Biodegradation of four polyolefin plastics in superworms (Larvae of Zophobas atratus) and effects on the gut microbiome.

Recent studies have demonstrated superworms (larvae of Zophobas atratus) ability to degrade polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC), and polypropylene (PP) within their digestive system. This study aimed to compare the ability of superworms to degrade the above four polyolefin plastics over a duration of 30 days. In this study, the degradation rate of PE was the highest, and the final average weight of superworms, as well as the final plastic mass loss consumed by them, significantly increased (73.38 % and 52.33 %, respectively) when PE was fed with wheat bran (1:1 [w/w]). FTIR and TGA indicated the occurrence of oxidation and biodegradation processes in the four polyolefin plastics when exposed to superworms. In addition, the molecular weights (Mw and Mn) of excreted polymer residues decreased by 3.1 % and 2.87 % in PE-fed superworms, suggesting that the depolymerization of PE was not entirely dependent on the gut microbial community. The analysis of the gut microbial communities revealed that the dominant microbial community were different for each type of plastic. The results indicate that the gut microbiome of superworms exhibited remarkable adaptability in degrading various types of plastics, and the intake preferences and efficiency of different plastics are associated with different dominant microbial community species.

Newly Woody Artificial Diet Reveals Antibacterial Activity of Hemolymph in Larvae of Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae).

The rearing of saproxylic insects in laboratory conditions is an important task for studying the biology of insects. Through understanding nutritional needs, it is possible to optimize beetle rearing in laboratory conditions. In this study, an artificial fungi-based diet (FD) was developed for the cultivation of the darkling beetle Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae) in laboratory conditions as a model object for studying the biology of saproxylophagous beetles. To assess the influence of the diet, a number of physiological parameters were measured, including development time, body size, and weight of all stages of the beetle's life cycle, as well as its immune status. The immune status of Z. atratus was assessed on the basis of larval hemolymph antibacterial activity against six different bacterial strains assessed using disk-diffusion and photometric tests. Our findings show that the FD reduces development time and boosts the immune status as compared to beetles reared on a standard diet (SD). Samples from FD-reared larvae had pronounced antibacterial activity as compared to samples from SD-reared larvae. This work is of fundamental importance for understanding the correlations between nutrition and development of saproxylic Coleoptera and is the first report on immune status regulation in this group of insects.

Understanding the Ecological Robustness and Adaptability of the Gut Microbiome in Plastic-Degrading Superworms (Zophobas atratus) in Response to Microplastics and Antibiotics.

Recent discoveries indicate that several insect larvae are capable of ingesting and biodegrading plastics rapidly and symbiotically, but the ecological adaptability of the larval gut microbiome to microplastics (MPs) remains unclear. Here, we described the gut microbiome assemblage and MP biodegradation of superworms (Zophobas atratus larvae) fed MPs of five major petroleum-based polymers (polyethylene, polypropylene, polystyrene, polyvinyl chloride, and polyethylene terephthalate) and antibiotics. The shift of molecular weight distribution, characteristic peaks of C═O, and metabolic intermediates of residual polymers in egested frass proved depolymerization and biodegradation of all MPs tested in the larval intestines, even under antibiotic suppression. Superworms showed a wide adaptation to the digestion of the five polymer MPs. Antibiotic suppression negatively influenced the survival rate and plastic depolymerization patterns. The larval gut microbiomes differed from those fed MPs and antibiotics, indicating that antibiotic supplementation substantially shaped the gut microbiome composition. The larval gut microbiomes fed MPs had higher network complexity and stability than those fed MPs and antibiotics, suggesting that the ecological robustness of the gut microbiomes ensured the functional adaptability of larvae to different MPs. In addition, Mantel's test indicated that the gut microbiome assemblage was obviously related to the polymer type, the plastic degradability, antibiotic stress, and larval survival rate. This finding provided novel insights into the self-adaptation of the gut microbiome of superworms in response to different MPs.

Effects of polymerization types on plastics ingestion and biodegradation by Zophobas atratus larvae, and successions of both gut bacterial and fungal microbiomes.

