Guido van den Thillart

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system

Significance Snake venoms are toxic protein cocktails used for prey capture. To investigate the evolution of these complex biological weapon systems, we sequenced the genome of a venomous snake, the king cobra, and assessed the composition of venom gland expressed genes, small RNAs, and secreted venom proteins. We show that regulatory components of the venom secretory system may have evolved from a pancreatic origin and that venom toxin genes were co-opted by distinct genomic mechanisms. After co-option, toxin genes important for prey capture have massively expanded by gene duplication and evolved under positive selection, resulting in protein neofunctionalization. This diverse and dramatic venom-related genomic response seemingly occurs in response to a coevolutionary arms race between venomous snakes and their prey. Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

The effects of cortisol administration on intermediary metabolism in teleost fish

Abstract 1. 1. This Mini Review deals with the metabolic consequences of administration of the hormone cortisol on proteins, carbohydrates and lipids in teleost fish. 2. 2. Many effects of administered cortisol on intermediary metabolism in fish have been reported: inhibition of protein synthesis and/or catabolism of tissue protein which result in higher availability of amino acids, induction of gluconeogenesis and of liver aminotransferases, hyperglycemia and glycogen deposition in the liver, induction of gluconeogenic enzymes, liberation of free fatty acids and deposition of liver lipids. All these effects are observed to a greater or less extent. However, the experimental data show that some effects are inconsistent. 3. 3. Some explanations for the inconsistencies are given.

Primitive duplicate Hox clusters in the European eel's genome.

The enigmatic life cycle and elongated body of the European eel (Anguilla anguilla L., 1758) have long motivated scientific enquiry. Recently, eel research has gained in urgency, as the population has dwindled to the point of critical endangerment. We have assembled a draft genome in order to facilitate advances in all provinces of eel biology. Here, we use the genome to investigate the eels complement of the Hox developmental transcription factors. We show that unlike any other teleost fish, the eel retains fully populated, duplicate Hox clusters, which originated at the teleost-specific genome duplication. Using mRNA-sequencing and in situ hybridizations, we demonstrate that all copies are expressed in early embryos. Theories of vertebrate evolution predict that the retention of functional, duplicate Hox genes can give rise to additional developmental complexity, which is not immediately apparent in the adult. However, the key morphological innovation elsewhere in the eels life history coincides with the evolutionary origin of its Hox repertoire.

Hypoxia tolerance of Amazon fish: Respirometry and energy metabolism of the cichlid Astronotus Ocellatus

Abstract As a result of regular flood pulses, the Amazon basin exhibits large annual changes in its chemical and physical parameters. Ecology and distribution of fish communities seem to be directed by seasonal and diurnal oxygen level oscillations. Amazon fish have developed strategies to thrive under these varying conditions. Astronotus ocellatus normally survives large fluctuations in oxygen availability in varzea lakes. Respirometric and metabolic changes in A. ocellatus were studied during exposure to stepwise declining oxygen levels. Respiration rates were continuously recorded. Haematologic and metabolic parameters (lactate, glucose, cortisol and free fatty acids (FFA)) were determined. A. ocellatus was found to be hypoxia tolerant; it survives more than 16 h of severe hypoxia (pO 2 ≤0.4 mg l −1 ) and even 4 h of complete anoxia at 28°C. Its routine metabolic rate is 10.8 mg O 2 h −1 per 100 g fresh weight. A significant decrease in standard metabolic rate (SMR) starts at 20% air saturation, whereas a significant change of blood lactate does not start until 6% air saturation in water. We suggest therefore that A. ocellatus responses to environmental hypoxia are based mainly on suppressed metabolic rate, whereas under deep hypoxia or anoxia partial compensation is obtained from anaerobic glycolysis.

Anaerobic production of carbon dioxide and ammonia by goldfish Carassius auratus (L.)

Abstract 1. 1. Groups of goldfish were held at complete anoxia at 20°C for about 10 hr. 2. 2. Before and during anoxia the production rates of CO2 and NH3 were measured. Both production rates were not influenced by the oxygen-availability. 3. 3. The anaerobic 14CO2 production after injection with [U−14C]glucose was 4 times the aerobic production. 4. 4. The influence of 10 hr of anoxia on whole fish levels of ammonia, carbon dioxide, lactate and glycogen was determined From the results can be concluded that glycogen is, at least partly, completely oxidized into CO2. 5. 5. The suggestion has been made that there must be an unknown electron acceptor involved in anaerobic energy production.

Anaerobic energy-metabolism of goldfish,Carassius auratus (L.)

SummaryThe concentrations of pyruvate, lactate, oxalo-acetate, aceto-acetate β-hydroxybutyrate, α-ketoglutarate, glutamate, NH4+, NAD+ and NADH were measured in goldfish tissues after previous conditioning to normal and anoxic (12h) conditions. For 11 different metabolites efficiency of different extraction methods was tested by means of internal standards. The recoveries were generally over 80%. The substrate/product couples of the reactions catalysed by lactate dehydrogenase, malate dehydrogenase, β-hydroxybutyrate dehydrogenase and glutamate dehydrogenase were used as redox parameters. In the lateral red muscle the redox state did not change during 12 h of anoxia. In the dorsal white muscle only the cytoplasmic redox state underwent a change, as indicated by the increase of the lactate/pyruvate ratio from 20 to 110. In liver both cytoplasm and mitochondria were reduced during anoxia. From the measured values the NAD+/NADH ratio was found to change only in white muscle, while the calculated free NAD+/NADH ratios were reduced in anoxic white muscle cytoplasm, anoxic liver mitochondria, and anoxic liver cytoplasm. Oxalo-acetate concentrations calculated from the equilibrium constants of lactate dehydrogenase and malate dehydrogenase were at least one order of magnitude smaller than the measured values. The data obtained from anoxic goldfish are in contrast to available data on other animals and support earlier reports which indicate that this animal has a special anaerobic metabolism. The results are discussed especially with respect to the role of ethanol as a sink for reducing equivalents.

