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Pigmentation appears first on the yolk sac and later on the body axis. Somitogenesis starts before the end of epiboly. At the anterior end of the embryo, the brain primordium, without visible morphological subdivisions, and the optic primordia, which evaginates from the future diencephalon part of the brain primordium are visible. The otic acoustic vesicle is forming in the posterior head region. At the somite stage, epiboly is finished and the entire yolk is covered by blastoderm. At the somite stage, the first melanophores appear on top of the yolk sac as well as in the posterior part of the embryo.

The tail extends further and starts to curl inside the chorion. At somite stage, the lens primordium can easily be seen. The eye and lens have expanded in size and the head thickens due to brain growth. Muscle contractions become more frequent. Because hatching is variable, it is not particularly useful as a staging index. The tail is still curled and the head is bent around the yolk. The rudiments of all organs are present and their morphogenesis organogenesis continues until the end of the first week of development.

Abbreviations: a, anus; ag, adhesive glands; at, atrium; dm, dorsal melanophore stripe ; em, eye melanophores; ffv, fin fold veins; fr, fin rays; gi, gills; he, heart; hb, hindbrain; hm, head melanophores; lj, lower jaw; mhb, midbrain-hindbrain boundary; mo, mouth opening; my, myomeres; ov, otic vesicle; pfb, pectoral fin bud; rp, rays primordia; sb, swim bladder; v, ventricle; vm, ventral melanophore stripe. Abbreviations: agp, adhesive gland primordium; at, atrium; av, atrio-ventricular valve; cb, cerebellum; dag, dorsal adhesive gland; fb, forebrain; gi, gills; he, heart; ht, heart tube; ir, iridophores; l, lens; lj, lower jaw; hm, head melanophores; mb, midbrain; mhb, midbrain-hindbrain boundary; ov, otic vesicle; pfb, pectoral fin bud; rh, rhombomeres; v, ventricle; vag, ventral adhesive gland.

Abbreviations: dm, dorsal melanophore stripe ; dx, dorsal xanthophores; hm, head melanophores; hx, head xanthophores; ffv, fin fold veins; fr, fin rays; my, myomeres; vm, ventral melanophore stripe.

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A-C, F, G. In contrast to zebrafish, but similarly to medaka, stickleback and other cichlids [ 24 , 26 , 29 , 44 ], the yolk has no posterior extension. In zebrafish, a small projection of the yolk extends posteriorly up towards the anal region [ 22 ]. Still, the yolk sac is not completely round, and forms a cone-like tip at the posterior end.

Molecular Isotopic Distribution Analysis (MIDAs) with Adjustable Mass Accuracy

Further posterior to the end of the yolk sac, the digestive tract and anus can be seen. Shortly after hatching, the adhesive gland apparatus forms. They are used by the larvae to attach to the substrate and to each other before they reach the free-swimming stage, as previously described in cichlids [ 29 , 45 , 46 ] and the cave fish Astyanax mexicanus [ 46 ]. Under the described laboratory conditions in petri dishes, larvae mainly stick to particles such as remnants of the chorion that remain in the dish after hatching. Thereby, groups of larvae may all connect to a single particle and group together.

The head starts to lift from the yolk. The pectoral fins can be seen as elongated blade-shaped tissues projecting dorsally from the yolk. Also, the heart can now be morphologically separated into the ventricular and atrial chamber, separated by the atrio-ventricular valve.


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More melanophores form dorsally and appear for the first time in the head region. Also the first few xanthophores start to appear after day six, and can be detected under UV light see Methods. They form both dorsally to the yolk and in the head region. The larva further increases in size, and the gills can be seen more clearly than in previous stages.

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Some of them project dendrites dorsally into the space between two myomeres. They appear colorless until day seven both in reflected and under transmitted light, and can only be detected using UV-light see Methods.


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The heart is now fully developed and can be divided into the sinus venosus, atrium, ventricle, and bulbus arteriosus [ 47 , 48 ]. Between days seven and eight, the swim bladder inflates and the larva begins to swim upright. The early development of the Midas cichlid is slower than that of teleost genetic models such as medaka and zebrafish.


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  4. We compared the homologous developmental stages to the South American cichlid Cichlasoma dimerus and the zebrafish Danio rerio. In particular, the rate of epiboly seems to be decelerated compared to zebrafish, a phenomenon that might be related to the large egg size. The influence of temperature on developmental rate is far greater than in zebrafish, where there is only a 1. In Midas cichlids, the difference is 1. Despite this, later developmental stages seem to be less affected by temperature, with only minor differences in hatching time, development of pectoral fins and mouth opening between C.

    The effects of temperature on developmental time. Developmental rates in D. Summary of Midas cichlid development and comparison to the development of other teleosts. B Comparison between this study and three further studies on teleosts including C. We describe the embryonic and larval development of Amphilophus xiloaensis as a representative of the Midas cichlid species complex. Midas cichlids are an excellent example of rapid adaptation and fast speciation in sympatry [ 49 , 50 ] and parallel evolution [ 19 , 51 ].

    The rate of divergence in many traits, including coloration and pigmentation, morphology of body shapes, lips, jaws and teeth as well as neural systems such as vision is much higher in cichlid fishes than in most other vertebrate groups [ 2 , 19 , 52 ]. Studies from other cichlids [ 21 , 41 , 54 - 60 ] and from sticklebacks [ 61 - 63 ] suggest that a few mutations of major effect are expected to play important roles in driving phenotypic richness and ecological diversity.

