Molecular-Weight-Dependent Degradation of Plastics: Deciphering Host-Microbiome Synergy Biodegradation of High-Purity Polypropylene Microplastics by Mealworms.

The biodegradation of polypropylene (PP), a highly persistent nonhydrolyzable polymer, by Tenebrio molitor has been confirmed using commercial PP microplastics (MPs) (Mn 26.59 and Mw 187.12 kDa). This confirmation was based on the reduction of the PP mass, change in molecular weight (MW), and a positive Δδ13C in the residual PP. A MW-dependent biodegradation mechanism was investigated using five high-purity PP MPs, classified into low (0.83 and 6.20 kDa), medium (50.40 and 108.0 kDa), and high (575.0 kDa) MW categories to access the impact of MW on the depolymerization pattern and associated gene expression of gut bacteria and the larval host. The larvae can depolymerize/biodegrade PP polymers with high MW although the consumption rate and weight losses increased, and survival rates declined with increasing PP MW. This pattern is similar to observations with polystyrene (PS) and polyethylene (PE), i.e., both Mn and Mw decreased after being fed low MW PP, while Mn and/or Mw increased after high MW PP was fed. The gut microbiota exhibited specific bacteria associations, such as Kluyvera sp. and Pediococcus sp. for high MW PP degradation, Acinetobacter sp. for medium MW PP, and Bacillus sp. alongside three other bacteria for low MW PP metabolism. In the host transcriptome, digestive enzymes and plastic degradation-related bacterial enzymes were up-regulated after feeding on PP depending on different MWs. The T. molitor host exhibited both defensive function and degradation capability during the biodegradation of plastics, with high MW PP showing a relatively negative impact on the larvae.

Differences in ingestion and biodegradation of the melamine formaldehyde plastic by yellow mealworms Tenebrio molitor and superworms Zophobas atratus, and the prediction of functional gut microbes.

Plastics biodegradation by insect larvae is considered as a new strategy for plastic wastes treatment. To uncover the biodegradation of a more complex chemical polymer of melamine formaldehyde (MF) by insect larvae, two worm species of yellow mealworm Tenebrio molitor and superworm Zophobas atratus were fed on MF foam as sole diet for 45 days with sole bran diet as control. Although the MF foam consumption by yellow mealworms of 0.38 mg/d/g-larvae was almost 40% higher than that by superworms of 0.28 mg/d/g-larvae, a similar decrease of survival rates in both species were obtained at about 58%, indicating the adverse effects on their growth. Depolymerization and biodegradation of MF foam occurred in both larval guts, but was more extensive in yellow mealworms. MF foam sole diet influenced gut bacterial and fungal microbiomes of both larvae species, which were assessed by Illumina MiSeq on day 45. Compared to the bran-fed group, both gut bacterial and fungal communities significantly changed in MF-fed groups, but differed in the two larvae species. The results demonstrated a strong association between the distinctive gut microbiome and MF foam degradation, such as unclassified Enterobacteriaceae, Hyphopichia and Issatchenkia. However, sole MF foam diet negatively influenced worms, like lower survival rates and gut abnormalities. In summary, MF foam could be degraded by both yellow mealworms and superworms, albeit with adverse effects. Gut microbes were strongly associated to MF foam degradation, especially the gut fungi.

Ingestion and biodegradation of disposable surgical masks by yellow mealworms Tenebrio molitor larvae: Differences in mask layers and effects on the larval gut microbiome.

During the COVID-19 pandemic, the usage and production of face masks considerably increased, resulting in large quantities of mask waste accumulating in the natural environment. To investigate whether masks of polypropylene (PP) material could be ingested and degraded by insect worms like PP foam plastic, yellow mealworms were provided with different layers of disposable surgical masks as sole diets for 30 d. Although mask layers, especially the middle layer of melt-blown filter, could be ingested by yellow mealworms, sole mask layer diets had adverse effects on the larval survival and growth. Analyses of Fourier transform infrared spectroscopy, differential scanning calorimeter and thermogravimetric, and gel permeation chromatography demonstrated the changes of functional groups, thermostability and molecular weights in frass compared to original masks, indicating the partial oxidation and degradation of masks. And the depolymerization of the middle layer of masks by yellow mealworms was different from that of other layers. The larval gut bacterial and fungal microbiomes were assessed by Illumina MiSeq, indicating that both of them shifted upon sole layer mask diets. Changes in relative abundances of dominant bacterial and fungal genera demonstrated the strong association between gut microbiome and mask degradation. For instance, unclassified Enterobacteriaceae was closely associated with outer layers degradation. Lactococcus and unclassified Ascomycota were responsible for middle layers degradation, while Lactococcus and Morganella for inner layers degradation. In conclusion, disposable surgical masks of PP material could be ingested and biodegraded by yellow mealworms. The diversities of gut bacterial and fungal microbiomes were associated with the differences in rigid crystalline structures of the layer masks.

