Discussion

Establishment and colonisation success, growth and development, reproductive performance and offspring fitness of root aphid A. lentisci on perennial ryegrass roots without and with endophytes were examined in this chapter, considering the possible impact of plant genotype and various plant biomass parameters.

The establishment success of > 75% achieved on endophyte-free perennial ryegrass in both the Biology I and II experiments was high, compared to the < 20% previously reported for A. lentisci neonates on barley and wheat seedlings (Wool & Kurzfeld-Zexer, 2008). It is possible that mature perennial ryegrass roots were more favourable in terms of root anatomy and/or physiology for establishment than the roots of young crop seedlings (see root usage and size discussion in Chapter 5, for example). Alternatively, other experimental aspects could have helped the starter nymphs of the Biology I and II experiments to establish, such as the comparatively drier environment of the agar- embedded roots. Condensation droplets were observed more than once to be a deadly trap for neonate root aphids (Appendix 7). Based on their observation of root age preferences in immature root aphids Popay and Cox (2016) hypothesised that actively growing plants would likely promote the development of root aphid populations. The positive effect of a large number of green leaves on the aphid establishment success during the Biology II experiment supported this view. Whether the two plant genotypes observed had an influence on the establishment success will need assessing in an experimental context with fewer confounding variables, in particular without large variations in plant age at aphid placement. The observed endophyte effect that aphids were significantly less likely to establish on AR37 symbionts, than on the alternatives (Biology II experiment), was consistent with previous observations by Popay and Cox (2016).

The colonisation successes of 30 to 71% achieved on endophyte-free plants in the Biology I, Biology II and Mature plant experiments corroborate what has been reported for A. lentisci too (Wool & Kurzfeld-Zexer, 2008). The reasons were likely the same as for the establishment success. Significant interactions by plant genotype-endophyte status group were detected for this variable in the Mature plant experiment. Aphids placed on S-AR37 symbioses were less successful in developing to colonies, than aphids on S-AR1 and N-CT plants in particular. Toxins produced in AR37 symbioses may partly explain the reduced colonisation success (Popay & Cox, 2016). However, biomass aspects may

also have had an effect. S-AR1 and N-CT symbioses had marginally faster growing shoots than S-AR37, and may thus have provided more food to the root aphids, considering that green shoot area growth and final root biomass were significantly correlated in the Mature plant experiment.

As with most aphid species (Dixon, 1973), A. lentisci moulted four times to maturity. Although the size of individual aphids increased at each ecdysis, the body length, abdominal width and their derived EP measurement did not differentiate between late instars (Table 3.5). These measurements are thus only conditionally useful when a classification has to be undertaken. If sizes in the field can be presumed similar to the ones reported during the Biology I experiment, some common criteria to separate mature from immature aphids could need reconsideration. The criterion ‘mature if > 1 mm body length’ (Popay & Cox, 2016) would indeed over-estimate the reproductive population of a sample considering that 21, 85 and 85% of all 2nd_{, 3}rd _{and 4}th _{instar specimen observed} exceeded this critical body length.

Two aspects complicated adult root aphid size analyses and results interpretation. Firstly, many root aphids grew further after the final moult and the first viviposition (Figure 3.3, Figure 3.5). Secondly, two clearly defined groups of adult size, ‘Large’ and ‘Small’, were observed in both Biology I and Biology II experiments. ‘Large’ aphids grew faster to a wider body frame than ‘Small’ aphids but receded in size towards the end of their adult life. Their predicted adult body length (Appendix 10, Tables A10.2.2.1. and A10.4.2.1) lay within the range reported in the literature for A. lentisci [e.g. 1.6 ± 1.1 mm (Podmore, 2015), 1.1 to 3.0 mm (Blackman & Eastop, 2000)]. By contrast, adults in the Small group maintained or slightly expanded their size up to their death. Their final adult length was below the typical range reported by Blackman and Eastop (2000). A figure by Schuett et al. (2015) [Figure 4; Schuett et al., 2015] suggested that the observed growth dichotomy may also exist in the pea aphid Acyrthosiphon pisum Harris. Further investigations will be necessary to gain insights into this phenomenon and its biological consequences. A link to aphid fecundity was obvious in both trials considering aphid size, however. As fecundity increases with body size or/and weight in many insect species (Honĕk, 1993), this observation could simply confirm that ‘Large’ aphids were generally more fecund than ‘Small’ ones. A part of an aphid’s reproductive potential is determined

before its birth, however (ovariole number; Section 1.2.4.3), and developing embryos are already present in an aphid long before it achieves maturity [telescoping generations (Kindlmann & Dixon, 1989)]. Thus, if ‘Large’ sizes were at least partly the result of the presence of many embryos, the two distinct growth patterns could have a “mechanical” explanation. Alternative explanations could involve possible modifications in abdominal structures with advancing age.

