Solanum nigrum Extract and Solasonine Affected Hemolymph Metabolites and Ultrastructure of the Fat Body and the Midgut in Galleria mellonella.

Glycoalkaloids, secondary metabolites abundant in plants belonging to the Solanaceae family, may affect the physiology of insect pests. This paper presents original results dealing with the influence of a crude extract obtained from Solanum nigrum unripe berries and its main constituent, solasonine, on the physiology of Galleria mellonella (Lepidoptera) that can be used as an alternative bioinsecticide. G. mellonella IV instar larvae were treated with S. nigrum extract and solasonine at different concentrations. The effects of extract and solasonine were evaluated analyzing changes in carbohydrate and amino acid composition in hemolymph by RP-HPLC and in the ultrastructure of the fat body cells by TEM. Both extract and solasonine changed the level of hemolymph metabolites and the ultrastructure of the fat body and the midgut cells. In particular, the extract increased the erythritol level in the hemolymph compared to control, enlarged the intracellular space in fat body cells, and decreased cytoplasm and lipid droplets electron density. The solasonine, tested with three concentrations, caused the decrease of cytoplasm electron density in both fat body and midgut cells. Obtained results highlighted the disturbance of the midgut and the fat body due to glycoalkaloids and the potential role of hemolymph ingredients in its detoxification. These findings suggest a possible application of glycoalkaloids as a natural insecticide in the pest control of G. mellonella larvae.

Fungal α-1,3-Glucan as a New Pathogen-Associated Molecular Pattern in the Insect Model Host Galleria mellonella.

Recognition of pathogen-associated molecular patterns (PAMPs) by appropriate pattern recognition receptors (PRRs) is a key step in activating the host immune response. The role of a fungal PAMP is attributed to ß-1,3-glucan. The role of α-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of α-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as ß-1,3-glucan recognition proteins, bound to A. niger α-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among α-1,3-glucan-interacting proteins. In response to α-1,3-glucan, a 4.5-fold and 3-fold increase in the gene expression of antifungal peptides galiomicin and gallerimycin was demonstrated, respectively. The significant increase in the level of five defense peptides, including galiomicin, corresponded well with the highest antifungal activity in hemolymph. Our results indicate that A. niger α-1,3-glucan is recognized by the insect immune system, and immune response is triggered by this cell wall component. Thus, the role of a fungal PAMP for α-1,3-glucan can be postulated.

Alteration of unfolded protein responses and autophagy signaling represented the molecular basis underlying saccharin toxicity to Drosophila (Diptera: Drosophilidae).

The purpose of this study was to develop a new control method for Drosophila using saccharin sodium dihydrate (saccharin), an artificial sweetener that is safe for humans and the environment, and to elucidate its mode of action. In this study, we confirmed that saccharin can dose-dependently inhibit the development of or kill vinegar flies (VFs) and spotted wing Drosophila (SWDs). In addition, we found that low concentrations of saccharin induced a similar effect as starvation in Drosophila, whereas high concentrations of saccharin induced changes in the unfolded protein response (UPR) and autophagy signaling that were unlike starvation and inhibited development or killed the VF and the SWD by performing real-time quantitative polymerase chain reaction analyses. Spinosad is a widely used plant protection agent for SWD control. When saccharin was cotreated with 0.25-1.0 ppm spinosad, an additive insecticidal activity was observed only at high concentrations of saccharin. However, when saccharin was cotreated with 2.0 ppm spinosad, an additive insecticidal activity was observed at low concentrations of saccharin. Taken together, alteration of UPR and autophagy signaling represented the molecular basis underlying saccharin toxicity to Drosophila and concurrent spraying of an insecticide with saccharin could enhance the insecticidal activities.

Honey bee queen health is unaffected by contact exposure to pesticides commonly found in beeswax.