Recent studies demonstrated that plastic degradation in Zophobas atratus superworms is related to the gut microbiota. To determine whether the biodegradation and gut-microbiota were influenced by ingested plastic polymerization types, foams of polypropylene (PP), polyurethane (PU) and ethylene vinyl acetate (EVA) were selected as representatives of polyolefins, polyester and copolymers, and the sole feedstock for superworms for 45 d. Both growth and survival rates of superworms were influenced by the type of plastic diet. Although the total consumptions of EVA- and PP-fed groups were similar at 29.03 ± 0.93 and 28.89 ± 1.14 mg/g-larva, which were both significantly higher than that of PU-fed groups (21.63 ± 2.18 mg/g-larva), the final survival rates of the EVA-fed group of 36.67 ± 10.41% exhibited significantly lower than that of the PP- and PU-fed groups of 76.67 ± 2.89% and 75.00 ± 7.07%, respectively, and even the starvation group of 51.67 ± 10.93%. The Illumina MiSeq results revealed similarities in the dominant gut bacterial communities between PU- and EVA-fed groups, with an increase in relative abundance of Lactococcus, but significant differences from the PP-fed groups, which had two predominant genera of unclassified Enterobacteriaceae and Enterococcus. Compared to bran-fed groups, changes in gut fungal communities were similar across all plastics-fed groups, with an increase in the dominant abundance of Rhodotorula. The abundance of Rhodotorula increased in the order of polyolefin, polyester, and copolymer. In summary, plastic ingestion, larval growth, and changes in gut bacterial and fungal community of superworms were all influenced by foam diets of different polymerization types, and especially influences on the gut microbiomes were different from each other.

Biodegradation of polyethylene and polystyrene by Zophobas atratus larvae from Bangladeshi source and isolation of two plastic-degrading gut bacteria.

Plastic pollution has become a major environmental concern globally, and novel and eco-friendly approaches like bioremediation are essential to mitigate the impact. Low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), and expanded polystyrene (EPS) are three of the most frequently used plastic types. This study examined biodegradation of these using Zophobas atratus larvae, followed by isolation and whole genome sequencing of gut bacteria collected from larvae frass. Over 36 days, 24.04 % LDPE, 20.01 % EPS, and 15.12 % LLDPE were consumed on average by the larvae, with survival rates of 85 %, 90 %, and 87 %, respectively. Fourier transform infrared spectroscopy (FTIR) analysis of fresh plastic types, consumed plastics, and larvae frass showed proof of plastic oxidation in the gut. Frass bacteria were isolated and cultured in minimal salt media supplemented with plastics as the sole carbon source. Two isolates of bacteria were sampled from these cultures, designated PDB-1 and PDB-2. PDB-1 could survive on LDPE and LLDPE as carbon sources, whereas PDB-2 could survive on EPS. Scanning Electron Microscopy (SEM) provided proof of degradation in both cases. Both isolates were identified as strains of Pseudomonas aeruginosa, followed by sequencing, assembly, and annotation of their genomes. LDPE- and LLDPE-degrading enzymes e.g., P450 monooxygenase, alkane monooxygenase, alcohol dehydrogenase, etc. were identified in PDB-1. Similarly, phenylacetaldehyde dehydrogenase and other enzymes involved in EPS degradation were identified in PDB-2. Genes of both isolates were compared with genomes of known plastic-degrading P. aeruginosa strains. Virulence factors, antibiotic-resistance genes, and rhamnolipid biosurfactant biosynthesis genes were also identified in both isolates. This study indicated Zophobas atratus larvae as potential LDPE, LLDPE, and EPS biodegradation agent. Additionally, the isolated strains of Pseudomonas aeruginosa provide a more direct and eco-friendly solution for plastic degradation. Confirmation and modification of the plastic-degrading pathways in the bacteria may create scope for metabolic engineering in the future.

Acinetobacter entericus sp. nov., isolated from the gut of plastic-eating insect larvae Zophobas atratus.

