Functions of Grasshopper Genitalia Revealed, in 3D, Via Correlative Microscopy

For their study recently published in the Journal of Morphology, Derek Woller and Hojun Song created an animated, visual overview of all the anatomical components of both sexes of Melanoplus rotundipennis that were reconstructed in 3D using micro-CT scanning.

By Derek A. Woller

Grasshoppers have often been used in entomology textbooks and classrooms as model organisms for studying basic insect morphology. This is most likely due to the presence of groundplan anatomy, their often-large size that allows for easier dissections, and their relative abundance in nature. Yet, there is one aspect of grasshopper morphology (and insects in general) that is still poorly understood despite over a century of scrutiny and careful observations: the function of genitalia.

To further clarify, we know what the various genitalic components are and their general roles, just not what they all specifically do during the process of copulation. This might be surprising to some because, after all, these are arguably the most important parts of a grasshopper (or any organism) and most animals would certainly cease to exist in their absence.

Over the years, numerous studies described and speculated on the general functions of grasshopper genitalia during copulation, but the majority examined only museum specimens, and almost all focused exclusively on male genitalia. Out of these, only five, each focusing on a different species or subspecies, made use of a physical framework of copulation (mainly with live specimens) to investigate function hypotheses of the external and internal genitalia of both sexes. For further perspective, there are 12,219 species of Caelifera (true grasshoppers) currently known worldwide, which means our collective knowledge on this subject is still quite meager.

However, my coauthor, Hojun Song, and I recently expanded on what is known on this subject by a considerable amount by publishing, in the Journal of Morphology, one of the most in-depth investigations yet into the functional morphology of insect genitalia during copulation. Building strongly on the insightful work done by previous grasshopper researchers, our specific focus was to elucidate the roles of male and female genitalic and other reproductive system components in the southeastern United States grasshopper species Melanoplus rotundipennis (Scudder, 1878) (Acrididae: Melanoplinae). Previous studies mainly relied on drawings and occasional photographs, but we accomplished our goals by using correlative microscopy, an imaging technique that combines multiple technologies. In this case, we synergized micro-computed tomography (micro-CT), digital single lens reflex (DSLR) camera photography with focal stacking, and scanning electron microscopy (SEM).

Examples of Correlative Microscopy Technologies

Three imaging technologies were synergized for the correlative microscopy approach of a study on the functional morphology of grasshopper genitalia: micro-computed tomography, digital single lens reflex camera photography with focal stacking, and scanning electron microscopy. (Photo credit: Derek A. Woller)

Increasing Our Collective Evolutionary Knowledge

Sexual selection is generally regarded as the main hypothesis for explaining the presence of divergent genitalia in closely-related species, and these differences most likely play a probable role in the speciation process. Therefore, gaining a clearer comprehension of functional morphology is integral to better understanding the evolution of genitalia. However, insect genitalia, as I am sure the majority of entomologists are aware, can be relatively complex (particularly in males) in terms of the number of structures involved in copulation and reproduction. This, combined with their often-hidden nature (in the case of internal genitalia), and other factors, makes studying genitalia during the act of copulation quite tricky. Luckily, advances in imaging technologies are now able to reveal what is actually happening during coitus.

One such technology is micro-CT scanning, which, with some patience and hard work, gives researchers the ability to reconstruct external and internal anatomy in three dimensions (3D). Although micro-CT has been popular for studying insect anatomy since its introduction into the field in 2002, relatively few studies have, so far, focused on investigating functional genitalic morphology in insects. In fact, prior to our study, only one other has even utilized Orthoptera and that was with a katydid (Tettigoniidae).

