Interactions between hawkmoths and flowering plants in East Africa: polyphagy and evolutionary specialization in an ecological context

Hawkmoths (Lepidoptera, Sphingidae) are considered important pollinators in tropical regions, but the frequency and degree of reciprocal specialization of interactions between hawkmoths and flowers remain poorly understood. Detailed observations at two sites in Kenya over a two‐year period indicate that adult hawkmoths are routinely polyphagous and opportunistic, regardless of their proboscis length. About 700 individuals of 13 hawkmoth species were observed visiting a wide range of plant species at the study sites, including 25 taxa that appear to be specifically adapted for pollination by hawkmoths. We estimate that 277 plant species in Kenya (c. 4.61% of the total angiosperm flora) are adapted for pollination by hawkmoths. Floral tube lengths of these plants have a bimodal distribution, reflecting the existence of two hawkmoth guilds differing in tongue length. Hawkmoths exhibited strongly crepuscular foraging patterns with activity confined to a 20‐min period at dusk and, in some cases, a similar period just before dawn. Corolla tube length appears to act as a mechanical filter as the longest‐tubed plants were visited by the fewest hawkmoth species and these were exclusively from the long‐tongued guild. Tube length showed a strong positive relationship with nectar volume, even after phylogenetic correction, which implies that plants with long corolla tubes are under selection to offer relatively large amounts of nectar to entice visits by polyphagous long‐tongued hawkmoths. Our study shows that diffusely co‐evolved pollination systems involving long‐tongued hawkmoths are clearly asymmetrical, with plants exhibiting a high degree of floral specialization, while hawkmoths exhibit polyphagous behaviour. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 199–213.     

Identification of Volicitin-related Compounds from the Regurgitant of Lepidopteran Caterpillars

Volicitin-related compounds were found in the oral secretion of the three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, and one sphingid species, Agrius convolvuli. Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine], N-(17-hydroxy-linoleoyl)-glutamine, N-linolenoylglutamine and N-linoleoylglutamine were identified in the secretion from the noctuid larvae. In secretions from the sphingid larvae, N-linolenoylglutamine and N-linoleoylglutamine were the main components. Furthermore, there were significant differences in the amounts of the N-acylamino acid conjugates in the secretions from the three noctuid species. These results suggest that the proportion of volicitin-related compounds in the regurgitant was species-specific.     

Pollination in Lilium sargentiae (Liliaceae) and the first confirmation of long‐tongued hawkmoths as a pollinator niche in Asia

The geographical distribution of pollinators is an important factor determining the biogeographical pattern of floral evolution and diversification. In Africa, a guild of plant species has converged in a floral syndrome for pollination by long‐tongued hawkmoths (predominantly Agrius convolvuli). It is hypothesized that such floral convergence could track the geographical distribution of long‐tongued hawkmoths, so it may not be confined to Africa. We investigated the pollination biology of Lilium sargentiae E. H. Wilson, which is endemic to China and exhibits traits suggestive of long‐tongued hawkmoth pollination. Lilium sargentiae was visited by A. convolvuli as well as small beetles (Nitidulidae) but pollination was mostly or totally effected by the moth. It was consistent with other hawkmoth‐pollinated plants in terms of floral tube length, nectar traits, tepal reflectance, and scent composition. We present the first experimental evidence for the hypothesis proposed above and for hawkmoth pollination in the widespread and ornamentally and economically important genus Lilium L. in a natural habitat. Our findings imply that long‐tongued hawkmoths (especially the extremely widespread A. convolvuli) as a distinctive pollinator niche may have underlain the evolution of long‐tubed (>8 cm) flowers across different continents.     

