Tribolium castaneum as a whole-animal screening system for the detection and characterization of neuroprotective substances.

Parkinson's disease (PD) is a movement disorder caused by the progressive loss of dopaminergic neurons. Natural antioxidants and plant extracts with neuroprotective properties offer a promising new therapeutic approach for PD patients, but a suitable large-scale screening system is required for their discovery and preclinical analysis. Here we used the red flour beetle (Tribolium castaneum ) as a whole-animal screening system for the detection and characterization of neuroprotective substances. Paraquat was added to the diet of adult beetles to induce PD-like symptoms, which were quantified using a novel positive geotaxis behavioral assay. These paraquat-induced behavioral changes were reduced in beetles fed on diets supplemented with l-dihydroxyphenylalanine, ascorbic acid, curcumin, hempseed flour, or the Chinese herb gou-teng. T. castaneum is, therefore, a valuable model for the screening of neuroprotective substances in chemical libraries and plant extracts and could be developed as a model for the preclinical testing of therapeutic candidates for the treatment of neurodegenerative diseases, such as PD.

Cross-Resistance: A Consequence of Bi-partite Host-Parasite Coevolution.

Host-parasite coevolution can influence interactions of the host and parasite with the wider ecological community. One way that this may manifest is in cross-resistance towards other parasites, which has been observed to occur in some host-parasite evolution experiments. In this paper, we test for cross-resistance towards Bacillus thuringiensis and Pseudomonasentomophila in the red flour beetle Triboliumcastaneum, which was previously allowed to coevolve with the generalist entomopathogenic fungus Beauveriabassiana. We combine survival and gene expression assays upon infection to test for cross-resistance and underlying mechanisms. We show that larvae of T.castaneum that evolved with B.bassiana under coevolutionary conditions were positively cross-resistant to the bacterium B. thuringiensis, but not P.entomophila. Positive cross-resistance was mirrored at the gene expression level with markers that were representative of the oral route of infection being upregulated upon B.bassiana exposure. We find that positive cross-resistance towards B. thuringiensis evolved in T.castaneum as a consequence of its coevolutionary interactions with B.bassiana. This cross-resistance appears to be a consequence of resistance to oral toxicity. The fact that coevolution with B.bassiana results in resistance to B. thuringiensis, but not P.entomophila implies that B. thuringiensis and B.bassiana may share mechanisms of infection or toxicity not shared by P.entomophila. This supports previous suggestions that B.bassiana may possess Cry-like toxins, similar to those found in B. thuringiensis, which allow it to infect orally.

Tribolium castaneum gene expression changes after Paranosema whitei infection.

BACKGROUND: Microsporidia are obligate parasites that possess some of the smallest eukaryotic genomes. Several insect species are susceptible to infections by microsporidian parasites. Paranosema whitei frequently infects young larvae of Tribolium castaneum and obligately kills the host whereupon transmission to subsequent hosts is accomplished via spores. P. whitei infection results in developmental arrest of T. castaneum, preventing larvae from pupation. The mechanisms underlying P. whitei virulence as well as the molecular underpinning of host defenses remain uncharacterized. In the present study, we evaluated gene expression differences of T. castaneum infected with the microsporidian parasite P. whitei. RESULTS: More than 1500 T. castaneum genes were differentially expressed after infection with P. whitei. Several important host pathways appeared to be differentially expressed after infection, where immune genes were among the highest differential expressed genes. Genes involved in the Toll pathway and its effectors were specifically upregulated. Furthermore, iron homeostasis processes and transmembrane transport appeared significantly altered after P. whitei infection. Krüppel homolog 1 (Kr-h1) and other genes of the juvenile hormone (JH) pathway appeared differentially expressed after parasite infection. In addition, a small number of long intergenic non-coding RNAs (lincRNAs) appeared differentially expressed after P. whitei infection. CONCLUSION: In this study we characterized for the first time using RNA-seq the immune response of T. castaneum to P. whitei. Other pathways (transmembrane transport, iron homeostasis, protein synthesis, JH) indicate possible alterations of the host by the parasite such as a possible developmental arrest caused by JH regulation. Furthermore we find evidence that some lincRNAs might be connected to defense as previously reported for other insect species.

Cryptic changes in immune response and fitness in Tribolium castaneum as a consequence of coevolution with Beauveria bassiana.

