Gustatory perception of phytoecdysteroids in Plodia interpunctella larvae

Phytoecdysteroids are steroidal compounds produced by various plants that disrupt growth and development of insects eating them. They exhibit an insecticidal activity on a number of insect pests such as Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). In this study, we further evaluated whether phytoecdysteroids deter larvae of this species from feeding, by using four phytoecdysteroid molecules, commonly occurring in plants: 20‐hydroxyecdysone (20E), ponasterone A (PonA), polypodine B (PolB), and makisterone A (MakA). Fourth instar P. interpunctella avoided contact with food pellets treated with these phytoecdysteroids in a dose‐dependent way (2–30 mm ). In order to test whether this avoidance was mediated by taste sensitivity, we recorded the responses of taste neurons located in the lateral and medial sensilla styloconica of the galea. At least one neuron responded to each of these compounds in both sensilla. The neuron located in the medial sensillum had a detection threshold of 10^?6 m for PonA, 10^?4 m for 20E and PolB, and 10^?3 m for MakA. The lateral sensillum neuron responded with less intensity and its detection threshold was 10 times higher than that of the medial sensillum neuron. These results indicate that phytoecdysteroids are detected as deterrent stimuli by P. interpunctella larvae and that small structural differences significantly affect their biological activity.     

Flavonoids from cabbage are feeding stimulants for diamondback moth larvae additional to glucosinolates: Chemoreception and behaviour

In caterpillars two styloconic contact chemoreceptors on the maxillary galea are assumed to contain the main taste receptors involved in host plant selection. The diamondback moth, Plutella xylostella L. is a specialist feeder of plants in the Brassicaceae, a plant family characterized by the biosynthesis of glucosinolates. We used pea (Pisum sativum L., Leguminosae) as a neutral non-host for a dual-choice leaf disc assay to quantify feeding stimulation by glucosinolates and flavonoids. Increasing concentrations of sinigrin resulted in significant preferences for sinigrin-treated leaf discs, with a threshold between 1 and 3 M. Millimolar concentrations of four of the five flavonol triglucosides likewise elicited a significant preference for flavonoid-treated leaf discs. A mixture of four flavonoids and sinigrin was significantly preferred over sinigrin-treated leaf discs alone. Vigorous unicellular electrophysiological responses of medial maxillary styloconic taste sensilla were observed in response to five glucosinolates (glucocapparin, sinigrin, glucobrassicin, glucoiberin, and gluconasturtiin). This medial taste neuron responded in a dose-dependent manner to a concentration series of sinigrin, with a threshold of response of ca. 1 M. The lateral sensillum styloconicum contained a neuron sensitive to sucrose, glucose, and fructose. However, no responses in the two types of maxillary styloconic sensilla to the phagostimulatory flavonoids could be detected, suggesting that other taste organs mediate chemoreception of flavonoids. We conclude that diamondback moth larvae employ a combination of biosynthetically distinct categories of feeding stimulants which allows for a higher degree of discriminatory ability than when this would be based on glucosinolates alone.     

Electrophysiological responses of gustatory sensilla of Mamestra brassicae (Lepidoptera,Noctuidae) larvae to three ecdysteroids: ecdysone, 20-hydroxyecdysone and ponasterone A

Specialised phytophagous Lepidoptera such as Bombyx mori and Pieris brassicae have contact chemoreceptors that perceive ecdysteroids at very low concentrations. This sensory perception allows them to feed on substrates with a high content of phytoecdysteroids. We have evaluated if a polyphagous insect like Mamestra brassicae does possess contact chemoreceptor cells that are sensitive to these molecules. Electrophysiological recordings were performed from contact chemoreceptors located on the maxilla. These receptors were stimulated with some sugars, amino acids and salts and with three ecdysteroids. Our results demonstrate that a specific cell within the lateral sensilla responds to 20-hydroxyecdysone and ponasterone A but not to ecdysone.     

