Metamorphosis revealed: time-lapse three-dimensional imaging inside a living chrysalis

Abstract

Studies of model insects have greatly increased our understanding of animal development. Yet, they are limited in scope to this small pool of model species: a small number of representatives for a hyperdiverse group with highly varied developmental processes. One factor behind this narrow scope is the challenging nature of traditional methods of study, such as histology and dissection, which can preclude quantitative analysis and do not allow the development of a single individual to be followed. Here, we use high-resolution X-ray computed tomography (CT) to overcome these issues, and three-dimensionally image numerous lepidopteran pupae throughout their development. The resulting models are presented in the electronic supplementary material, as are figures and videos, documenting a single individual throughout development. They provide new insight and details of lepidopteran metamorphosis, and allow the measurement of tracheal and gut volume. Furthermore, this study demonstrates early and rapid development of the tracheae, which become visible in scans just 12 h after pupation. This suggests that there is less remodelling of the tracheal system than previously expected, and is methodologically important because the tracheal system is an often-understudied character system in development. In the future, this form of time-lapse CT-scanning could allow faster and more detailed developmental studies on a wider range of taxa than is presently possible.

Quantitative X-ray tomography

“…Examples range from the degradation of rechargeable batteries during their operating life 44 to the metamorphosis of butterflies. 45 For quantitative work it is important to remember that the accuracy is significantly influenced by the spatial stability of the Xray focal spot 46,47 and that a stable position can take up to 2 hours to develop in certain circumstances. Rockett employed a UHV LaB 6 source to circumvent stability issues associated with tungsten sources 48 specifically for long timescale quantitative studies.…”

The unique sound production of the Death’s-head hawkmoth (Acherontia atropos (Linnaeus, 1758)) revisited

“…Only very few examples of in vivo X-ray imaging sequences of Lepidoptera exist; e.g. Lowe et al (2013) used an X-ray based time-lapse imaging approach to study pupa development in vivo. To our knowledge, the mammography system has not been used before to generate a photo series of live animals.…”

Rotational Scanning Electron Micrographs (rSEM): A novel and accessible tool to visualize and communicate complex morphology

“…These issues are further exacerbated in the case of complex or asymmetrical male genitalia, which are challenging to clearly and accurately describe in words. Multifocal, ‘stacked’ images of external structures or habitus are now standard in most taxonomic descriptions and recent advances in micro-computed tomography (µCT) and magnetic resonance imaging (MRI) promise to yield exciting new character data from both external and internal morphology (i.e., Hart et al 2003, Beutel et al 2008, Mietchen et al 2008); even from non-sclerotized specimens preserved in alcohol (Faulwetter et al 2013) or living specimens (Lowe et al 2013). Both µCT and MRI scans can be represented as rotating (Mietchen et al 2008), even interactive (Faulwetter et al 2013) animations and have dramatically increased the level of morphological data available to users in one ‘illustration’.…”