Erica Suchman

Densoviruses for control and genetic manipulation of mosquitoes.

Mosquito densoviruses (MDV) are parvoviruses that replicate in the nuclei of mosquito cells and cause the characteristic nuclear hypertrophy (densonucleosis) that gives them their name. Several MDV that differ in pathogenicity both in vitro and in vivo have been isolated. MDV have a number of features that make them potentially attractive as biological control agents for mosquito-borne disease. They are nonenveloped and relatively stable in the environment. They are highly specific for mosquitoes and they infect and kill larvae in a dose dependent manner in the aqueous larval habitat. Infected larvae that survive to become adult mosquitoes exhibit a dose-dependent shortening of lifespan and many do not survive longer than the extrinsic incubation period for arboviruses. Thus they may have a significant impact on transmission of pathogens. Infected females can transmit the virus vertically by laying infected eggs in new oviposition sites. Studies on how MDV affect populations are relatively limited. Population cage studies suggest that they will persist and spread in populations and limited field studies have shown similar preimaginal mortality in wild populations to that seen in laboratory studies. The availability of infectious clones of MDV genomes allows the development of densovirus vectors for expressing genes of interest in mosquito cells and mosquitoes. Recently short hairpin RNA expression cassettes that induce RNA interference have been inserted into densovirus genomes. These expression cassettes should be useful for both research and disease-control applications.

Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [ Afanasiev et al. (1994) Exp Parasitol 79: 322–339; Afanasiev et al. (1999) Virology 257: 62–72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139–159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations ( Afanasiev et al., 1999 ; Allen‐Muira et al. (1999) Virology 257: 54–61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day‐old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10–0.15 m inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.

Rhythms and synchronization patterns in gene expression in the Aedes aegypti mosquito

BackgroundAedes aegypti is arguably the most studied of all mosquito species in the laboratory and is the primary vector of both Dengue and Yellow Fever flaviviruses in the field. A large number of transcriptional studies have been made in the species and these usually report transcript quantities observed at a certain age or stage of development. However, circadian oscillation is an important characteristic of gene expression in many animals and plants, modulating both their physiology and behavior. Circadian gene expression in mosquito species has been previously reported but for only a few genes directly involved in the function of the molecular clock.ResultsHerein we analyze the transcription profiles of 21,494 messenger RNAs using an Ae. aegypti Agilent® microarray. Transcripts were quantified in adult female heads at 24 hours and then again at 72 hours and eight subsequent time points spaced four hours apart. We document circadian rhythms in multiple molecular pathways essential for growth, development, immune response, detoxification/pesticide resistance. Circadian rhythms were also noted in ribosomal protein genes used for normalization in reverse transcribed PCR (RT-PCR) to determine transcript abundance. We report pervasive oscillations and intricate synchronization patterns relevant to all known biological pathways.ConclusionThese results argue strongly that transcriptional analyses either need to be made over time periods rather than confining analyses to a single time point or development stage or exceptional care needs to be made to synchronize all mosquitoes to be analyzed and compared among treatment groups.

Short-hairpin RNA expressed from polymerase III promoters mediates RNA interference in mosquito cells

Putative U6snRNA polymerase III (PolIII) promoters were cloned from the Anopheles gambiae and Aedes aegypti genomes. The PolIII promoters were tested for their ability to express short‐hairpin RNA (shRNA) targeted to firefly luciferase and to mediate RNA interference (RNAi) knockdown of a co‐transfected luciferase reporter gene vector in AG‐55 Anopheles gambiae and ATC‐10 Aedes aegypti cells. Promoters capable of silencing expression of the co‐transfected luciferase plasmid by up to 95% in AG‐55 cells and up to 75% in ATC‐10 cells were identified. RNase protection experiments allowed detection of the 19 nt luciferase short‐interfering RNA (siRNA) in transfected cells. These findings indicate that mosquito U6snRNA gene promoters can be used for production of shRNA to induce the RNAi response in mosquito cells.