Contribution of the maxillary muscles to proboscis movement in hawkmoths (Lepidoptera: Sphingidae)--an electrophysiological study

The role of the maxillary muscles in the uncoiling and coiling movements of hawkmoths (Sphingidae) has been examined by electromyogram recordings, combined with video analysis. The maxillary muscles of adult Lepidoptera can be divided into two groups, galeal and stipital muscles. The galea contains two basal muscles and two series of oblique longitudinal muscles, which run through the entire length of the galea. Three muscles insert on the stipes, taking their origin on the tentorium and on parts of the cranium and gena, respectively. Proboscis extension is initiated by an elevation of the galea base caused by the basal galeal muscles. The actual uncoiling of the proboscis spiral is accompanied by rapid compressions of the stipites which are caused by two of the stipital muscles. The study provides strong support for the hypothesis that uncoiling is brought about by an increase of hemolymph pressure by the stipites forcing hemolymph into the galeae. Recoiling is caused by the contraction of both sets of oblique longitudinal galeal muscles supported by elasticity of the galea cuticle. Finally, the remaining stipital muscle pulls down the galea base which brings the coiled proboscis back to its resting position where it is held in the U-shaped groove of the labium without further muscle activity.     

Elsinochrome A production by the bindweed biocontrol fungus Stagonospora convolvuli LA39 does not pose a risk to the environment or the consumer of treated crops

Biological control as an alternative to chemical pesticides is of increasing public interest. However, to ensure safe use of biocontrol methods, strategies to assess the possible risks need to be developed. The production of toxic metabolites is an aspect which has so far largely been neglected in the risk assessment and the registration process for biocontrol products. We have evaluated the risks of elsinochrome A (ELA) and leptosphaerodione production by the fungus Stagonospora convolvuli LA39, an effective biocontrol agent used against bindweeds. The toxicity of the two metabolites to bacteria, protozoa, fungi and plants was evaluated in in vitro assays. The most sensitive bacteria and fungi were already affected at 0.01-0.07 microM ELA, whereas plants were far less sensitive. Leptosphaerodione was less toxic than ELA. Subsequently, it was investigated whether ELA is present in the applied biocontrol product or LA39-treated bindweed and crop plants. In plants ELA was never detected and in the biocontrol product the ELA concentration was far too low to have toxic effects even on the most sensitive organisms. We conclude that the production of ELA by biocontrol strain LA39 does not pose a risk to the environment or to the consumer.     

Cecropin D‐like antibacterial peptides from the sphingid moth,Agrius convolvuli

Two major antibacterial peptides were isolated and purified from immunized larval hemolymph of Agrius convolvuli. Acid extraction, gel filtration, ultrafiltration, and reversed‐phase FPLC were used for purification of peptides. These peptides had similar molecular mass and amino acid composition. Moreover, 21 of the first 23 N terminal residues were identical. The peptides were highly homologous with cecropin D in size and primary sequence, and named Agrius cecropin D1 and D2. The molecular masses of Agrius cecropin D1 and D2 were 3,879.39 and 3,839.27, respectively. In antibacterial and hemolytic assays, Agrius cecropin D showed potent antibacterial activities against a panel of Gram positive and negative bacteria without hemolytic activity against human red blood cells. Notably, our antibacterial assay revealed Agrius cecropin D possessed stronger or at least equivalent activities against B. megaterium than cecropin A. It suggests that Agrius cecropin D, which has an alternative structure from cecropin D, could be the model for the development of peptide antibiotics. Arch. Insect Biochem. Physiol. 41:178–185, 1999. © 1999 Wiley‐Liss, Inc.     

Biological Control of Hedge Bindweed (Calystegia sepium) with Stagonospora convolvuli Strain LA39 in Combination with Competition from Red Clover (Trifolium pratense)

In a maize cropping system where a living green cover suppresses many weeds, Calystegia sepium is able to escape control. In this paper we report the potential for biological control of C. sepium by using the bindweed pathogen Stagonospora convolvuli strain LA39 as a mycoherbicide in combination with competition by the green cover plant Trifolium pratense. In a greenhouse experiment, competition from shoots of T. pratense caused a strong reduction of the biomass of C. sepium, and combined competition from shoots and roots had the same effect. In a second, factorial greenhouse experiment, competition by T. pratense again reduced C. sepium biomass. However, S. convolvuli did not influence the number of leaves or the biomass of C. sepium in the greenhouse even though severe necrosis was observed on inoculated bindweed leaves. In contrast, in a 2-year field study, S. convolvuli caused severe disease and a strong reduction of C. sepium ground coverage in maize. Underseeding with T. pratense had no effect on disease severity, but T. pratense reduced ground coverage by C. sepium at one of eight samplings in the first year. In conclusion, S. convolvuli is useful in the field and, as shown in the greenhouse, a competitive green cover might improve biological control of C. sepium.     

