Immune Stimulation via Wounding Alters Chemical Profiles of Adult Tribolium castaneum.

Group-living individuals experience immense risk of disease transmission and parasite infection. In social and in some non-social insects, disease control with immunomodulation arises not only via individual immune defenses, but also via infochemicals such as contact cues and (defensive) volatiles to mount a group-level immunity. However, little is known about whether activation of the immune system elicits changes in chemical phenotypes, which may mediate these responses. We here asked whether individual immune experience resulting from wounding or injection of heat-killed Bacillus thuringiensis (priming) leads to changes in the chemical profiles of female and male adult red flour beetles, Tribolium castaneum, which are non-social but gregarious. We analyzed insect extracts using GC-FID to study the chemical composition of (1) cuticular hydrocarbons (CHCs) as candidates for the transfer of immunity-related information between individuals via contact, and (2) stink gland secretions, with analysis of benzoquinones as main active compounds regulating 'external immunity'. Despite a pronounced sexual dimorphism in CHC profiles, wounding stimulation led to similar profile changes in males and females with increases in the proportion of methyl-branched alkanes compared to naïve beetles. While changes in the overall secretion profiles were less pronounced, absolute amounts of benzoquinones were transiently elevated in wounded compared to naïve females. Responses to priming were insignificant in CHCs and secretions. We suggest that changes in different infochemicals after wounding may mediate immune status signaling in the context of both internal and external immune responses in groups of this non-social insect, thus showing parallels to social immunity.

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.

Paternal knockdown of tRNA(cytosine-5-)-methyltransferase (Dnmt2) increases offspring susceptibility to infection in red flour beetles.

Intergenerational effects from fathers to offspring are increasingly reported from diverse organisms, but the underlying mechanisms remain speculative. Paternal trans-generational immune priming (TGIP) was demonstrated in the red flour beetle Tribolium castaneum: non-infectious bacterial exposure of fathers protects their offspring against an infectious challenge for at least two generations. Epigenetic processes, such as cytosine methylation of nucleic acids, have been proposed to enable transfer of information from fathers to offspring. Here we studied a potential role in TGIP of the Dnmt2 gene (renamed as Trdmt1 in humans), which encodes a highly conserved enzyme that methylates different RNAs, including specific cytosines of a set of tRNAs. Dnmt2 has previously been reported to be involved in intergenerational epigenetic inheritance in mice and protection against viruses in fruit flies. We first studied gene expression and found that Dnmt2 is expressed in various life history stages and tissues of T. castaneum, with high expression in the reproductive organs. RNAi-mediated knockdown of Dnmt2 in fathers was systemic, slowed down offspring larval development and increased mortality of the adult offspring upon bacterial infection. However, these effects were independent of bacterial exposure of the fathers. In conclusion, our results point towards a role of Dnmt2 for paternal effects, while elucidation of the mechanisms behind paternal TGIP needs further studies.

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.

Oral Immune Priming Treatment Alters Microbiome Composition in the Red Flour Beetle Tribolium castaneum.

It is now well-established that the microbiome is relevant for many of an organism's properties and that its composition reacts dynamically to various conditions. The microbiome interacts with host immunity and can play important roles in the defenses against pathogens. In invertebrates, immune priming, that is, improved survival upon secondary exposure to a previously encountered pathogen, can be dependent upon the presence of the gut microbiome. However, it is currently unknown whether the microbiome changes upon priming treatment. We here addressed this question in a well-established model for immune priming, the red flour beetle Tribolium castaneum exposed to the entomopathogenic bacterium Bacillus thuringiensis (Bt). After priming treatments, the microbiota composition of beetle larvae was assessed by deep sequencing of the V1-V2 region of the bacterial 16S rRNA gene. We compared the effect of two established routes of priming treatments in this system: injection priming with heat-killed Bt and oral priming via ingestion of filtered sterilized bacterial spore culture supernatants. For oral priming, we used several strains of Bt known to vary in their ability to induce priming. Our study revealed changes in microbiome composition following the oral priming treatment with two different strains of Bt, only one of which (Bt tenebrionis, Btt) is known to lead to improved survival. In contrast, injection priming treatment with the same bacterial strain did not result in microbiome changes. Combined with the previous results indicating that oral priming with Btt depends on the larval microbiome, this suggests that certain members of the microbiome could be involved in forming an oral priming response in the red flour beetle.

Survival of the Sawfly Athalia rosae Upon Infection by an Entomopathogenic Fungus and in Relation to Clerodanoid Uptake.

Larvae of the turnip sawfly Athalia rosae are a pest of Brassicacae plants, as their feeding can cause defoliation of various crops of economic importance. The larvae and the adults of this sawfly species are known to take up different classes of chemical compounds from their respective host plants, with potentially deterrent functions against predators. In addition, compounds taken up by the adults, the clerodanoids, are known for their antimicrobial activity. These features could be a challenge to biocontrol strategies. Several natural enemies of A. rosae have been identified, targeting larval and pupal stages of A. rosae, which could potentially be used as biocontrol agents. However, targeting the adult stage of a larval pest in addition to targeting the juvenile stages may improve population control. In this study, we ask whether a strain of the entomopathogenic fungus Beauveria bassiana shows biological activity against A. rosae adults. We also investigate whether the behavior of clerodanoid uptake by the adults, which is commonly found, affects their survival in response to a B. bassiana exposure. We found a clear dose-response relationship, i.e., with increasing fungal conidia concentrations survival of A. rosae decreased. However, there was only a low incidence of mycelial growth and sporulation from A. rosae cadavers, indicating that either the fungus is not successfully developing inside this host, or it is not able to re-emerge from it. Clerodanoid uptake decreased the survival of healthy adults; however, it did not increase their survival to B. bassiana. Our results revealed that this strain of B. bassiana if applied alone is probably not suitable for biocontrol of this sawfly species, because A. rosae showed a high baseline resistance against this fungus. The behavior of clerodanoid uptake is unlikely to have evolved as a defense against this entomopathogenic fungus.

Zinc can counteract selection for ciprofloxacin resistance.

Antimicrobial resistance (AMR) has emerged as one of the most pressing threats to public health. AMR evolution occurs in the clinic but also in the environment, where antibiotics and heavy metals can select and co-select for AMR. While the selective potential of both antibiotics and metals is increasingly well-characterized, experimental studies exploring their combined effects on AMR evolution are rare. It has previously been demonstrated that fluoroquinolone antibiotics such as ciprofloxacin can chelate metal ions. To investigate how ciprofloxacin resistance is affected by the presence of metals, we quantified selection dynamics between a ciprofloxacin-susceptible and a ciprofloxacin-resistant Escherichia coli strain across a gradient of ciprofloxacin concentrations in presence and absence of zinc. The presence of zinc reduced growth of both strains, while ciprofloxacin inhibited exclusively the susceptible one. When present in combination zinc retained its inhibitory effect, while ciprofloxacin inhibition of the susceptible strain was reduced. Consequently, the minimal selective concentration for ciprofloxacin resistance increased up to five-fold in the presence of zinc. Environmental pollution usually comprises complex mixtures of antimicrobial agents. In addition to the usual focus on additive or synergistic interactions in complex selective mixtures, our findings highlight the importance of antagonistic selective interactions when considering resistance evolution.