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Zoological Institute, Department of Animal Ecology, Am Botanischen Garten 1–9, Christian-Albrechts-University of Kiel, D-24098 Kiel, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Larvae of saprophagous insects often have been suspected of being competitors of filamentous fungi on decaying organic matter, which negatively influence mold development. Of interest, the role of insects in determining fungal growth and the onset of sporulation largely has been ignored. I used Aspergillus niger and the vinegar fly Drosophila melanogaster as an ecological model system to analyze the influence of insect larvae on daily fungal growth and the start of conidiospore production. I used an artificial substrate to test whether the effect of larval density (one, five and 10 larvae) and inoculation date of the mold (2 and 3 d ahead of the addition of larvae) significantly altered fungal growth. Fungal growth (area covered by hyphal tissue of the artificial patch) was affected negatively by the number of larvae and by the time that elapsed between inoculation with fungal spores and transfer of larvae to the patches. Whereas one larva had only a minor effect on fungal growth, five or 10 larvae strongly hampered mold development. As time between inoculation with spores and introduction of fly larvae increased, mold increased, indicating a priority effect for the fungus. When 10 larvae were transferred at the same time as the patches were inoculated with spores, almost no mold was visible within the period of observation (after 12 d). In comparison with control treatment (no insect larvae), an increase in larval density caused an increasing delay of several days in the start of spore production. Thus only minor changes in the density of insect larvae and the time that larvae entered the patches after inoculation with spores had an enormous effect on fungal growth and spore production. Therefore insects co-occurring with mold on ephemeral resources might constitute an important biotic factor driving local fungal population dynamics. The mechanisms leading to the suppression of fungal growth and the evolutionary implications of insect-mold interactions are discussed.
Key words: Aspergillus, competition, ephemeral resources, insect-fungus interactions, life history evolution
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Marin et al 1995, Barnett and Hunter 1998, Marin et al 1998, Klein and Paschke 2004) and in turn may affect the onset of sporulation (Lin et al 1980) and the production of mycotoxins (e.g., Häggblom 1982, Hicks et al 1997, Calvo et al 2002). In the light of reproductive success relative to competing conspecifics or heterospecifics fungal growth and the start of spore production are important ecological traits determining the ability to disperse and to exploit new resource patches (Gilchrist et al in revision) (e.g. decaying plant materials). In addition to various abiotic factors influencing the growth of fungal populations, interactions with insects that exploit the same resource patches might decisively change the successful establishment of fungal colonies and the onset of sporulation. Hodge (1996) found a strong negative association between the presence of insect larvae and complete fungal cover on bananas and strawberries in field experiments. Moreover an increasing density of insect larvae seems to have remarkable negative effects on mold growth (Hodge and Arthur 1997, Wertheim et al 2002). Because food infections with mold have been shown to be harmful to saprophagous insect larvae (Atkinson 1981, Hodge et al 1999), especially at low larval densities (Rohlfs et al 2005), spatial aggregation of insect eggs/larvae across decaying fruits has been suggested to be an adaptation against fungal competitors (Wertheim et al 2002, Rohlfs and Hoffmeister 2003, Wertheim 2005). Given these observations, insects at varying densities might be an important biotic factor controlling mold growth and thereby driving local fungal population dynamics. Although evidence continues to be uncovered that insect-mold interactions occur on ephemeral resources, little is known about the type of interactions (competitive or trophic) or their role in the selection for specific adaptations, in both fungi and insects that reduce the negative effects of the corresponding antagonists (see also Dowd 1992). The aim of the study presented here was to analyze the effects of insect larvae on mold growth and sporulation. I used the vinegar fly Drosophila melanogaster and the fungus Aspergillus niger as a model system to study insect-mold interactions. Both organisms can be found on decaying plant materials on which the fungus frequently may interact with fly larvae. Because the time that has elapsed between substrate infection with mold and the arrival of ovipositing insect females might critically influence fungal growth, I tested whether the mold benefits from priority effects and becomes competitively dominant over the insect larvae. For this reason I also manipulated the time between substrate infection with spores and colonization by fly larvae.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experimental flies originated from a D. melanogaster strain collected in Sep 2003 near Kiel, Germany (ca 54°N, 10°E). Flies had been reared for 18 generations under constant laboratory conditions (22 C ± 0.5, 16 h photo period), on an artificial medium (30 g cornmeal, 30 g sugar, 30 g brewer’s yeast extract (Leiber, Germany) and Nipagin). To obtain experimental D. melanogaster larvae, a large population of flies (>1000) were allowed to lay eggs on Petri dishes containing a hard agar medium (22 g agar, 90 mL sugar beet syrup and 9.5 mL Nipagin per 500 mL water). Petri dishes were offered to the flies in a population cage (ca. 50 L), at ambient room temperature of 19–25 C. After ca. 16 h the agar dishes were removed from the cages and stored ca. 24 h under the same environmental conditions as for the experiments (see below). After this period almost 90% of the eggs had hatched and larvae were washed off the agar plate onto Mueller gauze with water, which I then used in the experiments. I transferred fly larvae to mold-infected patches with a fine brush.
