Mealworm Beetle Tenebrio Molitor Research Articles

Infection risk by oral contamination does not induce immune priming in the mealworm beetle (Tenebrio molitor) but triggers behavioral and physiological responses.

In invertebrates, immune priming is the ability of individuals to enhance their immune response based on prior immunological experiences. This adaptive-like immunity likely evolved due to the risk of repeated infections by parasites in the host's natural habitat. The expression of immune priming varies across host and pathogen species, as well as infection routes (oral or wounds), reflecting finely tuned evolutionary adjustments. Evidence from the mealworm beetle (Tenebrio molitor) suggests that Gram-positive bacterial pathogens play a significant role in immune priming after systemic infection. Despite the likelihood of oral infections by natural bacterial pathogens in T. molitor, it remains debated whether ingestion of contaminated food leads to systemic infection, and whether oral immune priming is possible is currently unknown. We first attempted to induce immune priming in both T. molitor larvae and adults by exposing them to food contaminated with living or dead Gram-positive and Gram-negative bacterial pathogens. We found that oral ingestion of living bacteria did not kill them, but septic wounds caused rapid mortality. Intriguingly, the consumption of either dead or living bacteria did not protect against reinfection, contrasting with injury-induced priming. We further examined the effects of infecting food with various living bacterial pathogens on variables such as food consumption, mass gain, and feces production in larvae. We found that larvae exposed to Gram-positive bacteria in their food ingested less food, gained less mass and/or produced more feces than larvae exposed to contaminated food with Gram-negative bacteria or control food. This suggests that oral contamination with Gram-positive bacteria induced both behavioral responses and peristalsis defense mechanisms, even though no immune priming was observed here. Considering that the oral route of infection neither caused the death of the insects nor induced priming, we propose that immune priming in T. molitor may have primarily evolved as a response to the infection risk associated with wounds rather than oral ingestion.

Side-effects of laser weeding: quantifying off-target risks to earthworms (Enchytraeids) and insects (Tenebrio molitor and Adalia bipunctata)

With challenges posed by chemical and mechanical weed control, there are now several research and commercial projects underway to develop autonomous vehicles equipped with lasers to control weeds in field crops. Recognition systems based on artificial intelligence have been developed to locate and identify small weed seedlings, and mirrors can direct a laser beam towards the target to kill the weed with heat. Unlike chemical and mechanical weed control, laser weeding only exposes a small area of the field for the treatment. Laser weeding leaves no chemicals in the field after the treatment or does not move the soil which may harm crop roots and non-target organisms. Yet, it is well-known that laser beams can harm living organisms; the effect on the environment and fauna should be studied before laser weeding becomes a common practice. This project aimed to study the effect of laser on some living non-target organisms. We investigated the effect of laser treatment on the mortality of two species of earthworms (Enchytraeus albidus and Enchytraeus crypticus), larvae, pupas, and beetles of yellow mealworm beetles (Tenebrio molitor) and the two-spotted lady beetle (Adalia bipunctata) for increasing dosages of laser energy. In all earthworms experiments except one, the mortality rates of the worms living in the uppermost soil layer of clay, sandy, and organic soil exposed to laser heating were not significantly different from the controls even with laser dosages up to 23.8 J mm-2. Laser doses sufficient to kill plants were lethal to the insects, and lower doses that did not kill plants, killed or harmed the insects across all life stages tested. The larger beetles survived higher doses than smaller. Laser weeding is a relatively new technology and not yet widely practiced or commercialized. Therefore, we do not discuss and compare the costs of the different weeding methods at this early stage of the development of the technology.

Herbicide exposure alters the effect of the enthomopathogen Beauveria bassiana on immune gene expression in mealworm beetles

