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The larvae of Hermetia illucens are among the most promising agents for the bioconversion of low-quality biomass, such as organic waste, into sustainable and nutritionally valuable proteins for the production of animal feed. Despite the great interest in this insect, the current literature provides information limited to the optimization of rearing methods for H. illucens larvae, with particular focus on their efficiency in transforming different types of waste and their nutritional composition in terms of suitability for feed production. Surprisingly, H. illucens biology has been neglected and a deep understanding of the morphofunctional properties of the larval midgut, the key organ that determines the extraordinary dietary plasticity of this insect, has been completely overlooked. The present study aims to fill this gap of knowledge. Our results demonstrate that the larval midgut is composed of distinct anatomical regions with different luminal pH and specific morphofunctional features. The midgut epithelium is formed by different cell types that are involved in nutrient digestion and absorption, acidification of the lumen of the middle region, endocrine regulation, and growth of the epithelium. A detailed characterization of the activity of enzymes involved in nutrient digestion and their mRNA expression levels reveals that protein, carbohydrate, and lipid digestion is associated to specific regions of this organ. Moreover, a significant lysozyme activity in the lumen of the anterior and middle regions of the midgut was detected. This enzyme, together with the strong acidic luminal pH of middle tract, may play an important role in killing pathogenic microorganisms ingested with the feeding substrate. The evidence collected led us to propose a detailed functional model of the larval midgut of H. illucens in which each region is characterized by peculiar features to accomplish specific functions. This platform of knowledge sets the stage for developing rearing protocols to optimize the bioconversion ability of this insect and its biotechnological applications.

Aphidius ervi (Hymenoptera, Braconidae) is an endophagous parasitoid of various aphid species, including Acyrthosiphon pisum (Homoptera, Aphididae), the model host used in the present study. Parasitized hosts show a marked increase of their nutritional suitability for the developing parasitoid larvae. This alteration of the biochemical and metabolic profile is due to a castration process mediated by the combined action of the venom, injected at the oviposition, and of the teratocytes, cells deriving from the dissociation of the embryonic membrane. Teratocytes produce and release in the host haemocoel two parasitism-specific proteins, which are of crucial importance for the development of their sister larvae. One of the proteins is a fatty acid binding protein (Ae-FABP), which shows a high affinity for C14–C18 saturated fatty acids (FAs) and for oleic and arachidonic acids. To better define the possible nutritional role of this protein, we have studied its immunolocalization profile in vivo and the impact on FA uptake by the epidermal and midgut epithelia of A. ervi larvae. During the exponential growth of A. ervi larvae, Ae-FABP is distributed around discrete lipid particles, which are abundantly present in the haemocoel of parasitized host aphids and in the midgut lumen of parasitoid larvae. Moreover, a strong immunodetection signal is evident on the surface of the two larval epithelia involved in nutrient absorption: the parasitoid midgut epithelium and the external epidermal layer. These two epithelia can effectively absorb radiolabelled myristic acid, but the FA transport rates are not affected by the presence in the medium of Ae-FABP. The protein appears to act essentially as a vector in the host haemolymph, transferring FAs from the digestion sites of host lipids to the growing parasitoid larvae. These data indicate that the proteins produced by A. ervi teratocytes may play complementary roles in the nutritional exploitation of the host.

Cell-Penetrating Peptides (CPPs) are short peptides that are able to translocate across the cell membrane a wide range of cargoes. In the past decade, different mammalian cell lines have been used to clarify the mechanism of CPPs penetration and to characterize the internalization process, which has been described either as an energy-independent direct penetration through the plasma membrane, or as endocytic uptake. Whatever the mechanism involved, the cell penetration properties of these peptides make their use very attractive as vector for promoting the cellular uptake of coupled bioactive macromolecules, such as peptides, proteins and oligonucleotides. Here we demonstrate, for the first time in insect, that cultured columnar cells from the larval midgut of Bombyx mori more readily internalize eGFP (enhanced Green Fluorescent Protein) when fused to CPP Tat. Tat-eGFP translocates across the plasma membrane of absorptive cells in an energy-independent and non-endocytic manner, since no inhibition of the fusion protein uptake is exerted by metabolic inhibitors and by drugs that interfere with the endocytic uptake. Moreover, the CPP Tat enhances the internalization of eGFP in the columnar cells of intact midgut tissue, mounted in a suitable perfusion apparatus, and the transepithelial flux of the protein. These results open new perspectives for effective delivery of insecticidal macromolecules targeting receptors located both within the insect gut epithelium and behind the gut barrier, in the hemocoel compartment.

