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Transcriptome profiles of Anopheles gambiae harboring natural low-level Plasmodium infection reveal adaptive advantages for the mosquito

ABSTRACT: Anopheline mosquitoes are the sole vectors for the Plasmodium pathogens responsible for malaria, which is among the oldest and most devastating of human diseases. The continuing global impact of malaria reflects the evolutionary success of a complex vector-pathogen relationship that accordingly has been the long-term focus of both debate and study. An open question in the biology of malaria transmission is the impact of naturally occurring low-level Plasmodium infections of the vector on the mosquito’s health and longevity as well as critical behaviors such as host-preference/seeking. To begin to answer this, we have completed a comparative RNAseq-based transcriptome profile study examining the effect of biologically salient, salivary gland transmission-stage Plasmodium infection on the molecular physiology of Anopheles gambiae s.s. head, sensory appendages, and salivary glands. When compared with their uninfected counterparts, Plasmodium infected mosquitoes exhibit increased transcript abundance of genes associated with olfactory acuity as well as a range of synergistic processes that align with increased fitness based on both anti-aging and reproductive advantages. Taken together, these data argue against the long-held paradigm that malaria infection is pathogenic for anophelines and, instead suggests there are biological and evolutionary advantages for the mosquito that drive the preservation of its high vectorial capacity.

SUBMITTER: Carr A

PROVIDER: S-EPMC8604914 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance.We introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae.The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK.MEK-ERK signaling in A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway.

Project description:Acetylcholinesterases (AChEs) catalyze the hydrolysis of acetylcholine, a neurotransmitter for cholinergic neurotransmission in animals. Most insects studied so far possess two AChE genes: ace-1 paralogous and ace-2 orthologous to Drosophila melanogaster ace. We characterized the catalytic domain of Anopheles gambiae AChE1 in a previous study (Jiang et al., 2009) and report here biochemical properties of A. gambiae AChE2 expressed in Sf9 cells. An unknown protease in the expression system cleaved the recombinant AChE2 next to Arg(110), yielding two non-covalently associated polypeptides. A mixture of the intact and cleaved AChE2 had a specific activity of 72.3 U/mg, much lower than that of A. gambiae AChE1 (523 U/mg). The order of V(max)/K(M) values for the model substrates was acetylthiocholine > propionylthiocholine ? acetyl-(?-methyl)thiocholine > butyrylthiocholine. The IC(50)'s for eserine, carbaryl, BW284C51, paraoxon and malaoxon were 1.32, 13.6, 26.8, 192 and 294 nM, respectively. A. gambiae AChE2 bound eserine and carbaryl stronger than paraoxon and malaoxon, whereas eserine and malaoxon modified the active site Ser(232) faster than carbaryl or paraoxon did. Consequently, the k(i)'s were 1.173, 0.245, 0.029 and 0.018 ?M(-1)min(-1) for eserine, carbaryl, paraoxon and malaoxon, respectively. Quantitative polymerase chain reactions showed a similar pattern of ace-1 and ace-2 expression. Their mRNAs were abundant in early embryos, greatly decreased in late embryos, larvae, pupae, and pharate adult, and became abundant again in adults. Both transcripts were higher in head and abdomen than thorax of adults and higher in male than female mosquitoes. Transcript levels of ace-1 were 1.9- to 361.8-fold higher than those of ace-2, depending on developmental stages and body parts. Cross-reacting polyclonal antibodies detected AChEs in adult brains, thoracic ganglia, and genital/rectal area. Activity assays, immunoblotting, and tandem mass spectrometric analysis indicated that A. gambiae AChE1 is responsible for most of acetylthiocholine hydrolysis in the head extracts. Taken together, these data indicate that A. gambiae AChE2 may play a less significant role than AChE1 does in the mosquito nervous system.

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Transcriptome profiles of Anopheles gambiae harboring natural low-level Plasmodium infection reveal adaptive advantages for the mosquito

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