Life in the cystic fibrosis upper respiratory tract influences competitive ability of the opportunistic pathogen Pseudomonas aeruginosa.

Understanding characteristic differences between host-associated and free-living opportunistic pathogens can provide insight into the fundamental requirements for success after dispersal to the host environment, and more generally into the ecological and evolutionary processes by which populations respond to simultaneous selection on complex interacting traits. We examined how cystic fibrosis (CF)-associated and environmental isolates of the opportunistic pathogen Pseudomonas aeruginosa differ in the production of an ecologically important class of proteinaceous toxins known as bacteriocins, and how overall competitive ability depends on the production of and resistance to these bacteriocins. We determined bacteriocin gene content in a diverse collection of environmental and CF isolates and measured bacteriocin-mediated inhibition, resistance and the outcome of competition in a shared environment between all possible pairs of these isolates at 25°C and 37°C. Although CF isolates encoded significantly more bacteriocin genes, our phenotypic assays suggest that they have diminished bacteriocin-mediated killing and resistance capabilities relative to environmental isolates, regardless of incubation temperature. Notably, however, although bacteriocin killing and resistance profiles significantly predicted head-to-head competitive outcomes, CF and environmental isolates did not differ significantly in their competitive ability. This suggests that the contribution of bacteriocins to competitive ability involves selection on other traits that may be pleiotropically linked to interference competition mediated by bacteriocins.

Superinfection interference between dengue-2 and dengue-4 viruses in Aedes aegypti mosquitoes.

OBJECTIVE: Dengue virus consists of four antigenically distinct serotypes (DENV 1-4) that are transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes. In many dengue-endemic regions, co-circulation of two or more DENV serotypes is fairly common increasing the likelihood for exposure of the two vectors to multiple serotypes. We used a model system of DENV-2 and DENV-4 to investigate how prior exposure of Aedes aegypti to one DENV serotype affects its susceptibility to another serotype. METHODS: Aedes aegypti mosquitoes were sequentially infected with DENV-2 and DENV-4 and the infection and dissemination rates for each virus determined. RESULTS: We found that prior infection of Ae. aegypti mosquitoes with DENV-4 rendered them significantly less susceptible to secondary infection with DENV-2. Although the results were not statistically significant, mosquitoes infected with DENV-2 were also less susceptible to secondary infection with DENV-4. The midgut dissemination and population dissemination rates for DENV-2 were significantly higher than those of DENV-4 when either virus was administered 7 days after administration of either a non-infectious blood meal or a blood meal containing a heterologous dengue serotype. CONCLUSION: These results demonstrate that superinfection interference between DENV serotypes is possible within Ae. aegypti mosquitoes, but its effect on DENV epidemiology may be dependent on the fitness of interacting serotypes.

Comparative Susceptibility of Ochlerotatus japonicus, Ochlerotatus triseriatus, Aedes albopictus, and Aedes aegypti (Diptera: Culicidae) to La Crosse Virus.

Invasive mosquito species can increase the transmission risk of native mosquito-borne diseases by acting as novel vectors. In this study, we examined the susceptibility of three exotic invasive mosquito species Aedes aegypti (L.), Ae. albopictus (Skuse), and Ochlerotatus japonicus (Theobald) to La Crosse virus (LACV) relative to the native primary vector Ochlerotatus triseriatus (Say). Adult females of the four mosquito species were orally challenged with LACV; incubated for 3, 5, 7, 9, or 11 d; and their midgut infection rates, dissemination rates, and effective vector competence were determined. Overall, Oc. japonicus (2.92) had the highest effective vector competence values, followed by Ae. albopictus (1.55), Ae. aegypti (0.88), and Oc. triseriatus (0.64). In addition, we assessed the relationship between mosquito size and LACV susceptibility for field-collected Oc. triseriatus and Oc. japonicus We hypothesized that smaller adults would be more susceptible to LACV; however, our results did not support this hypothesis. Infected Oc. triseriatus tended to be larger than exposed but uninfected females, while infected and uninfected Oc. japonicus were similarly sized. These findings suggest that Oc. japonicus, Ae. albopictus, and Ae. aegypti have significant potential to transmit LACV and more research is needed to uncover their potential role in LACV epidemiology.

Midgut fungal and bacterial microbiota of Aedes triseriatus and Aedes japonicus shift in response to La Crosse virus infection.

