Project description:Diapause is a reversible developmental arrest faced by many organisms in harsh environments. Annual killifish present this mechanism in three possible stages of development. Killifish are freshwater teleosts from Africa and America that live in ephemeral ponds, which dry up in the dry season. The juvenile and adult populations die, and the embryos remain buried in the bottom mud until the next rainy season. Thus, species survival is entirely embryo-dependent, and they are perhaps the most remarkable extremophile organisms among vertebrates. The aim of the present study was to gather information about embryonic diapauses with the use of a “shotgun” proteomics approach in diapause III and prehatching Austrolebias charrua embryos. Our results provide insight into the molecular mechanisms of diapause III. We detected a diapause-dependent change in a large group of proteins involved in different functions, such as metabolic pathways and stress tolerance, as well as proteins related to DNA repair and epigenetic modifications. Furthermore, we observed a diapauseassociated switch in cytoskeletal proteins. This first glance into global protein expression differences between prehatching and diapause III could provide clues regarding the induction/maintenance of this developmental arrest in A. charrua embryos. There appears to be no single mechanism underlying diapause and the present data expand our knowledge of the molecular basis of diapause regulation. This information will be useful for future comparative approaches among different diapauses in annual killifish and/or other organisms that experience developmental arrest.
Project description:We evaluate the global chromatin changes that occur during the developmental progression and development-to-diapause transition in a closely related set of killifish species.
Project description:The present study used microarray expression profiling to determine the effects of embryonic arsenic exposure. Fertilized killifish (Fundulus heteroclitus) eggs were exposed to 0, 5, 15, or 25ppm arsenic as sodium arsenite. To examine differentially expressed genes, the microarrays were probed using RNA obtained from the control and 25ppm-exposed killifish just after hatching. No differences were noted in survival or hatching success between any of the groups. After analysis, a set of 332 genes was found to accurately distinguish between the control and 25ppm exposure groups. Expression of several of the genes (CDBP1, Arts1, FetB, and Fbp7) was quantified by qPCR in the lower exposure groups and at earlier time points to examine temporal and dose-responsive expression patterns. These results will enable us to better understand how arsenic impacts development. Killifish eggs were fertilized, divided into petri dishes containing 40 eggs (n=10 replicate petri dishes), and cultured until hatch in 0 or 25 ppm arsenic as sodium arsenite. Four to five hatchlings within each petri dish were pooled to obtain RNA. A total of 20 arrays were probed, 10 with RNA from control fish and 10 with RNA from the arsenic-exposed fish.
Project description:Suspended animation (e.g. hibernation, diapause) allows organisms to survive extreme environments. But the mechanisms underlying the evolution of suspended animation states are unknown. The African turquoise killifish has evolved diapause as a form of suspended development to survive the complete drought that occurs every summer. Here, we show that gene duplicates – paralogs – exhibit specialized expression in diapause compared to normal development in the African turquoise killifish. Surprisingly, paralogs with specialized expression in diapause are evolutionarily very ancient and are present even in vertebrates that do not exhibit diapause. To determine if evolution of diapause is due to the regulatory landscape rewiring at ancient paralogs, we assessed chromatin accessibility genome-wide in fish species with or without diapause. This analysis revealed an evolutionary recent increase in chromatin accessibility at very ancient paralogs in African turquoise killifish. The increase in chromatin accessibility is linked to the presence of new binding sites for transcription factors, likely due to de novo mutations and transposable element (TE) insertion. Interestingly, accessible chromatin regions in diapause are enriched for lipid metabolism genes, and our lipidomics studies uncover a striking difference in lipid species in African turquoise killifish diapause, which could be critical for long-term survival. Together, our results show that diapause likely originated by repurposing pre-existing gene programs via recent changes in the regulatory landscape. This work raises the possibility that suspended animation programs could be reactivated in other species for long-term preservation via transcription factor remodeling and suggests a mechanism for how complex adaptations evolve in nature.
