Heat Shock Causes Lower Plasmodium Infection Rates in Anopheles albimanus.

The immune response of Anopheles mosquitoes to Plasmodium invasion has been extensively studied and shown to be mediated mainly by the nitric oxide synthase (NOS), dual oxidase (DUOX), phenoloxidase (PO), and antimicrobial peptides activity. Here, we studied the correlation between a heat shock insult, transcription of immune response genes, and subsequent susceptibility to Plasmodium berghei infection in Anopheles albimanus. We found that transcript levels of many immune genes were drastically affected by the thermal stress, either positively or negatively. Furthermore, the transcription of genes associated with modifications of nucleic acid methylation was affected, suggesting an increment in both DNA and RNA methylation. The heat shock increased PO and NOS activity in the hemolymph, as well as the transcription of several immune genes. As consequence, we observed that heat shock increased the resistance of mosquitoes to Plasmodium invasion. The data provided here could help the understanding of infection transmission under the ever more common heat waves.

Dietary and Plasmodium challenge effects on the cuticular hydrocarbon profile of Anopheles albimanus.

The cuticular hydrocarbon (CHC) profile reflects the insects' physiological states. These include age, sex, reproductive stage, and gravidity. Environmental factors such as diet, relative humidity or exposure to insecticides also affect the CHC composition in mosquitoes. In this work, the CHC profile was analyzed in two Anopheles albimanus phenotypes with different degrees of susceptibility to Plasmodium, the susceptible-White and resistant-Brown phenotypes, in response to the two dietary regimes of mosquitoes: a carbon-rich diet (sugar) and a protein-rich diet (blood) alone or containing Plasmodium ookinetes. The CHCs were analyzed by gas chromatography coupled to mass spectrometry or flame ionization detection, identifying 19 CHCs with chain lengths ranging from 20 to 37 carbons. Qualitative and quantitative changes in CHCs composition were dependent on diet, a parasite challenge, and, to a lesser extent, the phenotype. Blood-feeding caused up to a 40% reduction in the total CHC content compared to sugar-feeding. If blood contained ookinetes, further changes in the CHC profile were observed depending on the Plasmodium susceptibility of the phenotypes. Higher infection prevalence caused greater changes in the CHC profile. These dietary and infection-associated modifications in the CHCs could have multiple effects on mosquito fitness, impacts on disease transmission, and tolerance to insecticides.

Affinity purification of Plasmodium ookinetes from in vitro cultures using extracellular matrix gel.

Malaria transmission depends on the parasites' successful invasion of the mosquito. This is achieved by the ookinete, a motile zygote that forms in the blood bolus after the mosquito takes an infectious blood meal. The ookinete invades the midgut epithelium and strongly attaches to the basal lamina, differentiating into an oocyst that produces the vertebrate-invasive sporozoites. Despite their importance, the ookinete and the oocyst are the least studied stages of the parasite. Much of what we know about the ookinete comes from in vitro experiments, which are hindered by the concomitant contamination with blood cells and other parasite stages. Although methods to purify them exist, they vary in terms of yield, costs, and difficulty to perform. A method for ookinete purification taking advantage of their adhesive properties was herein developed. The method consists of covering any culture-suitable surface with extracellular matrix gel, after which the ookinete culture is incubated on the gel to allow for ookinete attachment. The contaminant cells are then simply washed away. This procedure results in purer and less stressed ookinete preparations, which, by the nature of the method, are ready for oocyst production. Furthermore, it allows for micro-purifications using only 1 µl of blood, opening the possibility to make axenic ookinete cultures without sacrificing mice.

DNA Methylation in Anopheles albimanus Modulates the Midgut Immune Response Against Plasmodium berghei.

Epigenetic mechanisms such as DNA methylation and histone post-translational modifications are fundamental for the phenotypic plasticity of insects during their interaction with the environment. In response to environmental cues, the methylation pattern in DNA is dynamically remodeled to achieve an epigenetic control of gene expression. DNA methylation is the focus of study in insects for its evolutionarily conserved character; however, there is scant knowledge about the epigenetic regulation in vector mosquitoes, especially during their infection by parasites. The aim of the present study was to evaluate the participation of DNA methylation in the immune response of Anopheles albimanus to a Plasmodium infection. For this, we first investigated the presence of a fully functional DNA methylation system in A. albimanus by assessing its potential role in larval development. Subsequently, we evaluated the transcriptional response to Plasmodium berghei of two mosquito phenotypes with different degrees of susceptibility to the parasite, in a scenario where their global DNA methylation had been pharmacologically inhibited. Our study revealed that A. albimanus has a functional DNA methylation system that is essential to larval viability, and that is also responsive to feeding and parasite challenges. The pharmacological erasure of the methylome with azacytidine or decitabine abolished the divergent responses of both mosquito phenotypes, leading to a transcriptionally similar response upon parasite challenge. This response was more specific, and the infection load in both phenotypes was lowered. Our findings suggest that DNA methylation may constitute a key factor in vector competence, and a promising target for preventing malaria transmission.

