The influence of natural enemies on wing induction in Aphis fabae and Megoura viciae (Hemiptera: Aphididae)

Previous studies have shown that the aphid species, Aphis fabae Scopoli and Megoura viciae Buckton, do not produce winged offspring in the presence of natural enemies, in contrast to results for the pea aphid (Acyrthosiphon pisum (Harris)) and the cotton aphid (Aphis gossypii Glover); but these studies did not involve exposing aphids directly to natural enemies. We exposed colonies of both A. fabae and M. viciae to foraging lacewing (Chrysoperla carnea (Stephens)) larvae and found that the predators did not induce winged morphs among offspring compared to unexposed controls. Colonies of A. fabae responded to an increase in aphid density with increasing winged morph production, while such response was not found for M. viciae. We suggest that different aphid species differ in their susceptibility to natural enemy attack, as well as in their sensitivity to contact.     

CLONAL VARIATION AND COVARIATION IN APHID RESISTANCE TO PARASITOIDS AND A PATHOGEN

Abstract The potential rate of evolution of resistance to natural enemies depends on the genetic variation present in the population and any trade-offs between resistance and other components of fitness. We measured clonal variation and covariation in pea aphids ( Acyrthosiphon pisum ) for resistance to two parasitoid species ( Aphidius ervi and A. eadyi ) and a fungal pathogen ( Erynia neoaphidis ). We found significant clonal variation in resistance to all three natural enemies. We tested the hypothesis that there might be trade-offs (negative covariation) in defensive ability against different natural enemies, but found no evidence for this. All correlations in defensive ability were positive, that between the two parasitoid species significantly so. Defensive ability was not correlated with fecundity. A number of aphid clones were completely resistant to one parasitoid ( A. eadyi ), but a subset of these failed to reproduce subsequently. We discuss the factors that might maintain clonal variation in natural enemy resistance.     

Aphid clonal resistance to a parasitoid fails under heat stress

Parasitoid virulence and host resistance are complex interactions depending on metabolic rate and cellular activity, which in aphids additionally implicate heritable secondary symbionts among the Enterobacteriaceae. As performance of the parasitoid, the aphid host and its symbionts may differentially respond to temperature, the success or failure of aphid parasitism is difficult to predict when temperature varies. We tested the hypothesis that resistance of the pea aphid Acyrthosiphon pisum to the parasitoid Aphidius ervi, which is linked to aphid secondary symbionts, may depend on temperature in several resistant and non-resistant aphid clonal lineages of different geographic origin and of known bacterial symbiosis, using experiments in controlled environments. Complete immunity to A. ervi at 20 degrees C in three different aphid clones whose symbiosis is characterized by the possession of Hamiltonella defensa reversed to high susceptibility at 25 degrees C and especially 30 degrees C, suggesting that the aphid's immune responses to the establishment and early development of the parasitoid is strongly reduced at moderately high temperatures. There was no evidence that a pea aphid control genotype that was susceptible to A. ervi at 20 degrees C could become more resistant as temperature increases, as has been suggested for insect fungal pathogens. By contrast, our results suggest that aphid clonal resistance to A. ervi and related parasitoids is characteristic of cool temperature conditions, similar to various other fitness attributes of aphids. Based on evidence that H. defensa symbionts characterized all three A. ervi resistant pea aphid clones studied, but was absent in control aphids that remained susceptible at all temperatures, we suggest that secondary symbiosis plays a key role in the heat sensitivity of aphid clonal resistance. Our study may also indicate that aphid natural control of variably susceptible host populations by aphid parasitoids is more likely at moderate to high temperatures.     

Biodiversity and biocontrol: emergent impacts of a multi-enemy assemblage on pest suppression and crop yield in an agroecosystem