Incubation.— – To test the effect of insect larvae on fungal growth, either one, five, or 10 first-instar larvae were transferred immediately after patches had been infected with A. niger spores (priority 0). Another set of first-instar larvae were transferred at 2 (priority 2) and 3 (priority 3) d after patches had been infected with spores. As a control one set of mold-infected patches was not treated with fly larvae. All experiments were carried out with 7–13 replicates per treatment. All replicates were set up on 1 d. During the experiment vials were stored in a cooled incubator (Rumed®, Series 3000) with a 16 h photoperiod and the this daily temperature profile: 18 C, 8 p.m.–10 a.m.; 20 C, 10 a.m.–12 a.m.; 22 C, 12 a.m.–6 p.m.; 20 C, 6 p.m.–8 p.m. Temperature was increased/decreased at 0.5 C/min.
Measurements.— – After fungal growth clearly was visible on the control patches (at day 3 after inoculation), I daily recorded mold development by taking photographs of each patch under identical light conditions with a camcorder (Sony® DCR-TRV345E) and the software package Pinnacle® Studio 8. Each picture was stored as an individual JPEG file. These pictures were used to record daily fungal growth. With SigmaScan® Pro5 I measured the diameter of each patch and the area covered by fungal hyphae. This let us calculate the proportion of the artificial substrate occupied by the fungus as a function of the treatment with fly larvae. All parameters were recorded until the 12th d after patches were inoculated with fungal spores. At that time the fly larvae started leaving the patch to pupate.
Statistical analysis.— –
I used repeated measures ANOVA in the GLM procedure of the SAS package (version 9.0). The proportion of a patch that was covered with fungal tissue was arcsine-square-root transformed. I tested the effect of priority, number of fly larvae, day, and interaction terms. Results of the type III test of the explanatory variables are shown. To compare the relative importance of the different factors in the model, I calculated "effect sizes" (
2 = factor sum of squares/[factor sum of squares + error sum of squares]) (Weiner et al 1997).
To test for the influence of the presence of insect larvae on the start of sporulation (the day on which the first mature conidiospores were visible), the mean time until the start of spore production and the proportion of fungi carrying spores 12 d after inoculation were recorded. I analyzed these data with the GENMOD procedure of SAS. I specified a normal distribution with an identity link function for the number of days until the first conidial heads were visible and a binomial distribution and a logit link function for the proportion of fungal patches carrying mature spores at the end of the experiment. Results of the type III analysis are shown. When the control treatment was tested repeatedly against treatment (e.g. control vs. number of larvae within one block of fungal priority), I used the Bonferroni correction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The presence of insect larvae had a strong negative effect on fungal growth; however, the strength of this influence depended on the number of larvae and on the priority of A. niger (FIG. 1, TABLE I). Despite the negative effect of even one larva on daily fungal growth, the mold was able to occupy 100% of the substrate area after 12 d (FIG. 1). In contrast five or 10 fly larvae more strongly hampered the growth of A. niger within priorities 0 and 2 (FIG. 1). When 10 larvae were transferred at the same time as the patches were inoculated with spores, no hyphal tissue could be observed during the course of the experiment (FIG. 1). Only when the mold was given a head start of 3 d over 10 fly larvae was it able to occupy the artificial patches completely at the end of the experiment (FIG. 1). Although DAY had the strongest effect on fungal growth, effect sizes for the number of Drosophila larvae, the developmental head start of mold over larvae, as well as their statistical interactions, indicate a strong impact on the expansion of mold colonies (TABLE I).
