Spermathecal variation in temperate Opiliones

Abstract

Most arachnid fertilization occurs internally, allowing for a variety of post-copulatory mechanisms to take place. Females are expected to exert some level of control over sperm fate when 1) the point of gametic fusion is particularly distant from the point of oogenesis, 2) the time of syngamy is significantly later than the time of mating, 3) sperm are non-motile, and/or 4) the morphology of females allows for selective containment of sperm. Many of these conditions are met in Opiliones (a.k.a. "harvesters," "harvestmen," or "daddy-longlegs"), where we have evidence of sexual antagonism, multiple mating, and delayed oviposition for a number of species. We used confocal laser scanning microscopy to capture and analyze images of harvester spermathecae, structures within the genitalia of female arthropods that store and maintain sperm after copulation. Spermathecal morphology may have critical function in controlling seminal movement. We anticipated that species with previously identified traits associated with sexual antagonism would also have thicker and/or relatively more complex spermathecae. We examined spermathecal morphology in thirteen species of Leiobunum and one species of Hadrobunus, which were collected from North America and Japan. Our results show that eight species had structures consisting of a single chamber with no or partial invagination, and the remainder had multiple cuticular invaginations producing 2-3 lumina within the spermathecae. Using phylogenetic multivariate comparative methods, we estimated a trend towards cross-correlation between conflict and spermathecal traits. Some, but not all, of the species with thicker, more complex spermathecae had morphological traits associated with sexual conflict (larger body size, thicker genital muscle). In conclusion, we discuss methods to elucidate spermathecal mechanism and sperm precedence in these species. Confocal microscopy allowed us to visualize internal structures difficult to interpret with two-dimensional brightfield microscopy, a technique that could be applied to the characterization of internal reproductive structures in other arthropods.

Keywords: Opiliones; Spermathecae; phylogenetic comparative methods; sexual conflict.

Fig. 1

Representative images of an Opiliones ovipositor and spermathecae on opposing ends of the spermathecal external wall thickness scale. (a) Maximum intensity projection of distal H. maculosus ovipositor showing location of paired spermathecae, vaginal opening, and sensilla; box around left spermatheca corresponds to high magnification image (b). (b) Single transverse optical section of H. maculosus spermatheca that represents a relatively thin spermathecal wall, posterior thickness measurements represented with dashed lines. (c) Single transverse optical section of L. formosum spermatheca that represents a relatively thick spermathecal wall, posterior thickness measurements represented with dashed lines. Anterior facing up, scale bar = 15 um.

Fig. 2

Representative images of spermatheca along the internal complexity scale, with lumina (L) and bifurcations (BF) labeled for complexity scale classification. The 5 um thick transverse, sagittal, and coronal sections of species that represent Levels 0, 1, 2, and 3 of spermathecal complexity, respectively, (a) L. euserratipalpe; (b) L. formosum; (c) L. bracchiolum; (d) L. verrucosum. Anterior facing up, scale bars = 20 um.

Fig. 3

DAPI-staining reveals the location of sperm within a L. formosum spermatheca. (a) Maximum intensity projection of select z-stack slices showing sperm (DAPI/blue channel) inside of spermatheca (autofluorescence/green channel); (b) surface rendering of green channel with dorsal slices showing the transverse spermathecal lumen; (c) surface rendering of blue channel showing the location of sperm cells throughout the spermatheca. Anterior facing up, scale bar = 20 um.

Fig. 4

Graphs of (a) spermathecal trait phylogenetic principal component 1 regressed against male conflict trait phylogenetic principal component 1 in Japanese species; (b) phylogenetic canonical correlation axes of spermathecal traits regressed against male and female conflict traits in North American species; (c) regression of log-transformed, size-corrected spermathecal thickness by conflict canonical axis 1, separated for species with and without female pregenital barrier. In (b) and (c), open circles indicate species where females have pre-genital chamber barriers, and closed circles indicate species without female pre-genital barriers. (d) Simplified phylogeny of study species after Burns and Tsurusaki (2016), Burns and Shultz (2015), and Hedin et al. (2012). Facultatively parthenogenetic taxa are indicated within shaded box. Numbering within tip labels signifies our spermathecal complexity ranking for the species, while open circle labeling indicates species where females have pre-genital chamber barriers, and closed circles indicate species without female pre-genital barriers.