JOURNAL OF MORPHOLOGY 278:334–359 (2017)

Investigating the Functional Morphology of Genitalia During Copulation in the Grasshopper Melanoplus rotundipennis (Scudder, 1878) via Correlative Microscopy

Derek A. Woller* and Hojun Song

Department of Entomology, Texas A&M University, College Station, Texas

ABSTRACT We investigated probable functions of the males, in terms of the number of components interacting genitalic components of a male and a involved in copulation and reproduction (Eberhard, female of the flightless grasshopper species Melanoplus 1985; Arnqvist, 1997; Eberhard, 2010). This rela- rotundipennis (Scudder, 1878) (frozen rapidly during tive complexity has often masked our ability to copulation) via correlative microscopy; in this case, by understand functional genitalic morphology, partic- synergizing micro-computed tomography (micro-CT) with digital single lens reflex camera photography with ularly during the copulation process. This process focal stacking, and scanning electron microscopy. To is difficult to examine intensely due to its often- assign probable functions, we combined imaging results hidden nature (internal components shielded from with observations of live and museum specimens, and view by external components) and because it is function hypotheses from previous studies, the majority often easily interrupted by active observation, but of which focused on museum specimens with few inves- some studies investigating function have manipu- tigating hypotheses in a physical framework of copula- lated genitalia as a way around such issues tion. For both sexes, detailed descriptions are given for (Briceno~ and Eberhard, 2009; Grieshop and Polak, each of the observed genitalic and other reproductive 2012; Dougherty et al., 2015). Adding further com- system components, the majority of which are involved in copulation, and we assigned probable functions to plexity to understanding function, several studies these latter components. The correlative microscopy have demonstrated that individual genitalic compo- approach is effective for examining functional morphol- nents may play different roles during copulation ogy in grasshoppers, so we suggest its use for other and evolve at different rates (Song and Wenzel, animals as well, especially when investigating body 2008; Song and Bucheli, 2010; Rowe and Arnqvist, regions or events that are difficult to access and under- 2011). Furthermore, when studying the evolution of stand otherwise, as shown here with genitalia and cop- genitalia, there has been a continued general bias ulation. J. Morphol. 278:334–359, 2017. VC 2017 Wiley toward examining the morphology of male genitalia Periodicals, Inc. for various reasons, including the difficulties in KEY WORDS: anatomy; insect; Acrididae; sexual selec- examining female genitalic morphology (Eberhard, tion; speciation 1985) (e.g., a common lack of rigidity), assumptions that female components do not appear to vary as much between conspecifics (which may also be true INTRODUCTION in some cases) (Eberhard, 1985; Ah-King et al., The evolution of animal genitalia is an active area of research in evolutionary biology and it is Additional Supporting Information may be found in the online generally accepted that sexual selection is the version of this article. major driving force that explains the observed divergence of genitalic components often found Contract grant sponsors: Orthopterists’ Society Research Grant among closely related species (especially in males (2012, to D.A.W.), and NSF Grant IOS-1253493 and Texas A&M University start-up (to H.S.). and particularly in insects), with these divergen- ces playing a potentially significant role in specia- *Correspondence to: Derek A. Woller, Minnie Belle Heep Center, tion (Eberhard, 1985; Arnqvist et al., 2000; Room 412, Campus MS 2475, College Station, TX 77843-2475. Arnqvist and Rowe, 2002; Hosken and Stockley, E-mail: [email protected] 2004; Ritchie, 2007; Hotzy et al., 2012; Richmond et al., 2012; Simmons, 2013). Insights into the Received 24 August 2016; Revised 18 November 2016; underlying mechanisms of this evolutionary pro- Accepted 3 December 2016. cess, however, are difficult to gain for a number of Published online 23 January 2017 in reasons. For one, insect genitalia are extremely Wiley Online Library (wileyonlinelibrary.com). diverse and often relatively complex, especially in DOI 10.1002/jmor.20642

VC 2017 WILEY PERIODICALS, INC. FUNCTIONAL MORPHOLOGY DURING COPULATION 335

Fig. 1. Melanoplus rotundipennis, abdomina of original male and female specimens frozen in copula compared to their 3D-reconstructions using micro-CT scanning (for all, female is above and male is below): A. left lateral view; B. right lateral view; C. left lateral view of 3D- reconstructions; D. right lateral view of 3D-reconstructions.

2014), and because differences in male components stacking and scanning electron microscopy (SEM), are typically more obvious (Eberhard, 1985; Arnqv- a method known as correlative microscopy (Caplan ist, 1997). However, male and female genitalia have et al., 2011), to investigate the copulation process presumably co-evolved, and, therefore, should be in the context: functional morphology. Compre- studied together. hending the function of morphology, principally We are currently in the midst of an insect (and how the male and female parts interact, is a key other invertebrates) morphology renaissance ever to better understanding genitalia evolution (Kelly since a pioneering study in by Hornschemeyer€ and Moore, 2016). The copulation process in et al. (2002) used micro-computed tomography insects has previously been difficult to study in (micro-CT) to examine beetle morphology. This fine detail because of the cryptic nature of interac- powerful imaging technology is non-invasive and tions between male and female genitalia and the non-destructive and has been used by many to invasive techniques that were necessary for exami- investigate the morphology of insect morphology nation. This frontier is ripe for exploration with in novel ways (e.g., Jongerius and Lentink, 2010; relatively few studies focusing on functional geni- Wipfler et al., 2011; Wojcieszek et al., 2012; talic morphology in insects (e.g., Eberhard and Breeschoten et al., 2013; Faulwetter et al., 2013; Ramirez, 2004; Briceno~ and Eberhard, 2009; Grie- Michalik et al., 2013; Beutel et al., 2014; Greco shop and Polak, 2012; Briceno~ et al., 2016; Rheber- et al., 2014; Simonsen and Kitching, 2014). One of gen et al., 2016), with even fewer utilizing micro- the primary benefits of micro-CT is the ability to CT to do so (e.g., Dougherty et al., 2015; Holwell build three-dimensional (3D) reconstructions of et al., 2015; Mattei et al., 2015; Schmitt and Uhl, any anatomical component included in the high- 2015). In terms of the order Orthoptera, only one resolution scanning process (Fig. 1A–D). For this other study of a similar nature has yet been study, we couple this technology synergistically undertaken (with Metrioptera roeselii (Hagenbach, with two other imaging methods, digital single 1822) (Ensifera: Tettigoniidae) in Wulff et al. lens reflex (DSLR) camera photography with focal (2015), but, to our knowledge, our study is the first

Journal of Morphology 336 D.A. WOLLER AND H. SONG (here defined as Alabama, North and South Caroli- na, Georgia, and Florida), and many are endemic to relatively small regions. Externally, all species resemble one another to a strong degree, but it is the male genitalia, primarily the aedeagus, that separate them due to their highly divergent forms (Hubbell, 1932). These collective factors make the Puer Group an excellent system in which to exam- ine the evolution of genitalia. Of all group members, M. rotundipennis was chosen for four reasons, the first being that it is the most widely distributed (in both Florida and southern Georgia), meaning that locating populations, normally quite abundant, was easy and integral to encouraging specimens to mate in the lab within habitat boxes (Fig. 2C). The second is that intraspecific variation in male genitalia, especially in the aedeagi, is negligible (Squitier et al., 1998), http://onlinelibrary.wiley.com/journal/ 10.1002/(ISSN)1097-4687/homepage/ForAuthors. html, which may also be true for female genitalia. Third, the species is one of the largest in the Puer Group with an average length of 15 mm in males and 20 mm in females and the aedeagus is males in also one of the most prominent, making this species one of the easiest to work with overall. Finally, the 58 named genitalic and other reproductive system components across both sexes (33 male, 25 female) of M. rotundipennis can arguably be labeled as the most complex in the Puer Group in terms of number and structural shape based on comparative observa- tions, making this species interesting to study. The general functions of grasshopper genitalia during copulation have been described and specu- lated on by numerous sources (Federov, 1927; Bol- dyrev, 1929; Snodgrass, 1935; Kyl, 1938; Roberts, 1941; Dirsh, 1956; Randell, 1963; Gregory, 1965; Otte, 1970; Pickford and Gillott, 1971; Whitman and Loher, 1984; Snodgrass, 1993; Eades, 2000; Chapman, 2012; Song and Marino-P~ erez, 2013), with the majority focused on museum specimens Fig. 2. Melanoplus rotundipennis,livephotosof:A. male; B. and many biased toward male anatomy. Only five female; C. in copula pair. of those studies (Boldyrev, 1929; Kyl, 1938; Grego- ry, 1965; Pickford and Gillott, 1971; Whitman and Loher, 1984) investigated hypotheses in a physical to use micro-CT to examine functional morphology framework of copulation to better examine the during the copulation process within the suborder largely cryptic interactions of the external and Caelifera (true grasshoppers). Although this study internal components of both sexes. For further is focused specifically on elucidating the functional perspective, there are 12,219 species of Caelifera role of genitalic morphology during the copulation currently known worldwide (Cigliano et al., 2016) of grasshoppers, we think that our findings and and each of the five previous framework studies methodology have wider implications for other ani- focused on four different species and one subspe- mals, especially arthropods. cies across two families of grasshoppers (Acrididae Our study subject, Melanoplus rotundipennis and Romaleidae). This means that our collective (Scudder, 1878) (Orthoptera: Caelifera: Acrididae: understanding of functional genitalic morphology Melanoplinae) (Fig. 2A–C) is a member of the Puer in grasshoppers is still quite poor, hence our deci- Group, a group of 24 small, flightless grasshoppers sion to also utilize a physical framework of copula- that reside in xeric habitats (e.g., scrub, pine flat- tion to investigate previous findings and posited woods, and sandhills). Additionally, they appear to hypotheses by answering two broad questions: 1) be strongly associated with scrubby oaks, are dis- what role(s) do the genitalic and other reproduc- tributed across the southeastern United States tive system components of males play during