A Gram-negative, non-motile and rod-shaped strain, BIT-DXN8T, was isolated from the gut of plastic-eating insect larvae Zophobas atratus. The taxonomic position of this new isolate was examined by using a polyphasic approach. A preliminary analysis based on the 16S rRNA gene sequence (1411 bp) indicated that the most similar strain to BIT-DXN8T was Acinetobacter bouvetii DSM 14964T (98.5%), followed by Acinetobacter haemolyticus CIP 64.3T (98.2%) and Acinetobacter pullicarnis S23T (98.2%). The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of five housekeeping genes (fusA, gyrB, recA, rplB and rpoB) and genome sequences, placed strain BIT-DXN8T in a separate lineage among the genus Acinetobacter of the family Moraxellaceae. The average nucleotide identity and digital DNA-DNA hybridization values of the strain when compared to all other species within the genus Acinetobacter were below 96 and 70 %, respectively. The physiological and biochemical tests confirm the affiliation of strain BIT-DXN8T to the present species within the genus Acinetobacter, but with some specific phenotypic differences. Therefore, strain BIT-DXN8T is considered to represent a novel species, for which the name Acinetobacter entericus sp. nov. is proposed. The type strain is BIT-DXN8T (=CCTCC AB 2022117T=KCTC 92696T).

Reactive Oxygen Species Triggered Oxidative Degradation of Polystyrene in the Gut of Superworms (Zophobas atratus Larvae).

Oxidative decomposition of polystyrene (PS) by insects has been previously demonstrated, yet little is known about the oxidation mechanism and its effect on the metabolism of plastics within the insect gut. Here, we demonstrate the generation of reactive oxygen species (ROS) in the gut of superworms (Zophobas atratus larvae) under different feeding trails, which in turn induced the oxidative decomposition of ingested PS. The ROS were commonly generated in the larva gut, and PS consumption resulted in a significant increase of ROS with a maximum ·OH of 51.2 µmol/kg, which was five times higher than in the bran feeding group. Importantly, scavenging of ROS significantly decreased the oxidative depolymerization of PS, indicating a vital role of ROS in effective PS degradation in the gut of superworms. Further investigation suggested that the oxidative depolymerization of PS was caused by the combinatorial effect of ROS and extracellular oxidases of gut microbes. These results demonstrate that ROS were extensively produced within the intestinal microenvironment of insect larvae, which greatly favored the digestion of ingested bio-refractory polymers. This work provides new insights into the underlying biochemical mechanisms of plastic degradation in the gut.

Whole genome assemblies of Zophobas morio and Tenebrio molitor.

Zophobas morio (=Zophobas atratus) and Tenebrio molitor are darkling beetles with industrial importance due to their use as feeder insects and their apparent ability to biodegrade plastics. High quality genome assemblies were recently reported for both species. Here, we report additional independent Z. morio and T. molitor genome assemblies generated from Nanopore and Illumina data. Following scaffolding against the published genomes, haploid assemblies of 462 Mb (scaffold N90 of 16.8 Mb) and 258 Mb (scaffold N90 of 5.9 Mb) were produced for Z. morio and T. molitor, respectively. Gene prediction led to the prediction of 28,544 and 19,830 genes for Z. morio and T. molitor, respectively. Benchmarking Universal Single Copy Orthologs (BUSCO) analyses suggested that both assemblies have a high level of completeness; 91.5 and 89.0% of the BUSCO endopterygota marker genes were complete in the Z. morio assembly and proteome, respectively, while 99.1 and 92.8% were complete in the T. molitor assembly and proteome, respectively. Phylogenomic analyses of four genera from the family Tenebrionidae yielded phylogenies consistent with those previously constructed based on mitochondrial genomes. Synteny analyses revealed large stretches of macrosynteny across the family Tenebrionidae, as well as numerous within-chromosome rearrangements. Finally, orthogroup analysis identified ∼28,000 gene families across the family Tenebrionidae, of which 8,185 were identified in all five of the analyzed species, and 10,837 were conserved between Z. morio and T. molitor. We expect that the availability of multiple whole genome sequences for Z. morio and T. molitor will facilitate population genetics studies to identify genetic variation associated with industrially relevant phenotypes.

Comparison on the effectiveness of Fourier transform infrared (FT-IR) and attenuated total reflection Fourier transform infrared (ATR-FT-IR) in characterizing plastics biodegradation by insect larvae.

