Study organisms
Pea aphids and defensive endosymbiont
Two clonal lineages of pea aphid were selected for the experiment, N116 and our Q1 isolate. The N116 aphid is of the biotype (K) as it was originally isolated from alfalfa Medicago sativa (L.) by Dr Julia Ferrari in Berkshire UK [40]. It has been a laboratory lineage for ca. 10 years and was provided to us by Dr Colin Turnbull of Imperial College London. Q1 is of the biotype (G) [69], which was established from one female of a population colonising pea plants (Pisum sativum L.) isolated from the quadrangle garden of the Faculty of Biology, Medicine and Health, University of Manchester. N116 is reported to have the heritable defensive endosymbiont H. defensa [55] that confers relative immunity to parasitoidism. By contrast, we established that Q1 was highly susceptible to being parasitoidised. As specified in the molecular analysis below, we surveyed the endosymbiont communities in each of the lineages. N116 and Q1 are ecologically distinct, derived from different geographic locations, have distinguishable life histories and susceptibility to the parasitoid wasp. They are, therefore, a good representative of the within-species genetic variability in the pea aphid. The aphids were reared on faba bean Vicia faba var minor (Harz) plants obtained from a local supplier, Manchester, UK, and maintained at 22–24 °C with a photoperiod of 16 h (light): 8 h (dark). Under temperate mesic conditions, aphids reproduce through parthenogenesis resulting in populations of genetically identical individuals.
Parasitoid wasp A. ervi
We purchased 250 mummies of aphids harbouring A. ervi juveniles from Koppert Biological Systems (UK). Unlike the non-parasitic males, the females of this solitary koinobiont parasitoid wasp are an efficient natural enemy and biocontrol agent of pea aphids [37, 70]. The female oviposits one egg in the viable aphid host. Subsequently, a larva hatches and parasitises the host consuming it internally whilst the parasitoid juvenile pupates, then develops into an adult that ecloses from the dead body of the host to resume the life cycle. Immediately upon their arrival, we separated the mummies into multiple 90 mm petri dishes, each dish containing a small ball of dental cotton, approximately 20 mm in diameter, which was saturated in 10% sucrose solution. The petri dishes were kept in the fridge at 10 °C to slow the rate of eclosion from the aphid mummy (i.e. the wasp puparium). The petri dishes were taken from the fridge hourly and checked for the eclosion of wasps; the sex of the emergent wasp was observed; if all the individuals were of the same sex, then they could be used in the next stage of the experiment. The females were always isolated and kept separately from the males to ensure the females were virgins prior to mating according to the quantitative genetic design explained below.
Intraguild predator C. carnea larva
The intraguild predator in our experiments was the aphid lion larva. The larvae were purchased from Ladybird Plant Care (UK) in tubes of approximately 300–500 individuals. The tube was emptied into a plastic container that contained some plant shoot parts with aphids as a provision and then kept in the fridge at 5 °C until they were needed; this was to slow the rate of metabolism and prevent the larvae from cannibalising each other. The larvae were used within 48 h of delivery or they were disposed of. As the wasps take ~ 11 days to emerge from the mummies, the aphid lion larvae (1st instars) were ordered so that they would arrive on day 10 ready to be used where applicable in the experiment as described below.
Experimental design
Haplodiploidy is the sex-determination system in the Hymenopteran parasitoid wasp A. ervi, meaning that males are the result of unfertilised eggs and hence haploid (1n), while females are diploid (2n) since they are produced from fertilised eggs [71]. Based on Khudr et al. (2013) [21], we mated randomly selected 34 male wasps (sires) with randomly selected female wasps (dams) to establish a quantitative genetic half-sibling design. Each of the sires was mated with a minimum of three dams, dependent on wasp availability right after their eclosion. We thus established sire-dam groups. Before the wasps were mated, they were isolated into Eppendorf tubes and inspected using a magnifying glass to observe abdomens and determine their sex; the female’s abdomen ends with a pronounced point (ovipositor) while the male’s abdomen is more rounded. The wasps were then put into the same tube by opening both tubes and putting them end to end. Once both wasps (sire and dam) had moved into the same tube, it was sealed with a small piece of foam. The mating wasps were monitored carefully until they completed copulation to ensure the corresponding sire inseminated the assigned dams. Copulation was checked to have occurred within two hours of eclosion. If copulation did not happen, the female wasps were disposed of because of the short window of time during which the otherwise arrhenotokous parthenogenetic female wasp will be usually receptive to mating [21, 71]. Once copulation was completed the foam was removed, the tubes were placed end to end, and we waited for the wasps to enter separate tubes before closing the lids and labelling the sire with its unique number (S1 – Sn), and the dams with the number of the associated sire they mated with plus their own unique number in order of mating (e.g. S1 D1 – Sn Dm). Figure 3 illustrates the experimental design.
