Aphids show interspecific and intraspecific variation in life history responses to host plant infection by the fungal pathogen Botrytis cinerea

The life histories of insect herbivores are affected by variation in host plant quality, with poor quality typically being associated with reduced herbivore fecundity, size and longevity. Plant pathogens are ubiquitous in nature and can alter host plant quality as experienced by insect herbivores. We asked how host plant infection by the widespread and economically important fungal pathogen Botrytis cinerea affected the life history traits of two aphid species. We found that the life history traits of the black bean aphid Aphis fabae were negatively affected by being reared on infected host plants, showing reduced fecundity, population growth rate, size, off‐plant survival time and development rate. In contrast, we found that pea aphids Acyrthosiphon pisum benefitted from being reared on infected plants, and that the degree of benefit varied between pea aphid clonal lines. This work suggests that the ecological and economic consequences of plant pathogen infection on the dynamics of aphid pests could be difficult to predict.

Botrytis spp. are globally important fungal plant pathogens, causing disease in >1400 plant species (Elad et al. 2016), including many economically important crops (Elad et al. 2004). Botrytis cinerea is an aggressive necrotrophic fungus that destroys host plants with necrotic lesions (Shaw et al. 2016) and is perhaps the most notorious species of this genus, causing dramatic losses in both pre-and post-harvest crops (Dean et al. 2012). Botrytis cinerea has been ranked as the second most important fungal pathogen in terms of its scientific and economic value (Dean et al. 2012).
Aphids are among the most important crop pests in temperate regions (van Emden & Harrington 2007), causing both direct damage to host plants and indirect damage by acting as vectors of plant viruses and by the production of honeydew, which can result in fungal infection and reduce photosynthesis (van Emden 2013). Aphids show both between and within species variation in their life history responses to environmental factors such as host plant quality (Service 1984;Stacey et al. 2002a), temperature (Stacey et al. 2002b;Stacey et al. 2003), and crowding (Hazell et al. 2005). Such variation will have economic and ecological consequences, affecting which species or genotypes are likely to benefit from such changes (Thompson 1988;Bolnick et al. 2011;Des Roches et al. 2018).
Fungal plant pathogen infection can alter host plant quality as experienced by herbivores by inducing biochemical defense responses inside the host plant. These biochemical responses can also negatively affect herbivorous insects (Fernandez-Conradi et al. 2018;Ederli et al. 2021) and indeed could also affect species at higher trophic levels (Ngah et al. 2018;Srisakrapikoop et al. 2020). Nevertheless, the effect of plant pathogen infection might also benefit some insect herbivores (Tack & Dicke 2013), and herbivores could differ in their responses to host plants infected by different plant pathogens (e.g. positive effects found for Aphis fabae feeding on Vicia faba infected by Uromyces viciae-fabae, but negative effects are seen when V. faba is infected by B. cinerea; Al-Naemi & Hatcher 2013).
What is not clear is whether different species of aphid or aphid genotypes differ in the life history consequences of feeding on the same host plant species infected by the same plant pathogen. We addressed these questions using two aphid species, the black bean aphid Aphis fabae and the pea aphid Acyrthosiphon pisum, and three clones of the latter species, asking whether host plant infection status influenced the size, fecundity, maturation time and off-plant survival of the study aphids.
Botrytis cinerea Pers.: Fr (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) was cultured on malt extract agar and incubated at 20 C under conditions of 12 h UV light : 12 h dark (LD 12:12) to encourage the fungus to produce spores.
The host plants, Vicia faba L. (Fabaceae, cv. Sutton dwarf), were individually grown in 1 L pots with peat compost (Clover ® , London, UK). When the plants had five true leaves, they were divided into two treatment groups. Plants in the infected group were treated with a 0.1 mL suspension of 1-month old B. cinerea (10 6conidia/mL) on the adaxial surfaces of the leaves using a paint brush. Uninfected plants were treated with distilled water in a similar manner. Plants were then kept individually in a sealed polythene bag at 20 C for 48 h to encourage spore germination.
A single black bean aphid Ap. fabae Scop. (Hemiptera: Aphididae) was collected from opium poppy Papaver somniferum L. (Papaveraceae), and three pea aphid Ac. pisum Harris (Hempitera: Aphididae) clones were collected from bird's-foot trefoil Lotus corniculatus L. (Fabaceae), from three widely separated locations, all in the Whiteknights campus of the University of Reading, UK. The two aphid species were identified and confirmed following Blackman and Eastop (2000). Aphid cultures were maintained as a monoclonal culture in separate insect cages and provided with either uninfected or infected V. faba plants for more than three generations before the experiments started to avoid confounding maternal effects. All work was carried out in a controlled environment room at 20 C, LD 16:8, 60% RH.
For the black bean aphid experiment, treatments comprised of five uninfected or five infected plants, each of which held eight aphids. Each aphid was confined individually in a clip cage (20 mm in diameter; Noble 1958) attached to individual leaflets (total 40 aphids/infection status; aphids were transferred from culture plants of the same infection status to avoid confounding maternal effects). Aphids were left to produce nymphs for 24 h, then all apart from one nymph were removed, which was allowed to grow to maturity and produce offspring.
Time to maturity was recorded and the number of offspring produced was then recorded every second day for 10 days. During each visit nymphs were removed to prevent competition. The intrinsic rate of increase (rm) was calculated from the formula rm = (c ln [Md]) / D, where c is a constant (0.738), Md is the number of offspring produced by the adult aphid in the D days of reproduction (Wyatt & White 1977).