The discovery that insect larvae can feed on foam plastics provided new exploration ideas and potential for plastic wastes biodegradation. In previous studies, both attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FT-IR) and conventional FT-IR have been used but no comparison has been done to evaluate the difference of effectiveness for the characterization of oxidization and biodegradation of plastics by insect larvae. To address this, foam plastics of polystyrene, polyurethane and polyethylene, as well as the frass of plastics-fed superworms Zophobas atratus were characterized using both FT-IR and ATR-FT-IR, and the differences were compared. For FT-IR, spectra were found to vary due to the difference in shape and thickness of the samples, as well as the moisture absorption of KBr. For ATR-FT-IR, although tests could be performed directly without pretreatment, the reflection with short wavelength could not deeply penetrate into the frass samples. Since the composition of plastics-fed larval frass is more complex than the original plastics, the spectra of FT-IR and ATR-FT-IR were observed significantly different. Therefore, the ATR-FT-IR was more effective in monitoring functional groups of original plastics, and be recommended to employ in combination with FT-IR for a more comprehensive characterization of plastics-fed larval frass in future studies.

A Novel ß-Hairpin Peptide Z-d14CFR Enhances Multidrug-Resistant Bacterial Clearance in a Murine Model of Mastitis.

The widespread prevalence of antimicrobial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) have gained comprehensive attention as one of the major alternatives to antibiotics. However, low antibacterial activity and high-cost production have limited the applications of natural AMPs. In this study, we successfully expressed recombinant Zophobas atratus (Z. atratus) defensin for the first time. In order to increase the antimicrobial activity of peptide, we designed 5 analogues derived from Z. atratus defensin, Z-d13, Z-d14C, Z-d14CF, Z-d14CR and Z-d14CFR. Our results showed that Z-d14CFR (RGCRCNSKSFCVCR-NH2) exhibited a broad-spectrum antimicrobial activity to both Gram-positive bacteria and Gram-negative bacteria, including multidrug-resistant bacteria. It possessed less than 5% hemolysis and 10% cytotoxicity, even at a high concentration of 1 mg/mL. Antimicrobial mechanism studies indicated that Z-d14CFR performed antimicrobial effect via inhibiting biofilm formation, disrupting bacterial membrane integrity and inducing cellular contents release. Furthermore, Z-d14CFR showed a great therapeutic effect on the treatment of multidrug-resistant Escherichia coli (E. coli) infection by enhancing bacterial clearance, decreasing neutrophils infiltration and the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) in a murine model of mastitis. Our findings suggest that Z-d14CFR could be a promising candidate against multidrug-resistant bacteria.

Sulfakinins influence lipid composition and insulin-like peptides level in oenocytes of Zophobas atratus beetles.

Insect sulfakinins are pleiotropic neuropeptides with the homology to vertebrate gastrin/cholecystokinin peptide family. They have been identified in many insect species and affect different metabolic processes. They have a strong influence on feeding and digestion as well as on carbohydrate and lipid processing. Our study reveals that sulfakinins influence fatty acids composition in Zophobas atratus oenocytes and regulate insulin-like peptides (ILPs) level in these cells. Oenocytes are cells responsible for maintenance of the body homeostasis and have an important role in the regulation of intermediary metabolism, especially of lipids. To analyze the lipid composition in oenocytes after sulfakinins injections we used gas chromatography combined with mass spectrometry and for ILPs level determination an immunoenzymatic test was used. Because sulfakinin peptides and their receptors are the main components of sulfakinin signaling, we also analyzed the presence of sulfakinin receptor transcript (SKR2) in insect tissues. We have identified for the first time the sulfakinin receptor transcript (SKR2) in insect oenocytes and found its distribution more widespread in the peripheral tissues (gut, fat body and haemolymph) as well as in the nervous and neuro-endocrine systems (brain, ventral nerve cord, corpora cardiaca/corpora allata CC/CA) of Z. atratus larvae. The presence of sulfakinin receptor transcript (SKR2) in oenocytes suggests that observed effects on oenocytes lipid and ILPs content may result from direction action of these peptides on oenocytes.

Biodegradation of foam plastics by Zophobas atratus larvae (Coleoptera: Tenebrionidae) associated with changes of gut digestive enzymes activities and microbiome.