Honey bee queen health is crucial for colony health and productivity, and pesticides have been previously associated with queen loss and premature supersedure. Prior research has investigated the effects of indirect pesticide exposure on queens via workers, as well as direct effects on queens during development. However, as adults, queens are in constant contact with wax as they walk on comb and lay eggs; therefore, direct pesticide contact with adult queens is a relevant but seldom investigated exposure route. Here, we conducted laboratory and field experiments to investigate the impacts of topical pesticide exposure on adult queens. We tested six pesticides commonly found in wax: coumaphos, tau-fluvalinate, atrazine, 2,4-DMPF, chlorpyriphos, chlorothalonil, and a cocktail of all six, each administered at 1, 4, 8, 16, and 32 times the concentrations typically found in wax. We found no effect of any treatment on queen mass, sperm viability, or fat body protein expression. In a field trial testing queen topical exposure of a pesticide cocktail, we found no impact on egg-laying pattern, queen mass, emergence mass of daughter workers, and no proteins in the spermathecal fluid were differentially expressed. These experiments consistently show that pesticides commonly found in wax have no direct impact on queen performance, reproduction, or quality metrics at the doses tested. We suggest that previously reported associations between high levels of pesticide residues in wax and queen failure are most likely driven by indirect effects of worker exposure (either through wax or other hive products) on queen care or queen perception.

Hormone-dependent activation and repression of microRNAs by the ecdysone receptor in the dengue vector mosquito Aedes aegypti.

Female mosquitoes transmit numerous devastating human diseases because they require vertebrate blood meal for egg development. MicroRNAs (miRNAs) play critical roles across multiple reproductive processes in female Aedes aegypti mosquitoes. However, how miRNAs are controlled to coordinate their activity with the demands of mosquito reproduction remains largely unknown. We report that the ecdysone receptor (EcR)-mediated 20-hydroxyecdysone (20E) signaling regulates miRNA expression in female mosquitoes. EcR RNA-interference silencing linked to small RNA-sequencing analysis reveals that EcR not only activates but also represses miRNA expression in the female mosquito fat body, a functional analog of the vertebrate liver. EcR directly represses the expression of clustered miR-275 and miR-305 before blood feeding when the 20E titer is low, whereas it activates their expression in response to the increased 20E titer after a blood meal. Furthermore, we find that SMRTER, an insect analog of the vertebrate nuclear receptor corepressors SMRT and N-CoR, interacts with EcR in a 20E-sensitive manner and is required for EcR-mediated repression of miRNA expression in Ae. aegypti mosquitoes. In addition, we demonstrate that miR-275 and miR-305 directly target glutamate semialdehyde dehydrogenase and AAEL009899, respectively, to facilitate egg development. This study reveals a mechanism for how miRNAs are controlled by the 20E signaling pathway to coordinate their activity with the demands of mosquito reproduction.

Lampaya Medicinalis Phil. decreases lipid-induced triglyceride accumulation and proinflammatory markers in human hepatocytes and fat body of Drosophila melanogaster.

BACKGROUND: Excess hepatic triglyceride (TG) accumulation (steatosis) commonly observed in obesity, may lead to non-alcoholic fatty liver disease (NAFLD). Altered regulation of intracellular lipid droplets (LD) and TG metabolism, as well as activation of JNK-mediated proinflammatory pathways may trigger liver steatosis-related disorders. Drosophila melanogaster is an animal model used for studying obesity and its associated disorders. In Drosophila, lipids and glycogen are stored in the fat body (FB), which resembles mammalian adipose tissue and liver. Dietary oversupply leads to obesity-related disorders, which are characterized by FB dysfunction. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in folk medicine of Chile to counteract inflammatory diseases. Hydroethanolic extract of lampaya (HEL) contains considerable amounts of flavonoids that may explain its anti-inflammatory effect. METHODS: We studied whether HEL affects palmitic acid (PA, C16:0) and oleic acid (OA; C18:1)-induced TG accumulation and proinflammatory marker content in HepG2 hepatocytes as well as impaired lipid storage and proinflammatory molecule expression in Drosophila melanogaster fed a high-fat diet (HFD). RESULTS: In HepG2 hepatocytes, exposure to OA/PA elevated TG content, FABP4, ATGL and DGAT2 expression, and the JNK proinflammatory pathway, as well as TNF-α and IL-6 production, while diminished FAS expression. These effects were prevented by HEL co-treatment. In Drosophila larvae fed a HFD, HEL prevented TG accumulation and downregulated proinflammatory JNK pathway activation. CONCLUSION: HEL effect counteracting OA/PA- and HFD-induced lipid accumulation and proinflammatory marker expression in HepG2 hepatocytes and Drosophila larvae may represent a preventive approach against hepatic steatosis and inflammation, associated to obesity and NAFLD.

