Sporadic feeding regulates robust food entrainable circadian clocks in blind cavefish.

The circadian clock represents a key timing system entrained by various periodic signals that ensure synchronization with the environment. Many investigations have pointed to the existence of two distinct circadian oscillators: one regulated by the light-dark cycle and the other set by feeding time. Blind cavefish have evolved under extreme conditions where they completely lack light exposure and experience food deprivation. Here, we have investigated feeding regulated clocks in two cavefish species, the Somalian cavefish Phreatichthys andruzzii and the Mexican cavefish Astyanax mexicanus, in comparison with the surface-dwelling zebrafish Danio rerio. Our results reveal that feeding represents an extremely strong synchronizer for circadian locomotor rhythmicity in subterranean cavefish. Indeed, we showed that consuming just one meal every 4 days is sufficient to entrain circadian rhythmicity in both cavefish species, but not in zebrafish. These profound adaptations to an extreme environment provide insight into the connections between feeding and circadian clocks.

Ontogeny of daily rhythms in the expression of metabolic factors in Nile tilapia (Oreochromis niloticus) kept at two different temperature regimes: Thermocycle and constant temperature.

The daily variations of temperature are one of the main synchronizers of the circadian rhythms. In addition, water temperature influences the embryonic and larval development of fish and directly affects their metabolic processes. The application of thermocycles to fish larvae has been reported to improve growth and the maturation of the digestive system, but their effects on metabolism are poorly understood. The aim of the present study was to evaluate the effect of two different temperature regimes, cycling versus constant, on the daily rhythms of metabolic factors of Nile tilapia (Oreochromis niloticus) larvae. For this purpose, fertilized eggs were divided into two groups: one reared in a 31 °C:25 °C day:night thermocycle (TCY) and another group maintained in a constant 28 °C temperature (CTE). The photoperiod was set to a 12:12 h light/dark cycle. Samples were collected every 4 h during a 24-h cycle on days 4, 8 and 13 post fertilization (dpf). The expression levels of alanine aminotransferase (alt), aspartate aminotransferase (ast), malic enzyme, glucose-6-phosphate dehydrogenase (g6pd), phosphofructokinase (pfk) and pyruvate kinase (pk) were analyzed by qPCR. Results showed that, in 13 dpf animals, most of the genes analyzed (alt, ast, malic, g6pd and pfk) showed daily rhythms in TCY, but not in the group kept at constant temperature, with most acrophases detected during the feeding period. An increase in nutrient metabolism around feeding time can improve food utilization and thus increase larval performance. Therefore, the use of thermocycles is recommended for tilapia larviculture.

Does exposure to moonlight affect day/night changes in melatonin and metabolic parameters in Amazonian fish?

Lunar cycle modulates the rhythmic activity patterns of many animals, including fish. The effect of the moonlight cycle on daily melatonin and metabolic parameters was evaluated in matrinxã (Brycon amazonicus) subjected to external natural lighting. Eighty juvenile were distributed in 4 tanks of 1m3 (20 fish/tank) and divided into two groups. One group was exposed to the full moon and the other group to the new moon for 30 days, which corresponds to the duration of the lunar period. At the end of the lunar phase, 6 fish from each group were anesthetized to collect blood, tissue and eye samples at midday and midnight. The comparison between the light and dark periods revealed a significant increase in plasma and ocular melatonin in the last period. However, there was no significant difference for plasma melatonin between moons. Ocular melatonin presented higher concentrations during the new moon. Glucose, total proteins, cortisol, liver glutathione and gill lipid peroxidation were higher in the full moon compared to in the new moon. Plasma triglyceride was higher during the night for the full moon, and the opposite was found for the new moon. Total cholesterol values were higher at night regardless the moon phase. Glutathione in the gills and lipid peroxidation in the liver showed no significant differences. These results highlight the importance of considering both the day and lunar cycles for melatonin and metabolic parameters in species of commercial interest and susceptible to stressful situations in rearing conditions.

Identification of Marine Biotechnology Value Chains with High Potential in the Northern Mediterranean Region.

