Nano/microparticles in conjunction with microalgae extract as novel insecticides against Mealworm beetles, Tenebrio molitor.

The intensive use of insecticides in global agricultural production has attracted much attention due to its many adverse effects on human health and the environment. In recent years, the utilization of nanotechnology has emerged as a tool to overcome these adverse effects. The aim of this work was to test different microparticles (zinc oxide (ZnO MPs) and silicon dioxide microparticles (SiO2 MPs)), and silver nanoparticles (Ag NPs) and to study their toxicity on a model organism, Tenebrio molitor. A comprehensive comparative study, which included more than a thousand mealworms divided into nine separate groups, was conducted. In addition to pure nano/microparticle solutions, the effect of particles mixed with the microalgae extract Chlamydomonas reinhardtii was also observed. Pure Ag NPs and SiO2 MPs resulted in larval mortality of more than 70% compared to that of pure ZnO MPs, in which the mortality rate was approximately 33%. A mixture of the algal extract with zinc oxide microparticles resulted in mortality that was double compared to that observed with pure ZnO MPs. In parallel, atomic absorption spectrometry (AAS) was used to determine the difference in the concentration of trace elements in the bodies of dead and live larvae.

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.

Dietary zinc enrichment reduces the cadmium burden of mealworm beetle (Tenebrio molitor) larvae.

The industrial production of Tenebrio molitor L. requires optimized rearing and processing conditions to generate insect biomass with high nutritional value in large quantities. One of the problems arising from processing is a tremendous loss in mineral accessibility, affecting, amongst others, the essential trace element Zn. As a feasible strategy this study investigates Zn-enrichment of mealworms during rearing to meet the nutritional requirements for humans and animals. Following feeding ZnSO4-spiked wheat bran substrates late instar mealworm larvae were evaluated for essential micronutrients and human/animal toxic elements. In addition, growth rate and viability were assessed to select optimal conditions for future mass-rearing. Zn-feeding dose-dependently raised the total Zn content, yet the Znlarvae/Znwheat bran ratio decreased inversely related to its concentration, indicating an active Zn homeostasis within the mealworms. The Cu status remained stable, suggesting that, in contrast to mammals, the intestinal Cu absorption in mealworm larvae is not affected by Zn. Zn biofortification led to a moderate Fe and Mn reduction in mealworms, a problem that certainly can be overcome by Fe/Mn co-supplementation during rearing. Most importantly, Zn feeding massively reduced the levels of the human/animal toxicant Cd within the mealworm larvae, a technological novelty of outstanding importance to be implemented in the future production process to ensure the consumer safety of this edible insect species.

Solanum Nigrum Fruit Extract Increases Toxicity of Fenitrothion-A Synthetic Insecticide, in the Mealworm Beetle Tenebrio Molitor Larvae.

Synthetic insecticides are widely used for crop protection both in the fields and in the food stored facilities. Due to their toxicity, and assumptions of Integrated Pest Management, we conducted two independent experiments, where we studied the influence of Solanum nigrum unripe fruit extract on the toxicity of an organophosphorus insecticide fenitrothion. In the first variant of the experiment, Tenebrio molitor larvae were fed with blended fenitrothion (LC50) and the extract in four concentrations (0.01, 0.1, 1 and 10%) in ratio 1:1 for 3 days. In the second variant, a two-day application of fenitrothion (LC40) was preceded by a one-day extract treatment. The first variant did not show any increase in lethality compared to fenitrothion; however, ultrastructure observations exhibited swollen endoplasmic reticulum (ER) membranes in the midgut and nuclear and cellular membranes in the fat body, after application of blended fenitrothion and extract. An increased amount of heterochromatin in the fat body was observed, too. In the second variant, pre-treatment of the extract increased the lethality of larvae, decreased the level of glycogen and lipids in the fat body and disrupted integrity of midgut cellular membranes. S. nigrum extract, applied prior to fenitrothion treatment can be a factor increasing fenitrothion toxicity in T. molitor larvae. Thus, this strategy may lead to decreased emission of synthetic insecticides to the environment.

Anticryptococcal activity of hexane extract from Spondias tuberosa Arruda and associated cellular events.

