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Feeding effects on gene expression of the hypertrehalosemic hormone in the cockroach, Blaberus discoidalis
Journal of Insect Physiology 44 (1998) 967–972
Feeding effects on gene expression of the hypertrehalosemic hormone in the cockroach, Blaberus discoidalis Danielle K. Lewis, J.Y. Bradfield, L.L. Keeley
*
Department of Entomology, Texas A and M University, Texas Agricultural Experiment Station, College Station, TX 77843-2475, USA Received 6 May 1997; received in revised form 16 October 1997
Abstract Feeding effects on hypertrehalosemic hormone (HTH) transcript levels in corpora cardiaca (CC) of adult females of the cockroach, Blaberus discoidalis were measured using dot blot hybridization. HTH transcript levels were nearly doubled in CC from females withheld from food and water for ten days compared to CC from fed females. The increase in HTH-mRNA was a response to starvation, not dehydration, and reversed within 2 days after exposure to food. HTH-mRNA was elevated in CC from fed insects that had their recurrent nerve severed, but low fecal output by insects with severed nerves indicated that feeding and digestion were impaired. Thus, the increased HTH synthesis likely resulted from starvation rather than disruption of neural regulation. CC from starved females that were refed with either solutions or agar that contained glucose did not show down-regulation of HTHmRNA. Likewise, injections of glucose or trehalose did not suppress HTH-mRNA levels in CC of starving insects. Down-regulation of the starvation-related increase in HTH-mRNA appears to be a response to consumption of a complex of nutrients and not to increased carbohydrates or mechanical aspects of feeding. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Adipokinetic hormone; Hypertrehalosemic hormone; Trehalose; Neurohormone gene expression; Blaberus discoidalis; Cockroach; Insect; Molecular cloning
1. Introduction The regulation of insect carbohydrate metabolism by neuropeptides of the adipokinetic hormone/red pigmentconcentrating hormone (AKH/RPCH) family is well documented (Bowers and Friedman, 1963; Anelli and Friedman, 1986; Wilps and Ga¨de, 1990; Ziegler et al., 1990; Lee and Keeley, 1994). In the tropical cockroach, Blaberus discoidalis, trehalose synthesis is stimulated by the hypertrehalosemic hormone ( = HTH; pQVNFSPGWGTa), a decapeptide of the AKH/RPCH family (Hayes et al., 1986). AKHs and HTHs regulate energy metabolism in insects, but it is unclear how AKH and HTH synthesis and secretion are, themselves, regulated. Cardioacceleratory hormones (CAH)-I and -II have hypertrehalosemic actions in the American cockroach, Periplaneta americana (Scarborough et al., 1984), and the syntheses of CAH-I and CAH-II by isolated corpora
* Corresponding author. Fax: 001 409 845 9727; E-mail: [email protected] 0022–1910/98/$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 3 4 - 1
cardiaca (CC) are reported to increase 5-fold in the presence of trehalose (Khan and Steele, 1992). This effect appears specific to trehalose and does not occur with glucose, except at high concentrations. Conversely, in Locusta migratoria, high levels of trehalose lower AKH titers and delay lipid mobilization (Cheeseman et al., 1976; Van Der Horst et al., 1979). Trehalose inhibits electrical activity in isolated CC from L. migratoria which may explain their reduced secretion of AKH (Bloemen et al., 1987). These results suggest that the circulating metabolites regulated by the AKHs and HTHs may have feedback effects over the synthesis and secretion of the hormones. HTH synthesis and secretion are highest during the first several days following the adult molt in both sexes of B. discoidalis, but after about a week, both processes decline by 60–70% and remain stable (Sowa et al., 1996). Furthermore, CC from starving females have 2fold higher rates of HTH synthesis than CC from fed females (Sowa et al., 1996). Results from age studies of HTH gene expression parallel the results from the synthesis and secretion studies (Lewis et al., 1997). The
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present research was undertaken to determine if starvation exerted its stimulatory effects on HTH biosynthesis at the level of gene expression, and if so, to determine if dietary carbohydrates or feeding reversed the starvation effects. The results showed that HTH-mRNA doubled in the CC of starved females and was suppressed by general nutrient intake, but not specifically by carbohydrate consumption.
