Periodic Report Summary 1 - MOULTING & CLIMATE (Integration of environmental signals in hormonal regulation of decapod crustaceans)
Project objectives
This project aims to improve our understanding for the causal relationship between long-term changes of environmental factors elicited by climate change, i.e. temperature and ocean acidification, and organismal performance of an ecologically and economically highly important group of aquatic arthropods, the decapod crustaceans. Physiological, biochemical and molecular methods are used to study how these environmental factors acutely and chronically affect hormonal regulation and the underlying cellular signalling mechanisms shaping the moulting event in relation to growth, acid-base and ion regulation in various life stages of species from temperate and subpolar latitudes. The species and their life stages are expected to show differences in their capacity for hormonal, acid-base and ion regulation, which may contribute to different thermal tolerances and sensitivities to climate change scenarios.
This project explicitly addresses drivers of the EU Strategy for Marine and Maritime Research, the Life+ Programme and the major theme “Environment (including Climate Change)” of the Cooperation Programme of FP7. The project is particularly useful in assessing and predicting vulnerabilities to the increasing environmental pressures on ocean ecosystems and biodiversity due to climate change.
Training in molecular cell biology and endocrinology at Colorado State University (CSU), Fort Collins, USA and Bodega Marine Laboratory (BML), UC Davis, USA significantly fosters the Marie Curie Fellow’s development and reintegration into the European Research Area and at the Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany.
Key objectives during the reporting period were
(1) to improve our understanding of how processes involved in moulting and growth are regulated in response to environmental factors (i.e. temperature) and
(2) to compare short-term and long-term effects of temperature on moult regulation to assess impacts of climate change on a crustacean model species.
The working hypothesis is that in the moulting gland (Y-organ) the mTOR (metazoan target of rapamycin) signalling pathway plays a major role in the regulation of production of moulting hormones (ecdysteroids), and that environmental stressors directly or indirectly inhibit the mTOR pathway, leading to a delay or inhibition of moulting (Figure 1).
Figure 1: Hypothetical model of the effect of acute and chronic temperature and CO2 exposure on moulting hormone production (ecdysteroidogenesis) in the moulting glands (Y-organs, YOs). During intermoult periodic MIH secretion from the XO/sinus gland complex in the eyestalk ganglia inhibits ecdysteroid synthesis in the YO via the MIH and mTOR signalling pathways. Reduction of MIH activates the YO, sensitivity of the YO to MIH decreases, and ecdysteroid synthesis starts in premoult. Progression through late premoult and ecdysis is hampered by thermal and CO2 stress induced by reduced oxygen and energy supply (reduced ATP:ADP ratio). This mismatch in energy supply and demand activates AMPK, which acutely inactivates enzymes involved in ecdysteroidogenesis. During chronic environmental stress, synthesis of these enzymes is reduced by inhibition of the mTOR pathway by AMPK. This results in only low levels of ecdysteroid secretion and inhibition of moulting. MIH: moult inhibiting hormone, mTOR: metazoan target of rapamycin, AMPK: AMP-dependent protein kinase.
Work performed
In a first approach, two shorter termed experiments were carried out using juvenile Dungeness crabs (Metacarcinus magister) from Bodega Harbour, CA, USA. The crabs were exposed to a range of constant seawater temperatures for up to two weeks to determine survival and progression of the moulting cycle as well as to collect haemolymph and tissue samples of crabs in four moult stages. A second group of crabs was incubated at an extremely high temperature for three days, a challenging yet sub-lethal condition. Again, haemolymph and tissues were sampled. In a follow-up study, juvenile M. magister were exposed to four water temperatures for up to 56 days and sampled at different moult stages to investigate long-term effects of temperature on moulting and growth. At the highest experimental temperature of this study, most of the crabs survived, but moulting was inhibited in a large portion of the animals.
Haemolymph ecdysteroid concentrations were determined using a competitive enzyme-linked immunosorbent assay (ELISA). In order to quantify the expression of genes possibly involved in moult regulation, first PCR cloning was used to obtain partial nucleotide sequences of M. magister AMPK α-subunit, AMPK γ-subunit, mTOR components (mTOR, Rheb, S6K, AKT), and the reference gene EF2, as well as Crustacean Hyperglycaemic Hormone (CHH) from eyestalk ganglia. Absolute quantification of mRNA expression of MIH, CHH, AMPK and mTOR signalling components in eyestalks (XO/sinus gland complex), and AMPK and mTOR components in Y-organs and heart was carried out using quantitative real-time PCR.
