A Cinderella Story

Keeping track of the literature isn’t easy, so Outside JEB is a monthly feature that reports the most exciting developments in experimental biology. Short articles that have been selected and written by a team of active research scientists highlight the papers that JEB readers can’t afford to miss. BITE FORCES iv Outside JEB SCIENCE TO GET ONE’S TEETH INTO How can you work out how hard a predatory mammal bites? Well, one way would be to place an instrumented prey item between the predator’s jaws, but this might impose an unacceptable risk to both life and limb of any intrepid investigator. A further problem arises if the animal you wish to investigate is extinct; clearly, any form of behavioural measurement becomes rather difficult. Undaunted by these complications, Stephen Wroe and coworkers sought to determine the scaling relationships of bite force and predator and prey body size in a variety of extant and extinct predatory mammals, in particular a number of marsupial predators. Wroe and his team hypothesised that bite forces should scale with both predator and prey size and that different modes of feeding, such as bone crunching versus only eating flesh, should be reflected in patterns of bite forces. To determine bite forces of different predators, the team examined the skulls of 39 predatory mammal species (eight of which are extinct). To calculate the maximal theoretical bite force for each species, the authors used a method that models jaws as simple levers. They estimated muscular power to the jaw by measuring the area of the muscular insertions on the jaw. Finally, they determined the distance between the jaw articulations, muscle attachments and the position of the canine and carnassial teeth. By calculating the torque applied to the jaw they derived estimates of the force exerted at the teeth. Wroe and his colleagues discovered some interesting patterns in the maximal bite forces of the taxa they examined, once they had corrected for body size. Contrary to THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY expectations, the team found that osteophagous (bone crunching) species have relatively lower maximal bite forces than non-osteophagous species. In general, marsupial predators produce higher bite forces than do placental mammals. The authors attribute this to placentals’ larger brains, which reduce the available skull surface area for muscle attachment. The implications for hunting performance are that placental predators require a more precise approach to seize their prey than do marsupial predators. The team also observed other differences between taxa; for example, they found a lower mean sizespecific bite force in cats than in canids. The authors suggest that possible reasons for this include cats having shorter jaws, more agile forelimbs to immobilize prey, and the possibility that cats recruit their neck muscles to increase bite forces. Perhaps the most interesting insights from this study are those regarding the extinct species, as these may allow researchers to reconstruct the ecological niches of these species. Of particular interest are the inferences that can be made about the predatory behaviour of extinct animals. For instance, the team found that the extinct marsupial lion Thylaceo carnifex appears to have produced very high bite forces, indicating that it was adapted to hunt large prey. Reconstructing animals’ feeding mechanics and behaviour by biomechanical modelling may provide fascinating insights into the life history and ecology of many enigmatic extinct predatory species. In presenting predictions from 39 species representing 7 families, the authors have certainly taken a significant step towards achieving this. 10.1242/jeb.01801 Wroe, S., McHenry, C. and Thomason, J. (2005). Bite club: comparative bite force in big biting mammals and the prediction of predatory behaviour in fossil taxa. Proc. R. Soc. B 272, 619-625. Richard Bonser University of Reading [email protected] Outside JEB GENES FOR THE LONG RUN Can the same genes both improve your aptitude for endurance exercise and lower your life expectancy? A recent study of mitochondrial DNA (mtDNA) in track athletes suggests that the answer may be ‘yes’. The mitochondrial genome is a small (17kb), circular chromosome that encodes 13 components of the electron transport chain, which generate the proton gradient necessary for aerobic ATP production. Mitochondrial DNA might therefore be expected to influence an individual’s ability to sustain long periods of exercise. To determine whether the mitochondrial genes of track athletes differ systematically from those of the population at large, Niemi and Majamaa analyzed the mtDNA sequences