Soluble tyrosinases from pharate pupal integument of the tobacco hornworm,Manduca sexta (L.)

Two soluble phenoloxidases were partially purified from pharate pupal integument of Manduca sexta by gel filtration or sucrose density gradient centrifugation. Thiourea was used to retard the formation of higher molecular weight aggregates. Subsequent analysis by gel filtration HPLC showed that the apparent molecular weights were about 300 kD and >700 kD. The smaller phenoloxidase was further purified by anion exchange HPLC. In comparative studies with mushroom tyrosinase and a fungal laccase, the Manduca phenoloxidases were identified as tyrosinases, since they exhibited monophenol mono-oxygenase activity (EC 1.14.18.1) and catechol oxidase activity (EC 1.10.3.1). Both enzyme preparations catalyzed ortho-hydroxylation of tyrosine at a relatively show rate, oxidized o-diphenols at a much faster rate than p-diphenols or monophenols, had broad pH optima from about pH 5.5–7.5, and were completely inhibited by 1 μM phenylthiourea, N-β-alanyldopamine (NBAD) and N-β-alanylnorepinephrine (NBANE), which are both abundant in pupal cuticle, were oxidized to o-quinones by the tyrosinases, with the former catecholamine oxidizing at a 10-fold higher rate than the latter. NBANE was synthesized from NBAD, apparently by spontaneous tautomerization of the o-quinone to a p-quinone methide, which then reacted with water to form the β-hydroxylated product. The possible role of tyrosinase in insect integument is discussed.     

Changes in ploidy level of epidermal cells during last larval instar of the tobacco hornworm, Manduca sexta

The relative DNA content of Manduca sexta abdominal epidermal nuclei during the final larval instar was measured by cytophotometry of whole-mount preparations of the epidermis. In the middle intrasegmental region, epidermal cells showed a ploidy level of 4C to 32C on the day of ecdysis. During the subsequent period of feeding, the proportion of higher ploidy cells, such as 16C and 32C, increased. This situation remained until the day of apolysis preceding pupal cuticle formation when mitoses reduced the cells to 2C, 4C, 8C and 16C, except for the pupal pock-mark cells, which increased to 32C or 64C. Metaphase cells showed various ploidy levels, correlated with the size of their mitotic figures. By contrast, in the anterior and posterior margin of a segment where no mitoses occurred, the cells continued to increase in ploidy throughout the instar.     

Carbohydrate and lipid metabolism during the last larval moult of the tobacco hornworm, Manduca sexta

Abstract. At 25°C and with a light regime of 17 h light and 7h dark, the last larval moult of the tobacco hornworm, Manduca sexta , lasts approximately 32 h, during which profound changes of metabolism were observed. At the onset of the moult, which coincides with the cessation of feeding, the proportion of active fat body glycogen phosphorylase increased from 10 (-2h) to 25–30% (Oh). A biphasic pattern with peak activities of 45–50% after t – 12 h and again just prior to the shedding of the cuticle (32 h) was subsequently observed. Haemolymph trehalose concentration decreased significantly from c. 35 (Oh) to 20mM (8h), but then recovered to an intermediate level (30mM; 12h). After completion of the moult, the trehalose concentration was 35–40 mM. The haemolymph glucose level in feeding fourth instar larvae was 4–5 mM, but decreased sharply before the onset of the moult to c. 1 mM, followed by a slow 6-fold increase over the next 20h. Prior to the shedding of the cuticle, the glucose level dropped again dramatically. The haemolymph lipid level increased slowly from an initial level of 1.2–1.4mg/ml during the early part of the moult, reaching a maximum of 1.8mg/ml after /= 16 h. Afterwards, a decrease of c. 50% was observed until ecdysis occurred. Oxygen consumption per animal decreased steadily from 30–35 μl/min pre-moult by approximately 70% to c. 10 μl/min but started to increase about 5 h before the animals resumed feeding.     

Excretory role of the midgut in larvae of the tobacco hornworm, Manduca sexta (L.).

Caterpillars of Manduca sexta use two distinct transport mechanisms for the excretion of dyes. One pump (Type A) has a high affinity for acid (anionic) dyes and occurs in the midgut and medial Malpighian tubules. Acid dyes accumulate rapidly in the lumen of the midgut while the Malpighian tubules appear to play only a minor role in the excretion of these dyes. The other pump (Type B) excretes basic (cationic) dyes and is located primarily in the proximal Malpighian tubules. Evidence is presented that hippuric acid competes with acid dyes for excretion by both midgut and Malpighian tubules. After the final-instar larva purges its gut the ability of the midgut and Malpighian tubules to excrete dyes gradually decreases. Sixty hours after the purge only the Malpighian tubules retain some dye excreting activity.     

Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta

While the larval midgut of Manduca sexta has been intensively studied as a model for ion transport, the developmental origins of this organ are poorly understood. In our study we have used light and electron microscopy to investigate the process of midgut epithelial cell differentiation in the embryo. Our studies were confined to the period between 56 and 95 hr of embryonic development (hatching is at 101 hr at 25 degrees C), since preliminary studies indicated that all morphologically visible differentiation of the midgut epithelium occurs during this time. At 56 hr the midgut epithelium is organized into a ragged pseudostratified epithelium. Over the next 10 hr, the embryo molts and the midgut epithelium takes on a distinctive character in which the future goblet and columnar cells can be identified. With further differentiation, closed vesicles in the goblet cells expand and subsequently communicate to the outside by way of a valve. The columnar cells form numerous microvilli on their apical surfaces that extend over the goblet cells. Both cell types form basal folds from a series of plasmalemmal invaginations. Differentiation occurs concurrent with a six-fold elongation of these cells.     

Prothoracicotropic hormone activity in the embryonic brain of the tobacco hornworm,Manduca sexta

Summary Head segments and brains were extirpated from embryos of the tobacco hornworm,Manduca sexta, extracted and the resulting extracts assayed for prothoracicotropic hormone (PTTH) activity on prothoracic glands from day 3 fifth instar larvae and day 0 pupae. Dose-response curves were generated and indicated the presence of PTTH activity in embryonic brains and head segments, suggesting a role(s) for this neurohormone during embryogenesis. Maximal PTTH activity was found in brains from embryos 117 h post-oviposition, just prior to hatching, but activity was also noted in head segments as early as 24 h postoviposition. These data on PTTH and those on ecdysteroids and juvenile hormones in embryos suggest that these 3 classes of hormones which control insect post-embryonic development, may also be involved in the regulation of developmental processes in the embryo.     

Carbohydrate metabolism during the pupal molt of the tobacco hornworm,Manduca sexta

During the pupal molt of the tobacco hornworm, Manduca sexta, the percentage of active fat body glycogen phosphorylase increased from 5–10 to 20%, but only for a period of 5 h prior to the molt. From the time of the appearance of two sclerotized dorsal bars to the time of the molt, the concentration of total hemolymph carbohydrates doubled to 100 mM trehalose. Initially, the glucose level was high (16 mM) when compared with feeding larvae (approximately 1 mM) but decreased to zero just prior to the molt. The amount of cuticular chitosan decreased from approximately 100 mg to 10 mg at pupation; the exuvia contained approximately 7 mg. While the levels of total lipids in hemolymph were not affected, the lipid content of the fat body decreased significantly prior to the molt but increased sharply thereafter. Fat body glycogen phosphorylase in pharate pupae and pupae of M. sexta was substantially activated by the Manduca adipokinetic peptide hormone, which in pharate pupae, produced the same response at 2 and 20 pmol per insect as in ligated larval abdomens. In pupae the response was clearly reduced. Using chilling to stimulate glycogen phosphorylase, it was found that the enzyme in pharate pupae and pupae responded both in vivo and in vitro as in ligated abdomens of larvae. Thus, a transition to the adult response seems to occur during the pupal and pharate adult development. © 1995 Wiley-Liss, Inc.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Iron binding proteins and their roles in the tobacco hornworm,Manduca sexta (L.)

Summary Manduca sexta larvae accumulate large amounts of iron during their larval feeding period. When⁵⁹Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle.Studies of the hemolymph disclosed two ironcontaining proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 g of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin inManduca sexta, the latter serving a storage function during development and possibly also a transport function.     

Acid/base transport across the midgut of the tobacco hornworm,Manduca sexta

Larval Lepidoptera generate a large pH gradient across the midgut epithelium. The in vitro rate of luminal alkalinization (J(OH-)) and hemolymph acidification (J(H+)) under nominally CO(2)-free conditions was measured in the three morphologically distinct regions of the tobacco hornworm midgut. Under open-circuit conditions, the highest J(OH-) and J(H+) was observed in the anterior section and the lowest was in the middle section. In all three sections the J(H+) was equal to J(OH-) indicating transepithelial movement of acid or base equivalents. Furthermore, the rate at which the midgut transported acid or base was the same under open- and short-circuit conditions, indicating that acid/base transport is an active process. Although the inhibitors, acetazolamide and ethoxyzolamide, inhibited the activity of carbonic anhydrase in tissue homogenates, they had no effect on J(OH-), J(H+), or transepithelial potential. Therefore, under the nominally CO(2)-free conditions of this study, it is unlikely that hydration of CO(2) and the formation of HCO(3)(-) is involved in luminal alkalinization.     

Voltage-current relation and K+ transport in tobacco hornworm (Manduca sexta) midgut

Summary Voltage-current curves for the isolated midgut of the tobacco hornworm were determined by transient and steady voltage clamping over the range of 200 to –200 mV. Over this range the transient method yields a linear relation while the steady method usually yields a curve consisting of two lines of differing slope which intersect at zero voltage. The difference between the results of the methods is due to a slow decline in total conductance which accompanies steady voltage clamping.Holding the midgut at short circuit increases the total conductance of the tissue in a manner consistent with increasing shunt conductance; this effect was seen in both diet-reared and leaf-reared animals.When potassium transport is inhibited by substitution of choline or sodium for potassium in bathing solution the total conductance decreases and the voltage-current curve intersects the normal curve in the hyperpolarizing region. Applying a simple equivalent circuit analysis to the results from partial or total potassium replacement suggests that the electromotive force of the potassium transport system is of the order of 140–190 mV. The conductance decrease during inhibition of potassium transport by transient anoxia is of similar magnitude, suggesting that a major effect of metabolic inhibition is to decrease the active conductance of the potassium transport pathway.     

