Effect of CO2 concentrations on entomopathogen fitness and insect-pathogen interactions

Numerous insect species and their associated microbial pathogens are exposed to elevated CO2 concentrations in both artificial and natural environments. However, the impacts of elevated CO2 on the fitness of these pathogens and the susceptibility of insects to pathogen infections are not well understood. The yellow mealworm, Tenebrio molitor, is commonly produced for food and feed purposes in mass-rearing systems, which increases risk of pathogen infections. Additionally, entomopathogens are used to control T. molitor, which is also a pest of stored grains. It is therefore important to understand how elevated CO2 may affect both the pathogen directly and impact on host-pathogen interactions. We demonstrate that elevated CO2 concentrations reduced the viability and persistence of the spores of the bacterial pathogen Bacillus thuringiensis. In contrast, conidia of the fungal pathogen Metarhizium brunneum germinated faster under elevated CO2. Pre-exposure of the two pathogens to elevated CO2 prior to host infection did not affect the survival probability of T. molitor larvae. However, larvae reared at elevated CO2 concentrations were less susceptible to both pathogens compared to larvae reared at ambient CO2 concentrations. Our findings indicate that whilst elevated CO2 concentrations may be beneficial in reducing host susceptibility in mass-rearing systems, they may potentially reduce the efficacy of the tested entomopathogens when used as biological control agents of T. molitor larvae. We conclude that CO2 concentrations should be carefully selected and monitored as an additional environmental factor in laboratory experiments investigating insect-pathogen interactions

Effets des aliments et bactéries probiotiques sur la santé de Tenebrio molitor et la résistance aux entomopathogènes

