Individual Ae Research Articles

Detection of Wolbachia in field‐collected Aedes aegypti mosquitoes from Jeddah, Saudi Arabia

AbstractRecent reports have disclosed the occurrence of Wolbachia in Aedes aegypti. Our study detected Wolbachia infection in Ae. aegypti by screening wild adult mosquitoes using two Wolbachia‐specific molecular markers. Overall, 444 adult Ae. aegypti mosquitoes were collected from April 2022 to October 2022 in Jeddah, Saudi Arabia. Each individual sample was processed and screened for the presence of Wolbachia using selected markers, the Wolbachia‐specific 16S rDNA and the Wolbachia surface protein gene (wsp), under optimized polymerase chain reaction (PCR) conditions, and sequenced. In total, 39 (8.78%) and 48 (10.81%) individual mosquito samples were determined to be infected with Wolbachia using the wsp and 16S rDNA markers, respectively. By utilizing two Wolbachia‐specific molecular markers, our study demonstrated the presence of Wolbachia from individual Ae. aegypti samples. Our results showed a low rate of Wolbachia infection and inferred that the detected strain belongs to supergroup B.

Extreme infectious titer variability in individual Aedes aegypti mosquitoes infected with Sindbis virus is associated with both differences in virus population structure and dramatic disparities in specific infectivity.

Variability in how individuals respond to pathogens is a hallmark of infectious disease, yet the basis for individual variation in host response is often poorly understood. The titer of infectious virus among individual mosquitoes infected with arboviruses is frequently observed to vary by several orders of magnitude in a single experiment, even when the mosquitoes are highly inbred. To better understand the basis for this titer variation, we sequenced populations of Sindbis virus (SINV) obtained from individual infected Aedes aegypti mosquitoes that, despite being from a highly inbred laboratory colony, differed in their titers of infectious virus by approximately 10,000-fold. We observed genetic differences between these virus populations that indicated the virus present in the midguts of low titer mosquitoes was less fit than that of high titer mosquitoes, possibly due to founder effects that occurred during midgut infection. Furthermore, we found dramatic differences in the specific infectivity or SI (the ratio of infectious units/viral genome equivalents) between these virus populations, with the SI of low titer mosquitoes being up to 10,000-fold lower than that of high titer mosquitoes. Despite having similar amounts of viral genomes, low titer mosquitoes appeared to contain less viral particles, suggesting that viral genomes were packaged into virions less efficiently than in high titer mosquitoes. Finally, antibiotic treatment, which has been shown to suppress mosquito antiviral immunity, caused an increase in SI. Our results indicate that the extreme variation that is observed in SINV infectious titer between individual Ae. aegypti mosquitoes is due to both genetic differences between virus populations and to differences in the proportion of genomes that are packaged into infectious particles.

Methods to Assess Blood and Nectar Meals in Aedes aegypti Mosquitoes.

Male and female Aedes aegypti mosquitoes survive by feeding on floral nectar for metabolic energy, but females require blood protein, obtained from biting a host, for egg development. Although males exclusively derive energy from nectar sugars, females must select the meal that best matches their present metabolic and reproductive needs. In females, blood and nectar promote independent feeding behaviors with distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. Understanding how male and female mosquitoes recognize, locate, and metabolize nutrients is essential for characterizing the survival and reproductive capabilities of this mosquito. Here, we provide an introduction to blood versus nectar feeding and methods to quantify nectar and blood meal sizes in individual Ae. aegypti mosquitoes. Precise quantification of meal size is crucial for ensuring consistency in assays that record events downstream of feeding behavior, including host attraction or fecundity.

Knockdown Resistance Mutations in the Voltage-Gated Sodium Channel of Aedes aegypti (Diptera: Culicidae) in Myanmar.

