Interactions of temperature and dietary protein concentration in growth and feeding of Manduca sexta caterpillars

Abstract .Temperature and the protein content of food affect rates of consumption and growth in herbivorous insects in different ways: reduced temperature typically reduces both consumption and growth rates, whereas reduced dietary protein typically increases consumption rate but either reduces or has no effect on growth rate. The interactions between temperature and dietary protein concentration in affecting consumption, growth and efficiency in fifth-instar caterpillars of Manduca sexta were studied, using both short-term (4 h) and long-term (duration of fifth stadium) experiments. The short-term experiments examined constant temperatures between 14 and 42°C, whereas the long-term experiments examined constant temperatures between 18 and 34°C; both experiments considered two levels of dietary protein. In both experiments, caterpillars had significantly higher consumption and frass production rates on low-protein compared with high-protein diets at each test temperature between 18 and 34°C, thereby compensating for the lower diet quality. In contrast, at more extreme temperatures (14 and 42°C) in the short-term studies, consumption and frass production rates were lower on low-protein compared with high-protein diets. As a result, there were substantial interactions between temperature and dietary protein for consumption and frass production rates in the short-term experiments, but not in the long-term experiments. These results suggest that interactions between temperature and dietary protein may emerge because of the failure of compensatory feeding responses at low and high temperatures. It is hypothesized that the failure of compensatory responses is more likely to occur under diurnally fluctuating temperatures than under a constant temperature with the same mean, and it is proposed that interactions between temperature and dietary protein for consumption are relevant to M. sexta and other caterpillars that experience wide diurnal fluctuations in temperature in the field.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

The feeding behaviour of caterpillars (Manduca sexta) on tobacco and on artificial diet

ABSTRACT. Feeding behaviour of fifth instar tobacco hornworm caterpillars, Manduca sexta (Johansen) (Lepidoptera; Sphingidae), eating tobacco or artificial diet, is quantitatively described. The insects grow at the same rate on both foods. There is no daily rhythm of feeding behaviour. For most insects, feeding on either food occurs in bouts with the lengths of interfeed gaps and of feeding bouts appearing to be distributed randomly. However, in many insects there is a strong correlation between the length of a feeding period and that of the preceding non-feeding period.
The proportion of time spent feeding on tobacco is much greater than on artificial diet. On tobacco, feeding periods are separated by shorter interfeed gaps than on the artificial diet, while the rate of bout initiation is similar on either food.
On both tobacco and artificial diet, the proportion of time spent feeding increases as the fifth stadium proceeds. This is due to both longer feeding bouts and shorter gaps. The rate of food acquisition within bouts does not change during the stadium.     

Juvenile hormone and ovarian growth in Manduca sexta

In Manduca sexta the major size increase of ovarian follicles is accomplished by two processes: (1) vitellogenesis in which follicular volume and dry weight increase simultaneously, and (2) hydration in which absorption of water by the oocyte accounts for an 80% increase in volume prior to chorion formation. Vitellogenic growth occurs in both a slow and rapid phase. Rapid vitellogenic growth is initiated only by follicles of a threshold size (1 mm) and is a juvenile hormone (JH)-dependent event. In the absence of JH follicles grow to 1 mm and then degenerate.     

Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta

In several insect species, serum lysozyme and antibacterial peptide concentration increases after injection of bacteria and other foreign substances. The purpose of this study was to characterize the specificity of this induction in the tobacco hornworm, Manduca sexta. By 48 h after injection of killed bacteria, lysozyme activity was approximately tenfold greater than in untreated insects. This maximal response was observed after injection of every bacterial species tested and after injection of purified cell walls of Micrococcus luteus. A variety of acellular particles, soluble molecules, and bacterial cell wall components were either poor lysozyme inducers or elicited no change in lysozyme concentration. The polysaccharide zymosan from yeast cell walls was a moderate lysozyme inducer. Peptidoglycan from M. luteus cell walls was found to induce lysozyme to a level as great or greater than whole cell walls. Small fragments of peptidoglycan generated by hen egg white lysozyme digestion were isolated, partially characterized, and shown to be good inducers of lysozyme as well as other antibacterial peptides. It appears that peptidoglycan provides a signal that initiates antibacterial responses in the insect.     

