Effect of short-term exposure to the strobilurin fungicide dimoxystrobin: Morphofunctional, behavioural and mitochondrial alterations in Danio rerio embryos and larvae.

Strobilurins, among the most used fungicides worldwide, are considered non-toxic to mammals and birds, but there is growing evidence that these compounds are highly toxic to aquatic species. Dimoxystrobin has been included in the 3rd Watch List of the European Commission, and it has been classified as very toxic to aquatic life. However, previous studies focused on acute toxicity and only two reports are available on its impact on fish, and none on its effects during the early life stages. Here, we evaluated for the first time the effects induced on zebrafish embryos and larvae by two dimoxystrobin sublethal concentrations (6.56 and 13.13 µg/L) falling in the range of predicted environmental concentrations. We demonstrated that short-term exposure to dimoxystrobin may exert adverse effects on multiple targets, inducing severe morphological alterations. Moreover, we showed enhanced mRNA levels of genes related to the mitochondrial respiratory chain and ATP production. Impairment of the swim bladder inflation has also been recorded, which may be related to the observed swimming performance alterations.

Morphological and Functional Alterations Induced by Two Ecologically Relevant Concentrations of Lead on Danio rerio Gills.

Lead (Pb), due to its high toxicity and bioaccumulation tendency, is one of the top three pollutants of concern for both humans and wildlife and occupies second place in the Priority List of Hazardous Substances. In freshwater fish, Pb is mainly absorbed through the gills, where the greatest accumulation occurs. Despite the crucial role of gills in several physiological functions such as gas exchange, water balance, and osmoregulation, no studies evaluated the effects of environmentally relevant concentrations of Pb on this organ, and existing literature only refers to high levels of exposure. Herein we investigated for the first time the molecular and morphological effects induced by two low and environmentally relevant concentrations of Pb (2.5 and 5 µg/L) on the gills of Danio rerio, a model species with a high translational value for human toxicity. It was demonstrated that Pb administration at even low doses induces osmoregulatory dysfunctions by affecting Na+/K+-ATPase and AQP3 expression. It was also shown that Pb upregulates MTs as a protective response to prevent cell damage. Modulation of SOD confirms that the production of reactive oxygen species is an important toxicity mechanism of Pb. Histological and morphometric analysis revealed conspicuous pathological changes, both dose- and time-dependent.

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.

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.

Eye morphology in four species of tiger beetles (Coleoptera: Cicindelidae).

Compound eyes undoubtedly represent the widespread eye architecture in the animal kingdom. The insects' compound eye shows a wide variety of designs, and insects use their visual capacity to accomplish several tasks, including avoiding enemies, searching for food and shelter, locating a mate, and acquiring information about the environment and its surroundings. Broad literature data support the concept that visual ability lies in the way the eyes are built. Since the resolution and sensitivity of the compound eye are partly determined by the density of the ommatidia and the size of the facets. Morphological parameters of the compound eyes could influence the function of the visual organ and its capacity to process information, also representing a sensitive indicator of different habitat demands. In this study, we compared compound eyes' parameters in four closely related species of tiger beetles to disclose differences arising from different habitats. Furthermore, to investigate whether there are consistent intersexual differences, we also compared the most relevant parameters of the eye in males and females of four selected species. Our results show sex-related and interspecific differences that occur in examined species.

Short-term effects of the strobilurin fungicide dimoxystrobin on zebrafish gills: A morpho-functional study.

Strobilurins represent the most widely used class of fungicides nowadays andare considered relatively non-toxic to mammals and birds but highly toxic to aquatic biota. Dimoxystrobin is one of the novel strobilurins, recently included in the 3rd Watch List of the European Commission as available data indicate that it could pose a significant risk to aquatic species. As yet, the number of studies explicitly assessing the impact of this fungicide on terrestrial and aquatic species is extremely low, and the toxic effects of dimoxystrobin on fish have not been reported. Here we investigate for the first time the alterations induced by two environmentally relevant and very low concentrations of dimoxystrobin (6.56 and 13.13 µg/L) in the fish gills. morphological, morphometric, ultrastructural, and functional alterations have been evaluated using zebrafish as a model species. We demonstrated that even short-term exposure (96 h) to dimoxystrobin alters fish gills reducing the surface available for gas exchange and inducing severe alterations encompassing three reaction patterns: circulatory disturbance and both regressive and progressive changes. Furthermore, we revealed that this fungicide impairs the expression of key enzymes involved in osmotic and acid-base regulation (Na+/K+-ATPase and AQP3) and the defensive response against oxidative stress (SOD and CAT). The information presented here highlights the importance of combining data from different analytical methods for evaluating the toxic potential of currently used and new agrochemical compounds. Our results will also contribute to the discussion on the suitability of mandatory ecotoxicological tests on vertebrates before the introduction on the market of new compounds.

The role of exposure window and dose in determining lead toxicity in developing Zebrafish.