of 141 elite Finnish long-distance runners and sprinters as well as 1060 Finnish control subjects. They found that certain groups of mtDNA sequences (called haplogroups, since there is only one copy of the chromosome per mitochondrion) occurred less frequently in the distance runners than in the sprinters and control subjects. In particular, no distance runner belonged to haplogroup K or subhaplogroup J2, whose combined frequency is at least 4.5% among control subjects and is even higher among sprinters. Since distance runners are heavily dependent on aerobic ATP production by their mitochondria, it isn’t surprising that their mtDNA differs from that of people with different exercise habits. For example, genes for protein isoforms that mildly impair ATP synthesis should presumably be rare among endurance athletes. What’s interesting, however, is that the haplogroups underrepresented among the distance runners are overrepresented among very old people. Previous work by the Niemi/Majamaa group and others has shown that members of haplogroup K and subhaplogroup J2 tend to live longer than people of other haplogroups. Is it plausible that the mitochondrial genes of haplogroup K and subhaplogroup J2 limit endurance performance but improve life expectancy? Niemi and Majamaa offer an intriguing hypothesis to explain this potential paradox: perhaps J2 and K are ‘uncoupling genomes’ that increase proton leak across the mitochondrial membrane. An elevated proton leak could certainly limit aerobic ATP production, and thus endurance performance. But it could also prevent the mitochondrial membrane potential from rising into the range (>140 mV) where production of reactive oxygen species (ROS) becomes high. ROS have been implicated in ageing, so limiting ROS production could contribute to a longer life. Although the link between ROS and ageing is still under investigation, several mouse studies have suggested that relatively ‘leaky’ mitochondria could promote longevity by minimizing the generation of ROS. Thus, if members of J2 and K haplotypes limit their ROS production, this could explain why they live longer than other people. If the rareness of J2 and K among endurance athletes is confirmed by additional work, the ‘uncoupling genome’ hypothesis should be tested with biochemical measurements of mitochondria isolated from representatives of different haplogroups. If the hypothesis is correct, mitochondria of haplogroup K and subhaplogroup J2 should have lower membrane potentials, lower ROS production rates and lower ATP production rates (relative to O2 consumption rates) than other mitochondria. Such studies would represent the completion of another lap in our race to understand the genetic basis of differences in athletic performance. 10.1242/jeb.01802 Niemi, A. K. and Majamaa, K. (2005). Mitochondrial DNA and ACTN3 genotypes in Finnish elite endurance and sprint athletes. Eur. J. Hum. Gen. 13, 965-969. Greg Crowther University of Washington [email protected] THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY CASTE SYSTEM MITOCHONDRIAL DNA v A CINDERELLA STORY In human societies, not everyone is born equal. The phrase ‘born with a silver spoon in the mouth’ refers to the fact that, at birth, some people find themselves in positions of privilege and wealth whereas others do not. Similar inequalities exist within animal societies and, in the case of some insects such as ants and bees, are taken to great extremes. Ants and bees have evolved eusociality – several generations live together in colonies, only one or a few individuals (queens) have offspring, and non-reproductive colony members, the workers, care for these offspring. Becoming a queen is not something that workers can aspire to. Although some sneaky workers do try to have offspring themselves, individuals born as workers cannot become queens. Nutrition is an important determinant of social class in many eusocial insect species. In some species, larvae destined to become queens are fed a particular substance (royal jelly in honey bees), whereas in other species queen larvae get more food than worker larvae. This latter system is found in the stingless bee Schwarziana quadripunctata. Now, an international team of researchers from Belgium, Britain and Brazil led by Tom Wenseleers has discovered that some stingless bees manage to beat the system and change their fate. The team investigated previous reports that dwarf queens occur in stingless bee colonies in addition to the normal, large queens. Wenseleers and his co-workers weighed the dwarf queens and showed that they are indistinguishable in weight from regular worker bees. In addition, dwarf queen larvae are raised in cells that are identical