Main results
The data support the hypothesis that the mTOR pathway plays a major role in the hormonal regulation of the moulting cycle during thermal acclimation of the studied animal model. As expected, moult interval was dependent on temperature. Moulting was delayed or inhibited outside the temperature range the juveniles currently experience in the environment of Bodega Harbour. Because moulting is imperative to long-term growth, this suggests that future climate change will result in changes of growth performance and/or habitat choice and distribution of the animals.
Expected final results and potential impact and use
Very little is known about the mechanisms by which environmental factors affect growth and development of decapod crustaceans on long time scales. Since climate change, a major global societal challenge, means that multiple factors change at the same time, interactive effects need to be investigated. This project addresses one of the greatest challenges to biologists in the 21st century: to understand the underlying mechanisms of perception of environmental change and subsequent transduction and combination to a physiological response. Decapod crustaceans are important components of ecological food webs as well as of fisheries and aquaculture economies. Understanding the hormonal regulation of moulting and growth is essential to be able to understand, project and mitigate impacts of climate change on native and invasive decapod species and on the ecosystems and economies that rely on them. So far, too few species of this very diverse group have been studied - especially from subpolar latitudes, in long-term experiments and in response to multiple factors like temperature and ocean acidification - to make for good predictions and management plans. In its integrative nature, this project is highly appropriate to tackle these shortcomings and to foster excellence of the European Research Area. It will “substantially strengthen the knowledge base for conservation and sustainable use of biodiversity, in the EU and globally”, an objective of FP7 formulated by the European Commission. This study elucidates mechanisms and develops methods that are also applicable in other important crustacean groups like krill and copepods of the zooplankton. The expected results will thus support high-level future research in this fascinating field within the marine sciences. The outcomes of this research will offer advanced methods to further investigate and predict effects of climate change in additional crustacean or even arthropod taxa.
This project aims to improve our understanding for the causal relationship between long-term changes of environmental factors elicited by climate change, i.e. temperature and ocean acidification, and organismal performance of an ecologically and economically highly important group of aquatic arthropods, the decapod crustaceans. Physiological, biochemical and molecular methods are used to study how these environmental factors acutely and chronically affect hormonal regulation and the underlying cellular signalling mechanisms shaping the moulting event in relation to growth, acid-base and ion regulation in various life stages of species from temperate and subpolar latitudes. The species and their life stages are expected to show differences in their capacity for hormonal, acid-base and ion regulation, which may contribute to different thermal tolerances and sensitivities to climate change scenarios.
This project explicitly addresses drivers of the EU Strategy for Marine and Maritime Research, the Life+ Programme and the major theme “Environment (including Climate Change)” of the Cooperation Programme of FP7. The project is particularly useful in assessing and predicting vulnerabilities to the increasing environmental pressures on ocean ecosystems and biodiversity due to climate change.
Training in molecular cell biology and endocrinology at Colorado State University (CSU), Fort Collins, USA and Bodega Marine Laboratory (BML), UC Davis, USA significantly fosters the Marie Curie Fellow’s development and reintegration into the European Research Area and at the Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany.
Key objectives during the reporting period were
(1) to improve our understanding of how processes involved in moulting and growth are regulated in response to environmental factors (i.e. temperature) and
(2) to compare short-term and long-term effects of temperature on moult regulation to assess impacts of climate change on a crustacean model species.
The working hypothesis is that in the moulting gland (Y-organ) the mTOR (metazoan target of rapamycin) signalling pathway plays a major role in the regulation of production of moulting hormones (ecdysteroids), and that environmental stressors directly or indirectly inhibit the mTOR pathway, leading to a delay or inhibition of moulting (Figure 1).
Figure 1: Hypothetical model of the effect of acute and chronic temperature and CO2 exposure on moulting hormone production (ecdysteroidogenesis) in the moulting glands (Y-organs, YOs). During intermoult periodic MIH secretion from the XO/sinus gland complex in the eyestalk ganglia inhibits ecdysteroid synthesis in the YO via the MIH and mTOR signalling pathways. Reduction of MIH activates the YO, sensitivity of the YO to MIH decreases, and ecdysteroid synthesis starts in premoult. Progression through late premoult and ecdysis is hampered by thermal and CO2 stress induced by reduced oxygen and energy supply (reduced ATP:ADP ratio). This mismatch in energy supply and demand activates AMPK, which acutely inactivates enzymes involved in ecdysteroidogenesis. During chronic environmental stress, synthesis of these enzymes is reduced by inhibition of the mTOR pathway by AMPK. This results in only low levels of ecdysteroid secretion and inhibition of moulting. MIH: moult inhibiting hormone, mTOR: metazoan target of rapamycin, AMPK: AMP-dependent protein kinase.
Work performed
In a first approach, two shorter termed experiments were carried out using juvenile Dungeness crabs (Metacarcinus magister) from Bodega Harbour, CA, USA. The crabs were exposed to a range of constant seawater temperatures for up to two weeks to determine survival and progression of the moulting cycle as well as to collect haemolymph and tissue samples of crabs in four moult stages. A second group of crabs was incubated at an extremely high temperature for three days, a challenging yet sub-lethal condition. Again, haemolymph and tissues were sampled. In a follow-up study, juvenile M. magister were exposed to four water temperatures for up to 56 days and sampled at different moult stages to investigate long-term effects of temperature on moulting and growth. At the highest experimental temperature of this study, most of the crabs survived, but moulting was inhibited in a large portion of the animals.
Haemolymph ecdysteroid concentrations were determined using a competitive enzyme-linked immunosorbent assay (ELISA). In order to quantify the expression of genes possibly involved in moult regulation, first PCR cloning was used to obtain partial nucleotide sequences of M. magister AMPK α-subunit, AMPK γ-subunit, mTOR components (mTOR, Rheb, S6K, AKT), and the reference gene EF2, as well as Crustacean Hyperglycaemic Hormone (CHH) from eyestalk ganglia. Absolute quantification of mRNA expression of MIH, CHH, AMPK and mTOR signalling components in eyestalks (XO/sinus gland complex), and AMPK and mTOR components in Y-organs and heart was carried out using quantitative real-time PCR.
Main results
The data support the hypothesis that the mTOR pathway plays a major role in the hormonal regulation of the moulting cycle during thermal acclimation of the studied animal model. As expected, moult interval was dependent on temperature. Moulting was delayed or inhibited outside the temperature range the juveniles currently experience in the environment of Bodega Harbour. Because moulting is imperative to long-term growth, this suggests that future climate change will result in changes of growth performance and/or habitat choice and distribution of the animals.
Expected final results and potential impact and use
Very little is known about the mechanisms by which environmental factors affect growth and development of decapod crustaceans on long time scales. Since climate change, a major global societal challenge, means that multiple factors change at the same time, interactive effects need to be investigated. This project addresses one of the greatest challenges to biologists in the 21st century: to understand the underlying mechanisms of perception of environmental change and subsequent transduction and combination to a physiological response. Decapod crustaceans are important components of ecological food webs as well as of fisheries and aquaculture economies. Understanding the hormonal regulation of moulting and growth is essential to be able to understand, project and mitigate impacts of climate change on native and invasive decapod species and on the ecosystems and economies that rely on them. So far, too few species of this very diverse group have been studied - especially from subpolar latitudes, in long-term experiments and in response to multiple factors like temperature and ocean acidification - to make for good predictions and management plans. In its integrative nature, this project is highly appropriate to tackle these shortcomings and to foster excellence of the European Research Area. It will “substantially strengthen the knowledge base for conservation and sustainable use of biodiversity, in the EU and globally”, an objective of FP7 formulated by the European Commission. This study elucidates mechanisms and develops methods that are also applicable in other important crustacean groups like krill and copepods of the zooplankton. The expected results will thus support high-level future research in this fascinating field within the marine sciences. The outcomes of this research will offer advanced methods to further investigate and predict effects of climate change in additional crustacean or even arthropod taxa.