In order to uncover the plastic types that superworm Zophobas atratus can degrade and the underlying changes associated with plastics consumption, three types of plastics including polystyrene (PS), polyethylene (PE) and polyurethane (PU) foam were used as sole feedstock to feed the superworm larvae for 35 days with bran as control. Compared to the control, PS- or PU-fed larvae showed 100% survival rates, the PE-fed and starvation larvae had decreased survival rates of 81.67% and 65%, respectively. Both plastics-fed and starvation groups showed decreased larvae weight. The consumption rates of PS, PE, and PU were 1.41, 0.30, and 0.74 mg/d/larva, respectively. The attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and differential scanning calorimeter and thermogravimetric (DSC-TGA) analyses demonstrated the changes of functional groups and thermostability in frass compared to plastic feedstocks, indicating the partial oxidation and degradation of plastics. Among the gut digestive enzymes tested, protease showed increased activities in all plastics-fed groups. Gut microbial communities displayed significant relative abundance changes such as increased abundances of Enterococcus in all plastic-fed groups, Citrobacter in PE-fed group, Dysgonomonas and Sphingobacterium in PS-fed group, and Mangrovibacter in PU-fed group. The latter 3 genera were reported for the first time. In summary, the results demonstrated that Z. atratus could efficiently degrade both PS and PU foam plastics, and the plastic degradation was associated with changes of gut microbial communities and digestive enzyme activities.

Biodegradation of low-density polyethylene and polystyrene in superworms, larvae of Zophobas atratus (Coleoptera: Tenebrionidae): Broad and limited extent depolymerization.

Larvae of Zophobas atratus (synonym as Z. morio, or Z. rugipes Kirsch, Coleoptera: Tenebrionidae) are capable of eating foams of expanded polystyrene (EPS) and low-density polyethylene (LDPE), similar to larvae of Tenebrio molitor. We evaluated biodegradation of EPS and LDPE in the larvae from Guangzhou, China (strain G) and Marion, Illinois, U.S. (strain M) at 25 °C. Within 33 days, strain G larvae ingested respective LDPE and PS foams as their sole diet with respective consumption rates of 58.7 ± 1.8 mg and 61.5 ± 1.6 mg 100 larvae-1d-1. Meanwhile, strain M required co-diet (bran or cabbage) with respective consumption rates of 57.1 ± 2.5 mg and 30.3 ± 7.7 mg 100 larvae-1 d-1. Fourier transform infrared spectroscopy, proton nuclear magnetic resonance, and thermal gravimetric analyses indicated oxidation and biodegradation of LDPE and EPS in the two strains. Gel permeation chromatography analysis revealed that strain G performed broad depolymerization of EPS, i.e., both weight-average molecular weight (Mw) and number-average molecular weight (Mn) of residual polymers decreased, while strain M performed limited extent depolymerization, i.e., Mw and Mn increased. However, both strains performed limited extent depolymerization of LDPE. After feeding antibiotic gentamicin, gut microbes were suppressed, and Mw and Mn of residual LDPE and EPS in frass were basically unchanged, implying a dependence on gut microbes for depolymerization/biodegradation. Our discoveries indicate that gut microbe-dependent LDPE and EPS biodegradation is present within Z. atratus in Tenebrionidae, but that the limited extent depolymerization pattern resulted in undigested polymers with high molecular weights in egested frass.

Myotropic activity of allatostatins in tenebrionid beetles.

Neuropeptides control the functioning of the nervous system of insects, and they are the most diverse signalling molecules in terms of structure and function. Allatostatins are pleiotropic neuropeptides that are considered potent myoinhibitors of muscle contractions in insects. We investigated the effects caused by three distinct allatostatins, Dippu-AST1 (LYDFGL-NH2 from Diploptera punctata), Grybi-MIP1 (GWQDLNGGW-NH2 from Gryllus bimaculatus) and Trica-ASTC (pESRYRQCYFNPISCF-OH from Tribolium castaneum) on contractile activity of the myocardium, oviduct and hindgut of two tenebrionid beetles, Tenebrio molitor and Zophobas atratus. Studies showed that all three peptides exerted myostimulatory effects on the oviduct and hindgut of the beetles, however they did not cause any effect on myocardium. The effects of Dippu-AST1, Grybi-MIP1 and Trica-ASTC were dose-dependent and tissue and species specific. The highest stimulatory effect was caused by Trica-ASTC, showing stimulation of approximately 82% at a 10-12 M concentration and 76% at a 10-11 M concentration for T. molitor and Z. atratus, respectively. The oviduct of T. molitor was more susceptible to allatostatins than that of Z. atratus. Dippu-AST1 showed the maximum stimulating effect at 10-11 M (57%), whereas Grybi-MIP 1 at 10-10 M caused a 41% stimulation. Trica-ASTC, in both species, showed a myostimulatory effect over the whole range of tested concentrations but was most potent at a 10-12 M concentration and caused a 54% and 31.9% increase in the frequency of contractions in the oviduct of T. molitor and Z. atratus, respectively. The results suggest that allatostatins may affect the regulation of egg movement within the oviducts and movement of food in the digestive tract of beetles and do not regulate directly the activity of heart, thus being good candidate compounds in neuropeptides based pest control agents in future research.

