Phospholipase A2 in hemocytes of the tobacco hornworm,Manduca sexta

On the hypothesis that prostaglandins and other eicosanoids mediate nodulation responses to bacterial infections in insects, we describe an intracellular phospholipase A₂ (PLA₂) in homogenates prepared from hemocytes collected from the tobacco hornworm, Manduca sexta. PLA₂ hydrolyzes fatty acids from the sn-2 position of phospholipids. Some PLA₂s are thought to be the first and rate-limiting step in biosynthesis of prostaglandins and other eicosanoids. The hemocyte PLA₂ activity was sensitive to hemocyte homogenate protein concentration (up to 250 μg protein/reaction), pH (optimal activity at pH 8.0), and the presence of a Ca²⁺ chelator. Like PLA₂s from mammalian sources, the hemocyte PLA₂ was inhibited by the phospholipid analog oleyoxyethyl phosphorylcholine. Whereas most intracellular PLA₂s require Ca²⁺ for catalytic activity, some PLA₂s, including the hemocyte enzyme, are Ca²⁺-independent. The hemocyte PLA₂ exhibited a preference for arachidonyl-associated substrate over palmitoyl-associated substrate. These findings show that M. sexta hemocytes express a PLA₂ that shows a marked preference for hydrolyzing arachidonic acid from phospholipids. The biological significance of this enzyme relates to cellular immune responses to bacterial infections. The hemocyte PLA₂ may be the first biochemical step in synthesis of the eicosanoids that mediate cellular immunity in insects. © 1996 Wiley-Liss, Inc.     

Studies on vitellogenin from the tobacco hornworm,Manduca sexta

In the tobacco hornworm moth, Manduca sexta, vitellogenin (Vg) is a very high-density (1.29 g/ml) phosphoglycolipoprotein containing 13% lipids, 3% carbohydrates, and 0.6% protein-bound phosphorus. Vitellogenin (M_r～500,000) has two apoproteins designated apoVg-l (M_r 177,000 ± 3,600) and apoVg-ll (M_r45,000 ± 5,000). ApoVg-l and apoVg-II can be dissociated with 6 M guanidine HCI and separated from each other by gel permeation chromatography. Immunoblotting experiments using antibodies against the apoproteins showed that apoVg-l and apoVg-II antigens were immunologically distinct polypeptides. Antibodies against Vg reacted only with apoVg-l. Antibodies against Vg and apoVg-l reacted with Vg in double immunodiffusion experiments, whereas antibodies against apoVg-II did not. These results suggest that in the native Vg molecule, apoVg-II is positioned inside the molecule away from the aqueous environment. Only apoVg-I contained covalently bound carbohydrate as shown by fluorescein isothiocyanateconjugated concanavalin A, periodate-Schiff reagent, and in vivo labeling with ³H-Man. In vivo labeling with ³²P-inorganic phosphate and chemical determination showed that apoproteins of both Vg and vitellin contain covalently bound phosphate groups.     

In vitro secretion of digestive phospholipase A2 by midguts isolated from tobacco hornworms,Manduca sexta

We report on secretion of phospholipase A₂ (PLA₂) by in vitro preparations of midguts isolated from tobacco hornworms, Manduca sexta. This enzyme is responsible for hydrolysis of fatty acids from the sn‐2 position of phospholipids, a necessary step in fatty acid absorption. The in vitro midgut preparations are competent to secrete PLA₂ into incubation buffer. Secretion began within the first 30 min of incubation and increased to a maximum at 8 h. We selected 2 h incubations because substantial loss of tissue integrity was observed after 8 h incubations. Using 2 h incubations, we recorded increased secretion of digestive PLA₂ from midguts incubated in buffer amended with diet or with yeast as a component of the diet. We also recorded small increases in secretion of PLA₂ from midguts incubated in buffer amended with a specific phospholipid, phosphatidylcholine. Midguts incubated in buffer amended with increased concentrations of phospholipid did not yield higher levels of PLA₂ activity. Lepidopteran midguts can be divided into three regions, and we recorded the highest secretion of PLA₂ from the middle region and lowest secretion from the anterior region. Because isolated midguts responded to food chemicals with increased secretion of digestive PLA₂, we suggest that secretion of digestive enzymes in tobacco hornworms is regulated by a prandial and/or paracrine mechanism, as suggested for digestive proteases in other insect species. Arch. Insect Biochem. Physiol. 42:179–187, 1999 .© 1999 Wiley‐Liss, Inc.     