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–level = 0.0167) and within priority 2 (F3,32 = 15.38, P < 0.0001; Bonferroni corrected –level = 0.0125) but not within priority 3 (F3,35 = 0.30, P = 0.8222; Bonferroni corrected –level = 0.0125). Whereas the mean time until the start of spore production increased with increasing larval density within priorities 0 and 2, it remained unaffected within priority 3 (FIG. 2). Twelve days after inoculation with A. niger spores, insect larval density had a strongly negative effect on the proportion of fungal patches carrying mature conidiospores within priority 0 (FIG. 2, F3,35 = 63.94, P < 0.0001; Bonferroni corrected –level = 0.0125). In contrast, larval density had only a slight influence on this parameter within priority 2 (F3,35 = 3.28, P = 0.0322; Bonferroni corrected –level = 0.0125) and almost no effect within priority 3 (F3,35 < 0.001, P >0.999; Bonferroni corrected –level = 0.0125) (FIG. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This negative impact on mold growth causes a strong delay in the start of spore production (FIG. 2). Because mold may compete both intra- and interspecifically for high quality resource patches with other fungi, including yeast (e.g. Gloer and Truckenbrod 1988, Filonow et al 1996, Ramakrishna et al 1996), bacteria and insects, any delay in the development of conidiospores may decisively diminish fungal reproductive success and hence the colonization of other suitable sites. Therefore, based on the results of this experimental study, insects, especially at high densities, may have the potential to control mold growth and thus may play an important role in local fungal population dynamics.
Mechanisms in insect-mold interactions on ephemeral resources.— –
Drosophilids are representative of a group of insects that feed on living yeast under natural conditions (Begon 1982), whereas feeding on mold has not yet been observed systematically (see Schatzmann 1977 for an incidental observation). Larvae feeding on patches with spore-producing fungi have a blackish gut content, which indicates the intake of spores (personal observation, Hodge and Arthur 1997). Although insect larvae might benefit from feeding on fungal tissue as a source of highly concentrated proteins (Dowd 1992), A. niger is clearly not a food source for D. melanogaster larvae because strong fungal growth can cause high mortality in the fly larvae (Rohlfs et al 2005), with no indication of any benefit to the larvae. Given the results of the experiments in this study, larvae control fungal growth only when the mold is at an early stage and/or the colonies are small (FIG. 1). Larval feeding (shoveling food with the mouth hooks) and locomotor behavior (crawling and digging) most likely destroys fungal hyphae (Hodge and Arthur 1997, Wertheim et al 2002). On the other hand larvae might devour the fungal tissue. With increasing age and/or size, the mold tissue might become more robust against larval feeding behavior and thereby are competitively superior to the insect larvae (Rohlfs et al 2005). As a consequence the larvae are isolated from the feeding substrate (personal observations). Simultaneously fungal secondary metabolites (e.g. mycotoxins) are likely to diffuse into the fly larval feeding substrate, which additionally might hamper larval development (see also Dowd 1992).
