Control of ecdysis by heat in Manduca sexta

Under constant-light conditions, adult Manduca sexta emerge approximately 5 hr after the middle of a 3 hr 3°C heat pulse. The insects can be entrained to a heat cycle in 3 days. Entrainment will decrease at approximately the same rate. Photic signals can modify the precision of the response but, when the insects are presented with conflicting photic and thermal signals, they respond to the thermal signal. During the 3 days of entrainment, the insects appear to hasten the time of eclosion as much as 6 hr to meet the thermal cycle, but the degeneration of the intersegmental muscles is not similarly hastened. The technique described can be applied to various physiological and behavioural studies.     

Uric acid excretion in larval Manduca sexta

The characteristic coating of frass of last-instar tobacco hornworms, Manduca sexta, reared on artificial diet, proved to be uric acid. Results indicated that uric acid is the major nitrogenous excretory product; during most of the larval feeding stage, 5–7% of the excreta (dry weight basis) was uric acid; only minute quantities of allantoic acid were present. The rate of uric acid excretion was linear for the periods when coated pellets were observed. Abrupt increases in uric acid resulted from delays in pellet expulsion associated with delays in feeding activity. A distinctive coating was not generated by penultimate instar larvae, but abrupt changes in uric acid content did occur, which suggests that the phenomenon of coated frass is directly related to a differential in uric acid concentration. The source of uric acid in the frass was the Malpighian tubule system. The transition period between feeding and the wandering stage was a time of rapid decrease in uric acid excretion; there were only low levels in the last fecal pellets and none in Malpighian ampullae of wandering-stage larvae. Since the first appearance of coated fecal pellets preceded the release of ecdysone by about 24 hr, the involvement of this hormone was not indicated.     

Ontogeny of electroantennogram responses in the moth, Manduca sexta

The antennae of the moth, Manduca sexta, and the sensilla and sensory neurons they contain, develop during metamorphosis from pupa to adult. To determine when, during their development, antennae become capable of generating electrical responses to various stimuli, we recorded the electroantennogram (EAG), believed to be the summed extracellular record of receptor potentials, from developing and mature antennae. Antennae from male and female moths are similarly responsive to trans-2-hexenal, while only males respond to Manduca sex pheromone; these two odorants presumably stimulate separate receptors. Mechanical stimulation also elicits and EAG response. EAG responses to olfactory and mechanical stimuli are detectable several days before eclosion but not until the neurons are morphologically and biochemically quite mature. Responses increase in magnitude until the end of metamorphosis and then change little during the first 3 days after emergence of the adult. Responses to different stimuli do not develop synchronously.     

Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae

The haemolymph of Manduca sexta larvae and pupae has been analyzed for proteins potentially associated with the bacterial defence response of the insect. Five proteins, M13, M18, M20, M23, and M24 in pupae, and M4, M11, M13, M18 and M23 in larvae, are induced by the injection of bacteria into the haemolymph. Proteins M4 and M11 are always present at high levels in uninjected pupae. Proteins M20 and M24 could not be induced in larvae. These proteins, as well as those not showing a response to bacterial challenge or injury, were also analyzed for presence of disulphide bonds and carbohydrate moieties, and their apparent molecular weights determined.     

Induction of hostplant specificity in the tobacco hornworm, Manduca sexta

The hostplant specificity of the tobacco hornworm, Manduca sexta, is not inherited but is induced. Newly hatched larvae are polyphagous and will feed on many kinds of non-hostplants although they are not able to grow on them. They become oligophagous when they are reared on solanaceous plants but remain polyphagous when reared on diet. The critical period for induction is in the first instar. The induced oligophagous behaviour can be reversed by forcing larvae to feed on diet.     

Flight performance of the moth, Manduca sexta, at variable gravity

The tethered and free flight of Manduca sexta were studied during period 1,2, and 0 times normal gravity (g) produced in an aeroplane by flying through parabolic trajectories. Moths in tethered flight did not change their aerodynamic output in response to increases or decreases in gravity. Some moths in free flight at 0 g maintained a position in the box by flying against a surface, or into the angle between two surfaces. In the absence of gravity as an orienting stimulus, the positive dorsophotic response to light was dominant. As the period of 0 g continued, moths were increasingly likely to periodically reduce the amplitude of their wingbeat and/or stop flying, for the equivalent of a few wingbeats. Only at 0 g, moths very occasionally spread their wings and floated freely for a few seconds. At 0 g moths retained control of rolling and yawing movements but stability in pitch was greatly reduced or absent.     

