Meiotic transmission of an in vitro-assembled autonomous maize minichromosome.

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes.

The Search for Novel Malaria Transmission-blocking Targets in the Mosquito Midgut.

The need for new malaria control strategies has led to increased efforts to understand more clearly the mosquito stages of Plasmodium. The absolute requirement of gamete maturation and fertilization, transformation of sedentary zygote to motile ookinete, ookinete interaction and invasion of gut epithelium, and the survival of the mosquito against immune attack suggest that numerous unidentified targets exist, which could be modified to achieve transmission-blocking of malaria. In the search for new transmission-blocking targets in the mosquito gut, Mohammed Shahabuddin, Stéphane Cociancich and Helge Zieler here summarize recent studies to identify the cellular and biochemical factors that affect the malaria parasite's development; in particular, factors influencing the early development of Plasmodium, receptor-mediated interactions between the parasite and the mosquito midgut, and the gut-associated immune responses directed against Plasmodium.

Seasonal malaria transmission and variation of anopheline density in two distinct endemic areas in Brazilian Amazonia.

Studies on seasonal anopheline fauna variation were performed in two distinct settlements in the State of Rondônia, Brazil: one at the Madeira River banks (Portuchuelo) with stable native Amazonian population; the other at an inland lumber-extracting farm (Urupá) in dry land, in which adults are mostly migrants. During a 6-yr period (1994-2000), 8,638 adult anophelines were collected: 2,684 in Urupá and 5,954 in Portuchuelo. Anopheles darlingi represented >95% of total mosquitoes caught.

Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes.

Malaria kills millions of people every year, and new control measures are urgently needed. The recent demonstration that (effector) genes can be introduced into the mosquito germ line to diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A.

Intron-dependent stimulation of marker gene expression in cultured insect cells.

We tested in a systematic fashion the effect of an intron on the level of luciferase expression in cultured C6/36 Aedes albopictus cells. The intron was inserted in both orientations, upstream and downstream of the luciferase coding region in two different luciferase expression vectors. The two parental luciferase expression vectors differed only in their promoters, one containing the Drosophila melanogaster actin5C promoter and the other the Autographa californica nuclear polyhedrosis virus hr5/ie1 enhancer/promoter.

A snake venom phospholipase A(2) blocks malaria parasite development in the mosquito midgut by inhibiting ookinete association with the midgut surface.

Oocyst formation is a critical stage in the development of the malaria parasite in the mosquito. We have discovered that the phospholipase A(2) (PLA2) from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus) inhibits oocyst formation when added to infected chicken blood and fed to mosquitoes. A similar transmission-blocking activity was demonstrated for PLA2s from the venom of other snakes and from the honeybee.

Invasion in vitro of mosquito midgut cells by the malaria parasite proceeds by a conserved mechanism and results in death of the invaded midgut cells.

Using an in vitro culture system, we observed the migration of malaria ookinetes on the surface of the mosquito midgut and invasion of the midgut epithelium. Ookinetes display constrictions during migration to the midgut surface and a gliding motion once on the luminal midgut surface. Invasion of a midgut cell always occurs at its lateral apical surface.

A tubular network associated with the brush-border surface of the Aedes aegypti midgut: implications for pathogen transmission by mosquitoes.

The mosquito Aedes aegypti is capable of transmitting a variety of pathogens to man and to other vertebrates. The midgut of this insect has been well-studied both as the tissue where the first contact occurs between ingested pathogens and the insect host, and as a model system for blood meal digestion in blood-sucking insects. To understand better the nature of the midgut surface encountered by parasites or viruses, we used scanning electron microscopy to identify the most prominent structures and cell morphologies on the luminal midgut surface.

Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines.

We have constructed a series of plasmid vectors for the expression of foreign genes in insects or insect cell lines. We incorporated the Drosophila hsp70 and actin 5C promoters, as well as the hr5 enhancer-driven baculovirus ie1 promoter, into plasmids that allow convenient cloning of heterologous genes into multiple cloning sites. We combined these promoters with either a short, double poly-adenylation site derived from the Heliothis virescens p63 chaperonin gene, or with a fusion of the small t intron with the early 3' untranslated region and poly-adenylation sites of SV40.

Plasmodium gallinaceum ookinetes adhere specifically to the midgut epithelium of Aedes aegypti by interaction with a carbohydrate ligand.

During the course of its development in the mosquito and transmission to a new vertebrate host, the malaria parasite must interact with the mosquito midgut and invade the gut epithelium. To investigate how the parasite recognizes the midgut before invasion, we have developed an in vitro adhesion assay based on combining fluorescently labelled ookinetes with isolated midgut epithelia from blood-fed mosquitoes. Using this assay, we found that Plasmodium gallinaceum ookinetes readily adhered to midguts of Aedes aegypti, mimicking the natural recognition of the epithelium by the parasite.

Adhesion of Plasmodium gallinaceum ookinetes to the Aedes aegypti midgut: sites of parasite attachment and morphological changes in the ookinete.

Plasmodium gallinaceum ookinetes adhered to Aedes aegypti midgut epithelia when purified ookinetes and isolated midguts were combined in vitro. Ookinetes preferentially bound to the microvillated luminal surface of the midgut, and they seemed to interact with three types of structures on the midgut surface. First, they adhered to and migrated through a network-like matrix, which we have termed microvilli-associated network, that covers the surface of the microvilli.

Plasmodium gallinaceum: fluorescent staining of zygotes and ookinetes to study malaria parasites in mosquito.

We have developed a fluorescent labeling procedure for staining the mosquito stages of Plasmodium gallinaceum. PKH26, a lipophilic dye, is efficiently and permanently incorporated into the membranes of zygotes and ookinetes. Stained zygotes undergo normal development into ookinetes; the stain does not interfere with ookinete mobility or ability to adhere to the mosquito midgut lumen.

Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein.

The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid.

Ability of Anopheles gambiae mosquitoes to transmit malaria during the dry and wet seasons in an area of irrigated rice cultivation in The Gambia.

The seasonality of malaria transmission was studied in a Gambian village situated in an area where rice was cultivated. Observations were made during two dry seasons, when pump-fed irrigation was used to grow rice, and in the intervening rainy season, when rice was cultivated using a combination of irrigated and rain-fed paddies. Clinical episodes of malaria were mainly confined to the months during and soon after the rainy season.