Simple SummaryKnockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) of mosquitoes confer resistance to pyrethroid insecticides. Analysis of kdr mutations in Aedes aegypti mosquitoes collected from five different townships in the Mandalay area, Myanmar, revealed high levels of validated kdr mutations in domains II and III of vgsc. Moreover, high frequencies of concurrent kdr mutations were also detected. The results of this study suggest that kdr mutations associated with pyrethroid resistance are widespread in the Ae. aegypti population of the study area. Our results provide a valuable molecular basis to understand the pyrethroid resistance status of the Ae. aegypti population in the area and underscore the need for an effective vector control program in Myanmar.Aedes aegypti is an important mosquito vector transmitting diverse arboviral diseases in Myanmar. Pyrethroid insecticides have been widely used in Myanmar as the key mosquito control measure, but the efforts are constrained by increasing resistance. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) are related to pyrethroid resistance in Ae. aegypti. We analyzed the patterns and distributions of the kdr mutations in Ae. aegypti in the Mandalay area of Myanmar. The segment 6 regions of domains II and III of vgsc were separately amplified from individual Ae. aegypti genomic DNA via polymerase chain reaction. The amplified gene fragments were sequenced. High proportions of three major kdr mutations, including S989P (54.8%), V1016G (73.6%), and F1534C (69.5%), were detected in the vgsc of Ae. aegypti from all studied areas. Other kdr mutations, T1520I and F1534L, were also found. These kdr mutations represent 11 distinct haplotypes of the vgsc population. The S989P/V1016G/F1534C was the most prevalent, followed by S989P/V1016V and V1016G/F1534C. A quadruple mutation, S989P/V1016G/T1520I/F1534C, was also identified. High frequencies of concurrent kdr mutations were observed in vgsc of Myanmar Ae. aegypti, suggesting a high level of pyrethroid resistance in the population. These findings underscore the need for an effective vector control program in Myanmar.

Effects of Aedes aegypti salivary protein on duck Tembusu virus replication and transmission in salivary glands

Duck Tembusu virus (DTMUV) infection is an arthropod-borne viral disease that affects many poultry species, including ducks, chickens, and geese. Aedes aegypti mosquito is an important vector of DTMUV. This study sought to determine whether any individual Ae. aegypti salivary protein modulated DTMUV replication in the mosquito salivary gland. Ae. aegypti salivary gland protein of 34 kDa (AaSG34) was found to be expressed explicitly in mosquito salivary glands and was upregulated following DTMUV infection. Thus, AaSG34 was silenced in mosquitoes via RNA interference using double strand RNA (dsRNA), and the mosquitoes were then infected with DTMUV to elucidate their effects on DTMUV replication and transmission. Transcripts of the DTMUV genome in salivary glands and virus titer in saliva were significantly diminished when AaSG34 was silenced, indicating that its presence enhances DTMUV replication in the salivary glands and DTMUV dissemination to saliva. Furthermore, the expression of antimicrobial peptides (AMPs) was upregulated upon AaSG34 silenced. Our results demonstrate that AaSG34 may play a vital role in the suppression of antiviral immune responses to enhance DTMUV replication and transmission. We thus provide new information on the effect of the AaSG34 salivary protein on DTMUV replication in Ae. aegypti as the mechanism of blocking virus transmission to the host.

Preliminary data from QUILT 3.055: A phase 2 multi-cohort study of N803 (IL-15 superagonist) in combination with checkpoint inhibitors (CPI).