The suppression of agricultural pests has often been proposed as an important service of natural enemy diversity, but few experiments have tested this assertion. In this study we present empirical evidence that increasing the richness of a particular guild of natural enemies can reduce the density of a widespread group of herbivorous pests and, in turn, increase the yield of an economically important crop. We performed an experiment in large field enclosures where we manipulated the presence/absence of three of the most important natural enemies (the coccinellid beetle Harmonia axyridis, the damsel bug Nabis sp., and the parasitic wasp Aphidius ervi) of pea aphids (Acyrthosiphon pisum) that feed on alfalfa (Medicago sativa). When all three enemy species were together, the population density of the pea aphid was suppressed more than could be predicted from the summed impact of each enemy species alone. As crop yield was negatively related to pea aphid density, there was a concomitant non‐additive increase in the production of alfalfa in enclosures containing the more diverse enemy guild. This trophic cascade appeared to be influenced by an indirect interaction involving a second herbivore inhabiting the system – the cowpea aphid, Aphis craccivora. Data suggest that high relative densities of cowpea aphids inhibited parasitism of pea aphids by the specialist parasitoid, A. ervi. Therefore, when natural enemies were together and densities of cowpea aphids were reduced by generalist predators, parasitism of pea aphids increased. This interaction modification is similar to other types of indirect interactions among enemy species (e.g. predator–predator facilitation) that can enhance the suppression of agricultural pests. Results of our study, and those of others performed in agroecosystems, complement the broader debate over how biodiversity influences ecosystem functioning by specifically focusing on systems that produce goods of immediate relevance to human society.     

The interplay between density- and trait-mediated effects in predator-prey interactions: a case study in aphid wing polymorphism

Natural enemies not only influence prey density but they can also cause the modification of traits in their victims. While such non-lethal effects can be very important for the dynamic and structure of prey populations, little is known about their interaction with the density-mediated effects of natural enemies. We investigated the relationship between predation rate, prey density and trait modification in two aphid-aphid predator interactions. Pea aphids (Acyrthosiphon pisum, Harris) have been shown to produce winged dispersal morphs in response to the presence of ladybirds or parasitoid natural enemies. This trait modification influences the ability of aphids to disperse and to colonise new habitats, and hence has a bearing on the population dynamics of the prey. In two experiments we examined wing induction in pea aphids as a function of the rate of predation when hoverfly larvae (Episyrphus balteatus) and lacewing larvae (Chrysoperla carnea) were allowed to forage in pea aphid colonies. Both hoverfly and lacewing larvae caused a significant increase in the percentage of winged morphs among offspring compared to control treatments, emphasising that wing induction in the presence of natural enemies is a general response in pea aphids. The percentage of winged offspring was, however, dependent on the rate of predation, with a small effect of predation on aphid wing induction at very high and very low predation rates, and a strong response of aphids at medium predation rates. Aphid wing induction was influenced by the interplay between predation rate and the resultant prey density. Our results suggests that density-mediated and trait-mediated effects of natural enemies are closely connected to each other and jointly determine the effect of natural enemies on prey population dynamics.     

Reciprocal effects of host plant and natural enemy diversity on herbivore suppression: an empirical study of a model tritrophic system

Many theoretical and empirical studies have shown that species diversity in a trophic level can impact the capture of limited resources in ways that cascade up or down a food web. Only recently, however, have ecologists begun to consider how diversity at multiple trophic levels might act in concert to have opposing or reinforcing effects on resource use. Here, we report the results of an empirical study of a model, tritrophic food web in which we manipulated the diversity of host plant species ( Medicago sativa , Trifolium pratense and Vicia faba ) and natural enemy species ( Harmonia axyridis , Coleomegilla maculata and Nabis sp.) of a widespread herbivorous pest (the pea aphid, Acyrthosiphon pisum ) in laboratory microcosms. We found that increasing natural enemy richness from one to three species increased the proportion of aphids consumed by 0.14. This effect of enemy diversity was due to facilitative interactions and/or a reduction in intraspecific competition in the more diverse assemblages. We also found an independent and additive main effect of host plant richness, with the proportion of aphids consumed by natural enemies decreasing by 0.14 in plant polycultures. A reduction in predator efficiency on a single host plant, Vicia faba , appeared to be responsible for this plant diversity effect. Aphid population sizes were, therefore, simultaneously determined by a top-down effect of natural enemy diversity, and an opposing bottom-up effect of host plant diversity that modified enemy–prey interactions. These results suggest that population sizes in nature, and biotic controls over insect pests, are influenced by species diversity at multiple trophic levels.     

