Cytotoxic and genotoxic effects of a pendimethalin-based herbicide in Apis mellifera.

Public concern about the effects of pesticides on non-target organisms has increased in the recent years. Nevertheless, there is a limited number of studies that address the actual toxic effects of herbicides on insects. This study investigated the side effects of herbicides on non-target organisms inhabiting agroecosystems and performing essential ecological and economic functions such as crop pollination. We analysed morphological alterations in the gut, Malpighian tubules and circulating haemocytes of Apis mellifera workers as markers of exposure effects. A commercial formulation of a pendimethalin-based herbicide (PND) was administered orally under laboratory conditions at a realistic concentration admitted in the field (330gL-1 of active ingredient., 4 L ha-1 for cereal and vegetable crops). The worker bees were exposed to a single application of PND for a period of one week, to simulate the exposure that can occur when foraging bees accidentally drink drops of contaminated water upon treatments. Histopathological analyses of the midgut, ileum and Malpighian tubules showed alterations over time (from 24 to 72 h after the beginning of exposure) such as loss of epithelial organisation, cellular vacuolisation and altered pyknotic nuclei as well as disruption of the peritrophic membrane over time. Semiquantitative analyses of the midgut showed a significant increase in the organ injury index 24 and 72 h after the initial exposure in PND-exposed bees compared to control bees. In addition, a change in positivity to Gram staining was observed in the midgut histological sections. A recovery of cytotoxic effects was observed one week after the initial exposure, which was favoured by the periodic renewal of the intestinal epithelium and the herbicide dissipation time. Cytochemical staining with Giemsa of haemocytes from PND-treated workers over 24 and 72 h showed significant nuclear alterations such as lobed or polymorphic nuclei and micronuclei compared to bees in the control group. These results show that the dose of PND used to protect crops from weeds can lead to significant cytotoxic and genotoxic effects in non-target organisms such as honey bees. In croplands, the sublethal effects on cell morphology can impair vital physiological processes such as nutrition, osmoregulation, and resistance to pathogens, contributing to the decline in biodiversity and abundance of species that play a prominent ecological role, such as pollinators.

Anatomical changes of Tenebrio molitor and Tribolium castaneum during complete metamorphosis.

In holometabolous insects, extensive reorganisation of tissues and cells occurs at the pupal stage. The remodelling of the external exoskeleton and internal organs that intervenes during metamorphosis has been traditionally studied in many insect species based on histological or ultrastructural methods. This study demonstrates the use of synchrotron X-ray phase-contrast micro-computed tomography as a powerful, non-destructive tool for in situ morphological observation of anatomical structures at the pupal stage in two Tenebrionid beetles, i.e. Tribolium castaneum and Tenebrio molitor, known as important pests, as well as emerging and promising models in experimental biology. Virtual sections and three-dimensional reconstructions were performed on both males and females at early, intermediate, and late pupal stage. The dataset allowed us to observe the remodelling of the gut and nervous system as well as the shaping of the female and male reproductive system at different pupal ages in both mealworm and red flour beetles. Moreover, we observed that the timing and duration pattern of organ development varied between the species analysed, likely related to the species-specific adaptations of the pre-imaginal stages to environmental conditions, which ultimately affect their life cycle. This research provides new knowledge on the morphological modifications that occur during the pupal stage of holometabolous insects and provides a baseline set of information on beetle metamorphosis that may support future research in forensics, physiology, and ecology as well as an image atlas for educational purposes.

The sperm structure of Clinidium canaliculatum (Costa): A contribution to the systematic position of Rhysodidae (Coleoptera: Carabidae).