Gain and loss of fluid in metamorphosing larvae of Manduca sexta

Larvae of Manduca sexta, (Sphingidae) increase their weight approx. 50% just before pupation and then secrete this fluid during the formation of their burrows. Time-lapse cinematography indicates that the fluid is ejected into the walls of the final burrow. It may offer some mechanical support; it is not particularly repellent to ants or mice, and it contains only small amounts of the alkaloids ingested from its preferred food plants. Comparison to other species indicates that the gain and loss of water is associated with burrowing behaviour; the fluid appears to be used in providing hydraulic pressure for burrowing, in forming and cementing the pupal chamber, and in acting as a CO₂ trap underground. The secretion is a hypertonic, highly alkaline solution containing KHCO₃ and small amounts of Na⁺, Ca²⁺, Mg²⁺, PO₄^?3 and some proteins. Haemolymph levels of K⁺ decrease, and those of Ca²⁺ increase, during the secretory phase. When radioactive calcium is injected into mature larvae, it appears promptly in the secretion. If however, the injection is given more than 24 hr before the animal begins secreting, then the calcium is bound to haemolymph protein and does not appear in the secretion.     

Effects of starvation and parasitism on foregut contraction in larval Manduca sexta

Larvae of Manduca sexta are parasitised by the braconid wasp, Cotesia congregata. In this study we examined whether contraction activity of the semi-isolated foregut was affected by parasitism. Parasitised larvae fed significantly less compared with unparasitised control larvae, therefore starved unparasitised animals were used as controls. Rate and force of foregut contraction in control caterpillars significantly increased with days of starvation. However, only contraction force in foreguts of parasitised larvae increased over time following infection. The presence of food in the foregut of caterpillars starved 7 days suggested that food moved anteriorly from the midgut and that contraction became antiperistaltic, but only normal peristalsis occurred in parasitised caterpillars. Rate and force of gut contractions may be controlled independently and starvation did not truly mimic the effects of the parasitoids. Dissection of caterpillars with emerged wasps indicated that 47% had a single wasp larva wedged between the brain and foregut. Removal of this wasp caused an increased rate of foregut contraction of the caterpillar. Brain removal resulted in an increased rate of foregut contraction only for unparasitised insects. Sectioning of the recurrent nerve temporarily eliminated foregut contraction, but the contraction began again in 250 s in parasitised caterpillars prior to wasp emergence, compared with over 500 s for unparasitised controls and parasitised caterpillars following wasp emergence.     

Juvenile hormone III biosynthesis by the larval corpora allata of Manduca sexta

A radioimmunoassay (RIA) for juvenile hormone III has been established which quantifies the biosynthesis of this hormone in vitro by the corpora allata of larvae and pupae of the tobacco hornworm, Manduca sexta. The specificity of the RIA for homologues and metabolites of juvenile hormone III was determined and it was found that the antibody was specific for juvenile hormone III and its acid. The juvenile hormone III RIA activity synthesized in vitro by corpora allata from day-5 last-instar larvae was identified as juvenile hormone III by high pressure liquid chromatography. The kinetics of hormone synthesis by corpora allata from selected stages during larval-pupal development revealed differential rates of synthesis, suggesting that juvenile hormone III may have a hormonal function in the larva and that regulation of its synthesis may occur. The significance of these developmental fluctuations in rates of juvenile hormone III synthesis by the corpora allata is discussed in relation to the haemolymph titres of the hormone.     

Photoperiodic induction of the pupal diapause in the tobacco hornworm, Manduca sexta

A study was made of photoperiodic induction of the facultative pupal diapause in the tobacco hornworm, Manduca sexta, reared on artificial diet in the laboratory. The species entered a prolonged diapause when the egg and larval feeding stages were reared in daily photoperiods of 13·5 hr or less. Diapause was induced in all insects at photoperiods ranging from 1 to 13 hr, and part of the population entered diapause at only 15 to 30 min of light per day. Photoperiods of 14 hr or more and continous darkness prevented diapause. Duration of diapause varied with the inductive photoperiod in which the hornworms were reared during the sensitive period. Insects reared in longer diapause-inducing photoperiods within a range of 12 to 13·25 hr remained in diapause longer than those reared in shorter photoperiods. There was no difference in the rate of larval development of hornworms reared in diapause-inducing vs diapause-preventing photoperiods. Temperatures of 26 to 30°C were most favourable for the photoperiodic induction of diapause; at 21°C, the critical photoperiod and incidence of diapause were decreased. Diapause induction was suppressed by low (18°C) and higher (33°C) temperatures. The number of inductive 12L:12D (light = 12 hr; dark = 12 hr) cycles required to induce diapause ranged from as few as 5 for some insects to as many as 12 for others when the post-inductive régimen was continuous light, but with insects previously held in continuous dark, as few as 2 12L:12D cycles during the last 2 days of larval feeding induced diapause in 38 per cent of the population. Only 3 to 4 cycles of 15L:9D during the final larval instar reversed inductive effects of 14 to 15 12L:12D cycles. Photoperiodic sensitivity extended from the late embryo to the end of larval feeding but showed considerable fluctuation during development with maximum sensitivity occurring just before egg hatch and during larval growth.Light breaks applied at different times during the dark period of 12L:12D cycles generated different response curves, depending on the number of cycles in which light breaks were repeated. When repeated for 6 cycles, a unimodal response curve was obtained; 10 cycles produced a bimodal curve and light breaks given for 18 cycles throughout the sensitive period averted diapause regardless of time of night applied. It is suggested that diapause is regulated by a photo- and thermolabile substance that accumulates during long nights (11 hr or more) and acts during the early pupal stage to inhibit the translocation and release of development-promoting neurosecretion from the brain.     

