzes the hydrolysis of the peptidoglycan of the cell walls of many bacteria. In insects, midgut lysozyme active in low pH is characteristic of cyclorrhaphous dipterans, in agreement with the fact that most of their larvae feed largely on bacteria. Insect digestive cellulases were studied in detail, including crystallography and resolution of their 3D structures.

Microorganisms play several roles in insects. However, in this chapter, we will consider only the roles of microbiota in insect digestion and nutrition. Bacteria are food for insects, exemplified by larval houseflies and blowflies, living in feces, decaying fruits, and corpses that are rich in bacteria. Microbiota, particularly bacteria, also play a role in the digestion of recalcitrant materials such as tannins, lignins, and humic substances. The last ones are complex molecules derived from residues occurring in soils. Bacterial action on those recalcitrant molecules is observed in termites and some beetles. Fungi are important players in lignocellulose digestion in termites of the subfamily Macrotermitinae. Sap-feeding insects usually lack essential nutrients, especially amino acids, which are supplied by microbial symbionts, such as those found in pea aphids. In diets poor in vitamins, symbiont microbiota may provide them for the insect, exemplified by the blood-feeder Rhodnius prolixus. Finally, gut microbiota help their hosts in detoxifying molecules that are characteristic of plant defenses or insecticides used in insect control.

The midgut of Zabrotes subfasciatus (Coleoptera) and other insects may have regions lacking a peritrophic membrane (matrix, PM) and covered with a jelly-like material known as peritrophic gel. This work was undertaken to test the hypothesis that the peritrophic gel is a vertebrate-like mucus. By histochemistry we identified mucins along the whole midgut, which contrasts with the known occurrence of PM only at the posterior midgut. We also analyzed the expression of the genes coding for mucus-forming mucins (Mf-mucins), peritrophins, chitin synthases and chitin deacetylases along the midgut and carcass (insect without midgut) by RNA-seq. Mf-mucins were identified as proteins with high O-glycosylation and multiple tandem repeats of Pro/Thr/Ser residues. Peritrophins were separated into PM proteins, cuticular proteins analogous to peritrophins (CPAPs) and ubiquitous-chitin-binding domain-(CBD)-containing proteins (UCBPs). PM proteins have at least 3, CPAP one or 3, and UCBPs have a varied number of CBDs. PM proteins are more expressed at midgut, CPAP at the carcass, and UCBP at both. The results showed that most PM proteins are mainly expressed at the posterior midgut, together with midgut chitin synthase and chitin deacetylase, and in agreement with the presence of PM only at the posterior midgut by visual inspection. The excretion of most midgut chitinase is avoided, suggesting that the shortened PM is functional. Mf-mucins are expressed along the whole midgut, probably forming the extracellular mucus layer observed by histochemistry. Thus, the lack of PM at anterior and middle midgut causes the exposure of a mucus, which may correspond to the previously described peritrophic gel. The putative functional interplay of mucus and PM is discussed. The major role of mucus is proposed to be tissue protection and of PM to enhancing digestive efficiency by allowing enzyme recycling.

Procedures to obtain reliable enzyme kinetic parameters are discussed. The classification of enzymes regarding their function (substrate specificity and mechanism, Enzyme Commission Nomenclature) and protein families (MEROPS, CAZy, and Brenda platforms) is presented. The mechanism of the enzymes acting on the major nutrient substrates is described. The substrates discussed are carbohydrates (starch, oligosaccharides, and disaccharides); peptides (proteins, oligopeptides, and dipeptides); and acylglycerol esters (triacylglycerols and phospholipids). The enzymes discussed in detail are: serine endopeptidases (trypsins, chymotrypsins, elastases); collagenases; carboxypeptidases; aminopeptidases; amylases; α-glucosidases; β-glucosidases; β-fructosidases; β-N-acetylhexosaminidases; α-mannosidases; α-galactosidases; myrosinases; trehalases; triacylglycerol lipases; and phospholipases. Lysosomal proteins recruited as digestive enzymes (cysteine- and aspartic-endopeptidases) and enzymes involved in the degradation of cell walls are the subject of specific chapters.

The findings regarding insect digestive features and phylogeny led to the proposal that the overall pattern of digestion of the insects derived from basic plans corresponding to ancestors. In the Neoptera ancestor, midgut digestive enzymes are translocated to the crop, where digestion starts, ending in the midgut. Enzymes active in initial, intermediate, and final digestion move freely among gut compartments. The excretion of digestive enzymes is decreased by midgut countercurrent fluxes caused by the secretion of water by the Malpighian tubules and its absorption back into the ceca. Condylognatha ancestors lost ceca, water-secreting regions, and acquired microvilli-associated membranes to facilitate nutrient absorption. The Holometabola insects have countercurrent fluxes of water caused by the secretion of fluid in the posterior midgut and its absorption into the ceca or, in their absence, by the anterior midgut. The Hymenoptera-Panorpoid ancestor has no enzymes in crop, only enzymes of initial digestion pass through the peritrophic membrane, but have midgut countercurrent fluxes like the Holometabola ancestors. The Cyclorrhapha ancestor acquired a highly acid middle midgut rich in lysozyme and cathepsin D to digest bacteria, and its countercurrent flux of water is absorbed in the middle posterior midgut. The Lepidoptera ancestor differs from the Hymenopteran-Panorpoid ancestor in having a highly alkaline midgut with goblet cells to excrete K+.