Biodegradation of additive-free polypropylene by bacterial consortia enriched from the ocean and from the gut of Tenebrio molitor larvae.

Accumulation of highly recalcitrant PP wastes has caused a serious environmental pollution. We evaluated the biodegradation of two types of additive-free PP polymers by microbial degraders from different environments. Two bacterial consortia, designated as PP1M and PP2G, were enriched from the ocean and from the guts of Tenebrio molitor larvae. Both consortia were able to utilize each of two different additive-free PP plastics with relatively low molecular weights (low molecular weight PP powder and amorphous PP pellets) as the sole carbon source for growth. After a 30-day incubation, several plastic characterization methods, including high-temperature gel permeation chromatography, scanning electron microscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry, were used to characterize the PP samples. The bio-treated PP powder was covered with tight biofilms and extracellular secretions with significantly increased hydroxyl and carbonyl groups and slightly decreased methyl groups. This suggested that degradation and oxidation had occurred. The altered molecular weights and the increased melting enthalpy and average crystallinity of the bio-treated PP samples all suggested that both consortia preferred to depolymerize and degrade the fractions with molecular weights of ≤34 kDa and the amorphous phase fractions of the two types of PP. Furthermore, low molecular weight PP powder was more susceptible to bacterial degradation compared to amorphous PP pellets. This study provides a unique example of different types of additive-free PP degradation by different culturable bacteria from the ocean and insect guts as well as a feasibility of PP waste removal in different environments.

Molecular Characterization and Functional Analysis of the Dipeptidyl Peptidase IV from Venom of the Ectoparasitoid Scleroderma guani.

Dipeptidyl peptidase IV (DPPIV) is a proline-specific serine peptidase that remains poorly investigated in terms of venom composition. Here, we describe the molecular characteristics and possible functions of DPPIV as a major venom component of the ant-like bethylid ectoparasitoid, Scleroderma guani, named SgVnDPPIV. The SgVnDPPIV gene was cloned, which encodes a protein with the conserved catalytic triads and substrate binding sites of mammalian DPPIV. This venom gene is highly expressed in the venom apparatus. Recombinant SgVnDPPIV, produced in Sf9 cells using the baculovirus expression system, has high enzymatic activity, which can be efficiently inhibited by vildagliptin and sitagliptin. Functional analysis revealed that SgVnDPPIV affects genes related to detoxification, lipid synthesis and metabolism, response to stimuli, and ion exchange in pupae of Tenebrio molitor, an envenomated host of S. guani. The present work contributes towards understanding the role of venom DPPIV involved in the interaction between parasitoid wasp and its host.

Mitigating Effects of Tenebrio molitor Larvae Powder Administration in Mice with Dextran Sodium Sulfate (DSS)- Induced Colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory bowel disease that affects people worldwide. The causes of UC are diverse, and symptoms include diarrhea, weight loss, anemia, rectal bleeding, and bloody stools. Tenebrio molitor larvae have recently gained attention as edible insects with various physiological and medical effects. Research on the anti-inflammatory effects of ingesting Tenebrio molitor larvae powder (TMLP) is being actively conducted. In this study, TMLP was administered to mice with dextran sodium sulfate (DSS)-induced colitis to investigate its effects in reducing colitis symptoms. METHODS: Mice were initially given 3% DSS in water to induce colitis and then feed containing 0%, 2%, or 4% TMLP. Pathologic changes in colon tissues were assessed by histology, and neutrophil levels were measured by myeloperoxidase (MPO) assay. Levels of IL-1ß, IL-6, and TNF-α were measured using real-time PCR and ELISA assays, and IκB and NF-kB protein levels were measured by western blotting. RESULT: Disease Activity Index (DAI) scores and MPO activity were reduced in TMLP-treated mice, and colon length increased as much as normal mice. Pathologic changes in the colon tissues of DSS-induced mice were attenuated, and the expression of inflammatory cytokine genes IL-1ß, IL-6, and TNF-α decreased. Concomitant decreases in the protein expression of IL-1ß and IL-6 were confirmed using ELISA. Western blotting revealed that levels of phosphorylated forms of IκB and NF-κB also decreased. CONCLUSION: These results show that feeding TMLP to DSS-induced mice inhibited the typical inflammatory pathway of colitis. Therefore, TMLP shows potential as a food additive that can help treat colitis.
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Immunological Roles of TmToll-2 in Response to Escherichia coli Systemic Infection in Tenebrio molitor.