Once mated, the inseminated dams were placed in their respective microcosms. The microcosms were constructed by removing the ends of a 2-L PVC bottle and attaching one end to the plant pot and covering the other with a fine nylon mesh (‘Non-Fray’, Insectopia, UK). Each microcosm contained a 3-week-old broad bean plant that had been infested with 30 third instars of N116 just before putting the wasp into the enclosure. To release the dam into the microcosm the top section was held in place over the plant (leaving a small gap on one side), the lid of the tube was opened and sealed with the end of a finger and then the tube was passed through the gap onto the soil. Once the inseminated wasp was inside the microcosm, the top section of the microcosm was secured to the plant pot using 48 mm wide polypropylene tape. The microcosms were placed, evenly spaced, into large trays, containing a shallow layer of water, in the growth chamber for eleven days. The conditions in the chamber were 22–24 °C with a 16 h (light):8 h (dark) photoperiod; the water level in the trays was maintained and the positions of the microcosms on the trays were randomised every other day. On the eleventh day, the microcosms were taken from the growth chamber, opened, and all the mummies present were removed from the plant and inner surfaces of the microcosm using a fine damp paintbrush. Each mummy was placed in a separate 35 mm petri dish that contained a small ball of dental cotton (approximately 10 mm in diameter) saturated with 10% sucrose solution and labelled with the associative sire-dam number. The petri dishes were left at room temperature on the lab bench and left until we observed eclosion. Once the progenies (sib and half-sib daughters denoting the intraspecific genetic variability of the parasitoid) had emerged from the aphid mummies, they were individually introduced into a microcosm with a 3-week-old faba bean plant that had been infested with 30 third instars of N116. The microcosms were sealed, and each of the introduced daughters (i.e. parasitoid genotype) was given 11 days to parasitise the provided aphid population leading to the production of mummies. We then censused the aphid population in each microcosm (mummified and healthy) and recorded the positions of the mummies off-plant versus on-plant. The whole procedure was simultaneously repeated for the Q1 lineage. As such, the wasp daughters (parasitoid genotype) represented the intraspecific genetic variation effects in the parasitoid wasp, whereas the within-species genetic variation in the pea aphid host was represented by the inclusion of the N116 and Q1 lineages.
The remainder of the generated parasitoid daughters were used to test the effect of the presence of the aphid lion as an intraguild predator (IGP) on aphid traits. After the introduction of the aphids (N116 or Q1) followed by the parasitoid daughter into the microcosm, as explained above, an aphid lion second-instar larva was transferred into the microcosm, on a fine paintbrush, onto the soil a few minutes after the wasp was added. The daughters (parasitoid genotype) that arose from each of the sire × dam mating groupings were numbered and then split randomly into one of two groups: without IGP (i.e. IGP absent) or with IGP (i.e. IGP present). Once the microcosm set up was completed, they were sealed and placed back into the growth chamber for eleven days at 22–24 °C with the 16 h:8 h photoperiod as above. The microcosms were randomised in the chamber and checked to ensure that they had enough water every other day. On the eleventh day, the microcosms were once again removed from the growth chamber, opened and the data were recorded. We recorded the total number of healthy aphids (non-mummified), the total number of mummies, and the distribution of the mummies within the microcosm (on versus off-plant), (Fig. 3). We were unable to create a fully factorial design with two aphid lineages and the presence or absence of a predator for each dam/sire combination. The differential survival in this multispecies system combined with the nature of the quantitative genetic design, and keeping all the parthenogenetic aphids at the same age, led to unbalanced sample sizes for a given aphid lineage, which, nevertheless, is sufficiently powered for the number of replicates. Overall, we were left with 118 parasitoid genotypes (daughters). Daughters were split into two groups, with one (n = 73) being provided with pea aphid N116 as provision, while the other (n = 45) was provided respectively with pea aphid Q1. Each of these two populations were further split into two groups, with one group exposed to intraguild predation by the aphid lion larva (n = 43, in the case of N116, and n = 15 in the case of Q1) and the other group not (n = 30, in the case of N116, and n = 30 in the case of Q1).
