epg5 knockout leads to the impairment of reproductive success and courtship behaviour in a zebrafish model of autophagy-related diseases.

BACKGROUND: Dysregulation of the autophagic flux is linked to a wide array of human diseases, and recent findings highlighted the central role of autophagy in reproduction, as well as an association between impairment of autophagy and behavioural disorders. Here we deepened on the possible multilevel link between impairment of the autophagic processes and reproduction at both the physiological and the behavioural level in a zebrafish mutant model. METHODS: Using a KO epg5 zebrafish line we analysed male breeding success, fertility rate, offspring survival, ejaculate quality, sperm and testes morphology, and courtship behaviour. To this aim physiological, histological, ultrastructural and behavioural analyses on epg5+/+ and mutant epg5-/- males coupled to WT females were applied. RESULTS: We observed an impairment of male reproductive performance in mutant epg5-/- males that showed a lower breeding success with a reduced mean number of eggs spawned by their WT female partners. The spermatogenesis and the ability to produce fertilising ejaculates were not drastically impaired in our mutant males, whereas we observed a reduction of their courtship behaviour that might contribute to explain their lower overall reproductive success. CONCLUSION: Collectively our findings corroborate the hypothesis of a multilevel link between the autophagic process and reproduction. Moreover, by giving a first glimpse on behavioural disorders associated to epg5 KO in model zebrafish, our results open the way to more extensive behavioural analyses, also beyond the reproductive events, that might serve as new tools for the molecular screening of autophagy-related multisystemic and neurodegenerative diseases.

Black goby territorial males adjust their ejaculate's characteristics in response to the presence of sneakers.

In many species, males can rapidly adjust their ejaculate performance in response to changing levels of sperm competition, an ability that is probably mediated by seminal fluid adaptive plasticity. In the black goby, Gobius niger, territorial males attach viscous ejaculate trails to the nest roof, from which sperm are slowly released into the water during the long-lasting spawning events. Sneaker males release their sperm in the vicinity of the nest, and territorial males try to keep them at a distance by patrolling their territory. We show here that territorial males' ejaculate trails released a higher proportion of their sperm in the presence of a single sneaker, but this proportion decreased when there were three sneakers, an effect that is most likely mediated by a change in the seminal fluid composition. Field observations showed that when multiple sneaking attempts occurred, territorial males spent more time outside the nest, suggesting that ejaculation rate and territory defence are traded-off. Altogether, these results suggest that the adjustment of sperm release from the ejaculate may be strategic, guaranteeing a more continuous concentration of the territorial male's sperm in the nest, although at a lower level, when he is engaged in prolonged territory defence outside the nest.

Going beyond conventional parameters to unveil sperm quality in fish: the use of fibre optic technology to assess mitochondrial respiratory performance.

Sperm fertilisation success depends on both intrinsic quality and the interactions with the surrounding reproductive fluids. In several fish species, these interactions have a variable effect on sperm performance. Although specific responses to reproductive fluids may depend on intrinsic differences in sperm quality, variations in the traditionally recorded sperm functional traits do not fully account for the observed patterns. New methods to enhance the evaluation of sperm quality may prove to be valuable at both applied and theoretical levels, by improving the breeding protocol of reared species and the understanding of mating success in sperm competition contexts. Here we develop a fibre optic-based technique, also adequate for small ejaculate samples, to test the role of mitochondrial respiratory efficiency in deciphering sperm performance variability. We purposely used as model the grass goby, Zosterisessor ophiocephalus, a fish with guard-sneaker mating tactics where the sperm in each male tactic have similar intrinsic qualities (velocity, viability, ATP content), but sneakers' sperm exploit territorial males' seminal fuid, overall displaying better fertilization ability. We found that sperm differed in their mitochondrial respiratory efficiency, which was higher in sneakers' sperm compared to territorial ones. This result draws the attention to an indicator of sperm quality that might be helpful in disentangling the mechanisms driving sperm-reproductive fluid interactions.

Contrasting life-history traits of two sympatric smooth-hound species: implication for vulnerability.

In this study, life-history traits (maximum and average size, size at maturity and fecundity) of two congeneric smooth-hounds, Mustelus mustelus and Mustelus punctulatus, which share a geographical distribution and experience a similar fishing exploitation, were estimated and compared between species. The results indicated a lower maximum and average size, a lower size at maturity and a higher fecundity in M. punctulatus compared with those in M. mustelus. Considering that these two species co-occur in the same areas and are caught by the same fishing gears, the results indicate a higher vulnerability to exploitation of M. mustelus compared with that of M. punctulatus.