Most mass reared insect can be exposed to entomopathogens with risk of infections, while the impact will depend notably on the environmental conditions, the nutritional feed quality and the microbiota composition of the insects. The yellow mealworm, Tenebrio molitor, is an edible insect mainly reared on cereal-based diets and other agricultural by-products, which can potentially expose the insect to entomopathogens as Bacillus thuringiensis (Btt) or Metarhizium brunneum. In this thesis question related to the evaluation on the impact of insect feed quality and the addition of two bacterial probiotics (alive/killed) on larval growth and susceptibility to two entomopathogens were addressed by performing in vivo experiments with a Btt strain active on Coleopteran larvae. The experimental protocols were based on free feeding method and larval densities values to mimicking the mass rearing environment. A first study contains results on Btt impact on yellow mealworm larval survival and growth and its persistence in larvae and frass. The results highlight direct correlation between larval body mass and entomopathogen susceptibility, larvae of higher mass being less susceptible. Effects on growth and feed conversion ratio of survivors showed significant impact for low mass larvae while less or no effect was recorded for higher mass larva. Btt was still recovered from larvae and frass 14 days after feeding with non-contaminated wheat bran, indicating a certain level of persistence, and therefore a potential risk in rearing facilities. A second aspect considered analysis on the bacterial microbiota composition and abundance to measure the impact on the microbiota and the persistence of the two probiotics and the bacterial pathogen. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactobacillus plantarum WJB were added in vital and deactivated form to wheat bran with and without egg-white powder, from egg hatching to early larval developmental stages. Overall, the presence of the entomopathogens was the significant variable that affected the larval growth. Along the analysis on insect performances, the bacterial microbiota composition by 16S rDNA sequencing pre-pathogen exposure (day 0), and at days 3 and 14 after inoculation with the pathogens was analysed. In all the samples, P. pentosaceus was detected from day 0 through-out the assay at the species level and Lb. plantarum was not detectable. The diet containing dried egg white showed no differences in relation to microbial richness after probiotic provision, suggesting a potential antimicrobial action of lysozymes on the insect microbial community. In the wheat bran diet, the effect of vital or deactivated probiotic provision was observed in terms of genera abundances and richness in the two diets. At day14, the bacterial community composition of the larvae was similar in all treatments, indicating that the pathogen did not establish at detectable level in the insect. A third aspect aimed to investigate to what level the feed composition (low and high protein content) and probiotic addition can influence larval growth and the protein and lipid composition of the larva. The experimental diets based on wheat bran, as in the former assays, were formulated by reducing the protein content by including potato starch. Overall, the chemical larval composition was influenced by the diet composition and not by probiotic provision. However, the provision of the probiotic strain resulted in larvae with FAMEs profiles characterized by an increment in MUFAs and PUFAs. The results highlight the possibility to obtain larvae with a fatty acid profile that is nutritionally more favorable for humans and animals. A Final discussion provides an overview on the obtained results in the context of insect a mass rearing and new technologies developed during these years. Perspectives based on omics-analyses approaches are suggested for unravelling host-microbiota and diet interactions.La plupart des insectes élevés en masse peuvent être exposés à des entomopathogènes avec des risques d'infection, tandis que l'impact dépendra notamment des conditions environnementales, de la qualité nutritionnelle de l'alimentation et de la composition du microbiote des insectes. Le ver de farine, Tenebrio molitor, est un insecte comestible principalement élevé avec des régimes à base de céréales et d'autres sous-produits agricoles, ce qui peut potentiellement exposer l'insecte à des entomopathogènes comme Bacillus thuringiensis (Btt) ou Metarhizium brunneum. Dans cette thèse, la question liée à l'évaluation de l'impact de la qualité de l'alimentation des insectes et de l'ajout de deux probiotiques bactériens (vivants/tués) sur la croissance larvaire et la sensibilité à deux entomopathogènes a été abordée en réalisant des expériences in vivo avec une souche Btt active sur les larves des Coléoptères. Les protocoles expérimentaux étaient basés sur une méthode d'alimentation libre et des densités larvaire imitant l'environnement d'élevage de masse. Une première étude concerne des résultats sur l'impact du Btt sur la survie et la croissance des larves du ver de farine ainsi que sur sa persistance dans les larves et les excréments. Les résultats mettent en évidence une corrélation directe entre la masse corporelle des larves et la sensibilité aux entomopathogènes ; les larves de masse plus élevée étant moins sensibles. Le Btt était encore présent dans les larves et des excréments 14 jours après avoir été nourri avec du son de blé non contaminé, ce qui indique un certain niveau de persistance, et donc un risque potentiel dans les installations d'élevage. Un deuxième aspect a porté sur l'analyse de la composition et de l'abondance du microbiote bactérien afin de mesurer l'impact sur le microbiote et la persistance des deux probiotiques et de l'agent pathogène bactérien. Deux espèces bactériennes probiotiques, Pediococcus pentosaceus KVL B19-01 et Lactobacillus plantarum WJB, ont été ajoutées sous forme vivante et désactivée au son de blé avec et sans poudre de blanc d'œuf, et ceci depuis l'éclosion des œufs jusqu'aux premiers stades de développement larvaire. Globalement la présence d'agents entomopathogènes était la variable la plus significative affectant la croissance larvaire. Parallèlement à l'analyse des performances des insectes, la composition du microbiote bactérien a été analysée par séquençage de l'ADNr 16S avant l'exposition aux agents pathogènes (jour 0), ainsi qu'aux jours 3 et 14 après l'inoculation des agents pathogènes. Dans tous les échantillons, P. pentosaceus a été détecté dès le jour 0 tout au long du test, contrairement à l'espèce et Lb. plantarum qui n'était pas détectable. Le régime alimentaire contenant du blanc d'œuf séché n'a montré aucune différence en ce qui concerne la richesse microbienne après l'apport de probiotiques, ce qui suggère une action antimicrobienne potentielle des lysozymes sur la communauté microbienne des insectes. Dans le régime alimentaire au son de blé, l'effet de l'apport de probiotiques vivants ou désactivés a été observé en termes d'abondance et de richesse des genres bactériens pour les deux régimes. Un troisième aspect visait à étudier dans quelle mesure la composition alimentaire (teneur faible et élevée en protéines) et l'ajout de probiotiques peuvent influencer la croissance larvaire et la composition protéique et lipidique de la larve. Dans l'ensemble, la composition chimique des larves était influencée par la composition du régime alimentaire et non par l'apport de probiotiques. Les résultats mettent en évidence la possibilité d'obtenir des larves avec un profil en acides gras nutritionnellement plus favorable pour l'humain et l'animal. Une discussion finale donne un aperçu des résultats obtenus et sur des perspectives basées sur des approches d'analyses omiques pour démêler les interactions hôte-microbiote et régime alimentaire

