Transcriptomic landscape of the interaction between the entomopathogenic fungus Beauveria bassiana and its tolerant host Tribolium castaneum revealed by dual RNA-seq.

Entomopathogenic fungi such as Beauveria bassiana are the only insect pathogens able to start the infection process by penetrating through the host cuticle. However, some insects try to avoid fungal infection by embedding their cuticle with antifungal compounds. This is the case of the red flour beetle Tribolium castaneum, which generates economical loss of great significance in stored product environments worldwide. In this study, T. castaneum adults were fed during different time periods (from 3 to 72 h) on B. bassiana conidia-covered corn kernels. The progression of fungal infection was monitored using the dual RNA-seq technique to reconstruct the temporal transcriptomic profile and to perform gene enrichment analyses in both interacting organisms. After mapping the total reads with the B. bassiana genome, 904 genes were identified during this process. The more expressed fungal genes were related to carbon catabolite repression, cation binding, peptidase inhibition, redox processes, and stress response. Several immune-related genes from Toll, IMD, and JNK pathways, as well as genes related to chitin modification, were found to be differentially expressed in fungus-exposed T. castaneum. This study represents the first dual transcriptomic approach to help understand the interaction between the entomopathogenic fungus B. bassiana and its tolerant host T. castaneum.

Diet supplementation with egg yolk powder fattens the beetle Tribolium castaneum.

Insects have become essential models in studying human metabolic diseases, mainly due to their low maintenance cost and available tools. Both mutations and modified diets induce metabolic states similar to human obesity and diabetes. Here, we explore the effect of a high-calorie, high-fat diet on the metabolism of the beetle Tribolium castaneum. Supplementation of the wheat flour diet with powdered egg yolk for 3 weeks increased the total triacylglycerol and accelerated larval development. In addition, this diet increased the triacylglycerol levels of adult beetles. However, this egg yolk supplementation did not alter the larvae's total glucose levels or lipogenic capacity and ATP citrate lyase activity. The diet also did not change the expression profile of several lipid and carbohydrate metabolism genes and insulin-like peptides. Thus, we conclude that the diet supplemented with egg yolk induces increased fat without causing diabetes phenotypes, as seen in other hypercaloric diets in insects.

Inorganic pyrophosphatase from the red flour beetle (Tribolium castaneum) modulates mitochondrial polyphosphate metabolism.

Polyphosphates (polyPs) have been found in all cell types examined to date and play diverse roles, depending on the cell type. In eukaryotic organisms, polyPs have been mainly investigated in mammalian cells, with few studies on insects. In this study, we investigated mitochondrial polyphosphate metabolism in the red flour beetle, Tribolium castaneum. Substrate specificity for different chain lengths demonstrated the presence of two exopolyphosphatase isoforms in mitochondria. T. castaneum mitochondrial polyP levels decreased after injection with soluble pyrophosphatase (Tc-sPPase) dsRNA, while the membrane exopolyphosphate activity increased. Mitochondrial respiration modulated exopolyphosphatase activity only in wild-type beetles. Tripolyphosphate was able to increase the F-ATPase activity in wild-type and Tc-sPPase RNAi beetles. We suggest that inorganic pyrophosphatase modulates polyphosphate metabolism in mitochondria and affects the link between mitochondrial activity and polyphosphate metabolism in T. castaneum.

Neurotoxic Effects of Linalool and ß-Pinene on Tribolium castaneum Herbst.

Effective, ethical pest control requires the use of chemicals that are highly specific, safe, and ecofriendly. Linalool and ß-pinene occur naturally as major constituents of the essential oils of many plant species distributed throughout the world, and thus meet these requirements. These monoterpenes were tested as repellents against Tribolium castaneum, using the area preference method, after four hours of exposure and the effect transcriptional of genes associated with neurotransmission. Changes in gene expression of acetylcholinesterase (Ace1), GABA-gated anion channel splice variant 3a6a (Rdl), GABA-gated ion channel (Grd), glutamate-gated chloride channel (Glucl), and histamine-gated chloride channel 2 (Hiscl2) were assessed and the interaction with proteins important for the insect using in silico methods was also studied. For linalool and ß-pinene, the repellent concentration 50 (RC50) values were 0.11 µL/cm² and 0.03 µL/cm², respectively. Both compounds induced overexpression of Hiscl2 gen in adult insects, and ß-pinene also promoted the overexpression of Grd and the Ace1 gene. However, ß-pinene and linalool had little potential to dock on computer-generated models for GABA-gated ion channel LCCH3, nicotinic acetylcholine receptor subunits alpha1 and alpha2, and putative octopamine/tyramine receptor proteins from T. castaneum as their respective binding affinities were marginal, and therefore the repellent action probably involved mechanisms other than direct interaction with these targets. Results indicated that ß-pinene was more potent than linalool in inducing insect repellency, and also had a greater capacity to generate changes in the expression of genes involved in neuronal transmission.

Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.