Effects of ovarian fluid on sperm traits and its implications for cryptic female choice in zebrafish

In polyandrous mating systems, females maintain the opportunity to bias male fertilization success after mating in a process known as cryptic female choice. Mechanisms of cryptic female choice have been described both in internal and external fertilizers, and may affect fertilization processes at different stages before, during, and after fertilization. In internal fertilizers, females have substantial control over sperm storage and fertilization, whereas in external fertilizers, female control is limited. A key factor proposed to mediate cryptic female choice is the fluid surrounding the eggs, the ovarian fluid, as it may directly affect sperm performance. Here, we studied the role of ovarian fluid in post-mating sexual selection using the zebrafish, Danio rerio. Firstly, we assessed how ovarian fluid affects sperm swimming performance compared with freshwater. We focused on sperm motility, velocity, swimming trajectory, and longevity, all traits associated with competitive fertilization success in externally fertilizing fish. In a second step, we used a North Carolina II design to explore female, male, and female x male effects by testing sperm motility of 2 males in the ovarian fluid of 2 females in a total of 11 blocks. Our results indicate that the ovarian fluid affects sperm performance differently from freshwater. Specifically, sperm velocity, motility, and longevity were higher in the ovarian fluid than in freshwater, whereas sperm linearity and beat cross frequency showed the opposite pattern. Moreover, these effects varied according to male, female, and male x female identities, supporting the potential for cryptic female choice mediated by ovarian fluid in this species

Ejaculates in competition: a sperm race influenced by the seminal fluid?