Evaluation of Ostrinia nubilalis (Lepidoptera: Crambidae) neonate preferences for corn and weeds in corn

Choice tests were conducted to determine feeding preferences of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), neonates for 15 species of plants. Percentage of neonates accepting (found on) each leaf disc after 24 h was measured using choice tests. Initially, nine species of plants were evaluated. The following year, 10 plant species were evaluated during O. nubilalis first generation and 11 species during the second generation. Pennsylvania smartweed, Polygonum pennsylvanicum (L.), had the highest percentage of neonates accepting leaf discs in both years. Other plants with high acceptance rates included swamp smartweed, Polygonum amphibium L.; velvetleaf, Abutilon theophrasti Medicus; cocklebur, Xanthium strumarium L.; and yellow foxtail, Setaria glauca (L.). Corn, Zea mays L., consistently had low percentages of neonates accepting leaf discs along with common waterhemp, Amaranthus rudis Sauer. Implications these results may have on O. nubilalis host plant selection in central Iowa's corn dominated landscape are considered.     

Identification of a gustatory receptor tuned to sinigrin in the cabbage butterfly Pieris rapae

Glucosinolates are token stimuli in host selection of many crucifer specialist insects, but the underlying molecular basis for host selection in these insects remains enigmatic. Using a combination of behavioral, electrophysiological, and molecular methods, we investigate glucosinolate receptors in the cabbage butterfly Pieris rapae. Sinigrin, as a potent feeding stimulant, elicited activity in larval maxillary lateral sensilla styloconica, as well as in adult medial tarsal sensilla. Two P. rapae gustatory receptor genes PrapGr28 and PrapGr15 were identified with high expression in female tarsi, and the subsequent functional analyses showed that Xenopus oocytes only expressing PrapGr28 had specific responses to sinigrin; when ectopically expressed in Drosophila sugar sensing neurons, PrapGr28 conferred sinigrin sensitivity to these neurons. RNA interference experiments further showed that knockdown of PrapGr28 reduced the sensitivity of adult medial tarsal sensilla to sinigrin. Taken together, we conclude that PrapGr28 is a gustatory receptor tuned to sinigrin in P. rapae, which paves the way for revealing the molecular basis of the relationships between crucifer plants and their specialist insects.     

Deficiency of gustatory sensitivity to some deterrent compounds in "polyphagous" mutant strains of the silkworm, Bombyx mori

Sawa-J, CSJ02 and N601 x C601 are selected mutant strains of Bombyx mori, which grow on various artificial diets or temporarily ingest various plant leaves. To examine the mechanisms mediating diet breadth of caterpillars, gustatory spike responses of the silkworms, called 'polyphagous" strains, were compared with normal strains, N137 x C146 and C02. There were notable differences in their feeding habits and in their sensitivity to salicin in deterrent cells in the maxillary medial styloconic sensilla and the epipharyngeal sensilla. By contrast, the deterrent cells of all strains responded similarly to strychnine nitrate in a dose-dependent manner. In additional comparisons of Sawa-J and N137 x C146, Sawa-J maxillary deterrent cells were significantly less sensitive to phloridzin, amygdalin and arbutin, but responded to some alkaloids and 20-hydroxyecdysone with similar or even higher firing rate. These results suggest that the deficiency of the sensitivity to some deterrent compounds on the deterrent cell of the polyphagous strains may be caused by mutant genes and affects the diet breadth of caterpillars.     

Diversity of detoxification pathways of ingested ecdysteroids among phytophagous insects

The metabolic pathways of ingested ecdysteroids have been investigated in three insect species, the aphid Myzus persicae and two Lepidoptera, Plodia interpunctella and Ostrinia nubilalis. M. persicae produces mainly a 22-glucoside conjugate, whereas P. interpunctella eliminates a mixture of 20E and its 3-oxo and 3-epi derivatives, both in free form and as conjugates with various fatty acids. O. nubilalis only produces fatty acyl ester conjugates. These data point out the great diversity of detoxification mechanisms used by phytophagous insects in order to overcome the potential harmful effects of ecdysteroids present in their food.     

