Carbohydrate metabolism during starvation in the silkworm Bombyx mori

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia.     

Involvement of adipokinetic hormone in the homeostatic control of hemolymph trehalose concentration in the larvae of Bombyx mori

Prior to wandering, 5th instar larvae of the silkworm, Bombyx mori, maintain constant hemolymph titers of trehalose. Head ligation of day 3, 5th instar larvae significantly decreased the hemolymph trehalose concentrations, but the concentrations did not decrease in starved larvae. After being diluted by replacement of larval hemolymph with insect Ringer's solution, the trehalose concentrations recovered the initial levels in 90 min in the non-ligated larvae, while they were not restored in 90 min in the neck-ligated larvae. These results suggest that a head factor(s) with hypertrehalosemic activity is involved in the homeostatic control of hemolymph trehalose concentration. When adipokinetic hormone (AKH) was injected into neck-ligated larvae, the trehalose concentrations increased in 2 h and decreased thereafter. Repeated injections of AKH every 4 h maintained the concentrations for 12 h. These findings suggest that AKH induces a hypertrehalosemic response and is involved in the homeostasis of hemolymph trehalose concentration in the larval feeding period.     

Identification of a hypertrehalosemic factor in Spodoptera exigua

Trehalose is a major blood sugar in insects with a range of physiological functions, including an energy source and a cryoprotectant. Hemolymph trehalose concentrations are tightly regulated according to physiological conditions. An insulin‐like peptide, SeILP1, downregulates hemolymph trehalose concentrations in Spodoptera exigua. Here, we identified a factor that upregulates hemolymph trehalose concentration in S. exigua. Hemolymph trehalose concentrations were significantly increased after immune challenge or under starvation in a time‐dependent manner. To determine endocrine factors responsible for the upregulation, stress‐associated mediators, such as octopamine, serotonin, or eicosanoids were injected, but they did not upregulate hemolymph trehalose. On the other hand, injection with Schistocerca gregaria adipokinetic hormone (AKH) significantly increased hemolymph trehalose concentration in S. exigua. During upregulation of hemolymph trehalose by AKH injection, trehalose degradation appeared to be inhibited because expression of trehalase and SeILP1 were significantly suppressed while that of trehalose phosphate synthase was not significantly changed. Interrogation of a Spodoptera genome database identified an S. exigua AKH‐like gene and its expression was confirmed. During starvation, its expression concentrations were increased, although RNA interference specific to the AKH‐like hypertrehalosemic factor (SeHTF) gene significantly prevented the upregulation of hemolymph trehalose concentrations during starvation. A synthetic peptide of SeHTF was prepared and injected into S. exigua larvae. At nanomolar concentration, the synthetic SeHTF peptide effectively upregulated hemolymph trehalose concentrations. Here we report a novel hypertrehalosemic factor in S. exigua (SeHTF).     

Proton gradient mediates hemolymph trehalose influx into aphid bacteriocytes

Aphids harbor proteobacterial endosymbionts such as Buchnera aphidicola housed in specialized bacteriocytes derived from host cells. The endosymbiont Buchnera supplies essential amino acids such as arginine to the host cells and, in turn, obtains sugars needed for its survival from the hemolymph. The mechanism of sugar supply in aphid bacteriocytes has been rarely studied. It also remains unclear how Buchnera acquires its carbon source. The hemolymph sugars in Acyrthosiphon pisum are composed of the disaccharide trehalose containing two glucose molecules. Here, we report for the first time that trehalose is transported and used as a potential carbon source by Buchnera across the bacteriocyte plasma membrane via trehalose transporters. The current study characterized the bacteriocyte trehalose transporter Ap_ST11 (LOC100159441) using the Xenopus oocyte expression system. The Ap_ST11 transporter was found to be proton-dependent with a K_m value ≥700 mM. We re-examined the hemolymph trehalose at 217.8 mM using a fluorescent trehalose sensor. The bacteriocytes did not obtain trehalose by facilitated diffusion along the gradient across cellular membranes. These findings suggest that trehalose influx into the bacteriocytes depends on the extracellular proton-driven secondary electrochemical transporter.     

