Histochemistry and transcriptomics of mucins and peritrophic membrane (PM) proteins along the midgut of a beetle with incomplete PM and their complementary function.

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.

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

A proteomic approach to identify digestive enzymes, their exocytic and microapocrine secretory routes and their compartmentalization in the midgut of Spodoptera frugiperda.

A proteomic approach was used to identify the digestive enzymes secreted by exocytosis and by microapocrine vesicles and enzyme midgut compartmentalization in Spodoptera frugiperda larvae. For this, proteomic analyses were performed in isolated midgut enterocyte microvillar membrane, in a fraction enriched in microapocrine vesicles (separated in soluble and membrane fractions), in the washings of the peritrophic membrane to isolate its loosely- and tightly-bound proteins, and in the peritrophic membrane contents. PM washings correspond to proteins extracted from the mucus layer surrounding PM. Serine endopeptidases (trypsins, chymotrypsins and serine endopeptidase homologs that have substitutions in the catalytic residues) and lipases are mainly secreted by exocytosis. Aminopeptidases are mainly microvillar enzymes and some are secreted membrane-bound to microapocrine vesicles, whereas carboxypeptidase isoforms follow different secretory routes. The results also showed that most polymer hydrolases (such as amylase and endopeptidases) are not retained in the ectoperitrophic fluid (found in PM washings but absent from PM contents). On the contrary, most enzymes involved in intermediate digestion (exemplified by carboxypeptidase and aminopeptidase) do not pass through the peritrophic membrane. Finally, the data revealed that the protein composition of PM includes peritrophins classified as peritrophic membrane proteins, PMP, and chitin deacetylase.

Recruitment of lysosomal cathepsins B, L and D as digestive enzymes in Coleoptera.

The recruitment of the lysosomal cathepsins B (CAB), L (CAL) and D (CAD) as luminal digestive enzymes was investigated in 3 species of beetles. Gene expression was determined by RNA-seq in different regions of the midgut and in the carcasses from the transcriptomes of Dermestes maculatus, Tenebrio molitor and Zabrotes subfasciatus. These data together with phylogenetic analyses, allowed us to identify the sequences of the gene coding for digestive and lysosomal CABs, CADs and CALs in T. molitor and Z. subfasciatus and observe the absence of digestive cathepsins in D. maculatus. Comparisons of structures based on the overall similarity of modelled structures were performed and subsite residues in the lysosomal and digestive CALs were identified by molecular docking. The data showed that S2 subsites are very variable, probably as an adaption to a luminal digestive role. The survey of sequences of the gene coding for cathepsins in the genomes of 13 beetle species from different phylogenetic groups showed that expansion of CAL and CAB genes occurred only in the Cucujiformia clade. Several digestive CABs have a reduced occluding loop, probably to act as digestive enzymes. Pollen-feeding was proposed to be the selective pressure to recruit cathepsins as digestive enzymes in Cucujiformia beetles.

Transcriptomic and proteomic analysis of the underlying mechanisms of digestion of triacylglycerols and phosphatides and absorption and fate of fatty acids along the midgut of Musca domestica.

Most dietary lipids are triacylglycerols (TAGs) and phosphatides that are digested by TAG lipases and phospholipases (PLIPs), respectively, originating fatty acids (FA). The genome of Musca domestica has genes coding for phospholipases A1 (1PLIP), A2 (2PLIP), B (BPLIP), and acid lipases (ALIP), as for proteins involved in activation, binding, and metabolism of FA, which expression in the larval midgut was evaluated by RNA-seq. Some of the codified proteins were identified in midgut microvillar-enriched membrane by proteomics. 1PLIPs are the most expressed PLIPs, mainly in anterior midgut whereas 2PLIPs, and BPLIP in middle and posterior midgut, and ALIPs between middle and posterior regions. Absorption of FAs is putatively accomplished by proteins involved in FA activation (acyl-CoA synthetases) found in microvillar-enriched membrane preparations. Furthermore, FA uptake could be enhanced by proteins that bind FAs (FA-binding proteins) and its activated form (acyl-CoA binding proteins) mainly expressed in posterior midgut. Activated FAs could have different fates: synthesis of diacylglycerol (DAG) and TAG through monoacylglycerol and glycerol-3-phosphate pathways; synthesis of phosphatides; energy source by ß-oxidation. Most genes coding for enzymes of those routes is expressed mainly at the end of posterior midgut. Data suggest that phosphatides are digested in anterior midgut by Md1PLIPs, releasing lysophosphatides that emulsify fats to be digested by MdALIPs in the middle and posterior midgut. Most resulting FAs is absorbed in the posterior midgut, where they follow the synthesis of DAG, TAG, and phosphatides or are oxidized along the midgut, mainly in highly metabolic middle and posterior midgut regions.