Introducing the Puer Group

Our study revolved around a small flightless grasshopper species, M. rotundipennis, a member of the Puer Group, which contains 24 species that are found in the xeric habitats (e.g., scrub, pine flatwoods, and sandhills) of the southeastern United States (here defined as Alabama, North and South Carolina, Georgia, and Florida). Furthermore, many are endemic to relatively small regions, but M. rotundipennis is one of the few exceptions to this, and being able to find it easily is one of the main reasons we chose to focus on it. Externally, all species resemble one another to a strong degree, but their male genitalia, specifically the internal aedeagus, are wildly divergent, forming the basis for the group’s species divisions. Collectively, these factors make the Puer Group an excellent system in which to examine genitalia evolution.

Based on our own investigations combined with previous studies, M. rotundipennis was found to possessgenitalia that are relatively complex compared to other Puer Group members, with 58 named genitalic and other reproductive system components across both sexes (33 male, 25 female). Of these, we were able to assign at least one probable function during copulation to all but one of the male components (the furculae, which are external and attached to the posterior dorsal margin of tergite 10) and to 13 of the female components. In addition to the imaging technologies previously mentioned, we were also able to glean insights into functions by observing mating in live specimens kept in habitat boxes in the lab in addition to closely-examining live and museum specimens under a microscope.

grasshoppers mating

In a study of the functional morphology of grasshopper genitalia, Melanoplus rotundipennis specimens were encouraged to mate inside a habitat box in the lab. Upon copulation, they were quickly frozen, dried, and scanned using micro-computed tomography. (Photo credit: Derek A. Woller)

Of the most use were the 3D reconstructions of the copulating specimens because they allowed for a very thorough understanding of the interactivity of the male and female components. This was made possible by encouraging multiple specimens to mate in their habitat boxes and then, within 30 minutes of penetration, isolating and freezing candidate specimens rapidly in a -80° C freezer, followed by dehydration via critical point drying, and, finally, scanning them with a micro-CT machine.

The Power of Combining Technologies

The correlative microscopy approach not only allowed us to confirm and support some long-standing hypotheses on the probable function of some components of grasshopper genitalia during copulation, but it also enabled novel observations and the discovery of novel anatomy. Examples of these observations include a male’s paraprocts acting as supports for a female’s subgenital plate, some internal male components being more flexible than previously understood, and several external and internal female components accommodating male components in interesting ways.

Novel anatomy includes the discovery of multiple sensory receptors on different internal male components, the presence of leaf-like projections on the male’s aedeagus that may be female stimulators or grippers, and an internal female component we named the “armor of bursa copulatrix,” which, as the name implies, encases the bursa copulatrix and may strengthen it for interactions with the male’s adeagus. In addition, its ribbed appearance may allow for expansion during the same interactions.

While it is undeniably clear that many of the reproductive system components (especially genitalic ones) of grasshoppers perform integral functions during copulation, many still require further investigation. My hope is that more entomologists take advantage of these imaging methods, because the applications for correlative microscopy, especially micro-CT technology, are myriad and are enabling greater exploration of the genitalic frontier of functional morphology. As an added bonus, micro-CT allows researchers to examine female genitalia in a non-invasive way, leaving the components intact in their natural positions, which can be invaluable information when attempting to identify (and even describe) species. The female genitalia of many insect species are often ignored for a number of reasons, such as the assumption that female components vary very little between conspecifics (which can also be true, of course). Male and female genitalia have presumably co-evolved, though, so they should be studied together when possible. Additionally, if anyone is interested in learning more about any aspects of this study, I would be delighted to discuss the details. Finally, I highly recommend you check out the interactive 3D PDF that we created as well (Adobe Reader required; see PDF instructions here).

Derek A. Woller is a Ph.D. candidate in the Song Lab of Insect Systematics and Evolution in the Department of Entomology at Texas A&M University. His research is focused on unraveling the evolutionary history of a fascinating group of scrub-lovin’ grasshoppers confined to xeric habitats in the southeastern United States. Additionally, he has recently joined the USDA (APHIS, PPQ, CPHST) as part of the Rangeland Grasshopper & Mormon Cricket Management Team in Phoenix, Arizona. Email: asilid@gmail.com or derek.a.woller@aphis.usda.gov

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