Marine (blue) biotechnology is an emerging field enabling the valorization of new products and processes with massive potential for innovation and economic growth. In the Mediterranean region, this innovation potential is not exploited as well as in other European regions due to a lack of a clear identification of the different value chains and the high fragmentation of business innovation initiatives. As a result, several opportunities to create an innovative society are being missed. To address this problem, eight Northern Mediterranean countries (Croatia, France, Greece, Italy, Montenegro, Portugal, Slovenia and Spain) established five national blue biotechnology hubs to identify and address the bottlenecks that prevent the development of marine biotechnology in the region. Following a three-step approach (1. Analysis: setting the scene; 2. Transfer: identification of promising value chains; 3. Capitalization: community creation), we identified the three value chains that are most promising for the Northern Mediterranean region: algae production for added-value compounds, integrated multi-trophic aquaculture (IMTA) and valorization aquaculture/fisheries/processing by-products, unavoidable/unwanted catches and discards. The potential for the development and the technical and non-technical skills that are necessary to advance in this exciting field were identified through several stakeholder events which provided valuable insight and feedback that should be addressed for marine biotechnology in the Northern Mediterranean region to reach its full potential.

Combined effects of rearing temperature regime (thermocycle vs. constant temperature) during early development and thermal treatment on Nile tilapia (Oreochromis niloticus) sex differentiation.

In nature, water temperature experiences daily variations known as thermocycles. Temperature is the main environmental factor that influences sex determination in most teleost fish. The purpose of this study was to examine the effects of rearing temperature (thermocycle (TC) vs. constant (CTE)) on development and a posterior thermal shock throughout the period of sex differentiation of Nile tilapia (Oreochromis niloticus). Embryos and larvae were kept under two temperature regimes: TC of 31 °C:25 °C day:night vs. CTE of 28 °C from 0 to 11 dpf. After this period, the larvae from each group were subjected to either heat treatment (HT, 36 °C for 12 days) or kept under the same rearing temperatures until 23 dpf (Control, C). Then all the groups remained at constant temperature until 270 dpf, when blood and gonads were collected. Larval samples were used to examine the expression of genes related to male (amh, ara, sox9a, dmrt1a) and female (cyp19a1a, foxl2, era) sexual differentiation. In juveniles, sex was characterized by histology, the gonadal expression of the genes involved in the sex steroid synthesis was analyzed by qPCR, and plasma testosterone (T) and estradiol (E2) levels were analyzed by ELISA. In larvae, daily TCs increased the survival rate against HT and up-regulated the expression of ovarian differentiation genes. In juveniles, TC + C induced a higher proportion of females and higher cyp19a1a expression compared to CTE + C. HT induced changes in the CTE group by up-regulating testicular differentiation genes and down-regulating female promoting genes, which did not occur in the TC group. Juveniles from TC + C group presented a higher proportion of females with higher E2 and cyp19a1a than CTE + HT. Fish from the CTE + HT group showed a higher percentage of males with highest T and amh. These findings indicate that daily TCs during larval development promote ovarian differentiation and diminish the masculinizing effects of HT.

Combined blue light and daily thermocycles enhance zebrafish growth and development.

In the wild, the light/temperature environment cyclically oscillates insofar as the temperature rises after dawn and drops after dusk. In the underwater photo-environment, light is filtered through the water column so that blue photons reach greater depths. This paper investigates the combined effects of both factors with two temperature regimes (constant temperature = 26°C, CTE vs. daily thermocycle = 28°C day:24°C night, TC) and three light wavelengths (white-W, blue-B, red-R) on Danio rerio embryos and larvae from fertilization to 30 days post-fertilization (dpf). It studied hatching rate, larval survival, growth, and food intake (gut content). It analyzed the expression of the genes involved in stress (crh), somatic growth (gh, ifg1a, igf2a), and food intake control (npy, agrp, ghrelin, orexin, mch1, mch2, grp, cck8) at 10 and 30 dpf. The results revealed that the lowest hatching rate was in R regardless of the temperature regime. The highest growth rate was for the larvae reared with B + TC, which was consistent with the highest expression values of the growth factors. The highest feeding and expression levels of the genes involved in food intake were for the larvae in B (regardless of the temperature regime) and W + TC. Conversely, the R + CTE combination obtained the worst growth and feeding results. These findings indicate that the best larval performance can be achieved with combinations of blue wavelengths and cyclic temperature regimes that come closer to those in the natural environment. These results should be considered when optimizing rearing protocols to improve the growth and welfare of the fish larvae.