to the cells in which worker larvae are raised, which are smaller than the large specialized cells in which the queen larvae develop. Dwarf queens aren’t a rare occurrence; when the team assessed the social class of 11 574 individual Outside JEB stingless bees from 19 colonies, they found six times more dwarf queens than normal queens. The team used a series of morphological measurements to identify adult dwarf queens and distinguish them from normal queens. They showed that the dwarf queens headed 12 out of a further 54 colonies, suggesting that the dwarf queens are able to reproduce. Although this may seem like a large proportion of colonies, it actually suggests that dwarf queens aren’t as successful as normal queens – 86% of queens reared were dwarf queens, but they only headed 22% of the colonies that the team studied. Wenseleers and his coworkers suggest that this discrepancy may be due to workers actively discriminating against the dwarf queens. Many questions remain about this intriguing system in which individuals seem able to choose whether to become queens. In particular, it would be interesting to know exactly which mechanisms determine the fate of stingless bee larvae. What role does nutrition play in determining their fate? If nutrition determines whether an individual becomes a queen or a worker, then some larvae destined to become workers are apparently overcoming nutritional limits and becoming queens despite their limited food supply. What physiological mechanisms control nutritional responses in this system? Recent advances in Drosophila have given us some places to start looking for answers, such as insulin receptor pathways. Perhaps these or other, as yet unknown, pathways play a role in the production of dwarf queens in stingless bee colonies. Whatever the case, this system has the potential to keep ecologists and physiologists busy for many years to come. 10.1242/jeb.01804 Wenseleers, T., Ratnieks, F. L. W., Ribeiro, M. de F., Alves, D. de A. and Imperatriz-Fonseca, V.-L. (2005). Working-class royalty: bees beat the caste system. Biol. Lett. 1, 125-128. Jeremy E. Niven University of Cambridge [email protected] CARDIAC REMODELLING vi PYTHON’S HEARTY MEAL Carnivorous reptiles exhibit a massive increase in oxygen demand following a meal to meet the increased metabolic demands associated with digestion. Inherently, this increase in oxygen demand places an extra demand on the cardiovascular system; the heart needs to work harder to transport more oxygen to the metabolically active digestive organs. Pythons appear to deal with this increased cardiac demand by substantially increasing the mass of their heart (cardiac hypertrophy) within two days of feeding. But just how do these snakes manage to pump up their heart’s mass? Andersen and colleagues at the University of California, Irvine, were interested in determining the cause of the cardiac hypertrophy following feeding in the python (Python molurus). They wanted to know if the increase in heart mass was due to increased protein synthesis (i.e. formation of new heart muscle) or a water shift between extracellular and intracellular compartments, leading to increased fluid content of the heart tissues. In order to investigate this, Andersen and colleagues obtained ventricles from three groups of pythons: (1) fasting (these snakes had been fasted for 28 days); (2) digesting (these animals had digested a large meal 2 days earlier); and (3) post-digestive (these pythons had digested a large meal 28 days earlier). For each of these groups, the team measured ventricular dry/wet mass ratio, the ventricle’s total protein, RNA and myofibrillar protein concentrations on a mass-specific basis, and the expression of messenger RNA for heavy-chain cardiac myosin, a contractile element of the heart. As they expected, the team observed a 40% increase in pythons’ ventricular mass during digestion. They identified several clues that this hypertrophy was due to de novo protein synthesis and not increased THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY fluid content of the heart. Primarily, the team found that the expression of messenger RNA for heavy-chain cardiac myosin increased significantly 2 days after feeding, indicating that digesting snakes synthesise myosin. Further, they discovered that the hearts’ mass-specific total protein, RNA and myofibrillar protein concentrations did not change during digestion. In other words, as the pythons’ hearts increased in mass after feeding, the ratio of protein to heart mass remained the same, indicating that new protein was being formed as the hearts expanded. This finding also ruled out increased water content as an explanation for the cardiac