Absorption of albumin by the midgut of a lepidopteran larva

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.     

The paracellular pathway in the lepidopteran larval midgut: modulation by intracellular mediators

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 Omega cm(2), 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 Ca(2+) caused a significant decrease of the shunt resistance and an increase of SJ permeability. The attenuation of Ca(2+) effect in the presence of the calcium channel blocker nifedipine indicated that the influx of external Ca(2+) into the cytoplasm was important for the opening of the SJ, as well as the release of Ca(2+) from the intracellular stores.     

Absorption of horseradish peroxidase in Bombyx mori larval midgut

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 degrees C was reduced to 27% of that measured at 25 degrees 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 J(max) relative to the HRP transcellular transport component was 121+/-24 pmol/cm(2)/h and the K(d) of the passage through the paracellular route was 1.9+/-0.3 microl/cm(2)/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.     

Functional significance of some particular amino acid residues in Bombyx mori pyridoxal kinase

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme for vitamin B₆ metabolism in animals. It catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal 5′-phosphate, an important cofactor for many enzymatic reactions. Bombyx mori PLK (BmPLK) is 10 or more residues shorter than mammalian PLKs, and some amino acid residues conserved in the PLKs from mammals are not maintained in the protein. Multiple sequence alignment suggested that amino acid residues Thr⁴⁷, Ile⁵⁴, Arg⁸⁸, Asn¹²¹ and Glu²³⁰ might play important roles in BmPLK. In this study, we used a site-directed specific mutagenesis approach to determine the functional significance of these particular amino acid residues in BmPLK. Our results demonstrated that the mutation of Asn¹²¹ to Glu did not affect the catalytic function of BmPLK. The corresponding site-directed mutants of Thr⁴⁷ to Asn, Ile⁵⁴ to Phe, and Arg⁸⁸ to Ile displayed a decreased catalytic efficiency and an elevated Km value for substrate relative to the wild-type value, and no enzyme activity could be detected in mutant of Trp²³⁰ to Glu. Circular dichroism analysis revealed that the mutation of Trp²³⁰ to Glu resulted in mis-folding of the protein. Our results provided direct evidence that residue Trp²³⁰ is crucial to maintain the structural and functional integrity of BmPLK. This study will add to the existing understanding of the characteristic of structure and function of BmPLK.     

The intestinal barrier in lepidopteran larvae: permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides

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.     

The amino acid/K symporters for neutral amino acids along the midgut of lepidopteran larvae: Functional differentiations

The functional properties of the K⁺-dependent symporter for neutral amino acids have been investigated in brush border membrane vesicles prepared from the anterior, middle and posterior portions corresponding to the three morphologically distinguishable regions of the midgut of Bombyx mori larvae. An intravesicular accumulation of leucine was driven by a K⁺-gradient in the three preparations, but vesicles from the posterior tract displayed much higher uptake and accumulation values. Kinetic analysis of leucine uptake, performed in experimental conditions which mimic as closely as feasible experimentally those occurring in vivo (Δψ = −90 mV, pH_in7.2/pH_out8.7, [K⁺]_out100 mM), evidenced that the affinity for the amino acid was similar along the midgut (150 μM), but V_max in the posterior region was more than 11-fold higher than that of the anterior-middle tract (11.3 ± 0.7 and 0.98 ± 0.07 nmol/7s/mg protein, respectively). Leucine uptake was remarkably influenced by extravesicular pH and by Δψ only in vesicles from the posterior midgut: a lowering of pH to 7.2 caused a sevenfold increase of K_m, whereas in the absence of Δψ, V_max decreased threefold. The selectivity sequence for the alkali cations was somewhat different in the two midgut regions, but K⁺ remained the most effective. In the posterior midgut, the selectivity for K⁺ was greatly enhanced when a transmembrane electrical potential was present. Leucine kinetics as a function of external potassium concentration was hyperbolic in the posterior and sigmoidal in the anterior-middle part. Inhibition of leucine uptake induced by a 20-fold excess of different amino acids suggested the presence in both midgut tracts of a broad specificity system for neutral amino acids, with many-but not all-features in common with the B^o system of mammal intestinal and renal epithelial brush borders. However, there are differences between the two midgut regions as regard to the ability of the symporters to recognize the different amino acids, which concern the side chain and the presence of the aromatic ring. Altogether these data suggest that two kinds of symporters for neutral amino acids, with different functional properties, are expressed in the anterior-middle and posterior regions of the lepidopteran midgut.     