2596 Background: There is currently a paucity of treatment options for checkpoint relapsed patients who have had an initial response but subsequently progress. N803, a novel IgG1 Fc-engineered IL-15-complexed protein may rescue checkpoint activity in a checkpoint independent manner via its selective enhancement of natural killer cell (NK) and CD8+ T cell number and function, without stimulation of T regs and MDSCs. Methods: QUILT 3.055, (NCT03228667) a phase 2b study of N803 plus investigator choice CPI in 11 tumor types: NSCLC, SCLC, Urothelial carcinoma, HNSCC, Merkel cell carcinoma, Melanoma (single PD-1/PD-L1 CPI or w/ ipilimumab), Renal cell carcinoma (RCC), Gastric cancer, Cervical cancer, Hepatocellular carcinoma, Microsatellite instability-high (MSI-H)/ mismatch repair deficient (dMMR) solid tumors, with a heterogeneous mix of prior therapies. We present interim data for 135 patients treated with CPI alone or in combination with chemotherapy as their most recent prior therapy. Trial inclusion required investigator assessed progression on last line of therapy, patients had either CR with relapse or partial response or stable disease for 6 months with progression as their most recent result of checkpoint therapy. Patients with hyperprogression or best initial response of progression were excluded. Subjects received N803 15mck/kg SC q 3 weeks in combination with the same checkpoint inhibitor on which they had their most recent progression. Results: Preliminary data from 135 patients (60% NSCLC) with treatment with checkpoint and N-803 following progression on the same checkpoint show CR 0%, PR 8%, Stable Disease 51%, Progression 29%, response unevaluable 12% to date. A PR or SD was seen in all subgroups. Median PFS 3.9 months (95% CI: 2.6,5.0). Median OS 13.8 months (95% CI: 11.8, 16.3) N-803 is well tolerated with grade 1-2 common N-803 treatment related adverse events (TRAE) were injection site reaction (68%), chills (32%) fatigue (26%), pyrexia (26%), flu-like illness (14%), nausea (12%) and no other individual AE > 10%. Grade 3 N-803 TRAE were 12% but no individual grade 3 AEs were greater than 5%. Conclusions: N803 demonstrates low toxicity in patients previously treated with CPI and promising efficacy of cessation of progression and induction of response and durable stable disease in patients who had previously progressed on a CPI containing regimen in multiple tumor types and different CPIs. Clinical trial information: NCT03228667.

Surveillance of dengue virus in individual Aedes aegypti mosquitoes collected concurrently with suspected human cases in Tarlac City, Philippines

BackgroundVector control measures are critical for the prevention and reduction of dengue virus (DENV) transmission. Effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito-borne infection. Mosquito-based virus surveillance programs typically rely on pool-based mosquito testing, although whether individual-based mosquito testing is a feasible alternative to this has not been widely studied. Applying an individual-based mosquito testing approach, we conducted a 1-month surveillance study of DENV in adult Aedes aegypti mosquitoes in homes of suspected dengue patients during the 2015 peak dengue season in Tarlac City, Philippines to more accurately assess the mosquito infection rate and identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients there.MethodsWe performed a one-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection and serotyping of DENV in patients and individual female Ae. aegypti mosquitoes. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENV serotypes in mosquitoes and patients at the genotype level.ResultsWe collected a total of 583 adult Ae. aegypti mosquitoes, of which we individually tested 359 female mosquitoes for the presence of DENV. Ten (2.8%) of the 359 female mosquitoes were positive for the presence of DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which was consistent with the serotypes concurrently found in infected patients. Sequencing and phylogenetic analyses of the detected DENV serotypes based on the partial sequence of the evelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely DENV-1 genotype IV, DENV-2 Cosmopolitan genotype, and DENV-4 genotype II.ConclusionsWe demonstrated the utility of a one-step multiplex real-time RT-PCR assay for the individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which are critical for dengue prevention and control.

Comparative quantitative safety profiles of PD-1 and PD-L1 checkpoint inhibitor monotherapies: A Bayesian model-based meta-analysis.