The systematic position and the phylogenetic relationship of Rhysodidae members is still debated, with some authors considering the group as a separate family of Adephaga, while for others they could be a subfamily of Carabidae. The group have morphological traits quite different from Carabidae and an aberrant behaviour compared to ground beetles being not predaceous. The sperm ultrastructure of C. canaliculatum was studied comparatively with other species of beetles, Carabidae in particular. The results indicate that the sperm structure of this species is similar to that of the Carabinae species. As in these species, C. canaliculatum has sperm conjugates with an apical conical cap protecting the heads and the initial region of flagella. This sperm appearance is also shared by another species of Rhysodidae, Omoglymmius hamatus. The material of the apical cap consists of an electron-dense material with a peculiar outer net configuration. Many species of Carabidae, however, can present a different type of sperm conjugation, the spermatostyle: a long rod-like structure where the individual sperms have only the most apical part inserted in the cortical area and the flagella are completely free. C. canaliculatum sperm are endowed with a mono-layered acrosome, a nucleus of variable shape along its length, a flagellum consisting of a typical axoneme 9 + 9+2, provided with 16 protofilaments in the tubular wall of accessory tubules, two asymmetric mitochondrial derivatives with the left one larger than the opposite one, and the right accessory body elongated and larger than the opposite one. These sperm characteristics, which are shared also by another member of the group, suggest the demotion of the family Rhysodidae to the subfamily Rhysodinae within Carabidae, a result also supported by recent molecular data.

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.

Triazine Herbicide and NPK Fertilizer Exposure: Accumulation of Heavy Metals and Rare Earth Elements, Effects on Cuticle Melanization, and Immunocompetence in the Model Species Tenebrio molitor.

The increasing use of agrochemicals, including fertilizers and herbicides, has led to worrying metal contamination of soils and waters and raises serious questions about the effects of their transfer to different levels of the trophic web. Accumulation and biomagnification of essential (K, Na, Mg, Zn, Ca), nonessential (Sr, Hg, Rb, Ba, Se, Cd, Cr, Pb, As), and rare earth elements (REEs) were investigated in newly emerged adults of Tenebrio molitor exposed to field-admitted concentrations of a metribuzin-based herbicide and an NPK blend fertilizer. Chemical analyses were performed using inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) supported by unsupervised pattern recognition techniques. Physiological parameters such as cuticle melanization, cellular (circulating hemocytes), and humoral (phenoloxidase enzyme activity) immune responses and mass loss were tested as exposure markers in both sexes. The results showed that NPK fertilizer application is the main cause of REE accumulation in beetles over time, besides toxic elements (Sr, Hg, Cr, Rb, Ba, Ni, Al, V, U) also present in the herbicide-treated beetles. The biomagnification of Cu and Zn suggested a high potential for food web transfer in agroecosystems. Gender differences in element concentrations suggested that males and females differ in element uptake and excretion. Differences in phenotypic traits show that exposure affects metabolic pathways involving sequestration and detoxification during the transition phase from immature-to-mature beetles, triggering a redistribution of resources between sexual maturation and immune responses. Our findings highlight the importance of setting limits for metals and REEs in herbicides and fertilizers to avoid adverse effects on species that provide ecosystem services and contribute to soil health in agroecosystems.

Anatomical study of the red flour beetle using synchrotron radiation X-ray phase-contrast micro-tomography.

Synchrotron X-ray phase-contrast microtomography (SR-PhC micro-CT) is well established, fast and non-destructive imaging technique for data acquisition that is currently being used to obtain new insights into insect anatomy and function in physiological, morphological and phylogenetic studies. In this study, we described in situ the internal organs of the red flour beetle Tribolium castaneum Herbst 1797, a widespread pest of cereals and stored food causing serious damage to the human economy. Two-dimensional virtual sections and volumetric reconstructions of the nervous, alimentary and reproductive systems were carried out in both sexes. The results provided a comprehensive overview of the morphological characteristics of this species, such as the different maturation stages of ovarioles and the realistic location, size and shape of internal organs. Given the great interest in this model species in experimental biology and forensic entomology, complete knowledge of the general anatomy is required for future functional applications in pest control and experimental studies. In addition, this study confirms SR-PhC micro-CT as a powerful and innovative tool in entomology, particularly suitable for small species and chitinized structures that are difficult to analyse using conventional dissection and histological methods.

Dinitroaniline herbicides: a comprehensive review of toxicity and side effects on animal non-target organisms.