Female comparative mate choice influences strategic male nesting strategy in the peacock blenny Salaria pavo.

Emerging evidence endorses the idea that comparative instead of rational choice is widespread among species and mating contexts. In a comparative scenario the attractiveness of a male is not absolute but depends on the attractiveness of the other males to whom he is compared by females. Therefore, a male may benefit from the ability to choose the appropriate context that enhances his attractiveness. Here we test for the occurrence of strategic male nesting strategies in the peacock blenny, Salaria pavo, a fish species in which females evaluate potential mates by comparing males. Our results show that the large majority of males choose the context that maximize their attractiveness by nesting close to less attractive neighbours. Overall, we suggest that, in the peacock blenny, comparative, non-directional female choice is associated to strategic male nesting strategies and this scenario may contribute to the maintenance of genetic variability of male sexually-selected traits.

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 performance. In the black goby, Gobius niger, an external fertilizer with guard-sneaker mating tactics and high sperm competition level, sneaker 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 the reproductive success of territorial males by enhancing their sperm performance and/or by decreasing that 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 fast as those of sneakers and with a similar fertilization rate. Moreover, both sneaker and territorial sperm remain unaffected by the seminal fluid of rival males. Thus, black goby males respond to the different level of sperm competition faced by differential allocation of 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.

Females' sampling strategy to comparatively evaluate prospective mates in the peacock blenny Salaria pavo.

Emerging evidence suggests the occurrence of comparative decision-making processes in mate choice, questioning the traditional idea of female choice based on rules of absolute preference. In such a scenario, females are expected to use a typical best-of-n sampling strategy, being able to recall previous sampled males based on memory of their quality and location. Accordingly, the quality of preferred mate is expected to be unrelated to both the number and the sequence of female visits. We found support for these predictions in the peacock blenny, Salaria pavo, a fish where females have the opportunity to evaluate the attractiveness of many males in a short time period and in a restricted spatial range. Indeed, even considering the variability in preference among females, most of them returned to previous sampled males for further evaluations; thus, the preferred male did not represent the last one in the sequence of visited males. Moreover, there was no relationship between the attractiveness of the preferred male and the number of further visits assigned to the other males. Our results suggest the occurrence of a best-of-n mate sampling strategy in the peacock blenny.

Multiple paternity and hybridization in two smooth-hound sharks.

Multiple paternity appears to be a common trait of elasmobranch mating systems, with its occurrence likely driven by convenience, due to females seeking to minimize the stress of male harassment. Here we use molecular markers to analyse the frequency of multiple paternity in two related viviparous sharks, Mustelus mustelus and Mustelus punctulatus. We first applied molecular methods to assign pregnant females, embryos and additional reference adults (N = 792) to one of the two species. Paternity analysis was performed using a total of 9 polymorphic microsatellites on 19 females and 204 embryos of M. mustelus, and on 13 females and 303 embryos of M. punctulatus. Multiple paternity occurs in both species, with 47% of M. mustelus and 54% of M. punctulatus litters sired by at least two fathers. Female fecundity is not influenced by multiple mating and in 56% of polyandrous litters paternity is skewed, with one male siring most of the pups. Genetic analyses also revealed hybridization between the two species, with a M. punctulatus female bearing pups sired by a M. mustelus male. The frequency of polyandrous litters in these species is consistent with aspects of their reproductive biology, such as synchronous ovulation and possible occurrence of breeding aggregations.

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.

Behavioural and immunological responses to an immune challenge in Octopus vulgaris.