Apoptotic and autophagic characteristics of perivisceral fat body remodeling of the greater wax moth Galleria mellonella and effects of juvenile hormone analog, fenoxycarb, on these processes.

In holometabolous insects, many tissues and organs such as the fat body and midgut undergo a remodeling process during metamorphosis. Larval fat body cells are eliminated by programmed cell death (PCD), while tissue cells that adapt to adult life are formed by stem cells. In this study, we analyzed the features of the remodeling period of Galleria mellonella fat body in terms of PCD types, apoptotic and autophagic cell death characteristics. Besides, the effects of juvenile hormone (JH) on these processes were evaluated under the modified hormonal conditions via applications of JH analog, fenoxycarb. Several hallmarks of apoptotic and autophagic cell death were analyzed by morphological, biochemical, and molecular methods. The results of the present study have ascertained that the degeneration process of larval cells occurs via autophagic cell death accompanied by caspase-3 activity during the pupal period and it is regulated by 20-hydroxyecdysone (20HE) mediated by ecdysone receptor B1 (EcR-B1). Increased activity of the acid phosphatase and upregulation of ATG6 and ATG8 in parallel with the formation of autophagosomes in the fat body of Galleria during the pupal period strongly indicated that autophagy was the key player in the remodeling processes.

Knockdown of CYP301B1 and CYP6AX1v2 increases the susceptibility of the brown planthopper to beta-asarone, a potential plant-derived insecticide.

The cytochrome P450 monooxygenases of insects play crucial roles in the metabolic detoxification of insecticides. Our previous finding showed that two cytochrome P450 genes, both CYP301B1 and CYP6AX1v2, in the BPH underwent overexpression due to ß-asarone. In this study, we investigated the molecular characteristics, expression patterns and functions of these two cytochrome P450 genes. The results showed that CYP301B1 had the highest expression level in the eggs, while CYP6AX1v2 was expressed in macropterous female adults. Moreover, the expression level of CYP301B1 in the head was higher than that in the integument, fat body and gut. The expression level of CYP6AX1v2 in the fat body and gut was higher than that in head and integument. Importantly, silencing CYP301B1 and CYP6AX1v2 separately could increase the sensitivity, resulting in significant higher mortality of BPH following treatment with ß-asarone. Our findings indicated that CYP301B1 and CYP6AX1v2 could contribute to the resistance of BPH to ß-asarone, and these two genes may be involved in the detoxification metabolism of ß-asarone in BPH.

Transmission Electron Microscopy as a Tool to Study the Toxicological Effects of Thiamethoxam in Workers of Atta sexdens (Myrmicinae, Attini).

Thiamethoxam is a neonicotinoid that has been used to control insect pests. The literature reports a few behavioral studies evaluating the toxic effect of thiamethoxam in ants; however, there are scarce studies at the cellular level. The present research evaluated the effects of thiamethoxam in labial (LG) and mandibular glands (MG), fat bodies (FB), and Malpighian tubules (MT) of workers of Atta sexdens, using transmission electron microscopy. The duct and secretory cells of LG were profoundly affected, then the production of saliva can be compromised, as well as its quality and subsequent use. In MG, reservoir and canaliculi cells presented slight alterations; however, MG secretory cells presented vacuoles containing lamellar structures, increased lipid production, and a large amount of mitochondria, which may lead to organ's malfunctioning. The FB cell alterations do not seem enough to cause significant changes that lead to cell death. Prominent changes in MT, such as loss of the electron-dense concentric ring, increased smooth endoplasmic reticulum, loss of basal infolds, vacuoles containing mineralized granules, and lamellar structures associated with mitochondria, suggest that their excretory function is compromised. In conclusion, thiamethoxam acts not only in the nervous system but also contributes to systemic toxicity on the target organism.