The widespread use of herbicides has increased concern about the hazards and risks to animals living in terrestrial and aquatic ecosystems. A comprehensive understanding of their effective action at different levels of biological organization is critical for establishing guidelines to protect ecosystems and human health. Dinitroanilines are broad-spectrum pre-emergence herbicides currently used for weed control in the conventional agriculture. They are considered extremely safe agrochemicals because they act specifically on tubulin proteins and inhibit shoot and root growth of plants. However, there is a lack of toxicity information regarding the potential risk of exposure to non-target organisms. The aim of the present review is to focus on side effects of the most commonly used active ingredients, e.g. pendimethalin, oryzalin, trifluralin and benfluralin, on animal non-target cells of invertebrates and vertebrates. Acute toxicity varies from slightly to high in terrestrial and aquatic species (i.e. nematodes, earthworms, snails, insects, crustaceans, fish and mammals) depending on the species-specific ability of tested organisms to adsorb and discharge toxicants. Cytotoxicity, genotoxicity and activation of oxidative stress pathways as well as alterations of physiological, metabolic, morphological, developmental and behavioural traits, reviewed here, indicate that exposure to sublethal concentrations of active ingredients poses a clear hazard to animals and humans. Further research is required to evaluate the molecular mechanisms of action of these herbicides in the animal cell and on biological functions at multiple levels, from organisms to communities, including the effects of commercial formulations.

Mass Spectrometry-Based Peptide Profiling of Haemolymph from Pterostichus melas Exposed to Pendimethalin Herbicide.

Pendimethalin-based herbicides are used worldwide for pre-emergence selective control of annual grasses and weeds in croplands. The endurance of herbicides residues in the environment has an impact on the soil biodiversity and fertility, also affecting non-target species, including terrestrial invertebrates. Carabid beetles are known as natural pest control agents in the soil food web of agroecosystems, and feed on invertebrates and weed seeds. Here, a mass spectrometry untargeted profiling of haemolymph is used to investigate Pterostichus melas metabolic response after to pendimethalin-based herbicide exposure. Mass spectrometric data are examined with statistical approaches, such as principal component analysis, for possible correlation with biological effects. Those signals with high correlation are submitted to tandem mass spectrometry to identify the associated biomarker. The time course exposure showed many interesting findings, including a significant downregulation of related to immune and defense peptides (M-lycotoxin-Ls4a, Peptide hormone 1, Paralytic peptide 2, and Serine protease inhibitor 2). Overall, the observed peptide deregulations concur with the general mechanism of uptake and elimination of toxicants reported for Arthropods.

Exploring Compound Eyes in Adults of Four Coleopteran Species Using Synchrotron X-ray Phase-Contrast Microtomography (SR-PhC Micro-CT).

Compound eyes in insects are primary visual receptors of surrounding environments. They show considerable design variations, from the apposition vision of most day-active species to the superposition vision of nocturnal insects, that sacrifice resolution to increase sensitivity and are able to overcome the challenges of vision during lightless hours or in dim habitats. In this study, Synchrotron radiation X-ray phase-contrast microtomography was used to describe the eye structure of four coleopteran species, showing species-specific habitat demands and different feeding habits, namely the saproxylic Clinidium canaliculatum (Costa, 1839) (Rhysodidae), the omnivorous Tenebrio molitor (Linnaeus, 1758) and Tribolium castaneum (Herbest, 1797) (Tenebrionidae), and the generalist predator Pterostichus melas italicus (Dejean, 1828) (Carabidae). Virtual sections and 3D volume renderings of the heads were performed to evaluate the application and limitations of this technique for studying the internal dioptrical and sensorial parts of eyes, and to avoid time-consuming methods such as ultrastructural analyses and classic histology. Morphological parameters such as the area of the corneal facet lens and cornea, interocular distance, facet density and corneal lens thickness were measured, and differences among the studied species were discussed concerning the differences in lifestyle and habitat preferences making different demands on the visual system. Our imaging results provide, for the first time, morphological descriptions of the compound eyes in these species, supplementing their ecological and behavioural traits.

Antennal sensilla in an anophthalmic wood-dwelling species, Clinidium canaliculatum, Costa 1839 (Coleoptera, Rhysodidae).