Non‐consumptive effects of a natural enemy on a non‐prey herbivore population

1. Predator–prey interactions have traditionally focused on the consumptive effects that predators have on prey. However, predators can also reduce the abundance of prey through behaviourally‐mediated non‐consumptive effects. For example, pea aphids (Acyrthosiphon pisum Harris) drop from their host plants in response to the risk of attack, reducing population sizes as a consequence of lost feeding opportunities. 2. The objective of the present study was to determine whether the non‐consumptive effects of predators could extend to non‐prey herbivore populations as a result of non‐lethal incidental interactions between herbivores and foraging natural enemies. 3. Polyculture habitats consisting of green peach aphids (Myzus persicae Sulzer) feeding on collards and pea aphids feeding on fava beans were established in greenhouse cages. Aphidius colemani Viereck, a generalist parasitoid that attacks green peach aphids but not pea aphids, was released into half of the cages and the abundance of the non‐host pea aphid was assessed. 4. Parasitoids reduced the population growth of the non‐host pea aphid by increasing the frequency of defensive drops; but this effect was dependent on the presence of green peach aphids. 5. Parasitoids probably elicited the pea aphid dropping behaviour through physical contact with pea aphids while foraging for green peach aphids. It is unlikely that pea aphids were responding to volatile alarm chemicals emitted by green peach aphids in the presence of the parasitoid. 6. In conclusion, the escape response of the pea aphid provided the opportunity for a parasitoid to have non‐target effects on an herbivore with which it did not engage in a trophic interaction. The implication is that natural enemies with narrow diet breadths have the potential to influence the abundance of a broad range of prey and non‐prey species via non‐consumptive effects.     

Resistance to a fungal pathogen and host plant specialization in the pea aphid

Herbivores that show host race formation on different plant species have proven to be valuable model systems for studying the evolution of specialization and speciation. Here, we use the pea aphid, Acyrthosiphon pisum, to investigate a possible link between specialization on two host plant species, Lotus uliginosus and Trifolium pratense, and resistance to a natural enemy, the fungal pathogen Erynia neoaphidis. Pea aphids collected on either plant species in the field showed in most cases poor survival on the alternate host plant. Furthermore, pea aphids specialized on T. pratense were very resistant to E. neoaphidis, whereas aphids specialized on L. uliginosus were susceptible. This susceptibility was not influenced by the actual food plant on which the assays were conducted. We discuss how selection from natural enemies may influence the process of specialization and race formation, and how specialization can affect the evolution of resistance.     

Aphid population dynamics: does resistance to parasitism influence population size?

1. Insect population size is regulated by both intrinsic traits of organisms and extrinsic factors. The impacts of natural enemies are typically considered to be extrinsic factors, however insects have traits that affect their vulnerability to attack by natural enemies, and thus intrinsic and extrinsic factors can interact in their effects on population size. 2. Pea aphids Acyrthosiphon pisum Harris (Hemiptera: Aphididae) in New York and Maryland that are specialised on alfalfa are approximately two times more physiologically resistant to parasitism by Aphidius ervi Haliday (Hymenoptera: Braconidae) than pea aphids specialised on clover. To assess the potential influence of this genetically based difference in resistance to parasitism on pea aphid population dynamics, pea aphids, A. ervi, and other natural enemies of aphids in clover and alfalfa fields were sampled. 3. Rates of successful parasitism by A. ervi were higher and pea aphid population sizes were lower in clover, where the aphids are less resistant to parasitism. In contrast, mortality due to a fungal pathogen of pea aphids was higher in alfalfa. Generalist aphid predators did not differ significantly in density between the crops. 4. To explore whether intrinsic resistance to parasitism influences field dynamics, the relationship between resistance and successful field parasitism in 12 populations was analysed. The average level of resistance of a population strongly predicts rates of successful parasitism in the field. The ability of the parasitoid to regulate the aphid may vary among pea aphid populations of different levels of resistance.     

Intra‐specific variation affects the structure of the natural enemy assemblage attacking pea aphid colonies

