We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.

Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti ( Aa ), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa , the malaria vector Anopheles gambiae ( Ag ), and the fruit fly Drosophila melanogaster ( Dm ). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.

A series of epidemiological explorations has suggested a negative association between national bacillus Calmette–Guérin (BCG) vaccination policy and the prevalence and mortality of coronavirus disease 2019 (COVID-19). However, these comparisons are difficult to validate due to broad differences between countries such as socioeconomic status, demographic structure, rural vs. urban settings, time of arrival of the pandemic, number of diagnostic tests and criteria for testing, and national control strategies to limit the spread of COVID-19. We review evidence for a potential biological basis of BCG cross-protection from severe COVID-19, and refine the epidemiological analysis to mitigate effects of potentially confounding factors (e.g., stage of the COVID-19 epidemic, development, rurality, population density, and age structure). A strong correlation between the BCG index, an estimation of the degree of universal BCG vaccination deployment in a country, and COVID-19 mortality in different socially similar European countries was observed (r2 = 0.88; P = 8 × 10−7), indicating that every 10% increase in the BCG index was associated with a 10.4% reduction in COVID-19 mortality. Results fail to confirm the null hypothesis of no association between BCG vaccination and COVID-19 mortality, and suggest that BCG could have a protective effect. Nevertheless, the analyses are restricted to coarse-scale signals and should be considered with caution. BCG vaccination clinical trials are required to corroborate the patterns detected here, and to establish causality between BCG vaccination and protection from severe COVID-19. Public health implications of a plausible BCG cross-protection from severe COVID-19 are discussed.

Mosquito midgut invasion by ookinetes of the malaria parasite Plasmodium disrupts the barriers that normally prevent the gut microbiota from coming in direct contact with epithelial cells. This triggers a long-lived response characterized by increased abundance of granulocytes, a subpopulation of hemocytes, circulating in the insect’s hemocoel, and enhanced immunity to bacteria that indirectly reduces survival of Plasmodium parasites upon reinfection. In mosquitoes, differentiation of hemocytes was necessary and sufficient to confer innate immune memory.

BackgroundIt has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown.ResultsWe have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized.ConclusionsIt is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

We present a detailed analysis of the interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei ookinetes during invasion of the mosquito by the parasite. In this mosquito, P.berghei ookinetes invade polarized columnar epithelial cells with microvilli, which do not express high levels of vesicular ATPase. The invaded cells are damaged, protrude towards the midgut lumen and suffer other characteristic changes, including induction of nitric oxide synthase (NOS) expression, a substantial loss of microvilli and genomic DNA fragmentation. Our results indicate that the parasite in¯icts extensive damage leading to subsequent death of the invaded cell. Ookinetes were found to be remarkably plastic, to secrete a subtilisin-like serine protease and the GPI-anchored surface protein Pbs21 into the cytoplasm of invaded cells, and to be capable of extensive lateral movement between cells. The epithelial damage in¯icted is repaired ef®ciently by an actin pursestring-mediated restitution mechanism, which allows the epithelium to`bud off' the damaged cells without losing its integrity. A new model, the time bomb theory of ookinete invasion, is proposed and its implications are discussed.

Comparison of the genomes and proteomes of the two diptera Anopheles gambiae and Drosophila melanogaster, which diverged about 250 million years ago, reveals considerable similarities. However, numerous differences are also observed; some of these must reflect the selection and subsequent adaptation associated with different ecologies and life strategies. Almost half of the genes in both genomes are interpreted as orthologs and show an average sequence identity of about 56%, which is slightly lower than that observed between the orthologs of the pufferfish and human (diverged about 450 million years ago). This indicates that these two insects diverged considerably faster than vertebrates. Aligned sequences reveal that orthologous genes have retained only half of their intron/exon structure, indicating that intron gains or losses have occurred at a rate of about one per gene per 125 million years. Chromosomal arms exhibit significant remnants of homology between the two species, although only 34% of the genes colocalize in small "microsyntenic" clusters, and major interarm transfers as well as intra-arm shuffling of gene order are detected.

Extracellular matrices in diverse biological systems are crosslinked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that the Immunomodulatory Peroxidase (IMPer), an enzyme secreted by the mosquito Anopheles gambiae midgut, and dual oxidase (Duox) form a dityrosine network that decreases gut permeability to immune elicitors and protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses.