The mgΔlpn mouse model for Marfan syndrome recapitulates the ocular phenotypes of the disease.

PURPOSE: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔlpn mouse model for MFS. METHODS: Eyes from mgΔlpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence. RESULTS: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes. CONCLUSIONS: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔlpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease.

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.

Insect midgut α-mannosidases from family 38 and 47 with emphasis on those of Tenebrio molitor.

α-Mannosidases are enzymes which remove non-reducing terminal residues from glycoconjugates. Data on both GH47 and GH38 (Golgi and lysosomal) enzymes are available. Data on insect midgut α-mannosidases acting in digestion are preliminary and do not include enzyme sequences. Tenebrio molitor midgut α-mannosidases were separated by chromatography into two activity peaks: a major (Man1) and a minor (Man2). An antibody generated against a synthetic peptide corresponding to a sequence of α-mannosidase fragment recognizes Man2 but not Man1. That fragment was later found to correspond to TmMan2 (GenBank access KP892646), showing that the cDNA coding for Man2 is actually TmMan2. TmMan2 codes for a mature α-mannosidase with 107.5 kDa. Purified Man2 originates after SDS-PAGE one band of about 72 kDa and another of 51 kDa, which sums 123 kDa, in agreement with gel filtration (123 kDa) data. These results suggest that Man2 is processed into peptides that remain noncovalently linked within the functional enzyme. The physical and kinetical properties of purified Man1 and Man2 are similar. They have a molecular mass of 123 kDa (gel filtration), pH optimum (5.6) and response to inhibitors like swainsonine (Man1 Ki, 68 nM; Man2 Ki, 63 nM) and deoxymannojirimycin (Man1 Ki, 0.12 mM; Man2 Ki, 0.15 mM). Their substrate specificities are a little different as Man2 hydrolyzes α-1,3 and α-1,6 bonds better than α-1,2, whereas the contrary is true for Man1. Thus, they pertain to Class II (GH38 α-mannosidases), that are catabolic α-mannosidases similar to lysosomal α-mannosidase. However, Man2, in contrast to true lysosomal α-mannosidase, is secreted (immunocytolocalization data) into the midgut contents. There, Man2 may participate in digestion of fungal cell walls, known to have α-mannosides in their outermost layer. The amount of family 38 α-mannosidase sequences found in the transcriptome (454 pyrosequencing) of the midgut of 9 insects pertaining to 5 orders is perhaps related to the diet of these organisms, as suggested by a large number of lysosomal α-mannosidase in the T. molitor midgut.

Quantitative T2 combined with texture analysis of nuclear magnetic resonance images identify different degrees of muscle involvement in three mouse models of muscle dystrophy: mdx, Largemyd and mdx/Largemyd.

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant-T2-measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.

Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three ß-glucosidases, one ß-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none ß-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single ß-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two ß-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of ß-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided.

Stromal cell derived factor-2 (Sdf2): a novel protein expressed in mouse.

The stromal derived factor (SDFs) family comprises a group of molecules generated by stromal cells. SDF1 and SDF4 are chemokines; SDF2 and SDF5 are not yet functionally and structurally defined. In human and mouse, Sdf2 has a paralogous gene, Sdf2l1, whose protein sequences are 78% similar and 68% identical. Human SDF2L1 is an endoplasmic reticulum-stress inducible-gene. In Arabidopsis thaliana, SDF2-like (39% and 37% amino acid sequence identity with Mus musculus Sdf2 and Sdf2l1) has also been implicated in activating the UPR in ER-stress. Here we have cloned, expressed and purified recombinant Sdf2 and raised an anti-Sdf2 antibody. We demonstrated that the protein is expressed in several tissues and is localized in the endoplasmic reticulum. We suggest that Sdf2, initially predicted as a secretory protein because it lacks the canonical ER retention signals in its C-terminal, could be ER-resident through accessory binding proteins or other amino acid sequence motifs, as suggested for the homolog protein SDF2-like. Furthermore, the crystal structure of SDF2-like from Arabidopsis thaliana is a typical ß-trefoil containing three MIR motifs; all hydrophobic residues considered important for maintaining the bottom layer of the ß-trefoil barrel seem to be conserved in the Sdf2 family. Multiple alignment using 43 sequences for SDF2 and 38 for SDF2L1 paralogous families also revealed a very similar residue conservation profile. Comparing the amino acid sequence and predicted 3D structure with other Sdf2-like proteins we suggest a role of mouse Sdf2 in the Unfolded Protein Response and ER-stress, similar to that of Sdf2l1 from human and mouse and SDF2-like from Arabidopsis thaliana. Chronic ER stress has been associated with many human diseases including cancer and diabetes. Identification of new factors associated with the ER stress pathway can help to identify and define key targets of this response.

Variant vicilins from a resistant Vigna unguiculata lineage (IT81D-1053) accumulate inside Callosobruchus maculatus larval midgut epithelium.

It has been demonstrated that variant vicilins are the main resistance factor of cowpea seeds (Vigna unguiculata) against attack by the cowpea beetle Callosobruchus maculatus. There is evidence that the toxic properties of these storage proteins may be related to their interaction with glycoproteins and other microvillar membrane constituents along the digestive tract of the larvae. New findings have shown that following interaction with the microvilli, the vicilins are absorbed across the intestinal epithelium and thus reach the internal environment of the larvae. In the present paper we studied the insecticidal activity of the variant vicilins purified from a resistant cowpea variety (IT81D-1053). Bioassays showed that the seeds of this genotype affected larval growth, causing developmental retardation and 100% mortality. By feeding C. maculatus larvae on susceptible and IT81D-1053 derived vicilins (FITC labelled or unlabelled), followed by fluorescence and immunogold cytolocalization, we were able to demonstrate that both susceptible and variant forms are internalized in the midgut cells and migrate inside vesicular structures from the apex to the basal portion of the enterocytes. However, when larvae were fed with the labelled vicilins for 24h and then returned to a control diet, the concentration of the variant form remained relatively high, suggesting that variant vicilins are not removed from the cells at the same rate as the non-variant vicilins. We suggest that the toxic effects of variant vicilins on midgut cells involve the binding of these proteins to the cell surface followed by internalization and interference with the normal physiology of the enterocytes, thereby affecting larval development in vivo.

The digestive system of the "stick bug" Cladomorphus phyllinus (Phasmida, Phasmatidae): a morphological, physiological and biochemical analysis.

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.

Midgut proteins released by microapocrine secretion in Spodoptera frugiperda.

Microapocrine vesicles bud from the lepidopteran midgut microvilli as double membrane vesicles. To identify the proteins secreted by this process, antibodies raised against isolated microapocrine vesicles from Spodoptera frugiperda were used for screening a midgut cDNA expression library. Positive clones were sequenced, assembled and N blasted against S. frugiperda sequences obtained by pyrosequencing midgut mRNA. This procedure led to the extension of microapocrine sequences that were annotated. A similar procedure was used to identify midgut microvillar proteins that necessarily are part of the microapocrine vesicle. Forty-eight proteins were associated with microvillar membranes. They pertain to 8 functional groups: digestive enzymes, peritrophic membrane, protection, transporters, receptors, secretory machinery, cytoskeleton and signaling, and unknown. Twenty-eight proteins are putatively secreted by microapocrine secretion. Most of them are digestive enzymes, but the list also includes proteins involved in protection and in peritrophic membrane formation. Among the identified digestive enzymes, aminopeptidases are typically microvillar and group into the classes 1, 2, 3, 5, and 6. There are two amylases secreted by microapocrine secretion: one is a digestive enzyme and the other is a transporter-like amylase with no clear function. One lipase has a predicted transmembrane loop, whereas the others are supposed to be secreted by microapocrine secretion and be digestive. Trypsin is membrane bound and is delivered by microapocrine secretion, but has no predicted features to bind membranes. It may remain bound through the signal peptide till be delivered into the midgut lumen. Proteins supposed to be involved in the microapocrine secretory machinery were: calmodulin, annexin, myosin 7a, and gelsolin 1. Their putative roles are discussed, but more research is necessary to settle this subject.

Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs.

Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46 °C) in the presence and absence of 10 mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28 kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila.

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents.

Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae).

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 µM); CAL2 (17 kDa, pHo 5.5, km 11 µM Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM l-Leucine-p-nitroanilide), α-glucosidase (90 kDa, pHo 5.0, km 5mM with p-nitrophenyl α-d-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans.

Description of a new species of bat-associated argasid tick (Acari: Argasidae) from Brazil.

A new species of argasid tick (Acari: Argasidae) is described from immature and adult specimens collected from several localities in Brazil. A complete morphological account is provided for all postembryonic life stages, i.e., larva, nymph, female, and male. Ornithodoros cavernicolous n. sp. is the 113(th) in the genus. Morphologically, the new species shares common features, e.g., presence of well-developed cheeks and legs with micromammillate cuticle, with other bat-associated argasid ticks included in the subgenus Alectorobius. In particular, the new species is morphologically related to Ornithodoros azteci Matheson, with which it forms a species group. Phylogenetic analysis based on the 16S rRNA gene sequences supports the placement of the new species within a large clade that includes other New World bat-associated argasids. However, the new species seems to represent an independent lineage within the genus Ornithodoros.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis.

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37°C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K(m)=37.53 mMS(-1)), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K(i)=0.196 nM), indicating that this protease is a potential target for pest control.

Characterization of a ß-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to ß-glucan-binding proteins.

Spodoptera frugiperda ß-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against ß-1,3-glucan (laminarin), but cannot hydrolyze yeast ß-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive ß-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of ß-1,3-glucanases and ß-glucan-binding protein supports the assumption that the ß-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived ß-1,3-glucanases by the loss of an extended N-terminal region and the ß-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut.

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midgut cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3.

Digestive morphophysiology of Gryllodes sigillatus (Orthoptera: Gryllidae).

The evolution of the digestive system in the Order Orthoptera is disclosed from the study of the morphophysiology of the digestive process in its major taxa. This paper deals with a cricket representing the less known suborder Ensifera. Most amylase and trypsin activities occur in crop and caeca, respectively. Maltase and aminopeptidase are found in soluble and membrane-bound forms in caeca, with aminopeptidase also occurring in ventriculus. Amaranth was orally fed to Gryllodes sigillatus adults or injected into their haemolymph. The experiments were performed with starving and feeding insects with identical results. Following feeding of the dye the luminal side of the most anterior ventriculus (and in lesser amounts the midgut caeca) became heavily stained. In injected insects, the haemal side of the most posterior ventriculus was stained. This suggested that the anterior ventriculus is the main site of water absorption (the caeca is a secondary one), whereas the posterior ventriculus secretes water into the gut. Thus, a putative counter-current flux of fluid from posterior to anterior ventriculus may propel digestive enzyme recycling. This was confirmed by the finding that digestive enzymes are excreted at a low rate. The fine structure of midgut caeca and ventriculus cells revealed that they have morphological features that may be related to their involvement in secretion (movement from cell to lumen) and absorption (movement from lumen to cell) of fluids. Furthermore, morphological data showed that both merocrine and apocrine secretory mechanisms occur in midgut cells. The results showed that cricket digestion differs from that in grasshopper in having: (1) more membrane-bound digestive enzymes; (2) protein digestion slightly displaced toward the ventriculus; (3) midgut fluxes, and hence digestive enzyme recycling, in both starved and fed insects.

Redescription of the female, description of the male, and several new records of Amblyomma parkeri (Acari: Ixodidae), a South American tick species.