Immunity is a key trait in host defence against parasites and is thus likely to be under selection during host-parasite coevolution. Broadly, the immune system consists of several lines of defence including physiological innate immunity, physical barriers such as the cuticle, avoidance behaviours and in some cases antimicrobial secretions. The defence conferring the highest fitness benefit may be situation specific and depend on the taxon and infection route of the parasite. We carried out a host-parasite coevolution experiment between the red flour beetle T. castaneum, which possesses a comprehensive immune system including the ability to secrete antimicrobial compounds into its environment, and the generalist entomopathogenic fungus Beauveria bassiana. We measured levels of external immunity (benzoquinone secretion) and an internal immune trait, phenoloxidase (PO) activity throughout and in F2 to beetles at the end of the experiment. Survival (a proxy for resistance) of F2 coevolved and control beetles exposed to the fungus was also measured. No change in external immunity or survival was observed as a consequence of host-parasite coevolution, however, PO responses in evolved beetles showed increased flexibility dependent on the route of infection of the parasite. This more flexible PO response appeared to result in beetle populations being better able to cope with the parasite, buffering their fitness during the course of the coevolution experiment. This represents a subtle but significant adaptation to the presence of a parasite over evolutionary time.

A Comparison of Wolbachia Infection Frequencies in Varroa With Prevalence of Deformed Wing Virus.

Wolbachia are widely distributed bacterial endosymbionts of arthropods and filarial nematodes. These bacteria can affect host fitness in a variety of ways, such as protecting hosts against viruses and other pathogens. Here, we investigate the possible role of Wolbachia in the prevalence of the deformed wing virus (DWV), a highly virulent pathogen of honey bees (Apis mellifera) that is transmitted by parasitic Varroa mites (Varroa destructor). About 180 Varroa mites from 18 beehives were tested for infection with Wolbachia and DWV. We first screened for Wolbachia using two standard primers (wsp and 16S rDNA), and found 26% of the mites to be positive for Wolbachia using the wsp primer and 64% of the mites to be positive using the 16S rDNA primer. Using these intermediate Wolbachia frequencies, we then tested for statistical correlations with virus infection frequencies. The analysis revealed a significant positive correlation between DWV and Wolbachia using the wsp primer, but no significant association between DWV and Wolbachia using the 16S rDNA primer. In conclusion, there is no evidence for an anti-pathogenic effect of Wolbachia in V. destructor, but weak evidence for a pro-pathogenic effect. These results encourage further examination of Wolbachia-virus interactions in Varroa mites since an increased vector competence of the mites may significantly impact disease outbreaks in honey bees.

Social dilemma in the external immune system of the red flour beetle? It is a matter of time.

Sociobiology has revolutionized our understanding of interactions between organisms. Interactions may present a social dilemma where the interests of individual actors do not align with those of the group as a whole. Viewed through a sociobiological lens, nearly all interactions can be described regarding their costs and benefits, and a number of them then resemble a social dilemma. Numerous experimental systems, from bacteria to mammals, have been proposed as models for studying such dilemmas. Here, we make use of the external immune system of the red flour beetle, Tribolium castaneum, to investigate how the experimental duration can affect whether the external secretion comprises a social dilemma or not. Some beetles (secretors) produce a costly quinone-rich external secretion that inhibits microbial growth in the surrounding environment, providing the secretors with direct personal benefits. However, as the antimicrobial secretion acts in the environment of the beetle, it is potentially also advantageous to other beetles (nonsecretors), who avoid the cost of producing the secretion. We test experimentally if the secretion qualifies as a public good. We find that in the short term, costly quinone secretion can be interpreted as a public good presenting a social dilemma where the presence of secretors increases the fitness of the group. In the long run, the benefit to the group of having more secretors vanishes and becomes detrimental to the group. Therefore, in such seminatural environmental conditions, it turns out that qualifying a trait as social can be a matter of timing.

Probiotic Enterococcus mundtii Isolate Protects the Model Insect Tribolium castaneum against Bacillus thuringiensis.

Enterococcus mundtii strains isolated from the larval feces of the Mediterranean flour moth Ephestia kuehniella show antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria. The in vitro probiotic characterization of one isolate revealed a high auto-aggregation score, a hydrophilic cell surface, tolerance for low pH, no hemolytic activity, and susceptibility to all tested antibiotics. We used the red flour beetle Tribolium castaneum, an established model organism, for the in vivo characterization of one probiotic E. mundtii isolate from E. kuehniella larvae. Tribolium castaneum larvae were fed orally with the probiotic isolate or the corresponding supernatant and then infected with either the entomopathogen Bacillus thuringiensis or Pseudomonas entomophila. Larvae exposed to the isolate or the supernatant showed increased survival following infection with B. thuringiensis but not P. entomophila. Heat treatment or treatment with proteinase K reduced the probiotic effect of the supernatant. However, the increased resistance attracts a fitness penalty manifested as a shorter lifespan and reduced fertility. T. castaneum has, pending on further research, the potential as an alternative model for the pre-screening of probiotics.