e15167 Background: Anti-cancer therapies with immune checkpoint inhibitors (ICIs) are associated with immune-mediated adverse events (imAEs). The objective of this study was to use a Bayesian model-based meta-analysis, to compare safety profiles of PD-1 and PD-L1 checkpoint inhibitor monotherapies. Methods: We performed an exhaustive search through PubMed and TrialTrove databases, ASCO and ESMO abstracts to identify relevant ICI safety data. A Bayesian meta-analysis (BMA) was performed for all-grade and high-grade (Grades 3-4) treatment-related AEs (trAEs) and immune-mediated AEs (imAEs). The analysis was performed for total AEs and individual AEs affecting different organs: dermatological (rash, pruritus), gastrointestinal (diarrhea, colitis), hepatic (AST, ALT), respiratory (pneumonitis), and endocrine (hyperthyroidism, hypothyroidism). Software STAN along with the brms package in R software were used for the BMA. Results: A total of 145 articles were identified, covering 30,737 patients in 153 cohorts treated with anti PD-1 and anti PD-L1 monotherapies. For total all-grade and grade 3&4 trAEs, anti PD-L1 demonstrated lower AE rates vs. anti PD-1 (65% vs. 69% and 12% vs. 16%, respectively). Results were similar for total all-grade and high-grade imAEs (19% vs. 26% and 4% vs. 7%, respectively). The analysis of all-grade individual trAEs showed that anti PD-L1 exhibited lower AE rates vs. anti PD-1 for rash (8% vs. 10.5%), pruritus (9% vs. 12%), diarrhea (8.5% vs. 11%), colitis (1.0% vs. 1.6%), AST (3.0% vs. 5.5%), ALT (3.5% vs. 5.6%), pneumonitis (3.0% vs. 3.9%), hyperthyroidism (0.8% vs. 4.0%) and hypothyroidism (6.0% vs. 8.0%). For grade 3&4 trAEs, differences between anti PD-L1 and anti PD-1 were: 0.6% vs. 1.3% for diarrhea, 0.7% vs. 1.4% for colitis, and 0.9% vs 1.6% for pneumonitis. All-grade imAEs were less frequent for anti PD-L1 vs. anti PD-1 for: rash (6.5% vs. 10%), colitis (1.0% vs. 2.0%), AST (3.5% vs. 3.9%), ALT (2.9% vs. 4.0%), pneumonitis (2.0% vs. 4.0%), hyperthyroidism (2.5% vs. 4.0%) and hypothyroidism (5.5% vs. 9.0%). Similar results were obtained for grade 3&4 imAEs incidences: rash (0.5% vs. 0.9%), diarrhea (0.5% vs. 1.5%), colitis (0.5% vs. 1.1%) and pneumonitis (0.9% vs. 1.4%). Conclusions: Significant differences in trAEs and imAEs rates were shown for anti PD-L1 vs. anti PD-1 monotherapies. Anti PD-L1 demonstrated more favorable safety profiles for total and individual AE rates. Notably, AE rates for pneumonitis and colitis were twice as high for anti PD-1 vs. anti PD-L1 therapies.

Blood Metabolism and Oxidative Stress in Zika Mosquitoes

Aedes aegypti mosquitoes are the primary vector of disease‐causing viruses such as yellow fever, Zika, dengue fever, and chikungunya. With increasing insecticide resistance, novel control approaches informed by an improved understanding of mosquito biology are urgently needed to reduce vector‐borne disease transmission. Our research aims to further understand the molecular and physiological mechanisms that underlie mosquito‐host interactions. Female mosquitoes feed on warm‐blooded vertebrate hosts to obtain blood nutrients required for egg development, but are subjected to extreme thermal stress when feeding and, upon digestion of the blood, must cope with high levels of oxidative stress. Increased knowledge of the biological mechanisms mosquitoes use to cope with these stressors may have important epidemiological consequences for humans. Using next‐generation sequencing methods, we have characterized rhythmic daily variations in the global transcriptome of female Ae. aegypti heads, which encompass the visual and olfactory sensory appendages mosquitoes rely on to sense host‐seeking cues, the mouthparts, and tissues immediately exposed to blood stressors when feeding. Here we describe daily rhythms in mosquito host‐seeking and blood‐feeding stress tolerance. We also investigated daily rhythms in the metabolism of blood digestion, as these mechanisms represent targets of opportunity for mosquito control. Using state‐of‐the‐art respirometry methods to quantify rhythms in the metabolism of blood digestion in female Ae. aegypti mosquitoes, we were able to observe individual ventilatory patterns and compare the metabolic rates at rest and during digestion. These are the first measurements reported on carbon dioxide release from individual Ae. aegypti female mosquitoes and, thus, these results provide new insights regarding mosquito respiratory and metabolic physiology. Analysis of the metabolic rate following the ingestion of various diets additionally allows us to evaluate the effects of diet composition, oxidative stress exposure, and blood protein digestion on mosquito metabolism. Since this work can be used to identify novel vector control approaches targeting mosquito blood meal processing, our results will inform future efforts to decrease mosquito‐borne disease.Support or Funding InformationUSDA National Institute of Food and Agriculture, Hatch project 1017860. The Company of Biologists Journal of Experimental Biology Travelling Fellowship.