Mechanistic studies of the degradation of juvenile hormone esterase in Manduca sexta

The mechanisms of degradation of juvenile hormone esterase (JHE) were investigated in larvae of the tobacco hornworm, Manduca sexta. JHE is removed from the hemolymph by the pericardial cells by receptor-mediated endocytosis and is ultimately degraded in the lysosomes. Immunoprecipitation experiments and native PAGE followed by Western blotting showed that JHE associates with a putative heat shock cognate protein (Hsp). Approximately 25% of the active JHE in the pericardial cell complex is associated with the putative Hsp 1 h postinjection of affinity purified JHE. Electron microscope analysis revealed that the putative Hsp is located in the trans-Golgi network of pericardial cells, where it is hypothesized to be involved in sorting of proteins destined for the lysosomes, from those destined for the cell membrane. Data acquired from immunoprecipitation and Western blotting experiments argue against the involvement of ubiquitin in the degradation of JHE. Injection of radiolabeled JHE into larvae of M. sexta followed by SDS-PAGE of pericardial cell homogenates revealed covalent binding of an unidentified protein to JHE in the pericardial cell complex. Arch. Insect Biochem. Physiol. 34:275–286, 1997. © 1997 Wiley-Liss, Inc.     

Absorption and storage of phosphorus by larval Manduca sexta

The role of phosphorus (P) in numerous important biological structures, coupled with the observation that P-content of many insect foods is disproportionately low, suggests that P may be a critical nutrient for growing insects — however, the few studies examining the effects of dietary P on insect performance have generally found only weak relationships. This mismatch may be reconciled by understanding the physiological mechanisms by which insects handle P. Here we describe P processing by larvae of Manduca sexta. When given un-manipulated leaves of a common host plant, Datura wrightii, fifth-instar larvae retained about 85% of P consumed; when given P-enriched leaves larvae retained only 25% of P consumed. Analysis of gut concentrations of P at four sites along the digestive tract, and in leaves and feces, indicates that the rectum is the primary site of P transport between the gut and body and that differences in P retention may be accounted for by differential rates of rectal P transport. Larvae given P-enriched leaves also showed an eightfold increase in the concentration of P in the hemolymph, primarily as α-glycerophosphate — but only a 12% increase in the concentration of P in body tissues, suggesting that hemolymph plays a central role in storage and buffering of P.     

Absorption and tissue distribution of cholesterol in Manduca sexta

In Manduca sexta larvae, radioactive free cholesterol is absorbed directly from the midgut into mucosal cells where it is stored both in the free form (87% in males and 93% in females) and esterified form (13% in males and 7% in females). Subsequently, cholesterol is transported to fat body via lipophorin in the hemolymph exclusively in the free form. In fat body, the distribution of cholesterol between the free and esterified form varied significantly between genders and developmental stages. Except for the larval stage, males and females were able to store cholesterol in both free and esterified forms in the fat body and in the adult stage cholesterol ester accounted for more than 75% of the stored cholesterol. Placement of radioactive cholesterol at different locations in the gut-foregut, midgut, and hindgut-demonstrated that the midgut is the site where cholesterol is absorbed and released into the hemolymph. Cholesterol-labeled lipophorin injected into larval hemolymph was cleared from the hemolymph with a half-life of 10.2 h. After 17 h, most of the cleared radioactivity was recovered in the fat body (38%). Arch.     