Immunolocalization of odorant-binding proteins in noctuid moths (Insecta, Lepidoptera)

Odorant-binding proteins were studied in the noctuid moths Agrotis segetum, Autographa gamma, Helicoverpa armigera, Heliothis virescens and Spodoptera littoralis using antisera raised against the pheromone-binding protein (PBP) and general odorant-binding protein 2 (GOBP2) of Antheraea polyphemus (Saturniidae). Proteins immunoreacting with these antisera were only found on the antennae and PBP and GOBP2 could be identified on western blots of males and females of all five species. PBPs were predominantly localized in sensilla trichodea and GOBP2 in sensilla basiconica, in good correlation with the stimulus specificity of the receptor cells in these sensilla. In H. armigera and H. virescens the majority of the s. trichodea immunoreacted with the antiserum against PBP of A. polyphemus; in A. segetum, A. gamma and S. littoralis, on the other hand, a high percentage of s. trichodea remained unlabelled. Probably, the PBP expressed in these sensilla is so different that it does not immunoreact with the antiserum used. Such a protein was found by native PAGE of antennal extracts of A. segetum and S. littoralis. These data correlate with the fact that the two heliothine species use pheromones with the same alkyl chain length as A. polyphemus, while the other three species use pheromones with shorter chains. In H. armigera, H. virescens, A. gamma and S. littoralis female antennae were also immunolabelled and a large number of PBP-expressing s. trichodea was consistently found. In S.littoralis this fits with the electrophysiologically recorded high pheromone sensitivity of female s. trichodea, whereas in females of H. armigera and H. virescens no or only weak responses to pheromone stimulation have been reported. Therefore, PBP expression in a sensillum does not necessarily imply pheromone sensitivity of its receptor cells.     

Critical role of desolvation in the binding of 20-hydroxyecdysone to the ecdysone receptor

The insect steroid hormone 20-hydroxyecdysone (20E) binds to its cognate nuclear receptor composed of the ecdysone receptor (EcR) and Ultraspiracle (USP) and triggers the main developmental transitions, in particular molting and metamorphosis. We present the crystal structure of the ligand-binding domains of EcR/USP in complex with 20E at 2.4A resolution and compare it with published structures of EcR/USP bound to ponasterone A (ponA). ponA is essentially identical to 20E but lacks the 25-OH group of 20E. The structure of 20E-bound EcR indicates that an additional hydrogen bond is formed compared with the ponA-bound receptor, yet, paradoxically, ponA has a significantly higher affinity for EcR than 20E. Theoretical studies based on docking and free energy methods lead to a rationale for understanding the difference in binding affinities between 20E and ponA. Results of the calculations indicate that the favorable contribution from the extra H-bond made by 25-OH of 20E is counterbalanced by its larger desolvation cost compared with that of ponA. The contribution of 25-OH to the binding affinity is further compared with those of 20- and 22-OH groups. Ligands that lack the 20- or 22-OH group are indeed known to bind less favorably to EcR than 20E, an effect opposite to that observed for ponA. The results indicate that their respective contributions to receptor-ligand complex stability reside mostly in their different contributions to solvation/desolvation. Together, the data demonstrate the critical role of ligand desolvation in determining binding affinity, with general implications for the binding of hormones to their cognate nuclear receptors.     

Specialist deterrent chemoreceptors enable Pieris caterpillars to discriminate between chemically different deterrents

Pieris butterflies (Lepidoptera: Pieridae) are specialist herbivores of cruciferous plants. They exploit glucosinolates, secondary plant metabolites chemotaxonomically characteristic for this plant family, as token stimuli. In addition to particular glucosinolates, some genera of the Cruciferae contain cardenolides, steroidal allelochemicals that act as potent feeding and oviposition deterrents to several Pieris species. We investigated the sensory mechanisms by which these compounds are perceived in larvae. Pieris caterpillars and many other lepidopterous species are endowed with so-called generalist deterrent receptors, that respond to a broad spectrum of secondary plant substances. In Pieris caterpillars we found a second type of deterrent chemoreceptor in maxillary styloconic taste sensilla. This neuron is very sensitive to cardenolides (threshold 0.1–0.3 M). The generalist deterrent receptor also responds to these substances but its threshold lies at 50–100× higher concentrations. In behavioural preference experiments Pieris brassicae L. caterpillars preferred cardenolide-treated cabbage leaf discs when confronted with a choice between them and a deterrent substance that does not occur in the Brassicaceae. The cardenolides acted as potent deterrents when offered against untreated cabbage leaf discs. This demonstrates that the balance of activity elicited in the two types of deterrent chemoreceptors determines the behavioural decision.     

