The paper below has been shortened and slightly modified.

References have been removed but are indicated by [R] in the text. Where necessary, methods are described in the text or figure legends. A glossary is provided after the discussion. Read the paper carefully and answer all ten questions at the end. Introduction Metabolic syndrome in humans is characterized by chronic changes in nutrient metabolism and distribution that often lead to obesity and type 2 diabetes (see glossary). Metabolic syndrome is commonly linked to chronic inflammation, an association that appears to arise from signaling molecules that function in both immune and metabolic pathways [R]. Inflammation stimulating signals emitted by excessive adipose tissue are presently under intense scrutiny as a causative factor for metabolic diseases [R], but a number of studies suggest that altered gut microbial composition can also trigger this type of inflammation [R] and affect host metabolism and obesity [R]. Here, we use an insect model to test the hypothesis that gut microbes can cause inflammation, abnormal metabolic activity and obesity. Research to date on metabolic syndrome has concentrated exclusively on mammals even though signaling molecules and pathways controlling energy homeostasis, immunity, and inflammation are highly conserved across animals, including insects. Insects have become valuable research models for many types of human disease [R], including recent efforts aimed at elucidating the genetic and physiological bases of metabolic homeostasis and dysregulation [R]. Libellula pulchella dragonflies are commonly infected with gregarine gut parasites (Microsporidia, Apicomplexa) of the genus Hoplorhynchus (Fig. 1). Gregarine parasites are historically viewed as relatively benign but more recently have been shown to affect fecundity and mortality of invertebrates [R]. Infected L. pulchella males show no external symptoms or internal lesions but have poor territoriality and mating success and lose the ability to adjust their flight-muscle energy consumption according to the quantity of their body fat reserves [R]. These observations led us to examine how parasitic infection affects metabolism and nutrient distribution in gregarine infected L. pulchella dragonflies.

Fig. 1. Infection of L. pulchella by gregarine protozoans, Hoplorhynchus. (a) Heavily infected L. pulchella midgut showing mature parasites. Ingested parasite spores enter the alimentary canal and develop in the midgut, attaching themselves to the lining of the midgut. (b) Scanning electron micrograph detailing a mature Hoplorhynchus sp. Scale bar = 100 m. Results Metabolic performance was first assessed by examining flight-muscle power output. We used single flight muscles that remained within the insect but were isolated mechanically from the rest of the thorax.

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Higher power output was found for flight muscles of healthy individuals compared with those with mature parasite infection but not for those with an immature infection.

Peak muscle performance depends in part on the ability to generate ATP from nutrient substrates. As occurs in vertebrates, insects show timedependence in how their flight muscles use fuel substrates. Carbohydrates are oxidized during the initial seconds to minutes of flight, followed by a transition to lipid oxidation if flight is sustained [R]. Typically, libellulid dragonflies display a mixture of brief and sustained flights, fueled by a mixture of carbohydrates and lipids [R]. Impaired utilization of oxidizable substrates generally results in a drop in muscle performance [R]. Because of the observed decrease in flight-muscle power output, we examined how parasitic infection is associated with changes in metabolic substrate utilization in vivo. We determined the ratio of CO2 emission to O2 consumption [i.e., the respiratory quotient (RQ)] by flight muscles undergoing a controlled regime of stressstrain cycles that closely match flight [R]. An RQ of 1.0 indicates pure carbohydrate metabolism, whereas values between 1 and 0.7 indicate partial to pure consumption of lipids [R]. We found that the carbohydrate and lipid utilization of muscles from healthy dragonflies (mean RQ = 0.87) was different (P = 0.001) from that of muscles from infected dragonflies, (mean RQ = 1.01; Fig. 3). Altered muscle fatty acid metabolism is a common result of infection and inflammation in mammals and is thought to be due to mitochondrial dysfunction [R]. Impaired fatty acid metabolism may lead to the accumulation of lipids in and around skeletal muscle tissue [R], an important feature in disease states such as obesity, insulin resistance, and type 2 diabetes [R].

Fig. 2. Effect of gregarine infection on dragonfly muscle contractile performance. Maximum flight-muscle power output for healthy (h) and infected individuals (n = 52) shows a significant effect of infection that is related to the maturity of the infection. Maximum power output is significantly lower (*, P< 0.0001, Students t-test) for individuals carrying a mature infection (mi) but not for those with an immature infection (ii). Error bars represent standard error of the mean. The absence of a difference between healthy and dragonflies with an immature infection indicates that these performance differences are not likely to arise from weaker animals being more susceptible to infection. Rather, flight-muscle performance appears to decline over time in response to changes in duration of exposure to developing gregarine parasites.