Endogenous peptide regulators of insect physiology and development are presently being considered as potential biopesticides, but their efficacy by oral delivery cannot be easily anticipated because of the limited information on how the insect gut barrier handles these kind of molecules. We investigated, in Bombyx mori larvae, the permeability properties of the two components of the intestinal barrier, the peritrophic membrane (PM) and the midgut epithelium, separately isolated and perfused in conventional Ussing chambers. The PM discriminated compounds of different dimensions but was easily crossed by two small peptides recently proposed as bioinsecticides, the neuropeptide proctolin and Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF), although their flux values indicated that the permeability was highly affected by their steric conformation. To date, there is very little functional data available on how peptides cross the insect intestinal epithelium, but it has been speculated that peptides could reach the haemocoel through the paracellular pathway. We characterized the permeability properties of this route to a number of organic molecules, showing that B. mori septate junction was highly selective to both the dimension and the charge of the permeant compound. Confocal images of whole-mount midguts incubated with rhodamine(rh)-proctolin or fluorescein isothiocyanate (FITC)-Aea-TMOF added to the mucosal side of the epithelium, revealed that rh-proctolin did not enter the cell and crossed the midgut only by the paracellular pathway, while FITC-Aea-TMOF did cross the cell apical membrane, permeating also through the transcellular route. © 2008 Elsevier Ltd. All rights reserved.

The mechanism responsible for fluorescein isothiocyanate (FITC)-albumin internalization by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time, and it appeared to be energy dependent, since it was strongly reduced by both low temperatures and metabolic inhibitors. Labeled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Michaelis constant value of 2.0 ± 0.6 μM. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalization experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homolog of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca2+for albumin internalization and was inhibited by gentamicin. FITC-albumin internalization was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalized in the intermicrovillar areas of the apical plasma membrane. The integrity of actin and microtubule organization was essential for the correct functioning of the endocytic machinery.

Insect immune defences rely on cellular and humoral responses targeting both microbial pathogens and metazoan parasites. Accumulating evidence indicates functional cross-talk between these two branches of insect immunity, but the underlying molecular mechanisms are still largely unknown. We recently described, in the tobacco budworm Heliothis virescens, the presence of amyloid fibers associated with melanogenesis in immune capsules formed by hemocytes, and identified a protein (P102) involved in their assembly. Non-self objects coated by antibodies directed against this protein escaped hemocyte encapsulation, suggesting that P102 might coordinate humoral and cellular defence responses at the surface of foreign invaders. Here we report the identification of a cDNA coding for a protein highly similar to P102 in a related Lepidoptera species, Spodoptera littoralis. Its transcript was abundant in the hemocytes and the protein accumulated in large cytoplasmic compartments, closely resembling the localization pattern of P102 in H. virescens. RNAi-mediated gene silencing provided direct evidence for the role played by this protein in the immune response. Oral delivery of dsRNA molecules directed against the gene strongly suppressed the encapsulation and melanization response, while hemocoelic injections did not result in evident phenotypic alterations. Shortly after their administration, dsRNA molecules were found in midgut cells, en route to the hemocytes where the target gene was significantly down-regulated. Taken together, our data demonstrate that P102 is a functionally conserved protein with a key role in insect immunity. Moreover, the ability to target this gene by dsRNA oral delivery may be exploited to develop novel technologies of pest control, based on immunosuppression as a strategy for enhancing the impact of natural antagonists. (C) 2014 Elsevier Ltd. All rights reserved.

Sugars are critical substrates for insect metabolism, but little is known about the transporters and epithelial routes that ensure their constant supply from dietary resources. We have characterized glucose and fructose uptakes across the apical and basolateral membranes of the isolated larval midgut of the aphid parasitoid Aphidius ervi. The uptake of radiolabeled glucose at the basal side of the epithelium was almost suppressed by 200 μM cytochalasin B, uninhibited by phlorizin, and showed the following decreasing rank of specificity for the tested substrates: glucose > glucosamine > fructose, with no recognition of galactose. These functional properties well agree with the expression of GLUT2-like transporters in this membrane. When the apical surface of the epithelium was also exposed to the labeled medium, a cation-dependent glucose uptake, inhibited by 10 μM phlorizin and by an excess of galactose, was detected suggesting the presence in the apical membrane of a cation-dependent cotransporter. Radiolabeled fructose uptakes were only partially inhibited by cytochalasin B. SGLT1-like and GLUT5-like transporters were detected in the apical membranes of the epithelial cell by immunocytochemical experiments. These results, along with the presence of GLUT2-like transporters both in the apical and basolateral cell membranes of the midgut, as we recently demonstrated, allow us to conclude that the model for sugar transepithelial transport in A. ervi midgut appears to be unexpectedly similar to that recently proposed for sugar intestinal absorption in mammals.