Detection of Wolbachia in field-collected Aedes aegypti mosquitoes in metropolitan Manila, Philippines

BackgroundRecent reports reveal the presence of Wolbachia in Ae. aegypti. Our study presents additional support for Wolbachia infection in Ae. aegypti by screening field-collected adult mosquitoes using two Wolbachia-specific molecular makers.MethodsA total of 672 Ae. aegypti adult mosquitoes were collected from May 2014 to January 2015 in Metropolitan Manila. Each individual sample was processed and screened for the presence of Wolbachia by selected markers, Wolbachia-specific 16S rDNA and its surface protein (wsp), under optimized PCR conditions and sequenced.ResultsTotals of 113 (16.8%) and 89 (13.2%) individual mosquito samples were determined to be infected with Wolbachia using the wsp and 16S rDNA markers, respectively. The Ae. aegpyti wsp sample sequences were similar or identical to five known Wolbachia strains belonging to supergroups A and B while the majority of 16S rDNA sample sequences were similar to strains belonging to supergroup B. Overall, 80 (11.90%) individual mosquito samples showed positive amplifications in both markers and 69% showed congruence in supergroup identification (supergroup B).ConclusionsBy utilizing two Wolbachia-specific molecular makers, our study demonstrated the presence of Wolbachia from individual Ae. aegypti samples. Our results showed a low Wolbachia infection rate and inferred the detected strains belong to either supergroups A and B.

Ranking ICME's efficiency for geomagnetic and ionospheric storms and risk of false alarms

A statistical analysis is undertaken on ICME's efficiency in producing the geomagnetic and ionospheric storms. The mutually-consistent thresholds for the intense, moderate and weak space weather storms and quiet conditions are introduced with an analytical model based on relations between the equatorial Dst index and geomagnetic indices AE, aa, ap, ap(τ) and the ionospheric Vσ indices. The ionosphere variability Vσ index is expressed in terms of the total electron content (TEC) deviation from the −15-day sliding median normalized by the standard deviation for the 15 preceding days. The intensity of global positive ionospheric storm, Vσp, and negative storm, Vσn, is represented by the relative density of anomalous ±Vσ index occurrence derived from the global ionospheric maps GIM-TEC for 1999–2016. An impact of total 421 ICME events for 1999–2016 on the geomagnetic and ionospheric storms expressed by AE, Dst, aa, ap, ap(τ), Vσp, Vσn indices and their superposition is analyzed using ICME catalogue by Richardson and Cane (2010) during 24 h after the ICME start time t0. Hierarchy of efficiency of ICME → storm relation is established. The ICMEs have a higher probability (22–25%) to be followed by the intense ionospheric and auroral electrojet storms at global and high latitudes as compared to the intense storms at middle and low latitudes (18–20%) and to moderate and weak storms at high latitudes (5–17%). At the same time ICMEs are more effective in producing the moderate storms (24–28%) at the middle and low latitudes as compared to the intense and weak storms at these latitudes (13–22%) and to moderate storms at high latitudes (8–17%). The remaining cases when quiet conditions are observed after ICMEs present higher chance for a false alarm. The risk factor for a false alarm can vary from 18% if the superposition of all indices is considered, to 51–64% for individual AE, Vσp and Vσn indices. The analysis indicates that the mutually-consistent thresholds can be successfully applied to the external sources of the geomagnetic and ionospheric storms other than ICME which present challenge for the further investigation.