Organization, ultrastructure, and development of midgut visceral muscle in larval Aedes aegypti

The midgut muscularis of larvae of the mosquito Aedes aegypti takes the form of a grid of longitudinal and circular muscle bands. The longitudinal and circular bands overlap at near right angles at many areas of intersection. The longitudinal bands run the length of the midgut. However, some bands of circular muscle, located in the anterior midgut, pass only partway around the gut. An unusual feature was observed at some regions where longitudinal and circular bands of muscle intersect: filaments oriented at near right angles to one another were present in the same membrane-bound fiber. These cruciform regions send contractile elements into both circular and longitudinal bands. The muscularis was fixed in a contracted state, so most of the sarcomeres are represented by complete overlap of myosin and lighter staining actin filaments. Features characteristic of supercontracting muscle, including perforated Z-lines, were seen in sarcomeres of circular muscle bands. Small invaginations resembling transverse tubules were present but a sarcoplasmic reticulum was not observed. While occasional cells that may be neurons or neurosecretory cells were observed, a network that might serve to coordinate the segmentation and peristaltic movement of the muscularis was not apparent.     

in silico identification of protein-protein interactions in Silkworm,Bombyx mori

The Domesticated silkworm, Bombyx mori, an economically important insect has been used as a lepidopteran molecular model next only to Drosophila. Compared to the genomic information in silkworm, the protein-protein interaction data are limited. Therefore experimentally identified PPI maps from five model organisms such as E.coli, C.elegans, D.melanogaster, H. sapiens, S. cerevisiae were used to infer the PPI network of silkworm using the well-recognized Interlog based method. Among the 14623 silkworm proteins, 7736 protein-protein interaction pairs were predicted which include 2700 unique proteins of the silkworms. Using the iPfam interaction domains and the gene expression data, these predictions were validated. In that 625 PPI pairs of predicted network were associated with the iPfam domain-domain interactions and the random network has average of 9. In the gene expression method, the average PCC value of the predicted network and random network was 0.29 and 0.23100±0.00042 respectively. It reveals that the predicted PPI networks of silkworm are highly significant and reliable. This is the first PPI network for the silkworm which will provide a framework for deciphering the cellular processes governing key metabolic pathways in the silkworm, Bombyx mori and available at SilkPPI (http://210.212.197.30/SilkPPI/).     

An immune-induced reeler protein is involved in the Bombyx mori melanization cascade

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.     

Analysis of BmNPV orf101 disruption: orf101 is essential for mediating budded virus production

In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.     

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.     

Cloning and characterization of an aromatic amino acid and leucine permease of Penicillium chrysogenum

The gene encoding the amino acid permease ArlP (Aromatic and leucine Permease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid permeases. The cDNA clone was used for expression of the permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid permease Gap1. Upon overexpression, an increase in the uptake of l-tyrosine, l-phenylalanine, l-tryptophan and l-leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched β-carbon atom.     

BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori

In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a ¹⁶⁹QACRG¹⁷³ sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells.     

Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition.

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.     

A new Bombyx mori larval ovarian cell line highly susceptible to nucleopolyhedrovirus

Lepidopteran cell lines constitute the backbone for studying baculoviral biology in culturo and for baculovirus vector based recombinant protein expression systems. In the present study, we report establishment of a new continuous cell line designated as DZNU-Bm-1 from larval ovaries of the silkworm, Bombyx mori. The cells were grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat inactivated B. mori haemolymph at 25+/-1 degrees C. A large number of attached epithelial-like and round refractive cells migrated from the explants and multiplied in the primary cultures. Both type of cells were subcultured initially for a few passages but after 10 passages the round refractive cells dominated the population, which could be subcultured continuously using MGM-448 medium with 10% FBS. The population doubling time of cell line was about 42h at 25+/-1 degrees C. The cell populations were largely diploids and triploids, while a few tetraploids and hexaploids were also observed. DNA profiles using Inter Simple Sequence Repeat (ISSR)-PCR and Simple Sequence Repeat (SSR) loci established the differences between DZNU-Bm-1 cell line and most widely used BmN cell line and the B. mori W-chromosome specific sequences confirmed the origin of DZNU-Bm-1 cell line to be from female silkworm. When cells were infected with free nonoccluded B. mori nucleopolyhedrovirus (BmNPV), the cell line was found to be highly susceptible with 92-94% of the cells harbouring BmNPV and having an average of 20-23 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV based baculoviral expression system and also for studying in culturo virus replication.     

Enhancement of Larval RNAi Efficiency by Over-expressing Argonaute2 in Bombyx mori

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.