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Title:Facultative mutualism between the navel orangeworm Amyelois transitella (Lepidoptera: Pyralidae) and Aspergillus flavus
Author(s):Bush, Daniel S
Advisor(s):Berenbaum, May R
Contributor(s):Hanks, Lawrence M; Miller, Andrew N
Department / Program:Entomology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):navel orangeworm
Aspergillus flavus
tree nuts
Abstract:The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is an economic pest of considerable importance by virtue of its habit of attacking damaged or overripe tree nuts and fruits in Californian orchards. Its economic impact is increased by its common association with the highly toxigenic fungus Aspergillus flavus Link. Despite the capacity of this fungus-insect association to damage a wide variety of tree crops, relatively little is known about the ecology of the interaction. I examined three aspects of this association to examine the possibility that the interaction represents a facultative mutualism. These studies are here presented in three chapters. First, to determine whether associating with the fungus allows the navel orangeworm to utilize its hostplants more efficiently, I conducted a series of laboratory bioassays to test if the presence of A. flavus decreases the toxicity of hostplant phytochemicals. Growth rates, mortality rates, and pupal weight for navel orangeworms were measured on an artificial potato dextrose agar (PDA) diet containing almond meal, in the presence and absence of the furanocoumarins xanthotoxin and bergapten, which occur in some fruit hostplant species. In the absence of furanocoumarins, larvae reached adulthood in 29.6 d on average in the presence of the fungus as opposed to 43.8 d in the absence of A. flavus (t = 16.06; df = 3; P < 0.001). Female and male pupae had on average five or six g more mass, respectively, when the larvae were fed with a diet containing fungus (t = 5.56; df = 3; P = 0.011 and t = 7.16; df = 3; P = 0.006, respectively). By contrast, the presence of furanocoumarins at natural concentrations in the diet (at concentrations determined by prior LC50 experiments) in the absence of the fungus extended the development time of the navel orangeworm by 14 to 22 d on average for bergapten and xanthotoxin, respectively (F = 1013.73; df = 3; P < 0.001). Pupal weights decreased as much as two-thirds in the presence of furanocoumarins (F = 328.09; df = 5; P < 0.001 for males, F = 83.00; df = 5; P < 0.001 for females), and mortality increased two-fold (F = 9.61; df = 5; P < 0.001). When navel orangeworms were raised on furanocoumarin-containing diets in the presence of the fungus, however, very little growth rate reduction was observed, and mortality returned to the levels observed on diets lacking furanocoumarins, although pupal weights remained depressed. These findings indicate that the presence of the fungus benefits navel orangeworm larvae by enhancing growth rate and survival, both directly and indirectly (by reducing the toxicity of host phytochemicals). Independent of its association with the navel orangeworm, A. flavus is an important plant pathogen that contributes to millions of dollars of crop loss annually. The adult navel orangeworm moth is a known vector of Aspergillus species. The competition between caterpillars and fungus for resources would seem to be a drawback of this partnership, as both organisms use the same plant materials for nutrition, but previous work has shown that A. flavus thrives in the presence of the navel orangeworm. In a second study, I assessed the growth of A. flavus in the presence of navel orangeworm larvae and in or on their frass by rearing larvae on an artificial diet in the presence and absence of the fungus. I found that the fungus grew ~2-fold faster on almond PDA in the presence of navel orangeworm larvae (t = 52.14; df = 19; P < 0.001). Additionally, I collected frass from larvae fed on standard lepidopteran diet and almond PDA and incorporated it into agarose diets to see if these diets could support fungal growth. On both frass diets, A. flavus grew rapidly, at rates intermediate between PDA and almond PDA. Therefore, A. flavus appears to use navel orangeworm larvae as both vectors and sources of nitrogen-rich substrate in the form of frass. I then collected frass from larvae that had fed on fungus-containing diets and examined it (at 800-2000X magnification) for the presence of fungal conidia, hyphae, or other intact fungal elements. In frass from both diets, I observed intact conidial heads, ornamented conidia, and septate hyphae. Moreover, conidia were isolated from this frass and transferred to PDA to assess viability. In some cases, the recovered conidia produced a viable colony, indicating that caterpillar excrement may be an alternative vehicle for fungal dispersal. If the interaction between the navel orangeworm and Aspergillus fungi represents a mutualistic relationship, it would be highly advantageous for the insect to be able to identify and seek out fungal colonies as well as spread them. I conducted a series of behavioral assays to assess whether larval and adult navel orangeworms can preferentially orient to the fungus. In an oviposition assay, I presented gravid female moths with a choice between Aspergillus-infected and uninoculated oviposition sites. Mating pairs were released in arenas with two PDA dishes, one inoculated with A. flavus, and one sterilized. On average, females laid 44.3 eggs on inoculated dishes and 12.7 on the uninoculated sites, with no oviposition elsewhere in the arena (t = 4.30; df = 2; P = 0.026). In addition, 62% of eggs on the sites with fungus present were fertilized, compared to only 26% on uninoculated sites (t = 4.30; df = 2; P =0.048). The differential rates of fertilization were unexpected. One possible explanation is that females preferentially lay greater numbers of unfertilized eggs on nutritionally deficient food sources to provide supplemental nutrients to hatching larvae of this cannibalistic species. To test this hypothesis, I conducted an assay comparing larval survivorship in the presence and absence of supplemental unfertilized eggs. I found that neonates provisioned with eggs survived 208.8 h on average, while starved neonates survived only 85.2 h (t = 2.78; df = 4; P = 0.002). Thus, navel orangeworms may be taking advantage of cannibalism to compensate for ovipositing on less nutritious hosts. These results are consistent with the hypothesis that the interaction between A. flavus and the navel orangeworm is a facultative mutualism.
Issue Date:2015-12-10
Rights Information:Copyright 2015 Daniel Bush
Date Available in IDEALS:2016-03-02
Date Deposited:2015-12

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