草地贪夜蛾幼虫对四种刺激物质的味觉感受和取食选择

【目的】为了筛选有效的草地贪夜蛾Spodoptera frugiperda幼虫取食激食素和抑制剂并探究其味觉感受机理,为生态防治草地贪夜蛾提供理论和实践上的依据。【方法】利用单感受器记录法测定草地贪夜蛾5龄第2天幼虫下颚外颚叶上中栓锥感器和侧栓锥感器对不同浓度的蔗糖、黑芥子苷、单宁酸和盐酸奎宁4种刺激物质的电生理反应,并采用二项叶碟法测定草地贪夜蛾幼虫对这些刺激物质的取食选择行为。【结果】草地贪夜蛾幼虫中栓锥感器和侧栓锥感器内均存在对蔗糖、黑芥子苷和单宁酸敏感的味觉受体神经元,但是神经元的活性随着刺激物的种类及浓度而变化。其中,两类感器内神经元对蔗糖和黑芥子苷的反应均呈现典型的浓度梯度反应。中栓锥感器内存在对盐酸奎宁敏感的味觉受体神经元,但是呈现逆浓度梯度的反应模式,侧栓锥感器内不存在对盐酸奎宁敏感的神经元。蔗糖显著诱导幼虫的取食行为,而盐酸奎宁、黑芥子苷和单宁酸均抑制幼虫的取食行为,且都呈现浓度梯度的抑制活性。【结论】草地贪夜蛾幼虫中栓锥感器和侧栓锥感器内均存在对取食激食素和抑制剂敏感的味觉受体神经元,但是两类感器不论在反应谱上还是敏感性上均存在差异。蔗糖可以作为取食激食素,盐酸奎宁、黑芥子苷和单宁酸可以作为取食抑制剂的有效候选物质。幼虫对蔗糖、黑芥子苷和单宁酸的味觉反应在一定程度上解释了其取食选择行为的味觉基础。本研究为草地贪夜蛾的生态防治提供了味觉信息。     

Comparative susceptibility of European corn borer, southwestern corn borer, and sugarcane borer (Lepidoptera: Crambidae) to Cry1Ab protein in a commercial Bacillus thuringiensis corn hybrid

One field strain each of the European corn borer, Ostrinia nubilalis (Hübner); southwestern corn borer, Diatraea grandiosella Dyar; and sugarcane borer, Diatraea saccharalis (F.); were collected from cornfields in northeastern Louisiana. Susceptibilities of the field strain and a corresponding laboratory strain of the three borer species to Cry1Ab protein in DK69-70 Bacillus thuringiensis (Bt) corn hybrid were determined by exposing neonates to intact leaf tissues from whorl stage plants or by feeding neonates or third instars on a meridic diet treated with different concentrations of Cry1lAb protein extracted from Bt corn leaves. Mortality and growth of larvae were evaluated after 2 and 4 d posttreatment in the bioassays by using intact leaf tissues or after 7 d in the bioassays by using diet incorporating Cry1Ab protein. D. saccharalis was the least susceptible species to Cry1Ab protein among the three species, followed by D. grandiosella, whereas O. nubilalis was most susceptible. The 2-d mortality of D. saccharalis neonates on intact Bt leaf tissues was lower than that of O. nubilalis and D. grandiosella. All neonates of O. nubilalis were killed on the diet treated with Cry1Ab protein at 0.5 and 1 mg/kg. The mortality of D. grandiosella was > 75% at 1 mg/kg, but it was < 6% for D. saccharalis at 1 mg/kg. The LC50 values of D. saccharalis were 3- and 11-fold higher than those of D. grandiosella and O. nubilalis, respectively. The LC90 values of D. saccharalis were 8- and 32-fold higher than those of D. grandiosella and O. nubilalis, respectively. Larval growth of the three species on Cry1Ab-treated diet was inhibited, but the inhibition was greater for O. nubilalis and D. grandiosella than for D. saccharalis. The lower susceptibility of D. saccharalis to Cry1Ab protein suggests that it is necessary to verify if a high-dose Bt corn for O. nubilalis and D. grandiosella is also a high dose for D. saccharalis.     