Adaptation and counter-adaptation.— –
Insect-mold interactions are assumed to have occurred over 400 000 000 y and in some cases are similar to the ecology of insects feeding on plants (insect-plant interactions), in which insects have adapted to feed and breed on plants, and plants have evolved chemical and physical counter-strategies to mitigate the negative impact of insect feeding (Strong et al 1984). Likewise various insects have adapted morphologically and physiologically to the use of fungi as a food source and breeding site (Hanski 1989, Lawrence 1989). However, given the results of the present study and the harmful effects of mold growth on the development of drosophilids (Atkinson 1981, Rohlfs et al 2005, Wertheim et al 2002), insect-mold interactions on ephemeral resources seem to be characterized by competition rather than by a trophic relationship. Although the true nature of how insect can suppress mold growth remains to be determined, I formulated specific hypotheses concerning the evolutionary inter-relationships between mold and insects on ephemeral resources. First, spatial egg aggregation and early arrival at the breeding site give the insects an advantage over mold if larval offspring are able to reduce substantially the expansion of competing fungi, as it has been shown in the present study. In this connection interactions with molds are one possible reason why drosophilids display aggregated egg-laying behavior and larvae achieve their highest survival probabilities at intermediate densities on decaying fruit (Rohlfs and Hoffmeister 2003). Similarly larvae would enhance their competitiveness over noxious molds if they display an aggregative behavior actively to suppress mold development within a patch on sites in which fungi start growing (Rohlfs 2005). Second, because fungi may suffer seriously from the presence of insect larvae (see Hodge [1996] for observations in the field), the production of secondary metabolites may have evolved as an adaptive response to competing insect larvae. Several well known mycotoxins have been demonstrated to have insecticidal properties (e.g. Cole and Rolinson 1972, Reiss 1975, Castillo et al 1999, Zhang et al 2003). Of interest, the role of mycotoxins in an ecological context is less well understood (Dowd 1992). In addition to chemical defense, the speed with which fungal colonies can cover decaying matter may be advantageous when they not only have to compete against other micro-organisms but also have to interact with insects. Competition with insects may further influence fungi to allocate limited resources to either growth or spore production (see Gilchrist et al in revision for a first theoretical approach to defining fungal fitness on food-limited patchy resources).
Both fungi and insects are essential for decomposition of organic matter, nutrient cycling and nutrient transport. Despite this vital role in ecosystem function, little is known about the biological mechanisms of insect-mold interactions and the way in which they drive decomposition rates and population dynamics. By using systems in which these interactions are confined to discrete resource patches (e.g. decaying plant tissues) I might be able to disentangle the ecological interactions between insects and molds, rather than a system in soils, which is spatially more difficult to manipulate. In conclusion further integration of the evolutionary ecology of insect life-history traits and the ecology of filamentous fungi might constitute an important field of research that might lead to fruitful cross-disciplinary discussions for both mycologists and entomologists.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 E-mail: rohlfs{at}zoologie.uni-kiel.de
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LITERATURE CITED |
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Ayerst G. 1969. The effects of moisture and temperature on growth and spore germination in some fungi. J Stored Products Res 5:127–141.[CrossRef]
Barnett HL, Hunter BB. 1998. Illustrated genera of imperfect fungi. 4th ed. St Paul, Minnesota: APS Press.
Begon M. 1982. Yeast and Drosophila. In: Ashburner M, Carson HL, Thompson Jr. JN, eds. The genetics and biology of Drosophila. London: Academic Press. p 345–384.
Calvo AM, Wilson RA, Bok JW, Keller NP. 2002. Relationship between secondary metabolism and fungal development. Microbiol Mol Biol Rev 66:447–459.
Castillo M-A, Moya P, Cantin A. Miranda MA, Primo J, Hernández E, Primo-Yúfera E. 1999. Insecticidal, anti-juvenile hormone, and fungicidal activities of organic extracts from different Penicillium species and their isolated active components. J Agric Food Chem 47:2120–2124.[CrossRef][Medline]
Cole M, Rolinson GN. 1972. Microbial metabolites with insecticidal properties. Appl Microbiol 24:660–662.[Medline]
Dowd PF. 1992. Insect interactions with mycotoxin-producing fungi and their hosts. In: Deepak B, Lillehoy EB, Arora DK, eds. Mycotoxins in ecological systems. New York: Marcel Dekker Inc. p 137–155.
Filonow AB, Vishniac HS, Anderson JA, Janisiewicz WJ. 1996. Biological control of Botrytis cinerea in apple by yeasts from various habitats and their putative mechanisms of antagonism. Biological Control 7:12–220.
Gilchrist MA, Sulsky DL, Pringle A. In revision. Fitness and optimal life-history strategies in filamentous fungi. Evolution.
Gloer JB, Truckenbrod SM. 1988. Interference competition among coprophilous fungi: production of (+)-isoepoxydon by Poronia punctata. Appl Environ Microbiol 54:861–864.
Häggblom P. 1982. Production of ochratoxin A in barley by Aspergillus ochraceus and Penicillium viridicatum: effect of fungal growth, time, temperature, and inoculum size. Appl Environ Microbiol 43:1205–1207.
Hanski I. 1989. Fungivory: fungi, insects and ecology. In: Wilding N, Collins NM, Hammond PM, Webber JF, eds. Insect-fungus interactions. London: Academic Press. p 25–61.
