Ann M. Fallon

Mosquito large subunit ribosomal RNA : simultaneous alignment of primary and secondary structure

We report the sequence and propose a secondary structure for the cytoplasmic large subunit (5.8S and 28S) ribosomal RNA of the mosquito, Aedes albopictus, in an aligned format that incorporates secondary structure comparisons with Homo sapiens, Drosophila melanogaster, and Escherichia coli ribosomal RNAs. This format facilitates comparison of subtle differences between models, allowing nucleotide by nucleotide analysis at each position of discrepancy. Comparison of the A. albopictus large subunit ribosomal RNA gene with those from other species revealed new compensatory base changes. The aligned format focuses attention to the specific contribution of the A. albopictus sequence by facilitating comparison with the sequence of another dipteran, D. melanogaster. This is the second report of a complete large subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a member of the lower Diptera (Nematocera).

Exploration of mosquito immunity using cells in culture

The propagation of immune-responsive cells in vitro has provided the basis for substantial contributions to our understanding of many aspects of the mammalian immune response. In contrast, the potential for exploring the innate immune response of insects using cultured cells is only beginning to be developed, particularly with various mosquito cell lines from the genera Aedes and Anopheles. Immune-reactive mosquito cell lines express various defensive factors, including transferrin, lysozyme, cecropin, defensin, and prophenoloxidase activities. In this review, we discuss insect immunity in the context of key concepts that have emerged in the study of the mammalian immune system, with emphasis on the properties of the cells that participate in the immune response. The nature of established cell lines and their contributions to our understanding of immune functions in humans and insects is described, with emphasis on our own work with the C7-10 and Aag-2 mosquito cell lines from Aedes albopictus and Aedes aegypti, respectively. Finally, we offer some speculation on further advances in insect immunology that may be facilitated by work with cells in culture.

Immunity proteins from mosquito cell lines include three defensin A isoforms from Aedes aegypti and a defensin D from Aedes albopictus.

An Aedes aegypti mosquito cell line, Aag‐2, exhibits a response to immune stimulation that is qualitatively similar to that of C7–10 cultured cells from the related mosquito, Aedes albopictus. Using SDS polyacrylamide gels, we found that a small peptide was preferentially induced by the treatment of growing cells with heat‐killed, Gram‐positive bacteria. By an analogy with other studies, this small peptide was postulated to be a member of the defensin family of insect immunity peptides. A differential display was used to obtain partial polymerase chain reaction products corresponding to mRNAs that were preferentially expressed in induced cells. One of these products, which contained the partial sequence of a defensin gene, was used to screen cDNA libraries from Ae. aegypti and Ae. albopictus cells. From Ae. aegypti cells, we found two previously described isoforms (A1 and A4) of mosquito defensin A, as well as a new isoform which we defined as A5. From Ae. albopictus cells, we found a new mature mosquito defensin, named D, which contains proline and isoleucine as the final amino acids. In both Ae. aegypti and Ae. albopictus cell lines, the expression of defensin mRNA was visible on Northern blots as early as 3 h after exposure to heat‐killed bacteria, and defensin mRNA abundance was maximal at 12–36 h after induction.

Expression of a heat-inducible gene in transfected mosquito cells

Abstract The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [ 35 S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.

Selective and Irreversible Inhibitors of Mosquito Acetylcholinesterases for Controlling Malaria and Other Mosquito-Borne Diseases

New insecticides are urgently needed because resistance to current insecticides allows resurgence of disease-transmitting mosquitoes while concerns for human toxicity from current compounds are growing. We previously reported the finding of a free cysteine (Cys) residue at the entrance of the active site of acetylcholinesterase (AChE) in some insects but not in mammals, birds, and fish. These insects have two AChE genes (AP and AO), and only AP-AChE carries the Cys residue. Most of these insects are disease vectors such as the African malaria mosquito (Anopheles gambiae sensu stricto) or crop pests such as aphids. Recently we reported a Cys-targeting small molecule that irreversibly inhibited all AChE activity extracted from aphids while an identical exposure caused no effect on the human AChE. Full inhibition of AChE in aphids indicates that AP-AChE contributes most of the enzymatic activity and suggests that the Cys residue might serve as a target for developing better aphicides. It is therefore worth investigating whether the Cys-targeting strategy is applicable to mosquitocides. Herein, we report that, under conditions that spare the human AChE, a methanethiosulfonate-containing molecule at 6 µM irreversibly inhibited 95% of the AChE activity extracted from An. gambiae s. str. and >80% of the activity from the yellow fever mosquito (Aedes aegypti L.) or the northern house mosquito (Culex pipiens L.) that is a vector of St. Louis encephalitis. This type of inhibition is fast (∼30 min) and due to conjugation of the inhibitor to the active-site Cys of mosquito AP-AChE, according to our observed reactivation of the methanethiosulfonate-inhibited AChE by 2-mercaptoethanol. We also note that our sulfhydryl agents partially and irreversibly inhibited the human AChE after prolonged exposure (>4 hr). This slow inhibition is due to partial enzyme denaturation by the inhibitor and/or micelles of the inhibitor, according to our studies using atomic force microscopy, circular dichroism spectroscopy, X-ray crystallography, time-resolved fluorescence spectroscopy, and liquid chromatography triple quadrupole mass spectrometry. These results support our view that the mosquito-specific Cys is a viable target for developing new mosquitocides to control disease vectors and to alleviate resistance problems with reduced toxicity toward non-target species.