Energy metabolism of swimming trout (Salmo gairdneri)

SummaryThe oxidation of 1-14C palmitate, 2-14C glucose, 1-14C lactate, 1-14C alanine, 1-14C leucine and 1-14C glutamate, injected via a cannula into the dorsal aorta, was determined in trout, either at rest, or during swimming at 80% of the maximum sustained speed. The oxygen consumption and the excretion rates of14CO2 as well as CO2 were measured.While the oxygen consumption of swimming trout was about twice as high as of resting trout, the oxidation rates of the injected tracers increased by up to 9 time. Despite the increased importance of blood borne substrates, the estimated contribution to total CO2 production is about 6% for the resting and 17% for the active trout. The majority of the oxidisable substrates must therefore be endogenous.The mobilization and oxidation rates of lactate, palmitate and leucine were particularly increased during swimming. During rest, palmitate and leucine oxidation rates are low. While oxidation rates of alanine and glutamate are intermediate, those of glucose were found to be extremely low, both at rest and during swimming. The measured RQ values for resting and swimming trout were 0.91 and 0.96 respectively, indicating that protein is the major fuel, since glucose oxidation seems of minor importance.

The effect of acclimation temperature on the activation energies of state III respiration and on the unsaturation of membrane lipids of goldfish mitochondria.

The influence of the acclimation temperature on the thermotropic behaviour of mitochondrial respiration and on the degree of unsaturation of mitochondrial membrane lipids has been studied. The mitochondria were isolated from red muscle, white muscle and liver of goldfish acclimated to 5, 20 and 30 degrees C. ADP-activated succinate oxidation was measured at different temperatures and resulted in non-linear Arrhenius-plots with breaks between 10 and 23 degrees C. As for the break-temperatures, there was found a shift downwards in preparations of decreased acclimation temperatures. This could be caused by a changed composition of membrane lipids and a simultaneous shift of the membrane phase transition temperature. Therefore, the fatty acid composition of all membrane preparations was analyzed. However, no consistent change of the degree of unsaturation due to a changed acclimation temperature could be found.

PCBs and the energy cost of migration in the European eel (Anguilla anguilla L.)

The effect of polychlorinated biphenyls (PCBs) on the energy consumption of fasting silver European eel (Anguilla anguilla L.) was studied over a 27-day period during which the animals were at rest or were swimming 800 km in Blazka swim tunnels. Three-year-old female hatchery eels (silver stage) between 73 and 80 cm long weighing around 1 kg were dosed intraperitoneally with PCBs at a nominal dosage of 10x the consumption standard as a mixture representative for planar (7 microg PCB126/kg eel), non-planar (5 mg PCB153/kg eel) and metabolizable PCBs (50 microg PCB77/kg eel) found in wild eel, or only with the vehicle (corn oil, 10 ml/kg eel). Four major observations were made: (1) PCB-exposed animals lose less weight compared to their unexposed controls; (2) PCB-concentrations on a lipid basis are 2.8-14 times higher in swimming compared to resting animals; (3) the standard metabolic rate is significantly lower in the PCB-exposed animals than in unexposed controls. In addition, PCB-exposure significantly reduces oxygen consumption during swimming, and starting at 400 km (18 days) this effect increases with time; (4) the relative spleen and liver weight significantly increased in the PCB-swim animals but not in the PCB-rest animals. The swimming animals lost about 75% more weight compared to resting animals and had about 50% lower plasma fat content. Hematocrit, haemoglobin, plasma pH, ion levels (sodium and potassium), and plasma lactate were not affected by PCB-exposure or swimming. Apparently, the current levels of PCBs and other dioxin-like compounds may seriously impair the reproduction of the European eel.

Physiology: Eel fat stores are enough to reach the Sargasso

It has long been assumed that the European eel (Anguilla anguilla) migrates to the Sargasso Sea — a region of the Atlantic Ocean between the Azores and the West Indies — to spawn1,2,3. During the past decade, however, the number of glass eels has inexplicably dropped4, and it has been suggested that a shortage of fat stores in adults, resulting from diminished food resources for juveniles in inland waters, may prevent the starving silver eels from reaching the spawning grounds4,5,6. But we find that the energetic cost of the 6,000-km migration is actually quite low, with 60% of the fat store remaining available for the developing gonads.It has long been assumed that the European eel (Anguilla anguilla) migrates to the Sargasso Sea — a region of the Atlantic Ocean between the Azores and the West Indies — to spawn. During the past decade, however, the number of glass eels has inexplicably dropped, and it has been suggested that a shortage of fat stores in adults, resulting from diminished food resources for juveniles in inland waters, may prevent the starving silver eels from reaching the spawning grounds. But we find that the energetic cost of the 6,000-km migration is actually quite low, with 60% of the fat store remaining available for the developing gonads.