Naoto Inui (Doctoral Student, Department of Biological Sciences)

Ryosuke Kanehara (Doctoral Student, Department of Biological Sciences)

Yu Yamaguchi (Doctoral Student, Department of Biological Sciences)

Toru Miura (Professor, Misaki Marine Biological Station)

Key points of the presentation

• Pill Bugs and Brachiopods are crustaceans, terrestrial isopods that emerged from the sea to the land about 300 million years ago. They possess lungs on their abdominal limbs for breathing in air.
• Detailed observation of the lung formation process in the bryozoan has revealed that the lungs form during the post-hatching larval stage, that the two pairs of lungs develop at different times, and that the epithelial tissues of the abdominal limbs become entrapped and a cuticular layer forms during the formation process.
• Further clarification of the formation mechanism and acquisition process of the lungs, which are air-breathing organs, is expected to deepen our understanding of how animals advance on land.

Summary of Presentation

Terrestrial isopods, such as the beetles and bollworms ^{(Note 1)}, are a particularly diverse group of crustaceans that have expanded and diversified on land. Many species in this group have air-breathing organs called lungs ^{(Note 2)} in their abdomens. Lungs are an important trait that played a key role in isopods' terrestrial expansion and diversification, but when and how they are formed during development has not been elucidated.

A research group led by Professor Toru Miura and graduate student Naoto Inui of the Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo, and their colleagues have revealed the formation process of the lungs for the first time using a species of bramble beetle that is easily collected near human houses. Observations using scanning electron microscopy, confocal laser microscopy, and tissue sections revealed that the lungs are formed during the post-hatching larval stage, that the two pairs of lungs develop at different times, and that epithelial incision and cuticular layer formation occur during formation.

This study provides fundamental results for studying the mechanism of lung formation and its evolutionary process, and is expected to deepen our understanding of the mechanism by which animals advance on land.

Presentations

Arthropods such as spiders, millipedes, and insects are the most diverse and prosperous animal group on land. Arthropods that expanded on land possess air-breathing organs such as book lungs and trachea, which are thought to have been acquired independently by the ancestors of each lineage during the process of moving from the water to land. However, the process and mechanism of how these air-breathing organs were acquired during the process of landward expansion are still unknown.

Terrestrial isopods, such as sowbugs and bollworms, are a group of arthropods that possess air-breathing organs called lungs on their abdominal limbs ^{(Note 3)} (Figure 1). Since there are various lineages of terrestrial isopods, ranging from species without lungs to those with well-developed lungs, they may provide excellent material for studying the evolution of air-breathing organs, but little has been known about the process of lung formation, which is fundamental to understanding the evolutionary process.

Figure 1: Lungs of the research material, a bracket worm.
The bollworm has five pairs of appendages on its abdomen, of which only the anterior two pairs form the lungs (yellow arrowheads).
(A) Ventral view of a bollworm. (B) Enlarged view of the abdomen.

The group led by Professor Toru Miura and graduate student Naoto Inui of the Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo, therefore observed the process of lung formation in detail morphologically and histologically using a bracket worm, which is easily collected around human houses. Using scanning electron microscopy, confocal laser microscopy, and tissue sections, they observed the morphology of the abdominal limbs where lungs are formed from the late embryonic stage to the larval stage after hatching, and made the following observations.
(1) Lung formation occurs during the post-hatching larval stage.
(2) Of the two pairs of lungs, the second pair of lungs is formed immediately after hatching, while the first pair of lungs is formed later, during the larval stage (Figure 2).
(3) Inside the abdominal limb where the lungs are formed, epithelial tissue undergoes incision and bifurcation before molting, forming a new internal cuticular layer (Figure 3).

Figure 2: Lung formation process in the bramble beetle.

Figure 3: Tissue changes during lung formation.
The second pair of lungs formed on the second abdominal limb is formed in the early juvenile stage and then emerges (white arrowheads).
The histological image in sagittal section (section at * in autofluorescence image) shows the epithelium is entrenched (yellow arrowhead).

These results suggest that terrestrial isopods, such as the dung beetles, acquired a new developmental program that caused the epithelial tissues of the abdominal limbs to become entrapped and deformed during the process of their expansion onto land. Based on the knowledge of the lung formation process obtained in this study, we plan to elucidate the molecular mechanism of lung formation in the future, which is expected to deepen our understanding of the evolution of air-breathing organs and the mechanism of terrestrial expansion in animals in the future.

This research was supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research, 18H04006, Toru Miura), Grant-in-Aid for Young Scientists from the JSPS Research Fellowship (22J20990, Naoto Inui), and Grant-in-Aid for Fostering Research from the Institute of Aquatic Invertebrate Research (IKU2021-04, Naoto Inui). The research was supported by a grant from the Japan Society of Fisheries Science (JSFS).

Journals

Journal Title	Arthropod Structure & Development
Title of paper	Pleopodal lung development in a terrestrial isopod, Porcellio scaber (Oniscidea)
Author(s)	Naoto Inui, Ryosuke Kimbara, Haruka Yamaguchi, Toru Miura*, Naoto Inui
DOI	https://doi.org/10.1016/j.asd.2022.101210

Glossary

1 Terrestrial isopods

Crustaceans belonging to the suborder Mollusca, suborder Isopoda, and most species are terrestrial. Includes hydrozoan beetles, bollworms, and ocellate beetles. ↑up

Note 2: Lungs

Some species of bramble beetles have air-breathing organs called pleopodal lungs on their abdominal limbs. The number and morphology of these organs vary greatly among species. There are also different names for these lungs, such as pseudotrachea and white body. They differ in evolutionary origin from the so-called "lungs" of humans and other vertebrates. ↑↑↑↑

Note 3: Ventral limb

An appendage formed on the abdomen. Brachiopods usually have 5 pairs. ↑↑