Looking into the puparium: Micro‐CT visualization of the internal morphological changes during metamorphosis of the blow fly, <i>Calliphora vicina</i>, with the first quantitative analysis of organ development in cyclorrhaphous dipterans

Abstract

Metamorphosis of cyclorrhaphous flies takes place inside a barrel-like puparium, formed by the shrinking, hardening and darkening of the third-instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro-computed tomography (micro-CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau-Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra-puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X-ray images and micro-CT virtual sections. The origin of this gas bubble has been largely unknown, but micro-CT virtual sections show that it is connected to one of the main tracheal trunks. Micro-CT virtual sections also provided enough resolution for determining the completion of the larval-pupal and pupal-adult apolyses, thus enabling an accurate timing of the different intra-puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra-puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre-helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.