The concept of innate immunity refers to the first-line host defense that serves to limit infection in the early hours after exposure to microorganisms. Recent data have highlighted similarities between pathogen recognition, signaling pathways, and effector mechanisms of innate immunity in Drosophila and mammals, pointing to a common ancestry of these defenses. In addition to its role in the early phase of defense, innate immunity in mammals appears to play a key role in stimulating the subsequent, clonal response of adaptive immunity.

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Phylogenetic Perspectives in Innate Immunity

Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

https://science.sciencemag.org/content/sci/298/5591/129.full.pdf

The Genome Sequence of the Malaria Mosquito Anopheles gambiae

Insect hemocytes and their role in immunity

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.

http://science.sciencemag.org/content/sci/298/5591/159.full.pdf

Immunity-Related Genes and Gene Families in Anopheles gambiae

Part 1 The importance of blood-sucking insects. Part 2 The evolution of the blood-sucking habit: prolonged close association with vertebrates morphological pre-adaptation for piercing. Part 3 Feeding preferences of blood-sucking insects: host choice host choice and species complexes. Part 4 Location of the host: the behavioural framework of host location appetitive searching activation and orientation attraction movement between hosts. Part 5 Ingestion of the blood meal: vertebrate haemostasis insect anti-haemostatic factors probing stimulants phagostimulants mouthparts blood intake. Part 6 Managing the blood meal: midgut anatomy the blood meal gonotrophic concordance nutrition host hormones in the blood meal partitioning of resources from the blood meal autogeny. Part 7 Host - insect interactions: insect distribution on the surface of the host morphological specializations for life on the host host immune responses to insect salivary secretions behavioural defences of the host density dependent effects on feeding success. Part 8 Transmission of parasites by blood-sucking insects: transmission routes specificity in vector-parasite relationships origin of vector parasite relationships.

Biology of Blood-Sucking Insects

The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.

https://science.sciencemag.org/content/sci/234/4776/607.full.pdf

Genetic Selection of a Plasmodium-Refractory Strain of the Malaria Vector Anopheles gambiae

Understanding the population structure of Plasmodium parasites is essential for malaria intervention. A survey of parasites in vectors and host infections was conducted in an area of intense mortality due to malaria in a captive penguin (Spheniscus demersus) colony, using a novel method for identification of Plasmodium species by amplification of ribosomal sequences in DNA or RNA. Three phylogenetically distinct groups of avian Plasmodium were detected in mosquitoes (Culex) collected at the study site (Baltimore Zoo, Baltimore, MD) during a period of high transmission. One of the three clades of Plasmodium was found to be prevalent in penguins monitored through the malaria transmission season and consistent with morphological identification as Plasmodium relictum. This parasite sequence was directly associated with the death of a penguin. Thus, a complete transmission cycle is defined at this site. Phylogenetic comparison of ribosomal sequences to an authenticated reference strain of Plasmodium relictum indicates that this is not the parasite causing death in the penguins, suggesting that different parasites may be morphologically indistinguishable.

Parasite diversity in an endemic region for avian malaria and identification of a parasite causing penguin mortality.

Analysis of restriction fragment length polymorphism (RFLP) is a powerful tool for analyzing linkage relationships in species where few genetic markers have been described and where conduct of crosses is difficult. It also permits integration of genetic and physical (cytogenetic) data when the probes have been mapped by in situ hybridization. To illustrate the utility of the method, and because some mutations of a diphenol oxidase gene might conceivably produce the malaria refractoriness phenotype of ookinete–oocyst encapsulation, backcrosses between two inbred lines of Anopheles gambiae Giles were carried out to determine the linkage relationship between the diphenol oxidase A2 (Dox) gene and the esterase locus associated with refractoriness to Plasmodium cynomolgi NIH. The Dox alleles were a Sal I restriction fragment length polymorphism visualized by probing Southern blotted DNA from portions of individual mosquitoes with a cloned Dox gene probe. The two genes were shown to segregate independently.

Use of a restriction fragment length polymorphism (RFLP) as a genetic marker in crosses of Anopheles gambiae (Diptera: Culicidae): independent assortment of a diphenol oxidase RFLP and an esterase locus.

The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain ofAnopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition. The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages. Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of bands which were restricted largely to adult male testes.

Douglas C. Seeley

Papers

Genetic Selection of a Plasmodium-Refractory Strain of the Malaria Vector Anopheles gambiae

Parasite diversity in an endemic region for avian malaria and identification of a parasite causing penguin mortality.

Use of a restriction fragment length polymorphism (RFLP) as a genetic marker in crosses of Anopheles gambiae (Diptera: Culicidae): independent assortment of a diphenol oxidase RFLP and an esterase locus.

The genetics and expression of an esterase locus in Anopheles gambiae