ABSTRACT In the last forty years, the historical notion of monogamous females has been gradually eroded away, and female multiple mating is now look as a common and ubiquitous phenomenon in nature, triggering theoretical and experimental attention to its biological implications and evolutionary consequences. Sexual selection is the evolutionary process that favours the increase in frequency of the genes that confer a reproductive advantage to the individuals carrying them. Polyandry implies that sexual selection may persist even after the copulation up to the point of fertilization, and in some cases beyond. In this scenario, male mating not necessarily results in successful insemination, but depends on the outcome of post-copulatory sexual mechanisms influencing the fertilization success, namely cryptic female choice and sperm competition. Female choice is the possibility for females to non-randomly bias paternity, favouring the ejaculate of the best quality male, in order to maximize their fitness. On the other hand, male-male competition results in sperm competition when the ejaculates of two or more males compete to fertilize the same set of eggs, as it has been firstly defined by Geoffrey Parker. When ejaculates overlap in space and time, differences in characteristics that are key factors for the fertilization success may lead one ejaculate to overcome the rivals, generating differential males’ reproductive success. This mechanism, investigated in both externally and internally fertilizing species, is a powerful evolutionary force moulding an amazing variety of behavioural, morphological and physiological traits. Ejaculates are costly to produce and, thus, sperm expenditure drawns the attention for how males, to increase their reproductive success, may modulate their investment in response to different sperm competition levels. Sperm competition is expected to influence those traits driving sperm fertilization capabilities in a specific context. To date, theoretical and empirical studies have primarily and only focused on how sperm characteristics, i.e. number and quality, affect the fertilization success of competing males. Increasing evidence are suggesting that predictions on the outcome of sperm competition should not revolve only around the sperm component of the ejaculate. The seminal fluid often makes up a large part of an ejaculate and it may influence paternity success both directly and indirectly. Indeed, seminal fluid is already known to enhances sperm performance in several species as well as to indirectly influence paternity success, by decreasing female receptivity, increasing oviposition rate and forming mating plugs. Seminal fluids may also play a frontline role in sperm competition by directly affecting rivals’ sperm performance. For instance, in promiscuous ants and bees, seminal fluid incapacitates the sperm of rival males, while in other insects, it improves equally the survival of own and other sperm. This suggests that, unless a self/non-self-recognition mechanism evolves, the function of seminal fluid to enhance own sperm performance can be exploited by the sperm of rival males. In particular, when a male can his reproductive role while mating with a female, if advantaged or disadvantaged, he could strategically allocate his ejaculate to maximize the reproductive success. Theoretical analyses, still waiting for experimental tests, posit that selection should favour phenotypic plasticity in male expenditure on both sperm and seminal fluid components, specifically influencing that/those that affect more the ejaculate competitive weight. Clear evidence still lack, likely because, up to the present study, models and experimental works considered mostly internal fertilizers, where it is difficult to attribute sperm and seminal fluid to a specific individual. I overcame these problems by using as model species two fishes with external fertilization, the grass goby (Zosterisessor ophiocephalus) and the black goby (Gobius niger) as they show a similar mating system and comparable levels of sperm competition, but potentially differ in the likelihood for seminal fluid to influence competition contexts. In both species males display territorial-sneaker mating tactics, where sperm competition risk varies according to the tactic adopted, with sneaker males experiencing the highest level producing a great number of sperm and less seminal fluid than territorial males. In the grass goby, sperm quality, in terms of velocity, viability and ATP content, does not vary between tactics, whereas, black goby sneakers produce sperm that are faster, more viable and richer in ATP than territorial males. In these two species, the dynamics of mating are potentially a crucial factor influencing the role of seminal fluid on the outcome of ejaculates competition. Indeed, grass goby sneakers enter inside the nest and may release their ejaculates in close proximity to those of the territorial male and to eggs. Thus, in this species I expected that the seminal fluid might have a competitive weigh, mediating sneaker and territorial ejaculates interplay. By contrast, in the black goby, sneakers are forced to release their ejaculate at the nest entrance and, thus, the opportunity for the mixing of territorial males’ and sneakers’ ejaculates does not occur or it is rare. On the basis of these preconditions, my PhD project pointed to i) verify in both species when and how the seminal fluid affects sperm performances, in terms of velocity, viability of own and rival sperm, making combinations of sperm and seminal fluid within and between males adopting different tactics; ii) deepen, in the grass goby, the proximate mechanisms driving sperm and seminal fluid interplay; iii) evaluate if the results from sperm performance give reliable insights on their fertilization ability and on the outcome of ejaculates competition in nature. Therefore, I performed in vitro fertilization tests, applying the same experimental design used in sperm performance trials. Secondly, considering that the paternity success of the grass goby has been already investigated from natural nests in a previous work, I concentrated on the black goby. I evaluated the fertilization success in the field through artificial nests located in natural breeding sites, by using molecular parentage analyses. In the grass goby, I found that sneaker’s sperm increase their performance, both in terms of velocity and fertilization rate, in presence of territorial male’s seminal fluid, while the performance of territorial male’s sperm is decreased in presence of sneaker’s seminal fluid. Appropriate control experiments demonstrate that this effect is not mediated by a self/non-self recognition mechanism. Investigating the proximate mechanisms driving sperm-seminal fluid cross interactions, we found that sneaker’ and territorial male’s ejaculates differ in seminal fluid protein content (quantitatively and qualitatively) and even in sperm quality, with sneaker sperm showing an higher oxygen consumption rate, a parameter rarely measured in sperm quality analyses. The deepening of sperm-seminal fluid proximate mechanisms is just at the beginning but I highlighted how the non-protein fraction of the seminal fluid (<3kDa) is crucial for sperm performance (velocity), despite even the protein fraction indicate a minor influence. In this species, sperm velocity results are perfectly mirrored by in vitro fertilization tests, hence sperm velocity is a reliable indicator of ejaculate fertilization ability. Considering the paternity success recorded in the field during a previous work, it seems that it is the territorial males nest guarding that finally determines their fertilization success, and thus, the distance at which sneakers are forced to release their ejaculates. Indeed, territorial males fertilization success positively correlate with their body size. In the black goby, where the ejaculates of competing males are released far from each other, seminal fluid does not affect the sperm performances of rival males, as expected. Despite the seminal fluid of territorial males significantly enhances their sperm speed, still sneaker sperm are significantly faster, regardless the seminal fluid present. Results from in vitro fertilization tests, apparently do not mirror sperm performance, since sneaker and territorial males fertilization rates do not significantly differ, probably because the low number of trials. However, I evidenced that sneaker and territorial males sperm differ in their swimming mode, with territorial male sperm moving in a significantly more linear trajectory. As a consequence, it could take the same time to sneaker and territorial male sperm to travel the same distance, even if those of sneaker have an higher speed. The analysis of the paternity distribution of territorial males in the field suggests that the distance at which sneakers are forced to release their ejaculates determines the number of eggs they fathered, as in the grass goby. Indeed, preliminary results from artificial nests in the field indicate that snakers stole more fertilizations close to the nest principal entrance, lowering the territorial male fertilization success in that area, but less in the rest of nest ceiling. However, territorial males parentage success is unexpectedly low, respect to that registered in the grass goby and across other fish species with a similar mating system. In addition, we found in two of four analysed nests few embryos sired by a neighbour territorial male. If the result would be confirmed by further analysis, it implies that territorial males may occasionally adopt sneaking behaviours, probably depending on the level of ejaculates competition determined by nests availability and male density. The territorial mating role would not appear as favoured as in other species with alternative mating tactics, especially considering that sneakers visits more than one nest. Further studies should be addressed to the investigation of territorial male paternity success along the breeding season. In both species, the spatial context in which the competition between ejaculates occurs proved to be important. The distance at which rival ejaculates are released determines the opportunity for the rival seminal fluid exploitation, and, consequently influences the strategy to maximize the fertilization success: through the number and/or the quality of the sperm, or taking advantage of the seminal fluid of a rival-tactic male. The seminal fluid proven itself to be one of the factor that may tip the balance in the ejaculates competition scenarios, that need to be investigated with a comprehensive multidisciplinary approach