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 337 copulation? Additionally, as noted previously, those components used specifically for copulation between a female morphology is frequently ignored, so female male and female. To aid in comprehension, the orientation of almost all of the DSLR and SEM images and 3D- genitalia was intentionally made a focus of this reconstructions was made uniform for both sexes (head facing study, both on their own and how they interact left) and reflects the natural positions of all anatomy. Finally, with male genitalic morphology, leading us to ask: the 3D-reconstructions of male components were colored in 2) what role(s) do the genitalic and other reproduc- shades of green, purple, and blue while female components tive system components of females play during were colored in shades of yellow, orange, pink, and red. copulation? Furthermore, the majority of previous Imaging and 3D-Reconstructions studies relied on standard dissections, drawings, and, occasionally, photographs, which were uti- Digital single lens reflex. Digital images of anatomical lized well to gather evidence to support observa- components of freshly collected and museum specimens were tak- en in the Song Laboratory of Insect Systematics and Evolution tions. Imaging technology, although, is continually using a Visionary Digital imaging system equipped with a Canon advancing, so we harnessed the combined power of EOS 6D DSLR camera combined with a 100 mm/65 mm lens (the three of these technologies (DSLR with focal stack- latter often coupled with a 23 magnifier) to take multiple images ing, SEM, and micro-CT) and our own observa- at different focal lengths. The resulting files were converted from RAW to TIFF format using Adobe Lightroom (v.4.4), stacked into tions of live and museum specimens for two a single composite image using Zerene Stacker (v.1.04), and then primary reasons: to investigate the general value Adobe Photoshop CS6 Extended was used to add a scale bar and of correlative microscopy and to decide if such an adjust light levels, background coloration, and sharpness. Photo- approach is more effective for examining genitalic graphs of live specimens were taken in the same lab in a simulat- ed habitat using a Canon EOS 6D Digital SLR Camera equipped morphology and function for at least grasshoppers. with a 100 mm lens and a Canon MT-24EX Macro Twin Lite Flash paired with two multi-directional halogen lights for addi- MATERIALS AND METHODS tional lighting. Photographs of the specimens used for micro-CT scanning were taken by a colleague in Germany using a KEY- Taxon Sampling ENCE VHX-2000 series digital microscope. Scanning electron microscopy. Genitalia of both sexes The present study is based on M. rotundipennis (Scudder, came from specimens that had been cleared in KOH as 1878) (Orthoptera: Caelifera: Acrididae: Melanoplinae). Utilized described above. All images were taken with a JEOL JSM- specimens (see Supporting Information for full locality informa- 6480 after genitalia were attached to standard metal stubs tion) mainly came from Florida with two from southern Georgia with carbon glue and coated with 30 nm of gold palladium. and included: museum, recently collected, and laboratory- Micro-CT. Scanning was performed on a copulating pair of observed within habitat boxes. Specimens in the latter category male and female specimens that were brought to the lab from were collected from different locations in Florida, with each box the field, kept within a habitat box with other specimens of containing up to 10 individuals of mixed sex ratios, mainly to both sexes, fed Romaine lettuce daily, observed until copulating, observe mating behavior. Specimen numbers and sexes used for carefully isolated into a smaller container, and then frozen rap- each imaging technique are as follows: 1) DSLR camera: idly in a 2808C freezer within 30 min of the male penetrating 10#,11$; 2) SEM: 5#,1$; 3) micro-computed tomography the female. Several other copulating pairs were also frozen this (micro-CT): 1#,1$. way and the set that looked the most intact was chosen to be the micro-CT subject. This specific pair was then prepared for Dissections the critical point drying process by removing the sample from the freezer and cutting through the entirety of their abdomens The internal genitalia and parts of the reproductive systems at or around segment 6. This smaller sample was then fully of male and female specimens were dissected from both freshly dehydrated over 24 hours within a 1.65 ml vial containing collected and museum specimens (rehydrated by being dipped 100% ethanol. The sample was transferred next to a special briefly into boiling water) and removed from the body using microvial punctured with a syringe for fluid/gas exchange and standard procedures for male grasshoppers (Hubbell, 1932) and run through a Tousimis Samdri-790 Semi-Automatic Critical female grasshoppers (Slifer, 1939) and the assistance of a Leica Point Drying Apparatus. Then, the final dried sample was MZ16 microscope system. Intact dissected components were put scanned using an X-radia 400 (Carl Zeiss X-ray Microscopy, in 0.65 ml vials containing a 10% KOH solution and placed into Pleasanton) with absorption contrast and a spatial resolution of a boiling water bath for up to an hour to clear away obstructing 2.6 mm. The resulting scans were mirrors of the sample. tissues. The specimens were then removed and further dissect- 3D-reconstructions. The micro-CT scanning process ed as necessary for examination and imaging. In the case of the resulted in a stack of 1,943 TIFF images that was then male, this meant fully separating the epiphallus from the ecto- imported into the program Amira (FEI, v. 6.0.1) and examined phallus and endophallus and, occasionally, the former from the in three two-dimensional (2D) planes (X-Y, X-Z, and Y-Z)to latter. For the female, this meant removing all exterior abdomi- locate all desired anatomical structures, which were then trans- nal segments to reveal hidden components. formed into three-dimensional (3D) reconstructions using the Segmentation Editor. The file sizes of the four halves of the full Terminology exterior terminalia of the male and female seen only in the interactive reconstruction were found too large to work with, Terminology was derived and synthesized (and, in some and thus, were made smaller with Resample module. Once cre- cases, modified) from a number of sources: Snodgrass, 1935, ated, each reconstruction was then isolated from the rest using 1993; Slifer, 1939, 1940a, 1940b, 1943; Roberts, 1941; Dirsh, the Surface View module and all were then exported as stereoli- 1956, 1965, 1973; Eades, 1961; Randell, 1963; Gregory, 1965; thography (STL) files. The program Blender (v.2.76) was then Uvarov, 1966; Ander, 1970; Amed egnato, 1976; Whitman and used to apply smoothing algorithms to the individual STL files Loher, 1984; Key, 1989; Stauffer and Whitman, 1997; Eades, as follows: for almost all anatomical components, the Smooth 2000; Gordh and Headrick, 2001; Chapman, 2012; and Song Vertex option was applied with “1” as the Smoothing value and and Marino-P~ erez, 2013. The reproductive system is defined “30” as the Repeat value, but, for the four halves of the termi- here as any anatomical components used by both sexes for the nalia, “0.5” was the Smoothing value and “5” was the Repeat production of offspring, with genitalia defined as a subset of value. Smoothed reconstructions were then exported as updated

Journal of Morphology 338 D.A. WOLLER AND H. SONG

Fig. 3. Melanoplus rotundipennis, 3D-reconstructions (in copula positions) of male and female components of genitalia and other parts of the reproductive system (male components are colored in shades of green, purple, and blue; female components are colored in shades of yellow, orange, pink, and red): A. dorsal view, posterior to right; B. left lateral view, posterior to right; C. right lateral view, posterior to left; D. posterior view; E. ventral view, posterior to right.

STL-files and imported back into Amira for color assignments Special features. The included video (Supporting Infor- using the Surface View module. Note that the smoothing pro- mation Fig. S1) is intended to be an animated, visual overview cess reduces general roughness, which can disconnect objects of the anatomical components that were reconstructed in 3D formerly tightly connected (e.g., see Fig. 4B,C: the base of the and was initially created using Amira’s Animation Director. spermathecal duct no longer touches the apex of the bursa Next, the video was exported as a series of image files and copulatrix). Blender was also used to mirror the STL-files imported into Windows Live Movie Maker (v. 2011) where it around the y-axis to match the original sample. All figures was mirrored around the y-axis to match the original sample, were assembled in Adobe Photoshop CS6 Extended and all transitions and titles were added, and it was then exported as reconstructions had their original backgrounds replaced with an MP4 file. a uniform color. Photos of the original sample were taken in The included interactive 3D-figure (Fig. 14) requires a 3D Germany once the micro-CT scanning process was completed PDF-enabled viewer such as Adobe Reader and is intended to in China. During the journey to Germany, the specimens simulate what we can see and do in Amira and to also assist shifted slightly, which is why they do not exactly match the the reader in better understanding how the anatomical compo- reconstructions (Fig. 1). nents of both sexes interact because 2D-images of such

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 339 components can be difficult to mentally place spatially in rela- tion to each other. The program PDF3D ReportGen (v. 2.13.0, Build 8317 364) was used to accomplish this by combining all STL files generated in Amira and a text file (of the type IV) containing associated titles for each component that was modi- fied from a template included with PDF3D. The color of each STL file was matched as closely as possible to the color of its 2D counterpart in the figures and video using PDF3D’s “pick screen color” option in the Visual Effects tab and clicking on the colors in the figures. Additionally, to reduce file size and optimize movement speed in the 3D-PDF, the following simplifi- cation options within PDF3D (Advanced tab) were employed, all with a value of 150,000: threshold triangles count, threshold line segments count, and threshold points count. Locations for placement of associated component titles were found using Amira by attaching the Line Probe module to each desired STL file, resulting in exact x, y, z coordinates. High-resolution versions of the static figures (TIFF), interac- tive 3D figure (PDF), video (MP4), 3D-reconstructions (STL – uncolored), and the micro-CT data used to build the 3D- reconstructions (TIFF) are available for download at Morpho- Bank (O’Leary and Kaufman, 2012) under Project P2517: http://morphobank.org/permalink/?P2517.

RESULTS All terminology used for the genitalic and other reproductive system components are included in this section with occasional modifications from original sources to aid in uniformity and ease of understanding. Additionally, definitions are given for those components that are either not obviously delimited in referenced figures or are potentially unique to this species and/or the Puer Group. Dis- coveries that are possibly novel are further elabo- rated on later. There are no intended statements of homology regarding terminology and given defi- nitions, and they may only apply to this species and/or the Puer Group. The genitalia and other reproductive system components of M. rotundipen- nis examined in this study are arguably the most complex compared to other Puer Group species, consisting of 58 named components (many paired) for both sexes combined, 33 for males and 25 for females. To aid in comprehension, after a brief overview of mating behavior for this species is giv- en, a detailed overview follows of the observed components of external and internal genitalia and other parts of the reproductive system with orien- Fig. 4. Melanoplus rotundipennis,leftlateral,cut-awayviews tation descriptions based on relative repose posi- of 3D-reconstructions (in copula positions) of male and female tions (i.e., when copulation is not occurring). The components of genitalia and other parts of the reproductive sys- tem (in all, posterior to right): A. male external and internal; B. majority of these components were transformed female external and internal (muscle tissue of spermathecal com- into 3D-reconstructions (e.g., Figs. 3, 4) and an plex removed); C. close-up view of combined male and female overview is provided separately for each sex, as (muscle tissue of spermathecal complex removed). well as for what components are touching while in copula (Figs. 3, 4C) solely based on the 3D- reconstructions. Additionally, further comprehen- Mating Behavior sion can be gained by watching the video of the Habitat boxes often contained up to 10 M. rotun- 3D-reconstructions (Supporting Information Fig. S1) dipennis individuals of mixed sex ratios, but mating and exploring the interactive 3D-reconstructions did not seem to occur until at least two males were (Fig. 14). in the same habitat box with at least one female and

Journal of Morphology 340 D.A. WOLLER AND H. SONG

Fig. 5. Melanoplus rotundipennis, 3D-reconstructions (in copula positions) and DSLR images of male external genitalic components: A. 3D, left lateral view, posterior to right; B. 3D, dorsal view, posterior downwards; C. DSLR, dorsal view; D. DSLR, left lateral view; E. DSLR, left later- al view, made translucent with KOH. then, occasionally, one would attempt copulation in a transferred to a private container for periodic obser- manner similar to what Bland (1987) observed in M. vations during which the pair would stay still or wan- childsi Otte, 2012 (2011) (formerly belonging to M. der around slowly. We also observed the femora of tequestae Hubbell, 1932) and what Otte (1970) both sexes vibrating intermittently along with period- observed in melanoplines in general. The male began ic pulsations of both abdomens. Despite a female his copulation attempt by moving slowly behind a seeming to be receptive, some males were unsuccess- female and then suddenly jumping on her back. The ful for unknown reasons, in either attempting to pull female would then often kick at him wildly with her down the female’s subgenital plate or inserting their hind tibiae and sometimes jump around the cage with adeagus. These rejected males would then dismount the male clinging tight. Usually, the female would and wander away, sometimes not trying to mate eventually calm down, allowing the male to position again for hours or days, if at all. his abdomen under hers, invert his supra-anal plate to expose his phallic complex, and attempt to mate Male: External and Internal Genitalia and with her. The general in copula positions for grass- Other Parts of the Reproductive System hoppers (e.g., Figs. 3, 4) are as follows: the male gains External (Fig. 5): Seven anatomical components access to the female’s internal genitalia by pulling are present, three of which are paired. The furculae down her subgenital plate with his epiphallus and (Figs. 4A, 5A–E) are prominent and extend from the then rotates his phallic complex anterodorsally posterior margin of tergite 10 (Fig. 5C–E) and around 908 (or more), inserting it through her vulva slightly across the base of the supra-anal plate (or (e.g., Chapman, 2012). After successful mounting, epiproct; Figs. 4A, 5A–E) that also emerges from defined here as a female allowing a male to insert his tergite 10. The cerci (Figs. 4A, 5A–E) extend poste- adeagus into her for an uninterrupted period, (Figs. riorly toward the cone-like pallium (Fig. 5C–E), the 2C) the M. rotundipennis pair were carefully apex of the subgenital plate (Fig. 5D,E). The

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 341

Fig. 6. Melanoplus rotundipennis, 3D-reconstructions (based on in copula positions, but rotated into repose positions) and a DSLR image of male internal genitalia and greater reproductive system components (posterior to right unless otherwise noted with one exception - spermatophore only present during copulation, so this component always points anteriorly): A. all components, quasi- dorsal view; B. epiphallus, dorsal view, posterior downwards; C. epiphallus, left lateral view; D. ectophallus, dorsal view; E. ectophal- lus, left lateral view; F. endophallus, dorsal view; G. endophallus, left lateral view; H. DSLR, discarded spermatophore, left lateral view; I. greater reproductive system, left lateral view; J. greater reproductive system, dorsal view. paraprocts (Figs. 4A, 5A–E) are in-between the Internal (Fig. 6A): Two main anatomical com- supra-anal plate and the cerci, also extending later- ponents are present: the genital chamber (cavity ally and posteriorly, and attach, via a membrane enclosed by the supra-anal plate (Figs. 4A, 5A–E) along the middle of their dorsal edge, to the base of dorsally and subgenital plate plus pallium (Fig. the cerci. The subgenital plate resembles the gener- 5C–E), laterally, posteriorly, and ventrally) and the al form found in many Acrididae species, almost phallic complex (Fig. 6A), which comprises three appearing to comprise two segments due to the subcomponents: epiphallus (Figs. 6B,C, 7F,G), ecto- medial transverse groove that can be stretched phallus (Figs. 6D,E, 8A), and endophallus (Figs. apart to a degree due to membranes in-between. 6F,G, 8A), each of which are divided further into