Effets des aliments et bactéries probiotiques sur la santé de Tenebrio molitor et la résistance aux entomopathogènes

Most mass reared insect can be exposed to entomopathogens with risk of infections, while the impact will depend notably on the environmental conditions, the nutritional feed quality and the microbiota composition of the insects. The yellow mealworm, Tenebrio molitor, is an edible insect mainly reared on cereal-based diets and other agricultural by-products, which can potentially expose the insect to entomopathogens as Bacillus thuringiensis (Btt) or Metarhizium brunneum. In this thesis question related to the evaluation on the impact of insect feed quality and the addition of two bacterial probiotics (alive/killed) on larval growth and susceptibility to two entomopathogens were addressed by performing in vivo experiments with a Btt strain active on Coleopteran larvae. The experimental protocols were based on free feeding method and larval densities values to mimicking the mass rearing environment. A first study contains results on Btt impact on yellow mealworm larval survival and growth and its persistence in larvae and frass. The results highlight direct correlation between larval body mass and entomopathogen susceptibility, larvae of higher mass being less susceptible. Effects on growth and feed conversion ratio of survivors showed significant impact for low mass larvae while less or no effect was recorded for higher mass larva. Btt was still recovered from larvae and frass 14 days after feeding with non-contaminated wheat bran, indicating a certain level of persistence, and therefore a potential risk in rearing facilities. A second aspect considered analysis on the bacterial microbiota composition and abundance to measure the impact on the microbiota and the persistence of the two probiotics and the bacterial pathogen. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactobacillus plantarum WJB were added in vital and deactivated form to wheat bran with and without egg-white powder, from egg hatching to early larval developmental stages. Overall, the presence of the entomopathogens was the significant variable that affected the larval growth. Along the analysis on insect performances, the bacterial microbiota composition by 16S rDNA sequencing pre-pathogen exposure (day 0), and at days 3 and 14 after inoculation with the pathogens was analysed. In all the samples, P. pentosaceus was detected from day 0 through-out the assay at the species level and Lb. plantarum was not detectable. The diet containing dried egg white showed no differences in relation to microbial richness after probiotic provision, suggesting a potential antimicrobial action of lysozymes on the insect microbial community. In the wheat bran diet, the effect of vital or deactivated probiotic provision was observed in terms of genera abundances and richness in the two diets. At day14, the bacterial community composition of the larvae was similar in all treatments, indicating that the pathogen did not establish at detectable level in the insect. A third aspect aimed to investigate to what level the feed composition (low and high protein content) and probiotic addition can influence larval growth and the protein and lipid composition of the larva. The experimental diets based on wheat bran, as in the former assays, were formulated by reducing the protein content by including potato starch. Overall, the chemical larval composition was influenced by the diet composition and not by probiotic provision. However, the provision of the probiotic strain resulted in larvae with FAMEs profiles characterized by an increment in MUFAs and PUFAs. The results highlight the possibility to obtain larvae with a fatty acid profile that is nutritionally more favorable for humans and animals. A Final discussion provides an overview on the obtained results in the context of insect a mass rearing and new technologies developed during these years. Perspectives based on omics-analyses approaches