Cultivation of monoxenous trypanosomatids: A minireview.

Trypanosomatids are obligatory parasites, some of which are responsible for important human and animal diseases, but the vast majority of trypanosomatids are restricted to invertebrate hosts. Isolation and in vitro cultivation of trypanosomatids from insect hosts enable their description, characterization, and subsequently genetic and genomic studies. However, exact nutritional requirements are still unknown for most trypanosomatids and thus very few defined media are available. This mini review provides information about the role of different ingredients, recommendations and advice on essential supplements and important physicochemical parameters of culture media with the aim of facilitating first attempts to cultivate insect-infesting trypanosomatids, with a focus on monoxenous trypanosomatids.

Stress tolerance in entomopathogenic nematodes: Engineering superior nematodes for precision agriculture.

Entomopathogenic nematodes (EPNs) are soil-dwelling parasitic roundworms commonly used as biocontrol agents of insect pests in agriculture. EPN dauer juveniles locate and infect a host in which they will grow and multiply until resource depletion. During their free-living stage, EPNs face a series of internal and environmental stresses. Their ability to overcome these challenges is crucial to determine their infection success and survival. In this review, we provide a comprehensive overview of EPN response to stresses associated with starvation, low/elevated temperatures, desiccation, osmotic stress, hypoxia, and ultra-violet light. We further report EPN defense strategies to cope with biotic stressors such as viruses, bacteria, fungi, and predatory insects. By comparing the genetic and biochemical basis of these strategies to the nematode model Caenorhabditis elegans, we provide new avenues and targets to select and engineer precision nematodes adapted to specific field conditions.

Environment-host-parasite interactions in mass-reared insects.

The mass production of insects is rapidly expanding globally, supporting multiple industrial needs. However, parasite infections in insect mass-production systems can lower productivity and can lead to devastating losses. High rearing densities and artificial environmental conditions in mass-rearing facilities affect the insect hosts as well as their parasites. Environmental conditions such as temperature, gases, light, vibration, and ionizing radiation can affect productivity in insect mass-production facilities by altering insect development and susceptibility to parasites. This review explores the recent literature on environment-host-parasite interactions with a specific focus on mass-reared insect species. Understanding these complex interactions offers opportunities to optimise environmental conditions for the prevention of infectious diseases in mass-reared insects.

Transcriptomic analysis of the adaptation to prolonged starvation of the insect-dwelling Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi is a digenetic unicellular parasite that alternates between a blood-sucking insect and a mammalian, host causing Chagas disease or American trypanosomiasis. In the insect gut, the parasite differentiates from the non-replicative trypomastigote forms that arrive upon blood ingestion to the non-infective replicative epimastigote forms. Epimastigotes develop into infective non-replicative metacyclic trypomastigotes in the rectum and are delivered via the feces. In addition to these parasite stages, transitional forms have been reported. The insect-feeding behavior, characterized by few meals of large blood amounts followed by long periods of starvation, impacts the parasite population density and differentiation, increasing the transitional forms while diminishing both epimastigotes and metacyclic trypomastigotes. To understand the molecular changes caused by nutritional restrictions in the insect host, mid-exponentially growing axenic epimastigotes were cultured for more than 30 days without nutrient supplementation (prolonged starvation). We found that the parasite population in the stationary phase maintains a long period characterized by a total RNA content three times smaller than that of exponentially growing epimastigotes and a distinctive transcriptomic profile. Among the transcriptomic changes induced by nutrient restriction, we found differentially expressed genes related to managing protein quality or content, the reported switch from glucose to amino acid consumption, redox challenge, and surface proteins. The contractile vacuole and reservosomes appeared as cellular components enriched when ontology term overrepresentation analysis was carried out, highlighting the roles of these organelles in starving conditions possibly related to their functions in regulating cell volume and osmoregulation as well as metabolic homeostasis. Consistent with the quiescent status derived from nutrient restriction, genes related to DNA metabolism are regulated during the stationary phase. In addition, we observed differentially expressed genes related to the unique parasite mitochondria. Finally, our study identifies gene expression changes that characterize transitional parasite forms enriched by nutrient restriction. The analysis of the here-disclosed regulated genes and metabolic pathways aims to contribute to the understanding of the molecular changes that this unicellular parasite undergoes in the insect vector.