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than those of healthy dragonflies (Fig. 4a). Our carbohydrate assay quantified total monosaccharide concentration (after chemical hydrolysis of disaccharides), which primarily reflects hemolymph trehalose (a disaccharide of glucose), the most abundant hemolymph carbohydrate in most insects [R]. Mass spectrometry measurements showed that the trehalose-to-glucose ratio in L. pulchella hemolymph is 150:1 (data not shown). Fig. 3. Substrate utilization by dragonfly flight muscles. Mean respiratory quotients (n = 22) for healthy (h) and infected (i) individuals during 30 seconds of continuous sinusoidal muscle contraction at 37 Hz. For infected individuals mean RQ = 1.01 and SE = 0.03), whereas for healthy individuals mean RQ = 0.87 and SE = 0.02. *, P = 0.001 (Students t-test). Error bars represent standard error of the mean. To determine whether lipid is distributed differently in healthy versus infected dragonflies, we determined how thoracic lipid, which is associated with the flight muscles, varied according to infection status. Gregarine-infected dragonflies had 26% more thoracic lipid (normalized to lipid content of the abdominal fat body) compared to healthy dragonflies (P=0.001). Furthermore, there was no effect of infection on hemolymph lipid content (P = 0.25) or lipid mobilization from the abdominal fat body (P = 0.96) in response to dragonfly adipokinetic hormone. We next investigated the effects of infection on carbohydrate metabolism. We found that infected dragonflies had hemolymph carbohydrate concentrations that were approximately twofold higher Dysglycemia is often attributed to tissue insulin resistance [R]. To test the hypothesis that the difference in hemolymph carbohydrate concentration between healthy and infected dragonflies is due to similarly impaired endocrine function, we analyzed the effect of bovine insulin on hemolymph carbohydrate concentrations. In samples collected 30 min after insulin injection, hemolymph carbohydrate concentrations decreased significantly in healthy but not infected dragonflies (Fig. 4b). The doubling of hemolymph carbohydrate concentration and impaired capacity of insulin to regulate carbohydrate metabolism implies that gregarine infection induces an insulin-resistant phenotype, which is yet another metabolic abnormality commonly ascribed to metabolic syndrome in mammals. Because the uptake of carbohydrate by flight muscles of infected dragonflies is apparently not disrupted (i.e., they readily oxidize carbohydrates; see RQ data in Fig 3), it appears that this insulin resistance is only partial or is localized in tissues other than flight muscles Interestingly, mean hemolymph carbohydrate concentration of infected dragonflies increased after insulin injection (i.e., a post-to-preinjection ratio >1.0; see Fig. 4b).

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This may have been caused by the known effect of insulin-like peptides on glycogen phosphorylase activity in the insect fat body [R], which releases carbohydrate into the hemolymph. The opposite response of infected versus healthy dragonflies indicates that the insulin-regulated net balance between carbohydrate uptake and release to the hemolymph was altered by gregarine infection. To determine whether the presence of gregarine parasites was responsible for the observed changes in carbohydrate metabolism, we simulated their presence by providing healthy dragonflies with drinking water containing trace amounts of excretory/secretory products (ESP)

obtained from live gregarines. The ESP containing solution was prepared by culturing parasites in saline solution for 45 minutes to allow the accumulation of soluble products. A control group of healthy individuals received water without ESP, and, after 2 days, we measured hemolymph carbohydrate concentrations from both groups. A 2day exposure to ESP caused a significant elevation in hemolymph carbohydrate concentration compared with the controls (Fig. 4c), similar to the difference between healthy and infected individuals in the field illustrated in Fig. 4a. These results indicate that factors released by gregarine parasites in the midgut can rapidly stimulate the observed changes in hemolymph carbohydrate concentration.

Fig. 4. Hemolymph carbohydrate metabolism in healthy (h) and infected (i) dragonflies. (a) Hemolymph carbohydrate concentration was significantly higher in infected dragonflies (P= 0.02; Students t-test, n = 16). (b) Injection of insulin caused a significant decrease in hemolymph carbohydrate levels of healthy individuals [i.e., post-/preinsulin injection ratio = 0.65 0.32 (mean SE); P = 0.04; paired t-test] and an increase in infected individuals (i.e., post-/preinsulin injection ratio = 1.80 0.32; P = 0.02; paired t-test, n = 16). (c) Exposure to parasite excretory/secretory products (ESP) for 2 days caused a significant increase (P = 0.02; Students t-test, n = 10) in hemolymph carbohydrate concentration in healthy individuals, similar to the difference seen between concentrations of healthy and infected dragonflies in the field. Absolute differences in carbohydrate levels between a and c most likely reflect the difference between dragonflies taken directly from the field (a) versus after a 48-h period of quiescence in the lab (c). Error bars represent standard error of the mean.

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Mammalian insulin resistance and obesity are commonly associated with infection and chronic inflammatory states [R], and it has been proposed that the development of these metabolic abnormalities could be mediated by activation of innate immune responses [R]. Many compounds released by parasites induce host immune responses and/or stimulate the production of inflammatory cytokines. These cytokines have downstream effects on signaling pathways such as the p38 mitogen activated protein kinase (MAPK of 38 kDa) pathway [R]. To determine whether dragonfly p38 MAPK signaling is affected by gregarine infection, we examined activation of p38 MAPK in the flight muscles. We probed Western blots of muscle homogenates from healthy and infected dragonflies with a polyclonal antibody made against human phosphorylated (i.e., activated) p38 MAPK. Included on these blots were samples of the same muscle homogenates that were exposed for 20 min to gregarine ESP. We found that p38 MAPK was chronically activated in flight muscles from infected dragonflies (lane 4) but not in muscles from healthy individuals (lane 1). Acute treatment of muscles from healthy individuals with ESP stimulated phosphorylation of p38 MAPK to levels indistinguishable from infected dragonflies (lanes 2 and 5) (Fig. 5). Discussion Dragonflies infected with noninvasive and seemingly benign gregarine parasites show symptoms typical of mammalian metabolic syndrome. Gregarine-infected dragonflies have reduced muscle performance; their muscles do not oxidize fatty acids, and lipid accumulates in their thorax; they have elevated levels of blood carbohydrates that do not respond normally to insulin; and they show signs of chronic systemic inflammation. Page 6 of 10

Fig. 5. Western blot of flight muscle homogenates. Homogenates were split into two equal portions, pretreated with saline (-) or ESP (+), and then probed with an antibody binding to activated p38 MAPK. Lanes 12 contain split homogenates from a healthy individual; lanes 4-5 contain split homogenates of muscle from an infected individual. Chronic activation of p38 MAPK is evident in the saline-treated muscle homogenate from the infected individual (lane 4) compared with salinetreated homogenate from the healthy individual (lane 1). p38 MAPK activation in the muscle homogenates from the healthy individual after 20-min treatment with parasite ESP (lane 2) was different from the untreated half of the same homogenate (lane 1) but was identical to the infected individual that was either treated with saline (lane 4) or with parasite ESP (lane 5). Lane 3 is a positive control (c+) consisting of human cell lysates treated with anisomycin, an activator of p38 MAPK. In addition to some nonspecific binding, dragonfly lanes show presence or absence of a band (arrows) identical in size to the positive control. That band is absent when muscle homogenates are pretreated with a specific inhibitor of p38 MAPK phosphorylation ( data not shown). The results shown are representative samples.

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Moreover, elevated blood carbohydrates and activation of p38 MAPK, a molecular marker of inflammation, can both be elicited in healthy dragonflies by exposing them to excretory/secretory products of the parasite. These results provide the insight that microbes residing in the intact lumen of the gut may provoke a suite of pathologies in the blood and in the muscles, i.e., across body compartments either directly or via signals emitted from the gut. Similar to mammals, infection in insects commonly induces an innate immune response that activates inflammatory pathways [R]. We have demonstrated that gregarine-infected dragonflies show signs of a chronic inflammatory state, which is widely recognized as a pathological feature underlying the origin of metabolic syndrome in mammals. Indeed, a recent study suggests that p38 MAPK activation due to inflammation or other stress can mediate inhibition of insulin signaling [R] and therefore play a potentially crucial role in the development of this key metabolic abnormality. Studies of mammalian metabolic disease generally focus on the role of inflammatory signals originating from excessive adipose tissue [R]. However, our studies on the interaction between gregarines and dragonflies indicate that there is a potential for other, nonadipose-derived inflammatory cues (i.e., microbes residing in the gut) to affect metabolic traits, including lipid metabolism, so that increased lipid deposition might be an end result rather than an initiator. The fact that gregarine infection is associated with impaired fatty acid oxidation by flight muscles and excessive thoracic lipid deposition identifies insects