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.     

Immunolocalization of a proton V-ATPase in ovarian follicles of the tobacco hornworm Manduca sexta

A monoclonal antibody, directed against an H⁺ translocating V-ATPase of the midgut of Manduca sexta, has been used for immunolocalization studies in ovarian follicles and testes of Manduca sexta. In testes, no distinct staining above background levels was observed. In vitellogenic follicles, V-ATPase immunoreactivity first appears in the cytoplasm of the trophocytes and then in the oocyte, but by far the strongest reaction is present in the region of the oolemma during endocytosis. All types of follicle cells surrounding both the oocyte and the trophocyte compartments show a distinct positive reaction. In the cylindrical follicle cells surrounding the oocyte, the immunoreactivity is clearly restricted to the basal part. Our results suggest an important role for V-ATPase in vitellogenin uptake in Manduca, similar to that suggested on electro-physiological grounds in Hyalophora cecropia. © 1995 Wiley-Liss, Inc.     

Midgut mitochondrial transhydrogenase in wandering stage larvae of the tobacco hornworm, Manduca sexta

Midgut mitochondria from fifth larval instar Manduca sexta exhibited a transhydrogenase that catalyzes the following reversible reaction: NADPH + NAD(+) <--> NADP(+) + NADH. The NADPH-forming transhydrogenation occurred as a nonenergy- and energy-linked activity. Energy for the latter was derived from the electron transport-dependent utilization of NADH or succinate, or from Mg++-dependent ATP hydrolysis by ATPase. The NADH-forming and all of the NADPH-forming reactions appeared optimal at pH 7.5, were stable to prolonged dialysis, and displayed thermal lability. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the NADPH --> NAD(+) and energy-linked NADH --> NADP(+) transhydrogenations, but not the nonenergy-linked NADH --> NADP(+) reaction. Oligomycin only inhibited the ATP-dependent energy-linked activity. The NADH-forming, nonenergy-linked NADPH-forming, and the energy-linked NADPH-forming activities were membrane-associated in M. sexta mitochondria. This is the first demonstration of the reversibility of the M. sexta mitochondrial transhydrogenase and, more importantly, the occurrence of nonenergy-linked and energy-linked NADH --> NADP(+) transhydrogenations. The potential relationship of the transhydrogenase to the mitochondrial, NADPH-utilizing ecdysone-20 monooxygenase of M. sexta is considered.     

Eicosanoids influence in vitro elongation of plasmatocytes from the tobacco hornworm, Manduca sexta

Nodule formation is the predominant insect cellular defense reaction to bacterial challenges, responsible for clearing the largest proportion of infecting bacteria from hemolymph circulation. Hemocyte spreading behavior is a critical step in the nodulation process. It has been suggested that eicosanoids mediate several steps in the process. However, the influence of eicosanoids on hemocyte spreading has not been investigated in detail. To test the hypothesis that eicosanoids mediate hemocyte spreading behavior, I treated larvae of the tobacco hornworm, Manduca sexta, with eicosanoid biosynthesis inhibitors and later assessed plasmatocyte elongation on glass slides. Plasmatocytes from larvae treated with dexamethasone did not elongate to the extent of plasmatocytes from untreated control larvae. The dexamethasone effect on plasmatocyte elongation was expressed in a dose-dependent manner and was reversed by injecting dexamethasone-treated larvae with the eicosanoid-precursor fatty acid, arachidonic acid. Palmitic acid, which is not substrate for eicosanoid biosynthesis, did not reverse the influence of dexamethasone on plasmatocyte elongation. Finally, plasmatocytes from larvae treated with a range of eicosanoid biosynthesis inhibitors did not elongate to the extent of plasmatocytes from control larvae. Plasmatocyte width did not appear to be influenced in this study. These findings strongly support the idea that insect plasmatocyte elongation is influenced by eicosanoids.     

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.     

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.     