Tyramine and its receptor TYR1 linked behavior QTL to reproductive physiology in honey bee workers (Apis mellifera).

Honey bees (Apis mellifera) provide an excellent model for studying how complex social behavior evolves and is regulated. Social behavioral traits such as the division of labor have been mapped to specific genomic regions in quantitative trait locus (QTL) studies. However, relating genomic mapping to gene function and regulatory mechanism remains a big challenge for geneticists. In honey bee workers, division of labor is known to be regulated by reproductive physiology, but the genetic basis of this regulation remains unknown. In this case, QTL studies have identified tyramine receptor 1 (TYR1) as a candidate gene in region pln2, which is associated with multiple worker social traits and reproductive anatomy. Tyramine (TA), a neurotransmitter, regulates physiology and behavior in diverse insect species including honey bees. Here, we examine directly the effects of TYR1 and TA on worker reproductive physiology, including ovariole number, ovary function and the production of vitellogenin (VG, an egg yolk precursor). First, we used a pharmacology approach to demonstrate that TA affects ovariole number during worker larval development and increases ovary maturation during the adult stage. Second, we used a gene knockdown approach to show that TYR1 regulates vg transcription in adult workers. Finally, we estimated correlations in gene expression and propose that TYR1 may regulate vg transcription by coordinating hormonal and nutritional signals. Taken together, our results suggest TYR1 and TA play important roles in regulating worker reproductive physiology, which in turn regulates social behavior. Our study exemplifies a successful forward-genetic strategy going from QTL mapping to gene function.

Effects of short- and long-term exposure to cadmium on salivary glands and fat body of soil centipede Lithobius forficatus (Myriapoda, Chilopoda): Histology and ultrastructure.

Cadmium (Cd) is the most widely studied heavy metal in terms of food-chain accumulation and contamination because it can strongly affect all environments (e.g., soil, water, air). It can accumulate in different tissues and organs and can affect the organism at different levels of organization: from organs, tissues and cells though cell organelles and structures to activation of mechanisms of survival and cell death. In soil-dwelling organisms heavy metals gather in all tissues with accumulation properties: midgut, salivary glands, fat body. The aim of this study was to describe the effects of cadmium on the soil species Lithobius forficatus, mainly on two organs responsible for gathering different substances, the fat body and salivary glands, at the ultrastructural level. Changes caused by cadmium short- and long-term intoxication, connected with cell death (autophagy, apoptosis, necrosis), and the crosstalk between them, were analyzed. Adult specimens of L. forficatus were collected in a natural environment and divided into three experimental groups: C (the control group), Cd1 (cultured in soil with 80 mg/kg of CdCl2 for 12 days) and Cd2 (cultured in soil with 80 mg/kg of CdCl2 for 45 days). Transmission electron microscopy revealed ultrastructural alterations in both of the organs analyzed (reduction in the amount of reserve material, the appearance of vacuoles, etc.). Qualitative analysis using TUNEL assay revealed distinct crosstalk between autophagy and necrosis in the fat body adipocytes, while crosstalk between autophagy, apoptosis and necrosis in the salivary glands was detected in salivary glands of the centipedes examined here. We conclude that different organs in the body can react differently to the same stressor, as well as to the same concentration and time of exposure. Different mechanisms at the ultrastructural level activate different types of cell death and with different dynamics.

Comet assay protocol for Bombus atratus fat body and pericardial cells (Hymenoptera, bombini) at a safe concentration of mercury.