Cryptococcosis is an opportunistic systemic mycosis whose treatment is limited to three drugs. In this work, we evaluated the antifungal activity of a hexane extract (HE) from Spondias tuberosa leaves against Cryptococcus neoformans and Cryptococcus gattii. Minimal inhibitory concentrations (MIC) were determined, and putative mechanisms were evaluated by flow cytometry. In addition, an in vivo infection assay was performed using Tenebrio molitor larvae. Treatment with HE inhibited the growth of standard and clinical isolates of C. neoformans and C. gattii (MICs ranging from 0.78 to 3.12mg/mL), significantly (P<0.05) increased mitochondrial superoxide anion levels, and induced mitochondrial membrane depolarization, loss of lysosomal membrane integrity, and phosphatidylserine externalization. The mean survival time of C. gattii-infected T. molitor larvae significantly (P<0.05) increased from 1.225 days in control to 3.067 and 3.882 days in HE-treated groups (78 and 156mg/kg, respectively). In conclusion, HE showed anticryptococcal activity, induced mitochondrial and lysosomal damage in yeast cells, and exhibited anti-infective action against C. gattii in T. molitor larvae.

Differentiated Effects of Secondary Metabolites from Solanaceae and Brassicaceae Plant Families on the Heartbeat of Tenebrio molitor Pupae.

The usage of insects as model organisms is becoming more and more common in toxicological, pharmacological, genetic and biomedical research. Insects, such as fruit flies (Drosophila melanogaster), locusts (Locusta migratoria), stick insects (Baculum extradentatum) or beetles (Tenebrio molitor) are used to assess the effect of different active compounds, as well as to analyse the background and course of certain diseases, including heart disorders. The goal of this study was to assess the influence of secondary metabolites extracted from Solanaceae and Brassicaceae plants: Potato (Solanum tuberosum), tomato (Solanum lycopersicum), black nightshade (Solanum nigrum) and horseradish (Armoracia rusticana), on T. molitor beetle heart contractility in comparison with pure alkaloids. During the in vivo bioassays, the plants glycoalkaloid extracts and pure substances were injected at the concentration 10-5 M into T. molitor pupa and evoked changes in heart activity. Pure glycoalkaloids caused mainly positive chronotropic effects, dependant on heart activity phase during a 24-h period of recording. Moreover, the substances affected the duration of the heart activity phases. Similarly, to the pure glycoalkaloids, the tested extracts also mainly accelerated the heart rhythm, however S. tuberosum and S. lycopersicum extracts slightly decreased the heart contractions frequency in the last 6 h of the recording. Cardioacceleratory activity of only S. lycopersicum extract was higher than single alkaloids whereas S. tubersoum and S. nigrum extracts were less active when compared to pure alkaloids. The most cardioactive substance was chaconine which strongly stimulated heart action during the whole recording after injection. A. rusticana extract which is composed mainly of glucosinolates did not significantly affect the heart contractions. Obtained results showed that glycoalkaloids were much more active than glucosinolates. However, the extracts depending on the plant species might be more or less active than pure substances.

Sublethal Effects of Solanum nigrum Fruit Extract and Its Pure Glycoalkaloids on the Physiology of Tenebrio molitor (Mealworm).

BACKGROUND: Solanaceae plants produce glycoalkaloids (GAs) that affect various physiological processes of herbivorous insects and they are being tested as potential alternatives for synthetic pesticides. They cause lethal and sublethal effects. Nevertheless, their mode of action remains unclear. Therefore, we examined the effects of Solanum nigrum fruit extracts and pure glycoalkaloids on a model beetle, Tenebrio molitor. METHODS: Plant extracts or pure alkaloids were added to the food of the larvae for three days. The lipid, glycogen, and protein content in the fat body and the midgut were determined, and the contractility of the heart, hindgut, and oviduct muscles was tested using the video-microscopy technique. Finally, the ultrastructure of the fat body and the midgut was observed using electron microscopy. RESULTS: No lethal effects were noted. Sublethal changes were observed in the content of biomolecules, malformations of organelles, chromatin condensation, and heart and oviduct contractility. The observed effects differed between the tested glycoalkaloids and the extract. CONCLUSIONS: Both the extract and pure GAs have a wide range of effects that may result in impaired development, food intake, and reproduction. Some early effects may be used as bioindicators of stress. The effects of the extract and pure alkaloids suggest that the substances produced by the plant may act additively or synergistically.

A dietary carotenoid reduces immunopathology and enhances longevity through an immune depressive effect in an insect model.