Food selection in adults and larvae of three species of Lepidoptera: a behavioural and electrophysiological study

Adults and larvae of Spodoptera littoralis, Heliothis virescens and Heliothis armigera were tested with a range of sugars, amino acids, sugar alcohols and allelochemics. Feeding behaviour was correlated with the electrophysiological responses of maxillary styloconic sensilla in the larvae and proboscis styloconic sensilla in the adult. The neural response was more vigorous in larvae than in adults but otherwise the spectra of responses were similar in the two life stages. Phagostimulants and antifeedants stimulated maximally different sensilla in larvae but not in adults. The responses of adult sensilla to sugars and amino acids were significantly correlated to behaviour in all three species, but only in H. armigera was there a similar correlation with the sugar alcohols and allelochemics.     

A neurophysiological study of sensitivity to a feeding deterrent in two sister species of Heliothis with different diet breadths

Heliothis subflexa and H. virescens are sister species that differ markedly in their hostplant specificity: the former is a specialist on one plant genus, the latter feeds on plants from many families. The behavioral threshold for rejection of deterrent chemicals is lower in larvae of H. subflexa than in those of H. virescens. In this paper, we examine the responses of the galeal styloconic sensilla of these larvae to stimulation by three chemicals, sucrose and inositol, which are phagostimulants, and sinigrin, a deterrent, in an attempt to determine the neural basis for the differences in feeding behavior between the species. The species difference could not be attributed to differences in firing rate of the deterrent-sensitive cells, differences in the ratio of responses to phagostimulants and deterrents, differences in the rates of adaptation of the sensory neurons, or differences in the extent of interactions between chemicals at the peripheral sensilla. We conclude that the differences between the species probably result from differences in processing sensory information within the central nervous system.     

Effects of intercropping with flowering plants on predation of Ostrinia nubilalis (Lepidoptera: Crambidae) eggs by generalist predators in bell peppers

Bell pepper plots intercropped with flowering plants were measured for improving biological control provided by natural enemies of the European corn borer [Ostrinia nubilalis (Hübner)]. The intercropped plants Dill, Anethum graveolens L.; coriander, Coriandrum sativum L.; and buckwheat, Fagopyrum escuelentum Moench; were established on the edge of two pepper plots and compared with nonintercropped control plots. Predation by the three species Orius insidiosus Say; Coleomegilla maculata DeGeer; and Chrysoperla, sp. Stephens, was monitored by installing O. nubilalis egg masses on sentinel plants in 2008, 2009, and 2010. To assess negative impacts of alternative prey on O. nubilalis egg predation, green peach aphid, Myzus persicae (Sulzer) populations were monitored via whole-plant counts in 2009 and 2010. Myzus persicae densities on pepper plants peaked in June or July and then declined rapidly. Predation on O. nubilalis eggs increased rapidly after aphid populations declined. Aphid populations were reduced in two out of three field-seasons in intercropped plots. Seasonal predation by O. insidiosus was significantly higher in the intercropped system four out of five field-seasons and one field season by C. maculata. Results indicate that biocontrol of O. nubilalis can be improved by intercropping with flowering plants, although this capacity may depend on the abundance of alternative prey.     