2. Materials and methods 2.1. Experimental animals B. discoidalis were reared in wood shavings in 40 l plastic buckets in a 12-h light, 12-h dark circadian cycle at 27 ± 2°C and fed Purina dog chow and water (Keeley et al., 1995). Males and females were separated at the adult molt. Starved insects were withheld from food and/or water from adult emergence until experimentation. 2.2. HTH-mRNA analysis Total RNA was extracted from 2 or 3 pairs of corpora cardiaca-corpora allata complexes ( = CC) (Puissant and Houdebine, 1990). Dot blot hybridization analysis was performed using a specific [32P]HTH-cDNA probe to quantify the HTH-mRNA (Lewis et al., 1997). Each replicate sample consisted of 2 or 3 CC pairs since dotblots are essentially linear for the HTH-mRNA present in a total RNA extract of 1 to 3 CC pairs (Lewis et al., 1997). Each preparation of probe is labeled with an independent lot of [32P] precursor. Therefore, each probe has a different specific activity and all treatment and control groups within an experiment must be probed simultaneously to compare results. Because a different probe is prepared for each experiment, it is not possible to compare absolute cpms between experiments. 2.3. Carbohydrate feeding Possible effects on HTH gene expression by food consumption, independent of nutrient content, were measured by feeding plain agar. Glucose-enriched agar was fed to determine if carbohydrate in the food or glucose passage through the gut regulated HTH gene expression. Three grams of agar (Gibco-BRL) were dissolved in 100 ml of boiled, distilled water and placed in petri dishes. Carbohydrate-enriched agar contained 3 grams of glucose per 100 ml of agar preparation (Siegert and Mordue, 1992). Females fed on agar in the form of 2 cm cubes. To prevent desiccation of the cubes, damp paper towels were placed on the bottom of the dish containing the agar. Agar cubes were replaced every two days. Studies were performed to determine if circulating
carbohydrates could down-regulate HTH-mRNA in the CC of starving females. 10% glucose and 50% trehalose were injected separately in 20 l volume. Female B. discoidalis contain approximately 800 l of hemolymph as determined with [14C]inulin (unpublished observation). Therefore, these doses of sugars should provide final hemolymph concentrations of 2.5 mg/ml for glucose or 12.5 mg/ml for trehalose which are equivalent or greater than the concentrations normally present for these carbohydrates (normal averages = 1 mg/ml glucose; 10 mg/ml trehalose). Thus, these treatments provided sufficient carbohydrate in the hemolymph of starved females to mimic or exceed the normal levels observed for fed insects. 2.4. Recurrent nerve section The recurrent nerve is part of the stomatogastric nervous system and runs along the dorsum of the esophagus connecting the frontal and ingluvial ganglia (Willey, 1961). The recurrent nerve was severed in day-0 females anesthetized with CO2. A longitudinal incision was made dorsally through the cervical membrane; the underlying tissues were moved to expose the esophagus and recurrent nerve; and the recurrent nerve was broken with microforceps. The wound was sealed with 1:1 (v:v) hot beeswax:petroleum jelly. Sham surgery controls underwent all surgical procedures to expose the recurrent nerve, but the nerve was left undamaged. 2.5. Statistics Mean values for experimental and control groups were based on 6 to 8 replicate samples. Means for experiments involving multiple variables were analyzed by ANOVA followed by a Student-Newman-Keuls multiple comparisons test to identify differences between individual means. Statistical analyses were performed using InStat 2.0 (GraphPad Software, San Diego, CA) for Macintosh computers.
3. Results 3.1. Starvation effects on HTH-mRNA HTH biosynthesis doubles in CC from female B. discoidalis held without food and water for 10 days (Sowa et al., 1996). In the present studies, HTH-mRNA content was measured in CC from females held for 10 days with various combinations of food and water to determine if the HTH transcript was also increased. The HTH-mRNA content was increased significantly in CC from females restricted from food, with or without water, and was low in CC from females provided food, with or without water
D.K. Lewis et al. / Journal of Insect Physiology 44 (1998) 967–972
Fig. 1. Effects of 10-day starvation and/or dehydration on HTHmRNA content in corpora cardiaca (CC) of adult female B. discoidalis. Day-0 females were separated and provided with or without food and/or water as indicated. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters above the data bars. Values represent mean ± SEM for 6 replicates using 2 CC per replicate.
(Fig. 1). Therefore, increased HTH-mRNA in the CC was a response to the absence of food, not water. 3.2. Effects of refeeding on HTH-mRNA Starved females were refed to determine if the elevated HTH-mRNA content in the CC was reversible. Females were starved for 11 days, then provided food for 2 days. Control females were either starved or fed for 13 days. Compared to CC from fed females, HTHmRNA content increased 73% in the CC of starved females, but only 13% in the CC from females that were starved, then refed (Fig. 2). These results show that the elevated HTH-mRNA associated with starvation was down-regulated to essentially normal levels by 2 days of feeding. 3.3. Recurrent nerve section and crop emptying Neural effects on HTH gene expression by food distention of the esophagus and/or crop was examined in B. discoidalis by severing the recurrent nerve in newly emerged adult females. Nerve-severed females were either provided food and water or withheld from food and water for 10 days. Sham-operated animals subjected to the same feeding conditions served as controls. Nervesevered insects were dissected to determine if they had
Fig. 2. Effects of feeding following 11-day starvation on HTHmRNA content in the corpora cardiaca (CC) of adult female B. discoidalis. Females were starved for 11 days and subsequently fed for 2 days. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters to the right of the data bars. Values represent mean ± SEM for 8 replicates using 2 CC per replicate.
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fed. Food was present in the crop of both unoperated and nerve-severed females. The HTH-mRNA increased between 2- and 3-fold in the CC of both fed and starved, nerve-severed females and in all the starved females as compared to CC of fed, unoperated females (Fig. 3). HTH-mRNA content in the CC of sham-operated females was intermediate between that of fed, unoperated controls and the nerve-severed and starved insects. These results suggest that the stomatogastric nervous system may monitor crop content or other mechanical actions of feeding to suppress HTH gene expression. Although food was present in the crop of all the nervesevered females, digestion could be impaired so that they were still starving which would result in elevated HTHmRNA. Therefore, fecal weights were measured to evaluate relative food consumption and digestion. Insects were deprived of food but permitted water for 5 days following surgery to empty the alimentary tract. Feeding was resumed on day 5 and fecal weights were measured at days 10 and 15. Total fecal mass of day-10, fed controls was 5 × greater than that of nerve-severed females, and twice that of sham-operated females (Table 1). Fecal weights of sham-operated females were comparable to fed controls by day 15; however, the fecal weight of 15-day-old nerve-severed females was still 67% below that of fed controls. Therefore, the increased HTH-mRNA in the CC of nerve-severed females likely resulted from covert starvation due to decreased crop emptying, and not from disruption of neural inhibition of HTH gene expression. 3.4. Effects of agar-feeding on HTH-mRNA Effects of food intake and neural stimulation of stretch receptors on HTH-mRNA was examined by feeding agar cubes for 12 days. The agar cubes were either plain or enriched with 3% glucose (Fig. 4), and both were consumed equally well. Females fed plain agar or glucoseenriched agar were compared with females fed a normal
Fig. 3. Effects of severing the recurrent nerve on HTH-mRNA content in the corpora cardiaca (CC) from adult female B. discoidalis. Day-0 females were treated as indicated for a period of 10 days. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters to the right of the data bars. Values represent mean ± SEM for 8 replicates using 2 CC per replicate.
