Studies on the fitness characteristics of wMel- and wAlbB-introgressed Aedes aegypti (Pud) lines in comparison with wMel- and wAlbB-transinfected Aedes aegypti (Aus) and wild-type Aedes aegypti (Pud) lines.

Wolbachia, an intracellular maternally transmitted endosymbiont, has been shown to interfere with the replication of dengue virus in Aedes aegypti mosquitoes. The Wolbachia-transinfected Ae. aegypti has been currently released in many countries to test its effectiveness in preventing the transmission of dengue virus. ICMR-Vector Control Research Centre in collaboration with World Mosquito Program Monash University, Australia, has generated two new Wolbachia-introgressed Ae. aegypti Puducherry (Pud) lines via backcrossing Ae. aegypti females of Australian (Aus) strains, infected with wMel and wAlbB Wolbachia with wild-type Ae. aegypti Puducherry (Pud) males. Wolbachia infections are known to induce a fitness cost and confer benefit on the host mosquito populations that will influence spread of the Wolbachia into native wild mosquito populations during the field release. Hence, the induced fitness cost or benefit/advantage in the two newly generated Ae. aegypti (Pud) lines was assessed in the laboratory in comparison with the wild-type Ae. aegypti (Pud) strain. In addition, maternal transmission (MT) efficiency, induced cytoplasmic incompatibility (CI), and insecticide resistance status of the two (Pud) lines were determined to assess the likely frequency of wMel and wAlbB infections in the native wild population after field invasion. The study shows that wMel and wAlbB infections did not induce any fitness cost on the two newly generated (Pud) lines. Rather, in terms of wing length, fecundity, egg hatch rate, and adult survival, the Wolbachia introgression conferred fitness benefits on the (Pud) lines compared to uninfected Wolbachia free wild Ae. aegypti population. wMel and wAlbB exhibited a high maternal transmission (99-100%) and induced nearly complete (98-100%) cytoplasmic incompatibility. Both the (Pud) lines were resistant to deltamethrin, malathion, DDT, and temephos, and the level of resistance was almost the same between the two lines as in the wild type. Overall, the stable association of wMel and wAlbB established with Ae. aegypti and the reproductive advantages of the (Pud) lines encourage a pilot release in the field for population replacement potential.

Sensitivity of wMel and wAlbB Wolbachia infections in Aedes aegypti Puducherry (Indian) strains to heat stress during larval development.

BACKGROUND: ICMR-Vector Control Research Centre, Puducherry, India, developed two colonies of Aedes aegypti infected with wMel and wAlbB Wolbacia strains called Ae. aegypti (Pud) lines for dengue control. The sensitivity of wMel and wAlbB strains in Ae. aegypti (Pud) lines to heat stress was studied. METHODS: wMel and wAlbB infected and uninfected Ae. aegypti larvae (first to fourth instars) were reared in the laboratory to adults at 26 °C, 30 °C, 36 °C and 40 °C constant temperatures and also 26-30 °C, 26-36 °C and 26-40 °C diurnal cyclic temperatures. The adults were tested for Wolbachia infection. Experiments were also carried out rearing the larvae under simulated field conditions in summer (April and June) under sunlight using fully open and half open bowls and also under sunlight and natural shade. RESULTS: At 36 °C and 40 °C constant temperatures, complete larval mortality was observed. At 30 °C and 26 °C, no larval mortality occurred, but Wolbachia density was relatively low in wMel infected males compared to control (maintained at 26 ± 1 °C). At diurnal cyclic temperature of 26-40 °C, Wolbachia density was reduced in males of both the (Pud) lines, but not in females. At 26-36 °C, reduction in Wolbachia density was observed in wMel males but not in wAlbB males. At 26-30 °C, no significant reduction in Wolbachia density was observed with wMel and wAlbB strains. In simulated field conditions (April), under sunlight, the daytime water temperature reached a maximum of 35.7 °C in both full and half open bowls. No larval mortality occurred. Wolbachia frequency and density was reduced in wMel-infected Ae. aegypti (Pud) males from both type of bowls and in females from full open bowls, and in wAlbB males from half open bowls. In June, rearing of larvae under sunlight, the first-instar larvae experienced a maximum daytime water temperature of > 38 °C that caused complete mortality. No larval mortality was observed in bowls kept under shade (< 32 °C). CONCLUSIONS: Exposure of larvae to higher rearing temperatures in the laboratory and simulated-field conditions reduced the densities of wMel and wAlbB strains particularly in males, but the impact was more pronounced for wMel strain. The actual effect of heat stress on the stability of these two Wolbachia strains needs to be tested under natural field conditions.