Developmental expression of heterotrimeric G proteins in the nervous system of Manduca sexta

The heterotrimeric G proteins are a conserved family of guanyl nucleotide-binding proteins that appear in all eukaryotic cells but whose developmental functions are largely unknown. We have examined the developmental expression of representative G proteins in the developing nervous system of the moth Manduca sexta. Using affinity-purified antisera against different G_α subunits, we found that each of the G proteins exhibited distinctive patterns of expression within the developing central nervous system (CNS), and that these patterns underwent progressive phases of spatial and temporal regulation that corresponded to specific aspects of neuronal differentiation. Several of the G proteins examined (including Gs_α and Go_α) were expressed in an apparently ubiquitous manner in all neurons, but other proteins (including Gi_α) were ultimately confined to a more restricted subset of cells in the mature CNS. Although most of the G proteins examined could be detected within the central ganglia, only Go_α-related proteins were seen in the developing peripheral nerves; manipulations of G protein activity in cultured embryos suggested that this class of G protein may contribute to the regulation of neuronal motility during axonal outgrowth. Go_α-related protein were also localized to the developing axons and terminals of the developing adult limb during metamorphosis. These intracellular signaling molecules may, therefore, play similar developmental roles in both the embryonic and postembryonic nervous system. © 1995 John Wiley & Sons, Inc.     

Influence of calcium on Manduca sexta plasmatocyte spreading and network formation

Plasmatocytes are a class of insect hemocytes important in the cellular defense response. In some species, they are phagocytic, protecting the insect from smaller pathogens. In many insects, they work in concert with other hemocytes (particularly other plasmatocytes and granular cells) to form nodules and to encapsulate foreign material. To perform these functions, plasmatocytes attach to, spread on, and surround suitable targets. Because of their importance, because we had previously observed that prolonged incubation of hemocytes in solutions containing the divalent cation chelator ethylenediaminetetraacetic acid (EDTA) inhibited plasmatocyte spreading, and because of the importance of divalent cations in many immune-related functions, we investigated the effect of calcium and magnesium on spreading of plasmatocytes from fifth instar Manduca sexta larvae. On glass slides, plasmatocytes spread more quickly and elongated in Grace's medium containing 5 mM calcium, compared to calcium-free medium. In the presence of calcium, plasmatocyte adhesion, spreading, and network formation were not visibly different in magnesium-free and magnesium-containing Grace's medium. Using immunomicroscopy with a monoclonal antibody specific for plasmatocytes, we measured the length and width of plasmatocytes incubated with several different concentrations of calcium. Plasmatocyte length positively correlated with calcium concentration to 5 mM (maximum concentration tested and approximately the hemolymph concentration). Mean plasmatocyte width was less in 0 and 5 mM calcium than in 0.05 or 0.5 mM calcium. On plastic, hemocytes survived longer than on glass (they survived beyond 24 h) and, in 5 mM calcium, formed an extensive network readily visible by phase-contrast microscopy. This network was never as extensive in the absence of calcium. Network formation in the absence of magnesium, but presence of calcium, resembled network formation in standard Grace's medium.     

Azadirachtin inhibits secretion of trypsin in midgut of Manduca sexta caterpillars: reduced growth due to impaired protein digestion

When given by injection to tobacco hornworm caterpillars, Manduca sexta, the allelochemical azadirachtin inhibits growth without reducing food intake. The growth reducing effect of azadirachtin is therefore in this case independent of the compound's well-known antifeedant effect. The cause of this reduced rate of growth is an increase in the costs associated with growth. These increased costs are largely a consequence of a decrease in the efficiency of utilisation of dietary nitrogen. This is associated with a drastic reduction in the activity of midgut trypsin. Azadirachtin has no effect on the activity of trypsin in vitro. Thus azadirachtin directly or indirectly inhibits the production of trypsin by the enzyme-secreting cells of the midgut wall; it is suggested that this is the cause of the increased costs and reduced rate of growth. The interesting parallel between this plant defence strategy and that of direct inhibition of herbivore proteinases by allelochemical proteinase inhibitors is discussed.     

