Experimental infection of bed bugs (Cimex lectularius L.) with Burkholderia multivorans.

The bacterium Burkholderia multivorans is an opportunistic nosocomial pathogen of humans. A previous study reported molecular detection of this bacterium in several specimens of the common bed bug (Cimex lectularius L.) collected from an elderly care facility in the U.S.A., raising questions about the possibility of vector-borne transmission. However, the ability of B. multivorans to colonize bed bugs and the ability of bed bugs to transmit the bacteria both remained untested. To resolve this knowledge gap, here we performed a set of experiments to examine the persistence and shedding of B. multivorans following ingestion by bed bugs in a blood meal. We isolated viable B. multivorans from the bodies of bed bugs for up to 13 days post-ingestion, but bacterial load substantially diminished over time. By 16 days post-ingestion, the bacteria could not be isolated. Further, B. multivorans was not shed in the saliva of infected bugs during feeding nor was it transmitted vertically from infected insects to their progeny. Based on these results, significant biological or mechanical transmission of B. multivorans to humans by bed bugs appears unlikely. Nonetheless, some viable bacteria were passively shed into the environment through defecation, a process which could potentially contribute to transmission through indirect contamination under rare circumstances.

Molecular detection of Rickettsia infection in field-collected bed bugs.

Bed bugs are now one of the most prevalent human-associated, blood-feeding pests in the urban world, but few studies of their association with human pathogens have been conducted since their resurgence. Here, we used PCR to screen samples of field-collected bed bugs (Cimex spp.) for the presence of Rickettsia bacteria and we describe the first detection of an uncharacterized Rickettsia in Cimex lectularius in nature. Rickettsia was detected in 5/39 (12.8%) of the bed bug samples tested. In particular, three pools from the USA and two individual insects from the UK were positive for Rickettsia DNA. Sequencing and analysis of a fragment of the citrate synthase gene (gltA) from positive samples from each country revealed that the Rickettsia detected in both were identical and were closely related to a Rickettsia previously detected in the rat flea Nosopsyllus laeviceps. Additional experiments indicated that the Rickettsia localizes to multiple tissues in the bed bug and reaches high titres. Attempts were made to infect mammalian cells in culture but these efforts were inconclusive. Our findings suggest that Rickettsia are secondary endosymbionts of bed bugs and have potential implications for both bed bug control and public health. However, further investigation is required to determine the pathogenicity of this Rickettsia, its transmission mechanisms, and its contributions to bed bug physiology.

Knockdown of mitogen-activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos.

Arthropod-borne infectious diseases are responsible for nearly 1.5 million deaths annually across the globe, with malaria responsible for >50% of these deaths. Recent efforts to enhance malaria control have focused on developing genetically modified Anopheles mosquitoes that are resistant to malaria parasite infection by manipulating proteins that are essential to the immune response. Although this approach has shown promise, the lack of efficient genetic tools in the mosquito makes it difficult to investigate innate immunity using reverse genetics. Current gene knockdown strategies based on small interfering RNA are typically labourious, inefficient, and require extensive training. In the present study, we describe the use of morpholino antisense oligomers to knockdown MEK-ERK signalling in the midgut of Anopheles stephensi through a simple feeding protocol. Anti-MEK morpholino provided in a saline meal was readily ingested by female mosquitoes with minimal toxicity and resulted in knockdown of total MEK protein levels 3-4 days after morpholino feeding. Further, anti-MEK morpholino feeding attenuated inducible phosphorylation of the downstream kinase ERK and, as predicted by previous work, reduced parasite burden in mosquitoes infected with Plasmodium falciparum. To our knowledge, this is the first example of morpholino use for target protein knockdown via feeding in an insect vector. Our results suggest this method is not only efficient for studies of individual proteins, but also for studies of phenotypic control by complex cell signalling networks. As such, our protocol is an effective alternative to current methods for gene knockdown in arthropods.