Full-genome dengue virus sequencing in mosquito saliva shows lack of convergent positive selection during transmission by Aedes aegypti.

Like other pathogens with high mutation and replication rates, within-host dengue virus (DENV) populations evolve during infection of their main mosquito vector, Aedes aegypti. Within-host DENV evolution during transmission provides opportunities for adaptation and emergence of novel virus variants. Recent studies of DENV genetic diversity failed to detect convergent evolution of adaptive mutations in mosquito tissues such as midgut and salivary glands, suggesting that convergent positive selection is not a major driver of within-host DENV evolution in the vector. However, it is unknown whether this conclusion extends to the transmitted viral subpopulation because it is technically difficult to sequence DENV genomes in mosquito saliva. Here, we achieved DENV full-genome sequencing by pooling saliva samples collected non-sacrificially from 49 to 163 individual Ae. aegypti mosquitoes previously infected with one of two DENV-1 genotypes. We compared the transmitted viral subpopulations found in the pooled saliva samples collected in time series with the input viral population present in the infectious blood meal. In all pooled saliva samples examined, the full-genome consensus sequence of the input viral population was unchanged. Although the pooling strategy prevents analysis of individual saliva samples, our results demonstrate the lack of strong convergent positive selection during a single round of DENV transmission by Ae. aegypti. This finding reinforces the idea that genetic drift and purifying selection are the dominant evolutionary forces shaping within-host DENV genetic diversity during transmission by mosquitoes.

The Effects of Larval Habitat Quality on Aedes albopictus Skip Oviposition.

Aedes albopictus is an invasive mosquito species that transmits human-disease-causing pathogens. It is a container-inhabiting species that oviposits in resource-limited habitats. To mitigate larval competition, Ae. albopictus females may choose to distribute eggs from a single gonotrophic cycle among multiple containers through skip oviposition. With the use of individual females released in indoor and outdoor caged trials, we evaluated the oviposition choices made by gravid Ae. albopictus offered larval habitats with different qualities. Our results demonstrate that Ae. albopictus performs skip oviposition and that the degree of egg distribution is related to the quality of the larval habitat. In a 4-choice arena, individual Ae. albopictus oviposited in fewer containers when presented with ovisites of high-quality larval habitat (uncrowded conditions) compared with oviposition in low-quality (crowded conditions) larval habitats. Additionally, the females selectively oviposited in high-quality habitats when offered both low- and high-quality habitats, but distributed eggs more evenly among multiple high-quality habitats. Our results have important implications for mosquito management plans that include the use of lethal ovitraps, as well as the role of this behavior in distribution of disease-causing pathogens.

Long-term Effects of Carbohydrate Availability on Mating Success of Newly Eclosed Aedes albopictus (Diptera: Culicidae) Males.

Sugar availability varies greatly in nature, and determining how this affects male mosquito fitness is essential for understanding population dynamics. We allowed male Aedes albopictus (Skuse) carbohydrate access for increasing intervals of time immediately after eclosion and we evaluated their fitness by comparing mortality, mating success, and sperm transfer. We compared individual male Ae. albopictus, which were offered water or 20% sucrose solution for 24, 48, or 72 h. As predicted, there were significant increases in fitness for each additional day of sucrose access. Following sugar exposure, we allowed males daily access to three virgin females. We assessed mating success through observation of spermatozoa in the female spermathecae. When individuals of the same age were compared, males with sugar access exhibited significantly greater mating success than water-treated males in all treatments. The total number of spermathecae filled by males with sugar access in the 48- and 72-h treatments was also significantly greater on some days; these were 3-5 d posteclosion in the 48-h treatment and 5-6 d posteclosion in the 72-h treatment. We conclude that extended sugar access at eclosion is important for maximizing fitness in male Ae. albopictus and should be applicable to sterile male release efforts, especially when laboratory-reared males suffered from other disadvantages. We recommend retaining adult males for 3 d posteclosion prior to release to improve their mating success in male release initiatives.