Isolation of Lysozyme from the Hemolymph of Sweet Potato Hornworm, Agrius convolvuli Larvae

ABSTRACT Lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. A new family member of insect lysozyme, an antibacterial peptide, has been isolated from the hemolymph of the fifth instar larvae of Agrius convolvuli . Vaccination was proceeded with E. coli K12 D21 (4x10⁶ cells of log phase) injection into the abdomen of the larvae. Agrius lysozyme was isolated by cation-exchange chromatography and RP-FPLC, and sequenced by HPLC system. It was observed that the purified Agrius lysozyme was heat-stable and had a molecular weight of approximately 15 kDa by SDS-PAGE. The N-terminal amino acid sequence of Agrius lysozyme is K-H-F-S-R-C-G-L-V-Q-E-L-F-W-Q-G-F-P with the highest similarity to that of Heliothis virescence , and it has been identified that the Agrius lysozyme belongs to c type lysozyme.     

ISSR fingerprinting for the assessment of the bindweed biocontrol agent Stagonospora convolvuli LA39 after field release

AIMS: To develop a molecular identification method based on ISSR fingerprints to monitor the fungal leaf pathogen Stagonospora convolvuli LA39 used to biologically control bindweeds after a field release. METHODS AND RESULTS: The developed method proved to be suitable to clearly distinguish LA39 from resident Stagonospora spp. and was applied in two field experiments. First, the environmental persistence of LA39 was assessed in an overwintering experiment. LA39 could be re-isolated from infected bindweed 1 year after field application, but with very low frequency of occurrence. Secondly, LA39 was applied in an area with natural bindweed infestation and re-isolated from infected bindweed. The dispersal of LA39 during one season was poor (4-5 m). CONCLUSIONS: ISSR fingerprinting has been shown to be a valuable tool to monitor the environmental fate of S. convolvuli in the field. It is concluded that an LA39-based mycoherbicide will have minimal environmental impact caused by the restricted mobility, poor proliferation and poor persistence over seasons of LA39. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies about the dispersal and survival of biocontrol agents after field release as well as the development of methods needed for this purpose are indispensable for a comprehensive risk assessment for biocontrol agents.     

Neuronal Cell Death of Abdominal Ganglia (A3) and Characterization of Neuron-Killing Factor in Ventral Nerve Cord from Sweet Potato Hornworm, Agrius convolvuli

ABSTRACT The central nervous system of Agrius convolvuli is composed of brain, followed by subesophageal ganglion, three thoracic ganglia, and eight abdominal ganglia in late larval stage. After metamorphic transition from larva to pupa, thoracic (T₁ and T₂) and abdominal ganglia (A₁ and A₂) are moved toward T₃ and fused together to construct composite ganglion, pterothoracic ganglion. The formation of composite ganglion is completed about 90% at 4 day and 100% at 7 day after pupation. Neuronal cell death was occurred significantly around 3 or 4 day after pupation and just after adult ecdysis. Although 170 neurons were detected 3 day before adult ecdysis, 24 cells were counted 5 day after adult ecdysis. Data of scanning and tandem electron microscope showed the symptom of cell death. In order to identify the mechanism of cell death in A. convolvuli , 1,200 ventral nerve cords were homogenized. Extracts were boiled for 3 minutes at 100°C and collected below 30,000 dalton of molecular mass. Each fraction from reverse phase column chromatography by HPLC system was tested in ventral nerve cord culture system, and fractions having killing activity in culture were isolated. By the addition of 20 hydroxyecdysone, actinomycin D, or cycloheximide into the culture medium, cell death was delayed significantly when compared to control group.