Development of a TaqMan qPCR assay for trypanosomatid multi-species detection and quantification in insects.

BACKGROUND: Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample. METHODS: A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major. RESULTS: The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host. CONCLUSIONS: These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings.

Sexual and developmental variations of ecto-parasitism in damselflies.

The prevalence and intensity of parasitism can have different fitness costs between sexes, and across species and developmental stages. This variation could arise because of species specific sexual and developmental differences in body condition, immunity, and resistance. Theory predicts that the prevalence of parasitism will be greater in individuals with poor body condition and the intensity of parasitism will be greater in individuals with larger body size. These predictions have been tested and verified in vertebrates. In insects, however, contradictory evidence has been found in different taxa. Here, we tested these predictions on two species of Agriocnemis (Agriocnemis femina and Agriocnemis pygmaea) damselflies, which are parasitized by Arrenurus water mite ectoparasites. We measured body weight, total body length, abdomen area and thorax area of non-parasitized damselflies and found body condition varied between males and females, between immature females and mature females and between A. femina and A. pygmaea. Then, we calculated the parasite prevalence, i.e., the absence or presence of parasites and intensity, i.e., the number of parasites per infected damselfly in eleven natural populations of both species. In line to our predictions, we observed greater prevalence in immature females than mature females but found no difference in parasite prevalence between males and females. Furthermore, we found that parasite intensity was higher in females than males and in immature females than mature females. Our result also showed that the frequency and intensity of parasitism varied between the two studied species, being higher in A. pygmaea than A. femina. Our study provides evidence that parasitism impacts sexes, developmental stages and species differentially and suggests that variation may occur due to sex, developmental stage, and species-specific resistance and tolerance mechanism.

Pathogen infection routes and host innate immunity: Lessons from insects.

Recent advances in insect-pathogen interactions have started to reveal the role of insect tissues and organs as natural infection routes for parasites and microbial pathogens. Here we summarize this information highlighting the micro- and macro-parasites that enter insects through distinct infection routes and link them to innate immune activity. We also examine whether the infection route determines the insect immune response and if the resulting immunological and physiological processes underpinning these different routes of infection are clearly distinct. Understanding how the infection route is associated with the robustness in insect host defense will help us identify conserved evolutionary and ecological patterns in order to design novel strategies for the management of destructive agricultural pests and disease vectors.

Pathogen within-host dynamics and disease outcome: what can we learn from insect studies?

Parasite proliferations within/on the host form the basis of the outcome of all infectious diseases. However, within-host dynamics are difficult to study in vertebrates, as it requires regularly following pathogen proliferation from the start of the infection and at the organismal level. Invertebrate models allow for this monitoring under controlled conditions using population approaches. These approaches offer the possibility to describe many parameters of the within-host dynamics, such as rate of proliferation, probability to control the infection, and average time at which the pathogen is controlled. New parameters such as the Pathogen Load Upon Death and the Set-Point Pathogen Load have emerged to characterize within-host dynamics and better understand disease outcome. While contextualizing the potential of studying within-host dynamics in insects to build fundamental knowledge, we review what we know about within-host dynamics using insect models, and what it can offer to our knowledge of infectious diseases.

The relevance of studying insect-nematode interactions for human disease.

Vertebrate-parasitic nematodes cause debilitating, chronic infections in millions of people worldwide. The burden of these so-called 'neglected tropical diseases' is often carried by poorer socioeconomic communities in part because research on parasitic nematodes and their vertebrate hosts is challenging and costly. However, complex biological and pathological processes can be modeled in simpler organisms. Here, we consider how insight into the interactions between entomopathogenic nematodes (EPN), their insect hosts and bacterial symbionts may reveal novel treatment targets for parasitic nematode infections. We argue that a combination of approaches that target nematodes, as well as the interaction of pathogens with insect vectors and bacterial symbionts, offer potentially effective, but underexplored opportunities.