Cathepsins L and B in Dysdercus peruvianus, Rhodnius prolixus, and Mahanarva fimbriolata. Looking for enzyme adaptations to digestion.

Cysteine peptidases (CP) play a role as digestive enzymes in hemipterans similar to serine peptidases in most other insects. There are two major CPs: cathepsin L (CAL), which is an endopeptidase and cathepsin B (CAB) that is both an exopeptidase and a minor endopeptidase. There are thirteen putative CALs in Dysdercus peruvianus, which in some cases were confirmed by cloning their encoding genes. RNA-seq data showed that DpCAL5 is mainly expressed in the anterior midgut (AM), DpCAL10 in carcass (whole body less midgut), suggesting it is a lysosomal enzyme, and the other DpCALs are expressed in middle (MM) and posterior (PM) midgut. The expression data were confirmed by qPCR and enzyme secretion to midgut lumen by a proteomic approach. Two CAL activities were isolated by chromatography from midgut samples with similar kinetic properties toward small substrates. Docking analysis of a long peptide with several DpCALs modeled with digestive Tenebrio molitor CAL (TmCAL3) as template showed that on adapting to luminal digestion DpCALs (chiefly DpCAL5) changed in relation to their ancestral lysosomal enzyme (DpCAL10) mainly at its S2 subsite. A similar conclusion arrived from structure alignment-based clustering of DpCALs based on structural similarity of the modeled structures. Changes mostly on S2 subsite could mean the enzymes turn out less peptide-bond selective, as described in TmCALs. R. prolixus CALs changed on adapting to luminal digestion, although less than DpCALs. Both D. peruvianus and R. prolixus have two digestive CABs which are expressed in the same extension as CALs, in the first digestive section of the midgut, but less than in the other midgut sections. Mahanarva fimbriolata does not seem to have digestive CALs and their digestive CABs are mainly expressed in the first digestive section of the midgut and do not diverge much from their lysosomal counterparts. The data suggest that CABs are necessary at the initial stage of digestion in CP-dependent Hemipterans, which action is completed by CALs with low peptide-bond selectivity in Heteroptera species. In M. fimbriolata protein digestion is supposed to be associated with the inactivation of sap noxious proteins, making CAB sufficient as digestive CP. Hemipteran genomes and transcriptome data showed that CALs have been recruited as digestive enzymes only in heteropterans, whereas digestive CABs occur in all hemipterans.

N-glycosylation in Spodoptera frugiperda (Lepidoptera: Noctuidae) midgut membrane-bound glycoproteins.

Spodoptera frugiperda is a widely distributed agricultural pest. It has previously been established that glycoproteins in the midgut microvillar membrane of insects are targets for toxins produced by different organisms as well as plant lectins. However, there is still little information about the N-glycome of membrane-bound midgut glycoproteins in Lepidoptera and other insect groups. The present study used mass spectrometry-based approaches to characterize the N-glycoproteins present in the midgut cell microvilli of Spodoptera frugiperda. We subjected midgut cell microvilli proteins to proteolytic digestion and enriched the resulting glycopeptides prior to analysis. We also performed endoglycosidase release of N-glycans in the presence of H218O determining the compositions of released N-glycans by MALDI-TOF MS analysis and established the occupancy of the potential N-glycosylation sites. We report here a total of 160 glycopeptides, representing 25 N-glycan compositions associated with 70 sites on 35 glycoproteins. Glycan compositions consistent with oligomannose, paucimannose and complex/hybrid N-glycans represent 35, 30 and 35% of the observed glycans, respectively. The two most common N-glycan compositions were the complex/hybrid Hex3HexNAc4dHex4 and the paucimannose structure that contains only the doubly-fucosylated trimannosylchitobiose core Hex3HexNAc2dHex2, each appearing in 22 occupied sites (13.8%). These findings enlighten aspects of the glycobiology of lepidopteran midgut microvilli.

