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In recent years, there has been a surge in interest in the effects of the microbiota on the host. Increasingly, we are coming to understand the importance of the gut microbiota in modulating host physiology, ecology, behavior, and evolution. One method utilized to evaluate the effect of the microbiota is to suppress or eliminate it, and compare the effect on the host with that of untreated individuals. In this study, we evaluate some of these commonly used methods in the model organism, Drosophila melanogaster. We test the efficacy of a low‐dose streptomycin diet, egg dechorionation, and an axenic or sterile diet, in the removal of gut bacteria within this species in a fully factorial design. We further determine potential side effects of these methods on host physiology by performing a series of standard physiological assays. Our results showed that individuals from all treatments took significantly longer to develop, and weighed less, compared to normal flies. Males and females that had undergone egg dechorionation weighed significantly less than streptomycin reared individuals. Similarly, axenic female flies, but not males, were much less active when analyzed in a locomotion assay. All methods decreased the egg to adult survival, with egg dechorionation inducing significantly higher mortality. We conclude that low‐dose streptomycin added to the dietary media is more effective at removing the gut bacteria than egg dechorionation and has somewhat less detrimental effects to host physiology. More importantly, this method is the most practical and reliable for use in behavioral research. Our study raises the important issue that the efficacy of and impacts on the host of these methods require investigation in a case‐by‐case manner, rather than assuming homogeneity across species and laboratories.

Tetrapods and fish have adapted distinct carbamoyl-phosphate synthase (CPS) enzymes to initiate the ornithine urea cycle during the detoxification of nitrogenous wastes. We report evidence that in the ureotelic subgenus of extremophile fish Oreochromis Alcolapia , CPS III has undergone convergent evolution and adapted its substrate affinity to ammonia, which is typical of terrestrial vertebrate CPS I. Unusually, unlike in other vertebrates, the expression of CPS III in Alcolapia is localized to the skeletal muscle and is activated in the myogenic lineage during early embryonic development with expression remaining in mature fish. We propose that adaptation in Alcolapia included both convergent evolution of CPS function to that of terrestrial vertebrates, as well as changes in development mechanisms redirecting CPS III gene expression to the skeletal muscle.

Although it is widely accepted that the cellular and molecular mechanisms of vertebrate cardiac development are evolutionarily conserved, this is on the basis of data from only a few model organisms suited to laboratory studies. Here, we investigate gene expression during cardiac development in the extremophile, non-model fish species, Oreochromis (Alcolapia) alcalica. We first characterise the early development of O. alcalica and observe extensive vascularisation across the yolk prior to hatching. We further investigate heart development by identifying and cloning O. alcalica orthologues of conserved cardiac transcription factors gata4, tbx5, and mef2c for analysis by in situ hybridisation. Expression of these three key cardiac developmental regulators also reveals other aspects of O. alcalica development, as these genes are expressed in developing blood, limb, eyes, and muscle, as well as the heart. Our data support the notion that O. alcalica is a direct-developing vertebrate that shares the highly conserved molecular regulation of the vertebrate body plan. However, the expression of gata4 in O. alcalica reveals interesting differences in the development of the circulatory system distinct from that of the well-studied zebrafish. Understanding the development of O. alcalica embryos is an important step towards providing a model for future research into the adaptation to extreme conditions; this is particularly relevant given that anthropogenic-driven climate change will likely result in more freshwater organisms being exposed to less favourable conditions.

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