Abstract 1. Intra‐specific variation in plant defence traits has been shown to profoundly affect herbivore community structure. Here we describe two experiments designed to test whether similar effects occur at higher trophic levels, by studying pea aphid–natural enemy interactions in a disused pasture in southern England. 2. In the first experiment, the numbers and identity of natural enemies attacking different monoclonal pea aphid colonies were recorded in a series of assays throughout the period of pea aphid activity. 3. In the summer assay, there was a significant effect of clone on the numbers of aphidophagous hoverfly larvae and the total number of non‐hoverfly natural enemies recruited. Clone also appeared to influence the attack rate suffered by the primary predator in the system, the hoverfly Episyrphus balteatus, by Diplazon laetatorius, an ichneumonid parasitoid. Colonies were generally driven to extinction by hoverfly attack, resulting in the recording of low numbers of parasitoids and entomopathogens, suggesting intense intra‐guild predation. 4. To further examine the influence of clonal variation on the recruitment of natural enemies, a second experiment was performed to monitor the temporal dynamics of community development. Colonies were destructively sampled every other day and the numbers of natural enemies attacking aphid colonies were recorded. These data demonstrated that clonal variation influenced the timing, abundance, and identity of natural enemies attacking aphid colonies. 5. Taken together, these data suggest that clonal variation may have a significant influence on the patterns of interactions between aphids and their natural enemies, and that such effects are likely to affect our understanding of the ecology and biological control of these insect herbivores.     

The effect of predators and hymenopterous parasites on population fluctuations of alfalfa aphids

Among alfalfa pests in Iran three aphid species, green alfalfa aphid Acyrthosiphon pisum Harris spotted alfalfa aphid Therioaphis trifolii forma maculata Buckton and blue alfalfa aphid, Acyrthosiphon kondoi Shinji are important pests. The green alfalfa aphid can be observed all along the growing season particularly from late May to mid June at Karaj climate conditions. During this period, the mean monthly maximum temperature and relative humidity were about 28 degrees C and 60-65% respectively. This aphid overwinters as nymph and viviparous female. Sexual forms and eggs could not be seen under field conditions. Spotted alfalfa aphid, Therioaphis trifolii fonna maculata is the most prevalent aphid in summer time, when the mean monthly maximum temperature and relative humidity are about 33-34 degrees C and 44-58% respectively. Sexual individuals have been observed in the laboratory but not in the field. Among predators (Coccinella septempunctata, Adonia variegata, Syrphus cinctus, S. corolae, S. grassulariae, Chrysoperla carnea and Nabis capsiformis) one coccinellid species, C. septempunctata, had greatest impact on fluctuations of population. Among hymenopterous parasitoids two species have been collected from alfalfa field they were Aphidius ervi and Praon palitans. These parasitoids destroyed a good percent of aphids and statistically proved to lower aphid populations significantly.     

Responses to aphid sex pheromones by the pea aphid parasitoids Aphidius ervi and Aphidius eadyi

Aphidius ervi and Aphidius eadyi, two parasitoids of the pea aphid Acyrthosiphon pisum, were attracted to components of the aphid sex pheromone in laboratory bioassays. Pre-test experience with host aphids in the presence of aphid sex pheromone did not affect the response of A. ervi to pheromone in a 4-way olfactometer, compared with that of naive parasitoids. Aphidius ervi females exposed only to the pheromone prior to testing did not respond in the olfactometer, suggesting habituation to the foraging cue by the parasitoid. In a wind tunnel, aphid sex pheromone increased the attraction of A. ervi to the plant-host complex (Vicia faba/A. pisum), suggesting an additive effect when two different foraging cues are present simultaneously.     

Genotype and temperature influence pea aphid resistance to a fungal entomopathogen

Abstract. The influence of temperature on life history traits of four Acyrthosiphon pisum clones was investigated, together with their resistance to one genotype of the fungal entomopathogen Erynia neoaphidis . There was no difference among aphid clones in development rate, but they did differ in fecundity. Both development rate and fecundity were influenced by temperature, but all clones showed similar responses to the changes in temperature (i.e. the interaction term was nonsignificant). However, there were significant differences among clones in susceptibility to the pathogen, and this was influenced by temperature. Furthermore, the clones differed in how temperature influenced susceptibility, with susceptibility rankings changing with temperature. Two clones showed changes in susceptibility which mirrored changes in the in vitro vegetative growth rate of E. neoaphidis at different temperatures, whereas two other clones differed considerably from this expected response. Such interactions between genotype and temperature may help maintain heritable variation in aphid susceptibility to fungal pathogen attack and have implications for our understanding of disease dynamics in natural populations. This study also highlights the difficulties of drawing conclusions about the efficacy of a biological control agent when only a restricted range of pest genotypes or environmental conditions are considered.     