Heavy metal contamination is recognized worldwide as a serious threat to human health and wildlife, and reducing their emissions is a priority of international and EU actions. Due to its persistence, high bioaccumulation tendency, and toxicity properties, lead (Pb) is one of the heavy metals of greatest concern. Even at low concentrations, lead induces various clinical and subclinical conditions in both humans and animals, and it has been included in the priority list of hazardous substances. In the present study, we used zebrafish's early stages as a model, given their well-acknowledged predictive value in the risk assessment of chemicals. This study was designed to investigate the morphological and morphometric alterations induced by Pb during zebrafish's early development and disclose the putative effects stage- and/or dose-dependent. We examined injuries induced by two environmentally relevant and extremely low concentrations of Pb (2.5 µg/L and 5 µg/L) during two exposure windows: early (between 1 and 7 dpf) and late (between 2 and 8 dpf). We clearly demonstrated that the incidence and severity of morphological abnormalities increased with increasing Pb dose and exposure time in both early and late-exposed groups. Furthermore, we revealed that malformation severity was significantly higher in the early exposed group than in the late exposure group at all exposure times and for both tested doses, thus highlighting the high sensitivity of zebrafish during the initial stages of development. The information presented in this paper emphasizes the effectiveness of morphological biomarkers in unveiling threatening situations and supports the role of zebrafish embryos and larvae in risk assessment and environmental monitoring.

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.

Circulating hemocytes from larvae and adults of Carabus (Chaetocarabus) lefebvrei Dejean 1826 (Coleoptera, Carabidae): cell types and their role in phagocytosis after in vivo artificial non-self-challenge.

Carabus lefebvrei Dejean 1826 is an helicophagous Italian endemic ground beetle that lives in central and south Apennines mountain forests, from lower altitudes to about 1500 m. In ground beetles, no morphofunctional data about immune system is available, even though they are well known both taxonomically and ecologically and they have been often used as indicators of the habitat quality due to their specificity to certain habitat types. In the current investigation the cellular population in the hemolymph of adult and third instar larvae of C. lefebvrei has been characterized by means of light and electron microscopy analysis and phagocytosis assays were performed in vivo by injection of 0.9 microm carboxylate-modified polystyrene latex beads in order to identify the hemocyte types involved in phagocytosis. Four morphotypes of circulating hemocytes were found both in larvae and in adults: prohemocytes, granulocytes, oenocytoids and plasmatocytes. After in vivo artificial non-self-challenge treatments, C. lefebvrei showed a non-specific immune response involving phagocytosis performed by plasmatocytes, both in adults and in larvae and by oenocitoids in larvae. In untreated animals, the hemocyte type presenting a firm phagocytic activity, the plasmatocytes, presented a percentage significantly higher in larvae than in adults, and after latex beads injections in larvae there was a tendency of significant difference in plasmatocyte percentage compared to controls injected with phosphate saline buffer. We think that these differences could be correlated with the peculiar morphology (less chitinization) and ecology of larval stages that are more sensitive to pathogens than adults.

Current knowledge on exocrine glands in carabid beetles: structure, function and chemical compounds.

Many exocrine products used by ground beetles are pheromones and allomones that regulate intra- and interspecific interactions and contribute to their success in terrestrial ecosystems. This mini-review attempts to unify major themes related to the exocrine glands of carabid beetles. Here we report on both glandular structures and the role of secretions in carabid adults, and that little information is available on the ecological significance of glandular secretions in pre-imaginal stages.

Morphometry of eyes, antennae and wings in three species of Siagona(Coleoptera, Carabidae).

In carabid beetles, physiological and behavioural characteristics reflect specific habitat demands and there is a strong correlation between body form and habit in species with different life style. In this study, we compared the morphometry and compound eye characteristics of three species of the genus Siagona: Siagona jenissoni, Siagona dejeani and Siagona europaea. These carabids have a stenotopic lifestyle in Mediterranean clayey soils, inhabiting the ground fissure system formed during the dry season. All species have a Mediterranean distribution and are nocturnal olfactory hunters, and are strict ant predators. For morphometric measurements, we considered body length (mm), wing length (mm), antenna length (mm), head width (mm), trochanter length (mm), number of ommatidia, eye surface area (mm2), ommatidia density (number of ommatidia/mm2 of eye surface area), head height (mm), thorax height (mm) and abdomen height (mm). The data revealed intersexual and interspecific differences. The three species differ in relative length of the antennae, density and number of ommatidia and relative trochanter length. Significant differences occurred in wing sizes, which are well developed in Siagona europaea, the only species capable of flight. When eye size is compared with other ground beetles of various lifestyles, Siagona shows pronounced "microphthalmy" an adaptation to subterranean life in clayey crevices of tropical and subtropical climates with a marked dry season.

The defensive secretion of Carabus lefebvrei Dejean 1826 pupa (Coleoptera, Carabidae): gland ultrastructure and chemical identification.

This study documents the defensive function of flavored humor secreted by the abdominal glands of Carabus lefebvrei pupae. The morphology and the ultrastructure of these glands were described and the volatile compounds of glands secretion were identified by gas chromatography/mass spectrometry. The ultrastructure analysis shows an acinose complex formed by about 50 clusters. Each cluster has 20 glandular units and the unit-composed of one secretory and one canal cell lying along a duct-belongs to the class 3 cell type of Quennedey (1998). In the cytoplasm, the secretory cell contains abundant rough endoplasmatic reticula, glycogen granules, numerous mitochondria, and many well-developed Golgi complexes producing electron-dense secretory granules. Mitochondria are large, elongated, and often adjoining electronlucent vesicles. The kind and the origin of secretory granules varying in size and density were discussed. The chemical analysis of the gland secretion revealed the presence of a mixture of low molecular weight terpenes, ketones, aldehydes, alcohols, esters, and carboxylic acids. Monoterpenes, especially linalool, were the major products. We supposed that ketones, aldehydes, alcohols, esters, and carboxylic acids have a deterrent function against the predators and monoterpenes provide a prophylaxis function against pathogens.