The decline of the Bombus population is closely related to the presence of environmental pollutants. Among these pollutants, trace metals represent a major concern, which includes mercury, a known genotoxic substance. The induction of genotoxicity may be demonstrated by the comet assay (a.k.a. single-cell gel electrophoresis), a simple and sensitive method for DNA damage estimating. The current work provided, for the first time, a protocol of comet assay for Bombus atratus using mercury as a standard chemical at safe concentrations according to the Environment National Council of Brazil, and the World Health Organization. Bees were collected and divided into three groups (n = 11 each), in which the exposed groups received a 0.2 ppb or a 1 ppb of mercury solution, and the control group received water. The bioassay was performed for 48 h at controlled temperature and humidity conditions, according to the OECD guideline toxicological test method for B. terrestris. The samples were stained with different dyes to observe the efficacy of each one. Variations of parameters in methodology, such as concentration and time of exposure to lysis solution as well as the electrophoretic process, allowed the observation of comets at different levels. DAPI and acridine orange presented an unstable fluorescence, and silver nitrate dye was more effective. Therefore, the comet assay was shown to be an effective method to evaluate genotoxic effects in bees. The obtained results may be helpful for the establishment of a suitable protocol for future genotoxicity assessment in neotropical bees using different doses of xenobiotics.

CDKI-73 is a Novel Pharmacological Inhibitor of Rab11 Cargo Delivery and Innate Immune Secretion.

Innate immunity is critical for host defence against pathogen and environmental challenge and this involves the production and secretion of immune mediators, such as antimicrobial peptides and pro-inflammatory cytokines. However, when dysregulated, innate immunity can contribute to multifactorial diseases, including inflammatory rheumatic disorders, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and even septic shock. During an innate immune response, antimicrobial peptides and cytokines are trafficked via Rab11 multivesicular endosomes, and then sorted into Rab11 vesicles for traffic to the plasma membrane and secretion. In this study, a cyclin-dependent kinase inhibitor CDKI-73 was used to determine its effect on the innate immune response, based on previously identified targets for this compound. Our results showed that CDKI-73 inhibited the delivery of Rab11 vesicles to the plasma membrane, resulting in the accumulation of large multivesicular Rab11 endosomes near the cell periphery. In addition to the effect on endosome delivery, CDKI-73 down-regulated the amount of innate immune cargo, including the antimicrobial peptide Drosomycin and pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFα). We concluded that CDKI-73 has the potential to regulate the delivery and secretion of certain innate immune cargo, which could be used to control inflammation.

Assessing the toxicant effect of spontaneously volatilized 4-vinylcyclohexane exposure in nymphs of the lobster cockroach nauphoeta cinerea.

Vinylcyclohexene (VCH) is an environmental contaminant well known for its ovotoxicant effects in several organisms. However, the mechanisms underlying the toxicity of VCH as well as its harmful effects toward other organs are until unclear. In this work, we assess some endpoint signals of toxicity induced by volatilized VCH exposure using nymphs of the lobster cockroach Nauphoeta cinerea. Nymphs were exposed to VCH via inhalation for 70 days. The levels of volatilized VCH were quantified by headspace gas chromatography and the concentration varied between 3.41 and 7.03 nmol/µl. VCH inhalation caused a reduction of 35% in the survival rate of the exposed animals. Nymphs exposed to volatilized VCH for 35 and 70 days had a reduction in the body weight gain of 1.8- and 2.6-fold, respectively with a reduction in dissected head, fat body, and maturing reproductive organs. The exposure did not change water consumption, excepting on the 20th day (with a 3-fold change) and decreased the food intake significantly. Regarding biochemical markers, we found that the activity of GST from the dissected organs was increased by volatilized VCH after both 35 and 70 days of exposure. The fat body presented the most prominent GST activity especially after 35 days of exposure with 1.6-fold higher than the control group. Exposure also caused an increase in RS levels in the fat body of 1.35-fold and 1.47-fold after 35 and 70 days, respectively and did not affect the activity of the AChE from the head. Our findings support the harmful impact of volatilized VCH inhalation, highlighting the cockroach N.cinerea as a valuable insect model to investigate environmental toxicants.

Impairment of the immune response after transcuticular introduction of the insect gonadoinhibitory and hemocytotoxic peptide Neb-colloostatin: A nanotech approach for pest control.