Journal of Morphology 342 D.A. WOLLER AND H. SONG

Fig. 7. Melanoplus rotundipennis, DSLR images of male internal genitalia and greater reproductive system components (posterior to right unless otherwise noted): A. all components, left lateral view; B. phallic complex, left lateral view; C. phallic complex, dorsal view; D. phallic complex, ventral view; E. phallic complex, posterior view; F. epiphallus, dorsal view, posterior downwards; G. epi- phallus, anterior view, dorsal downwards. more subcomponents. The epiphallus (Figs. 6B,C, 7F,G) do not. Additionally, the anterior projections 7F,G) consists of six subcomponents (five of which have what appear to be multiple sensory receptors are paired) and is the most dorsal part of the phal- (not shown here) scattered about dorsally with lic complex, resting above the ectophallus (Figs. more found in the median sclerotized cleft formed 6D,E, 8A) and endophallus (Figs. 6F,G, 8A), con- between the anterior projections and the ancorae. nected to these two components via muscles and The bridge (Figs. 6B, 7F,G) is thick and bends thick membranes. The ancorae (Figs. 4A, 6B,C, strongly inwards in the middle when viewed anteri- 7F,G) curve ventrally at a steep angle and the ante- orly while the lateral plates (Fig. 6B,C, 7F) are also rior projections (Figs. 6B, 7F,G) curve inwards thick, but unremarkable. The lophi (Figs. 4A, 6B,C, slightly while the posterior projections (Figs. 6B,C, 7F,G) are covered with minute ridges that resemble

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 343 fish scales that are oriented toward the anterior of the epiphallus (Fig. 9G). The ectophallus (Figs. 6D,E, 8A) is the middle phallic complex component that is firmly connected to, and largely surrounds, the endophallus (Figs. 6F,G, 8A) via thin membranes and consists of one primary component, the cingulum [that would resemble the letter “H” if flattened (Roberts, 1941)] with four subcomponents (only two of which are typically paired). The apodemes of cingulum (Figs. 4A, 6D,E, 7B,C,E, 9A) are of varying sizes and cur- vature while the zygoma (Figs. 4A, 6D,E, 7B,C, 9A) can be described as double-layered and complete, although atypical (because it slightly extends poste- riorly over the valves of aedeagus (Figs. 4A, 7B, 9B), but just ventral to it is an almost-identical sec- ond region that contains a medial gap. The rami (Figs. 4A, 6D,E, 7B,D, 9A) are well-developed, extend a little ways ventrally beyond the aedeagus, curve inwards slightly, and have what appear to be sensory receptors (not shown here) at the apex of the posterior region. The sheath of aedeagus (Figs. 4A, 6D,E, 7B–D, 9A) is a somewhat misleading term for what is observed in this species because it is only vaguely sheath-like. In fact, it would be bet- ter described as two individual lobes that extend a little ways beyond the apices of the rami and are lightly attached via membranes to the basal portion of the dorsal valves of aedeagus, but for ease of understanding, will be continued to be referred to as a single component. Additionally, the sheath of aedeagus curves upwards, almost reaching the top of the dorsal valves of aedeagus in some specimens (Fig. 7B), curve slightly inwards, and are also cov- ered with tiny spines arranged in overlapping rows that point anteriorly (Fig. 9F). The endophallus (Figs. 6F,G, 8A) is the most ven- tral part of the phallic complex and the primary por- tion that contains components of a species-specific nature, and consists of nine subcomponents (six of which are paired), with six more closely connected that belong to the greater reproductive system (Fig. 7A). The apodemes of endophallus (Figs. 4A, 6F,G, Fig. 8. Melanoplus rotundipennis, DSLR images and 3D- reconstructions (based on in copula positions, but rotated into 7B–E, 8B,D, 9A) are typical for acridids as is its repose positions) of male internal genitalic components (in all, whitish, opaque connective tissue (Figs. 4A, 6F,G, posterior to right): A. DSLR, separated ectophallus and endo- 7C,E, 8D), presumably, and, more than likely, so are phallus, left lateral view; B. DSLR, partially separated endo- the greater reproductive system components: ejacu- phallus, left lateral view; C. 3D, ventral valve of aedeagus, left lateral view; D. DSLR, partially separated endophallus, dorsal latory duct (Figs. 4A, 6I,J, 7A), ejaculatory sac view; E. 3D, ventral valves of aedeagus, dorsal view. (Figs. 4A, 6I,J, 7A), gonopore (not pictured because it is a hole connecting the sacs), and spermatophore sac (Fig. 6 I,J). The duct and sacs can still be very orientation is the opposite of other male internal difficult to see after dissection, being often obscured components because the phallic complex is rotated by vestiges of undissolved musculature (the whitish around 908 (or more) during copulation (e.g., Fig. coloration of the components blends in well with the 4A,C). The spermatophore comprises two compo- whitish musculature) and by their ethereal nature nents (anterior to posterior): 1) tube region (the and relative plasticity of their shapes; therefore, we body, which is lightly sclerotized; Fig. 4A, 6H–J) relied on the 3D-reconstructions to guide us to the and 2) reservoir (bulbous head; Fig. 6H–J). Addi- general shapes. The spermatophore (Fig. 4A, 6H–J) tionally, flexures (Figs. 6G, 7B, 8B, 9A, 10E) are is a temporary creation of the greater reproductive present and have obvious articulations (not labeled, system during copulation and, thus, its relative but see Figs. 6G, 7B, 8B, 10E) between them and

Journal of Morphology 344 D.A. WOLLER AND H. SONG

Fig. 9. Melanoplus rotundipennis, SEM images of male internal genitalic components: A. ectophallus and endophallus, left lateral view, posterior to right; B. dorsal and ventral valves of aedeagus, posterior view; C. close-up, lateral view of left ventral valve of aedeagus; D. close-up, left lateral view of dorsal valves of aedeagus; E. further magnified close-up, left lateral view of dorsal valves of aedeagus; F. sheath of aedeagus, quasi-left lateral view, posterior to right; G. right lophus of epiphallus, quasi-left lateral view. the aedeagus while the gonopore processes (Figs. are quite long and gently curve upwards when 6G, 7B,D, 8B, 10E) are near the apical end of the viewed laterally. For this species, both sets of apodemes of endophallus and slope dorsally toward valves can be imagined as nested structures in the flexures. which the concave dorsal valves almost fully The aedeagus, the intromittent organ, is highly encompass the tubular ventral valves. The dorsal species-specific and, here, both its dorsal valves valves are open along the entirety of their ventral (Figs. 4A, 6F,G, 7B–E, 8B,D, 9A,B, 10E) and ven- side, fused for more than 3=4 of their length with a tral valves (Figs. 4A, 6F,G, 7B-E, 8B–E, 9B, 10E) thin, median dorsal cleft appearing for the

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 345

Fig. 10. Melanoplus rotundipennis, 3D-reconstructions (A,B are in copula positions, C,D,E rotated into repose positions) of various genitalic components: A. complete male and female external, posterior view; B. partial male external and internal, plus female exter- nal, posterior view; C. partial male phallic complex, quasi-right lateral view, posterior to left; D. partial male phallic complex, quasi- ventral view, posterior downwards; E. endophallus, ventral view, posterior to right. remainder, and their entire outer surface is cov- Female: External and Internal Genitalia, and ered with microscopic leaf-like projections that are Other Parts of the Reproductive System anteriorly projected (Fig. 9A,D,E). The ventral External (Fig. 11): Nine anatomical compo- valves are subcylindrical, apically taper to points, nents are present (six of which are paired) and and are enclosed by the dorsal valves on all sides one has two subcomponents (one of which is except ventrally. However, the ventral valves can paired). The supra-anal plate (or epiproct) (Figs. sometimes extend a bit beyond the start of the 11A,B) is attached to tergite 10 (Fig. 11A,B) at its dorsal valves, and can also often be seen through base with the cerci (Fig. 11A,B) extending posteri- the walls of the dorsal valves, especially laterally, orly. The paraprocts (Figs. 11A,B) are similar to when sclerotization is light or a specimen has been fully cleared in KOH (e.g., Fig. 7B). Often, those in males, although larger and with a more these valves may appear to be attached for some rounded shape. Dorsal (Figs. 11A–C, 12B), inner length basally, but are actually only attached via (Fig. 11B), and ventral valves of ovipositor (Figs. light membranes. In fact, the valves can be fully 11A–D, 12A,B), and lateral basivalvular sclerites separated into two nearly identical halves, each (Figs. 11B,C, 12A,B) are unremarkable and resem- appearing to be smooth, but, on closer inspection, ble those of other melanoplines, if not the majority are actually quite rugose (Fig. 9C). Three length of acridids that lay eggs in soil (Stauffer and Whit- variations have been observed for the ventral man, 1997). Similarly, the subgenital plate (Figs. valves: they do not extend beyond the apex of the 4B, 11B–H), which often has species-specific char- dorsal valves (Figs 7B 9A), they extend a little acters, such as the shape of its posterior margin, ways beyond the apex (Fig. 8A), and they extend resembles that of all Puer Group species with the quite a bit beyond the apex (Figs. 7A, 9B). Due to margin possessing an ephemeral triangular projec- the unique shape of both valve sets, the phallot- tion that is attached firmly to the underside of the reme (not labeled, but see Figs. 7B, 8A,B), the egg guide, extending about 1=4 to 1=2 of its length channel formed by the inner space of these valves (partially seen in Fig. 10A,B). The subgenital plate through which the spermatophore (Fig. 4A, 6H–J) contains two subcomponents: the lophi receptacles travels, is, thus, unique to this species. The arch (Fig. 11E–H) and egg guide (Figs. 4B, 11D–H), of aedeagus (Figs. 6F,G, 7B, 8B,D, 10C) rises from both of which also resemble those of all Puer the median, dorsobasal region of the dorsal valves Group species. Additionally, the apical third of the of aedeagus. subgenital plate that is bent downwards into the

Journal of Morphology 346 D.A. WOLLER AND H. SONG

Fig. 11. Melanoplus rotundipennis, DSLR images and 3D-reconstructions (in copula positions) of female external genitalia and greater reproductive system components (posterior to right unless otherwise noted): A. DSLR, dorsal view; B. DSLR, left lateral view; C. DSLR, ventral view; D. DSLR, ventral view, made translucent with KOH; E. 3D, subgenital plate, posterior view; F. 3D, subgenital plate, quasi-left lateral view, posterior to right; G. DSLR, subgenital plate, dorsal view; H. DSLR, subgenital plate, ventral view. male’s genital chamber during copulation (Fig. 4B) by the apodemes of ovipositor (Fig. 12A,B) and ven- and contains the lophi receptacles is more heavily trally by the subgenital plate (Figs. 4B, 11B–H), sclerotized than the rest (Fig. 11G,H). with the spermathecal complex (Fig. 13H,I) as the Internal (Fig. 13A): Eight main anatomical most anterodorsal component, median oviduct (not components are present (three of which are paired) pictured) as the most ventral, and vulval sclerite and two have subcomponents. The genital chamber (Figs. 4B, 12A–E,G,H, 13B–E) as the most posterior. is the cavity containing the internal genitalia and The apodemes of ovipositor and anterior basivalvu- parts of the reproductive system, but can be difficult lar sclerites (Fig. 12A,B) are unremarkable and to delimit. Here, it seems to be delimited dorsally resemble those of other melanoplines.