Approximately 90% of the live adult size measurements conformed to the literature ranges for dead, mounted and thus possibly somewhat extended A. lentisci specimens (Blackman & Eastop, 1984; Cottier, 1953). Smaller adult sizes in the biology experiment I compared to the Biology II experiment (no aphid over 1.5 mm2 _{EP, marginally lower} average body size; Appendix 10, Section A10.4.4) may have been the result of high temperatures in the glasshouse (Dixon, 1984), or lower feed quality as the agar used in the Biology I experiment contained only a fraction of the nutrients provided to plants in the Biology II experiment (Section 2.3.1.3). That ‘Large’ aphids were associated with healthier plants in the Biology II experiment (Section 3.3.2.1) supports the latter explanation. The smaller adult size and the absence of large specimens in S-CT plants (Figure 3.6) may be explained by plant genotype-endophyte interactions. Rhopalosiphum padi L., for example, developed smaller adults on Lolium multiflorum Lam. with endophyte Epichloe occultans Schardl., than on NIL L. multiflorum plants (Bastias et al., 2017). On L. perenne with E. festucae var. lolii symbiont, R. padi showed no significant reaction in this regard, however (Meister et al., 2006). More research is needed to confirm this first report of a reduced growth response of root aphids in the presence of specific endophyte symbionts in their host plant.

The three to four weeks developmental time required by neonates to achieve reproductive maturity fell into the range reported by Wool and Sulami (2001) (< 1 month), but was longer than that recorded by Wool and Kurzfeld-Zexer (2008). While the checking schedule of the Biology I experiment likely masked any neonate size effects, the findings in the Biology II experiment agreed with the general observation that larger neonate aphids mature significantly earlier than smaller ones (Dixon, 1984). Plant characteristics influenced the development time too. However, an interpretation of the observed effects is challenging, considering various experimental and environmental

effects that likely interfered with aphid development [e.g. heat, possible water and nutrient stress for the green shoot biomass in the Biology I experiment; possible water stress, root lignification and various maternal origins of the neonates for the plant age at placement during the Biology II experiment]. In contrast to the observations by Meister et al. (2006) for Metopolophium dirhodum Walker and R. padi L on perennial ryegrass infected with a common-toxic strain of E. festucae var. lolii, time to reproductive maturity in the Biology II experiment was delayed in presence of some endophytes. The effect depended on the genotype of the plant the endophyte was associated with, however, and would need to be confirmed on a large number of aphids.

Long-lived aphids and the plants they dwelled on were exposed to heat stress towards the end of the Biology I experiment, when the temperatures in the glasshouse reached 38.0 to 50.2 °C for 0.2 to 3.7 h·day-1 _{on 14 occasions (Enders & Miller, 2016;} Hannaway et al., 1999; Skaljac, 2016). Handling was also a frequent cause of death. While this may explain the failure to fit an explanatory model to the mortality data of the Biology I experiment, it also suggested the longevity observed in the Biology II experiment was the better estimate for the potential lifespan of A. lentisci. With a median longevity of over two months at 17 to 18 °C (Figure 3.7, d) for an aphid on endophyte- free plants, A. lentisci was clearly a longer-lived aphid species than other root aphids and in general, above-ground feeding aphids (Kuo et al., 2006; National Pesticide Information Center, 2017; Tsai & Liu, 1998). The longevity of A. lentisci decreased significantly in the presence of endophytes during the Biology II experiment, a finding in line with observations for R. padi on L. perenne-E. festucae var. lolii (Meister et al., 2006) and L. multiflorum-E. occultans symbioses (Bastias et al., 2017). The data of the Biology II experiment indicated that the effects of AR37 and CT endophytes were possibly mitigated on plants with a larger number of green leaves. Because this plant trait is influenced by many factors (plant genotype [Chapter 4], plant age, water stress etc.) and influences, in turn, many other aspects (e.g. alkaloid production and distribution), its significance will need closer examination in further trials.