东方粘虫飞行初期糖类的动用和消耗

本文研究了静态和飞翔开始阶段东方粘虫Mythimna separta(Walker)飞翔肌、脂肪体及肠道内糖元和海藻糖以及血淋巴内海藻糖含量的动态变化。飞行1h后脂肪体内消耗糖元最多,约占总消耗糖类的80%-90%;其次是肠道组织的糖元消耗,飞翔肌内糖类含量是有限的,仅够飞翔最初时期(3min)使用。在此时期,飞翔肌海藻糖含量上升然后下降,脂肪体和肠道海藻糖经历下降和继而上升的变化。然而,血淋巴内海藻糖含量一直下降到流入和流出量持平。雄蛾飞翔肌糖类代谢水平高于雌娥飞翔肌。     

Feeding glucose or sucrose, but not trehalose, suppresses the starvation-induced premature pupation in the yellow-spotted longicorn beetle, Psacothea hilaris

Under 25 degrees C and a long-day photoperiod, starvation induces premature pupation in 4th instar Psacothea hilaris larvae exceeding a threshold weight of 180 mg, resulting in the formation of small but morphologically normal adults. To investigate possible mechanisms underlying this phenomenon, we first measured the hemolymph trehalose and glucose levels of starved larvae. When larvae were starved after 4 days of feeding (attaining the threshold weight), glucose levels decreased 4-fold within the next 24 h, while trehalose levels, after a temporary slight decrease, increased remarkably to reach a peak just before the prepupa stage. The effects of ingesting various nutrients on the developmental fate and the hemolymph sugar titers of starving larvae were then examined. Feeding on agar blocks containing sucrose or glucose totally suppressed the occurrence of premature pupation, while trehalose, fructose, casein and starch were ineffective. Feeding on glucose or trehalose resulted in a 6-fold decrease in hemolymph glucose levels and remarkably elevated trehalose levels. Since feeding on glucose and trehalose induced similar changes in hemolymph sugar titers but trehalose was not effective in suppressing premature pupation, glucose may have exhibited its effects via gustatory mechanisms.     

Flies without Trehalose

Living organisms adapt to environmental changes through metabolic homeostasis. Sugars are used primarily for the metabolic production of ATP energy and carbon sources. Trehalose is a nonreducing disaccharide that is present in many organisms. In insects, the principal hemolymph sugar is trehalose instead of glucose. As in mammals, hemolymph sugar levels in Drosophila are regulated by the action of endocrine hormones. Therefore, the mobilization of trehalose to glucose is thought to be critical for metabolic homeostasis. However, the physiological role of trehalose as a hemolymph sugar during insect development remains largely unclear. Here, we demonstrate that mutants of the trehalose-synthesizing enzyme Tps1 failed to produce trehalose as expected but survived into the late pupal period and died before eclosion. Larvae without trehalose grew normally, with a slight reduction in body size, under normal food conditions. However, these larvae were extremely sensitive to starvation, possibly due to a local defect in the central nervous system. Furthermore, Tps1 mutant larvae failed to grow on a low-sugar diet and exhibited severe growth defects on a low-protein diet. These diet-dependent phenotypes of Tps1 mutants demonstrate the critical role of trehalose during development in Drosophila and reveal how animals adapt to changes in nutrient availability.     

Bombyxin secretion in the adult silkmoth Bombyx mori: sex-specificity and its correlation with metabolism

Changes in the hemolymph bombyxin titer of the adult silkmoth Bombyx mori were investigated by time-resolved fluoroimmunoassay. Immediately after eclosion, hemolymph bombyxin titers were low in both males and females, and then increased steeply in males to a very high level and this high titer was maintained for at least 3 h, whereas the titer increment in females was small and transient. The difference in the change of bombyxin titer between males and females suggests that bombyxin is responsible for the regulation of physiological changes underlying sexually different activities of the adult moths. However, no evidence was obtained that bombyxin controls adult metabolism as far as the effects of bombyxin on the concentrations of carbohydrates and lipids in the hemolymph were investigated. The change in the hemolymph trehalose concentration was almost the same between sexes, and between intact and neck-ligated moths. Furthermore, bombyxin injection did not affect the hemolymph trehalose concentration nor trehalase activity in the muscle. Although the hemolymph lipid concentration rose after eclosion in males, it was not influenced by bombyxin. These results exhibit striking contrast to the results of our previous study, in which bombyxin showed hypotrehalosemic activity in the larval stage, thus indicating that the action of bombyxin changes during metamorphosis.     

