Sphingid Moth Research Articles

Cavitation in the embryonic tracheal system ofManduca sexta

Insect tracheae form during embryonic development and initially contain liquid, which impedes transport of oxygen and carbon dioxide. Only later do tracheae fill with gas and come to support high rates of gas flux. This liquid-to-gas transition is poorly understood. Using eggs of the sphingid moth Manduca sexta, we show that longitudinal tracheae in embryos fill with gas in less than 5 s, without invasion of external air, by a process of cavitation. Cavitation requires that tracheal liquids be under tension, and we propose two complementary processes for generating it. One likely, classical mechanism is tracheolar fluid absorption, first proposed by Wigglesworth. Our data support this mechanism in Manduca: after cavitation, liquids are progressively drawn out of finer tracheal branches. The second, previously unknown, mechanism is evaporative water loss across the eggshell, which leads both to declining egg volume and to a larger negative pressure potential of water. The pressure potential helps to drive rapid expansion of small bubbles nucleated near spiracles. Once bubbles are large enough to have displaced liquid across the diameter of a trachea, negative capillary pressure reinforces subsequent expansion of the bubble. Together with predictions from modern cavitation theory, our observations substantiate Wigglesworth's contention that gas filling is promoted by increasing hydrophobicity associated with tanning of the spiracles and major tracheal branches.

Water loss and gas exchange by eggs of Manduca sexta: Trading off costs and benefits

Like all terrestrial organisms, insect eggs face a tradeoff between exchanging metabolic gases (O2 and CO2) and conserving water. Here I summarize the physiology underlying this tradeoff and the ecological contexts in which it may be important. The ideas are illustrated primarily by work from my laboratory on eggs of the sphingid moth Manduca sexta. In particular, I discuss: (1) dynamic changes in metabolic demand and water loss during development; and (2) how the eggshell layers and embryonic tracheal system control the traffic of gases between the embryo and its environment. Subsequently, I identify three areas with interesting but unresolved issues: (1) what eggs actually experience in their microclimates, focusing particularly on the leaf microclimates relevant to eggs of M. sexta; (2) how egg experience influences whether or not hatchling larvae succeed in establishing feeding sites on host plants; and (3) whether Hetz and Bradley's [Hetz, S.K., Bradley, T.J., 2005. Insects breathe discontinuously to avoid oxygen toxicity. Nature 433, 516−519] oxygen toxicity hypothesis for discontinuous gas-exchange cycles applies to insect eggs.

Insect eggs at a transition between diffusion and reaction limitation: Temperature, oxygen, and water

In diverse animal taxa, eggs and embryos are incapable of transporting oxygen by convection. In such cases, internal oxygen distributions are determined jointly by rates of oxygen consumption and diffusive transport. Here we develop a mathematical model of oxygen consumption and transport in insect eggs, with the goal of understanding—for eggs in variable-temperature environments—the interactive effects of the two processes on development. We fit the model to previously published data on development time of eggs of a sphingid moth, Manduca sexta. The fitted coefficients suggest that eggs develop at a transition point between reaction- and diffusion-limitation. We test then this conclusion with independent data on development times of eggs distributed across a set of temperatures generated by a thermal gradient bar. Finally, we develop an extension of the model that considers tradeoffs between oxygen transfer to eggs versus water loss from them. The model results provide both a rationale for why development is often mass-transfer limited and a set of new predictions about oxygen–water tradeoffs.

Expression of UV-, blue-, long-wavelength-sensitive opsins and melatonin in extraretinal photoreceptors of the optic lobes of hawkmoths

Lepidopterans display biological rhythms associated with egg laying, eclosion and flight activity but the photoreceptors that mediate these behavioural patterns are largely unknown. To further our progress in identifying candidate light-input channels for the lepidopteran circadian system, we have developed polyclonal antibodies against ultraviolet (UV)-, blue- and extraretinal long-wavelength (LW)-sensitive opsins and examined opsin immunoreactivity in the adult optic lobes of four hawk moths, Manduca sexta, Acherontia atropos, Agrius convolvuli and Hippotion celerio. Outside the retina, UV and blue opsin protein expression is restricted to the adult stemmata, with no apparent expression elsewhere in the brain. Melatonin, which is known to have a seasonal influence on reproduction and behaviour, is expressed with opsins in adult stemmata together with visual arrestin and chaoptin. By contrast, the LW opsin protein is not expressed in the retina or stemmata but rather exhibits a distinct and widespread distribution in dorsal and ventral neurons of the optic lobes. The lamina, medulla, lobula and lobula plate, accessory medulla and adjacent neurons innervating this structure also exhibit strong LW opsin immunoreactivity. Together with the adult stemmata, these neurons appear to be functional photoreceptors, as visual arrestin, chaoptin and melatonin are also co-expressed with LW opsin. These findings are the first to suggest a role for three spectrally distinct classes of opsin in the extraretinal detection of changes in ambient light and to show melatonin-mediated neuroendocrine output in the entrainment of sphingid moth circadian and/or photoperiodic rhythms.