Juvenile hormone-specific esterases in the haemolymph of the tobacco hornworm, Manduca sexta

A new sensitive method for determining juvenile hormone (JH) hydrolysis has been developed which measures the release of tritiated methanol from JH labelled in the methyl ester group. Using this assay we investigated the interaction of JH with haemolymph esterases and haemolymph JH-binding protein. Haemolymph from fifth instar larvae of Manduca sexta contains two families of esterases which can be distinguished by their reactivity with diisopropylphosphorofluoridate (DFP). One group consists of general esterases which are capable of hydrolysing free JH but not JH complexed to the binding protein and are completely inhibited by low concentrations of DFP (10⁻⁴ M). The other group (JH-specific esterases), relatively DFP resistant, has little detectable general esterase activity but can hydrolyse JH bound to the binding protein as well as free JH. The major JH-esterase has a sedimentation coefficient of 4·98 S and a diffusion coefficient of 6·4 × 10⁻⁷ cm² sec⁻¹. The molecular weight calculated from these values is 6·7 × 10⁴. The general esterases are present throughout the larval stage, but the JH-specific esterases are barely detectable until the fourth day of the fifth instar when they suddenly appear at a high concentration. Since the general esterases cannot hydrolyse bound JH, one function of the binding protein is to protect JH during transport in the early instars, thus confirming that the binding protein is a true carrier of JH. In the late fifth instar prior to metamorphosis, however, JH-specific esterases appear in the haemolymph resulting in the hydrolysis of JH complexed to the carrier protein. Thus, by lowering JH titre, the JH-esterases play an important rôle in development in M. sexta.     

Neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta

The frontal ganglion of the tobacco hornworm, Manduca sexta (L.), was found to contain two neurosecretory (NS) cells (max dia = 40–45 μm). The cytoplasmic inclusions of the NS cells were stained purple with paraldehyde fuchsin, and marked fluctuations in amounts of NS material in the perikarya were observed, depending upon the developmental status of the insect. The perikarya of NS cells in the frontal ganglia of starved larvae and diapause pupae contained large accumulations of NS material, whereas feeding larvae and developing pharate adults showed relatively low amounts of neurosecretion. Electron microscopy revealed large accumulations of NS granules (dia = 80–240 nm) in the frontal ganglia of diapause pupae, but only slight accumulations of granules were seen in the NS cells of developing larvae and pharate adults.It was concluded that axonal transport and release but not synthesis is shut down during starvation and diapause, leading to accumulation of NS material in the perikarya. It is also suggested that the failure of many investigators to differentiate NS cells in the frontal ganglion of various insects may have been due to the selection of very active stages when the amount of available NS material was too low to be visualized by conventional staining techniques.     

The composition of larval-pupal moulting fluid in the tobacco hornworm, Manduca sexta

The concentrations of sodium, potassium, chloride, bicarbonate, total phosphate, labile phosphate, inorganic phosphate, protein, polypeptides, amino acids, trehalose, and glucose, as well as pH and osmotic pressure of larval-pupal moulting fluid and haemolymph were measured in the tobacco hornworm, Manduca sexta, during the secretion and resorption of moulting fluid. Moulting fluid is a mildly alkaline (pH 7.2), iso-osmotic (330 mOsm) potassium bicarbonate salt solution containing within it the sol form of moulting gel. Bicarbonate is the principal anion in moulting fluid. It is only a minor component of haemolymph. Significant differences in the concentrations of potassium, chloride, bicarbonate, non-labile phosphate, labile phosphate, inorganic phosphate, protein, polypeptide, amino acids, trehalose, and glucose indicate that the integumentary epithelium does not act as a semi-permeable membrane, and that free diffusion between moulting fluid and haemolymph can account for only a small fraction of the molecular species movement between these two fluids.     