NONSULFATED SULFAKININ CHANGES METABOLIC PARAMETERS OF INSECT FAT BODY MITOCHONDRIA.

We investigated the effect of neuropeptide, the nonsulfated sulfakinin (SK) Zopat-SK-1 (pETSDDYGHLRFa) on the mitochondrial oxidative metabolism in the Zophobas atratus larval fat body. Mitochondria were isolated from beetle fat bodies 2 and 24 h after hormone injection. The administration of 20 pmol of Zopat-SK-1 to feeding larvae led to decreased mitochondrial oxidative activities in larval fat body. Diminished activities of citrate synthase and the cytochrome pathway, that is, nonphosphorylating and phosphorylating respiration during succinate oxidation, were observed. However, the effect of Zopat-SK-1 was more pronounced in fat body of insects after 24 h since hormone application. In hormone-treated larval fat bodies, mitochondrial respiration was decreased at the level of respiratory chain and the TCA cycle as well as at the level of mitochondrial biogenesis, as indicated by decreased activities of mitochondrial marker enzymes in fat body homogenates. The inhibition of succinate oxidation may indicate the role of Zopat-SK-1 in the regulation of mitochondrial complex II activity. Moreover, decreased respiratory chain activity was accompanied by the reduced activity of mitochondrial energy-dissipating pathway, uncoupling protein 4. The observed decrease in mitochondrial oxidative metabolism may reflect the Zopat-SK-1-induced reduction in the metabolic rate of larval fat body linked to actual energetic demands of animal.

The activity of the nonsulfated sulfakinin Zopat-SK-1 in the neck-ligated larvae of the beetle Zophobas atratus.

Insect sulfakinins (SKs) are multifunctional neuropeptides structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK). It has been proposed that SKs play a role in modulating energy management in insects by interacting with adipokinetic hormone (AKH), the principle hormone controlling insect intermediary metabolism. To exclude head factors (including AKH) that influence the activity of the nonsulfated sulfakinin Zopat-SK-1 in the larvae of the beetle Zophobas atratus, ligature and in vitro bioassays were used. Our study showed that in the neck-ligated larvae, Zopat-SK-1 evoked a much more pronounced glycogenolytic effect in fat body tissue and a significantly higher hypertrahelosemic effect in hemolymph than in larvae without ligation. We found that the concentration of the sugar trehalose increased under hormonal treatment but no changes in glucose levels were observed. Under in vitro conditions, the maximal glycogenolytic effect of Zopat-SK-1 in fat body was observed at 10 pmol of hormone. Ligature and in vitro bioassays indicated that Zopat-SK-1 activity in the Z. atratus larvae is modulated by head signals and/or factors from the gastrointestinal tract. Our data indicate the existence of a brain-gastrointestinal axis that has a role in controlling of energy (carbohydrate) metabolism in the insect body. Moreover, these results, together with immunological evidence of a cholecystokinin-like (sulfakinin) receptor in the Z. atratus fat body, help us to better understand the SK signaling pathways and its physiological role in insect biology.

Cardioactive properties of Solanaceae plant extracts and pure glycoalkaloids on Zophobas atratus.