Separately, 80 7-day-old aphids (40 from each treatment) were randomly selected from cultures and transferred into individual Petri dishes without food or water and monitored every 8 h until death to yield off-plant survival time. Another 80 7-day-old aphids (40 from each treatment) were used to measure hind tibia length (Nicol & Mackauer 1999) under a high-performance stereomicroscope (Leica MZ9.5; Heerbrugg, Switzerland).
For the pea aphid experiment, 10 adult apterous pea aphids from each of the three clones were randomly selected from each of the base culture colonies feeding on uninfected and infected plants (60 aphids in total). Each aphid was placed into an individual clip cage (40 mm in diameter) directly onto an individual plant of the same colony infection status. Fecundity and intrinsic rate of increase were recorded in the same manner as described above, except the number of offspring were recorded every other day for 14 days.
In addition, aphid off-plant survival time, hind tibia size, days to maturity and the intrinsic rate of increase were also recorded again in a similar manner as for Ap. fabae. A total of 180 nymphs (30 from each clone and treatment) were allowed to grow for 7 days, reaching the 4th instar stage. When the individuals were transferred into a Petri dish without food or water, they were monitored every 12 h until death. Forty 7-day-old aphids (from each clone and treatment) were used to measure hind tibia length under a highperformance stereomicroscope (MZ9.5; Leica).
All statistical analyses were carried out on R 4.0.3 (R Core Team 2020). For the Ap. fabae experiment, as the data are not normally distributed, Wilcoxon rank sum tests were used to test for differences in hind tibia length and off-plant survival time between infected and uninfected plants. Initial examination of the data showed that the effect of nested data could be ignored as the variances resulting from different plants were very close to zero, and then the intrinsic rate of increase, fecundity and maturation time data could also be analyzed by using Wilcoxon rank sum tests.
In the Ac. pisum experiment the hind tibia length, off-plant survival time, maturation time and intrinsic rate of increase data were analyzed using ANOVA of aligned rank transformed using the ARTool package (Wobbrock et al. 2011) as data were not normally distributed. Fecundity data were analyzed by ANOVA using the car package (Fox & Weisberg 2019). Post hoc analyses with Tukey tests were analyzed using the emmeans package (Lenth 2019) or with the Mann-Whitney U-test to examine clonal variation within the infection status group.
There is increasing evidence showing that plant pathogens play important roles in determining the interaction between insects and their host plants, and indeed these effects can ramify through communities (Grunseich et al. 2020;Srisakrapikoop et al. 2020). In this study, we find that two aphid species respond in different directions to the same plant pathogen-host system. Host plant infection by B. cinerea caused negative indirect effects on Ap. fabae for all measured parameters, whereas infection caused positive indirect effects on Ac. pisum, and here the magnitude of these effects differed between aphid clones. The indirect effects of plant pathogen infection therefore differ both between and within aphid species.
Aphis fabae feeding on uninfected plants performed better than those feeding on infected plants in all measured parameters, which is consistent with a previous study with a similar system (Al-Naemi & Hatcher 2013). Acyrthosiphon pisum expressed the reverse pattern, benefitting from feeding on infected plants, and these effects were consistent, but differed in magnitude, across clones. The mechanisms underpinning the between species differences are not clear, given that both are generalist aphid species, which feed in a similar manner, and the host plant-pathogen treatment was controlled. The ultimate cause of this variation is worthy of more detailed study, as it suggests that the population dynamics of different aphid species might be differentially affected by pathogen infection.
Although the causes of the differences in response are unclear, we note that the indirect effects of B. cinerea on aphid life histories could be transmitted in two (nonindependent) pathways, either through a change in host nutrition and/or in host defense. The former is caused by a change in nitrogen content, which is decreased by fungal infection (Dulermo et al. 2009;Al-Naemi & Hatcher 2013), and the latter is induced resistance, where the plant responds to infection through two signaling routes, the salicylic acid (SA) and jasmonic acid (JA) pathways (Pieterse et al. 2014). The SA pathway is usually used by plants to respond to sucking/piercing herbivores (including aphids), whereas the JA pathway is upregulated in response to necrotrophic pathogens and chewing herbivores (Pieterse et al. 2014;Stout 2014), and these pathways are considered to trade-off against each other (Spoel et al. 2003;Brooks et al. 2005).
In this study, the plants had been first infected by B. cinerea before aphids were introduced, so it is likely that the JA pathway was triggered, suppressing the SA pathway (Leon-Reyes et al. 2010). Theoretically, aphids should benefit from this, but here Ap. fabae showed reduced performance when feeding on infected plants, perhaps because aphids are not only affected by outcomes of suppressing the SA pathway (Thaler et al. 2010), but also by the elicited JA pathway (Thaler et al. 2001;Goggin & Cooper 2005). Aphid species could therefore differ in their responses to changes in nutrition and plant defenses. In contrast, variation in the strength of within species responses between aphid clones is expected, given the importance of both genetic and endosymbiotic factors (Stacey et al. 2003;Hazell & Fellowes 2009;Heyworth et al. 2020).
This work is of considerable applied interest, as we show that the effects of infection by an economically very important plant pathogen on the life histories of two  related and also economically important aphid pest species differ. Feeding on B. cinerea-infected host plants benefits pea aphids (albeit the strength of this varies across clones), whereas black bean aphids are detrimentally affected by infected host plants. The cause of this difference between species is unclear. This could be the result of differences in the direct physiological effects of host plant quality on the developing aphids, or an indirect effect mediated by differences in other factors, such as the composition of the aphid's endosymbiont community. The links between plant pathogens and insect herbivores are rarely simple; elucidating the mechanisms that result in such unexpected contrasting effects would be of considerable value.