A secretory PLA2 associated with tobacco hornworm hemocyte membrane preparations acts in cellular immune reactions

We report on a secretory phospholipase A2 (sPLA2) associated with membrane-enriched fractions prepared from hemocytes of the tobacco hornworms, Manduca sexta. Virtually no PLA2 activity was detected in serum of immunologically naive or bacterially challenged hornworms. PLA2 activity was detected in cytosolic and membrane-enriched fractions prepared from hemocytes. PLA2 activity in the cytosolic fraction (1.2 pmol/mg/h) was approximately 4-fold greater than in the membrane-enriched fraction. The cytosol-associated PLA2 activity was strongly inhibited in reactions conducted in the presence of the specific cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP) but not in the presence of the sPLA2 inhibitor p-bromophenacyl bromide (BPB). Conversely, the membrane-associated PLA2 activity was inhibited in reactions conducted in the presence of BPB but not in the presence of MAFP. While the cytosol-associated PLA2 was independent of calcium, the membrane-associated sPLA2 required calcium for full catalytic activity. Hornworms treated with either BPB, MAFP or the glucocorticosteroid dexamethasone were severely impaired (by 50 to 80% relative to controls) in their ability to form nodules in reaction to bacterial challenge. However, the immune-impairing influence of the inhibitors was reversed by treating larvae with arachidonic acid, a precursor for eicosanoid biosynthesis. We infer that the biological significance of the sPLA2 (as well as the previously characterized cytosolic PLA2) relates to hydrolysis of polyunsaturated fatty acids from cellular phospholipids. Moreover, this enzyme may be the target of immunity-impairing factors from the bacterium Xenorhabdus nematophila. The fatty acids serve as precursors for the generation of eicosanoids responsible for mediating and coordinating cellular immune reactions to infection.     

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.     

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.

     

Correlation between glycerolipids and pheromone aldehydes in the sex pheromone gland of female tobacco hornworm moths,Manduca sexta (L.)

Analysis by TLC and HPLC revealed that the triacylglycerols comprise the most abundant lipid class in the sex pheromone glands of Manduca sexta females. Also, conjugated olefinic acyl analogs of the major pheromone aldehydes occur principally in the triacylglycerols. The amount of triacylglycerols with conjugated diene acyl moieties significantly decreased when the period of pheromone production was extended by 7 h beyond the normal period of pheromone production by 3 injections of pheromone biosynthesis activating neuropeptide (PBAN) at 3 h intervals. This decrease indicates that the triacylglycerols stored in the gland may serve as major sources of pheromone precursors in the biosynthesis of the sex pheromone aldehydes. Furthermore, analysis of pheromone aldehydes and triacylglycerols in the gland from moths treated with PBAN showed that the proportions of the triacylglycerols with conjugated diene moieties were closely correlated with the proportions of aldehydes found in the same gland. This correlation suggests that the proportions of fatty acids bound to certain triacylglycerols regulates the proportions of aldehydes in biosynthesis of the pheromone blend in M. sexta. © 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.

     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Identification of neuroendocrine cells producing a diuretic hormone in the tobacco hornworm moth,Manduca sexta

Summary Separate antisera were raised to the N- and C-terminal half of the diuretic hormone from Manduca sexta. Antisera against the two halves of this peptide recognized the same cells in M. sexta, and preabsorption of the antisera with the peptides used as antigens abolished the immunoreactivity, confirming their specificity. The antisera reacted with two median neurosecretory cells on each side of the protocerebral groove in larvae, and with a group of about 80 small median neurosecretory cells in the adult, as well as their axons to, and their axon terminals in, the corpora cardiaca. During the early pupal stages, small cells, which are possibly derived from a common neuroblast, differentiate into immunoreactive neurosecretory cells, which explains the large increase in cell numbers in the adult. In the sleepy sulphur butterfly, Eurema nicippe, homologous median neurosecretory cells in the adult were immunoreactive with both antisera.     

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.     