The antimicrobial roles of Toll-like receptors have been mainly identified in mammalian models and Drosophila. However, its immunological function in other insects has yet to be fully clarified. Here, we determined the innate immune response involvement of TmToll-2 encountering Gram-negative, Gram-positive, and fungal infection. Our data revealed that TmToll-2 expression could be induced by Escherichia coli, Staphylococcus aureus, and Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of Tenebrio molitor young larvae. However, TmToll-2 silencing via RNAi technology revealed that sole E. coli systemic infection caused mortality in the double-strand RNA TmToll-2-injected group compared with that in the control group. Further investigation indicated that in the absence of TmToll-2, the final effector of Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated, mainly E. coli post-insult. We showed that the expression of all AMP genes was suppressed in the main immune organ of insects, namely, fat bodies, in silenced individuals, while the relevant expressions were not affected after fungal infection. Thus, our research revealed the immunological roles of TmToll-2 in different organs of T. molitor in response to pathogenic insults.

Neuropeptidomes of Tenebrio molitor L. and Zophobas atratus Fab. (Coleoptera, Polyphaga: Tenebrionidae).

Neuropeptides are signaling molecules that regulate almost all physiological processes in animals. Around 50 different genes for neuropeptides have been described in insects. In Coleoptera, which is the largest insect order based on numbers of described species, knowledge about neuropeptides and protein hormones is still limited to a few species. Here, we analyze the neuropeptidomes of two closely related tenebrionid beetles: Tenebrio molitor and Zophobas atratus─both of which are model species in physiological and pharmacological research. We combined transcriptomic and mass spectrometry analyses of the central nervous system to identify neuropeptides and neuropeptide-like and protein hormones. Several precursors were identified in T. molitor and Z. atratus, of which 50 and 40, respectively, were confirmed by mass spectrometry. This study provides the basis for further functional studies of neuropeptides as well as for the design of environmentally friendly and species-specific peptidomimetics to be used as biopesticides. Furthermore, since T. molitor has become accepted by the European Food Safety Authority as a novel food, a deeper knowledge of the neuropeptidome of this species will prove useful for optimizing production programs at an industrial scale.

Comparative genomic analysis of ABC transporter genes in Tenebrio molitor and four other tenebrionid beetles (Coleoptera: Tenebrionidea).

ATP-binding cassette (ABC) transporters, one of the largest transmembrane protein families, transport a diverse number of substate across membranes. Details of their diverse physiological functions have not been established. Here, we identified 87 ABC transporter genes in the genomes of Tenebrio molitor along with those from Asbolus verrucosus (104), Hycleus cichorii (65), and Hycleus phaleratus (80). Combining these genes (336 in total) with genes reported in Tribolium castaneum (73), we analyzed the phylogeny of ABC transporter genes in all five Tenebrionids. They are assigned into eight subfamilies (ABCA-H). In comparison to other species, the ABCC subfamily in this group of beetles appears expanded. The expression profiles of the T. molitor genes at different life stages and in various tissues were also investigated using transcriptomic analysis. Most of them display developmental specific expression patterns, suggesting to us their possible roles in development. Most of them are highly expressed in detoxification-related tissues including gut and Malpighian tubule, from which we infer their roles in insecticide resistance. We detected specific or abundant expressions of many ABC transporter genes in various tissues such as salivary gland, ovary, testis, and antenna. This new information helps generate new hypotheses on their biological significance within tissues.

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production.

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

Selenium accumulation in protein fractions of Tenebrio molitor larvae and the antioxidant and immunoregulatory activity of protein hydrolysates.