The average aphid lifetime fecundity recorded in the Biology I experiment matched that reported in the literature (Wool & Kurzfeld-Zexer, 2008) but was likely lower, than it could have been due to the mothers dying early through handling and heat stress. The

nutrient stress experienced by the plants during the Biology I experiment could also have limited the reproductive potential of the root aphids. As for the longevity, the findings of the Biology II experiment were therefore considered a more accurate estimate of A. lentisci’s fecundity potential. These were in the range observed for aphids in general (National Pesticide Information Center, 2017). Experimental bias rendered any analysis of the lifetime fecundity difficult. Besides, the total lifetime fecundity of an aphid could be less relevant in the field than its reproductive rate in the first days, considering that natural agents are likely to result in an untimely death for many aphids (Adams & van Emden, 1972). The reproductive rate appeared, therefore, a better response variable to analyse in the present thesis. With average daily rates of 2 to > 8 offspring·mother-1_·day- 1_{, the mothers kept ex planta (Mature plant experiment; Table 3.11) were significantly} more fecund than the ones left to reproduce on plant roots (Biology I and II experiments, ~ 1 offspring·mother-1_·day-1_{; Table 3.9). This difference could have been the result of a} large body size developed in the cold outdoor conditions (Dixon, 1985). Large body sizes are known to be positively linked to daily reproductive rates in root aphids (Biology II experiment; Table 3.10) as well as in many other aphid and insect species (Adams & van Emden, 1972; Honĕk, 1993; Schuett et al., 2015; Traicevski & Ward, 2002). The sudden exposure to a warmer laboratory environment (19.6 ± 2.0 °C) could have caused or contributed to the higher reproduction rate, by accelerating all biological processes, embryo development and viviposition included. It is further possible that this was simply a reaction to starving. Accelerated maturation of the largest embryos has been observed in several aphid species when starved [e.g. Megoura viciae Buckton, Aphis craccivora Koch (Traicevski & Ward, 2002; Ward & Dixon, 1982). Actual starvation experiments on A. lentisci with imago dissection will be needed to confirm this explanation, however. Root aphids developing on young, strongly growing plants are likely more fecund as adults than aphids on plants of poorer quality, considering the decreasing reproductive rates on ageing plants and the positive effect of plants with high leaf appearance rates observed in the Biology II and Mature plant experiments, respectively. Which plant traits would per se be pivotal for root aphids could not be assessed from the data at hand, however. The reproductive response to specific endophytes can vary by aphid species (Meister et al., 2006). A. lentisci appeared to respond similarly to R. padi (Bastias et al., 2017; Meister et al., 2006), with smaller reproductive rates in S-CT endophyte symbioses in particular (Table 3.10, Table 3.11). The difference between S-CT and other plant

genotype-endophyte strain combinations was not always significant, however, an observation that in the specific case of the Mature plant experiment could have been related to seasonal endophyte development and activity patterns. During winter, endophyte hyphae numbers and alkaloid concentrations are indeed known to decrease (Christensen & Voisey, 2007; Hennessy et al., 2016; Hume & Cosgrove, 2005).

Offspring of more fecund mothers were larger (Table 3.8) and lived longer (Table 3.12, Table 3.13) than offspring of mothers with a low reproductive rate, supporting previous findings on aphids (Dixon, 1985, 1987; Traicevski & Ward, 2002). Maternal age also appeared significant for offspring fitness in the Biology II experiment only. Traicevski and Ward (2002) reported that nymphs produced late in a mother’s life tended to be larger than earlier-born offspring in several aphid species. This pattern was observed in the S-NIL group of the Biology II experiment only, however. In all other groups, the opposite effect prevailed [‘Lansing effect’ (Zehnder et al., 2007)]. These size changes could possibly explain the reduced offspring survival observed in neonates born to old mothers on S-CT and S-AR1 plants during the Biology II follow-up experiment. Decreasing plant quality during the experiment or maternal age effects (Zehnder et al., 2007) could also have had an effect. Young plants with good growth were beneficial for offspring fitness (Age effect for Biology II offspring in Table 3.12, 24-h-leaf regrowth for offspring on mature plants in Table 3.13). Plant genotype-endophyte groups appeared generally not to affect offspring survival (i.e. fitness) in the Biology II follow-up experiment. Only offspring of fecund mothers on AR1-infected plants lived longer than offspring of fecund S-NIL mothers. S-NIL-born neonates during the mature plant follow- up experiment were exceptionally long-lived compared to all other groups, however. Why this was so is unclear.

During the mature plant follow-up experiment, an unexpected variable of experimental bias was evident, the ‘collection period’. Aphids born in the second viviposition period were clearly shorter-lived than aphids born in the first one (Table 3.13). That could be an effect of maternal food deprivation, a phenomenon that has not yet been documented in root aphids. Whether such an effect would be observed in the field too by food withdrawal [e.g. when a herbicide is sprayed to eliminate an old sward before sowing a new pasture (Trafford & Trafford, 2011)] could be worth investigating.