Evolutionary trends of digestion and absorption in the major insect orders.

The spatial organization of digestion, which corresponds to the steps by which the ingested food is hydrolyzed in the different regions of the gut, was described in insects from the major insect orders. The pattern of digestion and absorption in the midgut shows a strong phylogenetic influence, modulated by adaptation to particular feeding habits. Based on this, basic digestive patterns were recognized and were proposed to represent the major ancestors from which the different orders evolved. The putative ancestors chosen to represent different points in the evolution from basal Neoptera to more derived orders were: Neoptera, Polyneoptera, Hemiptera, Hymenoptera-Panorpoidea (Diptera-Lepidoptera), Lepidoptera, and Cyclorrhapha. The basic plan of Neoptera was supposed to be alike that of Polyneoptera, which was hypothesized from studies performed in grasshoppers, crickets and from stick insects. For Holometabola, the basic plan was initially proposed from studies carried out in beetles, bees, nematocerous flies, common flies and also from moths. This review updates the physiological data supporting the putative midgut basic patterns by discussing available data on insects pertaining to different taxa and details the evolutionary trends of midgut function among the major insect orders. Furthermore, by using recent genomic and transcriptome data, this review discusses the few insects for which the spatial organization of midgut absorption is known.

Midgut fluxes and digestive enzyme recycling in Musca domestica: A molecular approach.

Insects are reported to have water midgut countercurrents fluxes powering enzyme recovery before excretion, usually known as enzyme recycling. Up to now there is a single, and very incomplete, attempt to relate transporters and channels with countercurrent fluxes. In this work, M. domestica midgut water fluxes were inferred from the concentration of ingested and non absorbable dye along the midgut, which anterior midgut was divided in two sections (A1, A2), the middle in one (M) and the posterior midgut in four (P1, P2, P3, and P4), which led to the finding of additional sites of secretion and absorption. Water is secreted in A1 and A2 and absorbed at the middle midgut (M), whereas in posterior midgut, water is absorbed at P2 and secreted in the other sections, mainly at P4. Thus, a countercurrent flux is formed from P4 to P2. To disclose the involvement of the known water transporters Na+:K+:2Cl- (NKCC) and K+:Cl- (KCC), as well as the water channels aquaporins in water fluxes, their expression was evaluated by RNA-seq analyses from triplicate samples of seven sections along the midgut. MdNKCC1 was expressed in A1, MdNKCC2 was expressed in M1 and P2 and MdKCC in middle and in the most posterior region, thus apparently involved in secretion, absorption and both, respectively. MdNKCC2, MdKCC and aquaporins MdDRIP1 and 2 were confirmed as being apical by proteomics of purified microvillar membranes. The role of NKCC and KCC on midgut water fluxes was tested observing the effect of the inhibitor furosemide. The change of trypsin distribution along the posterior midgut and the increase of trypsin excretion in the presence of furosemide lend support to the proposal that countercurrent fluxes power enzyme recycling and that the fluxes are caused by NKCC and KCC transporters helped by aquaporins.

Molecular mechanisms associated with acidification and alkalization along the larval midgut of Musca domestica.

To disclose the molecular mechanisms involved in luminal midgut buffering of M. domestica, we used RNA-seq analyses from triplicate samples of seven sections along the midgut to evaluate the expression levels of genes coding for selected manually curated protein sequences. Channels, pumps and transporters were confirmed as being apical by proteomics of purified microvillar membranes. Midgut pH determinations with a microsensor and a pH indicator were carried out in larvae in different diets with or without added compounds to evaluate the role of proteins in buffering. The data suggested that acidification occurs at middle midgut by the action of H+ V-ATPase with protons produced by carbonic anhydrase, followed by chloride ions transported by a K+Cl- symporter. K+ ions are recovered by an apical K+ channel and K+ homeostasis maintained by a basolateral Na+/K+-ATPase. Acidification is also affected by a Na+/H+ exchanger and a multidrug resistance protein. Posterior midgut alkalization results from the action of a NH3 transporter and H+-coupled peptide transporter, mainly in a diet rich in free peptides. A working model was proposed for the midgut luminal acidification and alkalization, as well as for mucosal protection against acid by a neutralized mucus layer.