PRODUCTION OF MOSQUITO DENSONUCLEOSIS VIRUSES BY AEDES ALBOPICTUS C6/36 CELLS ADAPTED TO SUSPENSION CULTURE IN SERUM-FREE PROTEIN-FREE MEDIA

SummaryTumor necrosis factor-α (TNF-α), cyclooxygenase (COX)-2, and prostaglandin (PG)E-2 play a critical role in the pathophysiology of arthritis. Tumor necrosis factor-α mediates induction of other cytokines, COX-2, PGs, and metallo-proteinases, which leads to cartilage degradation. We developed an in vitro human synoviocyte assay system for screening inhibitors of proinflammatory mediators in herbal extracts. Synoviocytes (5×105 cells/well) obtained during primary knee replacement from osteoarthritic patients were incubated with: control media alone or ginger extract (hydroxy-methoxy-phenyl compounds [HAPC]: EV.EXT® 77), 1 h before activation with 1 ng/ml TNF-α, 10 ng/ml interleukin-1β, or control media alone at 5% carbon dioxide, 37°C. Cell viability, TNF-α, COX-2, PGE-2, nuclear factor κB (NF-κB), and inhibitory subunit I kappa B-alpha (IκB-α) expression were analyzed by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blots. Ginger extract-HAPC (100 μg/ml) significantly inhibited the activation of TNF-α and COX-2 expression in human synoviocytes as well as suppressed production of TNF-α and PGE-2. Inhibition of TNF-α and COX-2 activation was accompanied by suppression of NF-κB and IκB-α induction. Using our in vitro assay, we discovered that the ginger extract blocks activation of proinflammatory mediators and its transcriptional regulator suggesting its mode of action. These observations indicate that ginger extract-HAPC offers a complementary and alternative approach to modulate the inflammatory process involved in arthritis.Abstract Mosquito densonucleosis viruses (MDVs) have the potential for use as biocontrol agents. To facilitate densovirus production, the Aedes albopictus mosquito cell line C6/36 was adapted to two commercially available serum-free protein-free media (SFPFM), Sf-900 II and Drosophila-SFM. Cells adapted more slowly to growth in Sf-900 II medium, but once adapted, they grew more rapidly and appeared healthier than cells growing in Drosophila-SFM. Cells that were adapted to growth in each of these SFPFM were tested for their ability to be transfected and infected with MDVs. The Sf-900 II–adapted cell line survived transfection and showed infection rates comparable with cells growing in L15 supplemented with 10% fetal bovine serum. Cells adapted to Drosophila-SFM were less infectable and did not survive transfection. Cells adapted to each of these SFPFM were adapted to growth in spinner flasks. Cells in Sf-900 II grew substantially better in spinner flasks than cells in Drosophila-SFM media. Cells grown in Sf-900 II could be frozen and, when thawed, could support the production of densonucleosis viruses in spinner flasks.

A Large Scale Laboratory Cage Trial of Aedes Densonucleosis Virus (AeDNV)

ABSTRACT Aedes aegypti (L.) (Diptera: Culicidae) the primary vector of dengue viruses (DENV1–4), oviposit in and around human dwellings, including sites difficult to locate, making control of this mosquito challenging. We explored the efficacy and sustainability of Aedes Densonucleosis Virus (AeDNV) as a biocontrol agent for Ae. aegypti in and among oviposition sites in large laboratory cages (>92 m3) as a prelude to field trials. Select cages were seeded with AeDNV in a single oviposition site (OPS) with unseeded OPSs established at varied distances. Quantitative real-time polymerase chain reaction was used to track dispersal and accumulation of AeDNV among OPSs. All eggs were collected weekly from each cage and counted. We asked: (1) Is AeDNV dispersed over varying distances and can it accumulate and persist in novel OPSs? (2) Are egg densities reduced in AeDNV treated populations? AeDNV was dispersed to and sustained in novel OPSs. Virus accumulation in OPSs was positively correlated with egg densities and proximity to the initial infection source affected the timing of dispersal and maintenance of viral titers. AeDNV did not significantly reduce Ae. aegypti egg densities. The current study documents that adult female Ae. aegypti oviposition behavior leads to successful viral dispersal from treated to novel containers in large-scale cages; however, the AeDNV titers reached were not sufficient to reduce egg densities.