    The mutation rate of Midas cichlids has been estimated to be between 6. Although most of these phenotypes arise later during ontogeny, differences might be already detectable on a subtle morphological and gene expression level — especially for craniofacial phenotypes associated with benthic-limnetic differences, as recently shown in other species e. To examine if inter-species morphological and gene expression differences are indeed already present at early stages of Midas cichlid development, a standardization of embryonic timing and a comprehensive - and comparative -staging system are necessary as a baseline for hypothesis-driven research in this field.

    This staging forms the basis for future comparative developmental work on different species of Midas cichlids and closely related Neotropical cichlids [ 16 ]. Since development is greatly influenced by temperature, easily recognizable landmarks, along with a standardized temperature-time protocol, must be defined.

    This will ease the collection of comparable stages for molecular biological experiments, such as in situ hybridization or RNA extraction. In Midas cichlids, embryonic traits such as the prominent adhesive glands and the early melanophore and xanthophore patterns differ from other model teleosts such as medaka and zebrafish. Adhesive glands have recently been studied in the cavefish Astyanax mexicanus divergence time approximately million years [ 77 ] and described in other cichlids [ 29 , 45 , 46 ], but are not present in medaka and zebrafish.

    The embryonic melanophore patterns we observe are very different from model teleosts such as medaka and zebrafish [ 22 , 23 , 44 ]. The prominent dorsal and lateral melanophore stripes are almost completely absent in Midas cichlid embryos, suggesting different migration patterns of neural crest cells, which are thought to generate all but the yolk melanophore in teleosts [ 78 ].

    It has been proposed that some melanophores migrate from the yolk to populate the embryo, especially in the ventral zone; however, there has been some controversy surrounding this claim [ 32 ]. Further histological analysis using neural crest markers could solve this controversy and clarify the genetic cause of the different melanophore patterns observed in Midas cichlids.

    The embryos of substrate brooders such as the Midas cichlid are also easier to use for genetic manipulations such as transgenesis than the massive, yolky eggs of mouth-brooding cichlids from the African Great Lakes, although these were the first species in which transgenesis was successfully performed [ 79 , 80 ]. Large clutch sizes and the slow development before the one-cell stage allow for the injection of considerable amounts of eggs.

    This method will allow researchers to transiently map the influence of gene overexpression or the activity of regulatory elements. Additionally, genetic manipulations by morpholinos or CRISPR-Cas, both of which have been shown to work in the Nile tilapia [ 81 , 82 ], might also be applicable in the Midas cichlid. Transcriptomic and genomic data sets, including a high-quality draft genome of A.

    One limitation is the long generation time - about nine to twelve months under laboratory conditions. Despite this drawback, it may still be possible to generate stable transgenes or knockouts, which would be relevant for experiments in Neotropical, substrate-brooding cichlids.

    Furthermore, the Midas cichlid could also serve as an excellent outgroup for the African cichlid species flocks, allowing for functional screens or assays of genes and cis-regulatory elements [ 83 , 84 ]. As shown here, Midas cichlids can be easily maintained, bred, stripped and raised in large numbers under laboratory conditions. It is still not fully understood which genes and mutations underlie the parallel evolution of traits and the quickly-evolving species richness exhibited by Midas cichlids. This study adds valuable information about the course of early development to help tackle questions about the molecular basis of phenotypic novelties from an evolutionary-developmental, evo-devo angle.

    The staging system in a representative Midas cichlid species will serve as a foundation for future experiments and ease interspecies comparisons. It will help to reproducibly select standardized developmental stages during development to analyze gene function and differences in gene expression and patterns. Midas cichlids have embryonic traits adhesive glands, melanophore patterns that differ from the classical developmental model teleosts, medaka and zebrafish.

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    It will be interesting to analyze the genetic causes of these differences. We also propose the Midas cichlid as a new model organism for evolutionary developmental research. In addition to the availability of genetic resources and the possibility to perform functional experiments, Midas cichlids and their adaptively relevant phenotypic diversity are well-described from an ecological and evolutionary standpoint. These advantages, taken together, make this system very attractive for evolutionary-developmental questions. Two to five pairs are usually kept per tank to minimize aggressive behavior while maximizing reproductive success.

    Gravel was used as a substrate for the tanks.

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    Cichlids are able to use the pots to hide, reducing stress and the frequency of attacks between fish. Pairs usually occupy one of the pots as their territory. If eggs are not removed by stripping, the female deposits her eggs on the inside of the pots, where the male fertilizes them. Specimens analyzed in this study were obtained both by regular spawning eggs can be easily removed from pots and stripping combined with in vitro fertilization. To obtain eggs, it is crucial that the fish are stripped at the right time. As soon as couples pair up and begin to defend their territory, the female must be checked daily for further behavioral and physiological changes.

    A few days before spawning, both the male and female become more aggressive. Females showing these signs were removed from the water with a net and the eggs were stripped by applying light pressure to the abdominal region anterior of the genital papilla, followed by a slight squeezing movement towards the genital pore. Eggs should come out easily; if not, the female is not yet ready to spawn. If only a few eggs come out, it is likely that the eggs are not yet mature. Between and eggs can be obtained using this method averaging around Females spawn regularly every four to six weeks throughout the year.

    Since there are no clear external signs to indicate the maturity of the males, we usually obtained sperm from one to three males. Stripping was performed using the same method as for females.

    Introduction

    After fertilization, eggs were kept for five minutes in the petri dish, which is sufficient for successful fertilization. Next, eggs were transferred into a new dish containing clean, autoclaved tank water. To visualize xanthophores in developing Midas cichlids, we used a modified version of the method described for zebrafish and African cichlids [ 85 , 86 ].