Understanding how midgut microbial communities of field-collected mosquitoes interact with pathogens is critical for controlling vector infection and disease. We used 16S rRNA and internal transcribed spacer sequencing to characterize the midgut bacterial and fungal communities of adult females of Aedes triseriatus and Aedes japonicus collected as pupae in tree holes, plastic bins and waste tires and their response to La Crosse virus (LACV) infection. For both mosquito species and across all habitat and virus treatments, a total of 62 bacterial operational taxonomic units (OTUs) from six phyla and 21 fungal OTUs from two phyla were identified. The majority of bacterial (92%) and fungal (71%) OTUs were shared between the mosquito species; however, several OTUs were unique to each species. Bacterial and fungal communities of individuals that took either infectious or noninfectious bloodmeals were less diverse and more homogeneous compared to those of newly emerged adults. Interestingly, LACV-infected A. triseriatus and A. japonicus had higher bacterial richness and lower fungal richness compared to individuals that took a noninfectious bloodmeal, suggesting that viral infection was associated with an increase in bacterial OTUs and a decrease in fungal OTUs. For both mosquito species, several OTUs were identified that had both high fidelity and specificity to mosquito midguts that were infected with LACV. Overall, these findings demonstrate that bacterial and fungal communities that reside in mosquito midguts respond to host diet and viral infection and could play a role in modulating vector susceptibility to LACV.

Culex pipiens and Culex restuans mosquitoes harbor distinct microbiota dominated by few bacterial taxa.

BACKGROUND: Mosquitoes host diverse microbial communities that influence many aspects of their biology including reproduction, digestion, and ability to transmit pathogens. Unraveling the composition, structure, and function of these microbiota can provide new opportunities for exploiting microbial function for mosquito-borne disease control. METHODS: MiSeq® sequencing of 16S rRNA gene amplicons was used to characterize the microbiota of adult females of Culex pipiens L. and Cx. restuans Theobald collected from nine study sites in central Illinois. RESULTS: Out of 195 bacterial OTUs that were identified, 86 were shared between the two mosquito species while 16 and 93 OTUs were unique to Cx. pipiens and Cx. restuans, respectively. The composition and structure of microbial communities differed significantly between the two mosquito species with Cx. restuans hosting a more diverse bacterial community compared to Cx. pipiens. Wolbachia (OTU836919) was the dominant bacterial species in Cx. pipiens accounting for 91% of total microbiota while Sphingomonas (OTU817982) was the dominant bacterial species in Cx. restuans accounting for 31% of total microbiota. Only 3 and 6 OTUs occurred in over 60% of individuals in Cx. pipiens and Cx. restuans, respectively. There was little effect of study site on bacterial community structure of either mosquito species. CONCLUSION: These results suggest that the two mosquito species support distinct microbial communities that are sparsely distributed between individuals. These findings will allow investigations of the role of identified microbiota on the spatial and temporal heterogeneity in WNV transmission and their potential application in disease control.

Container Type Influences the Relative Abundance, Body Size, and Susceptibility of Ochlerotatus triseriatus (Diptera: Culicidae) to La Crosse Virus.

Ochlerotatus triseriatus (Say), the primary vector of La Crosse virus (LAC), develops in a variety of natural and artificial aquatic containers where it often co-occurs with larvae of other mosquito species. We conducted a field study at two woodlots (South Farms and Trelease Woods) in Urbana, IL, to examine how container type influences vector abundance, body size, and susceptibility to LAC. Mosquito pupae were collected from tree holes, plastic bins, and waste tires, and eclosing adults were identified to species morphologically. Oc. triseriatus and Ochlerotatus japonicus (Theobald) females were orally challenged with LAC and midgut infection rate, disseminated infection rate, and body titer were determined by reverse-transcriptase real-time PCR. Oc. triseriatus was the dominant species collected in tree holes while Oc. japonicus and Culex restuans (Theobald) were mostly dominant in artificial containers. Female Oc. triseriatus and Oc. japonicus collected from plastic bins were significantly larger than those collected from tree holes or waste tires. Oc. japonicus females from South Farms were also significantly larger than those from Trelease Woods. Oc. triseriatus females collected from plastic bins and waste tires were significantly more susceptible to LAC infection relative to females collected from tree holes. In addition, Oc. triseriatus females from waste tires had significantly higher LAC titer relative to Oc. triseriatus from tree holes. For each container type and study site, wing length was not correlated to infection or dissemination rates. These findings suggest that the container type in which Oc.triseriatus develop may contribute to the spatial and temporal dynamics of LAC transmission.