Concerns have grown worldwide about the potentially far-reaching effects of herbicides on functional biodiversity in agroecosystems. Repeated applications over time can lead to accumulation of residues in soil, water, and food and may have negative impacts on non-target organisms. However, the effects of herbicide residues on interspecific relationships, such as host-pathogen interactions, are poorly studied. In this study, we evaluated the effects of two different concentrations of a commercial pendimethalin-based formulation (PND), the residual contamination (S, 13 ppm) in treated soils and the maximum residue level allowed by the European Commission in cereals (EU, 0.05 ppm). We tested the effect of PND on the biological interaction between the mealworm beetle Tenebrio molitor Linnaeus, 1758 and the entomopathogenic fungus Beauveria bassiana Vuillemin, 1912 (Bb, strain KVL 03–144) at two concentrations (LC50 5 × 105 conidia mL−1 and LC100 1 × 107 conidia mL−1). We checked the survival of beetles exposed to PND or/and inoculated with B. bassiana, the expression of four antimicrobial peptides (AMPs), and finally how PND affects in vitro germination of fungus. The exposure to PND had no significant effects on the survival of either control or Bb-exposed beetles. In the mealworm beetle, upregulation of gene expression of the inducible AMPs Tenecin 1, 2, and 4 was observed in PND-treated beetles after inoculation with Bb, while the levels of the non-inducible AMP Tenecin 3 were similar between treatments. In conclusion, our findings demonstrate that admitted residual doses of currently used herbicides modify an important component of the inducible immune response of an insect. This did not translate into an effect on the survival to B. bassiana in our system. However, residual doses of the herbicide at 13 ppm may temporarily affect fungal germination. These results raise questions about the compatibility of bioinsecticides with synthetic pesticides and the effects of herbicide residues on host-pathogen interactions.

Evaluation of alternative substrates for rearing the yellow mealworm Tenebrio molitor (L)

Larvae of the mealworm beetle Tenebrio molitor is commonly used as feed for pets and food for humans due to its rich nutrient contents. The beetle breeds prolifically and are reared in close proximity with their diet; known as substrates. However, the most commonly used substrate, wheat bran, is expensive making the rearing out of reach to many, especially in developing countries. This study evaluated the suitability of six other potential substrates; wheat flour, maize flour, Lucerne pellets, dog food, soya four and oats in comparison to wheat bran in order to explore a cost-effective alternative rearing substrate for mealworms. To achieve this, the mealworms were reared in a climate-controlled chamber and the total numbers, weight and the fecundity were determined for each substrate. Wheat bran and wheat flour produced the most mealworms, had the highest fecundity with wheat bran, Lucerne pellets an oat producing heavier worms than all other substrates. Maize flour, wheat flour and Lucerne, were found to be the most cost-effective alternative substrates for rearing mealworms with the cost per gram at 0.07, 0.05 and 0.04 US$ respectively. Both wheat flour and maize flour are easily accessible, have a long self-life thus ideal for both large-and small-scale production.Graphical

The females' response to male attractiveness: Mate choice, larvae production and differential brain protein expression

AbstractSexual Selection has been studied predominantly from a functional perspective with relatively limited investigation of the mechanistic basis of female choice. In this study, we evaluated female choice and larvae production in response to attractive or less attractive males and concurrently examined the protein profile in the brains of females, using the mealworm beetle Tenebrio molitor as a model system. Females were found to prefer males with high levels of pheromones, which requires the secretion of juvenile hormone (JH). Male attractiveness was enhanced using the JH analog methoprene, resulting in less female rejection and higher mating frequency and offspring production. Importantly, reproductive success was not due to differences in testis size or sperm production, or viability. Out of the 1833 proteins detected in the brain, only 32 were differentially expressed in females mating with attractive versus less attractive males. These proteins involved memory, metabolism, olfactory detection, reproduction, and movement. Although some brain proteins have been linked to mate choice in vertebrates, most of the differentially expressed molecules found in this study have not been previously reported concerning mate choice.

Insect meal in poultry feed: a potential protein source

The present study aims to conduct a review on the potential use of insect meal as an alternative protein source in poultry feed, particularly to serve the rearing of free-range chickens. Insects are already part of the diet of birds in their natural habitat, and the availability of low-cost alternative foods with low environmental impact is essential for the development of the activity. The review comprehended studies that used meals consisting of silkworm (Bombyx mori) chrysalis, earthworm (Eisenia foetida), housefly (Musca domestica), black soldier fly (Hermetia illucens) and mealworm beetle (Tenebrio molitor) to replace plant-based protein sources. In general, insect meals have a high content of crude protein and ether extract, as well as an essential amino acid profile suitable for poultry feeding. The addition of insect meal in poultry feed normally shows good results as to growth performance and egg production, without causing a negative effect on carcass characteristics, meat sensory quality and egg quality, presenting itself as an alternative protein source with good prospects for replacing plant-based sources in poultry feed.