Detection of Wolbachia from field collected Aedes albopictus Skuse in Malaysia.

Background & objectives:Wolbachia-based vector control strategies have been proposed as a mean to augment the existing measures for controlling dengue vector. Prior to utilizing Wolbachia in novel vector control strategies, it is crucial to understand the Wolbachia-mosquito interactions. Many studies have only focused on the prevalence of Wolbachia in female Aedes albopictus with lack of attention on Wolbachia infection on the male Ae. albopictus which also affects the effective expression of Wolbachia induced- cytoplasmic incompatibility (CI). In this study, field surveys were conducted to screen for the infection status of Wolbachia in female and male Ae. albopictus from various habitats including housing areas, islands and seashore.Methods:Adult Ae. albopictus (n=104) were collected using human landing catches and hand aspirator. Standard ovitraps were also set in the selected areas for five days and the larvae were identified to species level. All the collected Ae. albopictus were screened for the presence of Wolbachia using multiplex polymerase chain reaction (PCR) and gene sequencing of Wolbachia surface protein (wsp) gene.Results:A 100 per cent positivity of Wolbachia infection was observed for individual Ae. albopictus screened. For pooled mosquitoes, 73 of the 76 pools (female) and 83 of the 87 pools (male) were positive with Wolbachia infection. The wsp gene sequence of the Wolbachia strain isolated from individual and pooled mosquitoes showed a 100 per cent homology with Wolbachia sp. of Ae. albopictus isolated from various geographical regions. Phylogenetic analysis based on wsp gene fragments showed that the isolates were clustered into groups A and B, respectively.Interpretation & conclusions:The results indicated that Wolbachia infection was widespread in Ae. albopictus population both in female and male Ae. albopictus. All the infected females were superinfected with both A and B strains while the infected males showed a combination of superinfection of A and B strains and single infection of B strain.

Microsatellite-Based Parentage Analysis ofAedes aegypti(Diptera: Culicidae) Using Nonlethal DNA Sampling

To track Aedes aegypti (L.) egg-laying behavior in the field in Iquitos, Peru, we developed methods for 1) sampling DNA from live mosquitoes and 2) high through-put parentage analysis using microsatellite markers. We were able to amplify DNA extracted from a single hind leg, but not from the pupal exuvia. Removal of a leg from teneral females caused no significant changes in female behavioral or life history traits (e.g., longevity, blood feeding frequency, fecundity, egg hatch rate, gonotrophic cycle length, or oviposition behavior). Using a panel of nine microsatellite markers and an exclusion-based software program, we matched offspring to parental pairs in 10 Ae. aegypti test families in which parents originated from natural development sites in Iquitos. By mating known individuals in the laboratory, retaining the male, sampling the female's DNA before release, and collecting offspring in the field, the technique we developed can be used to genotype large numbers of Ae. aegypti, reconstruct family relationships, and track the egg-laying behavior of individual Ae. aegypti in nature.

Distribution of Acoustic Emission Parameters during Load Holding for CNG Vehicle Fuel Tank

Abstract This is a study on the distribution of acoustic emission parameters during a burst test for a type-II CNG vehiclefuel tank. A resonant AE sensor with a central frequency of 150 kHz was attached to the composite materials in the centerof the fuel tank. The pressure was increased from 30 to 100% of the expected burst pressure and was maintained for 10 minutesat each level. Damage at 70% of expected burst pressure occurred by various damage mechanisms including fiber breakageand delamination, while that of below 60% only occurred by matrix crack initiation and growth. The count, duration and risetime of the AE signal at 60% of the expected burst pressure are distributed below 500, 5000µs and 300µs, respectively. Then,at above 70% they increased with pressure by superimposing of individual AE signal generated at a nearby place. These resultsconfirmed that the analysis of the distribution of AE parameters is an effective tool for estimating damage of a CNG fuel tank.Key wordsacoustic emission, AE parameter, fuel tank, GFRP.

