Methods
Aphid, parasitoid and fungus cultures: A. pisum is
cyclically parthenogenetic which enables clonal lines to be produced and
maintained in the laboratory with continued exposure to a
long-day-length photoperiod. Lines used in this study were reared as
earlier described (Oliver et al. 2003) on Vicia faba Broad
Windsor seedlings in Percival biological incubators at 20 ± 1° C on a 16
h light (L):8 h dark (D) photoperiod. Lines hosting particular symbionts
were established by isolating a single parthenogenetic female in a petri
dish with V. fava leaves and allowed to reproduce. Offspring were
then regularly screened for all known pea aphid facultative symbionts
using previously published PCR-based diagnostics (Russell et al. 2013,
Martinez et al. 2014). An A. ervi culture was established from
commercially produced (Syngenta Bioline Ltd.) and field-collected wasps
from Dane County, WI USA, which are maintained on susceptible pea aphid
lines lacking facultative symbionts as previously reported (Oliver et
al. 2003). Adult parasitoids were kept at 20 ± 1° C under a 16 h L: 8 h
D h photoperiod and provided a diet of honey and water. P.
neoaphidis genotype ARSEF 2588 used in this study originated from the
USDA-ARS Collection of Entomopathogenic Fungal Culture and was
maintained on susceptible aphid lines (WI-48 and 5D-AB). Desiccated
fungal cadavers were stored at 4°C with low humidity for no more than
twelve weeks within an airtight container. Prior to experimental use,
cadavers were rehydrated to induce sporulation (described in later
section).
APV identification and sequencing: APV was first discovered in
our laboratory during an RNAseq study that compared five clonal lines
that hosted different strains of H. defensa (Chevignon,
Unpublished data). Following this discovery, we established a qPCR assay
to screen for the presence of APV in all of our laboratory cultures.
Briefly, individual aphids were chilled on ice for 5 min and homogenized
in lysis buffer (10mM Tris-Cl pH 8.2, 1mM EDTA, 25mM NaCl) in 0.2 mL
tubes. Samples were then centrifuged at 4°C for 20 min at 2200 x g.
After centrifugation, 11 µl of supernatant was transferred to new 0.2 mL
tubes which served as a template for complimentary DNA (cDNA) synthesis
using SuperScript IV First-Strand Synthesis Reaction Kit (Invitrogen®).
APV was the detected by real-time quantitative PCR (qPCR) using specific
primers: APV P1 diagF (ACCATCCGAACTTGAACA GG) and APV P1 diagR
(TGAAAGAACAACGCCTGTGA). Ten µl reactions were run using Quantabio
PerfeCTa SYBR Green FastMix chemistry and 0.5 µM of each primer on an
Analytik Jena qTower3 thermal cycler. Cycling
conditions were 95° C for 5 min; 40 cycles of 95° C for 10 s, 58° C for
10s, 72° C for 10 s, and a final extension at 72° C for 2min. For the 12
pea aphid lines testing positive for APV we Sanger sequenced a
~890 bp variable region of APV to examine strain
variation. Using cDNA template generated as described above, we
PCR-amplified a portion of the P1 ORF (Fig. 1A) using APVp1seqF (5’
GATTGCGGTTTTCCATTTGT 3’) and APVp1seqR (5’ GGGGTTTTGCCCTATAGCAT 3’).
PCRs were carried out in a 30 μl mixture using EconoTaq Plus master mix
(Lucigen, USA) that included 15 μl of Taq polymerase, 0.3 μM of forward
and reverse primers, 50 ng of template DNA and nuclease-free water up to
30 μl. The PCR conditions were 95 °C for 3 min (1 cycle), 95 °C for 30s,
60°C for 30 s and 72 °C for 1min (35 cycles), followed by 72 °C for 7
min. The amplified products were visually examined with electrophoresis
using a 1% agarose gel, with DNA products purified using a cycle pure
Kit (Omega Bio-Tek, USA) before submission to Eurofins Genomics for
sequencing.