Immunopathology corresponds to self-damage of the inflammatory response, resulting from oxidizing molecules produced when the immune system is activated. Immunopathology often contributes to age-related diseases and is believed to accelerate ageing. Prevention of immunopathology relies on endogenous antioxidant enzymes and the consumption of dietary antioxidants, including carotenoids such as astaxanthin. Astaxanthin currently raises considerable interest as a powerful antioxidant and for its potential in alleviating age-related diseases. Current in vitro and short-term in vivo studies provide promising results about immune-stimulating and antioxidant properties of astaxanthin. However, to what extent dietary supplementation with astaxanthin can prevent long-term adverse effects of immunopathology on longevity is unknown so far. Here, using the mealworm beetle, Tenebrio molitor, as biological model we tested the effect of lifetime dietary supplementation with astaxanthin on longevity when exposed to early life inflammation. While supplementation with astaxanthin was found to lessen immunopathology cost on larval survival and insect longevity, it was also found to reduce immunity, growth rate and the survival of non immune-challenged larvae. This study therefore reveals that astaxanthin prevents immunopathology through an immune depressive effect and can have adverse consequences on growth.

Insecticidal activity of garlic essential oil and their constituents against the mealworm beetle, Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae).

This study evaluated the insecticidal activity of garlic, Allium sativum Linnaeus (Amaryllidaceae) essential oil and their principal constituents on Tenebrio molitor. Garlic essential oil, diallyl disulfide, and diallyl sulfide oil were used to compare the lethal and repellent effects on larvae, pupae and adults of T. molitor. Six concentrations of garlic essential oil and their principal constituents were topically applied onto larvae, pupae and adults of this insect. Repellent effect and respiration rate of each constituent was evaluated. The chemical composition of garlic essential oil was also determined and primary compounds were dimethyl trisulfide (19.86%), diallyl disulfide (18.62%), diallyl sulfide (12.67%), diallyl tetrasulfide (11.34%), and 3-vinyl-[4H]-1,2-dithiin (10.11%). Garlic essential oil was toxic to T. molitor larva, followed by pupa and adult. In toxic compounds, diallyl disulfide was the most toxic than diallyl sulfide for pupa > larva > adult respectively and showing lethal effects at different time points. Garlic essential oil, diallyl disulfide and diallyl sulfide induced symptoms of intoxication and necrosis in larva, pupa, and adult of T. molitor between 20-40 h after exposure. Garlic essential oil and their compounds caused lethal and sublethal effects on T. molitor and, therefore, have the potential for pest control.

Toxicity of plant essential oils to different life stages of the poultry red mite, Dermanyssus gallinae, and non-target invertebrates.

Seven essential oils with potential as acaricides for use against the poultry red mite, Dermanyssus gallinae (De Geer) (Acari: Dermanyssidae), were selected for study. These products (essential oils of manuka, cade, pennyroyal, thyme, garlic, clove bud and cinnamon bark) were deployed against different life stages of D. gallinae in laboratory tests at the (lethal concentration) LC(50) level for adult mites. For all essential oils tested, toxicity to D. gallinae juveniles was as high as toxicity to adults, if not higher. However, at the LC(50) level determined for adults, some oils were ineffective in preventing hatching of D. gallinae eggs. The essential oils were also tested under laboratory conditions at their LC(90) levels for D. gallinae adults on two model non-target species, the brine shrimp, Artemia salina (L.), and the mealworm beetle, Tenebrio molitor (L.). Results showed that not all essential oils were as toxic to A. salina and T. molitor as they were to D. gallinae, suggesting that it may be possible to select certain oils for development as acaricides against D. gallinae that would have minimal impact on non-target organisms. However, the level of toxicity to A. salina and T. molitor was not consistent across the selected essential oils.

Repellence of plant essential oils to Dermanyssus gallinae and toxicity to the non-target invertebrate Tenebrio molitor.

With changes in legislation and consumer demand, alternatives to synthetic acaricides to manage the poultry red mite Dermanyssus gallinae (De Geer) in laying hen flocks are increasingly needed. These mites may cause losses in egg production, anaemia and even death of hens. It may be possible to use plant-derived products as D. gallinae repellents, especially if such products have a minimal impact on non-target organisms. An experiment was conducted with D. gallinae to assess the repellence of a range of plant essential oils, previously found to be of varying toxicity (relatively highly toxic to non-toxic) to this pest. Experiments were also undertaken to assess the toxicity of these products to mealworm beetles (Tenebrio molitor L.), a non-target invertebrate typical of poultry production systems. Results showed that all seven essential oils tested (manuka, thyme, palmarosa, caraway, spearmint, black pepper and juniper leaf) were repellent to D. gallinae at 0.14mg oil/cm(3) (initial concentration) during the first 2 days of study. Thyme essential oil appeared to be the most effective, where repellence lasted until the end of the study period (13 days). At the same concentration toxicity to T. molitor differed, with essential oils of palmarosa and manuka being no more toxic to adult beetles than the control. There was neither a significant association between the rank toxicity and repellence of oils to D. gallinae, nor the toxicity of oils to D. gallinae (as previously determined) and T. molitor.

