Electronic Supplementary Material
Material and Methods
Histological examination of seminal vesicles and spermatheca.
Mature males and mated queens were killed with chloroform and their abdomen was
dissected. The seminal vesicles (sometimes called vas deferens or accessory testes) and
the spermatheca were isolated and fixed for 20 min. at room temperature in formalin 10%
diluted in PBS (NaCl 145 mM, NaH2PO4/Na2HPO4 10 mM, pH 7.2). After dehydration with
ethanol 95% and butanol, tissues were embedded in paraffin. Sections (3µm) were cut with a
conventional microtome (Leica Microsystems) and placed onto Superfrost slides (Labonord).
Before staining, the slides were deparaffinized and rehydrated.
Fixed seminal vesicle and spermatheca samples were stained following a Masson’s
trichrome stain or a periodic acid-Schiff (PAS) stain according to Gabe [1]. The slides were
examined with a Leica DM2000 equipped with a camera (Leica Microsystems).
Supplementary Figures
Number of bundles
50
40
30
20
10
0
30
45
60
75
90
105
120
135
150
165
Estimated number of sperm per bundle
Supplementary Figure S1. Distribution of the number of spermatozoa per bundle.
The number of sperm cells per bundle was estimated from Scanning Electron Microscopy
(SEM) images. See Methods in the main text.
A
B
Supplementary Figure S2. Microscope pictures of sperm bundles in the seminal
vesicle of C. savignyi males.
A. Seminal vesicle stained with Masson's trichrome. Sperm bundles are present in the
seminal vesicle of mature males. Flagella are stained in red and sperm heads appear in dark
blue. The bundle cap is visible in turquoise. Scale bar : 10µm.
B. Seminal vesicle stained with periodic-acid Schiff's. Flagella and bundle caps are stained in
rose; spermatozoa nuclei appear in dark blue. Scale bar : 10µm.
Supplementary Movie 1. Sperm bundles swimming in in vitro fertilization medium.
Sperm flagella beat synchronously to propel bundles forward in a helical pattern.
References
1. Gabe N. 1968 Techniques Histologiques (Paris: Masson et Cie).