Hicks JK, Yu J-H, Keller NP, Adams TH. 1997. Aspergillus sporulation and mycotoxin production both require inactivation of the FadA Ga protein-dependent signaling pathway. EMBO Journal 16:4916–4923.[CrossRef][Medline]
Hodge S. 1996. The relationship between Drosophila occurrence and mould abundance on rotting fruit. Brit J Entomol Nat History 9:87–91.
Hodge S, Arthur W. 1997. Direct and indirect effects of Drosophila larvae on the growth of moulds. Entomologist 116:198–204.
Hodge S, Mitchell P, Arthur W. 1999. Factors affecting the occurrence of facilitative effects in interspecific interactions: an experiment using two species of Drosophila and Aspergillus niger. Oikos 87:166–174.[CrossRef]
Klein DA, Paschke MW. 2004. Filamentous fungi: the indeterminate lifestyle and microbial ecology. Microb Ecol 47:224–235.[Medline]
Lawrence JF. 1989. Mycophagy in the Coleoptera: feeding strategies and morphological adapations. In: Wilding N, Collins NM, Hammond PM, Webber JF, eds. Insect-fungus interactions. London: Academic Press. p 2–20.
Lin YC, Ayres JC, Koehler PE. 1980. Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus. Appl Environ Microbiol 40:333–336.
Marín S, Sanchis V, Saenz R, Ramos AJ, Vinas I, Magan N. 1998. Ecological determinants for germination and growth of some Aspergillus and Penicillium spp. from maize grain. J Appl Microbiol 84:25–6.[CrossRef][Medline]
Marín S, Sanchis V, Magan N. 1995. Water activity, temperature and pH effects on growth of Fusarium moniliforme and Fusarium proliferatum isolates from maize. Can J Microbiol 41:1063–1070.[Medline]
Ramakrishna N, Lacey J, Smith JE. 1996. Colonization of barley grain by Penicillium verrucosum and ochratoxin A formation in the presence of competing fungi. J Food Protect 59:1311–1317.
Reiss J. 1975. Insecticidal and larvicidal activities of the mycotoxins aflatoxin B1, rubratoxin B, patulin and diacetoxyscirpenol towards Drosophila melanogaster. Chem-Biol Interact 10:339–342.[CrossRef][Medline]
Rohlfs M. 2005. Clash of kingdoms or why Drosophila larvae positively respond to fungal competitors. Front Zool 2:2.[CrossRef][Medline]
Rohlfs M, Hoffmeister TS. 2003. An evolutionary explanation of the aggregation model of species coexistence. Proc R Soc London B (Suppl.) 270:S33–S35.
Rohlfs M, Obmann B, Petersen R. 2005. Competition with filamentous fungi and its implication for a gregarious lifestyle in insects living on ephemeral resources. Ecol Entomol 30:556–563.[CrossRef]
SAS Institute. 1999. SAS OnlineDoc, version 8. Cary, North Carolina: SAS Institute.
Schatzmann E. 1977. Früchte als natürliche Entwicklungs-substrate von Drosophiliden. Mitt Schweiz Entomol Ges 50:135–148.
Strong DR, Lawton JH, Southwood R. 1984. Insects on plants—Community patterns and mechanisms. Cambridge, Massachusetts: Harvard University Press.
Weiner J, Martinez S, Müller-Schärer H, Stoll P, Schmid B. 1997. How important are environmental maternal effects in plants? A study with Centaurea maculosa. J Ecol 85:133–142.[CrossRef]
Wertheim B. 2005. Evolutionary ecology of communication signals that induce aggregative behaviour. Oikos 109:117–124.[CrossRef]
Wertheim B, Marchais J, Vet LEM, Dicke M. 2002. Allee effect in larval resource exploitation in Drosophila: an interaction between density of adults, larvae and micro-organisms. Ecol Entomol 27:608–617.[CrossRef]
Zhang Y, Li C, Swenson DC, Gloer JB, Wicklow DT, Dowd PF. 2003. Novel antiisectan oxalicine alkaloids from two undescribed fungicolous Penicillium spp. Org Lett 5:773–776.[CrossRef][Medline]
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