Impact of an Alien Invasive Shrub on Ecology of Native and Alien Invasive Mosquito Species (Diptera: Culicidae).

We examined how leaf litter of alien invasive honeysuckle (Lonicera maackii Rupr.) either alone or in combination with leaf litter of one of two native tree species, sugar maple (Acer saccharum Marshall) and northern red oak (Quercus rubra L.), affects the ecology of Culex restuans Theobald, Ochlerotatus triseriatus Say, and Ochlerotatus japonicus Theobald. Experimental mesocosms containing single species litter or a mixture of honeysuckle and one of two native tree species litter were established at South Farms and Trelease Woods study sites in Urbana, IL, and examined for their effect on 1) oviposition site selection by the three mosquito species, and 2) adult production and body size of Oc. triseriatus and Oc. japonicus. There were no significant effects of study site and leaf treatment on Oc. japonicus and Oc. triseriatus oviposition preference and adult production. In contrast, significantly more Cx. restuans eggs rafts were collected at South Farms relative to Trelease Woods and in honeysuckle litter relative to native tree species litter. Significantly larger adult females of Oc. japonicus and Oc. triseriatus were collected at South Farms relative to Trelease Woods and in honeysuckle litter relative to native tree species litter. Combining honeysuckle litter with native tree species litter had additive effects on Cx. restuans oviposition preference and Oc. japonicus and Oc. triseriatus body size, with the exception of honeysuckle and northern red oak litter combination, which had antagonistic effects on Oc. triseriatus body size. We conclude that input of honeysuckle litter into container aquatic habitats may alter the life history traits of vector mosquito species.

Effect of Larval Competition on Extrinsic Incubation Period and Vectorial Capacity of Aedes albopictus for Dengue Virus.

Despite the growing awareness that larval competition can influence adult mosquito life history traits including susceptibility to pathogens, the net effect of larval competition on human risk of exposure to mosquito-borne pathogens remains poorly understood. We examined how intraspecific larval competition affects dengue-2 virus (DENV-2) extrinsic incubation period and vectorial capacity of its natural vector Aedes albopictus. Adult Ae. albopictus from low and high-larval density conditions were orally challenged with DENV-2 and then assayed for virus infection and dissemination rates following a 6, 9, or 12-day incubation period using real-time quantitative reverse transcription PCR. We then modeled the effect of larval competition on vectorial capacity using parameter estimates obtained from peer-reviewed field and laboratory studies. Larval competition resulted in significantly longer development times, lower emergence rates, and smaller adults, but did not significantly affect the extrinsic incubation period of DENV-2 in Ae. albopictus. Our vectorial capacity models suggest that the effect of larval competition on adult mosquito longevity likely has a greater influence on vectorial capacity relative to any competition-induced changes in vector competence. Furthermore, we found that large increases in the viral dissemination rate may be necessary to compensate for small competition-induced reductions in daily survivorship. Our results indicate that mosquito populations that experience stress from larval competition are likely to have a reduced vectorial capacity, even when susceptibility to pathogens is enhanced.

Sindbis virus interferes with dengue 4 virus replication and its potential transmission by Aedes albopictus.

BACKGROUND: Mosquitoes transmit a number of arboviruses associated with disease outbreaks in humans and other animals. The majority of medically important arboviruses belong to three families: Togaviridae, Flaviviridae and Bunyaviridae. Several members of these families have overlapping distributions and share common vectors, increasing the potential for arboviral coinfections. This study examined how two model viruses: Sindbis virus (SINV, Togaviridae: Alphavirus) and dengue-4 virus (DENV-4, Flaviviridae: Flavivirus) may interact in C6/36 Aedes albopictus cells and in the mosquito vector Ae. albopictus. METHODS: C6/36 cells were coinfected, superinfected, or singly infected with SINV and DENV-4 and the two viruses quantified at different time points. Four to seven day old adult females of Ae. albopictus were also fed blood containing one or both viruses and viral infection and dissemination rates determined. RESULTS: Sindbis virus suppressed replication of DENV-4 in C6/36 Ae. albopictus cells with greater inhibition occurring when the two arboviruses were inoculated simultaneously compared to sequentially. In addition, Ae. albopictus simultaneously exposed to both arboviruses had significantly lower DENV-4 infection and population dissemination rates compared to those exposed to DENV-4 alone. CONCLUSION: These results suggest that certain Alphaviruses may interfere with DENV-4 transmission by suppressing its replication and increasing vector refractoriness. The findings provide important insights into the potential contribution of mixed arboviral infections to DENV transmission dynamics.

