Revealing the respiratory system of the coffee berry borer (Hypothenemus hampei; Coleoptera: Curculionidae: Scolytinae) using micro-computed tomography

The coffee berry borer (Hypothenemus hampei) is the most economically important insect pest of coffee globally. Micro-computed tomography (micro-CT) was used to reconstruct the respiratory system of this species for the first time; this is the smallest insect (ca. 2 mm long) for which this has been done to date. Anatomical details of the spiracles and tracheal tubes are described, images presented, and new terms introduced. The total volume and the relationship between tracheal lumen diameter, length and volume are also presented. The total length of the tracheal tubes are seventy times the length of the entire animal. Videos and a 3D model for use with mobile devices are included as supplementary information; these could be useful for future research and for teaching insect anatomy to students and the public in general.This paper benefitted from the sub-award agreement S15192.01 between Kansas State University (KSU) and the University of Granada, as part of a USDANIFA Award 2014-70016-23028 to S.J. Brown (KSU), “Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease” (2015–2020)

Mechanical and cell-to-cell adhesive properties of aggregated Methanosarcina

The mechanical and adhesive properties as well as the turgor pressure of microbes play an important role in cell growth and aggregation. By applying AFM together with finite element modelling, one can determine the cell wall structural homogeneity, mechanical and cell-to-cell adhesive properties for aggregated Methanosarcina barked cells. This also allows a novel approach to determine in-aggregate turgor pressure determination. Analyzing the AFM force indentation response of the aggregates under loads less than 10 nN, our study reveals structural inhomogeneity of the polymeric part of the cell wall material and suggests that the cell wall consists of two layers of methanochondroitin (external: with a thickness of 3 +/- 1 nm and internal: with a thickness of 169 30 nm). On average, the hyperelastic finite element model showed that the internal layer is more rigid (mu = 14 +/- 4 MPa) than the external layer (mu = 2.8 +/- 0.9 MPa). To determine the turgor pressure and adhesiveness of the cells, a specific mode of indentation (under a load of 45 nN), aimed towards the centre of the individual aggregate, was performed. By modelling the AFM induced decohesion of the aggregate, the turgor pressure and the cell-to-cell adhesive interface properties could be determined. On average, the turgor pressure is estimated to be 59 +/- 22kPa, the interface strength is 78 +/- 12 kPa and the polymer network extensibility is 2.8 +/- 0.9 nm. We predict that internal cell wall comprised highly compressed methanochondroitin chains and we are able to identify a conceptual model for stress dependent inner cell wall growth. (C) 2014 Elsevier B.V. All rights reserved

Micro-CT imaging of live insects using carbon dioxide gas-induced hypoxia as anesthetic with minimal impact on certain subsequent life history traits

Abstract Background Live imaging of whole invertebrates can be accomplished with X-ray micro-computed tomography (micro-CT) at 10-100 μm spatial resolution. However, image quality could be compromised by the movement of live subjects, producing image artefacts. We tested the feasibility of using CO2 gas to induce temporary full-immobilization of sufficient duration to image live insects based on their ability to tolerate hypoxic conditions. Additionally, we investigated the effects of these prolonged hypoxic conditions on several life history traits of a lepidopteran species. Methods Live Colorado potato beetle (CPB) and true armyworm (TAW) adults were immobilized under a constant CO2 gas flow (0.5 L/min), and scanned using micro-CT (80 kVp; 450 μA). An L8 (24) orthogonal array (OA) was used to evaluate the effects of prolonged CO2-induced anesthesia on the recovery, longevity, and incidence of mating of TAW adults. The variable factors were age (immature and mature), sex (female and male), exposure time (3 and 7 h), and exposure regime (single and repeated). Results With this method, successful 3D reconstruction and visualizations of CPB and TAW adults were produced at 20 micron voxel spacing at an acceptable radiation dose and image noise level. From the inverse-square relationship found between the radiation doses and image noise levels, the optimal scanning protocol produced an entrance dose of 6.2 ± 0.04 Gy with images of 129.6 ± 5.1 HU noise level during a 2.7 h scan. Independent OA experiments indicated that CO2 gas did not result in death of exposed TAW adults, except when older males were exposed for longer durations. Exposure time and sex were more influential factors affecting recovery, longevity, and mating success than age and exposure regime following CO2 exposure. Conclusion We have demonstrated that using CO2 gas during micro-CT imaging effectively induces safe, repeatable, whole-body, and temporary immobilization of live insects for 3D visualizations without motion artefacts. Moreover, we have shown that exposed TAW individuals made a full recovery with very little impact on subsequent longevity, and mating success post hypoxia. This method is applicable to other imaging modalities and could be used for routine exploratory and time-course studies, for repeated scanning of live and intact individuals