The habit of feeding on slime moulds (Myxomycetes) commonly present in litter or dead wood requires specific morphological adaptations of the mouthparts and sensory structures involved in the search for habitat and food. In this study, the external morphology of antenna and its sensilla were studied using scanning electron microscopy in the saproxylic beetle, Clinidium canaliculatum, Costa 1839 (Coleoptera, Rhysodidae). Their moniliform antennae consist of a scape, pedicel, and nine flagellomeres. We identified seven different types of sensilla, according to their morphological characteristics: two types of sensilla chaetica (sc1 and 2), two types of sensilla basiconica (sb1 and 2), one type of sensilla campaniformia, one type of sensilla coeloconica, and Böhm sensilla. No sexual dimorphism was found regarding antennal morphology and sensilla type and distribution, except for the sensilla coeloconica. The functional role of these sensilla was discussed in relation to their external structure and distribution, and compared with the current knowledge on coleopteran sense organs. Results are basic information for further physiological and behavioral studies to identify their role in the selection of habitat, food, mates and oviposition sites.

Pygidial Glands in Carabidae, an Overview of Morphology and Chemical Secretion.

Predator community structure is an important selective element shaping the evolution of prey defence traits and strategies. Carabid beetles are one of the most diverse families of Coleoptera, and their success in terrestrial ecosystems is related to considerable morphological, physiological, and behavioural adaptations that provide protection against predators. Their most common form of defence is the chemical secretion from paired abdominal pygidial glands that produce a heterogeneous set of carboxylic acids, quinones, hydrocarbons, phenols, aldehydes, and esters. This review attempts to update and summarise what is known about the pygidial glands, with particular reference to the morphology of the glands and the biological function of the secretions.

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

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.

Pendimethalin-based herbicide impairs cellular immune response and haemocyte morphology in a beneficial ground beetle.

Herbicides have become the most commonly applied agrochemicals in agroecosystems. Thus, basic knowledge of their physiological effects on insects is needed, especially for understanding their impact on beneficial insect species. In this study, we evaluated the effect of a pendimethalin-based herbicide (PND) on the cellular immune response of the carabid beetle Harpalus (Pseudoophonus) rufipes (De Geer 1774) (Coleoptera, Carabidae), acting as biocontrol agent in agroecosystems. Total and differential haemocyte counts and phagocytosis assay, performed by injecting in vivo carboxylate-modified polystyrene latex beads, were measured in beetles exposed to a recommended field dose (4L per ha) of PND to evaluate the exposure effects over the time. The pattern of haemocyte subpopulations and the decrease of the phagocytic index after the exposure to PND suggested a lowering of P. rufipes ability to face an infection performing a cell-mediated response. PND was also found to cause cytotoxic effects on the haemocyte ultrastructure. Ultrastructural alterations such as irregular shape, large vacuolization of the cytoplasm, and condensation of marginated chromatin were recorded from 2d of exposure. The loss of RER, Golgi apparatus, mitochondria integrity and the swelling of the outer nuclear membrane found in some haemocytes suggested an interference of PND with the membrane permeability. Results indicated that the exposure to PND impairs the distribution, morphology and physiological functions of haemocytes causing a decrease of P. rufipes immunocompetence. Moreover, the sensitivity to herbicide exposure makes this species a suitable model and a useful bioindicator for monitoring exposure effects on non-target species. This study provides useful information to protect and preserve biodiversity of insects in agroecosystems.

Cardiometabolism as an Interlocking Puzzle between the Healthy and Diseased Heart: New Frontiers in Therapeutic Applications.

Cardiac metabolism represents a crucial and essential connecting bridge between the healthy and diseased heart. The cardiac muscle, which may be considered an omnivore organ with regard to the energy substrate utilization, under physiological conditions mainly draws energy by fatty acids oxidation. Within cardiomyocytes and their mitochondria, through well-concerted enzymatic reactions, substrates converge on the production of ATP, the basic chemical energy that cardiac muscle converts into mechanical energy, i.e., contraction. When a perturbation of homeostasis occurs, such as an ischemic event, the heart is forced to switch its fatty acid-based metabolism to the carbohydrate utilization as a protective mechanism that allows the maintenance of its key role within the whole organism. Consequently, the flexibility of the cardiac metabolic networks deeply influences the ability of the heart to respond, by adapting to pathophysiological changes. The aim of the present review is to summarize the main metabolic changes detectable in the heart under acute and chronic cardiac pathologies, analyzing possible therapeutic targets to be used. On this basis, cardiometabolism can be described as a crucial mechanism in keeping the physiological structure and function of the heart; furthermore, it can be considered a promising goal for future pharmacological agents able to appropriately modulate the rate-limiting steps of heart metabolic pathways.