Distribution of galling insects and their parasitoids on Caryocar brasiliense tree crowns / Distribuição de insetos galhadores e seus parasitoides em copas de pequizeiros (Caryocar brasiliense)

Caryocar brasiliense Camb. (Malpighiales: Caryocaraceae) is widely distributed in the Brazilian savanna and its fruits are used by humans for food, production of cosmetics, lubricants, and in the pharmaceutical industry. This plant is damaged by galling insects. Number of these galling insects and their parasitoids was recorded, in the field (galls) and in the laboratory (adults emerged from the galls), from three C. brasiliense crown heights, during three years. Numbers of adults of Eurytoma sp. (Hymenoptera: Eurytomidae), galling insect (younger attack) and Sycophila sp. (Hymenoptera: Eurytomidae) (a parasitoid of Eurytoma sp.), were greater on the apical parts of C. brasiliense tree crowns. Numbers and groups of Eurytoma sp. globoid galls (older attack) were higher in the median and basal crown parts. The numbers of Eurytoma sp. galls were higher on apical part of C. brasiliense tree crown and also of their parasitoids.

Caryocar brasiliense Camb. (Malpighiales: Caryocaraceae) é, amplamente, distribuída no cerrado brasileiro e seus frutos são utilizados para alimentação humana, produção de cosméticos, lubrificantes e na indústria farmacêutica, no entanto, é danificada por insetos galhadores. O número de insetos galhadores e seus parasitoides foram avaliados, em campo (galhas) e em laboratório (emergência de adultos das galhas), em três alturas do dossel de C. brasiliense, durante três anos. Os números de adultos Eurytoma sp. (Hymenoptera: Eurytomidae), inseto galhador (galhas novas) e de Sycophila sp. (Hymenoptera: Eurytomidae), parasitoide de Eurytoma sp., foram maiores na parte apical do dossel da copa de árvores de C. brasiliense. A quantidade de galhas globoides de Eurytoma sp., isoladas ou em grupo (galhas velhas), foi maior na parte mediana e basal da copa. Os números de adultos do galhador Eurytoma sp. e de seus parasitoides, que os seguem, foram maiores na parte apical da copa de árvores de C. brasiliense.

Potential of in vivo- and in vitro-cultured entomopathogenic nematodes to infect Lobesia vanillana (Lepidoptera: Tortricidae) under laboratory conditions.

Entomopathogenic nematodes (EPNs) have been successfully applied as biological control agents against above ground and soil stages of insect pests. However, for commercial application, it is crucial to mass culture these nematodes using in vitro liquid culture technology, as it is not attainable when using susceptible insects as hosts. Lobesia vanillana (Lepidoptera: Tortricidae) is regarded a sporadic pest of wine grapes in South Africa. The in vivo- and in vitro-cultured South African EPNs, Steinernema yirgalemense and Steinernema jeffreyense (Rhabditida: Steinernematidae), were evaluated against larvae and pupae of L. vanillana in laboratory bioassays. For larvae, high mortality was observed for all treatments: In vitro-cultured S. yirgalemense (98%) performed better than S. jeffreyense (73%), while within in vivo cultures, there was no difference between nematode species (both 83%). No significant difference was detected between in vivo- and in vitro cultures of the same nematode species. The LD50 of the in vitro-cultured S. yirgalemense, was 7.33 nematodes per larva. Mortality by infection was established by dissecting L. vanillana cadavers and confirming the presence of nematodes, which was > 90% for all treatments. Within in vitro cultures, both S. yirgalemense and S. jeffreyense were able to produce a new cohort of infective juveniles from L. vanillana larvae. Pupae, however, were found to be considerably less susceptible to EPN infection. This is the first study on the use of EPNs to control L. vanillana. The relative success of in vitro-cultured EPN species in laboratory assays, without any loss in pathogenicity, is encouraging for further research and development of this technology.

Insects and their pathogens in a changing climate.