Seminal fluid enhances competitiveness of territorial males' sperm in a fish with alternative male reproductive tactics.

The most common adaptation to sperm competition in males is represented by an increase in the sperm number and/or quality released at mating, to raise their probability of egg fertilization. However, rapidly mounting evidence highlights that seminal fluid may directly influence the competitive fertilization success of a male by affecting either own and/or rival sperm performances. In the black goby, Gobius niger, an external fertilizer with guard-sneaker mating tactics and high sperm competition level, sneaker males' ejaculates contain less seminal fluid and more sperm, that are also of better quality, than those of territorial males. However, territorial males, gain a higher paternity success inside natural nests. Here, we ask whether the seminal fluid can contribute to territorial males' reproductive success by enhancing their sperm performances and/or by decreasing those of sneaker males. Using sperm and seminal fluid manipulation and in vitro fertilization tests, we found that own seminal fluid influences the velocity and fertilization ability of sperm only in territorial males, making them as faster as those of sneakers and with similar fertilization rate. Moreover, both sneaker and territorial males' sperm remain unaffected by the seminal fluid of rival males. Thus, black goby males respond to the different level of sperm competition faced by differently allocating in sperm and non-sperm components of the ejaculate, with sneakers primarily investing in sperm of intrinsic high quality and territorial males relying on the effect of seminal fluid to increase the lower intrinsic quality of their sperm

Ownership dispersion and performance in cooperative banking

Different methodological approaches and hypotheses relative to the relationship between ownership structure and performances in cooperative banking generate contrasting findings, so motivating this innovative study which is grounded on an estimation approach allowing for the potential endogeneity of the membership base. Based on a sample of 241 Italian small cooperative banks over the 2013–2018 period, we find that bank profitability is positively affected by the membership as in the study conducted by Jones and Kalmi (2015) on Finnish cooperative banking and different from the comparable Austrian empirical research of Gorton and Schmid (1999). Unlike the latter we did not find an increasing exposure to agency costs as ownership dispersion grows and showed that greater membership raises individual bank financial stability, lowering the cost of credit risk