Glycoalkaloids, the biologically active secondary metabolites produced by Solanaceae plants, are natural defenses against animals, insects and fungi. In this paper, the effects of glycoalkaloids present in extracts of Solanaceae plants (potato, tomato and black nightshade) or pure commercial glycoalkaloids on the coleopteran Zophobas atratus F. were evaluated by in vitro and in vivo bioassays using heart experimental models. Each tested extract induced a dose-dependent cardioinhibitory effect. The perfusion of Zophobas atratus semi-isolated heart using the highest potato and tomato extract concentration (1 mmol/L) caused irreversible cardiac arrests, while extract from black nightshade produced fast but reversible arrests. Pure commercial glycoalkaloids caused similar but less evident effects compared with extracts. Our results showed that the bioactivity of tested compounds depended on their structure and suggested the existence of synergistic interactions when combinations of the main glycoalkaloids of potato and black nightshade were used for trials. Surprisingly, injection of tomato and potato extracts in 1-day-old pupae of Zophobas atratus induced reversible positive chronotropic effects and decreased the duration of the both phases (anterograde and retrograde) of the heart contractile activity. Furthermore, these extracts affected the amplitude of the heart contractions.

New metabolic activity of the nonsulfated sulfakinin Zopat-SK-1 in the insect fat body.

Insect sulfakinins are multifunctional neuropeptides homologous to vertebrate gastrin/cholecystokin (CCK) neuropeptide hormones. We investigated the action of the nonsulfated sulfakinin Zopat-SK-1 (pETSDDYGHLRFa) on the levels of chosen metabolites in the Zophobas atratus beetle fat body. Samples of fat body were collected 2h and 24h after hormone injection. The administration of 20pmol of Zopat-SK-1 to feeding larvae significantly increased concentrations of lipids and proteins and decreased the content of glycogen in fat body tissue in the 24h experimental group. In contrast, the only increase in total lipid concentration in prepupal fat bodies was observed 24h after Zopat-SK-1 treatment. Simultaneously, changes in the quality and quantity of free sugars in the hemolymph were measured. In larval hemolymph, a marked increase in free sugar concentration and a decrease in glucose content were observed 24h and 2h after Zopat-SK-1 application, respectively. No changes in the prepupal stage were observed. For the first time we show potent metabolic activity of sulfakinin in the fat body tissue of an insect. Our findings imply a physiological function of the nonsulfated form of sulfakinin in energy storage and release processes in fat body tissue of larvae and prepupae was indicated. We suggest a role for sulfakinin signaling in the regulation of energy metabolism in insect tissues.

Adipokinetic hormone induces changes in the fat body lipid composition of the beetle Zophobas atratus.

In insects, neuropeptide adipokinetic hormone (AKH) released from the corpora cardiaca mobilizes lipids and carbohydrates in the fat body. We examined the developmental differences in the action of Tenmo-AKH, a bioanalogue belonging to the adipokinetic/hypertrahelosemic family (AKH/HrTH), on the lipid composition of larval and pupal fat bodies in the beetle Zophobas atratus. Tenmo-AKH was administered to the beetle larvae and pupae either as a single dose or as two doses of 20 pmol during a 24h interval. Extracts of fat bodies were used to analyse the lipid composition by gas chromatography (GC) combined with mass spectrometry (GC-MS). Control extracts were analyzed using the same method. Fatty acids (FA) and fatty acid methyl esters (FAME) were the most abundant compounds in the fat bodies from both developmental stages. We observed significant differences in their concentrations following hormonal treatment. Tenmo-AKH also induced a distinct increase in larval sterols, fatty alcohols and benzoic acid.

UCP4 expression changes in larval and pupal fat bodies of the beetle Zophobas atratus under adipokinetic hormone treatment.

We investigated the influence of adipokinetic hormone (AKH), an insect neurohormone, on uncoupling protein 4 (ZaUCP4) expression and activity in larval and pupal fat body mitochondria of the beetle Zophobas atratus in relation to intermediary metabolism. Homologous Tenmo-AKH was administered to the beetle larvae and pupae as either a single dose or as two doses of 20pmol during a 24h interval. In the larval and pupal fat bodies, downregulation of ZaUCP4 expression at the mRNA and protein levels was observed 24h and 48h after AKH treatment, respectively. In both developmental stages, ZaUCP4 activity was lowered in fat body mitochondria 48h after AKH treatment. In the AKH-injected larvae, changes in ZaUCP4 expression were accompanied by the mobilization of carbohydrate reserves, no change in the concentration of total lipids and an increase in the free fatty acid level. In contrast, AKH had no effect on carbohydrate metabolism in the pupal fat body but induced lipid mobilization. It seems that AKH influences ZaUCP4 expression by triggering multiple events and that it has different physiological roles in controlling intermediary metabolism in the fat body of the beetle larvae and pupae.