are suggested for unravelling host-microbiota and diet interactions.La plupart des insectes élevés en masse peuvent être exposés à des entomopathogènes avec des risques d'infection, tandis que l'impact dépendra notamment des conditions environnementales, de la qualité nutritionnelle de l'alimentation et de la composition du microbiote des insectes. Le ver de farine, Tenebrio molitor, est un insecte comestible principalement élevé avec des régimes à base de céréales et d'autres sous-produits agricoles, ce qui peut potentiellement exposer l'insecte à des entomopathogènes comme Bacillus thuringiensis (Btt) ou Metarhizium brunneum. Dans cette thèse, la question liée à l'évaluation de l'impact de la qualité de l'alimentation des insectes et de l'ajout de deux probiotiques bactériens (vivants/tués) sur la croissance larvaire et la sensibilité à deux entomopathogènes a été abordée en réalisant des expériences in vivo avec une souche Btt active sur les larves des Coléoptères. Les protocoles expérimentaux étaient basés sur une méthode d'alimentation libre et des densités larvaire imitant l'environnement d'élevage de masse. Une première étude concerne des résultats sur l'impact du Btt sur la survie et la croissance des larves du ver de farine ainsi que sur sa persistance dans les larves et les excréments. Les résultats mettent en évidence une corrélation directe entre la masse corporelle des larves et la sensibilité aux entomopathogènes ; les larves de masse plus élevée étant moins sensibles. Le Btt était encore présent dans les larves et des excréments 14 jours après avoir été nourri avec du son de blé non contaminé, ce qui indique un certain niveau de persistance, et donc un risque potentiel dans les installations d'élevage. Un deuxième aspect a porté sur l'analyse de la composition et de l'abondance du microbiote bactérien afin de mesurer l'impact sur le microbiote et la persistance des deux probiotiques et de l'agent pathogène bactérien. Deux espèces bactériennes probiotiques, Pediococcus pentosaceus KVL B19-01 et Lactobacillus plantarum WJB, ont été ajoutées sous forme vivante et désactivée au son de blé avec et sans poudre de blanc d'œuf, et ceci depuis l'éclosion des œufs jusqu'aux premiers stades de développement larvaire. Globalement la présence d'agents entomopathogènes était la variable la plus significative affectant la croissance larvaire. Parallèlement à l'analyse des performances des insectes, la composition du microbiote bactérien a été analysée par séquençage de l'ADNr 16S avant l'exposition aux agents pathogènes (jour 0), ainsi qu'aux jours 3 et 14 après l'inoculation des agents pathogènes. Dans tous les échantillons, P. pentosaceus a été détecté dès le jour 0 tout au long du test, contrairement à l'espèce et Lb. plantarum qui n'était pas détectable. Le régime alimentaire contenant du blanc d'œuf séché n'a montré aucune différence en ce qui concerne la richesse microbienne après l'apport de probiotiques, ce qui suggère une action antimicrobienne potentielle des lysozymes sur la communauté microbienne des insectes. Dans le régime alimentaire au son de blé, l'effet de l'apport de probiotiques vivants ou désactivés a été observé en termes d'abondance et de richesse des genres bactériens pour les deux régimes. Un troisième aspect visait à étudier dans quelle mesure la composition alimentaire (teneur faible et élevée en protéines) et l'ajout de probiotiques peuvent influencer la croissance larvaire et la composition protéique et lipidique de la larve. Dans l'ensemble, la composition chimique des larves était influencée par la composition du régime alimentaire et non par l'apport de probiotiques. Les résultats mettent en évidence la possibilité d'obtenir des larves avec un profil en acides gras nutritionnellement plus favorable pour l'humain et l'animal. Une discussion finale donne un aperçu des résultats obtenus et sur des perspectives basées sur des approches d'analyses omiques pour démêler les interactions hôte-microbiote et régime alimentaire