as potential models for obesity in humans. Mammalian obesity is commonly thought to arise from mismatches between a persons energy consumption and activity level. Interestingly, infected dragonflies display a more sessile behavior, and their flight muscle performance is not adjusted according to the amount of energy resources stored as lipid, as is the case for flight muscles of healthy individuals [R]. A question that remains to be addressed is whether gregarine infection has initial and direct effects on behavior, followed by metabolic disruptions that are stimulated by the behavioral changes or, alternatively, whether the metabolic pathologies develop as a direct result of infection and inflammation from which the behavioral changes follow. The radical nature of the observed metabolic changes (i.e., total loss of lipid metabolism by the flight muscles) seem to favour the latter, but a more in-depth (in vivo) and timeresolved analysis is desirable. Given these observations of parasiteinduced metabolic syndrome in an insect, along with obesity and behavioral changes, it seems appropriate to ask whether changes in biotic environmental factors (e.g. intestinal microbial community composition) may cause metabolic disease in humans. A few studies have hinted at this type of internal environmental causation [R], and immunocompromised humans who are unable to clear infections of Cryptosporidium, the sister taxon of gregarines, show changes in RQ indicative of compromised ability to metabolize lipid [R]. Despite their obvious differences, insects and humans show a remarkable degree of conservation for molecular, cellular, and developmental mechanisms [R].

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Advances from invertebrate biology have often led to a better understanding of mammalian biology. For example, the discovery and dissection of mechanisms regulating innate immunity pathways in mammals were based on knowledge gleaned from Drosophila [R]. Pathways involved in the development of metabolic abnormalities may be similarly homologous, which opens the way for the use of non-mammalian systems as an additional tool to study causes and treatments for obesity and metabolic diseases. Glossary Adipokinetic hormone - small peptide hormone involved in regulating lipid mobilization and transport in the haemolymph of insects.

Bovine- derived from cattle/cows Dysglycemia- abnormal blood sugar concentration. Hemolymph- insect blood p38 MAPK pathway- an intracellular signaling pathway activated by inflammation Type 2 diabetes- metabolic disorder, associated with obesity, characterized by elevated blood glucose levels and resistance to insulin. Western blot- a method for separating a complex mixture of proteins based on size, followed by the detection of specific proteins using antibodies. Each band represents an individual protein.

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Questions 1. a) Provide a title for this paper using a maximum of 20 words. (5 marks) b) Suggest five additional keywords that do not replicate those in the title. (5 marks) 2. Write a summary of this paper, as it might be written by the authors, of between 150-200 words. (25 marks) Outline why insects are a good model system for studying metabolic syndrome. (10 marks) a) What do you deduce about the relative amounts of carbohydrate and lipid metabolism in the flight muscles of healthy compared to infected dragonflies? (5 marks) b) What effect does parasite infection have on the distribution and mobilization of lipids in the dragonflies? (5 marks) c) What do you think accounts for the observed changes in lipid distribution? (5 marks) 5. What reason is given for the almost two-fold difference in hemolymph carbohydrate concentration between healthy dragonflies in Figures 4(a) and 4(c)? (5 marks) Metabolic syndrome frequently leads to type 2 diabetes. What is the evidence that carbohydrate metabolism in Libellula, following parasite infection, is similar to that in human diseases, such as type 2 diabetes? (5 marks) a) What are the potential problems of using bovine insulin in the experiments described in this paper? (4 marks) b) How could you eliminate this problem? (2 marks) 8. a) Describe the hypothesis for the experiment where dragonflies were fed excretory-secretory products (ESP) derived from the parasites. (5 marks) b) What are the caveats of this hypothesis, ie. why might this experiment not produce the desired outcome? (5 marks)

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9.

Based on the data in Figure 5 it was concluded that Acute treatment of muscles from healthy individuals with ESP stimulated phosphorylation of p38 MAPK to levels indistinguishable from infected dragonflies (lanes 2 and 5). How could this experiment have been improved to strengthen the validity of this conclusion? (6 marks) a) What new hypothesis for the development of human metabolic syndrome is suggested by the experiments in this paper? (4 marks) b) Explain briefly how you would investigate if this hypothesis is correct. (4 marks)

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