Evolutionary trends of digestion and absorption in the major insect orders.

The spatial organization of digestion, which corresponds to the steps by which the ingested food is hydrolyzed in the different regions of the gut, was described in insects from the major insect orders. The data showed that the overall pattern of digestion depends more on the insect phylogeny than on the insect feeding habits. Based on this, basic digestive patterns were recognized and were proposed to represent the major ancestors from which the different orders evolved. The putative ancestors chosen to represent different points in the evolution from basal Neoptera to more derived orders were: Neoptera, Hemiptera, Hymenoptera-Panorpoidea (Diptera-Lepidoptera), Lepidoptera, Cyclorrhapha. This review updates the physiological data supporting the putative midgut basic patterns by discussing available data on insects pertaining to different taxa and details the evolutionary trends of midgut function among the major insect orders. Furthermore, by using recent genomic and transcriptome data, this review discusses the few insects for which the spatial organization of midgut absorption is known.

Detergent-resistant domains in Spodoptera frugiperda midgut microvillar membranes and their relation to microapocrine secretion.

The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery.

Recruited lysosomal enzymes as major digestive enzymes in insects.

The mass recruitment to the midgut contents of lysosomal proteolytic enzymes occurred in insects under three major selective pressures. Hemipteran (true bugs, aphids, and cicadas) ancestors lost their serine peptidases (SP) on adapting to feed on protein-free plant sap. When they returned to protein diets, their cathepsins L and B were recruited to replace their lost SP. Among beetles of the series Cucujiformia, cathepsins L were recruited to hydrolyze ingested plant inhibitors that affect their major SP and/or to deal with special seed proteins, such as prolamins. Larval flies have a very acid middle midgut and use cathepsin D to digest bacteria from their infected food. All the recruited enzymes originated from duplicated genes. The recruited digestive enzymes differ from their lysosomal counterparts in critical regions of their amino acid sequences that resulted in changes in substrate specificities and other kinetic properties. The discharge of digestive cathepsins in the midgut contents, instead of lysosomes, seems to be a consequence of their overexpression or the existence of new targeting signals. Their activation at the midgut contents occurs by an autoactivation mechanism or with the help of other enzymes or by a combination of both. The targeting to lysosomes of the insect lysosomal enzymes does not follow the mammalian mannose 6-phosphate route, but an incompletely known mechanism.

Domain structure and expression along the midgut and carcass of peritrophins and cuticle proteins analogous to peritrophins in insects with and without peritrophic membrane.

Most insects have a peritrophic membrane (matrix) (PM) surrounding the food bolus. This structure, similarly to the cuticle, is mainly composed of chitin and proteins. The main proteins forming PM are known as peritrophins (PMP), whereas some of the cuticle proteins are the cuticle proteins analogous to peritrophins (CPAP). Both proteins are composed of one or more chitin binding peritrophin-A domain (CBD) and no other recognized domain. Furthermore, insects containing PM usually have two chitin synthase (CS) genes, one mainly expressed in carcass and the other in midgut. In this work we identified PMP, CPAP and CS genes in the genome of insects from the Polyneoptera, Paraneoptera and Holometabola cohorts and analyzed their expression profile in different species from each group. In agreement with the absence of PM, we observed less CBD-containing proteins and only one CS gene in the genome of Paraneoptera species, except for the Phthiraptera Pediculus humanus. The lack of PM in Paraneoptera species was also confirmed by the micrographs of the midgut of two Hemiptera species, Dysdercus peruvianus and Mahanarva fimbriolata which agreed with the RNA-seq data of both species. Our analyses also highlighted a higher number of CBD-containing proteins in Holometabola in relation to the earlier divergent Polyneoptera group, especially regarding the genes composed of more than three CBDs, which are usually associated to PM formation. Finally, we observed a high number of CBD-containing proteins being expressed in both midgut and carcass tissues of several species, which we named as ubiquitous-CBD-containing proteins (UCBP), as their function is unclear. We hypothesized that these proteins can be involved in both cuticle and PM formation or that they can be involved in immune response and/or tracheolae formation.