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 347

Fig. 12. Melanoplus rotundipennis, DSLR and SEM images of female external and internal genitalia, and greater reproductive sys- tem components (posterior to right unless otherwise noted): A. DSLR, ventral view; B. DSLR, ventral view, posterior downwards; C. DSLR, bursa complex, dorsal view; D. DSLR, bursa complex, ventral view; E. DSLR, close-up of vulval sclerite, posterior view, dorsal upwards; F. DSLR, partially separated bursa complex, quasi-dorsal view; G. SEM, bursa complex, ventral view; H. SEM, close-up of posterior region of bursa complex, ventral view.

The spermathecal complex (Fig. 13H,I), too, is essen- detailed histological study by Ahmed and Gillott tially similar to other members of the Puer Group and (1982) called a “small discrete bundle of muscle fibers.” contains four subcomponents with one of these divided This is further supported by Lay et al. (1999) in which further into two more subcomponents. The entire com- it is noted that “longitudinal and transverse muscles plex is covered in what Snodgrass (1993) called a overlay” the entire complex. Here, we follow Gosalvez “muscular sheath” while Whitman and Loher (1984) et al. (2010) in calling it the more general “muscle referred to it as “glandular tissue” in Taeniopoda eques tissue” (partially seen in Figs. 12A, 13H). The sperma- (Burmeister, 1838) (Romaleidae) and which a more thecae comprises two subcomponents: preapical and

Journal of Morphology 348 D.A. WOLLER AND H. SONG

Fig. 13. Melanoplus rotundipennis, 3D-reconstructions (in copula positions) and a DSLR image of female internal genitalia and greater reproductive system components (posterior to right unless otherwise noted): A. all components, left lateral view; B. vulval sclerite, dorsal view; C. DSLR, vulval sclerite, posterior view, dorsal upwards; D. vulval sclerite, ventral view; E. bursa complex and vulval sclerite, left lateral, cut-away view; F. bursa complex, left lateral view; G. bursa complex, dorsal view; H. spermathecal com- plex, quasi-dorsal view; I. spermathecal complex, ventral view (muscle tissue of spermathecal complex removed). apical diverticula (Figs. 4B, 12A, 13H,I), with the for- the spermathecae and spermathecal duct, although mer very obvious, comparatively long, and slightly bul- sometimes long, were not observed to extend beyond bous, and the latter only occasionally slightly bulbous the basal arms of the subgenital plate (Fig. 3A–C,E), at its apex, but often resembling the spermathecal with spermathecal ducts typically compressed into duct (Fig. 4B, 12A, 13H,I), for which the spermathecae loosely tangled coils. The delimitation of the sperma- form the apex. The duct itself appears to vary in length thecal aperture seems clear here and is the slit con- to a degree (Fig. 12A, 13H,I) and is typically com- necting the spermathecal duct to the bursa copulatrix pressed into loosely tangled coils (Fig. 13H,I). Unlike (not labeled, but see Fig. 4B). in Melanoplus sanguinipes (Fabricius, 1798), M. rotun- The bursa complex (Fig. 13A,E) comprises two dipennis and all other examined Puer Group species subcomponents: the armor of bursa copulatrix do not have a U-shaped bend in the duct toward the (Figs. 4B, 12B–D,F,G, 13E–G) and the bursa copu- basal end (Pickford and Gillott, 1971). Furthermore, latrix (Figs. 4B, 12B–D,F,G, 13E–G), and has the for M. rotundipennis and other Puer Group members, overall appearance of a scorpion’s tail, with,

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 349 it is tough to the touch (of forceps), despite it not appearing to be heavily sclerotized as in other components, and it also does not possess the yel- lowish coloration often associated with sclerotiza- tion (Fig. 12F). The bursa copulatrix (Figs. 4B, 12B–D,F,G, 13E–G), conversely, does have the yel- lowish coloration (Fig. 12F), but is much weaker to the touch and only appears to be lightly sclero- tized, and to varying degrees among specimens. Overall, the bursa copulatrix is broadly tubular, but a portion just before its apex constricts into a narrower tube with its actual apex being quite bulbous (to varying degrees) and extending beyond the armor, sometimes quite extensively (Fig. 12B). The vulval sclerite (Figs. 4B, 12A–E,G,H, 13B- E) is found ventrally at the entrance to the bursa complex, also known as the vulva (not labeled, but Fig. 14. Melanoplus rotundipennis, interactive 3D-viewing see Fig. 12A–H), and often extending slightly window containing 3D-reconstructions and associated labels of beyond. Sclerotization levels for this component male and female components of genitalia and other parts of the vary between specimens (Figs. 12D,E,G,H, 13B) reproductive system of in copula specimens (male components are colored in maroon (Exterior) and shades of green, purple, and some may even be divided in two (Fig. 13C), and blue; female components are colored in white (Exterior) and but, in general, it is often plate-like, curves gently shades of yellow, orange, pink, and red). The 3D-PDF interface dorsally (lightly concave from an anterior view), is filled with useful abilities and we welcome exploration, but has two lateral arms that extend posteriorly, and here is a short guide to getting started quickly (the functionality sometimes has slight anterodorsal protrusions of this guide depends on your version of Adobe Reader): at the top of the viewing window is a toolbar with shortcuts, of which (Fig. 13C). Finally, the Comstock-Kellog glands the most useful is the one that resembles a phylogenetic tree (not pictured) do not appear to be as obvious as in called “Model Tree.” Clicking on this icon opens the Model Tree other Melanoplus species (Slifer and King, 1936; pane to the left of the viewing window and, from here, you can Slifer, 1940b), but are present in M. rotundipennis, check/uncheck boxes (clicking on “1”signsrevealsfulltree) associated with each component (name first, then body region, are of a typical shape, and almost translucent and then sex) as well as their associated labels (“LABELS-Ana- (made even fainter with KOH). tomical_Components,” naming scheme identical to components). In copula. External: During copulation, the These abilities are also present in the viewing window by right- apical third of the female’s subgenital plate is bent clicking, choosing “Part Options,” and then “Hide,” and this func- at an almost 908 angle ventrally, is wedged into tion can be applied to both components and labels. There are also 15 pre-set views (Left Lateral View is Home) that we think the male’s genital chamber, and is dominantly you may find interesting and these can be accessed from the resting atop his right paraproct and basal end of “View” dropdown menu of the viewing window toolbar, in a win- his right cercus, but is not quite touching his dow below the model tree pane, or by right-clicking and choosing folded-in supra-anal plate or furculae (Figs. 3C, “Views.” You may also change lighting schemes by clicking on the lamp icon (CAD Optimized Lights is default) in the viewing 10A,B). Additionally, the apical half of the female’s window toolbar. Finally, basic movements within the viewing egg guide is bent upwards and to the left (Fig. window are as follows: hold down left mouse button to rotate in 10A,B), most likely a result of encountering the any direction, scroll middle mouse button to zoom in and out, muscles that surround the male’s phallic complex. hold down right mouse button and move mouse forward and Finally, the male’s cerci are pressing slightly on back to more quickly zoom in and out (or hold Shift 1 left mouse button), and hold Ctrl 1 left mouse button to pan around. More the female’s abdomen toward the base of her later- movement abilities can be accessed from the far left arrowed al basivalvular sclerites (Fig. 1A–D). icon of the viewing window toolbar or by right-clicking and look- Internal: During copulation, the right lophus of ing in “Tools.” the male’s epiphallus is inserted for some distance into the female’s corresponding right lophus recep- tacle (Fig. 10B) while the majority of his aedeagus approximately, its apical fourth being curved back is inserted into her bursa complex, specifically on itself ventrally except in the 3D-reconstructions coming into direct contact with her bursa copula- (discussed later). Interestingly, this curvature was trix for some distance (Fig. 4C). The dorsal, con- observed to point left or right (Fig. 12B) depending cave surface of the female’s vulval sclerite is on the individual (and even within the same popu- entirely touching part of the basal, dorsal region lation) with no apparent pattern yet known. The of the male’s dorsal valves of aedeagus (Fig. 4C). armor of bursa copulatrix (Figs. 4B, 12B–D,F,G, Finally, the male’s spermatophore is emerging 13E–G) is an outer covering that surrounds the from between the apices of his dorsal and ventral majority of the inner bursa copulatrix, has a valves of aedeagus, so that the rest of its tube ribbed appearance (Fig. 12D,G) with flexible region is extended through the remainder of the membrane-like portions in-between. Furthermore, female’s bursa copulatrix. Here, the apex of the

Journal of Morphology 350 D.A. WOLLER AND H. SONG tube region has swollen to completely fill the into the more delicate areas of the male’s repro- interior of the bulbous apex of the bursa copula- ductive and digestive systems. trix, presumably because it is close to rupturing During observations of live copulation attempts (Fig. 4C). the cerci (Figs. 4A, 5A–E) were able to be flipped inwards and outwards, most likely due to muscu- DISCUSSION lar action assuming the four muscles mentioned by Snodgrass (1935) are present that attach the Functional Morphology cerci to both tergite 10 (Fig. 5C–E) and the supra- Unsurprisingly, but rarely demonstrated in anal plate. Although not observed directly, a possi- action, external and internal anatomical compo- ble reason for cerci inversion is to assist in scoop- nents of the male and female reproductive system, ing the phallic complex up and out of the genital mainly genitalic, in M. rotundipennis are involved chamber, with the possible assistance of the supra- in copulation, often appearing to possess multiple anal plate and paraprocts (Figs. 4A, 5A–E). In the functions. Herein, the probable functions of 45 of in copula 3D-reconstructions (Fig. 3A–E), the cerci the 58 named components will be discussed in are not inverted and are back in their original more detail as well as other items of interest, like positions, which must occur at some point during novel discoveries, starting, in general, with the the process of copulation, because the cerci have male (32 male components) and then moving to been observed to push against the female’s abdo- the female (13 female components; summarized in men, possibly for support during copulation and/or Table 1). Twelve female components were not to further prevent the female from leaving. Uvarov observed to be involved in copulation: tergite 10 (1966) and Chapman (2012) mention something (Fig. 11A,B), supra-anal plate (Fig. 11A,B), cerci similar occurring across Acrididae, but use the (Fig. 11A,B), paraprocts (Fig. 11A,B), median ovi- term “grip” instead, although it is unclear if this duct (not pictured), dorsal, inner, and ventral suggests the cerci are pushing against the female valves of ovipositor (Figs. 11A–D, 12A,B), apo- (as we observed) or are actually pulling her toward the male. Kyl (1938) too, says the cerci “grasp the demes of ovipositor (Fig. 12A,B), lateral basivalvu- abdomen of the female” in Melanoplus differentia- lar sclerites (Figs. 11B,C, 12A,B), anterior lis (Thomas, 1865), but then goes further by basivalvular sclerites (Fig. 12A,B), and Comstock- describing that the effect of this action pulls down Kellog glands (not pictured). Of these, the majority the egg guide, opening the genital chamber of the are most likely involved in laying eggs while female slightly. We know now that, more than others may be involved with structural support of likely, the epiphallus is the component responsible the abdomen, assist in waste excretion, and/or sen- for this in all grasshoppers, but Snodgrass (1935) sory reception (possibly even during copulation) seems to clarify the possible gripping role of the (Snodgrass, 1935; Eisner et al., 1966; Uvarov, cerci by noting that they “grasp the base of the 1966; Snodgrass, 1993; Stauffer and Whitman, subgenital plate of the female.” Boldyrev (1929) 1997). For the males, the lone exception was the goes further and actually calls cerci “organs of furculae (Figs. 4A, 5A–E), which were initially attachment,” specifically in reference to Locusta thought to be involved in copulation, but may not migratoria (Linnaeus, 1758), but also notes that be. Observations were based on correlative micros- their primary purpose is as “organs of orientation.” copy combining DSLR and SEM images with Furthermore, it appears there might be a mild micro-CT-based 3D-reconstructions, as well as invagination toward the basal end of the female’s observations of museum specimens and live speci- lateral basivalvular sclerites (Figs. 11B,C, 12A,B) mens, and inferences based on past publications that the apices of the cerci might be able to push on other species of Acrididae, some within the into slightly (Fig. 1A–D), but further investigation same genus. is needed to clarify this issue. Moreover, cerci in numerous insects have been Male: External and Internal Genitalia, and observed to be sensory structures, sensitive to Other Parts of the Reproductive System sounds, movement of air, and touch, primarily due External: All but one of the seven named exter- to the abundance of sensilla on them (Snodgrass, nal components that we observed appear to be 1935; Uvarov, 1966; Snodgrass, 1993; Gordh and involved in copulation. The primary probable func- Headrick, 2001; Chapman, 2012). So far, for the tion of the supra-anal plate, although, as in all Puer Group, the presence of such sensilla has yet other Acridoidea with eversible phallic complexes, to be confirmed, but the abundance of setae on the is to act as the protective dorsal surface (a “roof”) cerci of all species suggests it. Thus, it is for the genital chamber and its contents. When quite possible that if such sensilla are present, swung downwards, however, it may also serve as they might be used as contact receptors during an effective wall to ensure against the possibility copulation and/or may even have functions not of the female’s subgenital plate (Figs. 4B, 11B–H), involved in copulation. Moreover, grasshopper cer- or even debris, making its way further anterior ci, which are often species-specific (particularly in

Journal of Morphology TABLE 1. Summary of the probable functions of 45 (32 male, 13 female) of the 58 named genitalic and other reproductive system components/subcomponents involved in copu- lation in Melanoplus rotundipennis (Scudder, 1878), most of which have been reconstructed in 3D. For each function: 1 5 observed in this study and previous studies; 2 5 novel observation and/or hypothesis; 3 5 hypothesized based on our observations, and evidence or hypotheses from previous study(ies); 4 5 supported hypothesis from previous study(ies); 5 5 only observed in previous study(ies).