Wax secretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta

Scanning electron microscopy of the epicuticle of the tobacco hornworm, Manduca sexta, showed that pupae in diapause possess thicker wax layers than non-diapausing pupae. Extractions of the epicuticle with CHCl₃ established that diapausing pupae secreted over three times as much wax as non-diapausing pupae, and that the total period of wax secretion in diapausing pupae was two to three times longer than that of non-diapausing pupae. Apparently, the extra thickness of the wax layer of diapausing pupae protects the insect from desiccation during diapause. The deposition of additional wax may result from hormonal changes accompanying entry into diapause.     

Differential synthesis of bacteria-induced proteins of Manduca sexta larvae and pupae

The range of inducible antibacterial and other associated haemolymph proteins in Manduca sexta larvae and pupae was examined by high resolution two-dimensional (2D) isoelectric focusing-polyacrylamide gel electrophoresis. Twenty-two major inducible proteins were consistently resolved on gels of haemolymph from bacteria-injected larvae. Haemolymph from bacteria-injected pupae showed a different pattern of induced proteins. The proteins of the two stages include those which (i) are induced in both stages, (ii) those which are exclusively induced in either larvae or pupae, (iii) those which are inducible in larvae, but consititutively present in pupae, and, (iv) those which are induced in larvae, and which are present at intermediate levels but may be induced to higher levels in pupae.The antibacterial activity of the haemolymph from larvae and pupae was compared on acidpolyacrylamide gels, and the apparent M_r and pI of the inducible proteins determined. Certain of the inducible proteins appear to resemble the cecropin and attacin proteins of Hylophora cecropia.     

Transegmental electrical coupling between adjacent intersegemental muscles in the tobacco hawkmoth (Manduca sexta)

The lateral intersegmental muscles of pharate adult tobacco hawkmoths (Manduca sexta), exhibited electrical coupling across the segmental boundary. The degree of electrical coupling was constant throughout adult development. These muscle fibres did not appear to be dye coupled in that neither cobalt ions nor the flourescent dye Lucifer Yellow CH passed between cells. Electrical coupling was unaffected by cellular acidification with CO₂. Data are presented which suggest that this electrical coupling may be through the extracellular space rather than some membrane specialization. It is further speculated that many invertebrates may show this form of electrical coupling due to the metameric architecture of certain skeletal muscles.     

Scanning electron microscopy of the disruption of tobacco hornworm, Manduca sexta, midgut by Bacillus thuringiensis endotoxin

The ingestion of Bacillus thuringiensis crystal endotoxin by Manduca sexta causes the destruction of both goblet and columnar cells of the midgut. One hour after ingestion, the microvilli show pathological effects. Nearly complete destruction of the goblet and columnar cells has taken place after 4 hr exposure to the toxin.     

Lepidopteran anti-juvenile hormones: Effects on development of Apanteles congregatus in Manduca sexta

Parasitism by the braconid wasp Apanteles congregatus decreases the effectiveness of the anti-juvenile hormone agents ETB (ethyl 4-[2-{ittert-butyl carbonyloxy}bytoxy]benzoate) and fluoromevalonolactone (FMev) in inducing precocious metamorphosis of Manduca sexta larvae. Topical application of 1–200 μg ETB to parasitized third-instar larvae had no effect on either host or parasite development, whereas doses of 50μg or more ETB applied to unparasitized third-instar larvae caused formation of larval-pupal intermediates after the fourth instar. Parasitism also decreased the effectiveness of 100–200 μg FMev in causing metamorphosis at the moult following its application. In contrast to ETB, FMev disrupted development of the parasitoids. No wasps emerged when preterminal stage hosts were treated with FMev and the hosts formed larval-pupal intermediates. After treatment of terminal stage hosts with FMev, the number of emerging parasitoids was reduced by one-third. Precocene II (100 μg per larvae) had no effect on development of either M. sexta or A. congregatus.     

Azadirachtin affects growth and endocrine events in larvae of the tobacco hornworm, Manduca sexta

Injection of azadirachtin in freshly emerged last-instar larvae of Manduca sexta elicited different reactions according to the dose administered. At low doses, pupation occurred in most of the cases, but the resulting pupae were defective for the most part. Individuals treated with higher doses usually did not fully complete development, moulting to supernumerary larvae or dying as larvae (sometimes at the wandering stage) after varying periods of survival. The haemolymph ecdysteroid titre of individuals treated with 2 μg azadirachtin/g bodyweight showed characteristic changes which are presumed to cause the disorders in the last stages that normally lead to pupation. Injection of moulting hormone in azadirachtin-treated individuals at certain times during the penultimate stage elicited no reduction of the azadirachtin-induced effects. It is shown that azadirachtin is able to inhibit development even when individuals performed a complete moult after the treatment.