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Table 1 Effect of recurrent nerve severance on total fecal weight in female B. discoidalis No. of pellets
Total fecal mass (mg)
Treatment
Sample size
Day 10
Day 15
Day 10
Day 15
No treatment Sham-surgery Recurrent nerve severed
4 4 4
65 28 14
73 88 33
332 145 59
354 426 133
Females were provided water only for 5 days to empty the alimentary tract. On day 5, females were given both food and water. Measurements of total fecal weight were taken on days 10 and 15.
Fig. 4. Effects of feeding plain agar or glucose-enriched agar on HTH-mRNA in the corpora cardiaca (CC) of adult female B. discoidalis. Females were starved, fed normal diet, plain agar, or agar containing 3% glucose for 12 days. Each treatment group received water throughout the experiment. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters to the right of the data bars. Values represent mean ± SEM for 8 replicates using 2 CC per replicate.
dog food diet and with females held without food. All groups were permitted water. The HTH-mRNA content in the CC of females receiving both types of agar was 80% higher than for females fed dog food and was comparable to that of starved females. 3.5. Effects of glucose and trehalose on HTH-mRNA in vivo Possible regulatory actions by carbohydrates on HTHmRNA levels were examined further. Because HTH elevates hemolymph trehalose in cockroaches, high levels of glucose or trehalose in the food or the hemolymph may suppress HTH gene expression. The level of carbohydrates in the hemolymph may be monitored directly by neuro-endocrine systems, or glucose absorption through the gut during digestion may trigger specific gut receptors to stimulate or suppress the production of factors that regulate HTH gene expression. Females were starved for thirteen days then either fed a glucose solution for two days or injected daily, for two days, with 20 l of 10% glucose or 50% trehalose. A two-day exposure to carbohydrates should be sufficient to reverse the stimulus of starvation since this was the period of feeding needed to suppress HTH-mRNA content (Fig. 2). Only glucose was tested per os since com-
plex carbohydrates are degraded to monosaccharides during digestion (Friedman, 1985). Females that received the carbohydrate injections were also permitted water throughout the experiment. CC from females withheld from food (but allowed water) for 15 days contained twice as much HTH-mRNA as CC from insects that fed normally (Fig. 5). In all animals withheld from both food and water, the HTHmRNA content of the CC was elevated over that of fed females, but was lower than for insects that were starved but allowed water. No significant differences were noted in animals fed a glucose solution for 2 days when compared with controls that were starved with or without water. Furthermore, no significant differences were noted between insects that received injections of glucose or trehalose when compared to water-injected controls (Fig. 5). These results, along with the results from females fed glucose-enriched agar cubes indicated that the starvation increase in HTH-mRNA was not downregulated by increased levels of carbohydrates in either the diet or the hemolymph.
4. Discussion CC from adult female B. discoidalis held without food and water display a 2-fold higher rate of HTH biosynth-
Fig. 5. Effects of glucose and trehalose in vivo on HTH-mRNA content in corpora cardiaca (CC) from starved adult female B. discoidalis. Females were either fed a 10% glucose solution or injected with 20 l of 10% glucose or 50% trehalose for 2 days. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters to the right of the data bars. Values represent mean ± SEM for 8 replicates using 2 CC per replicate.
D.K. Lewis et al. / Journal of Insect Physiology 44 (1998) 967–972
esis than CC from animals that feed normally (Sowa et al., 1996). Elevation of HTH synthesis by starvation is not surprising since the role of HTH is to mobilize nutrient stores in the fat body for conversion to circulating metabolites. Increased HTH biosynthesis was confirmed in the present experiments by our observation of doubled HTH-mRNA content in CC from starved females compared to fed females (Fig. 1). The increases in HTH biosynthesis correlated with the absence of food, not water, and the starvation-related elevation in HTHmRNA reversed within 2 days of renewed access to food (Fig. 2). Experiments were undertaken to determine how feeding activity might regulate the steady-state levels of HTH-mRNA in the CC. Three possible effects of feeding were hypothesized. Mechanical effects by chewing, swallowing and crop or gut distention might be monitored by the stomatogastric nervous system. Digestive products might stimulate gut-wall receptors during absorption and cause release of factors from the gut or other tissues that signal the brain-CC to regulate HTH synthesis and secretion. Finally, hemolymph nutrients or metabolites might act directly on the brain-CC complex or stimulate other tissues to produce factors that suppress HTH synthesis-secretion. The stomatogastric nervous system transmits sensory inputs to the brain from the foregut and midgut. In L. migratoria, the stomatogastric nervous system is responsible for controlling neurosecretion by the CC following distention of the foregut (Bernays and Chapman, 1972). During feeding, swallowing movements serve as cues for the production of neurosecretory material in the medial neurosecretory cells and its release from the CC (Clarke and Langley, 1963). Information about these feeding movements is passed to the brain by the recurrent nerve-frontal ganglion and to the CC by the recurrent nerve-hypocerebral ganglion (Clarke and Langley, 1963). Severance of these neural pathways results in failure of the locusts to grow and molt. In L. maderae, the recurrent nerve innervates the proventricular valve and determines the rate at which food is released into the midgut (Engelmann, 1968). Crop emptying is reduced 50% when the recurrent or esophageal nerves are severed. Likewise, severance of the ventricular ganglion in Schistocerca gregaria or the esophageal or ingluvial ganglia in P. americana inhibit crop emptying (Clarke and Greenville, 1960; Davey and Treherne, 1963). In fifth instar larvae of M. sexta, the passage of glucose through the midgut wall is monitored through the frontal ganglion and exerts an inhibitory effect on brain neurosecretory cells that secrete allatotropin for juvenile hormone production (Bhaskaran et al., 1988). These studies demonstrate that the stomatogastric nervous system is central for monitoring and regulating actions related to feeding, digestion, and absorption. Severance of the recurrent nerve elevated HTH-
