Modifying insect population age structure to control vector-borne disease

Age is a critical determinant of the ability of most arthropod vectors to transmit a range of human pathogens. This is due to the fact that most pathogens require a period of extrinsic incubation in the arthropod host before pathogen transmission can occur. This developmental period for the pathogen often comprises a significant proportion of the expected lifespan of the vector. As such, only a small proportion of the population that is oldest contributes to pathogen transmission. Given this, strategies that target vector age would be expected to obtain the most significant reductions in the capacity of a vector population to transmit disease. The recent identification of biological agents that shorten vector lifespan, such as Wolbachia, entomopathogenic fungi and densoviruses, offer new tools for the control of vector-borne diseases. Evaluation of the efficacy of these strategies under field conditions will be possible due to recent advances in insect age-grading techniques. Implementation of all of these strategies will require extensive field evaluation and consideration of the selective pressures that reductions in vector longevity may induce on both vector and pathogen.

Exploring ionic liquids′ unique stimuli to elucidate uncharacterised cellular and molecular mechanisms in filamentous fungi

The emergence of new fungal pathogens, either of plants or animals, and the increasing number of reported cases of resistant human pathogenic strains to the available antifungal drugs reinforces the need for better understanding the biology of filamentous fungi. Conventional drugs target components of the fungal membrane or cell wall, therefore identifying novel intracellular targets, yet unique to fungi, is a global priority.(...)

Potential for entomopathogenic fungi to control Triatoma dimidiata (Hemiptera: Reduviidae), a vector of Chagas disease in Mexico

Introduction The use of entomopathogenic fungi to control disease vectors has become relevant because traditional chemical control methods have caused damage to the environment and led to the development of resistance among vectors. Thus, this study assessed the pathogenicity of entomopathogenic fungi in Triatoma dimidiata. Methods Preparations of 108 conidia/ml of Gliocladium virens, Talaromyces flavus, Beauveria bassiana and Metarhizium anisopliae were applied topically on T. dimidiata nymphs and adults. Controls were treated with the 0.0001% Tween-80 vehicle. Mortality was evaluated and recorded daily for 30 days. The concentration required to kill 50% of T. dimidiata (LC50) was then calculated for the most pathogenic isolate. Results Pathogenicity in adults was similar among B. bassiana, G. virens and T. flavus (p>0.05) and differed from that in triatomine nymphs (p=0.009). The most entomopathogenic strains in adult triatomines were B. bassiana and G. virens, which both caused 100% mortality. In nymphs, the most entomopathogenic strain was B. bassiana, followed by G. virens. The native strain with the highest pathogenicity was G. virens, for which the LC50 for T. dimidiata nymphs was 1.98 x108 conidia/ml at 13 days after inoculation. Conclusions Beauveria bassiana and G. virens showed entomopathogenic potential in T. dimidiata nymphs and adults. However, the native G. virens strain presents a higher probability of success in the field, and G. virens should thus be considered a potential candidate for the biological control of triatomine Chagas disease vectors.

Characterisation of microbial communities colonising the hyphal surfaces of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are symbiotic soil fungi that are intimately associated with the roots of the majority of land plants. They colonise the interior of the roots and the hyphae extend into the soil. It is well known that bacterial colonisation of the rhizosphere can be crucial for many pathogenic as well as symbiotic plant-microbe interactions. However, although bacteria colonising the extraradical AMF hyphae (the hyphosphere) might be equally important for AMF symbiosis, little is known regarding which bacterial species would colonise AMF hyphae. In this study, we investigated which bacterial communities might be associated with AMF hyphae. As bacterial-hyphal attachment is extremely difficult to study in situ, we designed a system to grow AMF hyphae of Glomus intraradices and Glomus proliferum and studied which bacteria separated from an agricultural soil specifically attach to the hyphae. Characterisation of attached and non-attached bacterial communities was performed using terminal restriction fragment length polymorphism and clone library sequencing of 16S ribosomal RNA (rRNA) gene fragments. For all experiments, the composition of hyphal attached bacterial communities was different from the non-attached communities, and was also different from bacterial communities that had attached to glass wool (a non-living substratum). Analysis of amplified 16S rRNA genes indicated that in particular bacteria from the family of Oxalobacteraceae were highly abundant on AMF hyphae, suggesting that they may have developed specific interactions with the fungi.

Insect trypanosomatids: the need to know more

Of ten recognized trypanosomatid genera, only two -- pathogenic Trypanosoma and Leishmania -- have been actively investigated for any length of time while the plant flagellates -- Phytomonas -- have recently begun to attract attention due to their role as agricultural parasites. The remaining genera that comprise parasites associated with insects have been largely neglected except for two or three containing popular isolates. This publication reviews current knowledge of trypanosomatids from insects.

