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.

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.

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.

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.

Gut microbiota plays a key role in physiological processes of insects, including nutritional metabolism, development, immunity and detoxification. Environmental stressors such as herbicides, used to optimize and improve crop yields, may interfere with the mutualistic relationships causing negative consequences for the host health. Dinitroaniline herbicides, for example pendimethalin, are used worldwide in pre‐emergence application to control grass and some broadleaf weeds. They target microtubules to arrest cell division and inhibit the development of roots and shoots. Effects of a pendimethalin‐based herbicide were assessed on the gut microbial community of Pterostichus melas italicus Dejean, 1828 (Coleoptera, Carabidae). The exposure effect was tested in vivo by using a recommended field rate (4 L per ha, 330 gL⁻¹ of active ingredient) and evaluating the variability of responses in 21 days, corresponding to the half‐life of pendimethalin. The 16S rRNA sequencing data showed that the gut lumen was dominated by Proteobacteria, Firmicutes, Fusobacteria, Tenericutes and Bacteroidetes. The exposure interfered with the bacterial community richness and diversity associated with the gut from 2 days after the treatment. The differential abundance analyses highlighted a shift involving Lactobacillaceae, Streptomycetaceae, Neisseriaceae, Ruminococcaceae and Enterobacteriaceae. An increase in species such as Enterobacter sp., Pseudomonas sp., Pantoea sp and Paracoccus sp. involved in the herbicide degradation was also recorded after 21 days of exposure. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis indicated that the exposure has effects on the most predicted functional categories of gut microbiota related to metabolic function including carbohydrate, amino acid and lipid metabolism. These results demonstrate that pendimethalin can impact microbial communities associated with generalist predators inhabiting croplands leading to severe implications for the species’ ecological role. Understanding the effects of herbicides such as pendimethalin on ground beetles may help to protect beneficial soil insects that have a crucial role in the ecosystem services.