Germ-line transformation of the South American malaria vector,Anopheles albimanus,with a piggyBac/EGFP transposon vector is routine and highly efficient

Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac-mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species.     

Ribosomal ITS2 sequence data for Anopheles maculipennis and An. messeae in northern Greece,with a critical assessment of previously published sequences

DNA sequences were generated for eight specimens of the Anopheles maculipennis complex from Florina in NW Greece, and identified to species on the basis of comparison with ITS2 sequences for members of the complex already in GenBank. The sequences revealed the presence of An. maculipennis and An. messeae in Florina. Problems with sequence reliability and accessibility of sequences generated in earlier studies of Palaearctic members of the complex are discussed.     

Infectivity of Plasmodium falciparum gametocytes to Anopheles arabiensis after treatment with sulfadoxine-pyrimethamine

Sulfadoxine-pyrimethamine induces increased gametocytaemia when used for treating Plasmodium falciparum malaria. Laboratory-reared Anopheles arabiensis mosquitoes were fed with blood from patients with post-therapeutic gametocytaemia using a membrane feeder. Fourteen days later the heads and thoraxes of 613 mosquitoes were negative for P. falciparum sporozoites by enzyme-linked immunosorbent assay.     

Natural swarming behaviour of the molecular M form of Anopheles gambiae

In Anopheles gambiae, as in most species of mosquitoes, mating is initiated in flight. The males aggregate in aerial swarms and conspecific females individually fly to these swarms where they mate with males. In this study, we investigated the swarming behaviour of A. gambiae and conducted 2 surveys in the rice field area of the Vallée du Kou in Burkina Faso in 1999 and 2002. A high number of anopheline mosquitoes were observed in this area and both molecular M and S forms of A. gambiae were found in sympatry. Swarms formed a few minutes after sunset in different places and no obvious markers were associated with their occurrence. However, swarms occurred close to cow herds generally in open flat areas, 2-3 m above the ground. Overall, 2829 anopheline mosquitoes were collected from 21 swarms composed primarily of males. A few specimens of Culex quinquefasciatus were collected from 3 swarms. Although both molecular M and S forms were found in sympatry in the village, swarms were composed almost exclusively of the molecular M form. This suggests that there are alternative swarming habits for both molecular M and S forms of A. gambiae in nature.     

Microfilarial periodicity of Wuchereria bancrofti in Vanuatu

A study on the relationship between the microfilarial periodicity of Wuchereria bancrofti and vector biting activity was carried out in Penama province, Vanuatu from February to April 1999, to enable the design of a more efficient strategy to control filariasis transmission. The microfilarial periodicities of 22 W. bancrofti antigen-positive volunteers were studied. Microfilariae (mf) were counted every hour for 24 h for 6 volunteers and every hour for 12 h (from 18:00 to 06:00) for 16 volunteers. At the same time as the preparation of mf test slides, indoor human landing catches of the vector mosquito, Anopheles farauti, were conducted to assess the vector biting activity. The time of peak microfilaraemia was 01:32 and the microfilarial periodicity index was 112.3, confirming the nocturnal periodicity of Wuchereria bancrofti in Vanuatu. Nearly all (98.5%) of the mf appeared during the time periods when A. farauti were collected. The timing of vector biting activity corresponded to the time of mf circulation.     

Identification of the vectors of lymphatic filariasis in the Lower Shire Valley, southern Malawi

An investigation of lymphatic filariasis vectors in Malawi is reported. Anopheles funestus, A. arabiensis, and A. gambiae sensu stricto had high rates of filarial infection (2.2-3.1%) and carried infective larvae. Anopheles funestus was the predominant species collected (77.6%) and was the primary vector during the study period of April to May 2002.     

Structure and evolution of the luciferin-regenerating enzyme (LRE) gene from the firefly Photinus pyralis

To study the structural features of genes for the luciferin-regenerating enzyme (LRE), the entire gene along with 524 bp of upstream sequence was determined from Photinus pyralis (Coleoptera: Lampyridae). The LRE gene revealed an open reading frame composed of five exons divided by four introns ranging in size from 47 to 904 bp. The deduced LRE amino acid sequence showed identity to senescence marker protein-30 (SMP30) from a number of insects and mammals including four putative SMP30 sequences from Anopheles gambiae. Gene structure comparisons showed some intron/exon site conservation with A. gambiae and mammalian SMP30 proteins but not Drosophila. LRE and luciferase sequence comparisons revealed two conserved putative luciferin-binding sites. The evolution of LRE was discussed in relation to its function.     

Molecular characterization of arrestin family members in the malaria vector mosquito, Anopheles gambiae

Olfaction influences many insect behaviours including mate seeking and host selection. The molecular machinery underlying insect olfactory systems is a G protein-coupled receptor pathway that, in addition to activation, requires adaptation for olfactory sensitivity and discrimination. We have previously identified ARR1 (henceforth AgARR1), a sensory arrestin from the malaria vector mosquito Anopheles gambiae that has been postulated to modulate olfactory adaptation. This report describes three additional arrestin family members including ARR2 (henceforth AgARR2), which is similar to previously characterized insect sensory arrestins and is expressed at significantly higher levels in the antennae of male vs. female A. gambiae mosquitoes. This finding is consistent with the hypothesis that AgARR2 may be important for the regulation of olfactory-driven behaviours particular to male mosquitoes.     

Genomic organization and immune regulation of the defensin gene from the mosquito, Anopheles gambiae

The defensin gene from the mosquito, Anopheles gambiae, is present as a single copy per haploid genome. Two exons, encoding a 102 residue preprodefensin, are separated by a 105 bp intron bounded by consensus splice sites. The upstream regulatory sequence includes a TATA box, arthropod initiator and numerous motifs homologous to insect and mammalian immune response elements. This promoter is capable of upregulation by immune challenge in cultured cells and activity is further stimulated by Gambif1, a mosquito Rel protein known to translocate to the nucleus and bind NF-kappa B sites in target promoters. Activity is inhibited by p50, a mammalian Rel protein that competitively binds NF-kappa B sites, and virtually abolished by p40, an avian I kappa B protein that inhibits nuclear translocation.     

Identification of the sibling species of the Anopheles maculipennis complex by heteroduplex analysis

The group of anopheline mosquitoes referred to as 'Anopheles maculipennis complex' includes the most important malaria vectors of the Palearctic Western region. The species belonging to this complex, however, are difficult or impossible to distinguish by morphological characters. To differentiate sibling palearctic species belonging to this complex, interspecific differences in the ITS2 sequences were used to set up a rapid and sensitive diagnostic tool based on heteroduplex analysis. The relative heteroduplex mobility allowed the following seven species to be readily distinguished: An. atroparvus, An. labranchiae, An. maculipennis s.s. , An. martinius, An melanoon, An. messeae and An. sacharovi.