EVIDENCE THAT ECDYSIS IN THE LARVAL COCKROACH,Periplaneta americana L. IS TRIGGERED BY AN INCREASE IN THE CONCENTRATION OF HEMOLYMPH SUGAR

Ecdysis in insects can be defined as shedding of the cuticle at the end of a larval stadium. This event can only occur after the peak titer of ecdysteroid in the hemolymph has returned to a low level. In the cockroach Periplaneta americana, ecdysis is strongly correlated with a rise in the concentration of trehalose and glucose in the hemolymph, leading to the idea that a causal relationship may exist between both events. The objective in this study was to determine if an increase in hemolymph sugar level would shorten the time to ecdysis in cockroach larvae with experimentally delayed ecdysis. The last larval stadium of P. americana averages 33.5 days but this increases significantly if the larva is injected with a small volume of saline. Injection of 10 μl of saline on day 20 and on four successive days lengthened the stadium by as much as 2 weeks. If, however, trehalose or glucose is incorporated into the saline, approximately 40% of the treated larvae undergo ecdysis at the same time as uninjected larvae. Injection of Peram‐AKH, the hypertrehalosemic hormone, also decreases the time for ecdysis to occur. This suggests that peak levels of ecdysteroid trigger the release of Peram‐AKH, which then leads to activation of trehalose synthesis. The results support the hypothesis that elevated hemolymph sugar is a contributing factor in the removal of ecdysteroid from the hemolymph.     

The potential of trehalose to replace insect hemolymph in artificial media for Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Insect additives have been shown to improve the value of artificial media for Trichogramma species, but at the same time maintain dependence on parallel cultures of host insects. In the present study, Trichogramma dendrolimi Matsumura was reared in vitro from egg to adult on artificial media with different contents of pupal hemolymph of Chinese oak silkmoth Antheraea pernyi (Guérin‐Méneville) and with supplements of distilled water, or of trehalose dissolved in water or in Grace's insect medium. The results indicated insect hemolymph was the key component of artificial medium. Developmental parameters, including rates of parasitism, pupation, adult emergence and normal adults, and numbers of produced adults, were increased on media supplemented with trehalose even when the proportion of pupal hemolymph was reduced. Two artificial media, the first containing 30% hemolymph and 10% trehalose in water with 98.9% parasitism rate, 77.7.0% pupation rate, 77.2% emergence rate, 80.0% normal adult rate and 333 produced adults, and the second containing 25% hemolymph and 15% trehalose in Grace's insect medium with 97.8% parasitism rate, 91.0% pupation rate, 85.2% emergence rate, 76.1% normal adult rate and 757 produced adults, were believed to hold potential to mass produce T. dendrolimi. The use of trehalose to partially replace pupal hemolymph in artificial medium of this and other Trichogramma species may contribute to a significant reduction in their production cost and may as such help to evade problems related to short supplies of lepidopteran eggs, which currently constitute the main factitious host for the mass rearing of the parasitoids.     

Sensitivities to juvenile hormone and ecdysteroid in the diapause larvae of Omphisa fuscidentalis based on the hemolymph trehalose dynamics index

The final instar larva of the bamboo borer, Omphisa fuscidentalis, is in diapause for 9 months from September to the following June. Trehalose and ecdysteroid concentrations in hemolymph were measured through the larval diapause period and in the pupal stage. The ecdysteroid concentration remained low until November, followed by a gradual increase to about 30 ng/ml in May. The trehalose concentration remained at levels ranging between 40-50 mM until May, and decreased to an almost undetectable level after pupation. Since a juvenile hormone analogue (JHA), methoprene, is capable of terminating diapause by stimulating larval prothoracic glands, we examined its effects on ecdysteroid and trehalose concentrations in larvae in December and February. The hemolymph ecdysteroid increased more quickly in February than in December, indicating that the sensitivity of the prothoracic glands to JHA increased towards the end of diapause termination. Similarly, hemolymph trehalose in February decreased within a few days after JHA application, while in December the decrease occurred in the third week. Exogenous 20-hydroxyecdysone (20E) caused a decrease in trehalose concentration in a dose-dependent manner. The effective dose of 20E, however, did not change from January until April, implying that the sensitivity of tissue(s) to 20E may not change until the end of diapause. Taken together, our results suggest that the sensitivities of tissues to JH and 20E do not increase simultaneously with the progress of diapause development and that termination of larval diapause is not associated simply with the restoration of hormone deficiencies.     