Field Validation of a Transcriptional Assay for the Prediction of Age of Uncaged Aedes aegypti Mosquitoes in Northern Australia

BackgroundNew strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior.Methodology/Principal FindingsWe produced “free-range” test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R2 value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy.Conclusions/SignificanceThe transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where mosquito survivorship or lifespan reduction are crucial to their success. The approximate cost of the method was US$7.50 per mosquito and 60 mosquitoes could be processed in 3 days. The assay is based on conserved genes and modified versions are likely to support similar investigations of several important mosquito and other disease vectors.

Detection of intergenomic chromosome rearrangements in irradiatedTriticum aestivum –Aegilops biuncialisamphiploids by multicolour genomic in situ hybridization

The frequency and pattern of irradiation-induced intergenomic chromosome rearrangements were analysed in the mutagenized (M0) and the first selfed (M1) generations of Triticum aestivum L. - Aegilops biuncialis Vis. amphiploids (2n = 70, AABBDDUbUbMbMb) by multicolour genomic in situ hybridization (mcGISH). mcGISH allowed the simultaneous discrimination of individual Ae. biuncialis genomes and wheat chromosomes. Dicentric chromosomes, fragments, and terminal translocations were most frequently induced by gamma-irradiation, but centric fusions and internal exchanges were also more abundant in the treated plants than in control amphiploids. Rearrangements involving the Ub genome (Ub-type aberrations) were more frequent than those involving the Mb genome (Mb-type aberrations). This irradiation sensitivity of the Ub chromosomes was attributed to their centromeric or near-centromeric regions, since Ub-type centric fusions were significantly more abundant than Mb-type centric fusions at all irradiation doses. Dicentrics completely disappeared, but centric fusions and translocations were well transmitted from M0 to M1. Identification of specific chromosomes involved in some rearrangements was attempted by sequential fluorescence in situ hybridization with a mix of repeated DNA probes and GISH on the same slide. The irradiated amphiploids formed fewer seeds than untreated plants, but normal levels of fertility were recovered in their offspring. The irradiation-induced wheat - Ae. biuncialis intergenomic translocations will facilitate the successful introgression of drought tolerance and other alien traits into bread wheat.

Further research on the production, longevity and infectivity of the zoospores of Leptolegnia chapmanii Seymour (Oomycota: Peronosporomycetes)

The effect of temperature on the production, survival and infectivity of zoospores of an Argentinean isolate of Leptolegnia chapmanii was determined under laboratory conditions. Production of zoospores of L. chapmanii in vitro and in vivo upon first and fourth instars larvae of the mosquito Aedes aegypti was studied at three different temperatures. Zoospores from infected larvae were infective to mosquito larvae for 51, 12, and 5 consecutive days when maintained at 25, 35, and 10 °C, respectively. Maximum zoospore production in infected fourth-instar larvae was 9.6 ± 1.4 × 10 4 zoosp/larva at 48 h at 25 °C. The average number of zoospores produced by individual fourth-instar Ae. aegypti larvae infected with L. chapmanii was 3.57 ± 0.46 × 10 5 zoospores during 6 consecutive days at 25 °C. Zoospore production in vitro was also affected by temperature with a maximum of zoospores ( n = 47,666/ml) produced at 25 °C. When zoospores produced in vitro were used as inoculum against Ae. aegypti larvae at 25 °C, larval mortality was recorded for 5 consecutive weeks. The encystment process for zoospores took 17–20 min; the germination of cysts (excystment) occurred 5 min after exposure in water to mosquito larvae. The minimal time of contact between zoospores and mosquito larvae to develop infection was two minutes. Infection took place by zoospore attachment onto and then penetration through the larval cuticle or by ingestion of cysts as was confirmed by histological studies. Temperature directly affected infectivity and production of zoospores in vivo and in vitro although L. chapmanii zoospores tolerate a wide range of temperatures.