Effects of plant flavonoids on Manduca sexta (tobacco hornworm) fifth larval instar midgut and fat body mitochondrial transhydrogenase

The reversible, membrane-associated transhydrogenase that catalyzes hydride-ion transfer between NADP(H) and NAD(H) was evaluated and compared to the corresponding NADH oxidase and succinate dehydrogenase activities in midgut and fat body mitochondria from fifth larval instar Manduca sexta. The developmentally significant NADPH-forming transhydrogenation occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg++-dependent ATPase. In general, the plant flavonoids examined (chyrsin, juglone, morine, quercetin, and myricetin) affected all reactions in a dose-dependent fashion. Differences in the responses to the flavonoids were apparent, with the most notable being inhibition of midgut, but stimulation of fat body transhydrogenase by morin, and myricetin as also noted for NADH oxidase and succinate dehydrogenase. Although quercetin inhibited or stimulated transhydrogenase activity depending on the origin of mitochondria, it was without effect on either midgut or fat body NADH oxidase or succinate dehydrogenase. Observed sonication-dependent increases in flavonoid inhibition may well reflect an alteration in membrane configuration, resulting in increased exposure of the enzyme systems to the flavonoids. The effects of flavonoids on the transhydrogenation, NADH oxidase, and succinate dehydrogenase reactions suggest that compounds of this nature may prove valuable in the control of insect populations by affecting these mitochondrial enzyme components.     

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.     

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta. There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O,O-diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.     

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.     

Regulation of fat body glycogen phosphorylase in larval tobacco hornworm,Manduca sexta

Activation and inactivation of fat body glycogen phosphorylase was investigated in ligated abdomens of larval Manduca sexta and in vitro. After maximal activation through Manduca adipokinetic hormone (AKH) or chilling, inactivation of glycogen phosphorylase commenced as soon as the stimulus for the activation was removed indicating that the enzyme system in the fat body is fine-tuned to low phosphorylase activities which is necessary to allow glycogen synthesis. In intact ligated abdomens phosphorylase can be activated repeatedly by either stimulus showing that the fat body system does not lose its responsiveness. It was impossible to achieve complete conversion of the inactive form of phosphorylase into the active form even after administration of AKH and simultaneous chilling. © 1992 Wiley-Liss, Inc.     

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.     

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.     

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.     

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.     

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.     

Changes in the chemical composition of fat body during the last larval instar of Manduca sexta.

The fat body increases in weight throughout the last larval instar in spite of the loss in total body weight during the wandering and prepupal stages. The protein content of fat body increases dramatically and is greatly responsible for the increase in fat body weight in the wandering and prepupal stages. Lipids do not contribute significantly to the fat body weight gain except during the feeding stage. The amount of total fat body RNA increases until wandering then drops abruptly in the prepupal stage. The total amount of fat body DNA peaks before the onset of wandering and prepupal stages.