Sex Body region Component Subcomponent Functions during copulation Male External (6) tergite 10 Attachment point for furculae, supra-anal plate, cerci, and paraprocts1 supra-anal plate Closes off genital chamber at rest1; may protect anterior body regions during copulation3; may assist in phallic complex eversion3 cerci Push against female abdomen to act as supports during copulation and/or to assist in prevention of female leaving3; inversion ability may assist in phallic complex eversion3; act as sensory structures5; support female subgenital plate when pulled into genital chamber2 COPULATION DURING MORPHOLOGY FUNCTIONAL paraprocts Act as supports for female subgenital plate2; may assist in cerci inversion3; may assist in phallic complex eversion3 pallium Covers and protects aedeagus1 subgenital plate Stretches to allow for phallic complex eversion1,2 Male Internal (26) genital chamber Encloses phallic complex1 phallic complex ancorae Hook onto and pull down female subgenital plate to gain access to vulva1; near-by (epiphallus) sensory receptors may aid in this action2 anterior projections May serve as structural support and/or muscle attachment site3; scattered sensory receptors may aid in ancorae’s action of pulling down female subgenital plate2 posterior projections May serve as structural support and/or muscle attachment site3 bridge May serve as structural support (can flex) and/or muscle attachment site3 lateral plates May serve as structural support and/or muscle attachment site3 lophi Pushed into female lophi receptacles to keep her subgenital plate pulled down for aedeagus insertion through her vulva2,4 phallic complex apodemes of cingulum General structural support for phallic complex1; attachment points for muscles5; (ectophallus) leverage point for aedeagus3 zygoma Lower portion receives arch of aedeagus1; extended portion of upper region pro- vides limited articulation and structural support for aedeagus3 rami May act as supports and attachment points for sheath of aedeagus3; Able to be lat- erally compressed inwards, which may tighten sheath of aedeagus for reason(s) unknown2; role of sensory receptors on posterior apices currently unknown2 sheath of aedeagus Covered in minute spines that may act as female-stimulators or as grippers2 phallic complex apodemes of endophallus Attachment points for muscles1; contractions of muscles separate gonopore process- (endophallus) es for transfer of spermatophore and may aid in pumping it into female5 connective tissue of apo- Acts like spring-loaded bellows to bring apodemes back to original position after demes of endophallus contractions of muscles2 flexures Act as support and leverage points for the aedeagus1 articulations Act as support and leverage points for the aedeagus1

ora fMorphology of Journal gonopore processes Laterally separate to open gonopore during contraction of muscles attached to apo- demes of endophallus5 dorsal valves of aedeagus Assist in forming phallotreme for spermatophore formation1; external surface cov- ered in microscopic leaf-like projections that act as female-stimulators or grippers3 ventral valves of Assist in forming phallotreme for spermatophore formation1; assist in shaping aedeagus spermatophore and guiding it into female3 phallotreme Channel between aedeagal valves that shapes spermatophore’s tube region1 arch of aedeagus Provides limited articulation and structural support for aedeagus3 351 ora fMorphology of Journal SONG H. AND WOLLER D.A. 352

Table 1. (continued).

Sex Body region Component Subcomponent Functions during copulation greater reproductive ejaculatory duct 1st step of spermatophore creation: uses accessory gland secretions to construct system reservoir, then tube region, and then begins to fill with semen5 ejaculatory sac 2nd step of spermatophore creation: enlarges reservoir/tube region and continues to fill with semen5 gonopore 3rd step of spermatophore creation: when opened, allows for transfer of spermato- phore from ejaculatory sac to spermatophore sac5 spermatophore sac 4th step of spermatophore creation: via gonopore, receives spermatophore and sur- rounding muscles pump semen-filled tube region into phallotreme while reser- voir stays within5 greater reproductive system: spermatophore tube region Primary portion used to transfer semen to female1 reservoir In spermatophore sac, continually adds semen to tube region until empty1

Female External (3) subgenital plate Blocks male’s access to vulva1; apical third fits into male’s genital chamber to allow him greater access to vulva by widening her genital chamber2,4 lophi receptacles Receive male lophi, possibly only one per copulation event2,4 egg guide Blocks access to vulva1; guides egg into intervalvular space5; provides support for male phallic complex to push against2 Female Internal (10) genital chamber Encloses internal components1 spermathecal complex muscle tissue of sperma- May assist in moving sperm back toward eggs for fertilization3 thecal complex spermathecal duct Duct through which sperm moves toward diverticula and then back for egg fertilization5 spermathecal aperture May serve as transfer point for sperm from spermatophore’s tube region, swelling within apex of bursa copulatrix, to spermathecal duct3 spermathecal complex: spermathecae preapical diverticulum Sperm receptacle5 apical diverticulum Sperm receptacle5 bursa complex armor of bursa copulatrix During interactions with male aedeagus - strengthens bursa copulatrix and/or rib- bing allows for expansion of bursa copulatrix2 bursa copulatrix Receives male aedeagus1; apex may alter size to accommodate swelling apex of spermatophore’s tube region and may be rupture point for spermatophore2,3 ; may contain nerve endings for stimulation by male or lined with material for gripping by male3 vulval sclerite Connects vulva to bursa copulatrix3; reinforces entrance to bursa copulatrix2; sup- port plate for male aedeagus2 vulva Opening to all other internal components1; receives male aedeagus1 FUNCTIONAL MORPHOLOGY DURING COPULATION 353 Melanoplus) may be important for appropriate keep their original positions when the supra-anal sensory stimulation and recognition of conspecific plate (Figs. 4A, 5A–E) is swung downwards into males by the female, a hypothesis in need of the genital chamber (presumably via muscular experimental investigation (Eberhard, 1985). action along tergite 10; Fig. 3C). Numerous mem- Finally, the cerci (at least the one on the right in bers of the Puer Group possess species-specific fur- this case), in conjuction with the paraprocts, culae, but others lack them entirely, and the same appear to assist in supporting the posterior portion observations can be applied to other Acrididae spe- of the apical third of the female’s subgenital plate cies, further suggesting that they may not play a when it is bent downwards into the male’s genital role in copulation. chamber (Figs. 3C, 10A). Internal: All the named internal components The paraprocts (Figs. 4A, 5A–E) may have at that we observed appear to be involved in copula- least two probable functions. The first one might tion. The genital chamber encloses the phallic be to assist in flipping the cerci down into the gen- complex (Fig 6A) until the male is ready to ital chamber due to their membranous connection attempt copulation. Then, the external genitalic (Fig. 5A,B). When the supra-anal plate is swung components are pushed out of the way (presum- down into the genital chamber, it pushes the para- ably via muscular action), so the phallic complex procts downwards as well and brings the cerci can be partially everted dorsally by muscular with them. This proposed function, supported by action and the inflation of surrounding mem- experimentation with freshly killed specimens in branes. The mechanism behind such inflation is the lab using fine-tipped forceps, is less likely, if currently unknown, but may be due to an increase muscles turn out to be present at the base of the in hemolymph-related hydrostatic pressure caused cerci. When the supra-anal plate was pushed by muscular action (Whitman and Loher, 1984; inwards with forceps, the paraprocts were as well Lawry, 2006; Chapman, 2012). Following that, the and pulled the cerci with them. Evidence con- epiphallus (Figs. 6B,C, 7F,G) is pushed higher tained in the 3D-reconstructions (Figs. 3C, 10A) than the other phallic complex components suggests the other possible function of the para- because it is the most dorsal and not directly con- procts (at least the one on the right in this case) is nected to the rest. The ancorae (Figs. 4A, 6B,C, the same as the cerci: support for the posterior 7F,G) of the epiphallus are then used as hooks portion of the apical third of the female’s subgeni- (maybe individually or together) to catch the pos- tal plate when it is bent downwards into the terior margin of the female’s subgenital plate male’s genital chamber. (Figs. 4B, 11B–H). This action pulls the subgenital The obvious extension of the pallium (Fig. 5C– plate down into the anterior of the male’s genital E) seems clear: because the aedeagus is one of the chamber, thus opening access to the female’s vul- longest in the Puer Group and is strongly curved va. More than likely, the sensory receptors on the upwards, it needs an extended cover for protec- anterior projections (Figs. 6B, 7F,G) and near the tion. When preparing for copulation, the rotators ancorae (mentioned earlier and possibly seen here of phallus and retractors of pallium muscle, specif- for the first time in acridids) play roles in this ically the posterior portion (muscle 266, specifi- action. In the 3D-reconstructions, also note that cally 266B in Eades, 2000) pulls the pallium the epiphallus is not symmetrically aligned with downwards, exposing the aedeagus’ apex. The the female’s subgenital plate, but, rather, skewed probable function of the subgenital plate (Fig. slightly toward the left (Fig. 10B). This is most 5D,E) is not unique to this species and also seems likely due to the fact that even though a male is clear: it is able to stretch ventroposteriorly to mounted on top of a female, he must lower his some degree via its medial membranous connec- abdomen below and to either side of a female’s tion, which, most likely, assists in moving the abdomen, resulting in asymmetrical copulation phallic complex somewhat toward the posterior of (Fig. 2C), which, in the case of the male used for the genital chamber, so the supra-anal plate, para- the 3D-reconstructions, was from the left side of procts, and cerci are able to swing inwards. the female (Figs. 1A–D, 14). As mentioned, the function of the furculae (Figs. After the apical third of the female’s subgenital 4A, 5A–E) during copulation is still unknown at plate is pulled down into the anterior of the male’s this time, although it is quite possible that they do genital chamber, the entire phallic complex is not play a role. If they do, although, one sugges- swung dorsally in a quick motion, continuing in a tion is that they might possess sensory receptors counterclockwise arc anteriorly with the majority (as seen on other male components) that might of the aedeagus (Figs. 4A, 7B,C, 9A) entering the assist in assessing the location of a female’s abdo- female’s bursa complex through the vulva (not men during copulation. However, preliminary labeled, but see Fig. 12A–H). Such a motion means SEM observations of the furculae of different Puer that all phallic complex components (previously Group species did not reveal their presence. described in a state of repose; e.g., Fig. 6A) have Regardless, it can be unquestionably stated that rotated around 908 (or more), resulting in orienta- the furculae in M. rotundipennis stay rigid and tions that can now be described as ventral/