Tachykinin-related peptides modulate immune-gene expression in the mealworm beetle Tenebrio molitor L.

Tachykinins (TKs) are a group of conserved neuropeptides. In insects, tachykinin-related peptides (TRPs) are important modulators of several functions such as nociception and lipid metabolism. Recently, it has become clear that TRPs also play a role in regulating the insect immune system. Here, we report a transcriptomic analysis of changes in the expression levels of immune-related genes in the storage pest Tenebrio molitor after treatment with Tenmo-TRP-7. We tested two concentrations (10–8 and 10–6 M) at two time points, 6 and 24 h post-injection. We found significant changes in the transcript levels of a wide spectrum of immune-related genes. Some changes were observed 6 h after the injection of Tenmo-TRP-7, especially in relation to its putative anti-apoptotic action. Interestingly, 24 h after the injection of 10–8 M Tenmo-TRP-7, most changes were related to the regulation of the cellular response. Applying 10–6 M Tenmo-TRP-7 resulted in the downregulation of genes associated with humoral responses. Injecting Tenmo-TRP-7 did not affect beetle survival but led to a reduction in haemolymph lysozyme-like antibacterial activity, consistent with the transcriptomic data. The results confirmed the immunomodulatory role of TRP and shed new light on the functional homology between TRPs and TKs.

Behavioural and physiological regulation of protein and carbohydrates in mealworm larvae: A geometric analysis

Protein-carbohydrate regulation in the larvae of the mealworm beetle (Tenebrio molitor L.) was analyzed using the Geometric Framework for nutrition. In this study, the ingestive and post-ingestive responses were measured from T. molitor larvae that were subjected to choice and no-choice experiments. In the choice experiment, T. moitor larvae were simultaneously presented with one of two protein-biased foods (p35:c7 or p28:c5.6) and one of two carbohydrate-biased foods (p7:c35 or p5.6:c28). T. molitor larvae selected protein and carbohydrate in a ratio close to 1:1 over the first 15 days since the start of the experiment (days 0–15), but exhibited preference for carbohydrate-biased food over the next 15 days. The average protein:carbohydrate ratio selected over days 0–30 was 1:1.24. In the no-choice experiment, T. molitor larvae were restricted to one of seven foods with different protein and carbohydrate content (p0:c42, p7:c35, p14:c28, p21:c21, p28:c14, p35:c7, or p42:c0). On the p0:c42 food, consumption was greatly suppressed and no larvae completed their development. Across a range of these foods except p0:c42, T. molitor larvae consistently over-ate the surplus nutrient in the foods and showed a pattern of nutrient balancing similar to that previously described for other nutritional generalists. Despite having consumed substantially different amounts and ratios of macronutrients as larvae, T. molitor pupae in the no-choice food treatments had similar body nutrient composition, suggesting the presence of strong homeostatic regulation for body nutrient growth. Larval survivorship was significantly lower on two extremely imbalanced foods (p7:c35 and p42:0) than on more balanced foods. T. molitor larvae reared on p7:c35 suffered reduced biomass growth and delayed development compared with those on foods with higher protein content.

Autophagy in Tenebrio molitor Immunity: Conserved Antimicrobial Functions in Insect Defenses.

The yellow mealworm beetle (Tenebrio molitor) has been exploited as an experimental model to unravel the intricacies of cellular and humoral immunity against pathogenic infections. Studies on this insect model have provided valuable insights into the phenotypic plasticity of immune defenses against parasites and pathogens. It has thus been possible to characterize the hemocoelic defenses of T. molitor that rely on the recognition of non-self-components of pathogens by pattern recognition receptors (PRRs). The subsequent signaling cascade activating pathways such as the NF-κB controlled by Toll and IMD pathways lead to the synthesis of antimicrobial peptides (AMPs), onset of hemocyte-driven phagocytosis, and activation of the prophenoloxidase cascade regulating the process of melanization. Nevertheless, the activation of autophagy-mediated defenses of T. molitor against the facultative intracellular gram-positive bacterium Listeria monocytogenes provides clear evidence of the existence of a cross-talk between autophagy and the IMD pathway. Moreover, the identification of several autophagy-related genes (Atgs) in T. molitor transcriptome and expressed sequence tag (EST) databases has contributed to the understanding of the autophagy-signaling cascade triggered by L. monocytogenes challenge. Providing further evidence of the cross-talk hypothesis, TmRelish has been shown to be required not only for regulating the synthesis of AMPs through the PGRP-LE/IMD pathway activation but also for the expression of Atgs in T. molitor larvae following L. monocytogenes challenge. Notably, L. monocytogenes can stimulate the T. molitor innate immune system by producing molecules recognized by the multifunctional PRR (TmPGRP-LE), which stimulates intracellular activation of the IMD pathway and autophagy. Considering the conservation of autophagy components involved in combating intracellular pathogens, it will be interesting to extrapolate a dynamic cross-talk model of immune activation. This review summarizes the most significant findings on the regulation of autophagy in T. molitor during L. monocytogenes infection and on the role of the innate immunity machinery, including the NF-κB pathway, in the control of pathogenic load.

