Laura Lyphout

Systematic Screening of Behavioral Responses in Two Zebrafish Strains

Wild-type (WT) zebrafish are commonly used in behavioral tests, but the term WT is not a precise description, and corresponds to many different strains (e.g., AB, TU, WIK, and others). Previous studies compared the physiological, behavioral, or metabolic characteristics of different zebrafish strains (indigenous WT populations versus laboratory WT strains). AB and TU are widely used, but at least one study has demonstrated behavioral differences between them. To choose the most appropriate strain for our experiments, we systematically screened behavioral responses of AB and TU fish in several assays. We analyzed the locomotion activity and responses to a light/dark challenge in adults and larvae, and exploratory behavior and color conditioning in adults. Differences were observed for all tests, the strains displaying particular behavior depending on the tests. As larvae, TU displayed a wider activity range than AB larvae at the onset of locomotor behavior; as adults, TU were more reactive to sudden light transitions and recovered the swimming activity faster in T-maze or homebase release in novel tank tests, whereas AB fish had more contrasted circadian rhythms and performed better in color learning. Strain-specific behavior should be considered when designing experiments using behavior.

Long-term food-exposure of zebrafish to PCB mixtures mimicking some environmental situations induces ovary pathology and impairs reproduction ability.

Although the use of polychlorinated biphenyls (PCBs) has been banned for several decades, they are still present in the environment and are occasionally mechanically released from sediment or transferred through the trophic chain. Field analyses have established correlations between exposure to PCBs and alterations in fish physiology including reproductive function. Experimental exposures have been mainly performed using dioxin-like PCBs or other congeners at very high concentrations. However, these studies are often difficult to relate to real-life conditions. In the present study, we performed a life-cycle exposure using zebrafish model and mixtures representative of some environmental situations in terms of doses, composition and containing mainly non dioxin-like congeners. Exposure was performed through diet which is the main contamination route in the field. We demonstrated a bioaccumulation of PCBs in males and females as well as a maternal transfer to the eggs. Survival, growth and organ size were similar for all conditions. Several reproductive traits were altered after exposure to a PCB-contaminated diet, including a reduction in the number of fertilized eggs per spawn as well as an increase of the number of poorly fertilized spawns. This latter observation was found irrespective of the sex of contaminated fish. This is related to modifications of ovary histology revealing a decrease of maturing follicles and an increase of atretic follicles in the ovaries of females exposed to PCBs. These results indicate that exposure to PCBs mixtures mimicking some environmental situations, including mainly non dioxin-like congeners, can lead to a dramatic reduction in the number of offspring produced by a female over a lifetime. This is of great concern for wild species living under natural conditions.

Long-term dietary-exposure to non-coplanar PCBs induces behavioral disruptions in adult zebrafish and their offspring

The use of polychlorinated biphenyls (PCBs) has been banned for several decades. PCBs have a long biological half-life and high liposolubility which leads to their bioaccumulation and biomagnification through food chains over a wide range of trophic levels. Exposure can lead to changes in animal physiology and behavior and has been demonstrated in both experimental and field analyses. There are also potential risks to high trophic level predators, including humans. A maternal transfer has been demonstrated in fish as PCBs bind to lipids in eggs. In this study, behavioral traits (exploration and free swimming, with or without challenges) of contaminated zebrafish (Danio rerio) adults and their offspring (both as five-day-old larvae and as two-month-old fish reared under standard conditions) were measured using video-tracking. Long-term dietary exposure to a mixture of non-coplanar PCBs was used to mimic known environmental contamination levels and congener composition. Eight-week-old fish were exposed for eight months at 26-28 °C. Those exposed to an intermediate dose (equivalent to that found in the Loire Estuary, ∑(CB)=515 ng g⁻¹ dry weight in food) displayed behavioral disruption in exploration capacities. Fish exposed to the highest dose (equivalent to that found in the Seine Estuary, ∑(CB)=2302 ng g⁻¹ dry weight in food) displayed an increased swimming activity at the end of the night. In offspring, larval activity was increased and two-month-old fish occupied the bottom section of the tank less often. These findings call for more long-term experiments using the zebrafish model; the mechanisms underlying behavioral disruptions need to be understood due to their implications for both human health and their ecological relevance in terms of individual fitness and survival.

Electronic individual identification of zebrafish using radio frequency identification (RFID) microtags.

SUMMARY Although individual electronic tagging using passive integrated acoustic (PIT) tags is established, it is mainly for fish >60 mm in length and is unsuitable for fish of <30 mm, like zebrafish. We used radio frequency identification (RFID) microtags (1 mm in diameter and 6 mm in length, with a mass of ~10 mg) to individually identify juvenile zebrafish (length 16–42 mm, mass 138–776 mg) for the first time, and studied the effects of intracoelomic implantation on fish survival and microtag loss, growth, spawning and exploratory behaviour. After 5.5 months, both high survival (82%) and low microtag loss (11%) were achieved. The smallest surviving fish weighed 178 mg, and success in microtag reading was 73% for the size class 350–450 mg (26 mm). Greater success was achieved when fish were larger at the time of tagging but no negative effects on growth were observed for any size class and some tagged fish spawned. No significant differences in behavioural responses could be detected between tagged fish and untagged controls after 2 months. Overall, the results suggest that the tagging method is highly suitable for fish as small as zebrafish juveniles. We think this method will provide significant advances for researchers of the ever-growing fish model community and more generally for all small-fish users. Tagging is essential when one needs to identify fish (e.g. particular genotypes with no external cue), to run longitudinal monitoring of individual biological traits (e.g. growth) or to repeat assays with the same individual at discrete points in time (e.g. behaviour studies). Such a method will find applications in physiology, genetics, behaviour and (eco)toxicology fields.