Effect of elevated atmospheric CO2 on oviposition behavior in Manduca sexta moths

AbstractMoths can detect changes in environmental carbon dioxide (CO2) with extremely high sensitivity, but the role of CO2 in the biology of these and other insects is not well understood. Although CO2 has been demonstrated to influence egg‐laying (oviposition) behavior of the pyralid moth Cactoblastis cactorum and nectar foraging of the sphingid moth Manduca sexta, information about the generalized role of CO2 in the behavioral biology of these species is lacking. Comparative data are necessary to properly assess how the behaviors of different species may be modified by steadily rising levels of greenhouse gases in the environment. Experiments carried out in Biosphere 2 addressed whether changes in ambient CO2 levels play a role in the oviposition behaviors of M. sexta moths. In the first series of experiments, oviposition was measured inside a flight cage with different levels of nearly ambient or elevated CO2 (400, 800 or 1200 ppm). For each concentration, hostplants used as oviposition sites were grown from seed at a CO2 level that matched the environment inside the flight cage. Under homogenous levels of CO2, we observed no significant difference in oviposition behavior at the concentrations tested. In a second series of experiments, two groups of hostplants, each surrounded by a mini free‐air CO2 enrichment (FACE) ring, were assembled inside a flight cage. In this choice test, a dynamic plume of artificially high CO2 was generated around one group of test plants, while ambient CO2 was released around the second (control) group. After eggs were counted on both plant groups, M. sexta females showed a small preference for ovipositing on the control plants. Therefore, in contrast to C. cactorum females tested under similar dynamic flow conditions, M. sexta female oviposition was not strongly inhibited by elevated CO2. To investigate this phenomenon further, we used electrophysiological recording and found that the CO2 receptor cells in M. sexta, unlike those in C. cactorum, are not readily affected by elevated levels of ambient CO2. These findings therefore suggest that elevated background levels of CO2 affect the physiology of the CO2 detection system of M. sexta to a lesser extent than that of C. cactorum, and this correlates well with the observed differences in oviposition behavior between the two species under elevated levels of environmental CO2. Hostplants of C. cactorum are crassulacean acid metabolism plants that generate nocturnal CO2 sinks on the cladode surfaces, whereas, M. sexta hostplants are nocturnal sources of respiratory CO2. We hypothesize that the abrupt and continuing increase in global ambient CO2 levels will differentially alter the behavior and physiology of moths that use CO2 sinks and sources as sensory cues to find hostplants.

Gregarious emergence of Macrostomion sumatranum (Hymenoptera: Braconidae; Rogadinae) from the mummified, full‐grown larvae of Theretra silhetensis (Lepidoptera: Sphingidae)

AbstractTwenty‐six to 160 adults of Macrostomion sumatranum emerged from a mummified full‐grown larva of a sphingid moth, Theretra silhetensis, on taro in Okinawa‐jima, Japan. This is the first host record of M. sumatranum. The sex ratio was female‐biased in a small brood but less so or even male‐biased in a large brood.

ATTRACTING ANTAGONISTS: DOES FLORAL NECTAR INCREASE LEAF HERBIVORY?

Traits that are attractive to mutualists may also attract antagonists, resulting in conflicting selection pressures. Here we develop the idea that increased floral nectar production can, in some cases, increase herbivory. In these situations, selection for increased nectar production to attract pollinators may be constrained by a linked cost of herbivore attraction. In support of this hypothesis, we report that experimentally supplementing nectar rewards in Datura stramonium led to increased oviposition by Manduca sexta, a sphingid moth that pollinates flowers, but whose larvae feed on leaf tissue. We speculate that nectar composition may provide information about plant nutritional status or defense that floral visitors could use to make oviposition decisions. Thus, selection by floral visitors and leaf herbivores may be inextricably intertwined, and herbivores may represent a relatively unexplored agent of selection on nectar traits.