The complex nature of climate change-mediated multitrophic interaction is an underexplored area, but has the potential to dramatically shift transmission and distribution of many insects and their pathogens, placing some populations closer to the brink of extinction. However, for individual insect-pathogen interactions climate change will have complicated hard-to-anticipate impacts. Thus, both pathogen virulence and insect host immunity are intrinsically linked with generalized stress responses, and in both pathogen and host have extensive trade-offs with nutrition (e.g., host plant quality), growth and reproduction. Potentially alleviating or exasperating these impacts, some pathogens and hosts respond genetically and rapidly to environmental shifts. This review identifies many areas for future research including a particular need to identify how altered global warming interacts with other environmental changes and stressors, and how consistent these impacts are across pathogens and hosts. With that achieved we would be closer to producing an overarching framework to integrate knowledge on all environmental interplay and infectious disease events.

Editorial: "Evolutionary Genetics of Insect Innate Immunity".

The insect innate immune system is under strong selection pressure to evolve resistance to pathogenic infections [...].

Insects dispersing taeniid eggs: Who and how?

Taeniosis/cysticercosis and echinococcosis are neglected zoonotic helminth infections with high disease burden caused by tapeworms which circulate between definitive and intermediate host reflecting a predator-prey interaction. Taeniid eggs can remain vital for months, allowing arthropods to mechanically transport them to intermediate hosts. However, the multiple routes that arthropods provide as carriers of taeniid eggs are still often unregarded or not considered. This review focuses on the prevalence and importance of arthropods as carriers and spreaders of taeniid eggs in the epidemiology of taeniosis/cysticercosis and echinococcosis. Current scientific knowledge showed a relevant role of houseflies (Muscidae), blowflies (Calliphoridae), dung beetles (Scarabaeoidea), darkling beetles (Tenebrionidae), ground beetles (Carabidae) and skin beetles (Dermestidae) in the spread of taeniid eggs in the environment, which may favor the infection of new hosts through the direct ingestion of an insect or of contaminated food and water. At last, key research challenges are highlighted, illustrating that further knowledge on the topic is needed to develop and improve guidelines and actions to prevent taeniid infections worldwide.

Microbiota, pathogens, and parasites as mediators of tritrophic interactions between insect herbivores, plants, and pollinators.

Insect-associated microbes, including pathogens, parasites, and symbionts, influence the interactions of herbivorous insects and pollinators with their host plants. Moreover, herbivory-induced changes in plant resource allocation and defensive chemistry can influence pollinator behavior. This suggests that the outcomes of interactions between herbivores, their microbes and host plants could have implications for pollinators. As epizootic diseases occur at high population densities, pathogen and parasite-mediated effects on plants could have landscape-level impacts on foraging pollinators. The goal of this minireview is to highlight the potential for an herbivore's multitrophic interactions to trigger plant-mediated effects on the immunity and health of pollinators. We highlight the importance of plant quality and gut microbiomes in bee health, and how caterpillars as model herbivores interact with pathogens, parasites, and symbionts to affect plant quality, which forms the centerpiece of multitrophic interactions between herbivores and pollinators. We also discuss the impacts of other herbivore-associated factors, such as agricultural inputs aimed at decreasing herbivorous pests, on pollinator microbiomes.

Influence of parasitoid-associated viral symbionts on plant-insect interactions and biological control.

Insect parasitoids have evolved symbiotic interactions with several viruses and thousands of parasitoid species have established mutualistic associations with polydnaviruses (PDVs). While PDVs have often been described as virulence factors allowing development of immature parasitoids inside their herbivore hosts, there is increasing awareness that PDVs can affect plant-insect interactions. We review recent literature showing that PDVs alter not only host physiology, but also feeding patterns and composition of herbivore's oral secretions. In turn PDV-induced changes in herbivore phenotype affect plant responses to herbivory with consequences ranging from differential expression of plant defense-related genes to wider ecological effects across multiple trophic levels. In this opinion paper we also highlight important missing gaps to fully understand the role of PDVs and other parasitoid-associated viral symbionts in a plant-insect interaction perspective. Because PDVs negatively impact performance and survival of herbivore pests, we conclude arguing that PDV genomes offer potential opportunities for biological control.

Development of Monoxenous Trypanosomatids and Phytomonads in Insects.