Intraguild interactions between the entomopathogenic fungus Pandora neoaphidis and an aphid predator and parasitoid at the population scale

The interactions that occur between the entomopathogenic fungus Pandora neoaphidis and a predator (Coccinella septempunctata) and a parasitoid (Aphidius ervi) were assessed in microcosm and polytunnel experiments. Transmission of P. neoaphidis to the pea aphid, Acyrthosiphon pisum, was enhanced in the presence of both C. septempunctata and A. ervi in microcosm experiments done under fixed abiotic conditions. In contrast, the reproductive success of A. ervi was reduced in the presence of P. neoaphidis. Despite the increased fungal transmission in the presence of C. septempunctata, there was no additional decrease in the aphid population indicating that P. neoaphidis is functionally redundant in the presence of the coccinellid. In polytunnel experiments the reproductive success of A. ervi was not affected by P. neoaphidis. These results do not support those of the microcosm and may be due to the more natural abiotic conditions in the polytunnel reducing the competitive advantage of the fungus. Microcosms therefore provide an arena in which the interactions between fungal pathogens and other aphid-natural enemies can be assessed however, further assessments at increased spatial scales under more natural abiotic conditions are also required to accurately determine the outcome of these interactions.     

Intraspecific heritable variation in life-history traits can alter the outcome of interspecific competition among insect herbivores

Competition is one of the most important biotic factors determining the structure of ecological communities. In this study, we show that there is variation in competitive ability between two clones of the pea aphid, Acyrthosiphon pisum, both of which out-compete a clone of the vetch aphid, Megoura viciae, in the laboratory. We tested whether this variation in competitive ability would alter the outcome of interspecific competition in the field. While one pea aphid clone followed the pattern set in the laboratory, out-competing the Megoura viciae clone, another showed the reverse effect with Megoura viciae dominating. These differences appear to be the result of variation in early population growth rate between the pea aphid clones, rather than predation, although predation did lead to the eventual extinction of colonies. We also questioned whether intra- and interspecific differences in predator escape behaviour could affect the outcome of competition in the field. All three clones responded similarly to the presence of foraging hoverfly larvae (Episyrphus balteatus), but the Megoura viciae clone dropped from the plant significantly less often in response to the presence of a foraging two-spot ladybird (Adalia bipunctata). This work provides evidence that intraspecific variation in competitive ability can alter the outcome of interspecific competitive interactions in nature and suggests that species–specific behavioural traits may have the potential to modify the outcome of these interactions.     

Coccinellid immigration to infested host patches influences suppression of Aphis glycines in soybean

Generalist natural enemies may be well adapted to annual crop systems in which pests and natural enemies re-colonize fields each year. In addition, for patchily-distributed pests, a natural enemy must disperse within a crop field to arrive at infested host patches. As they typically have longer generation times than their prey, theory suggests that generalist natural enemies need high immigration rates to and within fields to effectively suppress pest populations. The soybean aphid, Aphis glycines Matsumura, is a pest of an annual crop and is predominantly controlled by coccinellids. To test if rates of coccinellid arrival at aphid-infested patches are crucial for soybean aphid control, we experimentally varied coccinellid immigration to 1 m² soybean patches using selective barriers and measured effects on A. glycines populations. In a year with low ambient aphid pressure, naturally-occurring levels of coccinellid immigration to host patches were sufficient to suppress aphid populations, while decreasing coccinellid immigration rates resulted in large increases in soybean aphid populations within infested patches. Activity of other predators was low in this year, suggesting that most of the differences in aphid population growth were due to changes in coccinellid immigration. Alternatively, in a year in which alate aphids continually colonized plots, aphid suppression was incomplete and increased activity of other predatory taxa contributed to adult coccinellid predation of A. glycines. Our results suggest that in a system in which natural enemy populations cannot track pest populations through reproduction, immigration of natural enemies to infested patches can compensate and result in pest control.     

