Micro-CT data of complete metamorphosis process in Harmonia axyridis (2024)

Insect metamorphosis is the transformation process from the juvenile to the adult stage, playing a vital role in the insect lifecycle and encompassing extensive physical and biological changes. Holometabolous insects undergo complete metamorphosis and are characterized by a distinct pupal stage that exhibits profound internal structural transformations¹. This metamorphosis separates these from other insect groups, clearly delineating life stages and facilitating complex adaptations to various ecological niches^2,3,4. The group includes over 85% of insect species, which attests to the evolutionary success of the complete metamorphosis⁵. The investigation of the internal transformations associated with metamorphosis is a crucial focus of entomological studies, providing insights into the life cycle and developmental origin of this process.

The non-transparent nature of the body wall in many holometabolous insects makes it challenging to observe changes in their internal structures during development. To overcome this, various techniques were employed, including light microscopy, scanning electron microscope (SEM), confocal laser microscopy, and micro-computed tomography (micro-CT)⁶. Each method has specific benefits and limitations, such as the requirement for semi-transparent or removable body walls for light microscopy, and size constraints for SEM^7,8. In addition, confocal laser microscopy requires fluorescent labelling and slicing of the tissue^9,10. The miniaturization of X-ray imaging equipment has helped circumvent this problem, leading to the widespread application of micro-CT in studying the metamorphosis stages of insects across various orders. Previous studies provided important details on the prepupal stage of Calliphora vicina and Chrysopa pallens^11,12, while others mainly focused on changes occurring during the pupal stage^13,14,15,16. Notably, the prepupal stage, which marks the beginning of complete metamorphosis and is characterized by significant morphological changes, has remained understudied.

Phylogenetic analysis shows that the Coleoptera differentiated more than 280 million years ago, a split that predates other major holometabolous insect orders¹⁷. The larvae of some Coleopteran insects, particularly within the Coccinellidae, bear a closer resemblance to the wingless nymphs of hemimetabolous insects. This similarity reflects an intermediate evolutionary stage that makes Coleoptera insects particularly suited for research into the developmental origin of adult organs. However, the development and transformation of their internal organs have yet to be systematically documented.

The harlequin ladybird, Harmonia axyridis, is a native Asian insect and a significant biological control agent that is considered an invasive species globally^18,19, serving as a model for research in invasion biology²⁰. In recent years, the potential of this insect group as a model organism for genetic and developmental studies has also been recognized^21,22,23, making them an excellent material for studying the internal structural changes of Coleoptera insects during metamorphosis process.

The harlequin ladybird prepupal stage begins in the late phase of the fourth instar larva. As detailed in the Methods section, we started sampling every 4 hours from individuals entering the prepupal stage, which changed to 24 hours upon the start of the pupal stage (Fig.1a). This includes sampling of a total of five prepupal stages and four pupal stages, which were selected for micro-CT scanning and 3D modeling (Table1). Due to precision limitations, micro-CT is not able to delineate the organs of small insects in great detail. Hence, the primary focus of this study was the continuous changes in internal structures, including the brain, ventral nerve cord, digestive system, excretion system, and body wall muscles.

The insect central nervous system comprises two primary components: the brain and the ventral nerve cord. In harlequin ladybirds, brain volume gradually increases during the prepupal stages (PP1-PP5), followed by a rapid expansion from late prepupal (PP5) to the first day of pupa (Fig.1c). Maximum volume is achieved on the third day of the pupal stage (P3), after which growth plateaus (Fig.1c). At PP1, a dorsal view of the brain revealed kidney-shaped lobes with partial fusion of the protocerebrum (Fig.1e), while lateral views showed a small, downward-protruding tritocerebrum (Fig.1f). By PP5, the lobes elongated towards the mouthparts, paralleling each other (Fig.1g). Subsequently, on the first day of the pupal stage (P1), they expanded to both sides (Fig.1j). A ventral perspective showed an arch-shaped connection between the lobes (Fig.1l), which continued to extend to both sides and connected with the nerves of the optic lobes (Fig.1m). At the same time, the tritocerebrum enlarged and developed a noticeable protrusion towards the thoracic segments (Fig.1n).

At PP1, the larva contained a total of 12 ganglia in the ventral nerve cord, including one suboesophageal ganglion (SG), three thoracic ganglia (TG), and eight abdominal ganglia (AG), as observed from the lateral perspective (Fig.2). At PP5, the third TG and the first AG began to merge (Fig.2e) and completely fused into a larger one, with the second AG also integrating with it (Fig.2f). Eventually, the last three AGs fully merged, resulting in only seven distinct ganglia (Fig.2i). From PP1 to P5, and despite the decrease in ganglia, the volume of the ventral nerve cord increased (Fig.2j).

At PP1, the crop in the foregut was contracted, whereas the midgut appeared swollen (Fig.3a). The anterior section contracted in the hindgut, and the posterior section bulged. The Malpighian tubules are highly coiled at this stage, extending from the anterior part of the hindgut to the midgut, fully covering the latter (Fig.3a). Four hours later, the midgut had contracted, revealing a prominent bulge at its junction with the hindgut, which constituted the pylorus (Fig.3b). During the transition from PP4 to PP5, the midgut underwent marked swelling, creating an internal cavity, and the crop also enlarged (Figs.2d,e, 3d,e). Concurrently, the hindgut became coiled, while the Malpighian tubules reduced and could not fully envelope the midgut (Fig.3d,e).

Upon entering the pupal stage, the crop fully expanded, the midgut swelling reduced, and the hindgut was uncoiled (Fig.3f). From P1 to P5, the Malpighian tubules gradually disintegrated, eventually covering only the latter half of the midgut and becoming noticeably thinner, while the hindgut recoiled (Fig.3f–i). The intestinal volume decreased from the PP1 to PP4 stages, then stabilized in size, with a slight increase before eclosion occurred (Fig.3j).

Due to the impossibility of visually and computationally distinguishing muscle grayscale values in other appendages, we primarily focused on changes occurring in the body wall muscles of the trunk and flight muscles, as shown in Fig.4. The prepupa possesses four dorsal longitudinal muscles on the right side, extending from the abdomen to the thorax, as evident in the dorsal view. Owing to bilateral symmetry, there are eight dorsal longitudinal muscles in total. The ventral side possesses an equal number of longitudinal muscles as the dorsal side, with those nearer the midline being longer. Laterally, each body segment was interspersed with small muscle bundles, linking the external muscles of both the dorsal and ventral regions. From PP5 to P1, the thoracic body wall muscles rapidly degraded. By the late pupal stage P4, all body wall muscles in the thorax, except for the flight muscles, were virtually unobservable.

The indirect flight muscles (IFM) first emerged in the late prepupal stage (PP5) and are oriented perpendicularly to the dorsal plate. At P1, the direct flight muscles (DFM) appeared close to the lateral body wall. The IFMs, aligned parallel to the dorsal plate, become visible only on P2. The flight muscles compact in the day preceding eclosion, rendering each muscle bundle distinctly visible.

In summary, we employed micro-CT analysis to model and illustrate major internal structural changes during the metamorphic development of the harlequin ladybird, encompassing the transition from prepupal to late pupal stages. To our knowledge, this study presents the first 3D internal structural atlas for Coleoptera insects. The transformation processes of various organs were systematically described. The origins and time of emergence of various adult organs were determined. This extensive research provides an invaluable resource, significantly enhancing the understanding of insect metamorphosis and offering a helpful reference data for investigating complete metamorphosis in insects.