In this review, we summarize the current data on development of monoxenous trypanosomatids and phytomonads in various insects. Of these, Diptera and Hemiptera are the main host groups, and, consequently, most available information concerns their parasites. Within the insect body, the midgut and hindgut are the predominant colonization sites; in addition, some trypanosomatids can invade the foregut, Malpighian tubules, hemolymph, and/or salivary glands. Differences in the intestinal structure and biology of the host determine the variety of parasites' developmental and transmission strategies. Meanwhile, similar mechanisms are used by unrelated trypanosomatids, reflecting the limited range of options to achieve the same goal.

Arabidopsis natural variation in insect egg-induced cell death reveals a role for LECTIN RECEPTOR KINASE-I.1.

In Arabidopsis (Arabidopsis thaliana), a hypersensitive-like response (HR-like response) is triggered underneath the eggs of the large white butterfly Pieris brassicae (P. brassicae), and this response is dependent on salicylic acid (SA) accumulation and signaling. Previous reports indicate that the clade I L-type LECTIN RECEPTOR KINASE-I.8 (LecRK-I.8) is involved in early steps of egg recognition. A genome-wide association study was used to better characterize the genetic structure of the HR-like response and discover loci that contribute to this response. We report here the identification of LecRK-I.1, a close homolog of LecRK-I.8, and show that two main haplotypes that explain part of the variation in HR-like response segregate among natural Arabidopsis accessions. Besides, signatures of balancing selection at this locus suggest that it may be ecologically important. Disruption of LecRK-I.1 results in decreased HR-like response and SA signaling, indicating that this protein is important for the observed responses. Furthermore, we provide evidence that LecRK-I.1 functions in the same signaling pathway as LecRK-I.8. Altogether, our results show that the response to eggs of P. brassicae is controlled by multiple LecRKs.

ISSR markers to explore entomopathogenic fungi genetic diversity: Implications for biological control of tobacco pests.

Tobacco is one of the major industrial crops cultivated worldwide. Chemical control is the main method employed to reduce damage by insect pests. The use of entomopathogenic fungi represents an alternative to replace insecticides. The search for effective strains in the field constitutes a first step when developing a formulation. The objective of this work was to study genetic differences among isolates of entomopathogenic fungi obtained from tobacco grown soils using ISSR markers. The pathogenicity of the strains towards Helicoverpa gelotopoeon and Diabrotica speciosa was also assessed in order to search for a relationship between virulence and genetic diversity. Nineteen isolates were identified according to morphological features and molecular techniques as Beauveria bassiana (11) and Purpureocillium lilacinum (8). The diversity tree generated by ISSR analysis showed a high diversity among the strains. The pathogenicity towards H. gelotopoeon and D. speciosa was assessed and the logistic models generated showed that B. bassiana isolates LPSc1215 and LPSc1364 were the most pathogenic against both insect pests tested. In the diversity tree, these strains were grouped in a same cluster with a similarity level of approximately 85%, indicating a possible relationship between virulence and the band pattern generated.

Molecular Regulators of Entomopathogenic Nematode-Bacterial Symbiosis.

Entomopathogenic nematodes are parasitic organisms with an exceptional capacity to infect rapidly and efficiently a wide range of insect species. Their distinct pathogenic properties have established entomopathogenic nematodes as supreme biocontrol agents of insects as well as excellent models to simulate and dissect the molecular and physiological bases of conserved strategies employed by parasitic nematodes that cause infectious diseases in humans. The extreme infectivity of entomopathogenic nematodes is due in part to the presence of certain species of Gram-negative bacteria that live in mutualistic symbiosis during the infective juvenile stage, which forms the central part of the nematode life cycle. Both nematodes and their mutualistic bacteria are capable of interfering and undermining several aspects of the insect host innate immune system during the infection process. The mutualistic bacteria are also able to modulate other biological functions in their nematode host including growth, development, and reproduction. In this review, we will focus our attention on the mutualistic relationship between entomopathogenic nematodes and their associated bacteria to discuss the nature and distinct characteristics of the regulatory mechanisms, and their molecular as well as physiological components that control this specific biological partnership.