Adipokinetic hormone-induced mobilization of fat body triglyceride stores in Manduca sexta: role of TG-lipase and lipid droplets

Triglycerides (TG) stores build up in the insect fat body as lipid droplets at times of excess of food. The mobilization of fat body triglyceride (TG) is stimulated by adipokinetic hormones (AKH). The action of AKH involves a rapid activation of cAMP-dependent protein kinase (PKA). Recent in vitro studies have shown that PKA phosphorylates and activates the TG-lipase substrate, the lipid droplets. Conversely, purified TG-lipase from Manduca sexta fat body is phosphorylated by PKA in vitro but is not activated. This study was directed to learn whether or not AKH promotes a change in the state of phosphorylation of the lipase in vivo, and what are the relative contributions of cytosol and lipid droplets to the overall increase of lipolysis triggered by AKH. TG-lipase activity of fat body cytosols isolated from control and AKH-treated insects was determined against the native substrate, in vivo [3H]-TG radiolabeled lipid droplets, obtained from control and AKH-treated insects. The lipase activity of the system composed of AKH-cytosol and AKH-lipid droplets (11.1 +/- 2.1 nmol TG/min-mg) was 3.1-fold higher than that determined with control cytosol and lipid droplets (3.6 +/- 0.5 nmol TG/min-mg). Evaluation of the role of AKH-induced changes in the lipid droplets on lipolysis showed that changes in the lipid droplets are responsible for 70% of the lipolytic response to AKH. The remaining 30% appears to be due to AKH-dependent changes in the cytosol. However, the phosphorylation level of the TG-lipase was unchanged by AKH, indicating that phosphorylation of the TG-lipase plays no role in the activation of lipolysis induced by AKH.     

The effect of L-canavanine and L-canaline on the hemolymph amino acid composition of the tobacco hornworm,Manduca sexta (L.) (Sphingidae)

Tobacco hornworm larvae, Manduca sexta (L.) (Sphingidae), were administered L-canaline either by parenteral injection or by dietary consumption. The overt toxicity and the alteration of hemolymph amino acids caused by these nonprotein amino acids were evaluated. The LD₅₀ value for parenterally administered canavanine and canaline is 1.0 and 2.5 mg/g fresh body weight, respectively. A dietary concentration of 5.2 mM for canavanine and over 20 mM for canaline represent the respective LC₅₀ values. A large percentage of the larvae reared on diets supplemented with additional arginine, ornithine, or 2,4-diaminobutyric acid in addition to canavanine or canaline were unable to complete larval-pupal ecdysis. These toxic effects were associated with a decreased glutamic acid hemolymph titer and dramatically elevated ornithine. On the other hand, larvae administered canavanine or canaline alone, either by dietary consumption or parenteral injection, experienced less drastic developmental aberrations. These symptoms were in some cases correlated with increased ornithine and glutamic acid titers. Evidence is presented that even a canavanine- and canaline-sensitive insect such as M. sexta has a marked ability to eliminate these protective allelochemicals.     

Differentiation of roles of chemosensory organs in food discrimination among host and non-host plants by larvae of the tobacco hornworm, Manduca sexta

ABSTRACT. The contributions of olfactory and gustatory organs in food plant discrimination were examined in larvae of Manduca sexta (Johan.) (Lepidoptera, Sphingidae). Larvae, from which various chemosensory organs had been removed surgically, were tested for feeding preferences for a host, tomato ( Lycopersicon esculentum ); a weakly acceptable non-host, rape ( Brassica napus ); and an unacceptable non-host canna ( Canna generalis ), using a two-choice disc bioassay.
Removal of all known chemosensory organs resulted in failure to show discriminatory behaviour in a strictly chemosensory bioassay, indicating that all external chemosensory organs have been accounted for. The involvement of non-chemosensory organs results in residual discrimination for leaves by individuals with total chemosensory ablations.
Larvae possessing either olfactory or gustatory organs still exhibit normal preferences for tomato over rape. Gustatory (but not olfactory) organs are required for larvae to show normal preferences for tomato over canna; in fact, olfactory organs do not appear to participate in this decision.
To examine which if any of the plant species is being selected in two-choice tests, larvae were given a choice between each leaf species and a 'neutral' substance (wet filter paper). Both olfactory and gustatory organs are required for normal preferences for tomato, but either alone will suffice for rape. Only gustation is needed to select canna, and participation of either the epipharyngeal sensilla or a single medial sensillum styloconicum is sufficient to elicit complete rejection behaviour.
We conclude that, in larvae of M. sexta , the complement of chemosensory organs needed for food plant discrimination varies with the plant species sampled. Evidence is presented exposing a potential artefact of ablation experiments; extirpation of one sensory organ may affect the functioning of others nearby, even though they may appear normal by visual inspection.