The tick Amblyomma parkeri Fonseca and Aragão was described in 1952, based on female and immature ticks collected in the states of São Paulo and Santa Catarina, Brazil. Thereafter, there has been no further report of A. parkeri, and the male has remained unknown. Herein, we examined ticks collected on porcupines from a locality in the state of São Paulo. Some of the ticks were identified as Amblyomma longirostre (Koch, 1844), whereas others as A. parkeri, including male specimens, for which we provide the first description. We also provide additional reports of A. parkeri after examining collections of A. longirostre and Amblyomma geayi Neumann, 1899 from different tick collections. Morphological evidence to support the original description of A. parkeri is presented, supported by molecular analyses of portions of the 16S rRNA and 12S rRNA mitochondrial genes. Morphological particularities to separate A. parkeri, A. longirostre, and A. geayi are provided.

New reports of Antricola guglielmonei and Antricola delacruzi in Brazil, and a description of a new argasid species (Acari).

Adults of 3 tick species (Acari: Argasidae) identified as Antricola guglielmonei, Antricola delacruzi, and Carios rondoniensis n. sp. were collected on bat guano in a cave in the state of Rondônia, western Amazon, Brazil. Adults of C. rondoniensis possess a unique combination of characters that distinguish them from all described adults in the Argasidae, i.e., a large spiracular plate densely filled with small goblets, a well-developed flap covering the female genital opening, and palpi containing several tufts of long setae on articles 2 and 3. Unlike Ornithodoros or other Carios species, adults of C. rondoniensis have a scooplike hypostome devoid of denticles, as in Antricola spp. Conversely, the presence of a pair of long posthypostomal setae, and a slitlike transverse fissure at the capsule opening of the Haller's organ, are characters of C. rondonensis that are also found in species of Carios and Ornithodoros, but not in Antricola species. Molecular analyses inferred from a portion of the 16S rRNA mitochondrial gene indicate that C. rondoniensis is phylogenetically closest to species of Carios, followed by species of Antricola, and then Ornithodoros. Because the highest bootstrap value linking C. rondoniensis to Carios spp. was 62%, further phylogenetic studies are needed to better evaluate the taxonomic status of the former species.

Digestive enzyme compartmentalization and recycling and sites of absorption and secretion along the midgut of Dermestes maculatus (Coleoptera) larvae.

Bostrichiformia is the less known major series of Coleoptera regarding digestive physiology. The midgut of Dermestes maculatus has a cylindrical ventriculus with anterior caeca. There is no cell differentiation along the ventriculus, except for the predominance of cells undergoing apocrine secretion in the anterior region. Apocrine secretion affects a larger extension and a greater number of cells in caeca than in ventriculus. Ventricular cells putatively secrete digestive enzymes, whereas caecal cells are supposed to secrete peritrophic gel (PG) glycoproteins. Feeding larvae with dyes showed that caeca are water-absorbing, whereas the posterior ventriculus is water-secreting. Midgut dissection revealed a PG and a peritrophic membrane (PM) covering the contents in anterior and posterior ventriculus, respectively. This was confirmed by in situ chitin detection with FITC-WGA conjugates. Ion-exchange chromatography of midgut homogenates, associated with enzymatic assays with natural and synthetic substrates and specific inhibitors, showed that trypsin and chymotrypsin are the major proteinases, cysteine proteinase is absent, and aspartic proteinase probably is negligible. Amylase and trypsin occur in contents and decrease along the ventriculus; the contrary is true for cell-membrane-bound aminopeptidase. Maltase is cell-membrane-bound and predominates in anterior and middle midgut. Digestive enzyme activities in hindgut are negligible. This, together with dye data, indicates that enzymes are recovered from inside PM by a posterior-anterior flux of fluid outside PM before being excreted. The combined results suggest that protein digestion starts in anterior midgut and ends in the surface of posterior midgut cells. All glycogen digestion takes place in anterior midgut.