Rearing temperature conditions (constant vs. thermocycle) affect daily rhythms of thermal tolerance and sensing in zebrafish.

In the wild, the environment does not remain constant, but periodically oscillates so that temperature rises in the daytime and drops at night, which generates a daily thermocycle. The effects of thermocycles on thermal tolerance have been previously described in fish. However, the impact of thermocycles on daytime-dependent thermal responses and daily rhythms of temperature tolerance and sensing expression mechanisms remain poorly understood. This study investigates the effects of two rearing conditions: constant (26 °C, C) versus a daily thermocycle (28 °C in the daytime; 24 °C at night, T) on the thermal tolerance response in zebrafish. Thermal tolerance (mortality) was assessed in 4dpf (days post fertilization) zebrafish larvae after acute heat shock (39 °C for 1 h) at two time points: middle of the light phase (ML) or middle of the dark phase (MD). Thermal stress responses were evaluated in adult zebrafish after a 37 °C challenge for 1 h at ML or MD to examine the expression of the heat-shock protein (HSP) (hsp70, hsp90ab1, grp94, hsp90aa1, hspb1, hsp47, cirbp) and transient receptor potential (TRP) channels (trpv4, trpm4a, trpm2, trpa1b) in the brain. Finally, the daily rhythms of gene expression of HSPs and TRPs were measured every 4 h for 24 h. The results revealed the larval mortality rates and the expression induction of most HSPs in adult zebrafish brain reached the highest values in fish reared under constant temperature and subjected to thermal shock at MD. The expression of most HSPs and TRPs was mainly synchronized to the light/dark (LD) cycle, regardless of the temperature regime. Most HSPs involved in hyperthermic challenges displayed diurnal rhythms with their acrophases in phase with warm-sensing thermoTRPs acrophases. The cold-sensing trpa1b peaked in the second half of the light period and slightly shifted toward the dark phase anticipating the acrophase of cirpb, which is involved in hypothermic challenges. These findings indicated that: a) thermal shocks are best tolerated in the daytime; b) the implementation of daily thermocycles during larval development reduces mortality and stress-cellular expression of HSPs to an acute thermal stress at MD; c) daily rhythms need to be considered when discussing physiological responses of thermal sensing and thermotolerance in zebrafish.

Daily rhythms in the morphometric parameters of hepatocytes and intestine of the European sea bass (Dicentrarchus labrax): influence of feeding time and hepatic zonation.

The digestive system presents daily rhythms at both physiological and histological levels. Although cell morphology rhythms in mammals have been reported, they have scarcely been investigated in fish. The aim of the present research was to investigate the existence of daily rhythms in the morphology of cells in the liver and intestine of a teleost fish, the European sea bass (Dicentrarchus labrax), and how feeding time influences them. Regarding liver, we also focused on differences between the two metabolic zones: perivenous and periportal. For this purpose, fish were divided into two groups: fish fed once a day in the mid-light phase (ML) or the mid-dark phase (MD). After 1 month under each feeding regime, liver and intestine samples were collected every 4 h during a 24-h cycle, and different parameters were studied by light microscopy and image analysis. Daily rhythms occurred in most of the parameters evaluated in the liver. The effect of feeding time depended on the metabolic zone: the rhythms in the periportal zone were synchronized mainly by the light/dark cycle regardless of feeding time, whereas in the perivenous zone, rhythms were influenced more by feeding time. In the intestine, a daily rhythm in villi height was found with acrophases coinciding with feeding time in each group. These findings show for the first time the existence of cellular morphological rhythms in fish liver and intestine, and highlight the interactions between light and feeding cycles in the different metabolic zones of the liver.

Coping with exposure to hypoxia: modifications in stress parameters in gilthead seabream (Sparus aurata) fed spirulina (Arthrospira platensis) and brewer's yeast (Saccharomyces cerevisiae).