Changing academic culture to improve undergraduate STEM education

Improving undergraduate science, technology, engineering, and math (STEM) education requires faculty with the skills, resources, and time to create active learning environments that foster student engagement. Current faculty hiring, promotion, and tenure practices at many universities do not measure, reward, nor encourage faculty pursuit of these skills. A cultural change is needed to foster improvement.

Using Magnets and Classroom Flipping to Promote Student Engagement and Learning about Protein Translation in a Large Microbiology Class.

It is generally accepted within the education community that active learning is superior to traditional lecturing alone. Many science educators, however, are reluctant to give up classroom time for activities because they fear that they will not have time to cover as much content. Classroom flipping has been gaining momentum in higher education as one way to engage students in the classroom while still exposing students to the same volume of course content. The activity presented here demonstrates how flipping one lecture period can be used in conjunction with an engaging in-class activity to teach a concept that is often difficult for students to learn through lecture alone. Specifically, we asked students to view a lecture video on bacterial protein translation before coming to class. We then used the classroom period to conduct a hands-on activity that allowed students to interact with magnetic pieces representing the components of protein translation to generate a protein from a given piece of DNA. Survey data showed that students liked the flipped classroom format associated with this activity, but they would not want every class flipped, and they perceived that the hands-on protein translation activity helped them to learn the material. Preliminary summative assessment data showed that this activity may have been useful in helping students to achieve the fundamental learning outcome that students will be able to translate a protein from a given piece of bacterial DNA.

Video Lecture Capture Technology Helps Students Study without Affecting Attendance in Large Microbiology Lecture Courses

Recording lectures using video lecture capture software and making them available for students to watch anytime, from anywhere, has become a common practice in many universities across many disciplines. The software has become increasingly easy to use and is commonly provided and maintained by higher education institutions. Several studies have reported that students use lecture capture to enhance their learning and study for assessments, as well as to catch up on material they miss when they cannot attend class due to extenuating circumstances. Furthermore, students with disabilities and students from non-English Speaking Backgrounds (NESB) may benefit from being able to watch the video lecture captures at their own pace. Yet, the effect of this technology on class attendance remains a controversial topic and largely unexplored in undergraduate microbiology education. Here, we show that when video lecture captures were available in our large enrollment general microbiology courses, attendance did not decrease. In fact, the majority of students reported that having the videos available did not encourage them to skip class, but rather they used them as a study tool. When we surveyed NESB students and nontraditional students about their attitudes toward this technology, they found it helpful for their learning and for keeping up with the material.

Aedes aegypti Densonucleosis Virus Amplifies, Spreads, and Reduces Host Populations in Laboratory Cage Studies

ABSTRACT Aedes densonucleosis virus (family Parvoviridae, genus Brevidensovirus, AeDNV) is a mosquito pathogen that increases Aedes aegypti larval mortality and reduces adult life span. We conducted three laboratory population cage trials, each lasting 16–25 wk. We tested two broad hypotheses. First, Ae. aegypti raised in containers seeded with 108 AeDNV genome equivaIents/ml (geq/ml), a concentration feasible for field application, increase AeDNV to concentrations that cause significant adult and larval mortality. Second, infected female mosquitoes disperse AeDNV to uninfected larval habitats. In hypothesis 1, we addressed the rate at which infected larvae secrete virus, how AeDNV titers change in seeded containers over time, whether AeDNV decays over time, and whether AeDNV exposed populations are reduced. In hypothesis 2, we monitored AeDNV concentrations in novel containers after oviposition by infected females. Both hypotheses were supported. Larvae increased AeDNV, secreting virus at a rate of 2.14 × 106 geq/larva/d when exposed to 108 geq/ml. AeDNV titers reached an asymptote of 1010 geq/ml by week 10 in seeded containers. AeDNV decayed by 1 log every 4 d as indicated by a reduction in larval mortality. Adult population size was reduced in treated populations. Infected females dispersed AeDNV to novel containers, with titers reaching 108 geq/ml. The parameters were used in a Leslie-Lewis matrix model. This model predicted that AeDNV negatively affects Ae. aegypti densities and population structure and thus vectorial capacity.