Alien Abduction: Disruption of Native Plant‐Pollinator Interactions by Invasive Species

ABSTRACTThe indirect impacts of Shorea siamensis‐logging on the reproductive ecology of Dipterocarpus obtusifolius, a self‐incompatible butterfly‐, moth‐, and bird‐pollinated tree, were studied in tropical dry forest in Thailand. Pollinator activity at D. obtusifolius trees and subsequent seed production were recorded in three forest areas subject to differing intensities of S. siamensis extraction. The pollinator and plant understory communities in these areas were also noted. Forest areas subject to high S. siamensis extraction intensities had very high understory flowering plant cover, dominated by the exotic invader Chromolaena odorata. Activity of butterfly pollinators at D. obtusifolius trees decreased in these disturbed areas, although their abundance remained comparable to other forest areas subject to only moderate or no extraction. For sphingid moth pollinators, there was no difference across differentially disturbed forest areas in either abundance or in the proportion bearing pollen. Pollinator activity by birds increased at highly disturbed locations but was not sufficient to offset a decline in overall pollinator activity at D. obtusifolius canopies in areas of heavy 5. siamensis extraction. Thus, extraction of S. siamensis indirectly affected the pollination of D. obtusifolius, primarily by causing changes in the foraging behavior of butterfly pollinators rather than their abundance. A shift in the relative abundance of floral nectar resources from the canopy to the understory, a consequence of 5. siamensis extraction and invasion by C. odorata, led to a parallel shift in foraging location of the principal diurnal pollinators, the butterflies, toward the understory. Despite reduced pollination at disturbed sites, behavioral changes did not translate into a D. obtusifolius seed set effect, possibly because pollination by birds (or moths) at the disturbed site compensated for reduced butterfly pollination.

Alien Abduction: Disruption of Native Plant-Pollinator Interactions by Invasive Species1

ABSTRACT The indirect impacts of Shorea siamensis-logging on the reproductive ecology of Dipterocarpus obtusifolius, a self- incompatible butterfly-, moth-, and bird-pollinated tree, were studied in tropical dry forest in Thailand. Pollinator activity at D. obtusifolius trees and subsequent seed production were recorded in three forest areas subject to differing intensities of S. siamensis extraction. The pollinator and plant understory communities in these areas were also noted. Forest areas subject to high S. siamensis extraction intensities had very high understory flowering plant cover, dominated by the exotic invader Chromolaena odorata. Activity of butterfly pollinators at D. obtusifolius trees decreased in these disturbed areas, although their abundance remained comparable to other forest areas subject to only moderate or no extraction. For sphingid moth pollinators, there was no difference across differentially disturbed forest areas in either abundance or in the proportion bearing pollen. Pollina...

Effects of dietary variation on growth, composition, and maturation of Manduca sexta (Sphingidae: Lepidoptera)

Most studies linking dietary variation with insect fitness focus on a single dietary component and late larval growth. We examined the effects of variation in multiple dietary factors over most life stages of the sphingid moth, Manduca sexta. Larvae received artificial diets in which protein, sucrose, and water content were varied. The relationship between larval size, growth and consumption rates differed significantly across diets. Larvae on control and low-sucrose diets grew most rapidly and attained the largest pupal and adult sizes. Conversely, larvae on low-water and low-protein diets initially grew slowly, but accelerated in the fifth instar and became pupae and adults comparable to control animals in size. There were no fundamental differences in protein:carbohydrate consumption patterns or strategies among experimental diets and larval instars. However, inadequate dietary water appeared to be more important for early than late instar larvae. Larvae on all artificial diets showed increasing fat content throughout all stages, including wandering and metamorphosis. Compensatory feeding among low-water and low-protein larvae was correlated with significantly higher fat content in larvae, pupae and adults, whereas low-sucrose animals were substantially leaner than those on the control diet. These differences may have strong effects on adult physiology, reproduction, and foraging patterns.