Journal of Morphology 354 D.A. WOLLER AND H. SONG anteriorly projecting (e.g., Fig. 4A). In these new the phallic complex for copulation and back again in copula positions (Fig. 3A–E, 4A,C, 5A,B, 10A,B) to its resting state (Eades, 2000). Additionally, the is how descriptions of the probable function(s) of ectophallus acts as general structural support for the remaining components of the phallic complex the entire complex and most likely as a leverage will be described herein; referenced figures should point for the aedeagus, particularly during its be carefully examined to maximize comprehension. insertion into the female’s bursa complex (and Once rotation of the phallic complex has occurred, extraction) and throughout spermatophore trans- and (presumably) the aedeagus enters the female’s fer during copulation. This leverage suggestion bursa complex, at least one lophus on the male’s stems from the fact that the arch of aedeagus is epiphallus is able to be inserted into a female’s inserted into the gap in the lower region of the corresponding lophus receptacle of the subgenital zygoma (Figs. 7B, 10C) and it is quite possible plate (Fig. 10B). There is little doubt that this pin- that the upper region portion is extended to pro- ning action ensures that the female’s subgenital vide limited articulation and structural support plate stays down to allow for continued access by (acting as a plate to push against) for the aedea- the male to the female’s internal genitalia. The gus during the copulation process. scales observed on the lophi (Fig. 9G) all but con- For Melanoplinae, Eades (2000) made no men- firm this hypothesis, especially in light of the fact tion of any muscles being attached directly to the that said scales are directed anteriorly (posteriorly rami (Figs. 4A, 6D,E, 7B,D, 9A) and there do not during copulation) and would, thus, act as minia- seem to be any in this species (Fig. 7A), so it is ture friction anchors as a lophus is pushed into a strongly possible there are none meaning that the receptacle and pulled down and back. If a female rami may just be supports and attachment points were found to possess similar structures that face for the sheath of aedeagus (Figs. 4A, 6D,E, 7B–D, in the opposite direction the friction effect should 9A). Note, although, that the rami appear to be be even greater. The other three subcomponents of laterally compressed inwards during copulation as the epiphallus: anterior projections, posterior pro- captured in the 3D-reconstructions (Fig. 10C,D); jections, bridge, and lateral plates (Figs. 6B,C, such action may tighten the sheath around the 7F,G) appear to have no specialized function (a dorsal valves of aedeagus for an unknown reason possible exception are the scattered sensory recep- or may just be a by-product of muscle movement tors on the anterior projections) other than 1) to elsewhere. Additionally, it is currently unknown potentially act as structural support during copu- what role is played by the sensory receptors lation, in some cases being able to flex to a degree observed on the posterior apices of the rami. The as evident by the shape of the bridge, and 2) to probable function of the minute spines seen on the possibly also serve as attachment sites for muscles sheath of aedeagus (Fig. 9F) that are potentially (e.g. lateral plates: Eades, 2000). new to science is also unknown at this time, but, As mentioned previously, a male asymmetrically given their placement in relation to the female’s copulates with a female, so, like the epiphallus, anatomy (Fig. 4C), it is feasible that they either the ectophallus (Figs. 6D,E, 8A) and endophallus play a role in stimulating the region surrounding (Figs. 6F,G, 8A) are also asymmetrically aligned the female’s vulva (Eberhard, 1985) or act as addi- with the female’s internal genitalia. The particular tional grippers (possibly for gripping the ventral male used for the 3D-reconstructions entered the side of her abdomen), further securing the male’s female from her left side (Figs. 1A–D, 14), so the aedeagus to the female, but they do not appear to phallic complex components are therefore skewed be sensory receptors for the male’s use. slightly at an angle to the right meaning that the The subcomponents of the endophallus (Figs. aedeagus also enters the female’s bursa complex 6F,G, 8A–E, 10E), and the associated subcompo- at a slight angle (Fig. 3D). The fact that the phal- nents of the greater reproductive system (Figs. lic complex is able to be twisted a fair range man- 6H–J, 7A), serve numerous probable functions ually with forceps supports the idea that during copulation. The apodemes of endophallus asymmetrical entry is possible, but we do not (Figs. 4A, 6F,G, 7B–E, 8B,D, 9A) provide attach- think that it has been noted or demonstrated ment points for a number of muscles with many before. The probable functional role of the ecto- probable functions related to spermatophore pro- phallus overall is not fully clear compared to the duction and insemination (Snodgrass, 1935; Rob- other two phallic complex components. However, erts, 1941; Gregory, 1965; Hartmann, 1970; the apodemes of cingulum (Figs. 4A, 6D,E, 7B,C,E, Pickford and Gillott, 1971; Whitman and Loher, 9A) have a muscle attached to them called the pro- 1984; Snodgrass, 1993; Eades, 2000), but the tractors of cingulum (only found in Melanoplus) majority of these muscles will not be discussed that, when combined with the retractors of cingu- here as it is outside of this study’s scope. Insemi- lum muscle (attached to the zygoma: Figs. 4A, nation results from sperm contained within the 6D,E, 7B,C, 9A; muscles 267XC and 278, respec- semen contained in a male’s spermatophore (Figs. tively, in Eades, 2000) would most likely assist, in 4A, 6H–J) and the process of its creation can differ conjunction with other related muscles, in rotating from species to species, so it will only be briefly

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 355 touched on here, based on synthesized evidence between these subcomponents (not labeled, but see from studies of other grasshoppers, including some Figs. 6G, 7B, 8B, 10E) possibly function, as other Melanoplus species (Boldyrev, 1929; Snodgrass, phallic complex components appear to, as support 1935; Kyl, 1938; Roberts, 1941; Gregory, 1965; and leverage points for the dorsal and ventral Uvarov, 1966; Hartmann, 1970; Pickford and Gil- valves, allowing the aedeagus greater movement lott, 1971; Whitman and Loher, 1984; Snodgrass, and positioning ability (Whitman and Loher, 1993; Eades, 2000; Chapman, 2012). The first step 1984). in the process usually begins shortly after a male Based on the 3D-reconstructions, the aedeagus begins copulating with a female, which signals the can be inserted into the bursa complex almost transfer of accessory gland secretions to move into entirely, its apex reaching just to the bend in the the ejaculatory duct (Figs. 4A, 6I,J, 7A). These bursa complex, with essentially only the portion secretions are used to construct the spermato- touching the sheath of aedeagus left outside the phore’s reservoir (Fig. 6H–J), followed by its tube female (Fig. 4C). The mid-basal region of the dor- region (Figs. 4A, 6H–J), and then, it begins to fill sal valves of aedeagus appear to rest, and most with semen. Next, the reservoir and tube region likely push downwards, on the female’s vulval move to the ejaculatory sac (Figs. 4A, 6I,J, 7A) sclerite (Fig. 4C). Such support may assist the where they are enlarged and continue to fill with male in guiding his aedeagus into the bursa com- semen. Then, the adductor of endophallic apo- plex and additionally offer purchase during inser- demes (muscle 283 in Eades (2000), extending tion and extraction as well as spermatophore between the two apodemes of endophallus) is transfer. The tiny, leaf-like posterior-oriented pro- contracted, laterally separating the gonopore pro- jections observed all over the exterior of the dorsal cesses (Figs. 6G, 7B,D, 8B, 10E) and opening valves (Fig. 9A,D,E) were seemingly observed for the gonopore (not pictured). This transfers the only the second time in at least Acridoidea and spermatophore from the ejaculatory sac to the the first in Acrididae, despite Eades (2000) com- spermatophore sac (Fig. 6I,J) in which the sperma- menting that it was yet unknown if such micro- tophore’s reservoir will remain to continually add structures were confined to the species they were semen to the spermatophore’s tube region during originally found on (T. eques in Whitman and copulation until empty. Finally, via rhythmic con- Loher, 1984) or if they might be found in other tractions of the muscles surrounding the sperma- species of Acridomorpha. The probable function of tophore sac, the semen-filled tube region is these projections is not entirely clear here, but two pumped through the phallotreme (which further possible explanations are as follows: 1) as stimula- shapes the spermatophore into its final tubular tors for the interior of the female’s bursa copula- form), out of the aedeagus (Fig. 4A), and into the trix (Figs. 4B, 12B–D,F,G, 13E–G) as suggested female (Fig. 4C), followed by the eventual ejection broadly by Eberhard (1985) and/or 2) as friction- of the spermatophore from the body of both sexes structures for improved gripping while inserted on the completion of sperm transferal. The exact into the bursa copulatrix as suggested for T. eques sources of these muscular contractions are still by Whitman and Loher (1984). being debated, but it has been suggested that one The only functions of the ventral valves (Figs. 4A, or more of the following muscles are responsible: 6F,G, 7B–E, 8B–E, 9B, 10E) hypothesized at this adductor of endophallic apodemes, outer compres- time for this species are assisting in the shaping sors of ejaculatory sac (muscle 2813 in Eades, and guiding of the spermatophore (Figs. 4A, 6H–J). 2000), and/or inner compressors of ejaculatory sac As the semi-gelatinous spermatophore is exuded (muscle 284 in Eades, 2000). through the phallotreme during copulation it The probable function of the connective tissue of appears to mostly rest between the ventral valves, apodemes of endophallus (Figs. 4A, 6F,G, 7C,E, eventually passing further through them dorsally 8D), seemingly not previously identified, named, nearer to their apex, emerging beyond both sets of or described (composition is reminiscent of the valves, and then into the apical remainder of the female’s egg guide: Figs. 4B, 11D–H) by other bursa copulatrix (Fig. 4C). The ventral valves of studies, is not fully clear at this time. More than aedeagus, it should be noted, are quite soft and flex- likely, although, based on examination with for- ible compared to the much more rigid, and seeming- ceps and consideration, it serves to snap the apo- ly inflexible, dorsal valves, so it would make sense if demes back into their starting positions after each the primary, if not only, probable functions of the contraction by the adductor muscles (muscle 283 ventral valves for this species are spermatophore in Eades, 2000) that cover most of it, so that fur- shaping and guidance. Interestingly, in T. eques, the ther musculature is not needed to expand the apo- dorsal valves only possess transverse ridges while it demes again; in this way, the connective tissue is the ventral valves that have their exterior cov- would act in a similar manner as a spring-loaded ered in microstructures that are described as bellows. The flexures (Figs. 6G, 7B, 8B, 9A, 10E), backward-pointing spines. These spines serve mul- the membranes connecting them to the valves of tiple functions during copulation in T. eques, includ- aedeagus (Fig. 8A,B), and the articulations ing anchoring the aedeagus to the basal region of

