Thorough evaluation of normalization approaches is a fundamental aspect in real-time quantitative RT-PCR experiments to avoid artificial introduced intergroup variations. In our study, we tested three normalization strategies in an experimental data set derived from a toxicological exposure of Scophthalmus maximus to the peroxisome proliferator-activated receptor alpha (PPARα) agonist WY-14643. Juvenile turbots were exposed by repeated injections to 5. mg or 50. mg WY-14643/kg, and liver samples were taken at day 1, 7 and 21. Specifically, the mRNA expression of peroxiredoxin 5 (prdx5) was normalized to the cDNA content, to the mRNA expression of single reference genes (b-actin, b-act; elongation factor 1 α, ef1a; glyceraldehyde-3-phosphate dehydrogenase, gapdh; ribosomal protein L8, rpl8; tata-box binding protein, tbp; tubulin beta 2C chain, tubb2c; ubiquitin-conjugating enzyme E2L 3, ub2l3) or to a combination of multiple reference genes using geNorm, BestKeeper or NormFinder algorithms.Four single reference genes ( ef1a, rpl8, tubb2c, tbp) did not show any significant differences between the treatment groups over time, while significant intergroup variations were observed for cDNA content, gapdh, b-act and ub2l3. The normalization of prdx5 to the valid (not altered) single reference genes led to significant up-regulated ( prdx5/. rpl8), not-regulated ( prdx5/. ef1a; prdx5/. tbp) or down-regulated ( prdx5/. tubb2c) mRNA expression pattern. The multiple reference gene approaches resulted in different rankings and combinations of the most stable expressed reference genes (geNorm: ef1a>. rpl8>. b-act; BestKeeper: ub2l3>. gapdh>. ef1a; NormFinder: b-act>. ef1a). However, the normalization with the three multiple reference gene procedures demonstrated consistent expression pattern with a significant up-regulation of prdx5 in response to the higher concentration after 21. days.Concluding, even if not yet established as "gold" standard for expression profiling in environmental toxicology or physiology using freshwater or marine fish models, the multiple reference gene approach is recommended, since it eliminates any biased results, which represented the major flaw of single reference genes.

Em comparação com outros vertebrados, os peixes teleósteos têm um enorme potencial para produzir células novas no cérebro de animais adultos. Em contraste com os mamíferos, onde o processo de neurogénese adulta encontra-se restrito a duas áreas cerebrais, a zona subventricular (SVZ) e a zona subgranular parte do giro dentado do hipocampo, em peixes teleósteos foram descritas mais de 10 regiões neurogénicas. Através da marcação de células mitóticas com 5-bromo-2’-deoxiuridina (BrdU), foram caracterizadas as zonas proliferativas da Tilapia de Moçambique (Oreochromis mossanbicus). Nesta espécies, foram encontradas zonas proliferativas em regiões específicas do bolbos olfactivo, telecéfalo, região pré-optica, hipotálamo, tálamo, tecto óptico, torus longitudinalis, nas três divisões do cerebelo, valvula cerebelli, corpus cerebelli, e lobus caudalis e na região da medula, abrangendo assim toda a extensão cerebral. A localização destas zonas proliferativas parece ser extremamente conservada ao longo da taxonomia e até o número total de células produzidas parece ser mantido com pouca variação. Com um tempo de sobrevivência de 2 horas, foram encontrados na tilapia um total de 80.000 células novas em comparação com as 100.000 descritas para o peixe eléctrico Apteronotus leptorhynchus. Os nossos resultados sugerem que a actividade mitótica em regiões discretas do cérebro adulto são uma característica primitiva que tem sido conservada ao longo da evolução.