This study aimed to investigate the stress response of Sparus aurata specimens fed with nutraceutical aquafeed brewer's yeast (Saccharomyces cerevisiae) and spirulina (Arthrospira platensis). For that purpose, 96 (169.0 ± 2.8 g) animals were distributed randomly in 12 tanks (8 fish per tank, 4 replicates) and divided in 3 groups (D1, casein/gelatin, control; D2, brewer's yeast; D3, spirulina) and fed for 30 days. At the end of this period, fish from two replicates of each experimental diet were submitted to air exposure for 60 s while the fish from the other two replicates were maintained undisturbed (control). Afterwards, samples of blood, skin mucus, and head kidney were collected. The results revealed that after air exposure, cortisol, and glucose levels increased in the groups fed D1 (18.5 ± 2.6 mg/mL; 7.3 ± 0.6 mmol/L, respectively) and D2 (20.0 ± 6.2 mg/mL; 7.7 ± 0.6 mmol/L), but glucose not increased in fish fed D3 (13.7 ± 2.6 mg/mL; 5.5 ± 0.3 mmol/L). Lactate levels increased in all stressed groups, but in D1, its levels were significantly higher. After stress procedure, immunoglobulin M (IgM) levels in mucus increased only in fish fed D3 (0.1901 ± 0.0126 U/mL). Furthermore, there was a reduction in the expression of some genes involved in stress response (coxIV, prdx3, csfl-r, ucp1, and sod in fish fed D2 and D3). csf1 decreased only in stressed fish fed D2. However, cat increased in fish fed with D3. In summary, these findings points to the beneficial effects of spirulina and brewer's yeast to improve stress resistance in aquaculture practices of gilthead seabream.

Environmental Cycles, Melatonin, and Circadian Control of Stress Response in Fish.

Fish have evolved a biological clock to cope with environmental cycles, so they display circadian rhythms in most physiological functions including stress response. Photoperiodic information is transduced by the pineal organ into a rhythmic secretion of melatonin, which is released into the blood circulation with high concentrations at night and low during the day. The melatonin rhythmic profile is under the control of circadian clocks in most fish (except salmonids), and it is considered as an important output of the circadian system, thus modulating most daily behavioral and physiological rhythms. Lighting conditions (intensity and spectrum) change in the underwater environment and affect fish embryo and larvae development: constant light/darkness or red lights can lead to increased malformations and mortality, whereas blue light usually results in best hatching rates and growth performance in marine fish. Many factors display daily rhythms along the hypothalamus-pituitary-interrenal (HPI) axis that controls stress response in fish, including corticotropin-releasing hormone (Crh) and its binding protein (Crhbp), proopiomelanocortin A and B (Pomca and Pomcb), and plasma cortisol, glucose, and lactate. Many of these circadian rhythms are under the control of endogenous molecular clocks, which consist of self-sustained transcriptional-translational feedback loops involving the cyclic expression of circadian clock genes (clock, bmal, per, and cry) which persists under constant light or darkness. Exposing fish to a stressor can result in altered rhythms of most stress indicators, such as cortisol, glucose, and lactate among others, as well as daily rhythms of most behavioral and physiological functions. In addition, crh and pomca expression profiles can be affected by other factors such as light spectrum, which strongly influence the expression profile of growth-related (igf1a, igf2a) genes. Additionally, the daily cycle of water temperature (warmer at day and cooler at night) is another factor that has to be considered. The response to any acute stressor is not only species dependent, but also depends on the time of the day when the stress occurs: nocturnal species show higher responses when stressed during day time, whereas diurnal fish respond stronger at night. Melatonin administration in fish has sedative effects with a reduction in locomotor activity and cortisol levels, as well as reduced liver glycogen and dopaminergic and serotonergic activities within the hypothalamus. In this paper, we are reviewing the role of environmental cycles and biological clocks on the entrainment of daily rhythms in the HPI axis and stress responses in fish.

Daily rhythms of expression in reproductive genes along the brain-pituitary-gonad axis and liver of zebrafish.