Sequence analysis of a mosquito ribosomal protein rpL8 gene and its upstream regulatory region

The gene encoding Aedes albopictus ribosomal protein L8 was isolated using a cDNA probe. Based on the deduced amino acid sequence, rpL8 has a mass of 28,605 Da, a pl of 11.97, and contains 9.6% Arg and 11.9% Lys. The rpL8 gene spans 1229 nucleotides, and contains three exons measuring 73, 150, and 648 nucleotides. The first intron is 293 nucleotides long and interrupts an 85‐nucleotide untranslated leader sequence. The AUG codon is located 12 nucleotides downstream of the 5′‐end of the second exon. Separating the second and third exons is a 65‐nucleotide intron. The major transcription initiation site, identified by primer extension and polymerase stop reactions, mapped 378 nucleotides upstream from the AUG start codon; minor initiation sites were also detected. The DNA sequence upstream of the rpL8 gene was T‐rich, but conventional TATA and CAAT boxes were absent. This is the first molecular analysis of a mosquito ribosomal protein gene.

Optimization of gene transfer in cultured insect cells

The use of polybrene for the efficient transfection of cultured mosquito cells is described. Cells are transfected with purified plasmid DNA coding for a bacterial chloramphenicol acetyltransferase (CAT) gene regulated by sequences from aDrosophila heat shock protein promoter. The DNA is added to the cells in serumfree medium containing polybrene, which facilitates adsorption of DNA to the cell surface. Expression of the transfected gene is induced by subjecting recipient cells to heat shock conditions. CAT activity in lysates from transfected cells is assayed using a simple thin layer chromatographic procedure. Adaptation of the transfection protocol to other insect cell types is discussed.

Cloning and expression of three cecropin cDNAs from a mosquito cell line

We have characterized full‐length cDNAs encoding three isoforms of the antibiotic cecropin secreted by the C7–10 cell line from the mosquito, Aedes albopictus. The existence of two cecropin isoforms that differed from the previously described AalCecA was predicted by mass spectrometry and amino acid sequence analysis of peptides that eluted from reversed phase high performance liquid chromatography as a single peak just behind the previously described cecropin, AalCecA. Based on the amino acid sequence of the mature AalCecA peptide, we designed primers that amplified partial cDNAs encoding three different A. albopictus cecropins in reverse transcriptase polymerase chain reactions. Rapid amplification of cDNA ends was then used to complete the cDNA sequences of AalCecA, AalCecB and AalCecC, respectively. Each cDNA encoded a translation product containing a signal peptide, a pro region, and a mature cecropin peptide consistent with amino acid sequence data from chymotryptic digests. Although the mosquito cecropins shared 70–86% identity among each other, they shared only ∼40% identity to cecropins from Drosophila melanogaster. Each of the cecropins was expressed within 2 to 4 h after induction, and transcripts measuring 0.3 to 0.5 kb continued to accumulate over 24 h. The three cecropins were secreted in roughly equimolar proportions, and 30 to 90% of AalCecB was amidated at the terminal glycine residue. In contrast, amidated forms of AalCecA and AalCecC constituted a smaller proportion of these isoforms.

Nucleotide sequence of a mosquito 18S ribosomal RNA gene

We have sequenced an 18S ribosomal RNA gene from the mosquito, Aedes albopictus. Computer alignment of the 1950 nucleotide coding region (56% A + T) with 18S rRNA sequences from two insect and three vertebrate species revealed greater sequence divergence among the insects than among the vertebrates. Sequence alignments showed that variable region V4, which has been considered to be the most poorly conserved domain in the 18S rRNA gene, was better conserved among insects and vertebrates than was the V6 domain.

Mosquito ribosomal RNA genes: characterization of gene structure and evidence for changes in copy number during development.

Abstract A family of nine recombinant bacteriophages containing rRNA genes from cultured cells of the mosquito, Aedes albopictus , has been characterized by restriction mapping, Southern-blotting and S1-nuclease analyses. The 18S rRNA coding region measured 1800 bp and contained a conserved Eco RI site near the 3′-end. The 28S rRNA coding region was divided into α and β sequences, comprising 1750 and 2000 bp, respectively, which were separated by a 350 bp sequence that is removed from the rRNA precursor during processing. The entire rDNA repeat unit had a minimum length of 15.6 kb, including a nontranscribed spacer region that contained a series of Pvu I repeats upstream of the 18S rRNA coding sequence. During development of the mosquito, Aedes aegypti , the rRNA gene copy number per haploid genome increased from about 400 in larvae to about 1200 in adults.