Exposures of zebrafish through diet to three environmentally relevant mixtures of PAHs produce behavioral disruptions in unexposed F1 and F2 descendant

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment has increased very substantially over the last decades. PAHs are hydrophobic molecules which can accumulate in high concentrations in sediments acting then as major secondary sources. Fish contamination can occur through contact or residence nearby sediments or though dietary exposure. In this study, we analyzed certain physiological traits in unexposed fish (F1) issued from parents (F0) exposed through diet to three PAH mixtures at similar and environmentally relevant concentrations but differing in their compositions. For each mixture, no morphological differences were observed between concentrations. An increase in locomotor activity was observed in larvae issued from fish exposed to the highest concentration of a pyrolytic (PY) mixture. On the contrary, a decrease in locomotor activity was observed in larvae issued from heavy oil mixture (HO). In the case of the third mixture, light oil (LO), a reduction of the diurnal activity was observed during the setup of larval activity. Behavioral disruptions persisted in F1-PY juveniles and in their offspring (F2). Endocrine disruption was analyzed using cyp19a1b:GFP transgenic line and revealed disruptions in PY and LO offspring. Since no PAH metabolites were dosed in larvae, these findings suggest possible underlying mechanisms such as altered parental signaling molecule and/or hormone transferred in the gametes, eventually leading to early imprinting. Taken together, these results indicate that physiological disruptions are observed in offspring of fish exposed to PAH mixtures through diet.

Allometric relationship between body mass and aerobic metabolism in zebrafish Danio rerio.

The relationship between body mass (M) and metabolic rate was investigated through the assessment of active (R(A)) and standard (R(S)) metabolic rate at different life stages in zebrafish Danio rerio (5 day-old larvae, 2 month-old juveniles and 6 month-old adults). Scaling exponents and constants were assessed for standard (R(S)  = 0·273M(0·965) in mgO(2)  g(-1)  h(-1)) and active metabolic rate (R(A)  = 0·799M(0·926) in mgO(2)  g(-1)  h(-1)). These data provide the basis for further experiments regarding the effects of environmental factors on aerobic metabolism throughout the life cycle of this species.

Trophic contamination by pyrolytic polycyclic aromatic hydrocarbons does not affect aerobic metabolic scope in zebrafish Danio rerio

The effect of trophic exposure to pyrolitic polycyclic aromatic hydrocarbons (PAH) on aerobic metabolism of zebrafish Danio rerio was investigated. There were no significant differences in standard metabolic rate (SMR), active metabolic rate (AMR) or aerobic metabolic scope (AS) at any sublethal concentration of PAH in the diet of adult or juvenile fish. This suggests that under these experimental conditions, exposure to PAH in food did not influence aerobic metabolism of this species.

Fish Reproduction Is Disrupted upon Lifelong Exposure to Environmental PAHs Fractions Revealing Different Modes of Action

Polycyclic aromatic hydrocarbons (PAHs) constitute a large family of organic pollutants emitted in the environment as complex mixtures, the compositions of which depend on origin. Among a wide range of physiological defects, PAHs are suspected to be involved in disruption of reproduction. In an aquatic environment, the trophic route is an important source of chronic exposure to PAHs. Here, we performed trophic exposure of zebrafish to three fractions of different origin, one pyrolytic and two petrogenic. Produced diets contained PAHs at environmental concentrations. Reproductive traits were analyzed at individual, tissue and molecular levels. Reproductive success and cumulative eggs number were disrupted after exposure to all three fractions, albeit to various extents depending on the fraction and concentrations. Histological analyses revealed ovary maturation defects after exposure to all three fractions as well as degeneration after exposure to a pyrolytic fraction. In testis, hypoplasia was observed after exposure to petrogenic fractions. Genes expression analysis in gonads has allowed us to establish common pathways such as endocrine disruption or differentiation/maturation defects. Taken altogether, these results indicate that PAHs can indeed disrupt fish reproduction and that different fractions trigger different pathways resulting in different effects.

G.P.19 Collagen VI genes in zebrafish skeletal muscle: Implications for collagen VI-myopathies

Abstract Collagen VI (COLVI) is a heterotrimeric protein, ubiquitously expressed in connective tissues. COLVI plays different roles such as maintenance of structural integrity, cellular migration and survival. To date, six COLVI chains are recognized with two short (α1–2(VI)) and three long chains (α3–6(VI)). Little is known regarding the possible assembly of the newly characterized α4–6(VI) polypeptides with the short chains, and a putative compensation between the different chains. In humans, deficiency in α1–3(VI) due to mutations in the COL6A1–3 genes causes COLVI-myopathies a heterogeneous group of neuromuscular disorders. To study the development of muscular tissue as well as the early steps of the disease, we turned to the zebrafish model where we identified only two orthologs of the α 4–6(VI) chains: col6a4a and col6a4b . We showed that the genes encoding the short chains are expressed at higher levels, and that only col6a4b was differentially expressed during development. We created COLVI deficient zebrafish embryos using morpholinos that block splicing of col6a2 , col6a4a and col6a4b , thereby creating premature termination codons. By qPCR, we demonstrated that the targeted transcripts were degraded, likely by the nonsense mediated RNA decay. At the morphological level, all morphant embryos had a curved body and showed severely impaired motility. Birefringence analysis and whole mount immunohistochemistry showed alteration of the muscle structure, with disorganized fibers and U-shaped myosepta. These alterations were confirmed at the ultrastructural level by electron microscopy. In conclusion, col6a2 deficient embryos recapitulate the severe end of the COLVI-myopathy phenotypical spectrum, thereby confirming the importance of col6 genes in muscle development. Furthermore, the phenotypes associated with α4a and α4b deficiency may help orientate group(s) of patients to screen for mutations in the human genes.