An accessory olfactory pathway in Lepidoptera: the labial pit organ and its central projections in Manduca sexta and certain other sphinx moths and silk moths

Summary In the hawkmoth, Manduca sexta, the third segment of each labial palp contains a pit, which houses a densely packed array of sensilla. We have named this structure the labial pit organ (LPO). The sensilla within the pit are typical of olfactory receptors, characterized by a grooved surface, wall pores, and pore tubules. Axons arising from receptor cells that innervate these sensilla project bilaterally to a single glomerulus in each antennal lobe. We have compared this central projection with that in three other species of Manduca (M. quinquemaculata, M. dilucida, and M. lanuginosa) and in the silkmoths Antheraea polyphemus and Bombyx mori. A bilateral projection to a single glomerulus in each antennal lobe is present in all cases. We suggest that the LPO serves as an accessory olfactory organ in adult Lepidoptera.     

Pheromone-binding proteins contribute to the activation of olfactory receptor neurons in the silkmoths antheraea polyphemus and Bombyx mori

The sensilla trichodea of the silkmoth Antheraea polyphemus are innervated by three types of receptor neurons each responding specifically to one of three pheromone components. The sensillum lymph of these sensilla surrounding the sensory dendrites contains three different types of pheromone-binding proteins (PBPs) in high concentrations. The sensilla trichodea of the silkmoth Bombyx mori are supplied by two receptor neurons each tuned specifically to one of the two pheromone components bombykol and bombykal, but only one type of PBP has been found so far in these sensilla. Recombinant PBPs of both silkmoth species in various combinations with pheromone components were applied to the receptor neurons via tip-opened sensilla during electrophysiological recordings. Over a fairly broad range of pheromone concentrations the responses of the receptor neurons depended on both, the pheromone component and the type of the PBP. Therefore, the PBPs appear to contribute to the excitation of the receptor neurons. Furthermore, bombykal in combination with the expressed PBP of B. mori failed to activate the corresponding receptor neuron of B. mori, but did so if combined with one of the PBPs of A. polyphemus. Therefore, a still unknown binding protein involved in bombykal transport might be present in B. mori.     

Physiological host specificity: a model using the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and microsporidia of row crop and other stalk-boring hosts

We investigated vertical and horizontal transmission as means by which entomopathogenic microsporidia may be isolated in their hosts. Ostrinia nubilalis larvae were challenged with microsporidia isolated from other stalk-boring and row crop Lepidoptera and were susceptible to seven species. Two species were horizontally transmitted. A Nosema sp. from Eoreuma loftini was transmitted among O. nubilalis larvae but not among larvae of the E. loftini host. This species was also vertically transmitted to the offspring of infected O. nubilalis females. An rDNA sequence showed the E. loftini isolate to be Nosema pyrausta, a naturally occurring species in O. nubilalis. Our results suggest that both horizontal and vertical transmission provide physiological barriers to host switching in the microsporidia, thus restricting the natural host range.     

Gustatory responsiveness to pyrrolizidine alkaloids in the Senecio specialist, Tyria jacobaeae (Lepidoptera, Arctiidae)

Abstract.  Electrophysiological recordings from taste sensilla of the caterpillar Tyria jacobaeae with the pyrrolizidine alkaloids, characteristic compounds from their host plants, demonstrated sensitivity of a pyrrolizidine alkaloid-sensitive cell in the lateral galeal sensilla at concentrations as low as 1 × 10⁻¹¹ M. Another pyrrolizidine alkaloid-sensitive cell in the medial galeal sensilla responded at higher concentrations. Both pyrrolizidine alkaloid-cells were maximally sensitive to seneciphylline N -oxide and senecionine N -oxide. Seven other pyrrolizidine alkaloids were less stimulating. Monocrotaline N -oxide was the least stimulating. Observation experiments demonstrated that differences in sensitivity to different pyrrolizidine alkaloids at the electrophysiological level were correlated with differences in feeding behaviour; the first feeding bout was of longer duration on diet containing seneciphylline N -oxide than on diet containing monocrotaline N -oxide, and a plain diet was generally not accepted.