Morphological Characterisation of Haemocytes in the Mealworm Beetle Tenebrio molitor (Coleoptera, Tenebrionidae).

Simple SummaryTenebrio molitor is a pest of stored grain, causing considerable damage. However, its easy maintenance makes this species also interesting as a food source and as a model for physiological, immunological, ecological and evolutionary studies. We used light and transmission electron microscopy to study the morphology of circulating haemocytes. Prohaemocytes, plasmatocytes, granular cells and oenocytoids were described based on their morphological features and staining affinity. Results are a baseline for further study aimed to clarify the structure and function of haemocytes in insects.The immunocompetence of the mealworm beetle Tenebrio molitor has been well investigated at molecular and physiological levels, but information on morphological and functional characteristics of its immune cells (haemocytes) is still scarce and fragmentary. This study provides an updated overview of the morphology of circulating immune cells from mealworm beetle adults, using light and transmission electron microscopy. Based on their affinities for May–Grünwald Giemsa stain, haemocytes were defined as either eosinophilic, basophilic or neutral. Ultrastructural descriptions allowed to detect four main cell types in the haemolymph: prohaemocytes, plasmatocytes, granular cells and oenocytoids. The morphological plasticity of haemocytes and the evidence of mitotic circulating cells, intermediate cell stages, as well as autophagic activities suggest haemocyte proliferation, turnover and transdifferentiation as constantly active processes in the haemolymph. Cytochemical tests revealed differences in the distribution of carbohydrates among cell types underling the great plasticity of the immune response and the direct involvement of circulating immune cells in the resource allocation. In addition, our results provide a detailed morphological description of vesicle trafficking, macro- and microautophagy, apoptotic and necrotic processes, confirming the suitability of T. molitor haemocytes as a model for studying evolutionarily conserved cellular mechanisms.

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.

Deciphering the molecular mechanisms of mother-to-egg immune protection in the mealworm beetle Tenebrio molitor.

In a number of species, individuals exposed to pathogens can mount an immune response and transmit this immunological experience to their offspring, thereby protecting them against persistent threats. Such vertical transfer of immunity, named trans-generational immune priming (TGIP), has been described in both vertebrates and invertebrates. Although increasingly studied during the last decade, the mechanisms underlying TGIP in invertebrates are still elusive, especially those protecting the earliest offspring life stage, i.e. the embryo developing in the egg. In the present study, we combined different proteomic and transcriptomic approaches to determine whether mothers transfer a "signal" (such as fragments of infecting bacteria), mRNA and/or protein/peptide effectors to protect their eggs against two natural bacterial pathogens, namely the Gram-positive Bacillus thuringiensis and the Gram-negative Serratia entomophila. By taking the mealworm beetle Tenebrio molitor as a biological model, our results suggest that eggs are mainly protected by an active direct transfer of a restricted number of immune proteins and of antimicrobial peptides. In contrast, the present data do not support the involvement of mRNA transfer while the transmission of a "signal", if it happens, is marginal and only occurs within 24h after maternal exposure to bacteria. This work exemplifies how combining global approaches helps to disentangle the different scenarios of a complex trait, providing a comprehensive characterization of TGIP mechanisms in T. molitor. It also paves the way for future alike studies focusing on TGIP in a wide range of invertebrates and vertebrates to identify additional candidates that could be specific to TGIP and to investigate whether the TGIP mechanisms found herein are specific or common to all 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.