The brain-pituitary-gonadal (BPG) axis regulates the activation of the endocrine machinery that triggers reproduction, which is a typical rhythmic process. In this research we focused on investigating the daily expression rhythms of the key reproductive genes involved in the BPG axis and the liver of zebrafish. To this end, male and female zebrafish were subjected to a stimulating photoperiod with a 14 h light:10 h dark cycle. Brain, pituitary and gonads, as well as female liver samples, were taken every 4 h during a 24 h cycle. The results revealed that most genes exhibited statistically significant daily rhythms. Most of the brain reproductive genes (gnrh2, gnrh3, kiss1, kiss2 and gnrhr3) displayed a daily rhythm of expression with a nocturnal acrophase (between Zeitgeber Time [ZT] 14:34 h and ZT18:34 h, lights off at ZT = 14 h). The male kiss2 gene presented neither significant rhythms nor daily variations, while the male gnrh3 and female kiss2 genes exhibited diurnal peaks of expression at ZT06:34 h and ZT04:34 h, respectively. In contrast, the pituitary genes (fshß, lhß, gnrhr2) showed daily rhythms of expression with an acrophase during the light phase (between ZT02:10 h and ZT10:35 h). The female gnrhr3 gene exhibited neither significant rhythms nor daily variations. The male gnrhr3 gene presented a nocturnal acrophase (ZT14:32 h). The gonad genes (star, cyp17a1, 20ßhsd, lhr, fshr, cyp19a1a, foxl2, amh, dmrt1 and 11ßhsd) revealed statistically significant daily rhythms with nocturnal acrophases, except for female cyp17a1a (ZT06:21 h) and 20ßhsd (ZT05:19 h). Lastly, the female liver genes presented daily rhythms with a maximum peak of expression around the transition phase from darkness to light (ZT01:00 h for erα and at ZT23:09 h for vtg2). These findings are consistent with the daily reproduction rhythms displayed by zebrafish, which are timed by the reproductive axis. Considering that reproductive success is critical for survival of the species, the knowledge of the rhythms of the endocrine BPG machinery provides useful information to understand the reproduction process and to establish optimal protocols and conditions for reproductive treatments.

Daily rhythms after vaccination on specific and non-specific responses in Nile tilapia (Oreochromis niloticus).

We evaluated the daily changes in immunological and hematological factors in tilapia (Oreochromis niloticus) after an immunization period with a subsequent challenge. Experiments were divided into two phases: Phase 1 (immunization): 144 fish were distributed into two groups with 72 fish in six tanks. One group (T1) was immunized, comprising six vaccination time points (ZT schedule = ZT2 h, ZT6 h, ZT10 h, ZT14 h, ZT18 h, ZT22 h). The same schedule was applied to the other group, but with saline solution (non-vaccinated: T2). Both groups remained in the laboratory for 30 days (considered the immunization period). Phase 2 (challenge): on day 30, both vaccinated and non-vaccinated groups were challenged with Streptococcus agalactiae (2.0 × 107 CFU mL-1) following the same ZT schedule to stimulate the immune response without leading to widespread infection and mortality. On day 45, blood and head kidney samples were collected during the same ZT schedule. The variations in time of the following parameters within each group were evaluated: hematology, peroxidase activity, IgM, tnf-α3, tgf-ß1, il-1ß and il-12 gene expression. No significant mortality was observed for the groups or the ZT schedule (p > 0.05). Daily rhythms with diurnal acrophases were found in T2 for il12, tnf-α3 and tgf-ß1 expression gene, while the acrophases of the peroxidase level, hematocrit and thrombocytes were at nighttime (p < 0.05). In contrast, most of the parameters in the vaccinated tilapia showed no daily rhythms (p > 0.05), except IgM. For all the parameters, the interaction effect between time and treatment (vaccinated and non-vaccinated groups) depended on ZT. Our results reveal that the humoral and non-specific immune system displayed a circadian rhythm based on the light-dark cycle, which could be affected by the vaccination procedure in tilapia.

Effects of pinealectomy on the neuroendocrine reproductive system and locomotor activity in male European sea bass, Dicentrarchus labrax.