Project description:The present study used microarray expression profiling to determine the effects of embryonic arsenic exposure. Fertilized killifish (Fundulus heteroclitus) eggs were exposed to 0, 5, 15, or 25ppm arsenic as sodium arsenite. To examine differentially expressed genes, the microarrays were probed using RNA obtained from the control and 25ppm-exposed killifish just after hatching. No differences were noted in survival or hatching success between any of the groups. After analysis, a set of 332 genes was found to accurately distinguish between the control and 25ppm exposure groups. Expression of several of the genes (CDBP1, Arts1, FetB, and Fbp7) was quantified by qPCR in the lower exposure groups and at earlier time points to examine temporal and dose-responsive expression patterns. These results will enable us to better understand how arsenic impacts development.
Project description:Adult reproductive diapause is a powerful overwintering strategy for many continental insect species including bumblebees, which enables queens to survive several months through harsh winter conditions and then build new beehives in the following spring. There are few reports regarding the molecular regulatory mechanism of reproductive diapause in Bombus terrestris, which is an important pollinators of wild plants and crops, and our previous researches identified the conditions for reproductive diapause of year-round mass rearing. Here, we performed combined RNA sequencing transcriptomics and quantitative proteomic analyses in different development phases relate to reproductive diapause. According to the overall analysis, we found these differentially expressed proteins/genes act in the citrate cycle, insect hormone biosynthesis, insulin and mTOR signalling pathway. To get better sense of the reproductive diapause regulated mechanism, some genes regulated JH synthesis, insulin/ TOR signal pathway were detected, the BtRheb, BtTOR, BtVg and BtJHAMT had lower expression levels in diapause queens, and the JH III titers levels and some metabolic enzymes activities were significantly up-regulated in found post-diapause queens. After microinjected insulin-like peptides (ILPs) and JH analog (JHA), some indicators shows the significantly changes of hormones, cold tolerance substances, metabolic enzymes and reproduction. Along with other related researches, a reproductive diapause regulated model during B. terrestris year-round mass rearing process was establishment. This study contribute to a comprehensive view and the molecular regulate mechanism of productive diapause in eusocial insect.
Project description:The most common ladybird beetle, Coccinella septempunctata L., is an excellent predator of crop pests such as aphids and white flies, and it shows a wide range of adaptability, a large appetite and a high reproductive ability. In this study, we collected female adults in three different states, i.e., non-diapause, diapause and diapause termination, for transcriptome sequencing. The experimental insects consisted of three different states as follows: Non-diapause female insects were reared at 24±1°C, with a RH of 70±10% and a 16:8 h light: dark (L: D) photoperiod and collected after their first oviposition. Female adults in diapause were reared at 18±1°C at an RH of 70±10% and a 10:14-h (L:D) photoperiod. The experimental diapause insects were collected after 30 days. Diapause-terminated adults were transferred to another climatic cabinet with the 30-day diapause insects and reared under the same conditions as the non-diapause insects. After their first oviposition, the female insects were collected and stored at -80°C. Three biological replicates per treatment (non-diapause, diapause, diapause-terminated) were sequenced using Illumina HiSeq 2500.
Project description:Most northern insect species experience a period of developmental arrest, diapause, which enables them to survive over the winter and postpone reproduction until favorable conditions. We studied the timing of reproductive diapause and its long-term effects on the cold tolerance of Drosophila montana, D. littoralis and D. ezoana females in seasonally varying environmental conditions. At the same time we traced expression levels of 219 genes in D. montana using a custom-made microarray. We show that the seasonal switch to reproductive diapause occurs over a short time period, and that overwintering in reproductive diapause has long-lasting effects on cold tolerance. Some genes, such as Hsc70, Jon25bi and period, were upregulated throughout the diapause, while others, including regucalcin, couch potato and Thor, were upregulated only at its specific phases. Some of the expression patterns induced during the sensitive stage, when the females either enter diapause or not, remained induced regardless of the later conditions. qPCR analyses confirmed the findings of the microarray analysis in D. montana and revealed similar gene expression changes in D. littoralis and D. ezoana. The present study helps to achieve a better understanding of the genetic regulation of diapause and of the plasticity of seasonal responses in general. Custom made DNA microarray for Drosophila montana and D. virilis. Current experiment includes 8 samples (7 to 250 days old diapausing or non-diapausing D. montana females) with two or three biological replicates