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K+-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine > L-phenylglycine > L-methionine> L-phenylalanine > L-norleucine>> L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae. (C) 2002 Elsevier Science Ltd. All rights reserved

In the larval midgut of Bombyx mori a K + -dependent transporter for leucine and amino acids with a hydrophobic side chain is responsible for the absorption of most essential amino acids. We investigated if a modulation of its activity occurred as a result of starvation or after hormonal treatments. We measured amino acid uptake in brush border membrane vesicles (BBMV) purified from the anterior–middle (AM) and posterior (P) regions of the midgut in fifth instar larvae. Silkworms were either starved or topically treated with low dosages of fenoxycarb, a molecule often used as a juvenile hormone mimic. The maximal uptake value of K + -driven leucine transport was increased in BBMV of AM- and P-midgut regions of starved larvae. The initial uptake rates of serine and glutamine, two amino acids transported by the same cotransporter as leucine, were also increased. Leucine kinetics proved that V max was the kinetic parameter modified by starvation in both midgut regions. Topical applications of fenoxycarb at a dose of 2.5 fg/larva immediately after the fourth ecdysis, induced an increase of leucine initial uptake rates and of intravesicular accumulation of leucine in both AM- and P-BBMV. Kinetic analysis of leucine uptake indicated again that V max was increased in BBMV from both midgut regions in treated larvae.

Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12–18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch. Insect Biochem. Physiol. 48:190–198, 2001. © 2001 Wiley-Liss, Inc.

We have examined the type of inhibition exerted by an activated preparation of the Bacillus thuringiensis delta-endotoxin CrylAa on K+-dependent leucine transport into midgut brush border membrane vesicles or epithelial cells of the isolated midgut from Bombyx mori to study its possible interaction with the amino acid symporter. K+ permeability and the cation-dependent amino acid translocation into brush border membrane vesicles were evaluated by monitoring the fluorescence of the voltage-sensitive cyanine dye 3, 3'-dipropylthiadicarbocyanine iodide. The symporter ability to accept Na+ instead of K+ was exploited and the dissipation of an imposed inside-negative potential (K+ gradient inout and valinomycin) was registered in the presence of a Na+ gradient (outin) and of the amino acid. The fluorescence quenching dissipated more rapidly when the amino acid was present. Preincubation of brush border membrane vesicles with CrylAa caused a significant decrease of the amino acid-dependent recovery of fluorescence, whereas K+ permeability was sparely affected. In the isolated midgut, CrylAa inhibits leucine uptake as well as the transepithelial electrical potential difference. The strong inhibition exerted by the delta-endotoxin was observed also in the absence of potassium and the transepithelial electrical potential difference. The results obtained strongly suggest a direct interaction of CrylAa delta-endotoxin with the K+/amino acid symporter.

The larval midgut of the hymenopteran parasitoid Aphidius ervi accomplishes a large transport of nutrients from the lumen to the haemocoel, providing most of the organic molecules necessary for rapid insect development. l-amino acids in general, and leucine in particular, are efficiently accumulated in the larval body. We show here that the intact midgut of early 3rd instar larvae incubated in vitro can take up [3H]l-leucine from the basolateral side of the epithelium by transporters insensitive to the presence of monovalent cations. When the midgut is opened and the apical membrane of the absorbing epithelial cells is exposed to the medium containing radiolabelled leucine, a sodium-dependent uptake of the amino acid becomes apparent, disclosing the presence of a symport mechanism. Inhibition experiments of leucine uptake by a 100-fold excess of different amino acids, selected according to the properties of their side chain, revealed that this apical sodium-dependent mechanism is a broad spectrum transport system with a specialization for the absorption of aliphatic amino acids, that can also transfer glutamine and proline, but not phenylalanine, lysine and arginine. Altogether the experimental results obtained with intact- and open-gut preparations suggest that leucine transport across the basolateral membrane is mediated by both an uniporter and an obligatory amino acid exchange mechanism. © 2009 Elsevier Ltd. All rights reserved