Immune homeostasis to microorganisms in the guts of triatomines (Reduviidae): a review

Bacteria, fungi and parasites are in constant contact with the insect gut environment and can influence different aspects of the host gut physiology. Usually, some of these microorganisms develop and survive in the digestive tract. Therefore, the gut environment must be able to tolerate certain populations of these organisms for the establishment of interactions between non-pathogenic bacteria, parasites and the gut. This review provides a brief overview of the biological and molecular mechanisms that microorganisms use to interact with the gut epithelia in mosquitoes and speculates on their significances for the development of bacteria and Trypanosoma cruzi in the guts of triatomines.

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g-1)] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g-1, while the threshold for the ST was greater than 0.1.10³ CFU-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.

Targeted Gene Deletion and In Vivo Analysis of Putative Virulence Gene Function in the Pathogenic Dermatophyte Arthroderma benhamiae.

Dermatophytes cause the majority of superficial mycoses in humans and animals. However, little is known about the pathogenicity of this specialized group of filamentous fungi, for which molecular research has been limited thus far. During experimental infection of guinea pigs by the human pathogenic dermatophyte Arthroderma benhamiae, we recently detected the activation of the fungal gene encoding malate synthase AcuE, a key enzyme of the glyoxylate cycle. By the establishment of the first genetic system for A. benhamiae, specific ΔacuE mutants were constructed in a wild-type strain and, in addition, in a derivative in which we inactivated the nonhomologous end-joining pathway by deletion of the A. benhamiae KU70 gene. The absence of AbenKU70 resulted in an increased frequency of the targeted insertion of linear DNA by homologous recombination, without notably altering the monitored in vitro growth abilities of the fungus or its virulence in a guinea pig infection model. Phenotypic analyses of ΔacuE mutants and complemented strains depicted that malate synthase is required for the growth of A. benhamiae on lipids, major constituents of the skin. However, mutant analysis did not reveal a pathogenic role of the A. benhamiae enzyme in guinea pig dermatophytosis or during epidermal invasion of the fungus in an in vitro model of reconstituted human epidermis. The presented efficient system for targeted genetic manipulation in A. benhamiae, paired with the analyzed infection models, will advance the functional characterization of putative virulence determinants in medically important dermatophytes.

Pathogenic plant-microbe interactions. What we know and how we benefit

Plants, like humans and other animals, also get sick, exhibit disease symptoms, and die. Plant diseases are caused by environmental stress, genetic or physiological disorders and infectious agents including viroids, viruses, bacteria and fungi. Plant pathology originated from the convergence of microbiology, botany and agronomy; its ultimate goal is the control of plant disease. Microbiologists have been attracted to this field of research because of the need for identification of the agents causing infectious diseases in economically important crops. In 1878—only two years after Pasteur and Koch had shown for the first time that anthrax in animals was caused by a bacteria—Burril, in the USA, discovered that the fire blight disease of apple and pear was also caused by a bacterium (nowadays known as Erwinia amylovora). In 1898, Beijerinck concluded that tobacco mosaic was caused by a “contagium vivum fluidum” which he called a virus. In 1971, Diener proved that a potato disease named potato spindle tuber was caused by infectious RNA which he called viroid

Comparative genomics suggests that the human pathogenic fungus Pneumocystis jirovecii acquired obligate biotrophy through gene loss.

Pneumocystis jirovecii is a fungal parasite that colonizes specifically humans and turns into an opportunistic pathogen in immunodeficient individuals. The fungus is able to reproduce extracellularly in host lungs without eliciting massive cellular death. The molecular mechanisms that govern this process are poorly understood, in part because of the lack of an in vitro culture system for Pneumocystis spp. In this study, we explored the origin and evolution of the putative biotrophy of P. jirovecii through comparative genomics and reconstruction of ancestral gene repertoires. We used the maximum parsimony method and genomes of related fungi of the Taphrinomycotina subphylum. Our results suggest that the last common ancestor of Pneumocystis spp. lost 2,324 genes in relation to the acquisition of obligate biotrophy. These losses may result from neutral drift and affect the biosyntheses of amino acids and thiamine, the assimilation of inorganic nitrogen and sulfur, and the catabolism of purines. In addition, P. jirovecii shows a reduced panel of lytic proteases and has lost the RNA interference machinery, which might contribute to its genome plasticity. Together with other characteristics, that is, a sex life cycle within the host, the absence of massive destruction of host cells, difficult culturing, and the lack of virulence factors, these gene losses constitute a unique combination of characteristics which are hallmarks of both obligate biotrophs and animal parasites. These findings suggest that Pneumocystis spp. should be considered as the first described obligate biotrophs of animals, whose evolution has been marked by gene losses.