Journal of Morphology 356 D.A. WOLLER AND H. SONG the female’s spermathecal duct (a bursa copulatrix the sclerotization level lessens is structurally logi- is not present) and assisting in the removal of emp- cal, but also because the entire subgenital plate is ty spermatophores, so that more can be transferred quite long and would not fit into the male’s genital during a single copulation session (Whitman and chamber anyway. Loher, 1984). In contrast, in M. rotundipennis,itis In the 3D-reconstructions, the egg guide, which is the dorsal valves that contain the microstructures, apparently highly flexible, can be seen being bent which are far from being spines (we have described upwards and to the left near its middle (Fig. them as “leaf-like projections”) and are not as 10A,B). However, what the egg guide is pushing densely packed. Furthermore, it is doubtful that against is not shown and was not clearly observed, these microstructures could assist in spermato- but, based on its position, just anterior to the male’s phore removal because the dorsal valves are fused apodemes of cingulum (Fig. 4C), it can be inferred for almost their entire length as opposed to the to be the muscles that surround the ectophallus and independently articulating structures in T. eques. endophallus of the male’s phallic complex. The posi- Finally, the ventral valves appear to lack any sort of tion of the egg guide gives rise to three possibilities: ridges and only seem to be rugose (Fig. 9C), but, so 1) that it is simply random chance, 2) the egg guide far, only the apical third have been examined using provides some level of support for the male to push SEM. against because the subgenital plate is essentially As a whole, the phallic complex is consistent in hollow, or 3) a mixture of both meaning that the size and shape across the geographic range of the position can change, but the egg guide could still be species. Slight differences do exist, although, par- utilized by the male for support. ticularly in the lophi of epiphallus (Figs. 4A, 6B,C, Internal: The only named internal components 7F,G) and apodemes of cingulum (Figs. 4A, 6D,E, that we observed that appear to be involved in 7B,C,E, 9A; Squitier et al., 1998). Such differences copulation are the genital chamber, bursa complex in grasshoppers may be unique to an individual, (Fig. 13A,E), and vulval sclerite (Figs. 4B, 12A– population-specific [e.g., in Schistocerca lineata E,G,H, 13B–E). The genital chamber, other than Scudder, 1899 (Acrididae) (Song and Wenzel, containing the internal genitalia and other parts 2008)], or even simply developmental in nature of the greater reproductive system, seemingly [e.g., in S. americana (Drury, 1770) (Song, 2004)]. plays only a single, but important, role in copula- tion: without the subgenital plate being pulled Female: External and Internal Genitalia, and downwards by the male, thereby essentially wid- Other Parts of the Reproductive System ening the genital chamber dorsoventrally, he would not be able to access the vulva and begin External: The only named external components the copulation process. Collectively, the bursa com- that we observed that appear to be involved in copu- plex receives the male’s aedeagus (Figs. 4A, 7B,C, lation are the subgenital plate (Figs. 4B, 11B–H), 9A) and spermatophore (Fig. 4A, 6H–J), although its lophi receptacles (Fig. 11E–H), and, possibly, the neither directly contact the armor of bursa copula- attached egg guide [Figs. 4B, 11D–H; its primary trix (Figs. 4B, 12B–D,F,G, 13E–G). The ribbing of function appearing to be to guide an emerging egg the armor (Fig. 12D,G) probably serve one to two upwards into the intervalvular space (Snodgrass, functions, the first being reinforcement to 1935)]. The subgenital plate and its egg guide, in strengthen the bursa copulatrix (Figs. 4B, 12B– their normal state of repose, block entry to the vul- D,F,G, 13E–G) from either strong movements of va, which is centered just beneath the base of the the aedeagus during insertion and extraction as ventral valves of ovipositor (Figs. 11A–D, 12A,B). well as spermatophore transfer, or contact with The egg guide, at rest, passes dorsally between the the leaf-like projections that cover the male’s dor- ventral valves (Fig. 11D). When the male’s ancorae sal valves of aedeagus (Fig. 9A,D,E). The second (Figs. 4A, 6B,C, 7F,G) pull the apical third of the possible function could be expansion in that the female’s subgenital plate and egg guide downwards ribs may allow for the flexible membrane-like por- into his genital chamber, her vulva is exposed tions between them to stretch, akin to an accordi- through which the male is able to insert his aedea- on, in order for the bursa copulatrix to better gus into the female’s bursa complex (Fig. 4C). The accommodate aedeagi, perhaps of varying sizes, lophi receptacles then receive at least one lophus and their movements (and possibly even those of from the male’s epiphallus per copulation event (in the spermatophore). this case, the right lophus: Fig. 10B), which most The bursa copulatrix (Figs. 4B, 12B–D,F,G, likely acts as a friction anchor to keep the subgeni- 13E–G) appears to function as the primary recep- tal plate and egg guide from being retracted and, tacle for the male’s aedeagus (Figs. 4A, 7B,C, 9A) thus, close the vulva. Note again that the heavier and may be lined with nerve endings for the possi- sclerotization of the apical third of the subgenital ble purpose of stimulation (Eberhard, 1985) by the plate (Fig. 11G,H) is also the portion that is bent leaf-like projections on the aedeagus (Fig. downwards at an approximately 908 angle (Figs. 9A,D,E). Another possibility is that the bursa cop- 4B, 11E,F). The fact that a bend takes place where ulatrix is lined with rough material, possibly the

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 357 afore-mentioned light sclerotization (Fig. 12F), Before the 3D-reconstructions were completed, it thatthemaleisabletogripontoviafriction.The seemed apparent that the length and curvature of bulbousapexappearstobeabletoshiftinsize the aedeagus had evolved to fit better into the sim- (Fig. 12B) and may do so to accommodate the ilarly long and curved bursa complex (the fact that increase in size of the apex of the male’s sperma- it naturally bends left or right may not be a factor tophore (Figs. 4A,B, 6I,J) as semen is continually as it appears to be flexible: Fig. 12B). In fact, simi- added from the spermatophore’s reservoir into its larly matched structures between the sexes also tube region (Figs. 4A, 6H–J). Presumably, the appear to exist in many of the Puer Group species apex of the bursa copulatrix continues to expand dissected and examined, but all of these situations until the spermatophore’s apex ruptures (Fig. require much further investigation. The final 3D- 6H), most likely sending semen into the sperma- reconstructions of M. rotundipennis, although, thecal duct (Figs. 4B, 12A, 13H,I) through the while not necessarily disputing the idea of corre- spermathecal aperture where the sperm would sponding genitalic structures, do complicate mat- make their way toward the spermathecae (preapi- ters. This is because it appears that even if the cal and apical diverticula) (Figs. 4B, 12A, 13H,I). “tail” of the bursa complex was bent in the resting In addition to the observations of the 3D- ventral position the aedeagus would barely curve reconstructions, this hypothesis is based on evi- into it anyway since almost the entirety of its dence from previous studies on other species, two length has already been inserted (Fig. 4C). The also belonging to Melanoplus, in which a sperma- spermatophore, in the 3D-reconstructions, is the tophore (and in the non-Melanoplus species, the structure that moves the rest of the way through aedeagus) was introduced directly into the the bursa complex and into the apex of the bursa female’s spermathecal duct where it ruptured copulatrix (Fig. 4C); it seems possible the sperma- either further in or at the first narrow U-shaped tophore would do this regardless of “tail” orienta- bend, a structurally limiting situation similar to tion as it is essentially a tube being pushed what we have observed in M. rotundipennis (Kyl, through a slightly larger tube. Therefore, a reason 1938; Gregory, 1965; Pickford and Gillott, 1971; for the ventroposteriorly curving nature of the Whitman and Loher, 1984; Lay et al., 1999). The complex is still unclear, but because at least muscle tissue of spermathecal complex (partially population-level variation is known to exist (Squit- seen in Figs. 12A, 13H) is presumably responsible ier et al., 1998), it is plausible that variation in for moving the sperm back toward the eggs for the length of aedeagi also exists. Another possible the purpose of fertilization (Snodgrass, 1993). The reason for the length of the bursa complex is to vulval sclerite (Figs. 4B, 12A–E,G,H, 13B–E) may simply give the spermatophore more room to enter be an uncommon component in grasshopper geni- as it does not seem that the spermatophore would talia because it has only seemingly been identi- be able to move beyond the spermathecal aperture fied once previously in a distantly related genus, once its apex becomes engorged with semen. Praxibulus Bolıvar, 1906 (“dorsal sclerite of bursa As for why the 3D-reconstructions and the other copulatrix” in Key, 1989), and is only found in a imaging methods differ, we offer two plausible few other Puer Group species. In addition to explanations: 1) the freezing method used to cap- seemingly serving as reinforcement to the ture copulation also caused the “tail” and sperma- entrance to the bursa copulatrix and as the con- tophore to shift. We know this occurred with the nector between the latter and the vulva, this female’s valves of ovipositor (Fig. 11B) because the sclerite possibly also functions as a support plate dorsoventral widening observed here (Fig. 1A–D) for the male’s aedeagus to slide along and push occurs on all female specimens frozen at 2808C. against during insertion and extraction as well as Normally, based on multiple observations of lab spermatophore transfer. populations, the valves are kept in a “closed” state On the whole, the 3D-reconstructions of all com- during copulation and such forced widening may ponents of both sexes reflect the results gained also have affected musculature. This may also using DSLR and SEM imaging methods with one have shifted other linked components, although difference for an internal female component: the nothing other than the “tail” was noticed. 2) Pres- “tail” of the bursa complex (so-called here because sure from the inserted aedeagus and/or the of its overall resemblance to a scorpion’s tail as extruding spermatophore may have shifted the noted earlier) is bent ventrally at rest (e.g., Fig. “tail’s” position, meaning that the movement of 12B), but is bent dorsally when the aedeagus has this component is entirely natural. Clearly, as been inserted and the spermatophore is being with many other discussed items, this situation transferred (e.g., Fig. 4C). We know the entire bur- warrants further investigation. sa complex has not been rotated for two reasons: 1) the vulval sclerite (e.g., Figs. 12B, 13E) is ori- ACKNOWLEDGMENTS ented correctly and 2) the membranes anchoring the base of the bursa complex to the body (Fig. Thirty-one individuals and groups across the U.S. 12A) would not allow for full rotation, anyway. and the world made this study possible and we

Journal of Morphology 358 D.A. WOLLER AND H. SONG wish to thank them for their contributions: first and Breeschoten T, Clark DR, Schilthuizen M. 2013. Evolutionary foremost, for facilitating micro-CT scanning, photo- patterns of asymmetric genitalia in the beetle tribe Cycloce- phalini (Coleoptera: Scarabaeidae: Dynastinae). Contrib Zoo graphing the micro-CT sample, assisting with 82:95–106. Amira comprehension, and suggesting manuscript Briceno~ RD, Eberhard WG. 2009. Experimental demonstration improvements: B. Wipfler, Jena Institut fur€ Spe- of possible cryptic female choice on male tsetse fly genitalia. zielle Zoologie und Evolutionsbiologie mit Phyleti- J Insect Physiol 55:989–996. Briceno~ RD, Eberhard WG, Chinea-Cano E, Wegrzynek D, dos schem Museum, Friedrich-Schiller University, Santos Rolo T. 2016. Species-specific differences in the behav- Germany; micro-CT scanning assistance: S. Ge, ior of male tsetse fly genitalia hidden in the female during Institute of Zoology, Chinese Academy of Sciences, copulation. Ethol Ecol Evol 28:53–76. Beijing; critical point drying: S.M. Fullerton (1940– Caplan J, Niethammer M, Taylor IIRM, Czymmek KJ. 2011. 2014) and S.L. Kelly (who also helped collect speci- The power of correlative microscopy: Multi-modal, multi- scale, multi-dimensional. Curr Opin Struct Biol 21:686–693. mens), Department of Biology, University of Central Chapman RF. 2012. The Insects: Structure and Function, 5th Florida (UCF); SEM use, training, and scheduling: ed. New York: Cambridge University Press. p 959. Y. Sohn, K. Scammon, and K. Glidewell, Materials Cigliano MM, Braun H, Eades DC, Otte D. Orthoptera Species CharacterizationFacility,AMPAC,UCF;specimen File. Version 5.0/5.0. [11-5-2016]. Available at: http://Orthop- tera.SpeciesFile.org loans: M.F. O’Brien, Collections Manager, UMMZ’s Dirsh VM. 1956. The phallic complex in Acridoidea (Orthop- Insect Division and P. Skelley, FSCA Entomology tera) in relation to taxonomy. Trans R Ent Soc London 108: Section Administrator; field assistants (UCF under- 223–270. graduate/graduate students, friends, and family Dirsh VM. 1965. The African Genera of Acridoidea. New York: Cambridge University Press. 594 p. members) who collected specimens via Florida Dirsh VM. 1973. Genital organs in Acridomorphoidea (Insecta) Department of Environmental Protection, Division as taxonomic character. J Zool Syst Evol Res 11:133–154. of Recreation and Parks, Florida Park Service Dougherty LR, Rahman IA, Burdfield-Steel ER, Greenway Research/Collecting Permits #10031210 and EVG, Shuker DM. 2015. Experimental reduction of intromit- tent organ length reduces male reproductive success in a bug. 05281410 (chronological order): S. Gotham, C. Gale, Proc R Soc Lond, Ser B: Biol Sci 282:1–8. E. Kosnicki, E. Kerr-Woller (and for continued Eades DC. 1961. The terminology of phallic structures in the encouragement), P.H. and K.L. Woller, I.J. Stout, Cyrtacanthacridinae (Orthoptera, Acrididae). Entomol News and A.B. Orfinger; guides to the micro-CT popula- 72:141–149. tion:B.GochnourandH.Stephens;micro-CTdata Eades DC. 2000. Evolutionary relationships of phallic structures of Acridomorpha (Orthoptera). J Orthoptera Res 9:181–210. assistance and software training: T. Porri, Research Eberhard WG. 1985. Sexual Selection and Animal Genitalia. Associate with the Cornell Biotechnology Resource Massachusetts: Harvard University Press. 256 p. Center Imaging Facility; Amira Customer Support Eberhard WG. 2010. Rapid divergent evolution of genitalia: Specialists, especially K. Tinoco; PDF3D’s technical Theory and data updated. In: Leonard J, Cordoba-Aguilar A, editors. The Evolution of Primary Sexual Characters in Ani- support team; 3D PDF beta tester: M.L. Ferro; mals. Oxford, UK: Oxford University Press. pp 40–78. manuscript improvements: D.C. Eades, J.G. Hill, M. Eberhard WG, Ramirez N. 2004. Functional morphology of the Schmitt, and two anonymous reviewers. male genitalia of four species of Drosophila: Failure to con- firm both lock and key and male-female conflict predictions. Ann Entomol Soc Am 97:1007–1017. LITERATURE CITED Eisner T, Shepherd J, Happ GM. 1966. Tanning of grasshopper eggs by an exocrine secretion. Science 152:95–97. Ah-King M, Barron AB, Herberstein ME. 2014. Genital evolution: Faulwetter S, Vasileiadou A, Kouratoras M, Dailianis T, Why are females still understudied? PLoS Biol 12:1–7. Arvanitidis C. 2013. Micro-computed tomography: Introduc- Ahmed I, Gillott C. 1982. The spermatheca of Melanoplus san- ing new dimensions to taxonomy. ZooKeys 263:1–45. guinipes (Fabr.). I. Morphology, histology, and histochemistry. Federov SM. 1927. Studies in the copulation and oviposition of Int J Inver Rep 4:281–295. Anacridium aegyptium, L. (Orthoptera, Acrididae). Trans Ent Amed egnato C. 1976. Structure et evolution des genitalia chez Soc Lond 75:53–61. les Acrididae et familles apparentees. Acrida 5:1–15. Gordh G, Headrick D. 2001. A Dictionary of Entomology. Wal- Ander K. 1970. Orthoptera saltatoria. In: Tuxen SL, editor. lingford, UK & New York: CABI Publishing. pp 1–1032. Taxonomist’s Glossary of Genitalia in Insects, 2nd ed. Copen- Gosalvez J, Kjelland ME, Lopez-Fern andez C. 2010. Sperm hagen, Denmark: J. Jørgenson & Co. pp. 61–71. DNA in grasshoppers: Structural features and fertility impli- Arnqvist G. 1997. The evolution of animal genitalia: Distin- cations. J Orthoptera Res 19:1–10. guishing between hypotheses by single species studies. Biol J GrecoM,BellD,WoolnoughL,LaycockS,CorpsN,MortimoreD, Linn Soc 60:365–379. Hudson D. 2014. 3-D visualisation, printing, and volume deter- Arnqvist G, Rowe L. 2002. Correlated Evolution of male and mination oF the tracheal respiratory system in the adult desert female morphologies in water striders. Evolution 56:936–947. locust, Schistocerca gregaria. Entomol Exp Appl 152:42–51. Arnqvist G, Edvardsson M, Friberg U, Nilsson T. 2000. Sexual Gregory GE. 1965. The formation and fate of the spermato- conflict promotes speciation in insects. Proc Natl Acad Sci phore in the African migratory locust, Locusta migratoria USA 97:10460–10464. migratorioides Reiche and Fairmaire. Trans R Ent Soc Lon- Beutel RG, Friedrich F, Ge S-Q, Yang X-K. 2014. Insect Mor- don 117:33–66. phology and Phylogeny. Berlin, Germany & Boston: Walter de Grieshop K, Polak M. 2012. The precopulatory function of male Gruyter GmbH. 516 p. genital spines in Drosophila ananassae [Doleschall] (Diptera: Bland RG. 1987. Mating behavior of the grasshopper Melanoplus Drosophilidae) revealed by laser surgery. Evolution 66: tequestae (Orthoptera: Acrididae). Fla. Entomol 70:483–487. 2637–2645. Boldyrev BT. 1929. Spermatophore fertilization in the Migrato- Hartmann R. 1970. Experimental and histological studies on the ry Locust (Locusta migratoria L.). Izv prikl Ent (Rep App spermatophore formation of the grasshopper Gomphocerus Ent) 4:189–218. rufus L. (Orthoptera, Acrididae). Z Morph Tiere 68:140–176.

