We show that, in the malaria vector Anopheles gambiae, expression of Cecropin 1 is regulated by REL2, an NF-B-like transcription factor orthologous to Drosophila Relish. Through alternative splicing, REL2 produces a full-length (REL2-F) and a shorter (REL2-S) protein isoform lacking the inhibitory ankyrin repeats and death domain. RNA interference experiments show that, in contrast to Drosophila Relish, which responds solely to Gram-negative bacteria, the Anopheles REL2-F and REL2-S isoforms are involved in defense against the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli bacteria, respectively. REL2-F also regulates the intensity of mosquito infection with the malaria parasite, Plasmodium berghei. The adaptor IMD shares the same activities as REL2-F. Microarray analysis identified 10 additional genes regulated by REL2, including CEC3, GAM1, and LRIM1.innate immunity ͉ NF-B ͉ Relish ͉ Cecropin ͉ Imd

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Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) injected into mosquito embryos. Two G1 generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event in the injected G0 embryos. Genetic cross data suggest that the insertion site of the element either resulted in, or is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios in outcrosses to non-transformed individuals and failure to establish a pure-breeding line. The insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement in the injection technique at the end of the studies resulted in increased G0 hatching, transient expression and EGFP-expression rates among G1 progeny.

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The ability to manipulate the mosquito genome through germ-line transformation provides us with a powerful tool for investigating gene structure and function. It is also a valuable method for the development of novel approaches to combating the spread of mosquito-vectored diseases. To date, germ-line transformation has been demonstrated in several mosquito species. Transgenes are introduced into pre-blastocyst mosquito embryos using microinjection techniques that take a few hours, and progeny are screened for the presence of a marker gene. The microinjection protocol presented here can be applied to most mosquitoes and contains several improvements over other published methods that increase the survival of injected embryos and, therefore, the number of transformants. Transgenic lines can be established in approximately 1 month using this technique.

Various newly developed indicators of N deficiency, physiological state (approximate growth rate), and N source for growth were measured during five cruises to Dabob Bay, Washington from early spring to summer. Although nitrate and ammonium in the surface layer were depleted early in the spring, the plankton populations never became extremely N deficient, as indicated by high intracellular amino acid/protein ratios. However, growth rates, estimated from protein/DNA or RNA/DNA ratios, were usually low unless nitrate concentrations were high or had recently been high, as indicated by large intracellular nitrate pools or high nitrate reductase activities. High growth rates were observed during the spring bloom or as a result of the sporadic supply of nitrate to the euphotic zone, which was inferred from measurements of biochemical indicators on several cruises after the spring bloom. The sporadic supply of nitrate could account for the lack of N deficiency in these populations and mask diel periodicity in N utilization. These results demonstrate that biochemical indicators can be easily measured in the field and that variations in indicators such as intracellular amino acid/protein, protein/DNA, RNA/DNA ratios, NR activities and intracellular nitrate concentrations are an aid in understanding plankton dynamics.

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