The painted goby Pomatoschistus pictus emits courtship sounds (drums) that are important in reproductive outcome. The effect of temperature (14–22 °C) on courtship drum features was characterised. Pulse period (or rate) was linearly related with temperature (R2 = 0.83) presenting a Q10 of 2.13. Pulse period decreased by 1.95 ms per 1 °C and varied from 34 ms to 18.6 ms within the studied temperature range. Sound duration changed concomitantly from 1128 ms to 658 ms. Changes in sound duration were due to pulse period rather than pulse number, since the latter was not affected by temperature. Pulse duration was similarly affected by temperature, decreasing by 0.3 ms per 1 °C (R2 = 0.51, Q10 = 1.45) and changing from 10.0 ms at 14 °C to 7.5 ms at 22 °C. Drum emission rate, sound amplitude, fatigue and dominant frequency were also not related to temperature. The temperature effect on pulse rate likely reflects temperature-dependence of the vocal central pattern generator as observed in other animals. In contrast, shorter pulse durations likely result from temperature effects at the peripheral level of the vocal system, as muscle twitches typically shorten with rising temperatures. Because pulse rate is likely important in mate choice and conspecific recognition in fish, including P. pictus, future studies are needed to understand if temperature-related changes in pulse rate are coupled with changes in mate preference as found in insects and anurans.

Many fish species emit sounds in agonistic contexts. During direct confrontations sounds are typically produced during the display phase in conjunction with visual exhibitions. Here we studied sound production during territorial defence in captive painted gobies, Pomatoschistus pictus, and related acoustic parameters with male traits and the date of recording (Julian day, i.e., with the approach of the peak of the breeding season). Territorial males emitted drumming sounds during displays that involved darkening the chin and fins, spreading fins and quivering the body. Drums were trains of low frequency pulses (≈23 pulses) repeated every 27 ms and usually lasting under a second. Drums were produced in short sequences of sounds (bursts). All acoustic parameters differed significantly among males. Drum and burst duration, and drum number of pulses increased significantly with male size. Calling duration (including drum, burst duration and drum number of pulses) also increased significantly with Julian date and presented a high intra-male variability, suggesting that these parameters may also depend on the individual’s motivation. We provide the first report for agonistic sound production in sand gobies and give evidence that sound parameters contain information that can be used during mutual assessment in contests over territories.

Male reproductive success often depends on male attributes and resource quality. Here, we examined male preference for nest size in the Lusitanian toadfish, a nest-guarding fish with parental care.We also investigated the combined effect of male length and nest size in male breeding success. Approximately 80 shelters with five different sizes were placed on an intertidal zone of the Tagus estuary. Nests were checked every fortnight at spring low tides for occupation, nest-holder length, and number of eggs. Males did not select for the larger nests despite their availability but showed size assortative nest choice. Only nest size was a significant predictor of clutch size in occupied nests and it explained 60% of the variability in the number of obtained eggs. Male length and the interaction of male length and nest size did not have a significant effect on male reproductive success. These results suggest that although nest size is key to male's reproductive success, the choice for nest size results from a trade-off between accrued costs of offspring care or risk of a nest take-over and possible benefits of increased fitness.

Many teleost fishes use acoustic and visual signalling during courtship. Such displays may convey information about body condition. Here we experimentally altered body condition of sand goby (Pomatoschistus minutus) males to examine effects on acoustic and visual courtship and subsequent spawning decisions. Over two weeks, males fed in excess were fed daily, whereas food-deprived males were fed once a week. Females only spawned with males that produced courtship sound. However, there were no treatment effects on the occurrence of spawning and males fed in excess did not invest more in visual or acoustic courtship than food-deprived males. That said, males fed in excess built more well-covered nests, with more sand piled on top, compared to fooddeprived males. Male condition measured as lipid content differed significantly between treatments. However, only males fed in excess differed in lipid content from wild caught males, indicating that in nature, males are of similar condition to males in the low condition treatment group. Apart from the importance of courtship sound, the only male or female behaviour predicting reproductive success was if male displayed in the nest opening. Males often produce courtship sounds together with a visual display in this position. A female dark-eye display did not associate with reproductive success which, together with previous results, suggest a non-ornamental function of this trait. We conclude that male courtship sounds appear to be crucial in female mate choice, but the information content of the courtship sounds and how it relates to male condition remains elusive.

The function of fish sounds in territorial defence, in particular its influence on the intruder's behaviour during territorial invasions, is poorly known. Breeding Lusitanian toadfish males (Halobatrachus didactylus) use sounds (boatwhistles) to defend nests from intruders. Results from a previous study suggest that boatwhistles function as a 'keep-out signal' during territorial defence. To test this hypothesis we performed territorial intrusion experiments with muted Lusitanian toadfish. Males were muted by making a cut and deflating the swimbladder (the sound-producing apparatus) under anaesthesia. Toadfish nest-holder males reacted to intruders mainly by emitting sounds (sham-operated and control groups) and less frequently with escalated bouts of fighting. When the nest-holder produced a boatwhistle, the intruder fled more frequently than expected by chance alone. Muted males experienced a higher number of intrusions than the other groups, probably because of their inability to vocalise. Together, our results show that fish acoustic signals are effective deterrents in nest/territorial intrusions, similar to bird song.