Bugs in Bugs: The Role of Probiotics and Prebiotics in Maintenance of Health in Mass-Reared Insects

International audienceInteractions between insects and their microbiota affect insect behaviour and evolution. When specific microorganisms are provided as a dietary supplement, insect reproduction, food conversion and growth are enhanced and health is improved in cases of nutritional deficiency or pathogen infection. The purpose of this review is to provide an overview of insect–microbiota inter-actions, to review the role of probiotics, their general use in insects reared for food and feed, and their interactions with the host microbiota. We review how bacterial strains have been selected for insect species reared for food and feed and discuss methods used to isolate and measure the effectiveness of a probiotic. We outline future perspectives on probiotic applications in mass-reared insects

Effect of the rearing diet on gene expression of antimicrobial peptides in Hermetia illucens (Diptera: Stratiomyidae)

Insect proteins have been proposed for human and animal food production. Safeguarding the health status of insects in mass rearing allows to obtain high-quality products and to avoid severe economic losses due to entomopathogens. Therefore, new strategies for preserving insect health must be implemented. Modulation of the insect immune system through the diet is one such strategy. We evaluated gene expression of two antimicrobial peptides (one defensin and one cecropin) in Hermetia illucens (L.) (Diptera: Stratiomyidae) reared on different diets. Analyses were performed on prepupae and 10-day-old larvae reared on cereal- and municipal organic waste-based diets and on only prepupae reared on a cereal-based diet supplemented with sunflower, corn, or soybean oil. The inclusion of sunflower oil at different points in the cereal-based diet was also evaluated. Moreover, diet-driven differences in the inhibitory activity of the hemolymph were tested against Escherichia coli DH5α and Micrococcus yunnanensis HI55 using diffusion assays in solid media. Results showed that a municipal organic waste-based diet produced a significant overexpression of antimicrobial peptides only in prepupae. Inclusion of vegetable oils caused an upregulation of at least one peptide, except for the corn oil. Higher expression of both genes was observed when sunflower oil was added 5 days before pupation. All hemolymph samples showed an inhibitory activity against bacteria colonies. Our results suggest that municipal organic waste-based diet and vegetable oil-added diet may successfully impact the immune system of H. illucens. Such alternatives may also exist for other species of economic interest

Prognostic Significance of Grade Discrepancy Between Primary Tumor and Venous Thrombus in Nonmetastatic Clear-cell Renal Cell Carcinoma: Analysis of the REMEMBER Registry and Implications for Adjuvant Therapy

Background: Further stratification of the risk of recurrence of clear-cell renal cell carcinoma (ccRCC) with venous tumor thrombus (VTT) will facilitate selection of candidates for adjuvant therapy. Objective: To assess the impact of tumor grade discrepancy (GD) between the primary tumor (PT) and VTT in nonmetastatic ccRCC on disease-free survival (DFS), overall survival (OS), and cancer-specific survival (CSS). Design, setting, and participants: This was a retrospective analysis of a multi-institutional nationwide data set for patients with pT3N0M0 ccRCC who underwent radical nephrectomy and thrombectomy. Outcomes measurements and statistical analysis: Pathology slides were centrally reviewed. GD, a bidirectional variable (upgrading or downgrading), was numerically defined as the VTT grade minus the PT grade. Multivariable models were built to predict DFS, OS, and CSS. Results and limitations: We analyzed data for 604 patients with median follow-up of 42 mo (excluding events). Tumor GD between VTT and PT was observed for 47% (285/604) of the patients and was an independent risk factor with incremental value in predicting the outcomes of interest (all p < 0.05). Incorporation of tumor GD significantly improved the performance of the ECOG-ACRIN 2805 (ASSURE) model. A GD-based model (PT grade, GD, pT stage, PT sarcomatoid features, fat invasion, and VTT consistency) had a c index of 0.72 for DFS. The hazard ratios were 8.0 for GD = +2 (p < 0.001), 1.9 for GD = +1 (p < 0.001), 0.57 for GD = -1 (p = 0.001), and 0.22 for GD = -2 (p = 0.003) versus GD = 0 as the reference. According to model-converted risk scores, DFS, OS, and CSS significantly differed between subgroups with low, intermediate, and high risk (all p < 0.001). Conclusions: Routine reporting of VTT upgrading or downgrading in relation to the PT and use of our GD-based nomograms can facilitate more informed treatment decisions by tailoring strategies to an individual patient's risk of progression. Patient summary: We developed a tool to improve patient counseling and guide decision-making on other therapies in addition to surgery for patients with the clear-cell type of kidney cancer and tumor invasion of a vein