Sustainable farming of the mealworm Tenebrio molitor for the production of food and feed.

The farming of edible insects is an alternative strategy for the production of protein-rich food and feed with a low ecological footprint. The industrial production of insect-derived protein is more cost-effective and energy-efficient than livestock farming or aquaculture. The mealworm Tenebrio molitor is economically among the most important species used for the large-scale conversion of plant biomass into protein. Here, we review the mass rearing of this species and its conversion into food and feed, focusing on challenges such as the contamination of food/feed products with bacteria from the insect gut and the risk of rapidly spreading pathogens and parasites. We propose solutions to prevent the outbreak of infections among farmed insects without reliance on antibiotics. Transgenerational immune priming and probiotic bacteria may provide alternative strategies for sustainable insect farming.

Highly potent host external immunity acts as a strong selective force enhancing rapid parasite virulence evolution.

Virulence is often under selection during host-parasite coevolution. In order to increase fitness, parasites are predicted to circumvent and overcome host immunity. A particular challenge for pathogens are external immune systems, chemical defence systems comprised of potent antimicrobial compounds released by prospective hosts into the environment. We carried out an evolution experiment, allowing for coevolution to occur, with the entomopathogenic fungus, Beauveria bassiana, and the red flour beetle, Tribolium castaneum, which has a well-documented external immune system with strong inhibitory effects against B. bassiana. After just seven transfers of experimental evolution we saw a significant increase in parasite induced host mortality, a proxy for virulence, in all B. bassiana lines. This apparent virulence increase was mainly the result of the B. bassiana lines evolving resistance to the beetles' external immune defences, not due to increased production of toxins or other harmful substances. Transcriptomic analyses of evolved B. bassiana implicated the up-regulation of oxidative stress resistance genes in the observed resistance to external immunity. It was concluded that external immunity acts as a powerful selective force for virulence evolution, with an increase in virulence being achieved apparently entirely by overcoming these defences, most likely due to elevated oxidative stress resistance.

Coevolution of parasitic fungi and insect hosts.

Parasitic fungi and their insect hosts provide an intriguing model system for dissecting the complex co-evolutionary processes, which result in Red Queen dynamics. To explore the genetic basis behind host-parasite coevolution we chose two parasitic fungi (Beauveria bassiana and Metarhizium anisopliae, representing the most important entomopathogenic fungi used in the biological control of pest or vector insects) and two established insect model hosts (the greater wax moth Galleria mellonella and the red flour beetle Tribolium castaneum) for which sequenced genomes or comprehensive transcriptomes are available. Focusing on these model organisms, we review the knowledge about the interactions between fungal molecules operating as virulence factors and insect host-derived defense molecules mediating antifungal immunity. Particularly the study of the intimate interactions between fungal proteinases and corresponding host-derived proteinase inhibitors elucidated novel coevolutionary mechanisms such as functional shifts or diversification of involved effector molecules. Complementarily, we compared the outcome of coevolution experiments using the parasitic fungus B. bassiana and two different insect hosts which were initially either susceptible (Galleria mellonella) or resistant (Tribolium castaneum). Taking a snapshot of host-parasite coevolution, we show that parasitic fungi can overcome host barriers such as external antimicrobial secretions just as hosts can build new barriers, both within a relatively short time of coevolution.

When experimental selection for virulence leads to loss of virulence.

Information on virulence evolution is critical for understanding disease dynamics. Theory predicts that under certain evolutionary conditions virulence should increase; for example, during host-parasite coevolution. Although these theoretical predictions are supported by natural observations, tests of these hypotheses using experimental evolution have yielded confounding and contradictory results, with discrepancies often being seen among experiments. Here we provide a critical overview of experimental tests of hypotheses regarding virulence evolution and provide potential explanations for the contradictory results. We emphasise the key role of parasite transmission mechanisms that can explain many of the observed discrepancies among evolution experiments. Finally, we make suggestions for how evolution experiments could be conducted in the future to avoid potentially confounding factors.

Rapid evolution of virulence leading to host extinction under host-parasite coevolution.