Mechanistic and metabolic studies of sterol 24,25-double bond reduction in Manduca sexta

Larvae of Manduca sexta were used to obtain a cell-free sterol 24,25-reductase. From the midgut of fifth instar larvae fed a mixture of sitosterol and campesterol a microsome-bound 24,25-sterol reductase was prepared that transformed desmosterol (K_m, 3 μM), lanosterol (K_m, 18 μM), and cycloartenol (K_m, 33 μM), to cholesterol, 24,25-dihydrolanosterol, and cycloartanol, respectively. With desmosterol as substrate, the microsome-bound enzyme was found to incorporate tritium into cholesterol from 4S-tritium labelled NADPH. [24-²H]lanosterol was transformed by larvae to [24-²H]24,25-dihydrolanosterol (structure confirmed by mass spectroscopy (MS) and ¹H-nuclear magnetic resonance spectroscopy. A rationally designed inhibitor of 24,25-reductase activity, 24(R,S),25-epimino-lanosterol (IL), was assayed and found to be inhibitory with an I₅₀ of 2 μM. IL was supplemented in the diet of M. sexta with either sitosterol or stigmasterol and found to inhibit development (I₅₀ 60 ppm). The major sterol which accumulated in the IL-treated larvae was desmosterol, confirming the site of inhibition was reduction of the 24,25-bond. IL was converted to [2-³H]IL when fed to the larvae. [2-³H]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques. © 1996 Wiley-Liss, Inc.     

Characterizing psychophysical measures of discrimination thresholds and the effects of concentration on discrimination learning in the moth Manduca sexta

What is the spatial and temporal nature of odor representations within primary olfactory networks at the threshold of an animal's ability to discriminate? Although this question is of central importance to olfactory neuroscience, it can only be answered in model systems where neural representations can be measured and discrimination thresholds between odors can be characterized. Here, we establish these thresholds for a panel of odors using a Pavlovian paradigm in the moth Manduca sexta. Moths were differentially conditioned to respond to one odor (CS+) but not another (CS-) using undiluted odorants to minimize salience-dependent learning effects. At 24 and 48 h postconditioning, moths were tested for the presence of a conditioned response (CR) with a blank, then the CS+ and CS- (pseudorandomly) across a 5-log step series of increasing concentration. Results identified discrimination thresholds and established that differential CRs to the CS+ and CS- increased with stimulus concentration. Next, 3 separate groups of moths were differentially conditioned at either one-log step below, at, or one log step above the identified discrimination threshold. At 24 and 48 h postconditioning, moths were tested sequentially with a blank, the concentration used for conditioning, and then undiluted odor. Conditioning at one log step below the discrimination threshold established a CR, indicating both stimulus detection and learning, but was insufficient to establish evidence of discrimination. Moths conditioned at the discrimination threshold were able to discriminate but only when stimulated with undiluted odors, indicating learning, but discrimination measures were hampered. When conditioned above the discrimination threshold, moths had no difficulty in discriminating. These results establish methods for psychophysical characterization of discrimination and indicate that differential conditioning at lowered concentrations biases threshold measures.     

Induced responses in Nicotiana attenuata affect behavior and growth of the specialist herbivore Manduca sexta

Many plants employ induced responses against generalist herbivores. Specialist herbivores, however, may employ several mechanisms to overcome the negative effects of induced plant defenses. Here we test how the behavior and development of specialist Manduca sexta larvae are affected by induced responses in their natural host plant Nicotiana attenuata. On a spatial scale relevant to both the plant and the herbivore, we first determined how methyl jasmonate (MeJA)-induced responses, such as increased nicotine production, affect the tendency of larvae to leave induced plants. When larvae were allowed to move between two plants planted in one pot, they left an MeJA-treated plant faster than a control plant. When both plants in the pot were MeJA-treated, the larvae developed more slowly than when both plants were uninduced, or when the larvae had the opportunity to move to an uninduced neighbor. The sooner larvae moved from an MeJA-treated plant to an untreated neighbor, the larger the body mass they attained. This demonstrates that M. sexta larvae can compensate behaviorally for the deleterious effects of induced plant responses. These effects were observed in plants grown under both low and high N supply rates, though the effects were more pronounced under high N. To examine the consequences of the timing and the direction of the host plant switching behavior for larval development, neonate larvae were fed leaves excised from induced and uninduced plants. Larvae confined to MeJA-treated leaves had higher mortality rates and grew slower than larvae fed only control leaves. This demonstrates that MeJA-induced responses decrease growth and development of specialist herbivores that do not have the behavioral option of moving to an uninduced plant. The sooner the larvae were switched to MeJA-treated leaves, the slower their development compared to larvae fed only uninduced leaves. In contrast, the sooner larvae fed MeJA-treated leaves were switched to control leaves, the faster they developed. Again the effects of MeJA treatment were stronger in plants grown under high N supply. We propose that induced plants growing in close competition with an uninduced conspecific may offset the fitness costs of these induced responses and perhaps obtain a fitness benefit by motivating herbivores to move to their neighboring competitors. Received: 25 March 1999 / Accepted: 8 October 1999     