The seasonally changing photoperiod controls the timing of reproduction in most fish species, however, the transduction of this photoperiodic information to the reproductive axis is still unclear. This study explored the potential role of two candidate neuropeptide systems, gonadotropin-inhibitory hormone (Gnih) and kisspeptin, as mediators between the pineal organ (a principle transducer of photoperiodic information) and reproductive axis in male European sea bass, Dicentrarchus labrax. Two seven-day experiments of pinealectomy (Px) were performed, in March (end of reproductive season) and August (resting season). Effects of Px and season on the brain expression of gnih (sbgnih) and its receptor (sbgnihr), kisspeptins (kiss1, kiss2) and their receptors (kissr2, kissr3) and gonadotropin-releasing hormone (gnrh1, gnrh2, gnrh3) and the main brain receptor (gnrhr-II-2b) genes, plasma melatonin levels and locomotor activity rhythms were examined. Results showed that Px reduced night-time plasma melatonin levels. Gene expression analyses demonstrated a sensitivity of the Gnih system to Px in March, with a reduction in sbgnih in the mid-hindbrain, a region with bilateral connections to the pineal organ. In August, kiss2 levels increased in Px animals but not in controls. Significant differences in expression were observed for diencephalic sbgnih, sbgnihr, kissr3 and tegmental gnrh2 between seasons. Recordings of locomotor activity following surgery revealed a change from light-synchronised to free-running rhythmic behavior. Altogether, the Gnih and Kiss2 sensitivity to Px and seasonal differences observed for Gnih and its receptor, Gnrh2, and the receptor for Kiss2 (Kissr3), suggested they could be mediators involved in the relay between environment and seasonal reproduction.

Synchronization to light and mealtime of daily rhythms of locomotor activity, plasma glucose and digestive enzymes in the Nile tilapia (Oreochromis niloticus).

The light-dark cycle and feeding can be the most important factors acting as synchronizers of biological rhythms. In this research we aimed to evaluate synchronization to feeding schedule of daily rhythms of locomotor activity and digestive enzymes of tilapia. For that purpose, 120 tilapias (65.0±0.6g) were distributed in 12 tanks (10 fish per tank) and divided into two groups. One group was fed once a day at 11:00h (zeitgeber time, ZT6) (ML group) and the other group was fed at 23:00h (ZT18) (MD group). The fish were anesthetized to collect samples of blood, stomach and midgut at 4-hour intervals over a period of 24h. Fish fed at ML showed a diurnal locomotor activity (74% of the total daily activity occurring during the light phase) and synchronization to the feeding schedule, as this group showed anticipation to the feeding time. Fish fed at MD showed a disruption in the pattern of locomotor activity and became less diurnal (59%). Alkaline protease activity in the midgut showed daily rhythm with the achrophase at the beginning of the dark phase in both ML and MD groups. Acid protease and amylase did not show significant daily rhythms. Plasma glucose showed a daily rhythm with the achrophase shifted by 12h in the ML and MD groups. These results revealed that the feeding time and light cycle synchronize differently the daily rhythms of behavior, digestive physiology and plasma metabolites in the Nile tilapia, which indicate the plasticity of the circadian system and its synchronizers.

Daily Rhythms of the Expression of Key Genes Involved in Steroidogenesis and Gonadal Function in Zebrafish.

Fish present daily and seasonal rhythms in spawning and plasmatic levels of steroids that control reproduction. However, the existence of the rhythms of expression of the genes that underlie the endocrine mechanisms responsible for processes such as steroidogenesis and reproduction in fish have still been poorly explored to date. Here we investigated the daily pattern of the expression of key genes involved in sex steroid production that ultimately set the sex ratio in fish. Adult zebrafish were maintained under a 12:12 h light-dark cycle at a constant temperature of 27°C and were sampled every 4 h during a 24-hour cycle. The expression of key genes in the gonads and brains of female and male individuals were analyzed. In gonads, the expression of aromatase (cyp19a1a, ovarian aromatase) and the antimüllerian hormone (amh, testis) was rhythmic, with almost opposite acrophases: ZT 5:13 h (in the light phase) and ZT 15:39 h (at night), respectively. The expression of foxl2 (forkhead box L2) was also rhythmic in the ovary (acrophase located at ZT 5:02 h) and the expression of dmrt1 (doublesex and mab-3-related transcription factor 1) was rhythmic in testes (acrophase at ZT 18:36 h). In the brain, cyp19a1b (brain aromatase) and cyp11b (11beta-hydroxylase) presented daily differences, especially in males, where the expression peaked at night. These results provide the first evidence for marked time-of-the-day-dependent differences in the expression of the genes involved in sex ratio control, which should be considered when investigating processes such as reproduction, sex differentiation and steroidogenesis in fish.

