Effects of crowding and temperature on Wolbachia infection density among life cycle stages of Aedes albopictus

Species of the genus Wolbachia are a group of Rickettsia-like, maternally-inherited bacteria (gram negative), which cause various reproductive alterations in their arthropod and nematode hosts including cytoplasmic incompatibility (CI), male-killing, parthenogenesis and feminization. They can be divided into supergroups such as A and B based on phylogenetic analysis of 16S rDNA sequences. In this study, we examined the relative infection densities of Wolbachia strains among life cycle stages in the mosquito, Aedes albopictus in terms of crowding effect and temperature effect. A. albopictus is known to be superinfected with both A- and B-supergroup Wolbachia which cause CI. The relative Wolbachia densities within each individual mosquito were determined and quantified by using real-time quantitative PCR assay based on the wsp gene. We found that B-supergroup Wolbachia strain densities in this host species were consistently and significantly higher than in the A-supergroup. Larval crowding also reduced adult size of mosquitoes. Our results show clearly that the higher densities of mosquito larvae cause lower densities of Wolbachia strains. Examination of the effect of temperature on Wolbachia density in each stage of the mosquito clearly revealed a significant decrease in bacterial density following exposure to elevated temperature (37 °C) in both males and females.     

Diapause-specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization

In this study we probe the molecular events underpinning diapause observed in overwintering females of Culex pipiens. Using suppressive subtractive hybridization (SSH) we have identified 40 genes that are either upregulated or downregulated during this seasonal period of dormancy. Northern blot hybridizations have confirmed the expression of 32 of our SSH clones, including six genes that are upregulated specifically in early diapause, 17 that are upregulated in late diapause, and two upregulated throughout diapause. In addition, two genes are diapause downregulated and five remain unchanged during diapause. These genes can be categorized into eight functional groups: genes with regulatory functions, metabolically-related genes, those involved in food utilization, stress response genes, cytoskeletal genes, ribosomal genes, transposable elements, and genes with unknown functions.     

Stochastic eco-epidemiological model of dengue disease transmission by Aedes aegypti mosquito

We present a stochastic dynamical model for the transmission of dengue that takes into account seasonal and spatial dynamics of the vector Aedes aegypti. It describes disease dynamics triggered by the arrival of infected people in a city. We show that the probability of an epidemic outbreak depends on seasonal variation in temperature and on the availability of breeding sites. We also show that the arrival date of an infected human in a susceptible population dramatically affects the distribution of the final size of epidemics and that early outbreaks have a low probability. However, early outbreaks are likely to produce large epidemics because they have a longer time to evolve before the winter extinction of vectors. Our model could be used to estimate the risk and final size of epidemic outbreaks in regions with seasonal climatic variations.     

Inheritance of the photoperiodic response controlling imaginal summer diapause in the cabbage beetle, Colaphellus bowringi

Adults of the cabbage beetle Colaphellus bowringi display a summer diapause in response to the exposure of their larvae to long photoperiods. In the present study, the inheritance of the photoperiodic response controlling summer diapause in C. bowringi by crossing a high diapause strain (D strain) with a laboratory selected nondiapause strain (N strain) was investigated under different photoperiods at 22, 25 and 28 °C. The beetles in both reciprocal crosses and backcrosses showed a clear short-day response for the induction of diapause at all temperatures, similar to that of the D strain, suggesting that photoperiodic response of this beetle is heritable. The diapause incidences in the progeny from all the crosses under LD 15:9 or LD 12:12 at 25 °C suggest that genetic and genetic-environmental interactions are involved in diapause induction. The incidence of diapauses in F₁ progeny was significantly lower than that in the D × D strain but significantly higher than that in the N × N strain, indicating that the diapause capability is inherited in an incomplete dominant manner. The incidence of diapause was greater among the offspring of hybrid females when those females had a D strain mother or grandmother rather than a N strain mother or grandmother, indicating that maternal effects on diapause induction are stronger than paternal effects. The laboratory selected nondiapause strain also showed a short-day photoperiodic response at a low temperature of 22 °C, indicating that the photoperiodic photoreceptor and photoperiodic clock still function in the nondiapause strain.     

Present and future projections of habitat suitability of the Asian tiger mosquito,a vector of viral pathogens,from global climate simulation

Climate change can influence the transmission of vector-borne diseases (VBDs) through altering the habitat suitability of insect vectors. Here we present global climate model simulations and evaluate the associated uncertainties in view of the main meteorological factors that may affect the distribution of the Asian tiger mosquito (Aedes albopictus), which can transmit pathogens that cause chikungunya, dengue fever, yellow fever and various encephalitides. Using a general circulation model at 50 km horizontal resolution to simulate mosquito survival variables including temperature, precipitation and relative humidity, we present both global and regional projections of the habitat suitability up to the middle of the twenty-first century. The model resolution of 50 km allows evaluation against previous projections for Europe and provides a basis for comparative analyses with other regions. Model uncertainties and performance are addressed in light of the recent CMIP5 ensemble climate model simulations for the RCP8.5 concentration pathway and using meteorological re-analysis data (ERA-Interim/ECMWF) for the recent past. Uncertainty ranges associated with the thresholds of meteorological variables that may affect the distribution of Ae. albopictus are diagnosed using fuzzy-logic methodology, notably to assess the influence of selected meteorological criteria and combinations of criteria that influence mosquito habitat suitability. From the climate projections for 2050, and adopting a habitat suitability index larger than 70%, we estimate that approximately 2.4 billion individuals in a land area of nearly 20 million km² will potentially be exposed to Ae. albopictus. The synthesis of fuzzy-logic based on mosquito biology and climate change analysis provides new insights into the regional and global spreading of VBDs to support disease control and policy making.     