Eosinophils Restrict Diesel Exhaust Particles-induced Cell Proliferation of Lung Epithelial A549 Cells via Interleukin-13 Mediated Mechanisms: Implications for Tissue Remodeling and Fibrosis.

BACKGROUND: Diesel exhaust particles (DEPs) affect lung physiology and cause serious damage to the lungs. A number of studies demonstrated that eosinophils play a very important role in the development of tissue remodeling and fibrosis of the lungs. However, the exact mechanism of pathogenesis of tissue remodeling and fibrosis is not known. METHODS: Both in vitro and in vivo models were used in the study. HL-60 and A549 cells were also utilized in the study. 8 to 12 weeks old BALB/c mice were used for the in vivo study. Cell viability by MTT assay and RNA isolation by tri reagent was accomplished. mRNA expression of inflammatory genes was accomplished by real-time PCR or qPCR. Immunohistochemistry was done to assess the localization and expressions of proteins. One-way ANOVA followed by a post hoc test was done for the statistical analysis. Graph-Pad prism 5 software was used for statistical analysis. RESULTS: For the first time, we demonstrate that interleukin-13 plays a very important role in the development of tissue remodeling and fibrosis. We report that diesel exhaust particles significantly induce eosinophils cell proliferation and interleukin-13 release in in vitro culture conditions. Supernatant collected from DEP-induced eosinophils cells significantly restricts cell proliferation of epithelial cells in response to exposure to diesel exhaust particles. Furthermore, purified interleukin-13 decreases the proliferation of A549 cells, highlighting the involvement of IL- 13 in tissue remodeling. Notably, Etoricoxib (selective COX-2 inhibitor) did not inhibit the DEPtriggered release of interleukin-13, suggesting another cell signaling pathway. The in vivo exposure of DEP to the lungs of mice resulted in a high level of eosinophils degranulation as depicted by the EPX-1 immunostaining and altered level of mRNA expressions of inflammatory genes. We also found that a-SMA, fibroblast specific protein (FSP-1), has been changed in response to DEP in the mice lungs along with the mediators of inflammation. CONCLUSION: Altogether, we elucidated the mechanistic role of eosinophils and IL-13 in the DEP-triggered proliferation of lungs cells, thus providing an insight into the pathophysiology of tissue remodeling and fibrosis of lungs.

Seasonal abundance of Leptotrombidium deliense, the vector of scrub typhus, in areas reporting acute encephalitis syndrome in Gorakhpur district, Uttar Pradesh, India.

The rickettsial pathogen Orientia tsutsugamushi, causing scrub typhus, has been implicated as a major cause of acute encephalitis syndrome (AES) in many places in India including Gorakhpur district of Uttar Pradesh. Seasonal abundance of the principal vector mite of the pathogen, Leptotrombidium deliense, its animal hosts, and prevalence of infection on them are important attributes in the assessment of outbreaks of the disease. Hence, these aspects were investigated, seasonally, in rural villages of Gorakhpur district, where peak incidence of AES cases were reported. A total of 903 animals (rodents/shrews) was collected using 6484 Sherman traps in eight study villages (14% overall trap rate). A sum of 5526 trombiculid mites comprising 12 species was collected from 676 live rodents/shrews screened. Suncus murinus, the Asian house shrew was the predominant species (67%). Among trombiculids, the principal vector mite, L. deliense, was predominant (64.7%) and its infestation index (i.e., average number of chiggers per host animal) was 5.3. The L. deliense infestation index was higher during July to November with a peak in October. Out of 401 animal sera samples screened, 68% were positive for antibodies against O. tsutsugamushi. Of 465 blood samples tested by nested PCR, seven were positive for the 56 kDa gene of O. tsutsugamushi. In conventional PCR, 41 out of 265 samples were positive for the 60 kDa groEL gene of O. tsutsugamushi. Among the 5526 mite samples, tested as 352 pools through nested PCR, four pools were positive for 56 kDa gene. Phylogenetic analysis of 56 and 60 kDa genes confirmed circulation of Karp and TA678 (rodents) and TA678 (mite) serotypes of O. tsutsugamushi in Gorakhpur. Peak incidence of AES in Gorakhpur district occurs during the rainy season (July-October), coinciding with the peak abundance of L. deliense. These results indicate involvement of L. deliense as the vector mite transmitting the scrub typhus pathogen O. tsutsugamushi to humans in the rural areas of Gorakhpur district, India.