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Behavioral and spermatogenic hybrid male breakdown in Nasonia.

Clark ME, O'Hara FP, Chawla A, Werren JH - Heredity (Edinb) (2010)

Bottom Line: Several reproductive barriers exist within the Nasonia species complex, including allopatry, premating behavioral isolation, postzygotic inviability and Wolbachia-induced cytoplasmic incompatibility.Hybrid males produced in crosses between N. vitripennis and N. giraulti courted females at a reduced frequency (23-69%), compared with wild-type N. vitripennis and N. giraulti males (>93%).A strong effect of cytoplasmic (mitochondrial) background was also found in N. vitripennis and N. giraulti crosses; F2 hybrids with giraulti cytoplasm showing reduced ability at most stages of courtship.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA. mclark11@mail.rochester.edu

ABSTRACT
Several reproductive barriers exist within the Nasonia species complex, including allopatry, premating behavioral isolation, postzygotic inviability and Wolbachia-induced cytoplasmic incompatibility. Here we show that hybrid males suffer two additional reproductive disadvantages, an inability to properly court females and decreased sperm production. Hybrid behavioral sterility, characterized by a reduced ability of hybrids to perform necessary courtship behaviors, occurs in hybrids between two species of Nasonia. Hybrid males produced in crosses between N. vitripennis and N. giraulti courted females at a reduced frequency (23-69%), compared with wild-type N. vitripennis and N. giraulti males (>93%). Reduced courtship frequency was not a simple function of inactivity among hybrids. A strong effect of cytoplasmic (mitochondrial) background was also found in N. vitripennis and N. giraulti crosses; F2 hybrids with giraulti cytoplasm showing reduced ability at most stages of courtship. Hybrids produced between a younger species pair, N. giraulti and N. longicornis, were behaviorally fertile. All males possessed motile sperm, but sperm production is greatly reduced in hybrids between the older species pair, N. vitripennis and N. giraulti. This effect on hybrid males, lowered sperm counts rather than nonfunctional sperm, is different from most described cases of hybrid male sterility, and may represent an earlier stage of hybrid sperm breakdown. The results add to previous studies of F2 hybrid inviability and behavioral sterility, and indicate that Wolbachia-induced hybrid incompatibility has arisen early in species divergence, relative to behavioral sterility and spermatogenic infertility.

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Testes (left) and seminal vesicles (right) from parental strains (A-D) and F2 hybrids (E-F). (A - B) N. vitripennis (C - D) N. giraulti. (E – F) vitripennis-giraulti F2 [v]. (G – H) vitripennis-giraulti F2 [g]. arrows indicate nuclei bundle from late stage spermatocysts. Asterisks indicate apical end of testis. Scale bar = 20 μl.
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Figure 3: Testes (left) and seminal vesicles (right) from parental strains (A-D) and F2 hybrids (E-F). (A - B) N. vitripennis (C - D) N. giraulti. (E – F) vitripennis-giraulti F2 [v]. (G – H) vitripennis-giraulti F2 [g]. arrows indicate nuclei bundle from late stage spermatocysts. Asterisks indicate apical end of testis. Scale bar = 20 μl.

Mentions: Virgin males were aged between 48-72 hrs post eclosion. Male reproductive tracts (testes, seminal vesicles, accessory glands) were dissected in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween, 0.05% NaN3, pH7.5) on clean depression well slides. For each male, one seminal vesicle was carefully removed and placed alone in a depression well with TBST. The remainder of the reproductive tract was transferred to a tube containing fixative (3.7% formaldehyde in TBST). To count sperm, the single seminal vesicle was carefully opened with minuten pins and sperm liberated. The sperm and ruptured seminal vesicle were then transferred with 20 μl TBST into a 0.7 ml tube and vortexed for 30 seconds to evenly distribute the sperm. Sperm were then spotted onto lysine-coated slide in eight × 2 μl spots and allowed to adhere to the slide and air dry for 10-15 minutes under mild heat (approximately 30°). The resulting spots were typically about 5 mm in diameter. Each sperm spot was then fixed with a drop of 3.7% formaldehyde in TBST and allowed to air dry once again. Slides were washed in TBST and stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/ml) for 30 minutes and mounted with ProLong mounting media (Invitrogen) and covered with a coverslide. For each male, a minimum of 3 sperm spots (total 6 μl) were imaged with a Zeiss Z1 fluorescence microscope with 10X objective and captured with AxioVision software using a series of 8×8 overlapping images using both fluorescence (DAPI) and dark field microscopy. Sperm nuclei (DAPI) were manually counted using the Axioimage measurement function. Overlapping or ambiguous sperm nuclei were resolved by viewing dark field images. The total sperm count for each male was estimated as the total sperm counted in 6 ul × 20/3. A minimum of 10 males were analyzed for each parental line and hybrid cross (Figure 2). For each male analyzed, the second seminal vesicle and a single testis was similarly stained, mounted and imaged to confirm relative sperm production (seminal vesicle) and characterize testis development (Figure 3-5). Following the removal of the reproductive tract, males were decapitated and heads mounted on microscope slides on double-stick tape and inter-ocular distance measured to assay body size.


Behavioral and spermatogenic hybrid male breakdown in Nasonia.