Trade‐offs in host use by Manduca sexta: plant characters vs natural enemies

There are two principal native host plants for the sphingid moth Manduca sexta L. in south eastern Arizona: Datura wrightii (Solanaceae), and the more recently reported, nonsolanaceous host, Proboscidea parviflora (Martyniaceae). A comparative study on causes and rates of mortality was conducted, since this provided a “natural experiment” for examining potential trade‐offs in host use. The field data identified a dramatic trade‐off between plant quality and predation. D. wrightii appears to provide a high quality food source in growth experiments, and eggs laid on its leaves hatch at high rates. The eggs and larvae, however, underwent extremely high levels of predation and parasitism in the field, and fewer than 0.5% reached the final larval stage. Eggs laid on P. parviflora, on the other hand, suffered low levels of predation and virtually no parasitism, but many eggs did not hatch due to developmental abnormalities induced by the plant, while a large proportion of larvae died after a few days of feeding. Overall, 3% of the eggs reached the final stage on this plant. The maintenance of the two host plants cannot be explained by single factors, but rather by the outcome of multiple factors on the insect fitness, including plant quality, suitability for egg development, predation risk, plant availability and competition with other herbivores. Our data emphasize the importance of predation in shaping host‐plant ranges and support the idea of host‐specific predation rates and “enemy‐free” space.

Molecular Cloning of Bombyx Cerebral Opsin (Boceropsin) and Cellular Localization of Its Expression in the Silkworm Brain

We have cloned a cDNA for a novel opsin from the larval brain of the silkworm Bombyx mori in which the photoperiodic photoreceptor had been supposed to reside in the cephalic central nervous system (CNS). Its deduced amino acid sequence was composed of 381 amino acids and included amino acid residues highly conserved in insect visual pigments. This opsin belonged to the long wavelength photoreceptor group of insect opsins and showed the greatest degree of homology (84%) with the green visual photoreceptor in the sphingid moth. We have designated this Bombyx cerebral opsin as Boceropsin. Southern blotting experiments indicated that the Boceropsin gene is present in a single copy, and RT-PCR analysis revealed that Boceropsin mRNA is expressed in the larval brain but not in the subesophageal ganglion (Sg) or thoracic ganglion (Tg). Immunohistochemical analyses demonstrated that Boceropsin protein is present bilaterally in some defined cells localized in the brain of Bombyx larvae. This is the first report of expression of an opsin-based protein in CNS of an insect. The possibility that the Boceropsin functions as the photoperiodic receptive pigment in the silkworm is also discussed.

Development of highly nutritive culture media.

A highly nutritive culture medium (MGM-464) was developed for insect cell primary culture. The new medium consists of 6 inorganic salts, 4 organic acids, 21 amino acids, 3 sugars, 10 vitamins, and 8 other chemicals, including natural substances. The complete medium was generated by adding 20 ml fetal bovine serum to 100 ml MGM-464. The detail of the composition of the medium is given in a table, and the protocol to prepare the medium is described in the text. Among the 15 kinds of cultures made with MGM-464, embryonic cells from a walking stick and ovarian cells from the common white were subcultured more than 70 times, and embryonic cells of a chrysomelid beetle were subcultured more than 15 times. Other cultures could not be subcultured. However, embryonic cells from the commercial silkworm and a cockroach, ovarial cells from the commercial silkworm and a sphingid moth, nervous cells from the commercial silkworm and two sphingid moths, and cells from the dorsal vessel plus surrounding tissue of the commercial silkworm survived for several mo. The cells from the honeybee embryos, aphid embryos, and planthopper embryos were rather short-lived, and deteriorated after about 1 mo.

The fragility of extreme specialization: Passiflora mixta and its pollinating hummingbird Ensifera ensifera

Some of the most spectacular examples of coevolution between flowers and their pollinators are reflected in their morphologies. Tongue length of insects and bill lengths of nectar-feeding birds are some of the most significant characters in pollination studies (Kearns & Inouye 1993). As Darwin noted as early as 1862, the evolution of deep floral tubes or spurs and long tongues of flower visitors can be explained by runaway coevolution (Nilsson 1988). However, such a process has only been shown to be likely in a few cases, e.g. between Malagasy orchids and hawkmoths (Nilsson et al. 1985). The pollinator of the Malagasy orchid Angraecum sesquipedale, with a 30 cm nectar spur, is a sphingid moth with a tongue of a similar size (Darwin 1862, Nilsson 1998). However, most studies of such extreme pollination specialization also report that the interaction is asymmetrical, i.e. the pollinators interact with a guild of plants, whereas the plant often depends on only a few pollinators (Johnson & Steiner 1995).

Regional specialization in the eye of the sphingid moth Manduca sexta: blue sensitivity of the ventral retina.

