A research group led by Associate Professor Masaya Morita of Sesoko Station, the Tropical Biosphere Research Center at the University of the Ryukyus, along with researchers from Kyoto University and Osaka Metropolitan University, studied the cichlid fish Ophthalmotilapia ventralis, which inhabits Lake Tanganyika, an ancient lake in East Africa. By integrating observations of reproductive behavior, analysis of sperm motility, and gene expression analysis of seminal plasma protein (SPP120), they revealed that the intensity of sperm competition is associated with male sperm longevity and protein expression. This world-first achievement empirically demonstrates the relationship between sperm competition and sperm quality by combining field behavioral observations with molecular biology methods. The findings were published in Evolution.
(A) Courtship success rates (CSR) indicate the success rates of lekking to invite females to the bower.
(B)Bower density (BD) was estimated from the distance to the nearest bower. Females can collect sperm from many males in high density, but may be limited in collecting sperm from multiple males.
(C) Encounter rates of floating males (ERFM) represent the sneaking risk of floating males. Floating males sometimes transfer sperm to females during lekking.
Provided by University of the Ryukyus
Reproductive success in organisms is determined not only by mate acquisition but also by complex factors such as sperm competition and female choice. Particularly in fish, there are major differences in reproductive behavior and sperm characteristics between external fertilization, where eggs and sperm meet outside the body, and internal fertilization, where females receive male sperm inside their bodies.
"O. ventralis," a cichlid species inhabiting Lake Tanganyika, has a unique reproductive system in which males build nests called bowers on the lake bottom to attract females. These bowers are patch-like accumulations of sand that function as places where males transfer sperm to females and where females spawn. Females visit the bowers of multiple males and engage in behavior called sperm shopping, where they take sperm into their mouths. Afterward, females spawn in the bower and practice mouthbrooding, protecting the eggs by holding them in their mouths. It is here that the eggs first encounter the sperm held in the mouth and fertilization occurs. At this time, the female's mouth contains semen collected from multiple males, and it is believed that intense competition occurs among sperm over which will fertilize the eggs. Furthermore, sneaking behavior has been reported in which non-territorial males (floating males) without bowers intercept the courtship of territorial males with bowers to transfer sperm to females, further increasing the complexity of sperm competition.
In this situation, the ability to survive longer than other males' sperm and fertilize eggs greatly influences male reproductive success. The research group has revealed that the seminal plasma glycoprotein SPP120 contained in semen is involved in semen viscosity and retention in relation to sperm survival. In other words, SPP120 is expected to play an important role in how long sperm can maintain a fertilization-capable state in the female's mouth. In this sense, O. ventralis can be said to be an ideal model organism for understanding the evolution of sperm competition, in which female behavior, sperm traits, and the molecular mechanisms of seminal plasma proteins are intricately intertwined.
In this study, the researchers investigated the relationship between the unique reproductive ecology of O. ventralis and sperm and seminal plasma proteins. In particular, they conducted an integrated approach combining behavioral records from field diving observations, sperm motility analysis of observed individuals, and gene expression analysis of the seminal plasma glycoprotein SPP120 of observed individuals.
As a result, it was revealed that the higher the encounter rate with floating males and the higher the bower density, the longer the sperm longevity of territorial males, and that the higher the encounter rate with floating males and the higher the courtship success rate, the greater the gene expression level of SPP120. Furthermore, it was revealed that testis size, which is an indicator of energy investment in spermatogenesis itself, is also larger the higher the bower density. On the other hand, sperm swimming speed showed no correlation with any of these items.
In O. ventralis, while sperm shopping behavior was frequently observed, spawning occurred only very rarely. In other words, the fertilization process is thought to proceed in the female's mouth, where sperm from many males are mixed. Because the timing of female spawning is difficult for males to predict, long-term sperm survival becomes important for fertilization success. Males under more intense sperm competition (higher encounter rates with floating males and higher bower density) tended to have longer sperm longevity. In other words, it was shown that "living longer" rather than "swimming faster" is strongly related to reproductive success for sperm.
SPP120 is a protein believed to be involved in sperm motility and diffusion, and its expression level was associated with risk indicators for sneaking (encounter rate with floating males) and courtship success rate. This suggests that regulation at the gene level may be occurring in response to the intensity of sperm competition.
However, while sperm motility and SPP120 gene expression were both commonly associated with encounter rates with floating males, their relationships with courtship success rate and bower density differed. Testis size, which is treated as an indicator of the degree of sperm competition in many organisms, was related to bower density but showed no correlation with other items.
These results indicate that reproductive success in O. ventralis is determined by the combined action of behavior (female choice and male competition), sperm traits (longevity and quantity), and molecular-level regulation (protein expression). In particular, this study highlighted that this species has a unique fertilization environment that combines characteristics of both external and internal fertilization, and that sperm strategies adapted to this environment have evolved.
Journal Information
Publication: Evolution
Title: Variation in sperm motility and seminal plasma protein expression is shaped by pre- and post-mating sexual selection in the mouthbrooding cichlid (Ophthalmotilapia ventralis)
DOI: 10.1093/evolut/qpaf196
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