Identity and combinations of arbuscular mycorrhizal fungal isolates influence plant resistance and insect preference

1. Accumulating evidence indicates that plant resistance against above-ground herbivores can be affected by the presence of arbuscular mycorrhizal fungi (AMF) in association with the host plant. Little is known, however, about how AMF composition can influence herbivore choice to feed on a particular plant. 2. Unravelling the preference-performance hypothesis in a multitrophic context is needed to expand our knowledge of complex multitrophic interactions in natural systems. If given mycorrhizal fungal genotypes increase attractiveness for a herbivore (reduced plant resistance), then the benefits of increased unpalatability provided by the mycorrhizal fungi (increased plant resistance) might be outweighed by the increased herbivore recruitment. 3. This was addressed by designing three experiments to test the effects of different AMF genotypes, inoculated either alone or in combination, to measure intraspecific AMF effects on plant resistance and insect herbivore preference. Using strawberry (Fragaria vesca L.) plants that were colonised by eight different combinations of Rhizophagus irregularis isolates, we measured effects on plant growth, insect growth and survival, as well as feeding preferences of a generalist herbivore caterpillar (Spodoptera littoralis Boisduval). 4. Overall, it was found that: (i) AMF influenced plant resistance in an AMF genotype-specific manner; (ii) some AMF inoculations decreased insect performance; (iii) insects preferentially chose to feed more on leaves originating from non-mycorrhizal plants; but also that (iv) in a whole plant bioassay, insects preferentially chose the biggest plant, regardless of their mycorrhizal status. 5. Therefore, AMF-mediated trade-offs between growth and resistance against herbivores have been shown. Such trade-offs, particularly driven by plant attractiveness to herbivores, buffer the positive effects of the mycorrhizal symbiosis on enhanced plant growth.

Gene expression profiling in human pathogenic dermatophytes in vitro and in vivo

Objectives: Dermatophytes are highly specialized fungi which are the most common agents of superficial mycoses in humans and animals. The particular ability of these microorganisms to invade and multiply within keratinized host structures is presumably linked to their secreted keratinolytic activity, which is therefore a major putative virulence attribute of these fungi. The overall adaptation and transcriptional response of dermatophytes during protein degradation and/or infection is largely unknown. Methods: A Trichophyton rubrum cDNA microarray was developed and used for the transcriptional analysis of T. rubrum and Arthroderma benhamiae cells during growth on protein substrates. Moreover, the gene expression profile in A. benhamiae cells was monitored during infection of guinea pigs. Results: T. rubrum and A. benhamiae cells activate a large set of genes encoding secreted endo- and exoproteases during growth on soy and keratin. In addition, other specifically induced factors with potential implication in protein utilization were identified, e.g. multiple transporters, metabolic enzymes, transcription factors and hypothetical proteins with unknown function. Notably however, the protease gene expression profile in the fungal cells during infection was significantly different from the pattern elicited during in vitro growth on keratin. Conclusions: Our results suggest specific functions of individual proteases during infection, which may not be restricted to the degradation of keratin. This first, broad in vivo transcriptional profiling approach in dermatophytes gives new molecular insights into pathogenicity associated adaptation mechanisms that make these microorganisms the most successful causitive agents of superficial mycoses.

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection.

Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20-25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro -expressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.

Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Detection, transmission, pathogenicity and chemical treatment of fungi in Ceiba speciosa seeds

The aim of this work was to evaluate fungus association, transmission and pathogenicity, besides chemical seed treatment in Ceiba speciosa seeds from different regions of southern Brazil. Seven seed samples were used to do the germination test, fungus detection by blotter test and potato-dextrose-agar (PDA), fungus transmission and pathogenicity tests; besides, chemical seed treatments were tested. Germination ranged from 0 to 59,5%. The following fungi were associated in the seeds: Fusarium sp., Alternaria sp., Colletotrichum sp., Curvularia sp. and Pestalotia sp.; in addition, Fusarium sp. was found in all the samples. Alternaria sp. and Fusarium sp. were transmitted by seeds. The isolates of Alternaria sp., Colletotrichum sp. and Fusarium sp., were pathogenic to seedlings and seeds. The seed treatment with methyl tiophanate and the combination captan + methyl tiophanate reduced Fusarium sp. incidence.