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mRNA in CC from fed B. discoidalis females similar to starved females, but it also reduced fecal production 60– 80%. This latter finding indicated that nerve-severed animals were processing food much less effectively than the controls, and the rise in HTH-mRNA likely resulted from starvation, rather than by disruption of a feedingrelated, inhibitory neural feedback on the CC. In starved female B. discoidalis, refeeding for 2 days suppressed the elevated HTH-mRNA (Fig. 2); however, feeding plain or glucose-enriched agar did not lower the elevated HTH-mRNA. The results with plain agar verify that neural signals from chewing, swallowing, and crop stretching or emptying did not suppress starvationrelated HTH-mRNA in the CC, but required the presence of nutrients. Likewise, the results with glucose-enriched agar suggest that glucose alone is insufficient to suppress HTH gene expression either as a nutrient or as a signaling molecule for gut absorption of food. Finally, injections of glucose and trehalose failed to suppress HTH-mRNA levels of starved B. discoidalis. Therefore, hemolymph carbohydrates also failed to exert negative feedback on HTH gene expression. Some aspect of feeding is important for down-regulating HTH gene expression. The elevated HTH-mRNA in CC from starved females returned to normal levels within 2 days of renewed feeding. If the inhibitory signal for down-regulation of HTH gene expression is not a neural response to food consumption, it must reside within the nature of the food. In starving larvae of M. sexta, fat body glycogen phosphorylase activity is high but decreases within 2 min of refeeding sucrose-enriched agar (Siegert and Mordue, 1992). The rapidity of the decrease in phosphorylase activity occurs before sufficient carbohydrate could be digested and absorbed to increase hemolymph glucose levels, instead, release of a gut hormone that inactivates the phosphorylase was hypothesized. These results suggest that the gut can release hormonal factors to counteract the physiological effects of starvation. In L. migratoria, locustatachykinin induces AKH-I release from isolated CC (Na¨ssel et al., 1995), and in L. maderae, the midgut contains numerous cells immunoreactive for locustatachykinin (Muren and Nassel, 1996). Such a relationship suggests that gut factors that influence the secretion of AKH peptides could be released in response to feeding and a similar situation could exist in B. discoidalis to affect HTH synthesis and/or secretion. Our data do not identify the specific inhibitor for the starvation-related increase in HTH-mRNA. It is not neural detection of food consumption, the passage of glucose through the gut or increasing glucose or trehalose in the hemolymph. Only by eating a normal dog food diet was the starvation-related increase in HTH-mRNA suppressed. This suggests that the consumption of a complex of nutrients may be required to regulate HTH gene expression.
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Acknowledgements This research was supported by National Institutes of Health Grant No. AI 32942 and by National Science Foundation Grant No. DCB-9017757 and in part by National Science Foundation Grant No. DCB 9104536. The research was conducted by the Texas Agricultural Experiment Station. References Anelli, C.M., Friedman, S., 1986. Presence and regulation of trehalose in the body of adult Phormia regina. Archives of Insect Biochemistry and Physiology 3, 381–395. Bernays, E.A., Chapman, R.F., 1972. The control of changes in peripheral sensilla associated with feeding in Locusta migratoria (L.). Journal of Experimental Biology 57, 755–763. Bhaskaran, G., de la Garza, L.M., Barrera, P., Dahm, K.H., Dunton, R., 1988. The effect of frontal ganglionectomy on supernumerary molting in Manduca sexta. In: Sehnal, F., Zabza, A., Denlinger, D.L. (Eds.), Endocrinological Frontiers in Physiological Insect Ecology. Wroclaw Technical University Press, Wroclaw, pp. 681–684. Bloemen, R.E.B., De Vlieger, T.A., Beenakkers, A.M.T., 1987. The inhibitory effect of trehalose on electrical activity in the glandular lobe of in vitro corpus cardiacum of Locusta migratoria. Comparative Biochemistry and Physiology 87 (A), 193–195. Bowers, W.S., Friedman, S., 1963. Mobilization of fat body glycogen by an extract of corpus cardiacum. Nature 198, 685. Cheeseman, P., Jutsum, A.R., Goldsworthy, G.J., 1976. Quantitative studies on the release of locust adipokinetic hormone. Physiological Entomology 1, 115–121. Clarke, K.U., Greenville, G.J., 1960. Nervous control of movements in the foregut of Schistocerca gregaria Forsk. Nature 186, 98–99. Clarke, K.U., Langley, P., 1963. Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. and F. IV. The relationship between the stomatogastric nervous system and neurosecretion. Journal of Insect Physiology 9, 423–430. Davey, K.G., Treherne, J.E., 1963. Studies on crop function in the cockroach (Periplaneta americana L.). II. The nervous control of crop-emptying. Journal of Experimental Biology 40, 775–780. Engelmann, F., 1968. Feeding and crop emptying in the cockroach Leucophaea maderae. Journal of Insect Physiology 14, 1525–1531. Friedman, S., 1985. Intermediary metabolism. In: Blum, M.S. (Ed.), Fundamentals of Insect Physiology. John Wiley and Sons, New York, pp. 467–505. Hayes, T.K., Keeley, L.L., Knight, D.W., 1986. Insect hypertrehalosemic hormone: Isolation and primary structure from Blaberus discoidalis cockroaches. Biochemical and Biophysical Research Communications 140, 674–678.
