Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

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Wolbachia pipientis is an obligate intracellular bacterium found in a wide range of invertebrate taxa. While over ecological timescales Wolbachia infections are maintained by strict maternal inheritance, horizontal transfer events are common over evolutionary time. To be horizontally transferred between organisms, Wolbachia bacteria must pass through and survive an extracellular phase. We used BacLight live-dead staining, PCR, and fluorescence in situ hybridization to assess the ability for purified Wolbachia bacteria to survive in cell-free media. We found that purified Wolbachia bacteria were able to survive extracellularly for up to 1 week with no decrease in viability. While no replication was observed in the extracellular phase, purified Wolbachia bacteria were able to reinvade cells and establish stable infections at all time points. The ability of Wolbachia bacteria to survive outside host cells may increase the probability of successful horizontal transfer and the exploitation of new ecological niches. Our development of methods to purify and maintain viable Wolbachia bacteria from cultured cells will be useful for other researchers studying Wolbachia biology.Wolbachia pipientis is an obligate intracellular bacterium found in a wide range of invertebrate hosts, including arthropods and filarial nematodes (18). Wolbachia bacteria cause alterations in host reproductive phenotypes, including cytoplasmic incompatibility, feminization, parthenogenesis, male killing, fitness alterations, and obligate symbiosis (5, 18). There has been a great amount of recent interest in Wolbachia as an agent to manipulate pest and vector populations (13,14,15,16,18,22,27) and control diseases caused by filarial nematodes (19, 21) and as a system to address questions related to the ecology and evolution of symbiosis (3, 4, 23) and speciation events (2,20).Over ecological timescales, Wolbachia infections are maintained by strict maternal inheritance (18). However, comparative phylogenetics indicate that over evolutionary timescales, Wolbachia horizontal transfer events are common, resulting in closely related infections in very distantly related host taxa (18,28). Potential nonexclusive mechanistic hypotheses for Wolbachia transfer between nonrelated hosts include parasitoids, cofeeding on the same host plant, or predation events (11,17,24). In all cases, to be horizontally transferred between hosts, Wolbachia bacteria must spend some time outside the intracellular environment. In transferring infection from embryos to cell culture, Dobson and colleagues incidentally showed that Wolbachia bacteria must remain viable outside a host cell for a brief period of time (6). Recently, Frydman and colleagues (7) demonstrated that when injected into the abdomen of Drosophila melanogaster, Wolbachia remained viable in the extracellular hemolymph environment long enough to establish intracellular infection of the ovarian stem cells. These results suggest that the ability of Wolbachia bacteria to survive outside a host cell may greatly incre...