This article shows that nanodiamonds can transmigrate through the insect cuticle easily, and the doses used were not hemocytotoxic and did not cause inhibition of cellular and humoral immune responses in larvae, pupae and adults of Tenebrio molitor. The examination of the nanodiamond biodistribution in insect cells demonstrated the presence of nanodiamond aggregates mainly in hemocytes, where nanoparticles were efficiently collected as a result of phagocytosis. To a lesser extent, nanodiamond aggregates were also detected in fat body cells, while they were not observed in Malpighian tubule cells. We functionalized nanodiamonds with Neb-colloostatin, an insect hemocytotoxic and gonadoinhibitory peptide, and we showed that this conjugate passed through the insect cuticle into the hemolymph, where the peptide complexed with the nanodiamonds induced apoptosis of hemocytes, significantly decreased the number of hemocytes circulating in the hemolymph and inhibited cellular and humoral immune responses in all developmental stages of insects. The results indicate that it is possible to introduce a peptide that interferes with the immunity and reproduction of insects to the interior of the insect body by means of a nanocarrier. In the future, the results of these studies may contribute to the development of new pest control agents.

The increase of amino acids induces the expression of vitellogenin after spinning in the silkworm Bombyx mori.

Silkworms are economically important insects because of the value of their silk. After finishing silk spinning, silkworms begin another important physiological process, vitellogenesis. In this study, we explored the relationship between silk spinning and vitellogenin (BmVg) expression in silkworms. In silkworms with the silk fibroin heavy chain gene knocked-out, the concentration of amino acids in the hemolymph was found to be significantly higher than that in the wild type, and the expression of BmVg was advanced at day 7 of the fifth instar stage and 0 h after spinning. Furthermore, through culturing fat body in vitro with different substances treatment including glucose, trehalose, amino acids, 20-hydroxyecdysone, and insulin, we found that only amino acids could induce BmVg expression. RNA interference of BmTOR1 in female silkworms could down-regulate BmVg transcription, resulting in shortened egg ducts and smaller eggs relative to the control. Therefore, these results showed that amino acids could induce BmVg expression through the TOR signaling pathway. Fat body cultured with amino acids in vitro and experiments involving amino acids injected into the silkworm showed that the majority of main amino acids of silk protein could induce BmVg expression. These results suggested that BmVg expression is related to silk spinning and this study would lay a foundation for elucidating the stage specificity expression of BmVg.

Cytotoxicity of Piper aduncum (Piperaceae) essential oil in brown stink bug Euschistus heros (Heteroptera: Pentatomidae).

Euschistus heros (F.) (Hemiptera: Pentatomidae) is a soybean pest in Brazil, controlled with synthetic chemical insecticides, which may be harmful to the environment and humans, as well as to select pest resistant strains. The research for new pest control strategies such as the use of plant essential oils has been increased due to the selectivity and biodegradation of these molecules. The objective was to evaluate the cytological changes in the salivary glands, fat body and midgut of E. heros exposed to different concentrations of essential oil of Piper aduncum L. (Piperales: Piperaceae), which the main compounds were identified as myristicin 30.03%, aromadendrene 9.20%, dillapiole 8.43%, α-serinene 7.31%, tridecane 6.26%, γ-elemene 4.58% and o-cymene 4.20%. The essential oil of P. aduncum was toxic for E. heros with LD50 = 36.23 mg per insect and LD90 = 50.42 mg per insect. Cytological changes such as tissue disruption, increase in mitochondria population, and glycogen and lipid depletion occur in the fat body cells, whereas salivary glands and midgut are not affected by this essential oil. Results suggest that P. aduncum essential oil causes fat body cellular stress, which may compromise some physiological processes for the insect survival.

Anti-tumour effects of antimicrobial peptides, components of the innate immune system, against haematopoietic tumours in Drosophila mxc mutants.