Metabolic pathways for diacylglycerol biosynthesis and release in the midgut of larval Manduca sexta

The pathway for the synthesis of diacylglycerol in larval Manduca sexta midgut was studied. Fifth instar larvae were fed with [9,10–³H]–oleic acid–labeled triolein and the incorporation of the label into lipid intermediates was analyzed as a function of time. The results showed that the triacylglycerol was hydrolyzed to fatty acids and glycerol in the midgut lumen. In midgut tissue, the labeled fatty acids were rapidly incorporated into phosphatidic acid, diacylglycerol and triacylglycerol, but no significant labeling of monoacylglycerol was observed. Dual-labeling experiments were performed in order to characterize the kinetics of diacylglycerol biosynthesis in the midgut, its incorporation into hemolymph lipophorin and its clearance from hemolymph. The results were best described by a model in which the rate-limiting step in diacylglycerol biosynthesis was the uptake of fatty acid from the lumen of the midgut. Once in the cell the fatty acid was rapidly incorporated in phosphatidic acid and diacylglycerol. Diacylglycerol was converted to triacylglycerol or exported into hemolymph. The interconversion of diacylglycerol and triacylglycerol was fairly rapid, suggesting that triacylglycerol serves as a reservoir from which diacylglycerol can be produced. This mechanism permits the cell to maintain a low steady-state concentration of diacylglycerol and yet efficiently absorb fatty acids from the lumen of the midgut.     

Studies on binding and uptake of vitellogenin by follicles of the tobacco hornworm,Manduca sexta

An in vitro system for the uptake of ¹²⁵l-vitellogenin (VG) or vitellin into isolated follicles of the tobacco hornworm, Manduca sexta, is described. After incubation with ¹²⁵l-VG, follicles were disrupted and the internal yolk contents separated from the follicle membranes. The results showed that ¹²⁵l-VG was associated principally with the membranes (92%) after incubation at 4°C. However, at 27°C, ¹²⁵l-VG was mainly in the yolk (92%). Furthermore, trypsin treatment removed approximately 70% of VG bound to the follicles at 4°C. Labeled VG was shown to bind to sonicated follicle membranes with high specificity and affinity (K_D ? 1.3 × 10^?8 M). This binding was sensitive to pH and calcium concentration. The total binding sites were estimated at 4 × 10¹⁴ sites/g of membrane protein. Competition studies showed that binding of ¹²⁵l-VG to follicle membranes was blocked by excess unlabeled vitellin and deglycosylated vitellogenin but not by lipophorin (the major hemolymph lipoprotein), microvitellogenin, a female-specific protein (M_r ～ 31,000) found in both hemolymph and eggs, and the smaller vitellogenin subunit, apovitellogenin-II (M_r ～ 45,000). These results suggest that selective uptake of M. sexta VG from the hemolymph involves binding to specific receptors located on the follicle membranes.     

Effects of decapitation and PBAN injection on amounts of triacylglycerols in the sex pheromone gland of Manduca sexta (L.)