Synchrotron X-ray phase contrast micro tomography to explore the morphology of abdominal organs in Pterostichus melas italicus Dejean, 1828 (Coleoptera, Carabidae).

Micro-computer tomography imaging is a fast and non-destructive data acquisition technique which can replace or complement the traditional investigation methodologies used in entomology to study morphology. In this paper, Synchrotron Radiation X-ray Phase-Contrast micro tomography (SR-PhC micro-CT) was combined with histology and scanning electron microscopy (SEM) observations to describe the abdominal organs of Pterostichus melas italicus Dejean, 1828 (Coleoptera, Carabidae). This species was used as a representative model because of its ecological role as a generalist predator in agroecosystems. SR-PhC micro-CT allowed us to identify in situ abdominal structures including dorsal vessel, digestive tract with Malpighian tubules, male reproductive system, ganglia, fat bodies, pygidial glands, muscles and tracheae. The histology was performed to define the tissue organization of the digestive and reproductive systems. SR-PhC micro-CT and 3D rendering provided more accurate information on shape and size of organs than histological and SEM analyses, respectively. The finding of this study was to describe the anatomy and histology of organs involved in crucial life history traits, such as reproduction, nutrition and excretion. High quality images and the supplementary video represent a significant advance in knowledge of the carabid anatomy and are a baseline for future research.

Medium-term feasibility of the management of the invasive crayfish Procambarus clarkii with the sterile males release technique.

BACKGROUND: The sterile male release technique (SMRT) is a useful method applied for controlling invasive and pest species. However, the use of X-rays can lead to negative effects on the survival and health conditions of sterilized males. RESULTS: This study was set up to evaluate the functional integrity of physiological, morphological and behavioural responses in males of the red swamp crayfish, Procambarus clarkii (Girard, 1852), exposed to a dose of 40 Gy of ionizing radiation. Concerning physiological responses, the results showed that the irradiation dose, tested at 5, 12, 28, 35, 65, 99, 132 and 193 days after treatment, has no effects on glycaemic and plasmatic total protein levels measured as biomarkers for general stress indexes. Nevertheless, the significant reduction of circulating haemocytes and the basal levels of phenoloxidase (PO) activities recorded in 40-Gy irradiated crayfishes indicate that the exposure shrinks their capability to mount a rapid nonspecific response, and higher levels of plasmatic total PO activity indicate the ability to compensate and maintain an inducible response. Histological analyses performed at the end of the experiment showed no morphological damage in the testicular acini of irradiated males. Moreover, behavioural responses to two different water stimuli (vaporization and jet), measured at 15 and 45 days after the irradiation, were not modified in exposed crayfishes compared to the control group. CONCLUSIONS: These results confirm the validity of SMRT on young males when the breeding season is less than 4 months but exposure to X-ray should be repeated at mid-breeding season when temperatures allow a longer breeding season. © 2021 Society of Chemical Industry.

Agrochemical treatments as a source of heavy metals and rare earth elements in agricultural soils and bioaccumulation in ground beetles.

The continuous and extensive application of agrochemicals leads to the accumulation of heavy metals (HMs) and rare earth elements (REEs) in agricultural soils and their transfer in the food web with consequent relevant risks for human and ecosystem health. In this study, HM and REE concentrations were quantified in the soil of wheat crop fields conventionally managed in the agricultural areas of Sila Mountain (Southern Italy) and compared with the concentration in a field of wild herbs, used as control. Statistical analyses and principal component analysis suggested that the use of pesticides, herbicides and fertilizers contributes to the accumulation of HMs and REEs in the soil. Different accumulation patterns were recorded in treated fields as a consequence of the type and amount of agrochemical used and the crop rotation. The exposure risk associated with the transfer through the tropic levels of agroecosystem was carried out measuring the concentration of HMs and REEs in adults of Harpalus (Pseudoophonus) rufipes (De Geer, 1774) collected from each monitored site. Different accumulation patterns found in specimens from the monitored sites highlighted the ability of this generalist predator to regulate metal uptake under field conditions. The values of bioaccumulation factor (BAF) allow to defining the order of accumulation in P. rufipes which was classified as a macroconcentrator of Cd, Cu, Mg and Zn. Our results can supplement the limited information regarding the REE accumulation in soil invertebrates and may provide reference data for assessing potential environmental risks in croplands.