Journal of Morphology FUNCTIONAL MORPHOLOGY DURING COPULATION 359 Holwell GI, Kazakova O, Evans F, O’Hanlon JC, Barry KL. Schmitt M, Uhl G. 2015. Functional morphology of the copula- 2015. The functional significance of chiral genitalia: Patterns tory organs of a reed beetle and a shining leaf beetle (Coleop- of asymmetry, functional morphology and mating success in tera: Chrysomelidae: Donaciinae, Criocerinae) using X-ray the praying mantis Ciulfina baldersoni. PLoS One 10:1–14. micro-computed tomography. In: Jolivet P., Santiago-Blay J., Hornschemeyer€ T, Beutel RG, Pasop F. 2002. Head Structures Schmitt M. (Eds) Research on Chrysomelidae 5. ZooKeys 547: of Priacma serrata Leconte (Coleptera, Archostemata) 193–203. Inferred From X-ray Tomography. J Morphol 252:298–314. Simmons LW. 2013. Sexual selection and genitalia evolution. Hosken DJ, Stockley P. 2004. Sexual selection and genitalia Aust J Entomol 53:1–17. evolution. Trends Ecol Evol 19:87–93. Simonsen TJ, Kitching IJ. 2014. Virtual dissections through Hotzy C, Polak M, Ro JL, Arnqvist G. 2012. Phenotypic engi- micro-CT scanning: A method for non-destructive genitalia neering unveils the function of genital morphology. Curr Biol ‘dissections’ of valuable Lepidoptera material. Syst Entomol 22:2258–2261. 39:606–618. Hubbell TH. 1932. A revision of the Puer group of the North Slifer EH. 1939. The internal genitalia of female Acridinae, American genus Melanoplus, with remarks on the taxonomic Oedipodinae and Pauliniinae (Orthoptera, Acrididae). value of the concealed male genitalia in the Cyrtacanthacridi- J Morphol 65:437–469. nae (Orthoptera, Acrididae). Misc Publ Mus Zool, Univ Mich Slifer EH. 1940a. The internal genitalia of female Thrinchinae, 23:1–64. Batrachotetriginae, Pamphaginae and Pyrgomorphinae Jongerius SR, Lentink D. 2010. Structural analysis of a dragon- (Orthoptera, Acrididae). J Morphol 66:175–195. fly wing. Exp Mech 50:1323–1334. Slifer EH. 1940b. The internal genitalia of female Ommexechi- Kelly DA, Moore BC. 2016. The morphological diversity of nae and Cyrthacanthacridinae (Orthoptera: Acrididae). intromittent organs. Integr Comp Biol 56:630–634. J Morphol 67:199–239. Key KHL. 1989. Revision of the genus Praxibulus (Orthoptera: Slifer EH. 1943. The internal genitalia of some previously Acrididae). Invertebr Taxon 3:1–121. unstudied species of female Acrididae (Orthoptera). Kyl G. 1938. A study of copulation and the formation of sper- J Morphol 72:225–237. matophores in Melanoplus differentialis. Proc Iowa Acad Sci Slifer EH, King RL. 1936. An internal structure in the Cyrta- 45:299–308. canthacrinæ (Orthoptera, Acrididæ) of possible taxonomic val- Lawry JV. 2006. The Incredible Shrinking Bee: Insects as Mod- ue. J N Y Entomol Soc 44:345–348. els for Microelectromechanical Devices. London: Imperial Col- Snodgrass RE. 1935. The abdominal mechanisms of a grasshop- lege Press. per. Smithson Misc Collect 94:1–89. Lay M, Zissler D, Hartmann R. 1999. Ultrastructural and func- Snodgrass RE. 1993. Principles of Insect Morphology. New tional aspects of the spermatheca of the African Migratory York: Cornell University Press. 667 p. Locust Locusta migratoria migratorioides (Reiche and Fairm- Song H. 2004. Post-adult emergence development of genitalic aire) (Orthoptera: Acrididae). Int J Insect Morphol Embryol structures in Schistocerca Sta˚l and Locusta L. (Orthoptera: 28:349–361. Acrididae). Proc Entomol Soc Wash 106:181–191. Mattei AL, Riccio ML, Avila FW, Wolfner MF. 2015. Integrated Song H, Bucheli SR. 2010. Comparison of phylogenetic signal 3D view of postmating responses by the Drosophila mela- between male genitalia and non-genital characters in insect nogaster female reproductive tract, obtained by micro- systematics. Cladistics 26:23–35. computed tomography scanning. Proc Natl Acad Sci USA 112: Song H, Wenzel JW. 2008. Mosaic pattern of genital divergence 8475–8480. in three populations of Schistocerca lineata Scudder, 1899 Michalik P, Piacentini L, Lipke E, Ramırez MJ. 2013. The enig- (Orthoptera: Acrididae: Cyrtacanthacridinae). Biol J Linn Soc matic Otway odd-clawed spider (Progradungula otwayensis 94:289–301. Milledge, 1997, Gradungulidae, Araneae): Natural history, Song H, Marino-P~ erez R. 2013. Re-evaluation of taxonomic util- first description of the female and micro-computed tomogra- ity of male phallic complex in higher-level classification of phy of the male palpal organ. ZooKeys 335:101–112. Acridomorpha (Orthoptera: Caelifera). Insect Syst Evol 44: O’Leary MA, Kaufman SG. 2012. MorphoBank 3.0: Web appli- 241–260. cation for morphological phylogenetics and taxonomy. http:// Squitier JM, Deyrup M, Capinera JL. 1998. A new species of www.morphobank.org. Melanoplus (Orthoptera: Acrididae) from an isolated upland Otte D. 1970. A comparative study of communicative behavior in Peninsular Florida Fla. Entomol 81:451–460. in grasshoppers. Misc Publ Mus Zool, Univ Mich 141:1–169. Stauffer TW, Whitman DW. 1997. Grasshopper oviposition. In: Pickford R, Gillott C. 1971. Insemination in the migratory Gangwere SK, Muralirangan MC, Muralirangan M, editors. grasshopper, Melanoplus sanguinipes (Fabr.). Can J Zool/Rev The Bionomics of Grasshoppers, Katydids, and Their Can Zool 49:1583–1588. Kin. Wallingford, UK and New York: CAB International. pp. Randell RL. 1963. On the presence of concealed genitalic struc- 231–280. tures in female Caelifera (Insecta; Orthoptera). Trans Am Uvarov B. 1966. Grasshoppers and Locusts, Vol. 1. Cambridge, Entomol Soc 88:247–260. UK: The Syndics of the Cambridge University Press. 481 p. Rhebergen FT, Courtier-Orgogozo V, Dumont J, Schilthuizen M, Whitman DW, Loher W. 1984. Morphology of male sex organs Lang M. 2016. Drosophila pachea asymmetric lobes are part and insemination in the grasshopper Taeniopoda eques (Bur- of a grasping device and stabilize onesided mating. BMC Evol meister). J Morphol 179:1–12. Biol 16:1–24. Wipfler B, Machida R, Muller€ B, Beutel RG. 2011. On the head Richmond MP, Johnson S, Markow TA. 2012. Evolution of morphology of Grylloblattodea (Insecta) and the systematic reproductive morphology among recently diverged taxa in the position of the order, with a new nomenclature for the head Drosophila mojavensis species cluster. Ecol Evol 2:397–408. muscles of Dicondylia. Syst Entomol 36:241–266. Ritchie MG. 2007. Sexual selection and speciation. Annu Rev Wojcieszek JM, Austin P, Harvey MS, Simmons LW. 2012. Ecol, Evol Syst 38:79–102. Micro-CT scanning provides insight into the functional mor- Roberts HR. 1941. A comparative study of the subfamilies of phology of millipede genitalia. J Zool 287:91–95. the Acrididae (Orthoptera) primarily on the basis of their Wulff NC, Lehmann AW, Hipsley CA, Lehmann GUC. 2015. phallic structures. Proc Acad Nat Sci Phila 93:201–246. Copulatory courtship by bushcricket genital titillators Rowe L, Arnqvist G. 2011. Sexual selection and the evolution of revealed by functional morphology, mCT scanning for 3D genital shape and complexity in water striders. Evolution 66: reconstruction and female sense structures. Arthropod Struct 40–54. Dev 44:388–397.

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