Transcriptomic analyses uncover emerging roles of mucins, lysosome/secretory addressing and detoxification pathways in insect midguts.

The study of insect midgut features has been made possible by the recent availability of transcriptome datasets. These data uncovered the preferential expression of mucus-forming mucins at midgut regions that require protection (e.g. the acidic middle midgut of Musca domestica) or at sites of enzyme immobilization, particularly around the peritrophic membrane of Spodoptera frugiperda. Coleoptera lysosomal peptidases are directed to midgut lumen when over-expressed and targeted to lysosomes by a mechanism other than the mannose 6-phosphate-dependent pathway. We show that this second trend is likely conserved across Annelida, Mollusca, Nematoda, and Arthropoda. Furthermore, midgut transcriptomes of distantly related species reveal a general overexpression of xenobiotic detoxification pathways. In addition to attenuating toxicity of plant-derived compounds and insecticides, we also discuss a role for these detoxification pathways in regulating host-microbiota interactions by metabolizing bacterial secondary metabolites.

Molecular machinery of starch digestion and glucose absorption along the midgut of Musca domestica.

Until now there is no molecular model of starch digestion and absorption of the resulting glucose molecules along the larval midgut of Musca domestica. For addressing to this, we used RNA-seq analyses from seven sections of the midgut and carcass to evaluate the expression level of the genes coding for amylases, maltases and sugar transporters (SP). An amylase related protein (Amyrel) and two amylase sequences, one soluble and one with a predicted GPI-anchor, were identified. Three highly expressed maltase genes were correlated with biochemically characterized maltases: one soluble, other glycocalyx-associated, and another membrane-bound. SPs were checked as being apical or basal by proteomics of microvillar preparations and those up-regulated by starch were identified by real time PCR. From the 9 SP sequences with high expression in midgut, two are putative sugar sensors (MdSP4 and MdSP5), one is probably a trehalose transporter (MdSP8), whereas MdSP1-3, MdSP6, and MdSP9 are supposed to transport glucose into cells, and MdSP7 from cells to hemolymph. MdSP1, MdSP7, and MdSP9 are up-regulated by starch. Based on the data, starch is at first digested by amylase and maltases at anterior midgut, with the resulting glucose units absorbed at middle midgut. At this region, low pH, lysozyme, and cathepsin D open the ingested bacteria and fungi cells, freeing sugars and glycogen. This and the remaining dietary starch are digested by amylase and maltases at the end of middle midgut and up to the middle part of the posterior midgut, with resulting sugars being absorbed along the posterior midgut.

Active subsite properties, subsite residues and targeting to lysosomes or midgut lumen of cathepsins L from the beetle Tenebrio molitor.

Cathepsins L are the major digestive peptidases in the beetle Tenebrio molitor. Two digestive cathepsins L (TmCAL2 and TmCAL3) from it had their 3D structures solved. The aim of this paper was to study in details TmCAL3 specificity and properties and relate them to its 3D structure. Recombinant TmCAL3 was assayed with 64 oligopeptides with different amino acid replacements in positions P2, P1, P1' and P2'. Results showed that TmCAL3 S2 specificity differs from the human enzyme and that its specificities also explain why on autoactivation two propeptide residues remain in the enzyme. Data on free energy of binding and of activation showed that S1 and S2' are mainly involved in substrate binding, S1' acts in substrate binding and catalysis, whereas S2 is implied mainly in catalysis. Enzyme subsite residues were identified by docking with the same oligopeptide used for kinetics. The subsite hydrophobicities were calculated from the efficiency of hydrolysis of different amino acid replacements in the peptide and from docking data. The results were closer for S1 and S2' than for S1' and S2, indicating that the residue subsites that were more involved in transition state binding are different from those binding the substrate seen in docking. Besides TmCAL1-3, there are nine other cathepsins L, most of them more expressed at midgut. They are supposed to be directed to lysosomes by a Drosophila-like Lerp receptor and/or motifs in their prodomains. The mannose 6-phosphate lysosomal sorting machinery is absent from T. molitor transcriptome. Cathepsin L direction to midgut contents seems to depend on overexpression.