Molecular analysis
Healthy aphids from each microcosm were preserved in a cryogenic tube at − 195 °C, at The University of Manchester liquid nitrogen sample storage facility, for molecular analysis. The identification of the bacterial symbionts in the two lineages of pea aphid consisted of two parts: (1) the use of diagnostic PCR to confirm the presence or absence of the defensive symbiont H. defensa, and (2) 16 s rRNA gene sequencing for the identification of other symbionts. The aphid samples were surface-sterilised [72], then the DNA was extracted using ‘Qiagen DNAEasy Blood and Tissue Kit’ small insect supplementary protocol [72]. As the aphids are soft-bodied insects, we altered step 1 of the protocol slightly, rather than freezing them in liquid nitrogen and grinding them up in a pestle and mortar they were homogenised in a sterile microcentrifuge tube using a sterile disposable microcentrifuge tube homogenisation pestle. In step 3, the lysis time was increased from three to six hours and the rest of the protocol was followed with no further modifications. Subsequently, we ran a Diagnostic PCR [73]; the PCR reactions were visualised on a 1% agarose gel with SafeView Nucleic Acid Stain with Bioline HyperLadder™ 1 kb. Afterwards, we ran 16 s Gene Sequencing for a total of 70 samples (35 Q1 and 35 N116), which were sent for sequencing using GATC Biotech’s T7 sequencing primers. Once we had received the sequence data, both the vector sequences and the parts of the sequences that contained bases that were below the confidence threshold were removed. The sequences were then analysed using the NCBI ‘standard nucleotide BLAST’ (megablast) and the Nucleotide collection (nr/nt). The most closely related bacteria were selected based on the blast output and where they fall on the resulting distance tree of the results (Additional file 1, Molecular Analysis).
Statistics
The data on the parasitoid genotype with and without IGP were pooled because this enabled us to investigate the influence of the IGP on the outcome of the parasitoid genotype effect on aphid fitness (in terms of immunity to the parasitoid) and the behaviour of the aphid lineages. All statistical analyses were conducted using R [74] via RStudio [75]. Firstly, we tested the effects of parasitoid and aphid genetic variability in the absence or presence of IGP on aphid immunity ratio (IR: the proportion of aphids that was non-mummified [unparasitoidised] after 11 days of exposure to the parasitoid genotype relative to the entire population of aphids [healthy and mummified] per aphid lineage per microcosm). We applied a generalised linear mixed effect model (GLMMER1) with a Poisson family, R packages ‘car’ [76] and Ime4 [77]. The parsimonious model included the following explanatory variables as fixed effects: (1) sire (14 levels), (2) dam (8 Levels), (3) parasitoid genotype (daughter identity as per their sire × dam mating grouping that was the product of the quantitative genetic design; 118 daughters in total representing the parasitoid intraspecific genetic variation effect), (4) aphid lineage (two levels [N116, Q1]), (5) the interaction (parasitoid genotype × aphid lineage), (6) the interaction (parasitoid genotype × aphid lineage × Intraguild Predator presence (IGP [No, Yes])). The microcosm was modelled as a random effect.
Secondly, we analysed aphid behaviour as the proportion of aphid mummies off-plant relative to the total number of mummies in the microcosm. We used a generalised linear mixed-effect model (GLMMER2) with a Poisson family. Again, the parsimonious model included the following explanatory variables as fixed effects: (1) sire (13 levels), (2) parasitoid genotype (daughters’ identity as per their sire × dam mating grouping that was the product of the quantitative genetic design; 81 daughters in total representing the parasitoid intraspecific genetic variation effect), (3) aphid lineage (two levels [N116, Q1]), (4) Intraguild Predator (IGP) presence (two levels [No, Yes]), (5) the interaction (parasitoid genotype × aphid lineage), (6) the interaction (parasitoid genotype × aphid lineage × IGP). The microcosm was modelled as a random effect.