Circadian control of permethrin-resistance in the mosquito Aedes aegypti

Daily fluctuation of permethrin-resistance was found in adult mosquito Aedes aegypti, the major vector of dengue viruses in Taiwan. We hypothesized there is a relationship between resistance and the circadian clock. To test our hypothesis we correlated changes in the knock-down time (KT₅₀) response to permethrin with the expression of the pyrethroid-resistant gene CYP9M9 and the clock gene period (per) during a 12:12 h photoperiodic cycle. Rhythmic expression of per peaked at early scotophase of the light-dark cycle and at early subjective night in constant darkness. The values of KT₅₀ and the expression of CYP9M9 also exhibited circadian rhythms in both susceptible and permethrin-resistant mosquito strains, from which we inferred a link to the circadian clock. The KT₅₀ was significantly longer in the light than in the dark phase, and the level of CYP9M9 mRNA was maximal in early scotophase, dropped to a minimum in the midnight and then slowly increased through the photophase. Existence of a clock control over mosquito sensitivity to permethrin was further indicated by reduced expression of CYP9M9 and reduced mosquito resistance to permethrin after temporal silencing of the per gene. These data provide the first evidence on the circadian control of insect resistance to permethrin.     

Thermal response and reversibility of prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis undergoes prolonged larval diapause that is terminated by chilling and warming cycles. To examine the effects of warming temperatures and their duration on diapause termination, we exposed diapause larvae that had not been reactivated after chilling at 5 °C to 20 or 25 °C and chilled them again before incubation at 20 °C. With increasing warming duration at 20 °C, diapause termination after chilling increased and shorter chilling durations became effective. In contrast, few or no larvae warmed at 25 °C terminated diapause after chilling, irrespective of the warming duration. To investigate the effect of warming temperature on diapause intensity, larvae with diapause weakened by initial incubation at 20 °C after the first chilling were subsequently incubated at 15, 20, or 25 °C, then chilled at 5 °C before incubation at 20 °C. Diapause termination increased significantly after the larvae were treated at 15 or 20 °C but decreased significantly after they were treated at 25 °C. The intensification of prolonged diapause at 25 °C was reversed when the larvae were transferred to 20 °C. Diapause intensity in C. sikkimensis therefore decreases at 20 °C, increases at 25 °C, and can be reversed by alternately exposing diapause larvae to 20 and 25 °C. In C. sikkimensis, prolonged diapause does not always proceed in one direction, and its intensity fluctuates in response to ambient temperature conditions.     

Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes

The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes.     

Development of pooled suppression subtractive hybridization to analyze the pangenome of Staphylococcus aureus

We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.     

Detection of lysozyme-like enzymatic activity secreted by an immune-responsive mosquito cell line

Using molecular approaches, we have recently shown that the C7-10 mosquito cell line from Aedes albopictus, and the Aag-2 line from Aedes aegypti, secrete a variety of immune peptides into the culture medium, including cecropins, defensins, transferrin, and lysozyme. The diversity of these peptides makes it difficult to quantify the relative activities of each molecule, because possible synergistic interactions may occur. Using a microtiter plate assay with live bacteria, we now show that C7-10 cells secrete an activity that is more potent against the Gram-positive bacterium, Micrococcus luteus, than against Gram-negative Escherichia coli. This lysozyme-like activity is accompanied by production of a lytic zone in an agarose plate assay containing commercially available, lyophilized M. luteus. Properties of the lysozyme-like activity from C7-10 cells included a broad pH optimum from 5.5 to 6.5, and heat-sensitivity above 42 degrees C. Amounts of secreted activity increased during the initial 24h of incubation with heat-killed bacteria. During this induction, lysozyme-like activity was found primarily in the cell culture supernatant.     

Optimal control of Aedes aegypti mosquitoes by the sterile insect technique and insecticide

We present a mathematical model to describe the dynamics of mosquito population when sterile male mosquitoes (produced by irradiation) are introduced as a biological control, besides the application of insecticide. In order to analyze the minimal effort to reduce the fertile female mosquitoes, we search for the optimal control considering the cost of insecticide application, the cost of the production of irradiated mosquitoes and their delivery as well as the social cost (proportional to the number of fertilized females mosquitoes). The optimal control is obtained by applying the Pontryagin’s Maximum Principle.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.