The RNAseq data set that originally identified APV in some of our
cultures was used to assemble complete genomes for five of the infected
laboratory lines. In brief, this data set was generated by extracting
total RNA from 4th instar aphids from five clonal
lines (three biological replicates consisting of five individual aphids
per line) using the mirVana miRNA Isolation Kit (Ambion, Thermo Fisher
Scientific). After DNase treatment using the TURBO DNA-free Kit (Ambion,
Thermo Fisher Scientific) and ethanol precipitation in the presence of
glycogen, RNA templates were quality checked by the Georgia Genomics and
Bioinformatics Core using an Agilent 2100 Bioanalyzer (Agilent
Technologies) and Fragment Analyzer (Advanced Analytical). cDNA
libraries were then synthesized using the Kapa Stranded RNA-seq Library
Preparation Kit (Kapa Biosystems) and 75 bp paired-end sequenced using
Illumina NextSeq (150 Cycles). APV genomes were assembled by poolingA. pisum unaligned reads with APV reads for de novo assembly with
SPAdes v3.15.3 (Prjibelski et al. 2020) using the parameter rnaviral.
The first APV genome that was sequenced (NC_003780; Van der Wilk et al.
1997) was used as a reference to identify APV contigs using Minimap2
v2.22 with parameter ‘x splice’ (Li 2018). The resulting APV genomes
were then aligned using MAFFT v7.450 (Kuraku et al. 2013) in Geneious
Prime v2022.0.2 (https://www.geneious.com) with standard
parameters and compared to other sequenced APVs including: 1 from Europe
(AF024514) (Van der Wilk et al. 1997), six from China (MH301282 to
MH301287) and four related RNA viruses; YYSMMV1 (Sitobion
miscanthi virus 1; MK733235), two rosy apple aphid virus isolates
(Riboviria; DQ286292, MW929927), and one avian-associated Riboviria
(MT138201). The same viruses were also used to construct a
maximum-likelihood phylogenetic tree using PhyML v3.3.20180621 (Guindon
et al. 2010) implemented in Geneious Prime (v2022.0.2
https://www.geneious.com) with substitution model HK85 and100
bootstraps.
We further screened all lab colonies identified by qPCR to be APV
infected by sequencing an 890 bp region of the P1 ORF using APVp1seqF
(5’ GATTGCGGTTTTCCAT TTGT 3’) and APVp1seqR (5’ GGGGTTTTGCCCTATAGCAT
3’). PCRs were carried out in a 30 μl mixture using EconoTaq Plus master
mix (Lucigen, USA) that included 15 μl of Taq polymerase, 0.3 μM of
forward and reverse primers, 50 ng of template DNA and nuclease-free
water up to 30 μl. The PCR conditions were 95 °C for 3 min (1 cycle), 95
°C for 30s, 60°C for 30 s and 72 °C for 1min (35 cycles), followed by 72
°C for 7 min. Resulting products were visually examined on 1% agarose
gels and then purified using the cycle pure Kit (Omega Bio-Tek, USA)
before Sanger sequencing (Eurofins).
Experimental aphid lines: We engineered aphid lines that varied
in color, facultative symbiont status, and APV infection while
controlling for aphid and Buchnera genotypes (Table 1). Across
five aphid genotypes, each naturally susceptible to A. ervi andP. neoaphidis , we first produced 8 lines that were either green
or red color morphs that either lacked facultative symbionts or hostedR. insecticola or H. defensa (Parker et al. 2013, Doremus
et al. 2018). Lines hosting symbionts were generated by either
microinjecting H. defensa or R. insecticola into aphids
with no facultative symbionts or by selectively eliminating particular
facultative symbionts with antibiotics without harming Buchnera(Doremus and Oliver 2017). We then infected aphids from each of the
above lines with APV to produce a total of 16 lines (8 non-infected
lines (-APV), and 8 infected lines (+APV) (Table 1). +APV lines were
created by either: 1) collecting hemolymph from +APV aphids lacking
facultative symbionts and injecting it into aphids from each of our -APV
lines or 2) allowing aphids from our APV- lines to feed on a diet
inoculated with a homogenate prepared from +APV aphids (Van den Heuvel
et al. 1997). For the latter, 8-10 4th instar +APV
aphids were homogenized in 500 µl of aphid diet (Febvay et al. 1988) in
a 1.5 mL centrifuge tube. The homogenate was then spun down at 6000 x g
for 1 min to remove aphid debris followed by mixing the supernatant with
~1 ml of aphid diet. The mixture was then sandwiched
between two stretched parafilm layers on a 35mm x 10mm petri dish
followed by the addition of 10-15 second instar uninfected aphids that
were allowed to feed for 2 days. The aphids were then transferred to a
fresh V. faba plant to develop into adults. Each line used in
experiments was then generated from a single parthenogenetic female that
was maintained for a minimum of 8 generations before use any assay. We
confirmed bacterial symbiont and APV infection regularly after line
creation and before all experimental assays using previously described
PCR-based diagnostics.