Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

Background The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals. Results Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish. Conclusions The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration

†Kenyaichthyidae fam. nov and †Kenyaichthys gen. nov - First Record of a Fossil Aplocheiloid Killifish (Teleostei, Cyprinodontiformes)

The extant Cyprinodontiformes (killifishes) with their two suborders Cyprinodontoidei and Aplocheiloidei represent a diverse and well-studied group of fishes. However, their fossil record is comparatively sparse and has so far yielded members of the Cyprinodontoidei only. Here we report on cyprinodontiform fossils from the upper Miocene Lukeino Formation in the Tugen Hills of the Central Rift Valley of Kenya, which represent the first fossil record of an aplocheiloid killifish. A total of 169 specimens - mostly extraordinarily well preserved and a sample of ten extant cyprinodontiform species were studied on the basis of morphometrics, meristics and osteology. A phylogenetic analysis using PAUP was also conducted for the fossils. Both the osteological data and the phylogenetic analysis provide strong evidence for the assignment of the fossils to the Aplocheiloidei, and justify the definition of the new family dagger Kenyaichthyidae, the new genus dagger Kenyaichthys and the new species dagger K. kipkechi sp. nov. The phylogenetic analysis unexpectedly places dagger Kenyaichthys gen. nov. in a sister relationship to the Rivulidae (a purely Neotropical group),a probable explanation might be lack of available synapomorphies for the Rivulidae, Nothobranchiidae and Aplocheilidae. The specimens of dagger K. kipkechi sp. nov. show several polymorphic characters and large overlap in meristic traits, which justifies their interpretation as a species flock in statu nascendi. Patterns of variation in neural and haemal spine dimensions in the caudal vertebrae of dagger Kenyaichthys gen. nov. and the extant species studied indicate that some previously suggested synapomorphies of the Cyprinodontoidei and Aplocheiloidei need to be revised

Lack of essential fatty acids in live feed during larval and post-larval rearing: effect on the performance of juvenile Solea senegalensis

Despite the large progress obtained in recent years, Senegalese sole (Solea senegalensis) production of high quality juveniles is still a bottleneck. This paper examines the effect of larval and post-larval lipid nutrition on juvenile performance and quality. Four dietary treatments were tested: A—enriched Artemia spp. (EA); B—non-enriched Artemia spp. (NEA); C—EA during the pelagic larval period and NEA after larval settlement; D—50% EA and 50% NEA. Juvenile fatty acid profile at 60 days after hatching (DAH) clearly reflected the larval and post-larval diet composition. Feeding sole larvae on NEA (poor in lipids and essential fatty acids-EFA) had a negative effect, reducing growth (total length and dry weight) after 30 DAH and decreasing digestive enzyme activity at the end of the rearing period (60 DAH). However, relatively good performance compared to the EFA-richest treatment (A) was obtained when larvae were fed 50% EA and 50% NEA (D) or even EA only during the pelagic larval period followed by NEA after larval settlement (C). Malpigmentation was not affected by the dietary regimes and its incidence was very low. However, skeletal deformities were prevalent, particularly in the caudal complex, independently of diet. The results confirm that Senegalese sole appear to have lower larval EFA requirements than most cultured marine species and potentially even lower requirements during the post-larval stage. The importance of studying the impact of early nutrition on later juvenile stages was clearly highlighted in this study

Marine green macroalgae: a source of natural compounds with mineralogenic and antioxidant activities

Marine macroalgae represent a valuable natural resource for bioactive phytochemicals with promising applications in therapeutics, although they remain largely under-exploited. In this work, the potential of two marine green macroalgae (Cladophora rupestris and Codium fragile) as a source of bioactive phenolic compounds was explored, and antioxidant, mineralogenic, and osteogenic activities were evaluated. For each species, a crude hydroalcoholic extract (CE) was prepared by solid/liquid extraction and fractionated by liquid/liquid purification into an ethyl acetate fraction (EAF) enriched in phenolic compounds and an aqueous fraction (AF). Antioxidant activity, assessed through radical scavenging activity and reducing power assay, was increased in EAF fraction of both species and closely related to the phenolic content in each fraction. Mineralogenic activity, assessed through extracellular matrix mineralization of a fish bone-derived cell line, was induced by EAF fractions (up to 600 % for C. rupestris EAF). Quantitative analysis of operculum formation in zebrafish larvae stained with alizarin red S further confirmed the osteogenic potential of EAF fractions in vivo, with an increase of more than 1.5-fold for both C. fragile and C. rupestris fractions, similar to vitamin D (control). Our results demonstrated a positive correlation between phenolic fractions and biological activity, suggesting that phenolic compounds extracted from marine green macroalgae may represent promising molecules toward therapeutic applications in the field of bone biology.European Regional Development Fund (ERDF)-Atlantic Area Programme through MARMED project [2011-1/164]European Era-Net, Seas-Era program through the project INVASIVES [ANR-12-SEAS-0002-01]Portuguese Foundation for Science and Technology (FCT) [UID/Multi/04326/2013