Increasing experimental evidence indicates that ingested proteins can in part reach the haemocoel undegraded, but information on the mechanisms involved in protein transport across the insect gut is very limited, in spite of the implications that this may have on the development of novel delivery strategies of insecticide proteins targeting haemocoelic receptors. Here we contribute to this field of study, by focusing on horseradish peroxidase (HRP) transport through Bombyx mori larval midgut, isolated and perfused in vitro. The protein crossed the intestinal barrier in a time-dependent manner and the influx was linearly related to time between 30 and 90 min of incubation. HRP absorption was strongly affected by temperature and inhibition of cell metabolism: protein influx at 4 °C was reduced to 27% of that measured at 25 °C and was similarly inhibited by the metabolic inhibitor DNP. Transmission electron microscopy analysis of midgut columnar cells exposed to HRP showed the presence of the protein both in vesicular structures inside the cytoplasm and in the space between two adjacent absorptive cells, indicating the occurrence of both a transcellular and a paracellular permeation route. The analysis of HRP influx as a function of increasing protein concentration in the lumen supported this morphological indication. The Jmax relative to the HRP transcellular transport component was 121±24 pmol/cm2/h and the Kd of the passage through the paracellular route was 1.9±0.3 μl/cm2/h. The paracellular electrical resistance decreased in midguts exposed to HRP, indicating that its passage through this pathway was likely due to an alteration exerted on the junctional complex by the protein itself. The role of the cytoskeleton in HRP transport was investigated by assessing the impact of drugs affecting microtubules and actin filaments. © 2007 Elsevier Ltd. All rights reserved

The features of the paracellular pathway, an important route for the transfer of ions and molecules in epithelia, are in insects still poorly investigated and it has not yet been elucidated how the septate junction (SJ) acts as a transepithelial barrier. In this study, some properties of the paracellular pathway of Bombyx mori larval midgut, isolated in Ussing chambers, were determined and the modulation of SJ permeability by intracellular events disclosed. Diffusion potentials evoked by transepithelial gradients of different salts indicated that the junction bore weak negative charges and that the paracellular pathway was selective with respect to ion charge and size. In standard conditions, the transepithelial resistance was 28.2 ± 2.1 Ω cm2, a value indicating that the midgut is a low resistance epithelium. The modulation of midgut SJ by typical enhancers of mammalian tight junction permeability known to act on the cytoskeleton was studied by measuring the shunt resistance and the lumen-to-haemolymph flux of sucrose. An increase of the intracellular level of cAMP and Ca2+ caused a significant decrease of the shunt resistance and an increase of SJ permeability. The attenuation of Ca2+ effect in the presence of the calcium channel blocker nifedipine indicated that the influx of external Ca2+ into the cytoplasm was important for the opening of the SJ, as well as the release of Ca2+ from the intracellular stores. © 2006 Elsevier Inc. All rights reserved

In the last decade, the study of peptide and protein absorption by the insect gut has received increasing attention because of the considerable impact this information may have on the development of new delivery strategies for insecticide macromolecules targeting haemocoelic receptors. Available experimental evidence in vivo suggests that, in insects, peptides and proteins can cross the intestinal barrier reaching the haemocoel, but the functional bases of this absorption pathway have not yet been thoroughly investigated. The current knowledge of the mechanisms involved in protein and polypeptide absorption in animals derives from the extensive studies performed in mammalian polarised epithelial cells, where the transcellular transport of proteins by transcytosis has been demonstrated. In this process, proteins are internalised at one pole of the cell and transported by cytoplasmic vesicular traffic to the opposite plasma membrane domain, where they are released with unchanged biological activity. Here we report data on albumin translocation across the isolated midgut of Bombyx mori caterpillars perfused in vitro. The functional properties of the transepithelial transport of this protein are described and, since absorption prevails over secretion, its lumen-to-haemolymph flux is characterised. Low-temperature incubations nearly abolish the transepithelial transport, while the peculiar physiological features of the larval midgut, i.e. the high lumen positive transepithelial voltage and the luminal alkaline pH, do not affect the flux. The obtained results indicate that albumin crosses B. mori larval midgut by transcytosis. © 2005 Elsevier Ltd. All rights reserved