The innate immune response is the first line of defence against microbial infections. In Drosophila, two major pathways of the innate immune system (the Toll- and Imd-mediated pathways) induce the synthesis of antimicrobial peptides (AMPs) within the fat body. Recently, it has been reported that certain cationic AMPs exhibit selective cytotoxicity against human cancer cells; however, little is known about their anti-tumour effects. Drosophila mxcmbn1 mutants exhibit malignant hyperplasia in a larval haematopoietic organ called the lymph gland (LG). Here, using RNA-seq analysis, we found many immunoresponsive genes, including those encoding AMPs, to be upregulated in these mutants. Downregulation of these pathways by either a Toll or imd mutation enhanced the tumour phenotype of the mxc mutants. Conversely, ectopic expression of each of five different AMPs in the fat body significantly suppressed the LG hyperplasia phenotype in the mutants. Thus, we propose that the Drosophila innate immune system can suppress the progression of haematopoietic tumours by inducing AMP gene expression. Overexpression of any one of the five AMPs studied resulted in enhanced apoptosis in mutant LGs, whereas no apoptotic signals were detected in controls. We observed that two AMPs, Drosomycin and Defensin, were taken up by circulating haemocyte-like cells, which were associated with the LG regions and showed reduced cell-to-cell adhesion in the mutants. By contrast, the AMP Diptericin was directly localised at the tumour site without intermediating haemocytes. These results suggest that AMPs have a specific cytotoxic effect that enhances apoptosis exclusively in the tumour cells.

Morphological and immunohistochemical study of the midgut and fat body of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: noctuidae) treated with essential oils of the genus Piper.

Essential oils are a promising alternative to insecticides. We investigated the LD50 of oils extracted from Piper corcovadensis, P. marginatum, and P. arboreum after 48 h topical contact with Spodoptera frugiperda larvae using morphometry, histochemistry and immunohistochemistry of the midgut and fat body. Chromatography revealed that E-caryophyllene was the principal compound common to the Piper species. The essential oils of P. corcovadensis, P. marginatum and P. arboreum caused deleterious changes in the midgut of S. frugiperda larvae. P. corcovadensis oil produced the lowest LD50 and significant histopathological alterations including elongation of the columnar cells, formation of cytoplasmic protrusions, reduction in carbohydrate, increased apoptotic index and decreased cell proliferation. P. arboreum oil caused histopathological alterations similar to P. corcovadensis, but caused the highest rate of cell proliferation and increased regenerative cells, which indicated rapid regeneration of the epithelium. Our findings demonstrated the insecticidal potential of P. corcovadensis for control of S. frugiperda owing to the significant damage it inflicted on S. frugiperda midgut.

Fat body-specific vitellogenin expression regulates host-seeking behaviour in the mosquito Aedes albopictus.

The high vector competence of mosquitoes is intrinsically linked to their reproductive strategy because females need a vertebrate blood meal to develop large batches of eggs. However, the molecular mechanisms and pathways regulating mosquito host-seeking behaviour are largely unknown. Here, we test whether host-seeking behaviour may be linked to the female's energy reserves, with low energy levels triggering the search for a nutrient-rich blood meal. Our results demonstrate that sugar feeding delays host-seeking behaviour in the invasive tiger mosquito Aedes albopictus, but the levels of energy reserves do not correlate with changes in host-seeking behaviour. Using tissue-specific gene expression analyses, we show for the first time, to our knowledge, that sugar feeding alone induces a transient up-regulation of several vitellogenesis-related genes in the female fat body, resembling the transcriptional response after a blood meal. Specifically, high expression levels of a vitellogenin gene (Vg-2) correlated with the lowest host-seeking activity of sugar-fed females. Knocking down the Vg-2 gene via RNA interference (RNAi) restored host-seeking behaviour in these females, firmly establishing that Vg-2 gene expression has a pivotal role in regulating host-seeking behaviour in young Ae. albopictus females. The identification of a molecular mechanism regulating host-seeking behaviour in mosquitoes could pave the way for novel vector control strategies aiming to reduce the biting activity of mosquitoes. From an evolutionary perspective, this is the first demonstration of vitellogenin genes controlling feeding-related behaviours in nonsocial insects, while vitellogenins are known to regulate caste-specific foraging and brood-care behaviours in eusocial insects. Hence, this work confirms the key role of vitellogenin in controlling feeding-related behaviours in distantly related insect orders, suggesting that this function could be more ubiquitous than previously thought.