The correlation between triacylglycerols containing conjugated diene fatty acyl moieties and pheromone aldehydes in the sex pheromone glands of females of Manduca sexta was investigated. Females decapitated 15 h after adult emergence neither called nor produced pheromone during the natural period of pheromone production on the subsequent two nights. However, these females could be stimulated to produce sex pheromone for prolonged periods by repeated injection of synthetic pheromone biosynthesis activating neuropeptide (PBAN). Gas chromatographic analysis of methanolysis products of lipids extracted from the pheromone glands of decapitated and intact females showed no differences in the amounts of fatty acyl precursors of pheromone. High performance liquid chromatographic analysis of the triacylglycerols containing conjugated diene analogues of the pheromone components (diene TG), obtained 24 and 48 h after decapitation, showed that the total amounts of these components were not affected by decapitation. The amounts of all diene TG peaks declined significantly when decapitated females were stimulated to produce pheromone during a 7 h period by repeated injection of PBAN at 3 h intervals but recovered when pheromone production subsided. These results indicate that PBAN induces liberation of pheromone precursors from the triacylglycerols during pheromone biosynthesis but does not induce replenishment of this storage pool. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

三株烟草天蛾新细胞系的生长特性及重组蛋白表达

从尚未涉及的昆虫种类中建立新的细胞系能为基础研究和生物技术应用提供重要资源。本实验通过细胞培养技术, 建立了3株来源于鳞翅目昆虫烟草天蛾Manduca sexta卵组织的新细胞系, 分别命名为QB-Ms1-8, QB-Ms2-2和QB-Ms2-7。这3株细胞已经培养在TNM-FH培养基中, 28℃条件下传代培养了约50代, 大部分细胞呈梭形, 细胞群体倍增时间分别为51, 31和49 h。虽然这3株细胞系对苜蓿银纹夜蛾核型多角体病毒（Autographa californica multiple nuclear polyhedrosis virus, AcMNPV）不够敏感, 侵染后96 h感染率在33%~40%之间, 但是QB-Ms2-2细胞与BTI-Tn5B1-4细胞比较, 分泌型碱性磷酸酶（SEAP）活性表达更高。本研究从建立的3株烟草天蛾新细胞系中筛选出SEAP高表达的细胞系QB-Ms2-2, 为进一步细胞克隆和筛选提供了新资源。     

烟草天蛾几丁质酶的表达、纯化及多克隆抗体制备

将烟草天蛾Manduca sexta几丁质酶基因克隆入融合表达载体pET-28a,并在大肠杆菌E.coli BL21(DE3)中进行诱导表达。表达菌株经0.5 mmol/L IPTG诱导6～8 h后,几丁质酶表达并形成包涵体。在Ni²⁺-NTA亲和柱上经变、复性和纯化,得到可溶的几丁质酶。表达产物经Western 印迹鉴定。采用切胶回收的方法切割回收包涵体,并将回收产物免疫新西兰大白兔,ELISA检测抗体效价达1∶20 000。Western 印迹检测证明抗体特异性良好。     

Sexual differentiation in the CNS of the moth,Manduca sexta. I. Sex and segment-specificity in production,differentiation, and survival of the imaginal midline neurons

We analyzed the development of several sets of postembryonic sex-specific motoneurons in Manduca sexta which belong to a group of homologous lineage of neurons called the imaginal midline neurons (IMNs). Adult female oviduct motoneurons and male sperm duct motoneurons are IMNs that show similar anatomical features and differentiate during metamorphosis, despite appearing in different segments: A7 for oviduct neurons, A9 for sperm duct neurons. These cells are born at the same time and, initially, similar sets are found in A7 and A9 ganglia of larvae of both sexes. The dimorphic adult pattern is generated by sex-specific production and cell death. A7 IMNs differentiate in both sexes through early pupal stages, whereupon they disappear in the male and become the oviduct motoneurons in the female. A9 IMNs are overproduced in the male, and subsequent cell death reduces male cell number and eliminates the small complement of female cells; the surviving male cells develop into the sperm duct motoneurons. Similar IMN arrays are generated in nongenital ganglia, but show non-sex-specific fates. This suggests that both the sex of these cells and their segment of residence play major roles in their subsequent differentiation. 1994 John Wiley & Sons, Inc.