Behavioural and immunological changes consequent to stress and infection are largely unexplored in cephalopods, despite the wide employment of species such as Octopus vulgaris in studies that require their manipulation and prolonged maintenance in captivity. Here we explore O. vulgaris behavioural and immunological (i.e. haemocyte number and serum lysozyme activity) responses to an in vivo immune challenge with Escherichia coli lipopolysaccharides (LPS). Behavioural changes of immune-treated and sham-injected animals were observed in both sight-allowed and isolated conditions, i.e. visually interacting or not with a conspecific. Immune stimulation primarily caused a significant increase in the number of circulating haemocytes 4h after the treatment, while serum lysozyme activity showed a less clear response. However, the effect of LPS on the circulating haemocytes begins to vanish 24h after injection. Our observations indicate a significant change in behaviour consequent to LPS administration, with treated octopuses exhibiting a decrease of general activity pattern when kept in the isolated condition. A similar decrease was not observed in the sight-allowed condition, where we noticed a specific significant reduction only in the time spent to visually interact with the conspecific. Overall, significant, but lower, behavioural and immunological effects of injection were detected also in sham-injected animals, suggesting a non-trivial susceptibility to manipulation and haemolymph sampling. Our results gain importance in light of changes of the regulations for the use of cephalopods in scientific procedures that call for the prompt development of guidelines, covering many aspects of cephalopod provision, maintenance and welfare.

Tactic-specific differences in seminal fluid influence sperm performance.

Seminal fluid often makes up a large part of an ejaculate, yet most empirical and theoretical studies on sperm competition have focused on how sperm characteristics (number and quality) affect fertilization success. However, seminal fluid influences own sperm performance and may potentially influence the outcome of sperm competition, by also affecting that of rivals. As a consequence males may be expected to allocate their investment in both sperm and seminal fluid in relation to the potential level of competition. Grass goby (Zosterisessor ophiocephalus) is an external fertilizer with guard-sneaker mating tactics, where sperm competition risk varies according to the tactic adopted. Here, we experimentally manipulated grass goby ejaculates by separately combining sperm and seminal fluid from territorial and sneaker males. While sperm of sneaker and territorial males did not differ in their performance when they interacted with their own seminal fluid only, sperm of sneakers increased their velocity and fertilization rate in the presence of territorial males' seminal fluid. By contrast, sneaker males' seminal fluid had a detrimental effect on the performance of territorial males' sperm. Sperm velocity was unaffected by the seminal fluid of males employing the same tactic, suggesting that seminal fluid's effect on rival-tactic sperm is not based on a self/non-self recognition mechanism. Our findings show that cross interactions of sperm and seminal fluid may influence the fertilization success of competing ejaculates with males investing in both sperm and seminal fluid in response to sperm competition risk.

A male sexually dimorphic trait provides antimicrobials to eggs in blenny fish.

Predation and microbial infections are the major causes of natural mortality for early life stages of oviparous species. The parental traits reducing the effects of predation are rather well described, whereas antimicrobial mechanisms enhancing offspring survival are largely unexplored. In this paper, we report that a male sexually dimorphic trait, the anal glands, of the redlip blenny (Ophioblennius atlanticus atlanticus) and the peacock blenny (Salaria pavo), two fish species with paternal egg care, produce a mucus enriched with antimicrobial substances. Histological and histochemical analyses showed that the anal glands of these species are characterized by the massive presence of mucus-secreting cells. Anal gland extracts, from both the hydrophilic and the hydrophobic protein fraction, exhibited a lysozyme-like activity. Field observations demonstrated that redlip blenny males, while performing egg care, rub the anal region over the nest internal surface, probably facilitating the transfer of mucus to eggs. These results strongly indicate that this sexually dimorphic trait is involved in egg defence against microbial infections.

Variability of GnRH secretion in two goby species with socially controlled alternative male mating tactics.

Male reproductive phenotypic plasticity related to environmental-social conditions is common among teleost fish. In several species, males adopt different mating tactics depending on their size, monopolizing mates when larger, while parasitizing dominant male spawns when smaller. Males performing alternative mating tactics are often characterized by a strong dimorphism in both primary and secondary reproductive traits. According to studies on sex-changing species and on species where only one male morph is reproductively active, male alternative phenotypes are expected to vary also in gonadotropin-releasing hormone (GnRH) neurons in forebrain preoptic area (POA). Here, we compared the intra- and inter-sexual variations in number and size of GnRH neurons, along with gonads and male accessory structure investment, in two goby species, the grass goby, Zosterisessor ophiocephalus, and the black goby, Gobius niger, characterized by male alternative mating phenotypes. In both species, older and larger males defend nests, court and perform parental care, while younger and smaller ones try to sneak territorial male spawning. We found that grass goby and black goby have different patterns of GnRH expression. Grass goby presents a clear intra-sexual dimorphism in GnRH expression, related to the occurrence of alternative mating tactics, while in the black goby, only inter-sexual differences are observed. The inter- and intra-specific variability in the GnRH neurons in these two goby species is discussed in light of the differences in migratory behavior, nest type, and mating system.

