OCT for non-destructive examination of the internal biological structures of mosquito specimen

The Study of mosquitoes and their behavioral analysis are of crucial importance to control the alarmingly increasing mosquito-borne diseases. Conventional imaging techniques use either dissection, exogenous contrast agents. Non-destructive imaging techniques, like x-ray and microcomputed tomography uses ionizing radiations. Hence, a non-destructive and real-time imaging technique which can obtain high resolution images to study the anatomical features of mosquito specimen can greatly aid researchers for mosquito studies. In this study, the three-dimensional imaging capabilities of optical coherence tomography (OCT) for structural analysis of Anopheles sinensis mosquitoes has been demonstrated. The anatomical features of An. sinensis head, thorax, and abdomen regions along with internal morphological structures like foregut, midgut, and hindgut were studied using OCT imaging. Two-dimensional (2D) and three-dimensional (3D) OCT images along with histology images were helpful for the anatomical analysis of the mosquito specimens. From the concurred results and by exhibiting this as an initial study, the applicability of OCT in future entomological researches related to mosquitoes and changes in its anatomical structure is demonstrated

Anatomical damage caused by Bacillus thuringiensis variety israelensis in yellow fever mosquito Aedes aegypti (L.) larvae revealed by micro‑computed tomography

With micro-computed tomography techniques, using the single-distance phase-retrieval algorithm phase contrast, we reconstructed enhanced rendered images of soft tissues of Aedes aeqypti fourth instar larvae after Bti treatment. In contrast to previous publications based on conventional microscopy, either optical or electron microscopy, which were limited to partial studies, mostly in the form of histological sections, here we show for the first time the effects of Bti on the complete internal anatomy of an insect. Using 3D rendered images it was possible to study the effect of the bacterium in tissues and organs, not only in sections but also as a whole. We compared the anatomy of healthy larvae with the changes undergone in larvae after being exposed to Bti (for 30 min, 1 h and 6 h) and observed the progressive damage that Bti produce. Damage to the midgut epithelia was confirmed, with progressive swelling of the enterocytes, thickening epithelia, increase of the vacuolar spaces and finally cell lysis, producing openings in the midgut walls. Simultaneously, the larvae altered their motility, making it difficult for them to rise to the surface and position the respiratory siphon properly to break surface tension and breathe. Internally, osmotic shock phenomena were observed, resulting in a deformation of the cross-section shape, producing the appearance of a wide internal space between the cuticle and the internal structures and a progressive collapse of the tracheal trunks. Taken together, these results indicate the death of the larvae, not by starvation as a consequence of the destruction of the epithelia of the digestive tract as previously stated, but due to a synergic catastrophic multifactor process in addition to asphyxia due to a lack of adequate gas exchange.Consejería de Universidad, Investigación e Innovación of Junta de Andalucia (Spain)FEDER Programe through the research projects: “Caracterización de variantes de toxinas Cry activas frente a la Mosca de la Fruta del Mediterráneo (Ceratitis capitata) obtenidas mediante la tecnología del despliegue de proteínas en fagos” (B-BIO-081-UGR18)Búsqueda de nuevas toxinas Cry con actividad frente al ectoparásito de la abeja Varroa destructor mediante la evolución in vitro de proteínas y la técnica del despliegue de proteínas en fago” (A-BIO-424-UGR20

Utility of the Calliphora vicina (Diptera: Calliphoridae) pupal stage for providing temporal information for death invesigations

Blowflies (Diptera: Calliphoridae) are primary colonisers of cadavers; the ages of the eldest immature stages can be used to estimate minimum post-mortem interval (PMI). These estimates are obtained using calliphorid larvae, for which there are established and reliable preservation protocols and age estimation methods. The opposite is true for pupae; non-standardised crime-scene collection and preservation methods are employed, resulting in poorly preserved specimens for age estimation, which is often conducted using limited and inadequate research data. This has hindered the use of this sedentary, long-lasting stage of the blowfly lifecycle in PMI estimation. A multidisciplinary approach to age estimation of Calliphora vicina pupae was explored, including development of standard preservation protocols, with the aim of improving PMI estimation. Initial work involved the development of standardised egg collection protocols for the purpose of minimising variation in lifecycle length and precocious egg occurrence. This enabled quantification of pupal age error, which was subsequently applied to developmental timelines. Multiple preservation protocols were then trialled on pupae with the aim of retaining native morphological form and nucleic acid integrity for species identification and proposed age estimation methods. Optimal preservation methods for each analysis were suggested and the following universal preservative protocol proposed: pupae are pierced, hot-water-killed, and stored in 80% ethanol at - 20°C. Three methods of pupal age estimation were developed using changes in external morphology, histology and temporal gene expression. The external morphological development of 23 features was recorded from 1494 pupae. These data was used to create a Pupal Age Estimator tool, comprising a manual age-range correlation method and a regression equation for age estimation. Blind sample analysis indicated that age could be estimated to within 23 hours at 22°C, approaching the observed natural variation range. Internal morphological development of 42 pupae sampled at 24-hourly intervals was examined using histology and optical coherence tomography (OCT). Six additional features were identified as suitable age markers, however full analysis was limited by the inherent tissue loss due to sectioning and the low resolution of OCT. Finally, temporal gene expression levels of 42 pupae (selected at 24-hourly intervals) were quantified using qRT-PCR. Expression ratios were calculated between three developmentally expressed genes (Ecr, LSP-2 and Trp) and two housekeeping genes (EF1α and RP49). Regression analysis of these data indicated age estimation was possible to approximately 23 hours at 22°C. It can therefore be considered that the reliability and precision of PMI estimation using the C. vicina pupal stage is much improved from that possible at present. Pupal age estimation is critically dependent on appropriate preservation, now facilitated by the proposed standard protocols and by combining all age estimation methods presented here, a multidisciplinary approach can estimate C. vicina pupal age to within 23 hours at 22°C.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Quantifying the mechanical properties of skin in vivo and ex vivo to optimise microneedle device design

