APV transmission assays: To measure maternal transmission rates
and whether H. defensa impacts vertical transmission, we reared
APV-infected adult aphids of the same genotype with (ND18.H3) or without
(ND18) H. defensa individually in petri dishes (55mm x 15mm)
containing a single V. faba leaf. Adult aphids were monitored for
the production of offspring approximately every 30 min. Since APV can be
transferred from infected aphids to plants during feeding we replaced
fava leaves every 2-3 hours. Nymphs were collected 1-30 min after birth
then surface sterilized in a 1% bleach solution and transferred to a
new fava leaf containing Petri dish. Newborn aphids were individually
reared to adulthood to prevent possible aphid to aphid horizontal
transmission through the leaves. We allowed the first-generation cohort
to produce offspring and develop into third-fourth instar nymphs before
screening for APV infection using the diagnostics previously described.
Fisher’s Exact Test was used to compare rates of maternal APV
transmission among lines. To rule out rapid horizontal transmission in
our Petri dish arenas that would potentially inflate estimates of
vertical transmission, we also conducted a control assay, mimicking the
conditions described above by allowing single APV+ adults to feed on a
single V. faba leaf in a Petri dish. Adults were allowed to feed
continuously for 1 h before removing them and any offspring they
produced. We then added 8-10 second instar -APV aphids, which were
allowed to feed for 30 min on the leaves previously fed upon by +APV
before being separated and reared individually in petri dishes with a
fresh V. faba leaf. These aphids were then allowed to develop
into fourth instars and screened by PCR for the presence of APV.
Horizontal transmission of APV through plants was assessed by placing a
single V. faba in cup cages with three +APV aphids (donor) and
three -APV- aphids (recipient) which were distinguished by using 4
different donor and recipient lines lacking facultative symbionts that
differed in color (pink or green morphs). Cup cage arenas were
maintained at 20° C under 16 h light (L): 8 h dark (D) photoperiod.
Eight third or fourth instar donor and recipient aphids were then
collected after 1 or 3 weeks and screened by qPCR as described above to
assess APV infection status. We also conducted assays to determine if
oviposition by A. ervi could horizontally transfer APV from
infected to uninfected aphids. A female A. ervi was allowed to
oviposit into an +APV aphid and then immediately moved to a separate
arena and allowed to oviposit into three -APV aphids in rapid
succession. The three parasitized -APV aphids were identified by the
order in which oviposition occurred and then placed into separate petri
dishes with a single V. faba leaf. We allowed parasitized APV-
recipient aphids to develop into fourth instars before screening them
for APV infection as above.
Fitness measures: Aphid fecundity in different lines of +APV
and -APV aphids was estimated by allowing cohorts of five fourth instar
aphids to develop into adults on a single V. faba (equals 1
replicate). The number of offspring produced in each cup cage was
carefully removed and counted every 3 or 4 days. In total, there were 9
replicates for each aphid line. Aphid mortality was also recorded and
used to assess 50% survivorship. Aphid reproduction was analyzed using
Analyses of Variance (ANOVA) with Tukey’s HSD to compare means among
aphid lines. Aphid survival data was fit to a lognormal distribution to
estimate 50% survival time.
Enemy challenge assays: Cohorts of 20 aphids that were 48-72 h
old (second instars) were singly parasitized by a mated A. ervifemale and then placed onto a fresh V. faba plant in a cup cage
(=1 replicate). A total of 8 replicates were conducted for each
experimental aphid line (160 parasitized aphids per line). After
parasitism, cup cages were maintained at 20° C under a 16 h light:8 h
dark photoperiod. Ten days post-parasitism, we recorded the number of
aphids that survived, mummified (a pupating wasp), or both aphid and
wasp died (dual mortality) (Oliver et al. 2012). Results were then
analyzed by logistic regression analyses. Since parasitoid fitness is
often linked to host health, we measured hind tibia length to estimate
the size of A. ervi eclosing from APV+ and APV- aphids, which
served as a proxy for wasp quality (Godfray and Godfray 1994, van
Lenteren 2003). One day old adult A. ervi were frozen overnight
at -20°C and then dried at 60°C for 24 hours before measuring hind tibia
length using an Olympus SZX16 stereomicroscope equipped with CellSens
software (v. 1.4.1). Wasp tibia length was analyzed by One-way Analyses
of Variance (ANOVA) to compare mean tibia length between APV+ and APV-
aphid lines.
To assess whether APV infection affects fungal protection conferred byR. insecticola , we challenged aphids with P. neoaphidis as
previously described (Weldon et al. 2020). Ten cohorts of ten 9-day old
(early adult) aphids (total 100 aphids) from each R. insecticolaexperimental line (Table 1) were then exposed to two sporulating aphid
cadavers placed in a 35 mm diameter deep Petri dish with 1.5% agar for
90 minutes. Fungal plates were inverted over aphids to mimic a natural
spore shower and rotated every fifteen minutes between replicates to
normalize spore exposure. Each cohort was then placed onto a freshV. fava plant and kept at 20°C with 100% humidity (via an
unvented cup lid) for 24 hours under 16:8 L:D hour light cycle. After 24
hours, the unvented lid was replaced with a vented lid. Aphids were
monitored every twenty-four hours for ten days post-exposure for aphid
survival, dual mortality (aphid and pathogen), and fungal sporulation.
The results were analyzed using logistic regression.
APV and symbiont abundance: We estimated APV and symbiont
abundance by measuring genome copy number of each. Briefly, +APV and
-APV adult aphids from a given experimental line were placed in separate
cup cages with a fresh V. faba plant and allowed to reproduce for
approximately 24 hours. Thereafter, all adults were removed and
offspring were allowed to develop. Aphids were then sampled at 2, 4, 8
and 16 days old. APV genome copy number was then estimated by generating
cDNA templates from 6-8 aphids at each time point (biological
replicates) as described above followed duplicate qPCR (technical
replication) for each sample using APV-specific primers and reaction
conditions as described above. APV genome copy number per sample was
then estimated by plotting the data against a standard curve generated
by serial dilution of a plasmid containing the APV amplicon and
normalized using a single copy aphid gene (Ef-1α ). Relative
genome copy number for H. defensa was similarly determined at the
same time points using previously reported primers that amplify a region
of the H. defensa dnaK gene (Weldon et al. 2013, Martinez et al.
2014) while relative genome copy number for R. insecticola was
determined using primers designed during this study (Reg_dnaK_Q_F:
5’-TGGTGCAGCAAAAAGTG AAG-3’ and Reg_dnaK_Q_R:
5’-CACCCATGGTTTCAATACCC-3’) that amplify a region of the R.
insecticola dnaK gene. Cycle conditions for the R. insecticola primers
were 95° C for 5 min; 40 cycles of 95° C for 10 s, 60° C for 10 s, 72°
C for 10 s, and a final extension at 72° C for 2 min. Relative abundance
of each symbiont was then determined by the 2-(ΔCT)method (Livak and Schmittgen 2001). Results were log10 transformed, and
the distributions of symbiont titers in each experimental line at each
time point were checked for normality using the Goodness-of-fit test.
Transformed titers were then compared using ANOVA with Tukey’s post-hoc
Honest Significant Difference (HSD) test. Both analyses as well as all
other statistical tests performed during the study were performed using
JMP Pro v. 14.0 (SAS Institute Inc., Cary, NC).