De novo transcriptome sequencing and comparative analysis of midgut tissues of four non-model insects pertaining to Hemiptera, Coleoptera, Diptera and Lepidoptera.

Despite the great morphological diversity of insects, there is a regularity in their digestive functions, which is apparently related to their physiology. In the present work we report the de novo midgut transcriptomes of four non-model insects from four distinct orders: Spodoptera frugiperda (Lepidoptera), Musca domestica (Diptera), Tenebrio molitor (Coleoptera) and Dysdercus peruvianus (Hemiptera). We employed a computational strategy to merge assemblies obtained with two different algorithms, which substantially increased the quality of the final transcriptomes. Unigenes were annotated and analyzed using the eggNOG database, which allowed us to assign some level of functional and evolutionary information to 79.7% to 93.1% of the transcriptomes. We found interesting transcriptional patterns, such as: i) the intense use of lysozymes in digestive functions of M. domestica larvae, which are streamlined and adapted to feed on bacteria; ii) the up-regulation of orthologous UDP-glycosyl transferase and cytochrome P450 genes in the whole midguts different species, supporting the existence of an ancient defense frontline to counter xenobiotics; iii) evidence supporting roles for juvenile hormone binding proteins in the midgut physiology, probably as a way to activate genes that help fight anti-nutritional substances (e.g. protease inhibitors). The results presented here shed light on the digestive and structural properties of the digestive systems of these distantly related species. Furthermore, the produced datasets will also be useful for scientists studying these insects.

A physiologically-oriented transcriptomic analysis of the midgut of Tenebrio molitor.

Physiological data showed that T. molitor midgut is buffered at pH 5.6 at the two anterior thirds and at 7.9 at the posterior third. Furthermore, water is absorbed and secreted at the anterior and posterior midgut, respectively, driving a midgut counter flux of fluid. To look for the molecular mechanisms underlying these phenomena and nutrient absorption as well, a transcriptomic approach was used. For this, 11 types of transporters were chosen from the midgut transcriptome obtained by pyrosequencing (Roche 454). After annotation with the aid of databanks and manual curation, the sequences were validated by RT-PCR. The expression level of each gene at anterior, middle and posterior midgut and carcass (larva less midgut) was evaluated by RNA-seq taking into account reference sequences based on 454 contigs and reads obtained by Illumina sequencing. The data showed that sugar and amino acid uniporters and symporters are expressed along the whole midgut. In the anterior midgut are found transporters for NH3 and NH4+ that with a chloride channel may be responsible for acidifying the lumen. At the posterior midgut, bicarbonate-Cl- antiporter with bicarbonate supplied by carbonic anhydrase may alkalinize the lumen. Water absorption caused mainly by an anterior Na+-K+-2Cl- symporter and water secretion caused by a posterior K+-Cl- may drive the midgut counter flux. Transporters that complement the action of those described were also found.

Role of cathepsins D in the midgut of Dysdercus peruvianus.

Hemipteran ancestors probably lost their digestive serine peptidases on adapting to a plant sap diet. On returning to protein ingestion, these insects start using cathepsin (lysosomal) peptidases as digestive enzymes, from which the less known is cathepsin D. Nine of the ten cathepsin D transcribing genes found in Dysdercus peruvianus midgut are expressed exclusively in this tissue and only DpCatD10 is also expressed in other tissues. The main action of cathepsins D is in the first (V1) (from three, V1-3) midgut regions, where 40% of the total proteolytic activity was assigned to aspartic peptidases with an optimum pH of 3.5. The most expressed cathepsins D were identified in the midgut luminal contents by proteomics. The data indicate that D. peruvianus have kept a lysosomal gene expressed in all tissues and evolved another set of genes with a digestive function restricted to midgut. Digestive cathepsins D apparently complement the action of digestive cathepsin L and they are arguably responsible for the hydrolysis of cysteine peptidase inhibitors known to be present in the cotton seeds eaten by the insect, before they meet cathepsin L.