Clark ME, O'Hara FP, Chawla A, Werren JH - Heredity (Edinb) (2010)

Testes (left) and seminal vesicles (right) from parental strains (A-D) and F2 hybrids (E-F). (A - B) N. vitripennis (C - D) N. giraulti. (E – F) vitripennis-giraulti F2 [v]. (G – H) vitripennis-giraulti F2 [g]. arrows indicate nuclei bundle from late stage spermatocysts. Asterisks indicate apical end of testis. Scale bar = 20 μl.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2872237&req=5

Figure 3: Testes (left) and seminal vesicles (right) from parental strains (A-D) and F2 hybrids (E-F). (A - B) N. vitripennis (C - D) N. giraulti. (E – F) vitripennis-giraulti F2 [v]. (G – H) vitripennis-giraulti F2 [g]. arrows indicate nuclei bundle from late stage spermatocysts. Asterisks indicate apical end of testis. Scale bar = 20 μl.
Mentions: Virgin males were aged between 48-72 hrs post eclosion. Male reproductive tracts (testes, seminal vesicles, accessory glands) were dissected in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween, 0.05% NaN3, pH7.5) on clean depression well slides. For each male, one seminal vesicle was carefully removed and placed alone in a depression well with TBST. The remainder of the reproductive tract was transferred to a tube containing fixative (3.7% formaldehyde in TBST). To count sperm, the single seminal vesicle was carefully opened with minuten pins and sperm liberated. The sperm and ruptured seminal vesicle were then transferred with 20 μl TBST into a 0.7 ml tube and vortexed for 30 seconds to evenly distribute the sperm. Sperm were then spotted onto lysine-coated slide in eight × 2 μl spots and allowed to adhere to the slide and air dry for 10-15 minutes under mild heat (approximately 30°). The resulting spots were typically about 5 mm in diameter. Each sperm spot was then fixed with a drop of 3.7% formaldehyde in TBST and allowed to air dry once again. Slides were washed in TBST and stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/ml) for 30 minutes and mounted with ProLong mounting media (Invitrogen) and covered with a coverslide. For each male, a minimum of 3 sperm spots (total 6 μl) were imaged with a Zeiss Z1 fluorescence microscope with 10X objective and captured with AxioVision software using a series of 8×8 overlapping images using both fluorescence (DAPI) and dark field microscopy. Sperm nuclei (DAPI) were manually counted using the Axioimage measurement function. Overlapping or ambiguous sperm nuclei were resolved by viewing dark field images. The total sperm count for each male was estimated as the total sperm counted in 6 ul × 20/3. A minimum of 10 males were analyzed for each parental line and hybrid cross (Figure 2). For each male analyzed, the second seminal vesicle and a single testis was similarly stained, mounted and imaged to confirm relative sperm production (seminal vesicle) and characterize testis development (Figure 3-5). Following the removal of the reproductive tract, males were decapitated and heads mounted on microscope slides on double-stick tape and inter-ocular distance measured to assay body size.

Bottom Line: Several reproductive barriers exist within the Nasonia species complex, including allopatry, premating behavioral isolation, postzygotic inviability and Wolbachia-induced cytoplasmic incompatibility.Hybrid males produced in crosses between N. vitripennis and N. giraulti courted females at a reduced frequency (23-69%), compared with wild-type N. vitripennis and N. giraulti males (>93%).A strong effect of cytoplasmic (mitochondrial) background was also found in N. vitripennis and N. giraulti crosses; F2 hybrids with giraulti cytoplasm showing reduced ability at most stages of courtship.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, NY 14627, USA. mclark11@mail.rochester.edu

ABSTRACT
Several reproductive barriers exist within the Nasonia species complex, including allopatry, premating behavioral isolation, postzygotic inviability and Wolbachia-induced cytoplasmic incompatibility. Here we show that hybrid males suffer two additional reproductive disadvantages, an inability to properly court females and decreased sperm production. Hybrid behavioral sterility, characterized by a reduced ability of hybrids to perform necessary courtship behaviors, occurs in hybrids between two species of Nasonia. Hybrid males produced in crosses between N. vitripennis and N. giraulti courted females at a reduced frequency (23-69%), compared with wild-type N. vitripennis and N. giraulti males (>93%). Reduced courtship frequency was not a simple function of inactivity among hybrids. A strong effect of cytoplasmic (mitochondrial) background was also found in N. vitripennis and N. giraulti crosses; F2 hybrids with giraulti cytoplasm showing reduced ability at most stages of courtship. Hybrids produced between a younger species pair, N. giraulti and N. longicornis, were behaviorally fertile. All males possessed motile sperm, but sperm production is greatly reduced in hybrids between the older species pair, N. vitripennis and N. giraulti. This effect on hybrid males, lowered sperm counts rather than nonfunctional sperm, is different from most described cases of hybrid male sterility, and may represent an earlier stage of hybrid sperm breakdown. The results add to previous studies of F2 hybrid inviability and behavioral sterility, and indicate that Wolbachia-induced hybrid incompatibility has arisen early in species divergence, relative to behavioral sterility and spermatogenic infertility.

Show MeSH
Related in: MedlinePlus