Keeley, L.L., Sowa, S.M., Hesson, A.S., 1995. In vitro bioassay for hypertrehalosemic hormone-dependent trehalose biosynthesis by fat body from adult Blaberus discoidalis cockroaches. Archives of Insect Biochemistry and Physiology 28, 313–324. Khan, M.A., Steele, J.E., 1992. Enhancement of hypertrehalosemic hormone biosynthesis by trehalose in isolated cockroach (Periplaneta americana) corpora cardiaca in vitro. Journal of Insect Physiology 38, 659–664. Lee, Y.H., Keeley, L.L., 1994. Intracellular transduction of trehalose synthesis by hypertrehalosemic hormone in the fat body of the tropical cockroach Blaberus discoidalis. Insect Biochemistry and Molecular Biology 24 (5), 473–480. Lewis, D.K., Jezierski, M.K., Keeley, L.L., Bradfield, J.Y., 1997. Hypertrehalosemic hormone in a cockroach: molecular cloning and expression. Molecular and Cellular Endocrinolology 130, 101–108. Muren, J.E., Nassel, D.R., 1996. Radioimmunoassay determination of tachykinin-related peptide in different portions of the central nervous system and intestine of the cockroach Leucophaea maderae. Brain Research 739, 314–321. Na¨ssel, D.R., Passier, P.C.C.M., Elekes, K., Dircksen, H., Vullings, H.G.B., Cantera, R., 1995. Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca. Regulatory Peptides 57, 297–310. Puissant, C., Houdebine, L.-M., 1990. An improvement of the singlestep method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. BioTechniques 8, 148–149. Scarborough, R.M., Jamieson, G.C., Kalish, F., Kramer, S.J., McEnroe, G.A., Miller, C.A., Schooley, D.A., 1984. Isolation and primary structure of two peptides with cardioacceleratory and hyperglycemic activity from the corpora cardiaca of Periplaneta americana. Proceedings of the National Academy of Sciences, United States of America 81, 5575–5579. Siegert, K.J., Mordue, W., 1992. Regulation of fat body glycogen phosphorylase activity during refeeding in Manduca sexta larvae. Archives of Insect Biochemistry and Physiology 19, 225–235. Sowa, S.M., Lu, K.-H., Park, J.H., Keeley, L.L., 1996. Physiological effectors of hyperglycemic neurohormone biosynthesis in an insect. Molecular and Cellular Endocrinololgy 123, 97–105. Van Der Horst, D.J., Van Doorn, J.M., Beenakkers, A.M.T., 1979. Effects of the adipokinetic hormone on the release and turnover of haemolymph diglycerides and on the formation of the diglyceridetransporting lipoprotein system during locust flight. Insect Biochemistry 9, 627–635. Willey, R.B., 1961. The morphology of the stomodeal nervous system in Periplaneta americana (L.) and other Blattaria. Journal of Morphology 108, 219–247. Wilps, H., Ga¨de, G., 1990. Hormonal regulation of carbohydrate metabolism in the blowfly Phormia terraenovae. Journal of Insect Physiology 36, 441–449. Ziegler, R., Eckart, K., Law, J.H., 1990. Adipokinetic hormone controls lipid metabolism in adults and carbohydrate metabolism in larvae of Manduca sexta. Peptides 11, 1037–1040.
Feeding effects on gene expression of the hypertrehalosemic hormone in the cockroach, Blaberus discoidalis Danielle K. Lewis, J.Y. Bradfield, L.L. Keeley
*
Department of Entomology, Texas A and M University, Texas Agricultural Experiment Station, College Station, TX 77843-2475, USA Received 6 May 1997; received in revised form 16 October 1997
Abstract Feeding effects on hypertrehalosemic hormone (HTH) transcript levels in corpora cardiaca (CC) of adult females of the cockroach, Blaberus discoidalis were measured using dot blot hybridization. HTH transcript levels were nearly doubled in CC from females withheld from food and water for ten days compared to CC from fed females. The increase in HTH-mRNA was a response to starvation, not dehydration, and reversed within 2 days after exposure to food. HTH-mRNA was elevated in CC from fed insects that had their recurrent nerve severed, but low fecal output by insects with severed nerves indicated that feeding and digestion were impaired. Thus, the increased HTH synthesis likely resulted from starvation rather than disruption of neural regulation. CC from starved females that were refed with either solutions or agar that contained glucose did not show down-regulation of HTHmRNA. Likewise, injections of glucose or trehalose did not suppress HTH-mRNA levels in CC of starving insects. Down-regulation of the starvation-related increase in HTH-mRNA appears to be a response to consumption of a complex of nutrients and not to increased carbohydrates or mechanical aspects of feeding. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Adipokinetic hormone; Hypertrehalosemic hormone; Trehalose; Neurohormone gene expression; Blaberus discoidalis; Cockroach; Insect; Molecular cloning
1. Introduction The regulation of insect carbohydrate metabolism by neuropeptides of the adipokinetic hormone/red pigmentconcentrating hormone (AKH/RPCH) family is well documented (Bowers and Friedman, 1963; Anelli and Friedman, 1986; Wilps and Ga¨de, 1990; Ziegler et al., 1990; Lee and Keeley, 1994). In the tropical cockroach, Blaberus discoidalis, trehalose synthesis is stimulated by the hypertrehalosemic hormone ( = HTH; pQVNFSPGWGTa), a decapeptide of the AKH/RPCH family (Hayes et al., 1986). AKHs and HTHs regulate energy metabolism in insects, but it is unclear how AKH and HTH synthesis and secretion are, themselves, regulated. Cardioacceleratory hormones (CAH)-I and -II have hypertrehalosemic actions in the American cockroach, Periplaneta americana (Scarborough et al., 1984), and the syntheses of CAH-I and CAH-II by isolated corpora
* Corresponding author. Fax: 001 409 845 9727; E-mail: [email protected] 0022–1910/98/$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 3 4 - 1
cardiaca (CC) are reported to increase 5-fold in the presence of trehalose (Khan and Steele, 1992). This effect appears specific to trehalose and does not occur with glucose, except at high concentrations. Conversely, in Locusta migratoria, high levels of trehalose lower AKH titers and delay lipid mobilization (Cheeseman et al., 1976; Van Der Horst et al., 1979). Trehalose inhibits electrical activity in isolated CC from L. migratoria which may explain their reduced secretion of AKH (Bloemen et al., 1987). These results suggest that the circulating metabolites regulated by the AKHs and HTHs may have feedback effects over the synthesis and secretion of the hormones. HTH synthesis and secretion are highest during the first several days following the adult molt in both sexes of B. discoidalis, but after about a week, both processes decline by 60–70% and remain stable (Sowa et al., 1996). Furthermore, CC from starving females have 2fold higher rates of HTH synthesis than CC from fed females (Sowa et al., 1996). Results from age studies of HTH gene expression parallel the results from the synthesis and secretion studies (Lewis et al., 1997). The
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present research was undertaken to determine if starvation exerted its stimulatory effects on HTH biosynthesis at the level of gene expression, and if so, to determine if dietary carbohydrates or feeding reversed the starvation effects. The results showed that HTH-mRNA doubled in the CC of starved females and was suppressed by general nutrient intake, but not specifically by carbohydrate consumption.