Insect growth regulatory effects of linear diterpenoids and derivatives from Baccharis thymifolia.

Linear diterpenes isolated from aerial parts of Baccharis thymifolia ( 1- 3) were tested for insect growth inhibitory activity against Tenebrio molitor larvae. Compounds 4- 16 were prepared by various chemical transformations. The diterpenes 6,10-( E,E)-thymifodioic acid ( 1), the butenolide 3, and the derivatives 4, 11, and 14 produced developmental deficiencies in assays using topical application on fifth instar larvae of T. molitor. Compound 3 is a new natural product.

Insect growth regulatory effects of some extracts and sterols from Myrtillocactus geometrizans (Cactaceae) against Spodoptera frugiperda and Tenebrio molitor.

A methanol extract from the roots and aerial parts of Myrtillocactus geometrizans (Cactaceae) yielded peniocerol 1, macdougallin 2, and chichipegenin 3. The natural products 1, 2 their mixtures, MeOH and CH(2)Cl(2) extracts showed insecticidal and insect growth regulatory activity against fall armyworm [Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae)], an important insect pest of corn, and [Tenebrio molitor (Coleoptera)], a pest of stored grains in Mexico. The most active compounds were 1, 2, and a mixture (M(2)) of 1 and 2 (6:4). All these extracts, compounds and the mixture had insect growth regulating (IGR) activity between 5.0 and 50.0 ppm and insecticidal effects between 50 and 300 ppm in diets. The extracts were insecticidal to larvae, with lethal doses between 100 and 200 ppm. These compounds appear to have selective effects on the pre-emergence metabolism of Coleoptera, because in all treatments of the larvae of T. molitor, pupation were shortened and this process show precociousness in relation to controls. In contrast to S. frugiperda larvae, onset of pupation was noticeably delayed. Emergence in both cases was drastically diminished. In both pupae and in the few adults that were able to emerge, many deformations were observed. The results of these assays indicated that the compounds were more active than other known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. Peniocerol, macdougallin and chichipegenin, as well as mixtures of these substances, may be useful as natural insecticidal agents.

Incubation temperature as a modifying factor on survival of Tenebrio molitor reared in selenium-containing media.

Newly emerged Tenebrio molitor were reared at 4, 25, and 37 degrees C in nutrient media supplemented with sodium selenate (0.0125, 0.0200, 0.0500, and 0.1000%). Ten insects comprised each group. Controls were maintained in unsupplemented medium at the same temperatures as the experimental groups. Survival percentages were determined. Survival curves were estimated at given times postincubation. Controls at 4 and 37 degrees C showed an increased lethality compared to those insects at 25 degrees C. Data indicate that 4 degrees C had a protective effect on survival for insects reared in media containing the three highest concentrations of selenium. For insects at 37 degrees C, killing was striking and equivalent for all groups, irrespective of media supplementation.

Naturally occurring insect growth regulators. II. Screening of insect and plant extracts as insect juvenile hormone mimics.

Ethereal extracts prepared from the larvae, pupae, or eggs of 10 species of insects and from various parts of 343 species of higher plants were screened for juvenilizing effects against Tenebrio molitor and Oncopeltus fasciatus. Activity in both species was shown by an extract of the larvae of the stable fly, Stomoxys calcitrans, whereas an extract of the pupae was active in O. fasiatus only. Extracts of two plant species (Echinacea angustifolia roots and Chamaecyparis lawsoniana seeds) showed high juvenilizing activity in T. MOLITOR, AND EXtracts of five plant species (Clethra alnifolia stems, leaves, and fruits, Sassafras albidum roots and root bark, Eucalyptus camaldulensis stems and bark, Pinus rigida twigs and leaves, and Iris douglasiana roots, stems, and fruits) were highly active in O. fasciatus an extract of Tsuga canadensis leaves showed lower activity in this insect. Extracts of 16 species of plants showed high insecticidal activity (mortality) in O. fasciatus but lacked juvenilizing properties in both species of test insects.