A novel protein that binds juvenile hormone esterase in fat body tissue and pericardial cells of the tobacco hornworm Manduca sexta L

Juvenile hormone esterase degrades juvenile hormone, which acts in conjunction with ecdysteroids to control gene expression in insects. Circulating juvenile hormone esterase is removed from insect blood by pericardial cells and degraded in lysosomes. In experiments designed to characterize proteins involved in the degradation of juvenile hormone esterase, a pericardial cell cDNA phage display library derived from the tobacco hornworm moth Manduca sexta L. was constructed and screened for proteins that bind juvenile hormone esterase. A 732-base pair cDNA encoding a novel 29-kDa protein (P29) was isolated. Western and Northern analyses indicated that P29 is present in both pericardial cell and fat body tissues and is expressed in each larval instar. In immunoprecipitation experiments, P29 bound injected recombinant juvenile hormone esterase taken up by pericardial cells and native M. sexta juvenile hormone esterase in fat body tissue, where the enzyme is synthesized. Binding assays showed that P29 bound juvenile hormone esterase more strongly than it did a mutant form of the enzyme with mutations that perturb lysosomal targeting. Based on these data, we propose that P29 functions in pericardial cells to facilitate lysosomal degradation of juvenile hormone esterase.     

Sequential structural changes in the fat body of the tobacco hornworm, Manduca sexta, during the fifth larval stadium

Light and electron microscopy revealed a series of structural changes that occur in the fat body of the tobacco hornworm, Manduca sexta, during the fifth, i.e. the final, larval stadium. At each developmental stage studied, the cells of the fat body were homogeneous in structure. We found no evidence suggesting the presence of more than one type of fat body cell. Our structural data are consistent with published observations on biochemical activities of M. sexta fat body at particular developmental stages. Specific points of agreement include: (a) acquisition of Golgi complex (GC) and rough endoplasmic reticulum (RER) concomitant with the time of major protein production; (b) loss of many cellular organelles (such as GC and RER) as protein production drastically decreases; (c) accumulation of protein granules and urate granules after the onset of wandering (i.e. during the pre-pupal period); (d) accumulation of lipid and glycogen throughout the feeding period. In addition we found that (a) the plasma membrane reticular system (PMRS) developed during the period when protein secretion was great; (b) the PMRS was lost abruptly at the onset of wandering; and (c) the nucleus changed in shape from being roughly spherical to elliptoid in the pre-pupal stage. We found that the structure of M. sexta fat body is similar to that published for other Lepidoptera. However, it differs from that of Heliothis zea in that regional differences are not obviously apparent.     

PBAN regulation of pheromone biosynthesis in female tobacco hornworm moths,Manduca sexta (L.)

Manduca sexta females that were decapitated produced no pheromone during the scotophase following decapitation, indicating that they were free of pheromone biosynthesis activating neuropeptide (PBAN). When deuterated hexadecanoic or (Z)-11-hexadecenoic acid was applied to the sex pheromone glands of decapitated or intact females of the same age, and allowed to incubate in vivo for 24 h, deuterium labeled Δ-11- and Δ-10, 12-unsaturated 16-carbon fatty acids were produced in both types of females. Injection of PBAN into intact or decapitated females 23 h after application of labeled acids had no effect on the production of unsaturated labeled fatty acids. However, deuterium labeled aldehydes were produced only in females that were injected with PBAN. Therefore, in this species, PBAN activates the process by which fatty acyl precursors in the pheromone gland are converted into the pheromonal aldehydes. © 1995 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.

     

Autophagic and apoptotic features during programmed cell death in the fat body of the tobacco hornworm (Manduca sexta)

Two major pathways of programmed cell death (PCD)--the apoptotic and the autophagic cell death--were investigated in the decomposition process of the larval fat body during the 5th larval stage of Manduca sexta. Several basic aspects of apoptotic and autophagic cell death were analyzed by morphological and biochemical methods in order to disclose whether these mechanisms do have shared common regulatory steps. Morphological examination revealed the definite autophagic wave started on day 4 followed by DNA fragmentation as demonstrated by agarose gel electrophoresis and TUNEL assay. By the end of the wandering period the cells were filled with autophagic vacuoles and protein granules of heterophagic origin and the vast majority of the nuclei were TUNEL-positive. No evidence was found of other aspects of apoptosis, e.g. activation of executioner caspases. Close correlation was disclosed between the onset of autophagy and the nuclear accumulation of the ubiquitin-proteasome system.