The transdermal delivery of therapeutics is limited to only a few molecules due to the outermost layer of skin, the stratum corneum, which acts as a barrier against the ingress of substances into the body. Microneedle arrays, which are commonly between 70μm and 900μm in length, have been developed as a method of promoting drug and vaccine delivery by creating microperforations in the stratum corneum to increase transport into the skin. The design of microneedle devices has significantly developed over recent years to allow for the delivery of numerous compounds into in vivo and ex vivo skin. Microneedle devices are now beginning to be taken away from the laboratory and towards clinical use but to achieve this it is desirable that all microneedles within the device penetrate skin in vivo to a sufficient depth. As microneedle devices have been extensively tested in cadaver tissue, a greater understanding of the mechanical properties of skin in vivo and ex vivo is required and to hypothesise whether animal models such as murine skin ex vivo serves as an appropriate model for human skin ex vivo. Measurements were performed on human skin in vivo by applying small cylindrical and spherical indenters to the volar aspect of the forearm on 7 volunteers. The average Young’s Modulus of the skin was 39.64kPa and 65.86kPa when applying the spherical and cylindrical indenters respectively. In a series of tensile measurements performed at three load axis orientations using ex vivo samples from human and murine donors, it was found that the key variation was attributed to the deformation experienced at initial low loads. This was shown to be significantly longer for human skin with an average of 5.10mm, when compared with murine skin which had an average of 1.61mm (p<0.05). Histological examination showed that human skin was far thicker, with an increased volume of dermal tissue, compared with murine skin, and this anatomical variation may have been the main reason why human and murine skin exhibited different mechanical properties. Finite element models (FEMs) were established of skin indentation in vivo, which incorporated the epidermis, dermis and hypodermis, and of human and murine skin in tension. Appropriate boundary conditions and mesh densities were implemented and the geometries were taken from real life measurements where possible. The Ogden material model of hyperelasticity was chosen to represent the skin layers for the FEM of skin indentation and an anisotropic material was used to describe human and murine skin in tension by adapting the Weiss et al model of transverse isotropy. Inverse finite element analysis was then used to match the FEMs with the experimental measurements. The multilayered FEM of skin was correlated against the in vivo indentation tests where model and experimental fit gave average root mean squared errors (R2 ave) of between 0.00103 and 0.0488 for the 7 volunteers. The optimal material parameters showed correlations with experimental measurements, where volunteers 1, 6 and 7 were shown to have the stiffest skin through Young’s Modulus calculations, which was reflected in the increased nonlinearity of the parameters extracted for the hypodermal layer. A stronger agreement between model and experiment for the anisotropic model of human and murine skin in tension was shown where the R2 ave was between 0.0038 and 0.0163. Again, model and experimental observations were shown to correlate where there was a significant difference (p<0.05) between 6 of the 14 average material parameters (C2, C3,1, λ1, C3,2, C3,3, λ3) when comparing human to murine skin. The multilayered FEM of human skin in vivo was further validated by modelling the application of a single microneedle to skin, prior to penetration. The model was then correlated against in vivo measurements performed on one of the volunteers and it was found that the model provided a good approximation for the experimental measurements. Using the multilayered FEM of human skin indentation, it was possible to model the deflection of the skin during the application of a pressure load comparable to microneedle array application. This allowed for the development of several curved microneedle arrays which aimed to distribute the load over all microneedles to potentially create uniform skin penetration by all those within the array. The microneedles were manufactured simply and quickly using wire cutting technologies from stainless steel and tested in human skin in vivo and in ex vivo samples of human and murine skin, where methylene blue was applied to identify any microchannels created by the microneedles. Preliminary measurements taken from murine skin ex vivo were discounted as microchannel staining was not possible. Analyses performed on human skin ex vivo showed penetration at high loads (4-5N) for all four microneedle array designs and the microneedle array with the smallest curvature (0.95mm) had the most consistent puncture for all microneedles, however puncture in vivo was difficult to characterise using approach developed. Therefore further work is required to assess more volunteers and donors. This study has highlighted the great differences in the mechanical properties of human and murine skin, suggesting that murine skin is not an appropriate model to assess microneedle puncture. It has also shown that the underlying tissues and hypodermis play a pivotal role in microneedle insertion mechanics