Although numerous types of organisms have been used to enrich selenium, a low-cost and efficient organism is yet to be identified. This study aimed to develop a new means of selenium enrichment using Tenebrio molitor larvae. Our results indicated that the total selenium content in larvae was increased 83-fold to 54.21 ± 1.25 µg/g, and of this content, organic selenium accounted for over 97% after feeding the larvae with 20 µg/g of sodium selenite. Selenium was distributed unequally in the protein fraction with following order: alkali-soluble protein-bound selenium (36.32%) > salt-soluble protein-bound selenium (19.41%) > water-soluble protein-bound selenium (17.03%) > alcohol-soluble protein-bound selenium (3.21%). Additionally, 81% of the selenium within the soluble proteins was distributed in subunits possessing molecular weights of <40 kDa. After hydrolysis by alcalase, the protein hydrolysate of selenium-enriched larvae possessing 75% selenium recovery exhibited stronger antioxidant and immunoregulatory activities than those of regular larvae.

Optimization of a Simplified and Effective Analytical Method of Pesticide Residues in Mealworms (Tenebrio molitor Larvae) Combined with GC-MS/MS and LC-MS/MS.

An effective analytical method was optimized for residues including chlorpyrifos-methyl, deltamethrin, fenoxanil, thiobencarb and fludioxonil in mealworms, the larval form of Tenebrio molitor. They are listed for pest control during wheat cultivation and can be found in wheat-bran feed for growing mealworms in South Korea. Analytes were extracted using acetonitrile and salt packet. Four clean-up methods ((1) MgSO4 + 25 mg PSA + 25 mg C18; (2) MgSO4 + 50 mg PSA + 50 mg C18; (3) EMR-lipidTM tube; and (4) 10 mL n-hexane) were investigated and the method (1) was selected due to its robustness. Low-temperature precipitation of fat and proteins improved the recoveries. Recoveries from the Method (1) were satisfying with 70-120% with <20% relative SD at a spiking level of 0.01 mg/kg. With the simultaneous sample preparation, fenoxanil, thiobencarb and fludioxonil were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and chlorpyrifos-methyl and deltamethrin by gas chromatography tandem mass spectrometry (GC-MS/MS). Quantification limits for LC-MS/MS and GC-MS/MS were 0.5 and 2.5 µg/L, respectively. No pesticides of interest were detected in 30 real samples collected across the nation. However, the data can be provided for establishing maximum residue limits for the pesticides in mealworms in response to the positive list system.

Identification of sulfakinin receptors (SKR) in Tenebrio molitor beetle and the influence of sulfakinins on carbohydrates metabolism.

Sulfakinins (SKs) are pleiotropic neuropeptides commonly found in insects, structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK) neuropeptides. SKs together with sulfakinin receptors (SKRs) are involved in sulfakinin signaling responsible for variety of biological functions, including food intake or fatty acid metabolism. In the present study, we determined the distribution of SKRs in Tenebrio molitor larvae and characterized the impact of nonsulfated and sulfated SKs on carbohydrates and insulin-like peptides (ILPs) level in beetle hemolymph. Our results indicate the presence of both sulfakinin receptors, SKR1 and SKR2, in the nervous system of T. molitor. The distribution of SKR2 in peripheral tissues was more widespread than SKR1, and their transcripts have been found in fat body, gut and hemolymph. This is also the first evidence for SKRs presence in insect hemocytes indicating immunotropic activity of SKs. Moreover, in the present study, we have demonstrated that SKs regulate ILPs and carbohydrates level in insect hemolymph, and that sulfation is not crucial for peptides activity. Our study confirms the role of SKs in maintaining energy homeostasis in beetles.

Biosurfactants Induce Antimicrobial Peptide Production through the Activation of TmSpatzles in Tenebrio molitor.