In silico identification and expression analyses of Defensin genes in the mealworm beetle Tenebrio molitor

AbstractDefensins are a major family of antimicrobial peptides that serve as the innate immune defense of both vertebrates and invertebrates. Due to their antimicrobial, chemotactic, and regulatory activities, Defensins have been exploited for their therapeutic potential. Insect Defensins are cysteine‐rich and contain an N‐terminal loop, α‐helix, and antiparallel β‐sheet, forming a “cysteine‐stabilized alpha beta (CSαβ)” or “loop–helix‐sheet” structure. In this study, we identified the full‐length open reading frame (ORF) sequences of Defensin (TmDef) and Defensin‐like (TmDef‐like) genes from the mealworm beetle Tenebrio molitor using in silico methods. TmDef and TmDef‐like genes encode the peptides of 72 and 71 amino acid residues, respectively. TmDefensin is comprised of a Defensin domain and the TmDefensin‐like is comprised of a signal peptide of 21 amino acid residues. Phylogenetic analysis revealed close similarities of TmDefensin with the Defensin of Acalolepta luxuriosa of the longhorn beetle family. The expression of TmDef mRNA was found to be greater than that of TmDef‐like mRNA and was mostly expressed in the pupal and adult stages. Tissue distribution showed high expression of TmDef‐like mRNA in larval hemocytes, gut, integument, and fat body, while in adults, the expression was high in gut and hemocytes. Following bacterial and fungal stimulation in vivo, TmDef was upregulated at 24 h post‐infection in whole body, fat body, and hemocytes of the larvae. Even TmDef‐like mRNA was upregulated in the gut and hemocytes at 12 and 9 h post‐infection respectively. These results suggest that TmDef and TmDef‐like genes play important roles in protecting T. molitor from microbial contact.

Individual variation in cardiotoxicity of parotoid secretion of the common toad, Bufo bufo, depends on body size – first results

Anurans secrete a wide diversity of toxins from skin glands to defend themselves against predators and pathogens. Bufonids produce potent poison in parotoid macroglands located in the postorbital region. Parotoid secretion is a rich source of bioactive compounds with cardiotoxic, cytotoxic and hemolytic activity. Poison content and toxicity may vary between species, populations, and among conspecifics inhabiting the same area. In the present paper, we pre-analyzed the individual variation in cardiotoxicity of parotoid extract of common toads (Bufo bufo Linnaeus, 1758) and impact of body mass (BM), snout to vent length (SVL), and body condition (BC) of toad on the poison toxicity. We hypothesized that large toads produce poison with higher cardiotoxicity than smaller ones. Parotoid extract was fractionated by reverse phase chromatography, and then in vitro physiological bioassays were carried out on the semi-isolated hearts of the mealworm beetle (Tenebrio molitor Linnaeus, 1758) to determine cardiotoxicity of the whole poison and separated fractions. Generalized linear mixed models were used to determine effects of BM, SVL, and BC on the poison toxicity. We recorded significant changes in the insect heart contractility after treatment with the whole poison and separated fractions. We found an individual variation in cardiotoxicity of the parotoid extract which was explained by the body size of toad. Poison of smaller toads displayed a negative, whereas poison of larger toads positive, chronotropic effect on the heart contractility. Thus, we conclude that the effectiveness of parotoid secretion in repelling predators may vary depending on the toad individual size.

Synthesis of the Mealworm Tenebrio molitor L. Pheromone

Diastereoselective allylation of (R)-2,3-O-cyclohexylideneglyceraldehyde with 3-(bromomethyl)but-3-enoate, followed by reduction with zinc, gave homoallylic alcohol which was subjected to lactonization. Opening of the lactone ring in the latter afforded methyl (3S)-5,5-dimethoxy-3-methylpentanoate with a high yield and enantioselectivity. This compound is a valuable intermediate product in the synthesis of pheromone of the mealworm beetle Tenebrio molitor L., a pest of stored cereals.

Comparative Mortality and Adaptation of a Smurf Assay in two Species of Tenebrionid Beetles Exposed to Bacillus thuringiensis.

