Blowfly puparia in a hermetic container: survival under decreasing oxygen conditions.
ABSTRACT: Despite widely accepted standards for sampling and preservation of insect evidence, unrepresentative samples or improperly preserved evidence are encountered frequently in forensic investigations. Here, we report the results of laboratory studies on the survival of Lucilia sericata and Calliphora vomitoria (Diptera: Calliphoridae) intra-puparial forms in hermetic containers, which were stimulated by a recent case. It is demonstrated that the survival of blowfly intra-puparial forms inside airtight containers is dependent on container volume, number of puparia inside, and their age. The survival in both species was found to increase with an increase in the volume of air per 1 mg of puparium per day of development in a hermetic container. Below 0.05 ml of air, no insect survived, and above 0.2 ml of air per 1 mg of puparium per day, survival reached its maximum. These results suggest that blowflies reveal a single, general pattern of survival under decreasing oxygen conditions and that this pattern is a product of number of developing insects, their age and the initial amount of available air. Implications for forensic entomology are discussed.
Project description:Minimum post-mortem interval (minPMI) estimates often rely on the use of developmental data from blow flies (Diptera: Calliphoridae), which are generally the first colonisers of cadavers and, therefore, exemplar forensic indicators. Developmental data of the intra-puparial period are of particular importance, as it can account for more than half of the developmental duration of the blow fly life cycle. During this period, the insect undergoes metamorphosis inside the opaque, barrel-shaped puparium, formed by the hardening and darkening of the third instar larval cuticle, which shows virtually no external changes until adult emergence. Regrettably, estimates based on the intra-puparial period are severely limited due to the lack of reliable, non-destructive ageing methods and are frequently based solely on qualitative developmental markers. In this study, we use non-destructive micro-computed tomography (micro-CT) for (i) performing qualitative and quantitative analyses of the morphological changes taking place during the intra-puparial period of two forensically relevant blow fly species, Calliphora vicina and Lucilia sericata, and (ii) developing a novel and reliable method for estimating insect age in forensic practice. We show that micro-CT provides age-diagnostic qualitative characters for most 10% time intervals of the total intra-puparial period, which can be used over a range of temperatures and with a resolution comparable to more invasive and time-consuming traditional imaging techniques. Moreover, micro-CT can be used to yield a quantitative measure of the development of selected organ systems to be used in combination with qualitative markers. Our results confirm micro-CT as an emerging, powerful tool in medico-legal investigations.
Project description:Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6?hours with full containers, 36?hours with full containers, 36?hours with container three quarters full of medium, and 36?hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36?hours if the container is full.
Project description:The proliferation of artificial container habitats in urban areas has benefitted urban adaptable mosquito species globally. In areas where mosquitoes transmit viruses and parasites, it can promote vector population productivity and fuel mosquito-borne disease outbreaks. In Britain, storage of water in garden water butts is increasing, potentially expanding mosquito larval habitats and influencing population dynamics and mosquito-human contact. Here we show that the community composition, abundance and phenology of mosquitoes breeding in experimental water butt containers were influenced by urbanisation. Mosquitoes in urban containers were less species-rich but present in significantly higher densities (100.4±21.3) per container than those in rural containers (77.7±15.1). Urban containers were dominated by Culex pipiens (a potential vector of West Nile Virus [WNV]) and appear to be increasingly exploited by Anopheles plumbeus (a human-biting potential WNV and malaria vector). Culex phenology was influenced by urban land use type, with peaks in larval abundances occurring earlier in urban than rural containers. Among other factors, this was associated with an urban heat island effect which raised urban air and water temperatures by 0.9°C and 1.2°C respectively. Further increases in domestic water storage, particularly in urban areas, in combination with climate changes will likely alter mosquito population dynamics in the UK.