Context-dependent evaluation of prospective mates in a fish

Female choice is often assumed to be based on absolute preference, driven by a threshold value of mate attractiveness. However, increasing evidence suggests that females may instead perform a comparative evaluation of prospective mates, possibly incurring in violation of rational decision rules (e.g. independence from irrelevant alternative, IIA). A prototypical case is the ‘asymmetrically dominated decoy’ effect where the preference for a target option over a competitor is altered by the addition of an irrelevant alternative. Here, we test for this effect in the peacock blenny Salaria pavo. Females, in binary test (i.e. focal option dyad differing in body size and extension of a yellow spot), strongly preferred one of the options. The effect of decoys, asymmetrically dominating the focal options for either yellow spot extension or body size, varied according to the initially preferred trait and the decoy type. Indeed, the addition of a decoy caused a shift in preference only when the decoy exhibited the intermediate expression of the trait less preferred initially. By contrast, females did not modify their preference in the presence of the decoy for their preferred trait. Although females’ evaluation was context-dependent, the violation of IIA was clearly observed only with respect to the initially less preferred trait. This does not exclude that females are in any case using comparative decision rules. Indeed, when faced with three alternatives, two of which are proportionally closer to each other than to the third one, they might not be able to discriminate among them, perceiving stimulus absolute magnitude

Thermo-optical numerical modal analysis of multicore fibers for high power lasers and amplifiers

Lasers for modern industrial and research applications are required to provide a high average beam power and at the same time be reliable, efficient, and compact. Rare-earth doped single-mode fiber lasers are the most promising solution. Large mode area fibers are effectively used to reduce nonlinear effects and scaling up the beam power which is now bounded by thermal effects. A new cutting-edge approach to the issue involves the use of Multi-Core Fibers (MCFs), coherently combining several lower power beams into a higher power one, and thus pushing the threshold of nonlinearities and transverse mode instabilities to higher power. The amplification process involves heat generation in the doped cores due to quantum defect, which propagates radially and creates a temperature gradient across the fiber cross-section. Even though the cores are optically uncoupled, the refractive index gradient due to thermo-optical effects could cause cross-talk and core mode coupling. In this work, we numerically analyze the performances of 9-core MCFs for high power fiber lasers by taking into account the coupling and bending effects due to the heat load generated by the quantum defect between pump and laser radiation. MCFs show very low sensitivity to heat load and bending, with effectively single-mode behaviour up to 15 um core diameter (effective area 181 um2) and down to 35 um pitch

Chalcogenide suspended-core fibers for supercontinuum generation in the mid-infrared

Chalcogenide suspended core fibers are a valuable solution to obtain supercontinuum generation of light in the mid-infrared, thanks to glass high transparency, high index contrast, small core diameter and widely-tunable dispersion. In this work the dispersion and nonlinear properties of several chalcogenide suspended core mi-crostructured fibers are numerically evaluated, and the effects of all the structural parameters are investigated. Optimization of the design is carried out to provide a fiber suitable for wide-band supercontinuum generation in the mid-infrared

Highly nonlinear chalcogenide suspended-core fibers for applications in the mid-infrared

Due to their unique dispersion and nonlinear properties, chalcogenide suspended-core fibers, characterized by a few micrometer-sized core suspended between large air-holes by few small glaß struts, are excellent candidates for mid-infrared applications. In the present study the influence of the main croß-section characteristics of the chalcogenide suspended-core fibers on the dispersion curve and on the position of the zero-dispersion wavelength has been thoroughly analyzed with a full-vector modal solver based on the finite element. In particular, the design of suspended-core fibers made of both As2S3 and As2Se3 has been optimized to obtain dispersion properties suitable for the supercontinuum generation in the mid-infrared

Detection of Hepatitis E Virus Antibodies in Domestic and Wild Animal Species in Central Italy

Hepatitis E virus (HEV) is known for its zoonotic potential. Although several mammalian species have been indicated as possible viral reservoir, the host range of the infection is partially defined. In this work serum samples collected from wild brown hares, red deer, wild rabbits, cattle living in semi-wild state and wild boar-hunting dogs were tested by a multi-species ELISA assay. Only sera from red deer (5.6%), wild rabbit (38.5%) and wild-boar hunting dogs (14.3%) scored positive. The investigation indicated the circulation and the high endemicity of HEV in various animal species in Central Italy, and the importance that these species can play in the epidemiology of infection