Bacillus thuringiensis is a spore-forming bacterium which infects insect larvae naturally via the oral route. Its virulence factors interact with the epithelium of the digestive tract of insect larvae, disrupting its function and eventually leading to the death of susceptible hosts. The most cited B. thuringiensis killing mechanism is the extensive damage caused to the insect midgut, leading to its leakage. The mortality caused by B. thuringiensis has been shown to vary between serovars and isolates, as well as between host life stages. Moreover, whether susceptibility to B. thuringiensis-induced gut leakage is generalized to all host species and whether there is individual variation within species is unclear. In this study, we adapted a non-invasive “Smurf” assay from Drosophila melanogaster to two species of tenebrionid beetles: The mealworm beetle Tenebrio molitor and the red flour beetle Tribolium castaneum, during exposure to B. thuringiensis. We highlight a differential mortality between two age/size classes of T. molitor larvae, as well as different killing dynamics between B. thuringiensis var. tenebrionis and var. tolworthi in T. castaneum. The Smurf assay did not reveal a high occurrence of extensive gut disintegration in both host species upon ingestion during B. thuringiensis exposure.

The effect of mating on the sex pheromone system of the yellow mealworm beetle, Tenebrio molitor L. (Coleoptera: Tenebrionidae)

The mating behavior of the yellow mealworm beetle (Tenebrio molitor L.) is mediated by several pheromones, including a female-produced male attractant, (R)-(+)-4-methylnonanol (4-MNol); a male-produced female attractant, (Z)-3-dodecenyl acetate; a male-produced anti-aphrodisiac; and at least one copulation pheromone. The objective of this study was to investigate the effect of mating on this complicated pheromone system. Glass rod bioassays were conducted to determine the response of virgin adult male beetles to the surface extracts of adult female beetles, prepared from virgin females, females allowed to mate once (extracted 0, 0.5, 1 or 2 h after mating), and females allowed to mate over an extended period of time (0, 1, or 21 h). Mating caused an immediate but temporary inhibition of the ability of the female extracts (FEs) to attract the males. This wore off relatively quickly, since FEs prepared 0.5–1 h after mating were as attractive as the virgin FE. Two hours after mating a second inhibition of attraction was observed, which was likely due to a drop in the titer of 4-MNol. Gas chromatographic analyses confirmed that the level of 4-MNol dropped after mating, from ∼40 ng per virgin female to below our levels of detection in mated females. A single mating event, or the 1 h extended mating, did not significantly affect the ability of the FEs to elicit copulation behavior (CB) from the males. However, FEs prepared after an extended mating (21 h) were significantly less able to attract males and to elicit CB. This is the first in depth study of the effect of a single and extended mating on sex pheromone production by a coleopteran species. In the long term, a greater understanding of the regulation of pheromone production could be useful for the manipulation of pest populations.

Safety evaluation of the entomopathogenic bacterium Brevibacillus laterosporus for the green lacewing Chrysoperla agilis (Neuroptera: Chrysopidae)

The safety of the entomopathogenic bacterium Brevibacillus laterosporus for the natural insect predator Chrysoperla agilis was evaluated in this study. For this purpose, laboratory bioassays were conducted exposing different larval instars and adults of the chrysopid to bacterial spore preparations, in order to evaluate the possible effects on survival, longevity, immature development, and adult reproductive performance. The sub-lethal effects were investigated by feeding the bacterium directly to adults and larvae of C. agilis or to mealworm beetles (Tenebrio molitor) used as hosts for chrysopids (tritrophic interaction). Direct feeding of B. laterosporus spores to different lacewing larvae instars and to adults did not cause mean mortality levels significantly different from untreated control, and slight though not significant effects of treatments were generally observed on insect longevity, development, fecundity and egg hatching. In the case of lacewing larvae feeding on treated mealworm beetles, adult emergence percentage was reduced approximately 12%, in comparison with untreated control. Based on these results, the use of B. laterosporus for pest management in the agroecosystem, appears to be compatible with chrysopids.

TmDorX2 positively regulates antimicrobial peptides in Tenebrio molitor gut, fat body, and hemocytes in response to bacterial and fungal infection

Dorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor (TmDorX2). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. TmDorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli, the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2-knockdown larvae exposed to E. coli, S. aureus, or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of dsTmDorX2-injected larvae upon E. coli challenge. After C. albicans and S. aureus challenge of dsTmDorX2-injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2-silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.