Sperm ultrastructure in several species of Carabidae beetles (Insecta, Adephaga) and their organization in spermatozeugmata.

Sperm structure of seven species from different Carabidae tribes was studied. Carabus preslii and Carabus granulatus interstitialis form sperm bundles (spermatozeugmata) in which the heads of conventional insect sperm cells are embedded in the apical cap (spermatostyle), leaving the posterior flagella free. On the contrary, Pterostichus morio, Pterostichus melas, Pterostichus melanarius and Amara aulica form complex spermatozeugmata bearing conspicuous axial spermatostyles associated with lateral groups of sperm. Individual sperm cells are contained in chambers delimited by laminar extensions of the cortical region of the spermatostyle. Demetrias atricapillus shares the general spermatozeugma structure with the above species, but the anterior spermatostyle region has a different structure and posteriorly, the lateral groups of sperm are separated from the spermatostyle but remain connected to it by peduncles consisting of extensions of the cortical region of the spermatostyle. The sperm of the species examined in the study consist of a short, flat acrosomal cap and, with the exception of the Carabus species, show long nuclei which extend parallel to the axonemes, along the flagella. Two relatively small mitochondrial derivatives and two small accessory bodies flank the axoneme. These bodies become very thick in the posterior flagellar region of D. atricapillus. The study revealed different spermatozeugma models and different sperm organization in the Carabidae family.

Impact of agrochemicals on non-target species: Calathus fuscipes Goeze 1777 (Coleoptera: Carabidae) as model.

Carabid beetles are important in the biological control of arable crop pests. Agricultural practices can produce over time a delayed toxic effect at the organismal and population levels and can compromise the survival on these species. In this research, we quantified the cumulative sublethal effect on body size, Malpighian tubules and immune responses in Calathus fuscipes adults living in the potato field and exposed to lambda-cyhalothrin and cymoxanil-based commercial formulates. Reductions of morphological parameters such as body, pronotum and elytron in both males and females from the potato field indicated that the pre-imaginal stages (larvae and pupae) suffer the sublethal effects of exposure to the larvicide control action of lambda-cyhalothrin. Ultrastructural alterations recorded in Malpighian tubules at the level of plasma membrane, mitochondria and nucleus indicated the reduction of the detoxification capability. The basal phenoloxidase and lysozyme-like enzyme activities have measured as markers of immune competence. Spectrophometric analyses showed that the chronic exposure in field causes an increase of basal phenoloxidase enzyme activity, while the lytic activity of haemolymph was not affected. As a result, the use of larvicides and fungicides have a harmful effect on beneficial species such C. fuscipes living in the soil of potato fields. These morphological and physiological results recorded at the organismal level can provide useful information of effects at the population and community levels to preserve the biodiversity of agroecosystem.

Exosomes: Ultrastructural evidence in epithelial cells of Malpighian tubules.

Exosomes in Malpighian tubules of Calathus fuscipes (Coleoptera: Carabidae) were investigated under transmission electron microscopy. Ultrastructural analyses showed a wide number of multivesicular bodies localized in the apical portion of epithelial cells. Each multivesicular body encloses from 15 to 80 intraluminal vesicles (about 50nm in diameter), originating through inward budding of late endosomes that package molecules into luminal membrane-bound structures. Subsequently they are released as exosomes through exocytosis of multivesicular bodies into the extracellular space after fusion with plasma membrane. Our results are the base for further investigation on the role of exosomes in functional polarization of tubule cells and on cell-to-cell communication in insects.