The compound eye of the tobacco hornworm moth Manduca sexta contains green-, blue-, and ultraviolet-sensitive photoreceptors. Electroretinogram spectral-sensitivity measurements were recorded from different regions of the retina in order to broadly map the distribution of the three receptor types. The relative contribution of the three receptors to spectral-sensitivity curves was estimated by fitting theoretical curves based on the absorption spectra of the three rhodopsins. This analysis indicated that the dorsal retina is green and ultraviolet dichromatic, with green-sensitive cells greatly predominating. The ventral retina is trichromatic with a substantial population of blue- and ultraviolet-sensitive receptors. We previously showed that flower visitation for nectar feeding is mediated mainly by blue-sensitive cells. Their localization in the ventral retina seems an appropriate adaptation of the receptor mosaic, since the moths hover above flowers as they feed.

Presence of cardenolides and ursolic acid from oleander leaves in larvae and frass of Daphnis nerii

Cardenolide glycosides from larvae of the sphingid moth Daphnis nerii reared on oleander leaves, and those from their frass, were examined by HPLC and then isolated preparatively. Most of the cardenolide triosides in the leaves were detected as their corresponding monosides in the larval and frass extracts. From the frass, adynerin was obtained in large amounts, along with nine cardenolide monosides. A decrease of oleandrin due to deacetylation and an increase of adynerin in the frass are discussed.

The advantages of an unsteady panel method in modelling the aerodynamic forces on rigid flapping wings

This paper responds to research into the aerodynamics of flapping wings and to the problem of the lack of an adequate method which accommodates large-scale trailing vortices. A comparative review is provided of prevailing aerodynamic methods, highlighting their respective limitations as well as strengths. The main advantages of an unsteady aerodynamic panel method are then introduced and illustrated by modelling the flapping wings of a tethered sphingid moth and comparing the results with those generated using a quasi-steady method. The improved correlations of the aerodynamic forces and the resultant graphics clearly demonstrate the advantages of the unsteady panel method (namely, its ability to detail the trailing wake and to include dynamic effects in a distributed manner).

Expression of opsin mRNA in normal and vitamin A deficient retinas of the sphingid moth Manduca sexta.

Two distinct opsin-encoding cDNAs, designated MANOP1 and MANOP2, were isolated as 3' fragments from the sphingid moth Manduca sexta. They were obtained by reverse transcription of retinal RNA and amplification with the polymerase chain reaction (PCR) using a degenerate primer designed to an amino-acid sequence conserved in arthropod opsins. The cDNA fragments labelled bands at approximately 1.8 kb on Northern blots of retinal RNA extracts. Levels of opsin message were compared in retinas from normal moths, whose diets were fortified with carotenoid precursors of the Manduca rhodopsin chromophore, 3-hydroxyretinal, and those reared on carotenoid/retinoid (vitamin A) deficient diets. The chromophore-depleted retinas contained more opsin mRNA;this was particularly true for MANOP2. Thus, the chromophore is not required for opsin gene transcription in Manduca.

Comparison of the Aerodynamic Forces on a Flying Sphingid Moth with Those Predicted by Quasi-Steady Theory

A camera and strain-gauge probe were used to record the wing beat and instantaneous vertical and horizontal forces on a sphingid moth flying in a wind of 3.36 m s⁻¹. The data were averaged over several wing beats to remove beat-to-beat variation. The moth was producing suffcient upward force to balance its weight of 14.7 mN and approximately sufficient thrust to balance its drag. The film of the wing beat was used to predict the wing inertial and virtual mass forces. The experimental aerodynamic force was obtained by subtracting the inertial force from the recorded force. The film was also used to predict the quasisteady aerodynamic forces due to wing translation and rotation. The vertical experimental aerodynamic and total predicted (quasi-steady plus virtual mass) forces have a similar shape, but the experimental downstroke peak (70 mN) was larger than predicted (47 mN). The horizontal recorded forces are smaller than the vertical forces, and wing inertia is a large component. However, a small aerodynamic thrust in the downstroke and a larger thrust of about 25 mN in the upstroke are both predicted and recorded. The experimental aerodynamic power output was between 21.6 and 30.0 W kg⁻¹ body mass or between 72 and 100 W kg⁻¹ of flight muscle.

Calystegines in Solanum and Datura species and the death's-head hawk-moth ( Acherontia atropus)

Polyhydroxylated tropane alkaloids, not hitherto recorded from the potato ( Solanum tuberosum), were detected in the leaves and tubers of this plant. They are potent inhibitors of glycosidases and may be responsible for neurological disorders in livestock. These alkaloids were also extracted from a sphingid moth and an ithomiine butterfly, the larvae of which feed on Solanum.