2. Materials and methods 2.1. Experimental animals B. discoidalis were reared in wood shavings in 40 l plastic buckets in a 12-h light, 12-h dark circadian cycle at 27 ± 2°C and fed Purina dog chow and water (Keeley et al., 1995). Males and females were separated at the adult molt. Starved insects were withheld from food and/or water from adult emergence until experimentation. 2.2. HTH-mRNA analysis Total RNA was extracted from 2 or 3 pairs of corpora cardiaca-corpora allata complexes ( = CC) (Puissant and Houdebine, 1990). Dot blot hybridization analysis was performed using a specific [32P]HTH-cDNA probe to quantify the HTH-mRNA (Lewis et al., 1997). Each replicate sample consisted of 2 or 3 CC pairs since dotblots are essentially linear for the HTH-mRNA present in a total RNA extract of 1 to 3 CC pairs (Lewis et al., 1997). Each preparation of probe is labeled with an independent lot of [32P] precursor. Therefore, each probe has a different specific activity and all treatment and control groups within an experiment must be probed simultaneously to compare results. Because a different probe is prepared for each experiment, it is not possible to compare absolute cpms between experiments. 2.3. Carbohydrate feeding Possible effects on HTH gene expression by food consumption, independent of nutrient content, were measured by feeding plain agar. Glucose-enriched agar was fed to determine if carbohydrate in the food or glucose passage through the gut regulated HTH gene expression. Three grams of agar (Gibco-BRL) were dissolved in 100 ml of boiled, distilled water and placed in petri dishes. Carbohydrate-enriched agar contained 3 grams of glucose per 100 ml of agar preparation (Siegert and Mordue, 1992). Females fed on agar in the form of 2 cm cubes. To prevent desiccation of the cubes, damp paper towels were placed on the bottom of the dish containing the agar. Agar cubes were replaced every two days. Studies were performed to determine if circulating
carbohydrates could down-regulate HTH-mRNA in the CC of starving females. 10% glucose and 50% trehalose were injected separately in 20 l volume. Female B. discoidalis contain approximately 800 l of hemolymph as determined with [14C]inulin (unpublished observation). Therefore, these doses of sugars should provide final hemolymph concentrations of 2.5 mg/ml for glucose or 12.5 mg/ml for trehalose which are equivalent or greater than the concentrations normally present for these carbohydrates (normal averages = 1 mg/ml glucose; 10 mg/ml trehalose). Thus, these treatments provided sufficient carbohydrate in the hemolymph of starved females to mimic or exceed the normal levels observed for fed insects. 2.4. Recurrent nerve section The recurrent nerve is part of the stomatogastric nervous system and runs along the dorsum of the esophagus connecting the frontal and ingluvial ganglia (Willey, 1961). The recurrent nerve was severed in day-0 females anesthetized with CO2. A longitudinal incision was made dorsally through the cervical membrane; the underlying tissues were moved to expose the esophagus and recurrent nerve; and the recurrent nerve was broken with microforceps. The wound was sealed with 1:1 (v:v) hot beeswax:petroleum jelly. Sham surgery controls underwent all surgical procedures to expose the recurrent nerve, but the nerve was left undamaged. 2.5. Statistics Mean values for experimental and control groups were based on 6 to 8 replicate samples. Means for experiments involving multiple variables were analyzed by ANOVA followed by a Student-Newman-Keuls multiple comparisons test to identify differences between individual means. Statistical analyses were performed using InStat 2.0 (GraphPad Software, San Diego, CA) for Macintosh computers.
3. Results 3.1. Starvation effects on HTH-mRNA HTH biosynthesis doubles in CC from female B. discoidalis held without food and water for 10 days (Sowa et al., 1996). In the present studies, HTH-mRNA content was measured in CC from females held for 10 days with various combinations of food and water to determine if the HTH transcript was also increased. The HTH-mRNA content was increased significantly in CC from females restricted from food, with or without water, and was low in CC from females provided food, with or without water
D.K. Lewis et al. / Journal of Insect Physiology 44 (1998) 967–972
Fig. 1. Effects of 10-day starvation and/or dehydration on HTHmRNA content in corpora cardiaca (CC) of adult female B. discoidalis. Day-0 females were separated and provided with or without food and/or water as indicated. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters above the data bars. Values represent mean ± SEM for 6 replicates using 2 CC per replicate.