hypertrophy; if the increased heart mass was due to an increase in fluid content, these mass-specific protein concentrations would have decreased. Finally, they found that ventricular dry/wet mass ratio did not differ between fasted and fed snakes, providing further evidence that the larger hearts were not due to increased water content. The team concluded that the cardiac hypertrophy observed in digesting pythons is due to the synthesis of new contractile protein. Additionally, the team showed that the increase in heart mass during digestion was a fully reversible process. The mass of post-digestive snakes’ hearts was similar to the mass of fasted snakes’ hearts. Thus, following a meal, a python can rapidly increase its heart size by 40% and then decrease it again within 28 days. In comparison with mammalian species, in which comparable increments in ventricular size take weeks to develop, this cardiac remodelling occurs very rapidly. As such, Andersen and colleagues stress that this natural, rapidly occurring and fully reversible cardiac hypertrophy could provide a useful model for investigating the mechanisms that lead to cardiac remodelling and growth in other animals. 10.1242/jeb.01805 Andersen, J. B., Rourke, B. C., Caiozzo, V. J., Bennet, A. E. and Hicks, J. W. (2005). Postprandial cardiac hypertrophy in pythons. Nature 434, 37-38. Jonathan A. W. Stecyk University of British Columbia [email protected] Outside JEB EAT LITTLE, DIE LATE? The ‘free radical’ theory of ageing, which relates to the production of harmful, lifetime-reducing reactive oxygen species, currently receives much attention. This hypothesis suggests that animals live longer when they reduce their caloric intake, because the associated drop in their metabolic rate leads to a decreased production of reactive oxygen species. Thus, to disentangle the relationship between metabolic rate and reactive oxygen species, experiments involving food-restricted animals are necessary. But, as animals also lose weight during fasting, an important question is whether caloric restriction directly affects metabolism or, alternatively, whether any changes in metabolic rate may simply be attributed to weight loss and different organ composition in thinner animals. To address this issue, Colin Selman from the Department of Aging and Geriatric Research at the University of Florida and his colleagues in Florida and Scotland investigated the energy expenditure and body morphology of well-fed and foodrestricted Fischer rats. The team expected caloric restriction to result in a drop in the rodents’ metabolic rate. Restricting the food intake of 12 young and old rats by 40%, they tried to uncover how caloric restriction, and thus a lowered metabolic rate, affects the ageing process. Simultaneously, they measured total daily energy expenditure using the doubly labelled water method both in ad libitumfed and food-restricted rats from the two age classes. To assess the impact of morphology and lean tissue mass on the rats’ total energy budgets, the team sacrificed all the rats and performed intensive organ morphometric analyses. month-old juveniles. The team was surprised to find that older food-restricted rats (aged 26 months) expended just as much energy as their ad libitum-fed counterparts of the same age. Selman and his team attributed this to the fact that the ad libitum food intake of elderly rats declined from the beginning of the food intake measurements until the end of the experiment, so that they ended up eating about the same amount as the foodrestricted rats. Thus, the energy expenditure of well-provisioned rats was not different from that of undernourished rats. The authors generated an interesting model to predict the energy demands of rats with different food intakes, based on the rats’ morphological variation, and compared these predicted energy demands with their observed data. They found that food-restricted rats spent significantly more energy than the authors predicted from the rats’ altered morphology. Selman and his colleagues concluded that both young and old underfed rats have a significantly increased metabolic rate when taking into account the rats’ altered body condition. At first sight, these results contradict the free radical theory of ageing, as old rats with high levels of energy expenditure presumably have to cope with more free radicals. Since previous studies on mice and dogs have revealed that increased energy expenditure is associated with increased longevity, the authors suggest that the free radical theory of ageing should be reassessed. Can fasting rats provide us with clues to the secret of a longer life? 10.1242/jeb.01806 Selman, C., Phillips, T., Staib, J. L., Duncan, J. S., Leeuwenburgh, C. and Speakman, J. R. (2005). Energy expenditure of calorically restricted rats is higher than predicted from their altered body composition. Mech. Ageing Develop. 