It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) host regulation by the parasitoid larva induces in the aphid haemolymph major changes of the titer of nutritional compounds such as proteins, acylglycerols and free amino acids, in order to meet the stage-specific demands of the developing larva. Since little is known about how the larva absorbs these mobilized nutritional resources, nutrient absorption by larval stages of A. ervi was studied. In 2nd instar larvae, leucine was ten-fold accumulated in the haemocoel, and tyrosine and glutamine two-fold. Glucose and fructose were readily absorbed and fructose was extensively metabolized by larval tissues. In 3rd instars, the presence of a number of larvae that did not ingest the incubation medium enabled us to determine the respective amounts of substrate absorbed by the epidermis and the midgut. An accumulation of leucine in the haemocoel was observed only when midgut cells were involved in absorption, while the amino acid concentration within body fluids never exceeded that of the incubation medium when the uptake was performed only by epidermal cells. The immunofluorescence analysis, the mutual inhibition exerted on labeled glucose or fructose uptakes by a 100-fold excess of the sugars and the strong inhibition of uptakes induced by 0.2mM cytochalasin B support the expression of facilitative GLUT2-like transporters in the apical and basal cell membranes of midgut epithelial cells. Taken together, these results prove that both midgut and epidermis are involved in nutrient absorption throughout the parasitoid development, that GLUT2 transporters are responsible for glucose and fructose uptakes and that the chemical gradient that favors the passive influx of the two sugars is maintained by their conversion to other substrates.

Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of several aphid species of economic importance, widely used in biological control. The definition of a suitable artificial diet for in vitro mass production of this parasitoid is still an unresolved issue that, to be properly addressed, requires a deeper understanding both of its nutritional needs and of the functional properties of the larval epithelia involved in nutrient absorption. The experimental evidence presented in this paper unequivocally demonstrates that the uptake of sugars and amino acids takes place through the body surface of the larval stages of A. ervi. These nutrients are efficiently absorbed by the larval epidermis, but the transport rate progressively declines over time. The epidermis exhibits a cross-reactivity to antibodies raised against the mammalian facilitative glucose transporter GLUT2 and the sodium cotransporter SGLT1. The analysis of sugar transport sensitivity to specific inhibitors indicates the involvement of GLUT2-like transporters, while a role for SGLT1-like transporters is not supported. The peculiar pathways of nutrient absorption in A. ervi larvae further corroborate the general idea that the pre-imaginal stages of endophagous koinobiont Hymenoptera, like Metazoan parasites, show a high degree of physiological integration with their hosts.

The K+-dependent symporter for leucine and other neutral amino acids expressed along the midgut of the silkworm Bombyx mori operates with best efficiency in the presence of a steep pH gradient across the brush-border membrane, with external alkaline pH values up to 11, and an electrical potential difference (Δψ) of ∼200 mV. Careful determinations of leucine kinetics as a function of external amino acid concentrations between 50 and 1,000 μM, performed with brush-border membrane vesicles (BBMV) obtained from the middle and posterior midgut regions, revealed that the kinetic parameter affected by the presence of a ΔpH was the maximal rate of transport. The addition of Δψ caused a further marked increase of the translocation rate. At nonsaturating leucine concentrations in the solution bathing the external side of the brush-border membrane, leucine accumulation within BBMV and midgut cells was not only driven by the gradient of the driver cation K+and Δψ but occurred also in the absence of K+. The ability of the symporter to translocate the substrate in its binary form allows the intracellular accumulation of leucine in the absence of K+, provided that a pH gradient, with alkaline outside, is present. The mechanisms involved in this accumulation are discussed.

We investigated the kinetics of leucine influx as a funtion of external substrate concentration between 0.03 and 16 mM in brush-border membrane vesicles (BBMV) prepared from the middle region of Bombyx mori larval midgut. A detailed kinetic analysis of leucine uptake led to the identification, in parallel with the K+-dependent symporter for neutral amino acids, of a K+-independent, low-affinity, high-capacity system. The parameter values of the Michaelis constant (7.12 mM) and maximal rate of transport (4.48 nmol ⋅ 7 s−1⋅ mg protein−1) were not influenced by an external alkaline pH nor by a transmembrane electrical potential difference. The uniporter is poorly specific, as it displayed the following rank of preference: Leu, His, Val, Ile, Phe, Ser > Lys, Arg, Gln > Pro, 2-amino-2-norbornane-carboxylic acid, Ala, Gly. The kinetic analysis performed in BBMV prepared from the posterior midgut portion indicates that the low-affinity, high-capacity uniporter is present along the entire length of the silkworm larval midgut with similar expression and functional properties.