(Fig. 1). Therefore, increased HTH-mRNA in the CC was a response to the absence of food, not water. 3.2. Effects of refeeding on HTH-mRNA Starved females were refed to determine if the elevated HTH-mRNA content in the CC was reversible. Females were starved for 11 days, then provided food for 2 days. Control females were either starved or fed for 13 days. Compared to CC from fed females, HTHmRNA content increased 73% in the CC of starved females, but only 13% in the CC from females that were starved, then refed (Fig. 2). These results show that the elevated HTH-mRNA associated with starvation was down-regulated to essentially normal levels by 2 days of feeding. 3.3. Recurrent nerve section and crop emptying Neural effects on HTH gene expression by food distention of the esophagus and/or crop was examined in B. discoidalis by severing the recurrent nerve in newly emerged adult females. Nerve-severed females were either provided food and water or withheld from food and water for 10 days. Sham-operated animals subjected to the same feeding conditions served as controls. Nervesevered insects were dissected to determine if they had
Fig. 2. Effects of feeding following 11-day starvation on HTHmRNA content in the corpora cardiaca (CC) of adult female B. discoidalis. Females were starved for 11 days and subsequently fed for 2 days. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters to the right of the data bars. Values represent mean ± SEM for 8 replicates using 2 CC per replicate.
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fed. Food was present in the crop of both unoperated and nerve-severed females. The HTH-mRNA increased between 2- and 3-fold in the CC of both fed and starved, nerve-severed females and in all the starved females as compared to CC of fed, unoperated females (Fig. 3). HTH-mRNA content in the CC of sham-operated females was intermediate between that of fed, unoperated controls and the nerve-severed and starved insects. These results suggest that the stomatogastric nervous system may monitor crop content or other mechanical actions of feeding to suppress HTH gene expression. Although food was present in the crop of all the nervesevered females, digestion could be impaired so that they were still starving which would result in elevated HTHmRNA. Therefore, fecal weights were measured to evaluate relative food consumption and digestion. Insects were deprived of food but permitted water for 5 days following surgery to empty the alimentary tract. Feeding was resumed on day 5 and fecal weights were measured at days 10 and 15. Total fecal mass of day-10, fed controls was 5 × greater than that of nerve-severed females, and twice that of sham-operated females (Table 1). Fecal weights of sham-operated females were comparable to fed controls by day 15; however, the fecal weight of 15-day-old nerve-severed females was still 67% below that of fed controls. Therefore, the increased HTH-mRNA in the CC of nerve-severed females likely resulted from covert starvation due to decreased crop emptying, and not from disruption of neural inhibition of HTH gene expression. 3.4. Effects of agar-feeding on HTH-mRNA Effects of food intake and neural stimulation of stretch receptors on HTH-mRNA was examined by feeding agar cubes for 12 days. The agar cubes were either plain or enriched with 3% glucose (Fig. 4), and both were consumed equally well. Females fed plain agar or glucoseenriched agar were compared with females fed a normal
Fig. 3. Effects of severing the recurrent nerve on HTH-mRNA content in the corpora cardiaca (CC) from adult female B. discoidalis. Day-0 females were treated as indicated for a period of 10 days. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters to the right of the data bars. Values represent mean ± SEM for 8 replicates using 2 CC per replicate.
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Table 1 Effect of recurrent nerve severance on total fecal weight in female B. discoidalis No. of pellets
Total fecal mass (mg)
Treatment
Sample size
Day 10
Day 15
Day 10
Day 15
No treatment Sham-surgery Recurrent nerve severed
4 4 4
65 28 14
73 88 33
332 145 59
354 426 133
Females were provided water only for 5 days to empty the alimentary tract. On day 5, females were given both food and water. Measurements of total fecal weight were taken on days 10 and 15.
Fig. 4. Effects of feeding plain agar or glucose-enriched agar on HTH-mRNA in the corpora cardiaca (CC) of adult female B. discoidalis. Females were starved, fed normal diet, plain agar, or agar containing 3% glucose for 12 days. Each treatment group received water throughout the experiment. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters to the right of the data bars. Values represent mean ± SEM for 8 replicates using 2 CC per replicate.
dog food diet and with females held without food. All groups were permitted water. The HTH-mRNA content in the CC of females receiving both types of agar was 80% higher than for females fed dog food and was comparable to that of starved females. 3.5. Effects of glucose and trehalose on HTH-mRNA in vivo Possible regulatory actions by carbohydrates on HTHmRNA levels were examined further. Because HTH elevates hemolymph trehalose in cockroaches, high levels of glucose or trehalose in the food or the hemolymph may suppress HTH gene expression. The level of carbohydrates in the hemolymph may be monitored directly by neuro-endocrine systems, or glucose absorption through the gut during digestion may trigger specific gut receptors to stimulate or suppress the production of factors that regulate HTH gene expression. Females were starved for thirteen days then either fed a glucose solution for two days or injected daily, for two days, with 20 l of 10% glucose or 50% trehalose. A two-day exposure to carbohydrates should be sufficient to reverse the stimulus of starvation since this was the period of feeding needed to suppress HTH-mRNA content (Fig. 2). Only glucose was tested per os since com-
plex carbohydrates are degraded to monosaccharides during digestion (Friedman, 1985). Females that received the carbohydrate injections were also permitted water throughout the experiment. CC from females withheld from food (but allowed water) for 15 days contained twice as much HTH-mRNA as CC from insects that fed normally (Fig. 5). In all animals withheld from both food and water, the HTHmRNA content of the CC was elevated over that of fed females, but was lower than for insects that were starved but allowed water. No significant differences were noted in animals fed a glucose solution for 2 days when compared with controls that were starved with or without water. Furthermore, no significant differences were noted between insects that received injections of glucose or trehalose when compared to water-injected controls (Fig. 5). These results, along with the results from females fed glucose-enriched agar cubes indicated that the starvation increase in HTH-mRNA was not downregulated by increased levels of carbohydrates in either the diet or the hemolymph.
