Differential proteome profiling of bacterial culture supernatants reveals candidates for the induction of oral immune priming in the red flour beetle.

Most organisms are host to symbionts and pathogens, which led to the evolution of immune strategies to prevent harm. Whilst the immune defences of vertebrates are classically divided into innate and adaptive, insects lack specialized cells involved in adaptive immunity, but have been shown to exhibit immune priming: the enhanced survival upon infection after a first exposure to the same pathogen or pathogen-derived components. An important piece of the puzzle are the pathogen-associated molecules that induce these immune priming responses. Here, we make use of the model system consisting of the red flour beetle (Tribolium castaneum) and its bacterial pathogen Bacillus thuringiensis, to compare the proteomes of culture supernatants of two closely related B. thuringiensis strains that either induce priming via the oral route, or not. Among the proteins that might be immunostimulatory to T. castaneum, we identify the Cry3Aa toxin, an important plasmid-encoded virulence factor of B. thuringiensis. In further priming-infection assays we test the relevance of Cry-carrying plasmids for immune priming. Our findings provide valuable insights for future studies to perform experiments on the mechanisms and evolution of immune priming.

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.

The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor.

Introduction: Upon infection, insect hosts simultaneously express a cocktail of antimicrobial peptides (AMPs) which can impede pathogen colonization and increase host fitness. It has been proposed that such a cocktail might be adaptive if the effects of co-expressed AMPs are greater than the sum of individual activities. This could potentially prevent the evolution of bacterial resistance. However, in vivo studies on AMPs in combination are scarce. Attacins are one of the relatively large AMP families, which show anti-Gram-negative activity in vitro. Material and methods: Here, we used RNA interference (RNAi) to silence three members of the Attacin family genes in the mealworm beetle, Tenebrio molitor: (TmAttacin1a (TmAtt1a), TmAttacin1b (TmAtt1b), and TmAttacin2 (TmAtt2) both individually and in combination. We then infected T. molitor with the Gram negative entomopathogen Pseudomonas entomophila. Results: We found that survival of the beetles was only affected by the knockdown of TmAttacin1b, TmAttacin2 and the knockdown of all three Attacins together. Triple knockdown, rather than individual or double knockdowns of AMPs, changes the temporal dynamics of their efficiency in controlling the colonization of P. entomophila in the insect body. Discussion: More precisely, AMP gene expression influences P. entomophila load early in the infection process, resulting in differences in host survival. Our results highlight the importance of studying AMP-interactions in vivo.

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.

Comparative Mortality and Adaptation of a Smurf Assay in two Species of Tenebrionid Beetles Exposed to Bacillus thuringiensis.

Bacillus thuringiensis is a spore-forming bacterium which infects insect larvae naturally via the oral route. Its virulence factors interact with the epithelium of the digestive tract of insect larvae, disrupting its function and eventually leading to the death of susceptible hosts. The most cited B. thuringiensis killing mechanism is the extensive damage caused to the insect midgut, leading to its leakage. The mortality caused by B. thuringiensis has been shown to vary between serovars and isolates, as well as between host life stages. Moreover, whether susceptibility to B. thuringiensis-induced gut leakage is generalized to all host species and whether there is individual variation within species is unclear. In this study, we adapted a non-invasive "Smurf" assay from Drosophila melanogaster to two species of tenebrionid beetles: The mealworm beetle Tenebrio molitor and the red flour beetle Tribolium castaneum, during exposure to B. thuringiensis. We highlight a differential mortality between two age/size classes of T. molitor larvae, as well as different killing dynamics between B. thuringiensis var. tenebrionis and var. tolworthi in T. castaneum. The Smurf assay did not reveal a high occurrence of extensive gut disintegration in both host species upon ingestion during B. thuringiensis exposure.

A constitutively expressed antifungal peptide protects Tenebrio molitor during a natural infection by the entomopathogenic fungus Beauveria bassiana.