Changes along the male reproductive axis in response to social context in a gonochoristic gobiid, Zosterisessor ophiocephalus (Teleostei, Gobiidae), with alternative mating tactics.

Sexual selection has given rise, in several taxa, to intrasexual variation in male phenotype. While evolutionary studies have provided explanations of the adaptive function of this dramatic male phenotypic diversity, the proximate control of its expression has still to be completely understood. Several observations, primarily from sex-changing species, indicated a major role of social interactions in reproductive axis regulation and consequently in the expression of alternative male phenotypes. Here we documented changes along the male reproductive axis in response to social context in a gonochoristic species, the grass goby Zosterisessor ophiocephalus, where fully functional alternative male mating tactics appear to be expressed as an ontogenetic gradient. In the grass goby, larger and older males dig a nest and perform parental care, while smaller males sneak fertilization during territorial male spawning. Territorial males are characterized by a higher number of gonadotropin-releasing hormone (GnRH) neurons in forebrain preoptic area, smaller testes, larger seminal vesicles, and viscous ejaculates that last longer and contain fewer sperm than those of sneakers. To experimentally investigate the role of social factors in inducing changes along the male reproductive axis, sneakers were tested in two different situations: nesting alone or with ripe females. Sneakers that mated and performed parental care showed dramatic changes in brain, reproductive apparatus morphology, and ejaculate traits. GnRH-immunoreactive cells in forebrain preoptic area increased in number, reaching values typical of wild-caught parental males. Testes size decreased while seminal vesicle size increased and ejaculates showed lower sperm densities. These results were discussed within the framework of the social transduction hypothesis, which predicts that social experience should mediate, through a cascade of internal processes, shifts between morphs throughout life.

Individual adjustment of sperm expenditure accords with sperm competition theory.

Sperm competition theory predicts that males should strategically allocate their sperm reserves according to the level of sperm competition, defined as the probability that the sperm of two males compete for fertilizing a given set of ova. Substantial evidence from numerous animal taxa suggests that, at the individual level, sperm expenditure increases when the risk of sperm competition is greater. In contrast, according to the "intensity model" of sperm competition [Parker, G. A., Ball, M. A., Stockley, P. & Gage, M. J. G. (1996) Proc. R. Soc. London Ser. B 263, 1291-1297], when more than two ejaculates compete during a given mating event, sperm expenditure should decrease as the number of competing males increases. Empirical evidence supporting this prediction, however, is still lacking. Here we measured sperm expenditure in two gobiid fishes, the grass (Zosterisessor ophiocephalus) and black goby (Gobius niger), in which up to six sneakers can congregate around the nest of territorial males and release their sperm when females spawn. We show that, in accordance with theory, sneaker males of both species release fewer sperm as the number of competitors increases.

The juxtatesticular body of jawfishes (Teleostei, Opistognathidae): Comparative morphology and fine structure.

A peculiar gland, the juxtatesticular body (JTB), ductless and consisting of follicles, had previously been discovered in males of two Opistognathus species (Teleostei, Opistognathidae). In this paper, we describe (1) the general morphology of the JTB in an additional two Opistognathus species, O. aurifrons and O. macrognathus, comparing it with that of the previously described species, and (2) the fine structure of the JTB of Opistognathus whitehurstii and O. maxillosus. Interspecific variability occurs both in the general organization of this gland and in the number of follicular cells. Fine structural analysis of the JTB, both in O. whitehurstii and O. maxillosus, reveals strong similarities with thyroid follicular cells, suggesting a similar pattern of synthesis and secretion. JTB follicular cells are arranged as a monolayered epithelium that surrounds a follicular lumen; they show a polarity in organelle distribution and membrane specialization typical of secreting cells. On the basis of their cytological and histochemical characteristics we propose that JTB follicular cells perform two major types of secretory activities: the secretion of a glycoprotein from the apical part of the cells into the follicular lumen and the endocrine, or paracrine, secretion of a still unknown substance(s) from the basal part of the cell either into the extrafollicular space or the blood or both. A hypothesis concerning the functional cycle of JTB follicle is also discussed. © 1995 Wiley-Liss, Inc.