4. Discussion CC from adult female B. discoidalis held without food and water display a 2-fold higher rate of HTH biosynth-
Fig. 5. Effects of glucose and trehalose in vivo on HTH-mRNA content in corpora cardiaca (CC) from starved adult female B. discoidalis. Females were either fed a 10% glucose solution or injected with 20 l of 10% glucose or 50% trehalose for 2 days. Significant differences (P = 0.05) between means are indicated by dissimilar lower-case letters to the right of the data bars. Values represent mean ± SEM for 8 replicates using 2 CC per replicate.
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esis than CC from animals that feed normally (Sowa et al., 1996). Elevation of HTH synthesis by starvation is not surprising since the role of HTH is to mobilize nutrient stores in the fat body for conversion to circulating metabolites. Increased HTH biosynthesis was confirmed in the present experiments by our observation of doubled HTH-mRNA content in CC from starved females compared to fed females (Fig. 1). The increases in HTH biosynthesis correlated with the absence of food, not water, and the starvation-related elevation in HTHmRNA reversed within 2 days of renewed access to food (Fig. 2). Experiments were undertaken to determine how feeding activity might regulate the steady-state levels of HTH-mRNA in the CC. Three possible effects of feeding were hypothesized. Mechanical effects by chewing, swallowing and crop or gut distention might be monitored by the stomatogastric nervous system. Digestive products might stimulate gut-wall receptors during absorption and cause release of factors from the gut or other tissues that signal the brain-CC to regulate HTH synthesis and secretion. Finally, hemolymph nutrients or metabolites might act directly on the brain-CC complex or stimulate other tissues to produce factors that suppress HTH synthesis-secretion. The stomatogastric nervous system transmits sensory inputs to the brain from the foregut and midgut. In L. migratoria, the stomatogastric nervous system is responsible for controlling neurosecretion by the CC following distention of the foregut (Bernays and Chapman, 1972). During feeding, swallowing movements serve as cues for the production of neurosecretory material in the medial neurosecretory cells and its release from the CC (Clarke and Langley, 1963). Information about these feeding movements is passed to the brain by the recurrent nerve-frontal ganglion and to the CC by the recurrent nerve-hypocerebral ganglion (Clarke and Langley, 1963). Severance of these neural pathways results in failure of the locusts to grow and molt. In L. maderae, the recurrent nerve innervates the proventricular valve and determines the rate at which food is released into the midgut (Engelmann, 1968). Crop emptying is reduced 50% when the recurrent or esophageal nerves are severed. Likewise, severance of the ventricular ganglion in Schistocerca gregaria or the esophageal or ingluvial ganglia in P. americana inhibit crop emptying (Clarke and Greenville, 1960; Davey and Treherne, 1963). In fifth instar larvae of M. sexta, the passage of glucose through the midgut wall is monitored through the frontal ganglion and exerts an inhibitory effect on brain neurosecretory cells that secrete allatotropin for juvenile hormone production (Bhaskaran et al., 1988). These studies demonstrate that the stomatogastric nervous system is central for monitoring and regulating actions related to feeding, digestion, and absorption. Severance of the recurrent nerve elevated HTH-
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mRNA in CC from fed B. discoidalis females similar to starved females, but it also reduced fecal production 60– 80%. This latter finding indicated that nerve-severed animals were processing food much less effectively than the controls, and the rise in HTH-mRNA likely resulted from starvation, rather than by disruption of a feedingrelated, inhibitory neural feedback on the CC. In starved female B. discoidalis, refeeding for 2 days suppressed the elevated HTH-mRNA (Fig. 2); however, feeding plain or glucose-enriched agar did not lower the elevated HTH-mRNA. The results with plain agar verify that neural signals from chewing, swallowing, and crop stretching or emptying did not suppress starvationrelated HTH-mRNA in the CC, but required the presence of nutrients. Likewise, the results with glucose-enriched agar suggest that glucose alone is insufficient to suppress HTH gene expression either as a nutrient or as a signaling molecule for gut absorption of food. Finally, injections of glucose and trehalose failed to suppress HTH-mRNA levels of starved B. discoidalis. Therefore, hemolymph carbohydrates also failed to exert negative feedback on HTH gene expression. Some aspect of feeding is important for down-regulating HTH gene expression. The elevated HTH-mRNA in CC from starved females returned to normal levels within 2 days of renewed feeding. If the inhibitory signal for down-regulation of HTH gene expression is not a neural response to food consumption, it must reside within the nature of the food. In starving larvae of M. sexta, fat body glycogen phosphorylase activity is high but decreases within 2 min of refeeding sucrose-enriched agar (Siegert and Mordue, 1992). The rapidity of the decrease in phosphorylase activity occurs before sufficient carbohydrate could be digested and absorbed to increase hemolymph glucose levels, instead, release of a gut hormone that inactivates the phosphorylase was hypothesized. These results suggest that the gut can release hormonal factors to counteract the physiological effects of starvation. In L. migratoria, locustatachykinin induces AKH-I release from isolated CC (Na¨ssel et al., 1995), and in L. maderae, the midgut contains numerous cells immunoreactive for locustatachykinin (Muren and Nassel, 1996). Such a relationship suggests that gut factors that influence the secretion of AKH peptides could be released in response to feeding and a similar situation could exist in B. discoidalis to affect HTH synthesis and/or secretion. Our data do not identify the specific inhibitor for the starvation-related increase in HTH-mRNA. It is not neural detection of food consumption, the passage of glucose through the gut or increasing glucose or trehalose in the hemolymph. Only by eating a normal dog food diet was the starvation-related increase in HTH-mRNA suppressed. This suggests that the consumption of a complex of nutrients may be required to regulate HTH gene expression.
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