Antimicrobial peptides have been well studied in the context of bacterial infections. Antifungal peptides have received comparatively less attention. Fungal pathogens of insects and their hosts represent a unique opportunity to study host-pathogen interactions due to the million of years of co-evolution they share. In this study, we investigated role of a constitutively expressed thaumatin-like peptide with antifungal activity expressed by the mealworm beetle Tenebrio molitor, named Tenecin 3, during a natural infection with the entomopathogenic fungus Beauveria bassiana. We monitored the effect of the expression of Tenecin 3 on the survival of infected hosts as well as on the progression of the fungal infection inside the host. Finally, we tested the activity of Tenecin 3 against B. bassiana. These findings could help improving biocontrol strategies and help understanding the evolution of antifungal peptides as a defense mechanism.

Evolution of defence cocktails: Antimicrobial peptide combinations reduce mortality and persistent infection.

The simultaneous expression of costly immune effectors such as multiple antimicrobial peptides is a hallmark of innate immunity of multicellular organisms, yet the adaptive advantage remains unresolved. Here, we test current hypotheses on the evolution of such defence cocktails. We use RNAi gene knock-down to explore, the effects of three highly expressed antimicrobial peptides, displaying different degrees of activity in vitro against Staphylococcus aureus, during an infection in the beetle Tenebrio molitor. We find that a defensin confers no survival benefit but reduces bacterial loads. A coleoptericin contributes to host survival without affecting bacterial loads. An attacin has no individual effect. Simultaneous knock-down of the defensin with the other AMPs results in increased mortality and elevated bacterial loads. Contrary to common expectations, the effects on host survival and bacterial load can be independent. The expression of multiple AMPs increases host survival and contributes to the control of persisting infections and tolerance. This is an emerging property that explains the adaptive benefit of defence cocktails.

Trans-generational Immune Priming Protects the Eggs Only against Gram-Positive Bacteria in the Mealworm Beetle.

In many vertebrates and invertebrates, offspring whose mothers have been exposed to pathogens can exhibit increased levels of immune activity and/or increased survival to infection. Such phenomena, called "Trans-generational immune priming" (TGIP) are expected to provide immune protection to the offspring. As the offspring and their mother may share the same environment, and consequently similar microbial threats, we expect the immune molecules present in the progeny to be specific to the microbes that immune challenged the mother. We provide evidence in the mealworm beetle Tenebrio molitor that the antimicrobial activity found in the eggs is only active against Gram-positive bacteria, even when females were exposed to Gram-negative bacteria or fungi. Fungi were weak inducers of TGIP while we obtained similar levels of anti-Gram-positive activity using different bacteria for the maternal challenge. Furthermore, we have identified an antibacterial peptide from the defensin family, the tenecin 1, which spectrum of activity is exclusively directed toward Gram-positive bacteria as potential contributor to this antimicrobial activity. We conclude that maternal transfer of antimicrobial activity in the eggs of T. molitor might have evolved from persistent Gram-positive bacterial pathogens between insect generations.

Differential expression and costs between maternally and paternally derived immune priming for offspring in an insect.

1. When parasitized, both vertebrates and invertebrates can enhance the immune defence of their offspring, although this transfer of immunity is achieved by different mechanisms. In some insects, immune-challenged males can also initiate trans-generational immune priming (TGIP), but its expressions appear qualitatively different from the one induced by females similarly challenged. 2. The existence of male TGIP challenges the traditional view of the parental investment theory, which predicts that females should invest more into their progeny than males. However, sexual dimorphism in life-history strategies and the potential costs associated with TGIP may nevertheless lead to dissymmetric investment between males and females into the immune protection of the offspring. 3. Using the yellow mealworm beetle, Tenebrio molitor, we show that after parental exposure to a bacterial-like infection, maternal and paternal TGIP are associated with the enhancement of different immune effectors and different fitness costs in the offspring. While all the offspring produced by challenged mothers had enhanced immune defence, only those from early reproductive episodes were immune primed by challenged fathers. 4. Despite the fact that males and females may share a common interest in providing their offspring with an immune protection from the current pathogenic threat, they seem to have evolved different strategies concerning this investment.