126, 783-793. Teresa Valencak Veterinary University Vienna [email protected] OSMOTIC STRESS AGEING vii SALTY WORMS? Osmotic stress is a frequent experience, both for organisms and the cells that make them up. An organism’s ability to osmoregulate successfully is key to its survival, and mechanisms of osmoregulation are intensively studied by some authors in this journal. A recent paper from Kevin Strange’s group uses the tiny nematode worm to implicate a very unexpected gene in the ability to survive hypertonic stress, linking osmoregulation to the ageing process. Caenorhabditis elegans is a tiny soildwelling nematode that has become a favoured model organism for genetics. It might seem unlikely that useful physiology could be performed on something only a few tens of microns in diameter, but Strange nonetheless showed that C. elegans could readily adapt to hyper- or hypotonic environments. Previous work had shown that a class of C. elegans mutants that altered the organism’s ability to form a long-lived, stress-tolerant larval stage, called the dauer larva, could greatly alter the animal’s resistance to thermal, oxidative or hypoxic stress. Strange reasoned that this might also extend to osmotic stress and, sure enough, showed that one such mutant, daf-2, conferred greatly increased survival on agar plates containing up to 400 mmol l–1 NaCl. daf-2 encodes the worm’s insulin receptor, so the implication is clear; disrupting insulin signalling actually enhances survival under stress. There are other players in the insulin signalling cascade, so to explore the genes responsible for stress tolerance in C. elegans, the authors tried these other genes singly and in combination. Mutations in age-1, a gene implicated in longevity that encodes PI3-kinase, showed a similar salt resistance phenotype to daf-2. However, mutations in daf-16, also identified in As expected, Selman and his colleagues found that food-restricted rats expended less energy than their well-fed conspecifics. However, this was only the case for 6THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY Outside JEB viii screens for genes that affect lifespan, could suppress these effects in double mutants carrying daf-2 and daf-16 or carrying age-1 and daf-16. That is, normal daf-16 is required for daf-2 or age-1 mutants to be effective at conferring salt resistance. Overexpression of daf-16, which leads to increased longevity, also increases salt tolerance. So longevity, ageing and resistance to osmotic and other stressors are all interlinked. RNAi (particularly easy in C. elegans), several of the hsps were shown to be necessary for successful adaptation to high salt. Inspired by this result, the authors screened a further 222 target genes of daf16 by RNAi and found that 10 of them were also important in salt response. Two of these were trehalose-6-phosphate synthases, which catalyse the formation of trehalose, an important osmolyte in many organisms. variety of genes that allow the animal to adapt to stress more easily. It is interesting to note that these genes are presently the darlings of the ageing research community, suggesting that ageing and osmoregulation may be linked. And as these genes are conserved right across to humans, there is the prospect that work on the tiny worm may have uncovered a general mechanism for response to hypertonic stress. daf-16 encodes a FOXO transcription factor, which switches on other genes, and other workers had recently published a microarray list of genes upregulated by daf-16 over-expression. Some might be candidates for increasing survival after salt stress. Conspicuous in the list were the heat shock protein (hsp) genes, which protect organisms from stress-induced damage; by knocking these down with In summary, the authors propose that in normal worms, insulin signalling through the daf-2 insulin receptor activates the age-1 PI3 kinase, which in turn normally phosphorylates the daf-16/FOXO transcription factor to keep it out of the nucleus, stopping it from activating downstream genes. In mutants of insulin signalling, daf-16 is able to enter the nucleus, allowing it to switch on a 10.1242/jeb.01803 © 2005 The Company of Biologists Limited THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY Lamitina, S. T. and Strange, K. (2005). Transcriptional targets of DAF-16 insulin signaling pathway protect C. elegans from extreme hypertonic stress. Am. J. Physiol. Cell Physiol. 288, C467-C474. Julian A. T. Dow University of Glasgow [email protected]