感染布氏白僵菌后油松毛虫血淋巴中海藻糖酶活性、海藻糖和葡萄糖含量的变化

为了揭示病原真菌白僵菌Beauveria侵染昆虫过程中如何利用虫体内糖类物质作为自身营养, 本研究测定了布氏白僵菌Beauveria brongniartii (Sacc.) Petch （2382菌株）感染油松毛虫Dendrolimus tabulaeformis Tsai et Liu幼虫后, 虫体血淋巴中酸性海藻糖降解酶活性及海藻糖和葡萄糖含量的变化。油松毛虫4龄幼虫感染菌株孢子悬浮液后, 血淋巴中酸性海藻糖降解酶的活性明显高于对照组, 感染后第3天酶活性达到最大值（0.2786 U/mg）, 此后第4-6 天酶活性逐渐降低; 染菌后的6 d中, 血淋巴中海藻糖含量显著低于对照组, 同样在感染后第4天其含量逐渐降低, 第6天时降到最低值。相比之下, 处理组血淋巴中的葡萄糖含量显著高于对照组; 处理组其含量在第1-3天内呈现快速升高趋势, 在第3天达到最大值（7.7615 mmol/L）, 然后逐渐降低。结果说明, 白僵菌侵入昆虫血淋巴后, 菌株代谢产生酸性海藻糖降解酶, 将血淋巴中的海藻糖水解成为葡萄糖, 然后为真菌利用, 破坏了虫体内的血糖平衡, 这是一个相互连接的生理代谢和生化反应过程。     

A trehalose 6-phosphate synthase gene of the hemocytes of the blue crab, Callinectes sapidus: cloning, the expression, its enzyme activity and relationship to hemolymph trehalose levels

Trehalose in ectoderms functions in energy metabolism and protection in extreme environmental conditions. We structurally characterized trehalose 6-phosphate synthase (TPS) from hemocytes of the blue crab, Callinectes sapidus. C. sapidus Hemo TPS (CasHemoTPS), like insect TPS, encodes both TPS and trehalose phosphate phosphatase domains. Trehalose seems to be a major sugar, as it shows higher levels than does glucose in hemocytes and hemolymph. Increases in HemoTPS expression, TPS enzyme activity in hemocytes, and hemolymph trehalose levels were determined 24 h after lipopolysaccharide challenge, suggesting that both TPS and TPP domains of CasHemoTPS are active and functional. The TPS gene has a wide tissue distribution in C. sapidus, suggesting multiple biosynthetic sites. A correlation between TPS activity in hemocytes and hemolymph trehalose levels was found during the molt cycle. The current study provides the first evidence of presence of trehalose in hemocytes and TPS in tissues of C. sapidus and implicates its functional role in energy metabolism and physiological adaptation.     

Correlation between concentration of hemolymph nutrients and amount of fat body consumed in lightly and heavily parasitized hosts (Pseudaletia separata)

Two states of parasitization in the Pseudaletia separata-Cotesia kariyai system were examined: one that was lightly parasitized and one that was heavily parasitized. We predicted that the consumption of fat body and hemolymph nutrients depends on the number of parasitoid larvae in the host. Lightly parasitized hosts (average clutch size+/-S.E.: 42.5+/-16.2, N=15) and heavily parasitized hosts (average clutch size+/-S.E.: 230.2+/-8.8, N=15) were prepared artificially. Eight days after parasitization, perivisceral fat body was depleted in the heavily parasitized host, although peripheral fat body was not yet consumed, but by day 10 most of the peripheral fat body was consumed. In lightly parasitized hosts, perivisceral fat body was not consumed by day 10. The parasitoid larvae deplete the perivisceral fat body first and then consume the peripheral fat body in the heavily parasitized host. The amount of trehalose, the major carbohydrate in the hemolymph, was related to the number of parasitoid larvae developing in the host. In a heavily parasitized host, trehalose concentrations remained low. However, in lightly parasitized hosts, the amount of trehalose increased 8 days after parasitization and then decreased by day 10. Protein and total lipid concentrations in the hemolymph of the heavily parasitized host were significantly lower than in lightly parasitized host on day 10, suggesting that the large number of parasitoid larvae depleted the fat body and hemolymph nutrients by day 10. High concentrations of total lipid on day 8 and 10 in lightly parasitized hosts and on day 8 in heavily parasitized host are likely to be attributed to the teratocytes.     

Hemolymph of Anopheles stephensi from noninfected and Plasmodium berghei-infected mosquitoes. 3. Carbohydrates.

Determinations were made of carbohydrates in hemolymph collected from adult female mosquitoes (Anopheles stephensi). First the hemolymph was fractionated by extraction and precipitation procedures, after which qualitative and quantitative determinations of carbohydrates were made by thin layer chromatography. The most abundant sugars found in the hemolymph were glucose and trehalose, though maltose, glucuronic acid, and inositol could be found after the mosquitoes took blood meals. After the mosquitoes ingested a noninfected blood meal, their hemolymph sugar levels rose almost 4-fold. There was less of an increase following a blood meal infected with the rodent malaria parasite, Plasmodium berghei. Depletion of sugars in the hemolymph of infected mosquitoes may result from direct utilization of sugar by the malaria parasite developing within the mosquito.     