The importance of antennae for pea aphid wing induction in the presence of natural enemies

The pea aphid Acyrthosiphon pisum Harris has been shown to produce an increasing proportion of winged morphs among its offspring when exposed to natural enemies, in particular hoverfly larvae, lacewing larvae, adult and larval ladybirds and aphidiid parasitoids. While these results suggest that wing induction in the presence of predators and parasitoids is a general response of the pea aphid, the cues and mechanisms underlying this response are still unclear. Tactile stimuli and the perception of chemical signals as well as visual signals are candidates for suitable cues in the presence of natural enemies. In this paper the hypothesis that the aphids' antennae are crucial for the wing induction in the presence of natural enemies is tested. Antennae of pea aphids were ablated and morph production was scored when aphids were reared either in the presence or the absence of predatory lacewing larvae over a six-day period. Ablation of antennae resulted in a drastic drop in the proportion of winged morphs among the offspring, both in the presence and the absence of a predator whereas predator presence increased wing induction in aphids with intact antennae, as reported in previous experiments. The results show that antennae are necessary for wing induction in the presence of natural enemies. Critical re-examination of early work on the importance of aphid antennae and tactile stimuli for wing induction suggests that a combination of tactile and chemical cues is likely to be involved not only in predator-induced wing formation but also for wing induction in response to factors such as crowding in the aphid colony.     

Temperature checks the Red Queen? Resistance and virulence in a fluctuating environment

Numerous studies have revealed genetic variation in resistance and susceptibility in host–parasite interactions and therefore the potential for frequency‐dependent selection (Red Queen dynamics). Few studies, if any, have considered the abiotic environment as a mediating factor in these interactions. Using the pea aphid, Acyrthosiphon pisum, and its fungal pathogen, Erynia neoaphidis, as a model host–parasite system, we demonstrate how temperature can mediate the expression of genotypic variation for susceptibility and virulence. Whilst previous studies have revealed among‐clone variation in aphid resistance to this pathogen, we show that resistance rankings derived from assessments at one temperature, are not conserved across differing temperature regimes. We suggest that variation in environmental temperature, through its nonlinear impact on parasite virulence and host defence, may contribute to the general lack of evidence for frequency‐dependent selection in field systems.     

Competition and dispersal in the pea aphid: clonal variation and correlations across traits

Abstract.  1. The presence of an across-species trade-off between dispersal ability and competitive ability has been proposed as a mechanism that facilitates coexistence. It is not clear if a similar trade-off exists within species. Such a trade-off would constrain the evolution of either trait and, given appropriate selection pressures, promote local adaptation in these traits.
2. This study found substantial levels of heritable variation in competitive ability of the pea aphid, Acyrthosiphon pisum Harris (Homoptera: Aphididae), measured in terms of relative survival when reared with a single clone of the vetch aphid, Megoura viciae Buckton (Homoptera: Aphididae).
3. Pea aphids can move to new patches by either flying (longer distance dispersal) or walking (local dispersal) from plant to plant. There was considerable clonal variation in dispersal ability, measured in terms of the proportion of winged offspring produced, and ability to survive away from their host plant.
4. Winged individuals showed longer off-plant survival times than wingless forms of the same pea aphid clone.
5. There was no evidence of a relationship between clonal competitive ability and either measure of dispersal ability, although the power of the test is limited by the number of pea aphid clones used in the trial.
6. However, there was a positive correlation between clonal fecundity and the proportion of winged offspring produced. Although speculative, it is suggested that clones that are more likely to either overwhelm their host plant or attract higher numbers of natural enemies as a result of having higher fecundity are more likely to produce winged morphs.     

Apparent competition between two species of aphid via the fungal pathogen Erynia neoaphidis and its interaction with the aphid parasitoid Aphidius ervi

Abstract 1. Motivated by a community study on aphids and their fungal pathogens, three hypotheses were tested experimentally to investigate the influence of the fungal pathogen, Erynia neoaphidis Remaudière and Hennebert, on aphid population and community ecology.
2. Field experiments were performed in 2 years to test whether two susceptible aphid species on different host plants might interact through the shared fungal pathogen. No strong pathogen-mediated indirect interactions (apparent competition) between populations of pea aphid Acyrthosiphon pisum Harris and nettle aphid Microlophium carnosum Buckton were detected.
3. In the first of the field experiments, pea aphids exposed to the fungus showed a weak tendency to produce more winged dispersal morphs than control populations not exposed to the fungus. In a laboratory test, however, no support was found for the hypothesis that the presence of volatiles from fungus-infected cadavers promotes production of winged offspring.
4. The response of the pea aphid parasitoid Aphidius ervi Halliday to colonies containing hosts infected 1 and 3 days previously was assessed. Wasps initiated fewer attacks on 1-day-old infected colonies than on healthy colonies, with the numbers on 3-day-old fungus-infected colonies intermediate.