BACKGROUND: Host-parasite coevolution is predicted to result in changes in the virulence of the parasite in order to maximise its reproductive success and transmission potential, either via direct host-to-host transfer or through the environment. The majority of coevolution experiments, however, do not allow for environmental transmission or persistence of long lived parasite stages, in spite of the fact that these may be critical for the evolutionary success of spore forming parasites under natural conditions. We carried out a coevolution experiment using the red flour beetle, Tribolium castaneum, and its natural microsporidian parasite, Paranosema whitei. Beetles and their environment, inclusive of spores released into it, were transferred from generation to generation. We additionally took a modelling approach to further assess the importance of transmissive parasite stages on virulence evolution. RESULTS: In all parasite treatments of the experiment, coevolution resulted in extinction of the host population, with a pronounced increase in virulence being seen. Our modelling approach highlighted the presence of environmental transmissive parasite stages as being critical to the trajectory of virulence evolution in this system. CONCLUSIONS: The extinction of host populations was unexpected, particularly as parasite virulence is often seen to decrease in host-parasite coevolution. This, in combination with the increase in virulence and results obtained from the model, suggest that the inclusion of transmissive parasite stages is important to improving our understanding of virulence evolution.

Odoriferous Defensive stink gland transcriptome to identify novel genes necessary for quinone synthesis in the red flour beetle, Tribolium castaneum.

Chemical defense is one of the most important traits, which endow insects the ability to conquer a most diverse set of ecological environments. Chemical secretions are used for defense against anything from vertebrate or invertebrate predators to prokaryotic or eukaryotic parasites or food competitors. Tenebrionid beetles are especially prolific in this category, producing several varieties of substituted benzoquinone compounds. In order to get a better understanding of the genetic and molecular basis of defensive secretions, we performed RNA sequencing in a newly emerging insect model, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae). To detect genes that are highly and specifically expressed in the odoriferous gland tissues that secret defensive chemical compounds, we compared them to a control tissue, the anterior abdomen. 511 genes were identified in different subtraction groups. Of these, 77 genes were functionally analyzed by RNA interference (RNAi) to recognize induced gland alterations morphologically or changes in gland volatiles by gas chromatography-mass spectrometry. 29 genes (38%) presented strong visible phenotypes, while 67 genes (87%) showed alterations of at least one gland content. Three of these genes showing quinone-less (ql) phenotypes - Tcas-ql VTGl; Tcas-ql ARSB; Tcas-ql MRP - were isolated, molecularly characterized, their expression identified in both types of the secretory glandular cells, and their function determined by quantification of all main components after RNAi. In addition, microbe inhibition assays revealed that a quinone-free status is unable to impede bacterial or fungal growth. Phylogenetic analyses of these three genes indicate that they have evolved independently and specifically for chemical defense in beetles.

Paternally derived immune priming for offspring in the red flour beetle, Tribolium castaneum.

1. Parasitized females in mammals, fish and birds can enhance the immune defence of their offspring by transferring specific antibodies for the embryo. Likewise, social insect mothers transfer immunity despite the fact that invertebrates lack antibodies. 2. Female trans-generational immune priming is consistent with parental investment theory, because mothers invest more into rearing their offspring than fathers. However, when immune priming is not directly linked to parental care, as is often the case in insects that abandon their eggs after oviposition, both sexes might benefit from protecting their offspring. 3. Using the red flour beetle, Tribolium castaneum, we show that after parental exposure to heat-killed bacteria, trans-generational immune priming occurs through fathers as well as mothers. 4. This novel finding challenges the traditional view that males provide only genes to their offspring in species without paternal care, and raises the possibility of a division of tasks with respect to immune protection between parents.

Immune function and parasite resistance in male and polymorphic female Coenagrion puella.

BACKGROUND: Colour polymorphisms are widespread and one of the prime examples is the colour polymorphism in female coenagrionid damselflies: one female morph resembles the male colour (andromorph) while one, or more, female morphs are described as typically female (gynomorph). However, the selective pressures leading to the evolution and maintenance of this polymorphism are not clear. Here, based on the hypothesis that coloration and especially black patterning can be related to resistance against pathogens, we investigated the differences in immune function and parasite resistance between the different female morphs and males. RESULTS: Our studies of immune function revealed no differences in immune function between the female morphs but between the sexes in adult damselflies. In an experimental infection females infected shortly after emergence showed a higher resistance against a fungal pathogen than males, however female morphs did not differ in resistance. In a field sample of adult damselflies we did not find differences in infection rates with watermites and gregarines. CONCLUSION: With respect to resistance and immune function 'andromorph' blue females of Coenagrion puella do not resemble the males. Therefore the colour polymorphism in coenagrionid damselflies is unlikely to be maintained by differences in immunity.