Biosurfactant immunomodulatory activities in mammals, nematodes, and plants have been investigated. However, the immune activation property of biosurfactants in insects has not been reported. Therefore, here, we studied the defense response triggered by lipopeptides (fengycin and iturin A), glycolipids (rhamnolipid), and cyclic polypeptides (bacitracin) in the coleopteran insect, mealworm Tenebrio molitor. The in vitro antimicrobial activities against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans) were assessed by mixing these pathogens with the hemolymph of biosurfactant-immune-activated larvae. E. coli growth was remarkably inhibited by this hemolymph. The antimicrobial peptide (AMP) induction results also revealed that all biosurfactants tested induced several AMPs, exclusively in hemocytes. The survivability analysis of T. molitor larvae challenged by E. coli (106 CFU/µL) at 24 h post biosurfactant-immune activation showed that fengycin, iturin A, and rhamnopid significantly increased survivability against E. coli. Biosurfactant-induced TmSpatzles activation was also monitored, and the results showed that TmSpz3 and TmSpz-like were upregulated in the hemocytes of iturin A-injected larvae, while TmSpz4 and TmSpz6 were upregulated in the fat bodies of the fengycin-, iturin A-, and rhamnolipid-injected larvae. Overall, these results suggest that lipopeptide and glycolipid biosurfactants induce the expression of AMPs in T. molitor via the activation of spätzle genes, thereby increasing the survivability of T. molitor against E. coli.

Identification, Localization in the Central Nervous System and Novel Myostimulatory Effect of Allatostatins in Tenebrio molitor Beetle.

Allatostatins (ASTs) are pleiotropic insect neuropeptides that are potent myoinhibitors of muscle contractions. In this study, we identified and immunolocalized peptides from the MIP/AST and PISCF/AST families in the nervous system of a model beetle, Tenebrio molitor. Neurons containing MIPs were immunolocalized in the brains of adults and the ventral nerve cords of larvae, pupae and imagines of this species as well as in the retrocerebral complex. PISCFs were immunolocalized in the ventral nerve cord of all stages as well as the brain of the adult beetle. Faint signals were also observed in the corpus allatum but not in the corpus cardiacum. The results allowed us to deduce the sequences of three neuropeptides belonging to MIP/ASTs, Tenmo-MIP4-NWGQFGXWa, Tenmo-MIP5-SKWDNFRGSWa and Tenmo-MIP6-EPAWSNLKGIWa, and one peptide from the PISCF/AST family, QSRYXQCYFNPISCX. Furthermore, we showed for the first time myostimulatory action of endogenous MIP/ASTs. Tenmo-MIP5 caused dose-dependent stimulation of the contractile activity of the beetle oviduct muscles, showing a sigmoidal curve up to 81.20% at the 10-8 M concentration, and the EC50 value for the myostimulatory effect of this peptide was 8.50 × 10-12 M. This is the first report of myostimulatory action of an endogenous myoinhibitory peptide in insect muscles.

The roles of mucus-forming mucins, peritrophins and peritrophins with mucin domains in the insect midgut.

Most insects have a gut lined with a peritrophic membrane (PM) consisting of chitin and proteins, mainly peritrophins that have chitin-binding domains. The PM is proposed to originate from mucus-forming mucins (Mf-mucins), which acquired a chitin-binding domain that interlocked with chitin, replacing mucus in function. We evaluated the expression of Mf-mucins and peritrophins by RNA-sequencing (RNA-seq) throughout the midgut of four distantly related insects. Mf-mucins were identified as proteins with high o-glycosylation and a series of uninterrupted Pro/Thr/Ser residues. The results demonstrate that the mucus layer is widespread in insects, and suggest that insect Mf-mucins are derived from those found in other animals by the loss of the cysteine knot and von Willebrand domains. The data also support a role of Mf-mucins in protecting the middle midgut of Musca domestica against acidic buffers. Mf-mucins may also produce a jelly-like material associated with the PM that immobilizes digestive enzymes in Spodoptera frugiperda. Peritrophins with a domain similar to Mf-mucins may be close to the ancestor of peritrophins. Expression data of peritrophins and chitin synthase genes throughout the midgut of M. domestica, S. frugiperda and Tenebrio molitor indicated that peritrophins were incorporated along the PM, according to their preferential sites of formation. Finally, the data support the view that mucus has functions distinct from the PM.

Antioxidant and Anti-Inflammatory Activities of Hydrolysates and Peptide Fractions Obtained by Enzymatic Hydrolysis of Selected Heat-Treated Edible Insects.

