Jake Lewis, an entomologist in the Environmental Science and Informatics Section at the Okinawa Institute of Science and Technology (OIST), is fascinated by weevils, a diverse group of beetles that includes many species with elephant trunk-like mouthparts (called a rostrum). Weevils provide various ecosystem services such as pollination and decomposition, but some species are serious pests known to decimate crop fields and timber forests.

Using x-ray microtomography, a 3D imaging technique that uses x-rays to visualize cross sections of the internal structure of objects, Lewis and his collaborators digitally removed scales that cover the cuticle of the weevils. They found that the underlying cuticle differs significantly between species and can therefore be used for taxonomic and classification purposes. Using this technique in combination with traditional light microscopy and DNA barcoding, they discovered, described, and named 12 new weevil species from Japan, Malaysia, Vietnam, and Taiwan. These species range from 1.5 – 3.0 mm in length and are comparatively quite small weevils.

Two of these new species are present in Japan: Aphanerostethus magnus (Oo-daruma-kuchikakushi-zoumushi) and Aphanerostethus japonicus (Nippon-daruma-kuchikakushi-zoumushi). One of these, Aphanerostethus japonicus, is also found in Yanbaru National Park, Okinawa. This is the first time x-ray microtomography has been used to remove obscuring scales to examine underlying differences in morphology for taxonomic purposes. The findings from this study have been published in the journal Zookeys.

The researchers showed that removing scales using x-ray microtomography reveals significant morphological differences between species, which cannot be easily observed using other methods. Consequently, this technique may gain more popularity as a tool for identifying new insect species, especially those covered in scales or debris.

Lewis, OIST’s Insect Collection Manager and lead author of the paper, examined specimens from collections in Canada, Germany, Japan, Malaysia, Taiwan, and the Netherlands. One of the primary goals was to investigate the use of x-ray microtomography as a tool in weevil taxonomy. The genus Aphanerostethus was poorly studied in the past, but many undescribed species were discovered in museum collections around the world, including the two new species from Japan.

Multiple methods to find new species

The researchers used traditional methods such as light microscopy and dissections to observe differences between species, including the scales along the elytra (back), leg spines, and the shape of the rostral canal (a canal that protects the rostrum). They also used DNA barcoding to analyze their genes and create a phylogenetic tree of eight of the species. Some species were not as easy to separate based on morphology alone, but as the gene sequences differ between species, the phylogenetic tree was informative and provided additional evidence of new species.

Although the above methods are standard practice in taxonomy, the researchers’ use of X-ray microtomography was novel and was successfully used to examine the structure of not only the hidden cuticle, but also the hindwings. Aphanerostethus weevils have lost the ability to fly due to the gradual reduction of their hindwings, however the degree of reduction was shown to differ between species. Normally, specimens would have to be dissected to view the hindwings, but because x-ray microtomography allows for non-destructive examination of internal anatomy, it is invaluable when working with rare or precious specimens that cannot be dissected or altered.

The presence of partially reduced wings in some species offers a fascinating glimpse into the ongoing process of evolutionary change: “Some species have almost completely lost their hindwings, while others still have non-functional half-wings with remaining vein patterns. The differing degrees in hindwing loss is not only useful for taxonomy and systematics, but also shows how different species within the same group can be at different stages of losing a historically highly important organ that played a crucial role in insect evolution,” Lewis explained. 

Investing in Japan’s natural heritage

The discovery of new weevil species can be challenging for two main reasons. Firstly, weevils are incredibly diverse, making complete cataloging time consuming and tedious. Secondly, many weevil species are highly host-specific, may only inhabit very particular microhabitats, and may only be active for a short period of time as adults. For example, some species feed on a single tree species and may only occupy a certain part of a tree, such as the canopy. Furthermore, some species of weevils are strictly nocturnal and rarely observed during the daytime. 

This extreme specialization and variation in natural history means that unless researchers investigate at night and day, across seasons, and focus on specific parts of many different plant species, they will inevitably overlook certain species.  

Dr. Dan Warren, a research fellow at the Gulbali Institute for Applied Ecology and former leader of the Environmental Science and Informatics Section, emphasized the importance of investing in specimen collections: “These specimen collections are crucial for discovering new species and documenting biodiversity changes, both from human activities and natural cycles. They are essential tools for scientific research and conservation biology,” he stated. “Without proper support for them and the people who maintain them, we risk losing irreplaceable information on species and ecosystems, potentially before we even discover them.” 

“These new weevil species are part of Japan's natural heritage, and although still poorly known ecologically, discovering and naming them is the first step towards an understanding of their biology,” Lewis added. Protected areas like Yanbaru National Park, home to the newly discovered A. japonicus, are essential to the protection of the island’s rich and endemic biodiversity.