Sublethal effects of atrazine and glyphosate on life history traits of Aedes aegypti and Aedes albopictus (Diptera: Culicidae).

Although exposure of mosquito larvae to agricultural chemicals such as herbicides is common and widespread, our understanding of how these chemicals affect mosquito ecology and behavior is limited. This study investigated how an environmentally relevant concentration of two herbicides, atrazine and glyphosate, affects mosquito life history traits. One hundred and fifty (150) first instar Aedes (Stegomyia) aegypti (L.) or Aedes (Stegomyia) albopictus (Skuse) larvae were reared in 1.6 L of live oak leaf (Quercus virginiana) infusion in the presence (5 mg/L) or absence (0 mg/L) of atrazine or glyphosate. The containers were monitored daily to determine the emergence rates, sex ratio, male and female emergence times, and female body size. Emergence rates of A. aegypti from atrazine treatment were significantly higher relative to either glyphosate or control treatments (A. aegypti: atrazine = 93 ± 6% (±95% CI), glyphosate = 82 ± 5%, control = 78 ± 5%), while emergence rates of A. albopictus in atrazine treatments were significantly higher than in glyphosate treatments but not in controls (A. albopictus: atrazine = 84 ± 5 %, glyphosate = 76 ± 4%, control = 78 ± 4%). For both mosquito species, a sex ratio distortion with male bias was observed in control and glyphosate treatments, but not in atrazine treatments (A. aegypti: atrazine = 0.90 ± 0.17 (±SE), glyphosate = 1.63 ± 0.21, control = 1.69 ± 0.26; A. albopictus: atrazine = 1.09 ± 0.08, glyphosate = 1.88 ± 0.12, control = 1.37 ± 0.11). Emergence times for both sexes of the two mosquito species were significantly longer in atrazine treatments compared to glyphosate or control treatments (A. aegypti: females: atrazine = 11.20 ± 0.50 (days ± 95 % CI), glyphosate = 9.71 ± 0.23, control = 9.87 ± 0.21; males: atrazine = 9.46 ± 0.27, glyphosate = 8.80 ± 0.25, control = 8.85 ± 0.24; A. albopictus: females: atrazine = 17.40 ± 1.70, glyphosate = 12.4 ± 0.40, control = 12.5 ± 0.30; males: atrazine = 12.96 ± 0.41, glyphosate = 10.48 ± 0.24, control = 10.64 ± 0.37). For A. albopictus but not A. aegypti, adult females from atrazine treatment had significantly longer wing lengths compared to those from glyphosate or control treatments (A. albopictus: atrazine = 3.06 ± 0.07 (mm ± 95% CI), glyphosate = 2.80 ± 0.07, control = 2.83 ± 0.06). These results demonstrate the potential for atrazine, a widely used herbicide, to influence epidemiologically relevant life history traits of mosquitoes.

Effect of mixed infections of Sindbis and La Crosse viruses on replication of each virus in vitro.

Mixed viral infections are ubiquitous in natural populations and may have significant but unpredictable biological and epidemiological consequences. These infections may be acquired simultaneously (coinfection) or result from two single infections at different time intervals (superinfection). This study investigated the effect of mixed infections of two mosquito-borne viruses, Sindbis (SINV) and La Crosse (LACV), on the replication of each virus in vertebrate baby hamster kidney (BHK) and invertebrate Aedes albopictus (C6/36) cell lines. SINV and LACV were inoculated onto confluent monolayers of each cell type either singly, simultaneously, or with a 2-h delay between single inoculations and titers for each virus were quantified by qRT-PCR. The titers for both viruses were significantly higher in BHK compared to C6/36 cells. Mixed infections suppressed replication of both viruses in BHK cells except for one superinfection treatment where inoculation of SINV ahead of LACV did not result in a reduction in SINV titer relative to single infection treatment. In C6/36 cells mixed infections had no effect on LACV replication, while coinfection enhanced SINV replication. These results demonstrate the potential for mixed viral infections to modify arbovirus transmission and pathogenesis.