Acoustic signals are sexual ornaments with an established role on mate choice in several taxa, but not in fish. Recent studies have suggested that fish vocal activity may signal male quality and influence male's reproductive success but experimental evidence is lacking. Here we made two experiments to test the hypothesis that vocal activity is essential for male breeding success in a highly vocal fish, the Lusitanian toadfish. We first compared the reproduction success between muted and vocal males. In a second experiment we related male reproduction success with acoustic activity and male quality, including biometric, condition and physiological features. As a proxy for reproductive success we tallied both total number and number of sired eggs, which were correlated. Muting experiments showed that successful mating was dependent on vocalizing. In addition, the number of eggs was positively associated with the male's maximum calling rate. In the second experiment male's reproductive success was positively associated with male condition and negatively related with circulating androgen levels and relative gonad mass, but was not associated with vocal activity. Differences in results may be related with nest design which could have influenced mate choice costs and intra-sexual competition. In the muting experiment nests had a small opening that restrained the large nest-holder but allowed smaller fish, such as females, to pass while in the second experiment fish could move freely. These experiments suggest that a combination of factors, including vocal activity, influence reproductive success in this highly vocal species.

Signal attributes should show different degrees of variability depending on the information to be conveyed. Species identity is usually associated with stereotyped features of a signal, whereas other types of information such as individual quality and motivation are associated with signal plasticity. Lusitanian toadfish males form aggregations during the breeding season and emit a tonal advertisement call (the boatwhistle) to attract mates to their nests. We test the hypothesis that the boatwhistle can convey information both on individual identity and motivation by checking how signal parameters vary with time. We study how the physical (tide level) and social (calling alone or in a chorus) environments and male calling rate affect this advertisement signal and how all these external and internal factors (environment, social and male motivation) blend to modulate the Lusitanian toadfish’s advertisement call. Boatwhistles of each male were very stereotyped in short periods of time (minutes), but intra-male signal variability greatly increased in a longer time scale (days). Nevertheless, significant differences among males could still be found even in a long time scale. Pulse period was the acoustic feature that most contributed to discriminate among males. Tide level and male calling rate modulated boatwhistle characteristics, and there was a differential effect of tide on call attributes depending on male calling rate. Social acoustic environment only affected calling rate. These results suggest that inter-individual differences in call characteristics and call plasticity may mediate both male– male assessment and mate choice.

Blenniid fish exhibit a polygynandric mating system with parental care restricted to males. Nest-holder males defend a breeding territory centered on their nest, usually a crevice or hole in a rocky substrate, to which they attract females to spawn. Females, on the other hand, must search for nests in order to spawn and usually are the choosy sex, producing several sequential egg batches and broods during the breeding season. Therefore, male blennies are more site-attached than females. This situation offers an opportunity to investigate potential neural correlates of intraspecific differences in selective pressures for different spatial abilities in these species. Since the dorsolateral telencephalon has been considered a teleost homologue of the mammalian hippocampus, we predicted that the spatial abilities required for females to locate and return accurately to nests of males may have produced a sex difference in the size of the telencephalic nuclei involved in spatial abilities, biased towards females. To test this hypothesis, we assessed the home ranges and measured the size of the dorsolateral telencephalon of both sexes during the breeding season in two blenniid species, the shanny (Lipophrys pholis) and the Azorean rock-pool blenny (Parablennius parvicornis) . We chose these two species because they differ in the degree of chemical communication they use, and this could also lead to differences in telencephalic areas. As predicted, in both species females present considerably larger home ranges paralleled by larger dorsolateral ventral telencephalic nuclei (DLv) than males. Other telencephalic nuclei that were measured did not show any sex difference in size. These results suggest that the DLv is involved in spatial abilities in blenniid fish and that sexual selection may be promoting this divergence as already described for mammals and birds.