Project description:Reforestation is needed globally to help restore degraded sites, combat desertification, protect watersheds, and provide forest products. This involves planting forest tree seedlings grown in local nurseries, but technologies to produce quality seedlings are lacking in developing countries. Modern nursery containers used to propagate seedlings have internal-surface barriers (ribs or ridges) or side-slits to prevent root spiraling. These are cost prohibitive or unavailable in developing countries and so polybags (plastic bags) are more commonly used, despite their tendency to produce seedlings with deformed root systems that have less potential to establish on field sites. Discarded plastic bottles, which are readily available worldwide, may be a feasible alternative for seedling propagation. We conducted two experiments to assess the potential of repurposed plastic beverage bottles to grow quality trees: 1) Container Comparison-to evaluate Arizona walnut (Juglans major [Toor.] Heller) and Afghan pine (Pinus eldarica Medw.) seedling root and shoot development in two plastic bottle types compared to modern nursery containers and polybags, and 2) Bottle Modification-to examine the effects of root spiraling prevention techniques (side-slits, internal-ridges, and control) and container opacity (green, black, and clear) on Afghan pine seedling morphological attributes. Nursery growth and first-year seedling field performance were evaluated for both experiments. In experiment one, seedlings of both species had fewer spiraled roots in bottle containers compared to polybags. Arizona walnut had more fibrous root systems in polybags, while Afghan pine root system fibrosity was greatest in bottle containers. First-year field performance of both species was not affected by container type. In experiment two, less spiraled roots occurred in containers with air-slits and interior-ridges compared to the control. The effects of container opacity on seedling morphology were inconsistent. Root spiral prevention and opacity had no influence on Afghan pine one-year survival, field height and diameter, with the exception of opacity for height growth, whereby seedlings grown in green containers were taller than those grown in black containers, but seedlings grown in clear containers were similar to both. Our results provide the first evidence that plastic bottle containers may provide an effective alternative for production of high quality seedlings, which may benefit agroforestry, reforestation, restoration, and conservation programs in developing countries.
Project description:The mosquito virus vector Aedes (Ae.) aegypti exploits a wide range of containers as sites for egg laying and development of the immature life stages, yet the approaches for modeling meteorologically sensitive container water dynamics have been limited. This study introduces the Water Height and Temperature in Container Habitats Energy Model (WHATCH'EM), a state-of-the-science, physically based energy balance model of water height and temperature in containers that may serve as development sites for mosquitoes. The authors employ WHATCH'EM to model container water dynamics in three cities along a climatic gradient in México ranging from sea level, where Ae. aegypti is highly abundant, to ~2100 m, where Ae. aegypti is rarely found. When compared with measurements from a 1-month field experiment in two of these cities during summer 2013, WHATCH'EM realistically simulates the daily mean and range of water temperature for a variety of containers. To examine container dynamics for an entire season, WHATCH'EM is also driven with field-derived meteorological data from May to September 2011 and evaluated for three commonly encountered container types. WHATCH'EM simulates the highly nonlinear manner in which air temperature, humidity, rainfall, clouds, and container characteristics (shape, size, and color) determine water temperature and height. Sunlight exposure, modulated by clouds and shading from nearby objects, plays a first-order role. In general, simulated water temperatures are higher for containers that are larger, darker, and receive more sunlight. WHATCH'EM simulations will be helpful in understanding the limiting meteorological and container-related factors for proliferation of Ae. aegypti and may be useful for informing weather-driven early warning systems for viruses transmitted by Ae. aegypti.
Project description:This study examined household risk factors and prevalence, abundance, and distribution of immature Aedes aegypti and Aedes albopictus, and their association with socioeconomic and ecological factors at urban zonal and household levels in the city of Dhaka, Bangladesh. During the 2011 monsoon, 826 households in 12 randomly selected administrative wards were surveyed for vector mosquitoes. Results revealed that the abundance and distribution of immature Ae. aegypti and Ae. albopictus, and pupae-per-person indices did not vary significantly among the zones with varied socioeconomic status. Of 35 different types of identified wet containers, 30 were infested, and among the 23 pupae-positive container types, nine were defined as the "most productive" for pupae including: disposable plastic containers (12.2% of 550), sealable plastic barrels (12.0%), tires (10.4%), abandoned plastic buckets (9.6%), flower tub and trays (8.5%), refrigerator trays (6.5%), plastic bottles (6.4%), clay pots (4.9%), and water tanks (1.6%). When the function of the containers was assessed, ornamental, discarded, and household repairing and reconstruction-related container categories were found significantly associated with the number of pupae in the households. The purpose of storing water and income variables were significant predictors of possession of containers that were infested by vector mosquitoes.