Synchronization to light and mealtime of the circadian rhythms of self-feeding behavior and locomotor activity of white shrimps (Litopenaeus vannamei).

The role of light and feeding cycles in synchronizing self-feeding and locomotor activity rhythms was studied in white shrimps using a new self-feeding system activated by photocell trigger. In experiment 1, shrimps maintained under a 12:12h light/dark (LD) photoperiod were allowed to self-feed using feeders connected to a photoelectric cell, while locomotor activity was recorded with a second photocell. On day 30, animals were subjected to constant darkness (DD) for 12days to check the existence of endogenous circadian rhythms. In the experiment 2, shrimps were exposed to both a 12:12h LD photoperiod and a fixed meal schedule in the middle of the dark period (MD, 01:00h). On day 20, shrimps were exposed to DD conditions and the same fixed feeding. On day 30, they were maintained under DD and fasted for 7days. The results revealed that under LD, shrimps showed a clear nocturnal feeding pattern and locomotor activity (81.9% and 67.7% of total daily food-demands and locomotor activity, respectively, at nighttime). Both feeding and locomotor rhythms were endogenously driven and persisted under DD with an average period length (τ) close to 24h (circadian) (τ=24.18±0.13 and 23.87±0.14h for locomotor and feeding, respectively). Moreover, Shrimp showed a daily food intake under LD condition (1.1±0.2gday(-1) in the night phase vs. 0.2±0.1gday(-1) in the light phase). Our findings might be relevant for some important shrimp aquaculture aspects, such as developing suitable feeding management on shrimp farms.

Follow-me: A new start-and-stop method for visual animal tracking in biology research.

Very frequently, research in biology and ethology requires visual tracking of live animals, such as insects, rodents and fish. The challenge is particularly difficult in the aquatic environment because of the light scattering effect of water: reflections and refractions, low contrast and resolution images, noise, and also due to the unpredictable animal behavior. This paper describes a new tracking method that is based in start and stop detection. When the animals stop or move very slowly, a precise tracker based on AdaBoost classifiers is applied. Otherwise, when they move faster, a more efficient process based on background detection is used. A control system is responsible for deciding which method should be used in each instant. The experimental results using sea cucumbers (Echinodermata, Holothuroidea), zebrafish and rodents, show the efficiency and accuracy of the proposed method, which is able to deal with complex situations.

Effect of lighting conditions on zebrafish growth and development.

In the underwater environment, the properties of light (intensity and spectrum) change rapidly with depth and water quality. In this article, we have described how and to what extent lighting conditions can influence the development, growth, and survival of zebrafish. Fertilized eggs and the corresponding larvae were exposed to different visible light wavelengths (violet, blue, green, yellow, red, and white) in a 12-h light-12-h dark (LD) cycle until 30 days posthatching (dph), when the expression of morphometric parameters and growth (igf1a, igf2a)- and stress-related (crh and pomca) genes were examined. Another group of larvae was raised under constant darkness (DD) until 5 or 10 dph, after which they were transferred to a LD of white light. A third group remained under DD to investigate the effects of light deprivation upon zebrafish development. The results revealed that the hatching rate was highest under blue and violet light, while total length at 30 dph was greatest under blue, white, and violet light. Red light led to reduced feeding activity and poor survival (100% mortality). Larvae raised under constant white light (LL) showed a higher proportion of malformations, as did larvae raised under LD violet light. The expression of growth and stress factors was upregulated in the violet (igf1a, igf2a, pomca, and chr) and blue (igf2a) groups, which is consistent with the higher growth recorded and the higher proportion of malformations detected under the violet light. All larvae kept under DD died before 18 dph, but the survival rates improved in larvae transferred to LD at 5 dph and at 10 dph. In summary, these findings revealed that lighting conditions are crucial factors influencing zebrafish larval development and growth.