DNA Synthesis Is Activated in Mosquitoes and Human Monocytes During the Induction of Innate Immune Memory.

Endoreplication is a cell cycle program in which cells replicate their genomes without undergoing mitosis and cytokinesis. For the normal development of many organisms (from fungi to humans) and the formation of their organs, endoreplication is indispensable. The aim of the present study was to explore whether endoreplication and DNA synthesis are relevant processes during the induction of trained innate immunity in human monocytes and in the Anopheles albimanus mosquito cell line. During the induction of trained immunity in both models, endoreplication markers were overexpressed and we observed an increase in DNA synthesis with an augmented copy number of genes essential for trained immunity. Blocking DNA synthesis prevented trained immunity from being established. Overall, these findings suggest that DNA synthesis and endoreplication are important mechanisms involved in inducing innate immune memory. They have probably been conserved throughout evolution from invertebrates to humans.

[Evolution and phylogeny of B lymphocytes]. / Evolución y filogenia de los linfocitos B.

B lymphocytes are one of the most important cell types involved in the immune response of mammals. The origin and evolution of this cellular type is unknown, but the B lymphocyte bona fide appeared first in fish. In this review we analize the principal components of the immune response of invertebrates, their phylogenetic distribution and the permancence of some properties that allowed the emergence of the B lymphocyte. We started from the idea that many of the components that characterize the B lymphocyte are found distributed among the invertebrates, however, it is in the B lymphocyte, where all these components that give this type of cell its identity, converged. The actual knowledge we have in regards of the lymphocytes comes, in the most part, from physiological studies in mammals, being the mice the more representative. The origin of the B lymphocyte, its alternative mechanisms for generating receptor diversity, its immune effector response, and the generation of memory, require an evolutionary and multidisiplinary approach for its study.

Los linfocitos B son unos de los tipos celulares más importantes de la respuesta inmunitaria de los mamíferos. El origen y evolución de este tipo celular se desconocen, pero el linfocito B bona fide aparece en peces. En esta revisión se analizan los principales componentes de la respuesta inmunitaria en invertebrados, su distribución filogenética y la permanencia de algunas propiedades que permitieron el surgimiento del linfocito B. Se parte de la idea de que muchos de los componentes que caracterizan a los linfocitos B están distribuidos desde los invertebrados; sin embargo, es en el linfocito donde se integran todos estos componentes que le dan identidad a este tipo celular. El conocimiento actual de los linfocitos proviene, en su mayor parte, del estudio de la fisiología en mamíferos y como mayor representante el ratón. El origen del linfocito B, sus mecanismos alternativos de generación de diversidad de receptores, su respuesta inmunitaria efectora y la generación de memoria, requieren para su estudio de un abordaje multidisciplinario y con enfoque evolutivo.

Injury and immune response: applying the danger theory to mosquitoes.

The insect immune response can be activated by the recognition of both non-self and molecular by-products of tissue damage. Since pathogens and tissue damage usually arise at the same time during infection, the specific mechanisms of the immune response to microorganisms, and to tissue damage have not been unraveled. Consequently, some aspects of damage caused by microorganisms in vector-borne arthropods have been neglected. We herein reassess the Anopheles-Plasmodium interaction, incorporating Matzinger's danger/damage hypothesis and George Salt's injury assumptions. The invasive forms of the parasite cross the peritrophic matrix and midgut epithelia to reach the basal lamina and differentiate into an oocyst. The sporozoites produced in the oocyst are released into the hemolymph, and from there enter the salivary gland. During parasite development, wounds to midgut tissue and the basement membrane are produced. We describe the response of the different compartments where the parasite interacts with the mosquito. In the midgut, the response includes the expression of antimicrobial peptides, production of reactive oxygen species, and possible activation of midgut regenerative cells. In the basal membrane, wound repair mainly involves the production of molecules and the recruitment of hemocytes. We discuss the susceptibility to damage in tissues, and how the place and degree of damage may influence the differential response and the expression of damage associated molecular patterns (DAMPs). Knowledge about damage caused by parasites may lead to a deeper understanding of the relevance of tissue damage and the immune response it generates, as well as the origins and progression of infection in this insect-parasite interaction.