Project description:<h4>Objective</h4>To test whether community mobilization adds effectiveness to conventional dengue control.<h4>Design</h4>Pragmatic open label parallel group cluster randomized controlled trial. Those assessing the outcomes and analyzing the data were blinded to group assignment. Centralized computerized randomization after the baseline study allocated half the sites to intervention, stratified by country, evidence of recent dengue virus infection in children aged 3-9, and vector indices.<h4>Setting</h4>Random sample of communities in Managua, capital of Nicaragua, and three coastal regions in Guerrero State in the south of Mexico.<h4>Participants</h4>Residents in a random sample of census enumeration areas across both countries: 75 intervention and 75 control clusters (about 140 households each) were randomized and analyzed (60 clusters in Nicaragua and 90 in Mexico), including 85,182 residents in 18,838 households.<h4>Interventions</h4>A community mobilization protocol began with community discussion of baseline results. Each intervention cluster adapted the basic intervention-chemical-free prevention of mosquito reproduction-to its own circumstances. All clusters continued the government run dengue control program.<h4>Main outcome measures</h4>Primary outcomes per protocol were self reported cases of dengue, serological evidence of recent dengue virus infection, and conventional entomological indices (house index: households with larvae or pupae/households examined; container index: containers with larvae or pupae/containers examined; Breteau index: containers with larvae or pupae/households examined; and pupae per person: pupae found/number of residents). Per protocol secondary analysis examined the effect of Camino Verde in the context of temephos use.<h4>Results</h4>With cluster as the unit of analysis, serological evidence from intervention sites showed a lower risk of infection with dengue virus in children (relative risk reduction 29.5%, 95% confidence interval 3.8% to 55.3%), fewer reports of dengue illness (24.7%, 1.8% to 51.2%), fewer houses with larvae or pupae among houses visited (house index) (44.1%, 13.6% to 74.7%), fewer containers with larvae or pupae among containers examined (container index) (36.7%, 24.5% to 44.8%), fewer containers with larvae or pupae among houses visited (Breteau index) (35.1%, 16.7% to 55.5%), and fewer pupae per person (51.7%, 36.2% to 76.1%). The numbers needed to treat were 30 (95% confidence interval 20 to 59) for a lower risk of infection in children, 71 (48 to 143) for fewer reports of dengue illness, 17 (14 to 20) for the house index, 37 (35 to 67) for the container index, 10 (6 to 29) for the Breteau index, and 12 (7 to 31) for fewer pupae per person. Secondary per protocol analysis showed no serological evidence of a protective effect of temephos.<h4>Conclusions</h4>Evidence based community mobilization can add effectiveness to dengue vector control. Each site implementing the intervention in its own way has the advantage of local customization and strong community engagement.<h4>Trial registration</h4>ISRCTN27581154.
Project description:Because no dengue vaccine or antiviral therapy is commercially available, controlling the primary mosquito vector, Aedes aegypti, is currently the only means to prevent dengue outbreaks. Traditional models of Ae. aegypti assume that population dynamics are regulated by density-dependent larval competition for food and little affected by oviposition behavior. Due to direct impacts on offspring survival and development, however, mosquito choice in oviposition site can have important consequences for population regulation that should be taken into account when designing vector control programs.We examined oviposition patterns by Ae. aegypti among 591 naturally occurring containers and a set of experimental containers in Iquitos, Peru. Using larval starvation bioassays as an indirect measure of container food content, we assessed whether females select containers with the most food for their offspring. Our data indicate that choice of egg-laying site is influenced by conspecific larvae and pupae, container fill method, container size, lid, and sun exposure. Although larval food positively influenced oviposition, our results did not support the hypothesis that females act primarily to maximize food for larvae. Females were most strongly attracted to sites containing immature conspecifics, even when potential competitors for their progeny were present in abundance.Due to strong conspecific attraction, egg-laying behavior may contribute more to regulating Ae. aegypti populations than previously thought. If highly infested containers are targeted for removal or larvicide application, females that would have preferentially oviposited in those sites may instead distribute their eggs among other suitable, previously unoccupied containers. Strategies that kill mosquitoes late in their development (i.e., insect growth regulators that kill pupae rather than larvae) will enhance vector control by creating "egg sinks," treated sites that exploit conspecific attraction of ovipositing females, but reduce emergence of adult mosquitoes via density-dependent larval competition and late acting insecticide.
Project description:We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n = 12), cemetery urns (n = 23), and miscellaneous containers that included two tree holes (n = 19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species.
Project description:A variety of insect and arachnid species are able to remain submerged in water indefinitely using plastron respiration. A plastron is a surface-retained film of air produced by surface morphology that acts as an oxygen-carbon dioxide exchange surface. Many highly water repellent and hydrophobic surfaces when placed in water exhibit a silvery sheen which is characteristic of a plastron. In this article, the hydrophobicity of a range of commercially available water repellent fabrics and polymer membranes is investigated, and how the surface of the materials mimics this mechanism of underwater respiration is demonstrated allowing direct extraction of oxygen from oxygenated water. The coverage of the surface with the plastron air layer was measured using confocal microscopy. A zinc/oxygen cell is used to consume oxygen within containers constructed from the different membranes, and the oxygen consumed by the cell is compared to the change in oxygen concentration as measured by an oxygen probe. By comparing the membranes to an air-tight reference sample, it was found that the membranes facilitated oxygen transfer from the water into the container, with the most successful membrane showing a 1.90:1 ratio between the cell oxygen consumption and the change in concentration within the container.