Comparative study of pineal clock gene and AANAT2 expression in relation to melatonin synthesis in Atlantic salmon (Salmo salar) and European seabass (Dicentrarchus labrax).

The photoreceptive teleost pineal is considered to be essential to the generation, synchronisation and maintenance of biological rhythms, primarily via melatonin release. The role of internal (circadian clock) and external (light) signals controlling melatonin production in the fish pineal differs between species, yet the reasons underpinning this remain largely unknown. Whilst in salmonids, pineal melatonin is apparently regulated directly by light, in all other studied teleosts, rhythmic melatonin production persists endogenously under the regulation of clock gene expression. To better understand the role of clocks in teleost pineals, this study aimed to characterise the expression of selected clock genes in vitro under different photoperiodic conditions in comparison to in vivo in both Atlantic salmon (Salmo salar) and in European seabass (Dicentrarchus labrax) (in vitro 12L:12D), a species known to display endogenous rhythmic melatonin synthesis. Results revealed no rhythmic clock gene (Clock, Period 1 &2) expression in Atlantic salmon or European seabass (Clock and Period 1) pineal in vitro. However rhythmic expression of Cryptochrome 2 and Period 1 in the Atlantic salmon pineal was observed in vivo, which infers extra-pineal regulation of clocks in this species. No rhythmic arylalkylamine N-acetyltransferase 2 (Aanat2) expression was observed in the Atlantic salmon yet in the European seabass, circadian Aanat2 expression was observed. Subsequent in silico analysis of available Aanat2 genomic sequences reveals that Atlantic salmon Aanat2 promoter sequences do not contain similar regulatory architecture as present in European seabass, and previously described in other teleosts which alludes to a loss in functional connection in the pathway.

Circadian rhythms of embryonic development and hatching in fish: a comparative study of zebrafish (diurnal), Senegalese sole (nocturnal), and Somalian cavefish (blind).

During early development, most organisms display rhythmic physiological processes that are shaped by daily changes in their surrounding environment (i.e., light and temperature cycles). In fish, the effects of daily photocycles and their interaction with temperature during early developmental stages remain largely unexplored. We investigated the existence of circadian rhythms in embryonic development and hatching of three teleost species with different daily patterns of behavior: diurnal (zebrafish), nocturnal (Senegalese sole), and blind, not entrained by light (Somalian cavefish). To this end, fertilized eggs were exposed to three light regimes: 12 h of light: 12 h of darkness cycle (LD), continuous light (LL), or continuous darkness (DD); and three species-appropriate temperature treatments: 24°C, 28°C, or 32°C for zebrafish and cavefish and 18°C, 21°C, or 24°C for sole. The results pointed to the existence of daily rhythms of embryonic development and hatching synchronized to the LD cycle, with different acrophases, depending on the species: zebrafish embryos advanced their developmental stage during the light phase, whereas sole did so during the dark phase. In cavefish, embryogenesis occurred within 24 h post fertilization (hpf) at the same pace during day or night. The hatching rhythms appeared to be controlled by a clock mechanism that restricted or "gated" hatching to a particular time of day/night (window), so that embryos that reached a certain developmental state by that time hatch, whereas those that have not wait until the next available window. Under LL and DD conditions, hatching rhythms and the gating phenomenon persisted in cavefish, in zebrafish they split into ultradian bouts of hatching occurring at 12-18-h intervals, whereas in sole DD and LL produced a 24-h delay and advance, respectively. Hatching rates were best under the LD cycle and the reported optimal temperature for each species (95.2±2.7% of the zebrafish and 83.3±0.1% of the cavefish embryos hatched at 28°C, and 93.1±2.9% of the sole embryos hatched at 21°C). In summary, these results revealed that hatching rhythms in fish are endogenously driven by a time-keeping mechanism, so that the day and time of hatching are determined by the interplay between the developmental state (temperature-sensitive) and the circadian clock (temperature-compensated), with the particular phasing being determined by the diurnal/nocturnal behavior of the species.