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.

To induce the water solubility of hexagonal boron nitride (h-BN), we exfoliated and functionalized bulk h-BN with hydroxyl groups (h-BN-OH-n). Short-term studies showed that h-BN-OH-n induced low cytotoxicity in different models: insect haemocytes (in vivo), human erythrocytes and mouse fibroblasts (in vitro). We also demonstrated that Alexa Fluor 647-h-BN-OH-n administered topically to the insects passed through the cuticle barrier and was phagocytosed by haemocytes. Nanoflakes did not affect the haemocyte cell membrane and did not interfere with the phagocytosis of latex beads. Long-term immunoassays showed that h-BN-OH-n, despite not inducing haemocytotoxicity, impaired nodulation, the most important cellular immune response in insects. The haemocytes exposed to h-BN-OH-n and then to bacteria differed in morphology and adhesiveness from the haemocytes exposed only to bacteria and exhibited the same morphology and adhesiveness as the control haemocytes. The h-BN-OH-n-induced decrease in nodulation can therefore result from the reduced ability of haemocytes to recognize bacteria, migrate to them or form microaggregates around them, which can lead to dysfunction of the immune system during pathogen infection. Long-term in vivo studies with animal models are still necessary to unambiguously confirm that h-BN is biocompatible and useful for application as a platform for drug delivery or for bioimaging.

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A stock culture of T. molitor L. was maintained at the Department of Animal Physiology and Development as described previously SUMMARY The gonadoinhibitory peptide hormone Neb-colloostatin was first isolated from ovaries of the flesh fly Neobellieria bullata. This 19-mer peptide is thought to be a cleaved product of a collagen-like precursor molecule that is formed during remodelling of the extracellular matrix. In this study, we report that upon injection of picomolar and nanomolar doses, this peptide exerts a proapoptotic action on haemocytes of Tenebrio molitor adults, as visualized by changes in morphology and viability. The F-actin cytoskeleton was found to aggregate into distinctive patches. This may be responsible for the observed inhibition of adhesion of haemocytes and for the stimulation of filopodia formation. However, Neb-colloostatin injection did not induce the formation of autophagic vacuoles. Our results suggest that physiological concentrations of Neb-colloostatin play an important role in controlling the quantity and activity of haemocytes in insect haemolymph. They also suggest that during periods in which Nebcolloostatin is released, this peptide may cause a weakening of the insectsʼ immune system. This is the first report that exposure to a peptide hormone causes apoptosis in insect haemocytes.

Insect overwintering is one of the most astonishing phases of the insect life cycle. Despite vast amounts of knowledge available about the physiological mechanisms of this phenomenon, the impact of stress factors on insect immune system functioning during the winter is still unknown. The aim of this study is to analyze how low temperatures influence the immune system of the beetle Nicrophorus vespilloides. The results show that the beetle's immune system is differently modulated by cold induced in laboratory settings than that which occurs in natural conditions. Among beetles cultured in conditions similar to summer, low temperatures, did not influence the number of circulating haemocytes, phenoloxidase activity, haemocytes morphology, and percentage ratio of haemocyte types. In these beetles, differences were noted only in the ability of haemocytes to perform phagocytosis. Individuals acclimated in natural conditions in autumn had a higher level of humoral response and a different percentage ratio of haemocyte types. During the winter period, the number of haemocytes in the beetles decreased, but the percentage ratio of phagocytic haemocytes increased. Furthermore, we noted an increase of phenoloxidase activity. Our study also showed mitotic divisions of haemocytes in haemolymph collected from burying beetles after cold exposure and from burying beetles collected from natural conditions during autumn and winter. Differences in response to low temperatures in laboratory conditions and the natural environment suggest that the simultaneous presence of other stress factors during winter such as desiccation and starvation have a significant influence on the activity of burying beetle's immune system.