Circadian rhythm of trehalose in the face fly Musca autumnalis de Geer

Trehalose levels were determined over two 24 hr spans in groups of face fly adults 3-4 days after emergence from the puparium. Face fly pupae were placed in rearing chambers at 27 degrees C in a staggered light-dark regimen, LD 16:8, so that at a given clock hour, samples could be obtained at several different hours after lights on (HALO). Trehalose was determined in hemolymph collected from a puncture in the intersegmental membrane of the abdomen. Treated hemolymph samples were passed through a Bio-Rad Amino 5-S disaccharide column and a Waters 410 refractive index detector was used to differentiate among sugars. The circadian acrophase derived by cosinor analysis in hemolymph trehalose (when the values were 25.49 and 26.86 micrograms/microliters on the first and second days respectively) occurred at -226 degrees (ca 15 HALO) and the bathyphase at 24 HALO. The mesor = 11.82 micrograms/microliters trehalose, the amplitude = 8.57 micrograms/microliters trehalose and the P-value for presence of a rhythm was 0.003. Based on these data, differences between control and test flies in a bioassay of hypertrehalosemic activity would be most easily observed at 0-8 HALO, while exogenous hypotrehalosemic activity would be best assayed at 12-20 HALO.     

Growth and hemolymph trehalose levels of Heliothis zea larvae fed diets containing various sugars

• 1.1. The amount of sugar required for growth of Heliothis zea larvae on a chemically defined diet was determined.
• 2.2. Larvae grew well on fructose, galactose, sucrose, trehalose and raffinose diets but not on diets containing more than 0.5% glucose.
• 3.3. A starch diet did not promote rapid larval growth.
• 4.4. Hemolymph trehalose levels in 12-day-old larvae ranged from none to 45μmoles/ml.
• 5.5. A method for analysis of hemolymph trehalose by gas chromatography is described.

     

RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica

Hypertrehalosemic hormone (HTH) is a neuropeptide within the adipokinetic hormone (AKH) family that induces a release of trehalose from fat body into hemolymph in a number of insects. In this study, we first showed that female adult German cockroach, Blattella germanica, displayed a cyclic fluctuation of hemolymph trehalose levels correlated to the maturation of oocytes in the reproductive cycle. After cloning the HTH cDNA from the German cockroach (Blage-HTH), expression studies indicated that Blage-HTH mRNA showed the cyclic changes during the first reproductive cycle, where peak values occurred in 8-day-old virgin female cockroaches, which were going to produce oothecae. The functions of Blage-HTH were studied using RNA interference (RNAi) to knockdown its expression. Adult virgin females of B. germanica injected with Blage-HTH dsRNA increased hemolymph trehalose levels in the late period of vitellogenesis more slowly than control. Furthermore, RNAi of Blage-HTH delayed oviposition time and some (10%) individuals did not produce the first ootheca until 15 days after eclosion, whereas the control group produced ootheca before 9 days in all cases.     

Orcadian Rhythm of Trehalose in the Face Fly Musca Autumnalis De Geer

Trehalose levels were determined over two 24 hr spans in groups of face fly adults 3-4 days after emergence from the puparium. Face fly pupae were placed in rearing chambers at 27° C in a staggered light-dark regimen, LD 16:8, so that at a given clock hour, samples could be obtained at several different hours after lights on (HALO). Trehalose was determined in hemolymph collected from a puncture in the intersegmental membrane of the abdomen. Treated hemolymph samples were passed through a Bio-Rad Amino S-S disaccharide column and a Waters 410 refractive index detector was used to differentiate among sugars. The circadian acrophase derived by cosinor analysis in hemolymph trehalose (when the values were 25.49 and 26.86μg/μ¹ on the first and second days respectively) occurred at -226° (ca 15 HALO) and the bathyphase at 24 HALO. The mesor = 11.82μg/μ¹ trehalose, the amplitude = 8.57/μg/μ¹ trehalose and the P-value for presence of a rhythm was 0.003. Based on these data, differences between control and test flies in a bioassay of hypertrehalosemic activity would be most easily observed at 0-8 HALO, while exogenous hypotrehalosemic activity would be best assayed at 12-20 HALO.