This study investigated the effect of heat treatment of edible insects on antioxidant and anti-inflammatory activities of peptides obtained by in vitro gastrointestinal digestion and absorption process thereof. The antioxidant potential of edible insect hydrolysates was determined as free radical-scavenging activity, ion chelating activity, and reducing power, whereas the anti-inflammatory activity was expressed as lipoxygenase and cyclooxygenase-2 inhibitory activity. The highest antiradical activity against DPPH• (2,2-diphenyl-1-picrylhydrazyl radical) was noted for a peptide fraction from baked cricket Gryllodes sigillatus hydrolysate (IC50 value 10.9 µg/mL) and that against ABTS•+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical) was the highest for raw mealworm Tenebrio molitor hydrolysate (inhibitory concentration (IC50 value) 5.3 µg/mL). The peptides obtained from boiled locust Schistocerca gregaria hydrolysate showed the highest Fe2+ chelation ability (IC50 value 2.57 µg/mL); furthermore, the highest reducing power was observed for raw G. sigillatus hydrolysate (0.771). The peptide fraction from a protein preparation from the locust S. gregaria exhibited the most significant lipoxygenase and cyclooxygenase-2 inhibitory activity (IC50 value 3.13 µg/mL and 5.05 µg/mL, respectively).

High stability and biological activity of the copper(II) complexes of alloferon 1 analogues containing tryptophan.

Copper(II) complex formation processes between the alloferon 1 (Allo1) (HGVSGHGQHGVHG) analogues where the tryptophan residue is introducing in the place His residue H1W, H6W, H9W and H12W have been studied by potentiometric, UV-visible, CD and EPR spectroscopic, and MS methods. For all analogues of alloferon 1 complex speciation have been obtained for a 1:1 metal-to-ligand molar ratio and 2:1 of H1W because of precipitation at higher (2:1, 3:1 and 4:1) ratios. At physiological pH7.4 and a 1:1 metal-to-ligand molar ratio the tryptophan analogues of alloferon 1 form the CuH-1L and/or CuH-2L complexes with the 4N binding mode. The introduction of tryptophan in place of histidine residues changes the distribution diagram of the complexes formed with the change of pH and their stability constants compared to the respective substituted alanine analogues of alloferon 1. The CuH-1L, CuH-2L and CuH-3L complexes of the tryptophan analogues are more stable from 1 to 5 log units in comparison to those of the alanine analogues. This stabilization of the complexes may result from cation(Cu(II))-π and indole/imidazole ring interactions. The induction of apoptosis in vivo, in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active were the H12W peptide and Cu(II)-H12W complex formed at pH7.4.

Subcellular partitioning of cadmium and zinc in mealworm beetle (Tenebrio molitor) larvae exposed to metal-contaminated flour.

By studying the internal compartmentalization of metals in different subcellular fractions we are able to better understand the mechanisms of metal accumulation in organisms and the transfer of metals through trophic chains. We investigated the internal compartmentalization of cadmium (Cd) and zinc (Zn) in mealworm beetle (Tenebrio molitor) larvae by breeding them in flour contaminated with either Cd at 100, 300 and 600mgkg(-1), or Zn at 1000 and 2000mgkg(-1). We separated the cellular components of the larvae into 3 fractions: the S1 or cytosolic fraction containing organelles, heat-sensitive and heat-stable proteins, the S2 or cellular debris fraction and the G or metal-rich granule fraction. The concentration of Cd and Zn in each fraction was measured at 0, 7, 14 and 21 days of being fed the flour. The concentration of Cd in the flour affected the concentration of Cd measured in each larval subcellular fraction (p≤0.0001), while the concentration of Zn in the flour only affected the Zn concentration in the S2 and G fractions (p≤0.02). Both Cd and Zn concentrations in mealworms remained relatively constant during the exposure (days 7, 14 and 21) in all three fractions, but the Cd concentrations were much higher than those found in larvae before the exposure (day 0). The concentration of Cd in the flour, however, did not affect the percentage of Cd in the S1 fraction. The contribution of Cd in the G fraction to the total Cd amount was similar (30-40%) in all Cd treatments. The percentage of Zn in all three fractions was not affected by the concentration of Zn in the flour and the relative contributions of each subcellular fraction to the total burden of Zn remained generally constant for both control and treated larvae. In general, larvae sequestered approximately 30% of Cd and Zn in the S1 fraction, which is important for the transport of metals to higher trophic levels in a food web.

Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures.

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes.