Aphid Facultative Symbionts Aid Recovery of Their Obligate Symbiont and Their Host After Heat Stress

ORIGINAL RESEARCH

published: 19 March 2020

doi: 10.3389/fevo.2020.00056

Frontiers in Ecology and Evolution | www.frontiersin.org 1 March 2020 | Volume 8 | Article 56

Edited by:

Cesar A. Cardenas,

Instituto Antártico Chileno

(INACH), Chile

Reviewed by:

Hugo Mathé-Hubert,

Université de Lorraine, France

Alison Karley,

The James Hutton Institute,

United Kingdom

Christoph Vorburger,

Swiss Federal Institute of Aquatic

Science and Technology, Switzerland

*Correspondence:

Julia Ferrari

[email protected]

†

These authors have contributed

equally to this work

‡

Present address:

Melanie R. Smee,

Microbiology Department, Cornell

University, Ithaca, NY, United States

Specialty section:

This article was submitted to

Population and Evolutionary

Dynamics,

a section of the journal

Frontiers in Ecology and Evolution

Received: 18 October 2019

Accepted: 26 February 2020

Published: 19 March 2020

Citation:

Heyworth ER, Smee MR and Ferrari J

(2020) Aphid Facultative Symbionts

Aid Recovery of Their Obligate

Symbiont and Their Host After Heat

Stress. Front. Ecol. Evol. 8:56.

doi: 10.3389/fevo.2020.00056

Aphid Facultative Symbionts Aid

Recovery of Their Obligate Symbiont

and Their Host After Heat Stress

Eleanor R. Heyworth

†

, Melanie R. Smee

†‡

and Julia Ferrari

Department of Biology, University of York, York, United Kingdom

Environmental conditions affect insect ﬁtness, with many species constrained by speciﬁc

temperature ranges. Aphids are limited to temperate climates and it is hypothesized

that this is partly due to their heat-susceptible obligate nutritional symbiont Buchnera.

Aphids often carry additional facultative symbionts which can increase the host’s ﬁtness

after heat stress. Here we used the pea aphid (Acyrthosiphon pisum) and three of

its facultativ e endosymbionts (Candidatus Regiella insecticola, Candidatus Fukatsuia

symbiotica (X-type; PAXS), and Candidatus Hamiltonella defensa) to investigate how

these species respond to heat stress and whether their presence affects the ﬁtness of

the host or the obligate symbiont. We exposed aphid lines to a single high temperature

event and measured lifetime fecundity and population densities of both obligate and

facultative symbionts. Heat shock reduced aphid fecundity, but for aphids infected with

two of the facultati ve symbionts (Regiella or Fukatsuia), this reduction was less than in

uninfected aphids. The population density of Buchnera was also reduced after heat

shock, and only recovered in aphids infected with Regiella or Fukatsuia but not in

uninfected aphids or those with Hamiltonella. Although heat shock initially reduced the

densities of two of the facultative symbionts (Hamiltonella and Fukatsuia), all facultative

symbiont densities recovered by adulthood. Two of the facultative symbionts tested

therefore aided the recovery of the obligate symbiont and the host, and we discuss

possible underlying mechanisms. Our work highlights the beneﬁcial effects of protective

symbionts on obligate symbiont recovery after heat stress and how facultative symbionts

may affect the wider ecological community.

Keywords: Acyrthosiphon pisum, Buchnera aphidicola, facultative symbiont, heat stress, insect symbionts,

quantitative PCR, symbiosis

INTRODUCTION

It is well-established that infection with bacterial symbionts can aﬀect an insect host’s biology.

Reproductive ﬁtness, insect behavior, immune pathway function, and responses to natural

enemies may all be inﬂuenced by the presence of endosymbionts (

Dion et al., 2011; Gerardo

and Parker, 2014; Vorburger, 2014; Martinez et al. , 2015). By improving the ecological

ﬁtness of a host through raising its immunity to natural enemies, or by enhancing its

tolerance to environmental stress, a vertically transmitted symbiont increases its own ﬁtness

(

Oliver et al., 2005; Brownlie and Johnson, 2009).

Heyworth et al. Symbionts Aid Recovery From Heat-Shock

Rising global temperatures are already aﬀecting insect

populations; there is evidence of range shifts (Parmesan and

Yohe, 2003), changes in phenology (Walther et al., 2002) and

interactions with predators and parasitoids (Harrington et al.,

1999; Schmitz and Barton, 2014

). For several insect groups

infection with various bacterial symbionts has been shown to

enhance resistance to temperature stress (Corbin et al., 2017).

These eﬀects may be direct symbiont-mediated host protection

(Montllor et al., 2002; Neelak anta et al., 2010; Brumin et al., 2011)

or indirect eﬀects of temperature on the symbiont itself (Chen

et al., 2009; Bordenstein and Bordenstein, 2011).

There are a number of hypotheses for the mechanisms

underlying indirect symbiont-mediated protection from heat.

Infection with symbionts has been shown to incre ase the

expression of immune system genes (Laughton et al., 2013),

and it is hypothesized that this immune response controls the

bacteria, restricting growth or location and protecting the host

from microbial over-proliferation (

Kwong et al., 2017; Maire

et al., 2018). It may also bestow temperature tolerance as a by-

product. For example, infection of Rickettsia in whiteﬂies leads

to the upregulation of stress-response genes in the host and

thus increases survival of the insect under heat shock (Brumin

et al., 2011); similarly, insects infected with bacterial symbionts

often produce more immune cells than t h ose that are uninfected

(Schmitz et al., 2012; Weiss et al., 2012; Laughton et al., 2013; Kim

et al., 2015). There are close links between insect responses to heat

and to infection–many heat shock proteins are chaperones that

aid protein production and refolding post-stress, and they may

also enhance immune responses (Young et al., 1993).

Instead of indirectly aﬀecting the host’s stress or immune

responses, symbionts may themselves produce and release heat

shock proteins or metabolites that directly protect the host or

other microbes that the host depends on (i.e., obligate symbionts)

(Burke G. et al., 2010). Obligate symbionts are often a thermal

“weak link” and more susceptible to temperature extremes than

their hosts (Corbin et al., 2017; Shan et al., 2017; Zhang et al.,

2019). Shielding an obligate symbiont from thermal damage

would therefore beneﬁt both the host and all of its symbionts. For

example, in pea aphids that experience heat shock, the density of

the obligate nutritional symbiont Buchnera aphidicola is usually

reduced, but is maintained at near normal levels in aphids that

carry the facultative symbiont Candidatus Serratia insecticola

(hereafter Serratia) (

Burke G. et al., 2010). It is also plausible

that facultative symbionts might be directly protecting the host

by replacing an obligate symbiont t hat is no longer able to

perform its function. When the obligate symbiont Buchnera is

removed using antibiotics at benign temperat ures, Serratia in pea

aphids moved into the bacteriocytes vacated by Buchnera and

subsequently allow the stressed aphid to survive and reproduce

(Koga et al., 2003, 2007).

Pea aphids, Acyrthosiphon pisum, are a model system for

understanding how facultative symbionts protect their hosts

from thermal stress. They and their obligate symbiont are

typically intolerant to heat in laboratory populations (

Dixon

et al., 1987; Dunbar et al., 2007

), but three of t heir eight potential

facultative symbionts (Serratia, Candidatus Fukatsuia symbiotica

and Candidatus Hamiltonella defensa; hereafter Fukatsuia and

Hamiltonella, respectively) are known to improve survival or

reproduction after heat shock (

Montllor et al., 2002; Koga et al.,

2003; Russell and Moran, 2006; Heyworth and Ferrari, 2015). The

obligate symbiont Buchnera synth esizes essential amino acids

for the host, which are required for aphids to thrive on their

imbalanced diet of plant phloem sap (

Douglas, 1998). Buchnera

has a highly reduced genome (Moran, 1996; Gómez-Valero et al.,

2007) and some genotypes are susceptible to hig h temperatures;

under heat st ress, just ﬁve protective heat shock proteins are

deployed (Wilcox et al., 2003) compared to over 75 in its free

living relative Escherichia coli (Carruthers and Mi nion, 2009) and

during severe heat shock Buchnera can be killed.

While the costs and beneﬁts of infection are being explored

in a broad spectrum of insect species, relatively little is known

about how diﬀerent facultative symbionts confer increased

heat tolerance to their hosts, and how these mechanisms vary

depending on symbiont species. Understanding how insects

can and will respond to increases in temperature is vital to

accurately model current and future populations. We investigate

whether three common facultative symbionts of the pea aphid

[Candidatus Regiella insecticola (hereafter Regiella), Fukatsuia

and Hamiltonella] protect the host and how they respond to heat

stress themselves. Importantly, we test whether t he protection

from the eﬀects of heat co-occur with the protection of the

obligate symbiont Buchnera. We test whether the facultative

symbionts directly protect Buchnera, allow it to recover after heat

stress or protect the host by replacing Buchnera and whether this

mode of protection is similar for all tested facultative symbionts.

MATERIALS AND METHODS

Aphids and Symbionts

Rapid, asexual reproduction results in clonal lines of aphids that

can be kept indeﬁnitely under long-day conditions. This allows

the manipulation of facultative symbiont presence through

antibiotic curing or artiﬁcial infections while maintaining

an essentially identical aphid genotype. Two pea aphid

genotypes were used for this study, both collected from the

UK (Supplementary Table S1). Genotype 218 was collected

naturally infected with Fukatsuia and Hamiltonella, and was

cured more than a year before use. This was achieved by

feeding young aphids with broad bean leaves suspended in a

tube of antibiotic solution (0.5% Gentomicin, 1% Ampicillin,

0.5% Cefotaxime in distilled water) over 4 days (

McLean

et al., 2011). Genotype 2 00 was collected naturally uninfected,

harboring no known facultative symbionts. All aphid lines

were screened for Hamiltonella, Regiella, Serratia symbiotica,

Fukatsuia, Spiroplasma sp., Rickettsia sp., and Rickettsiella viridis

following protocols in Tsuchida et al. (2010) and Ferrari et al.

(2012) to ensure that they had the appropriate symbiont

infection and were not infected with any other known facultative

symbionts. The symbiont infections were regularly checked

to detect possible contamination. The symbiont-speciﬁc PCR

primers can be found in Supplementary Table S2. The PCR mix

comprised 6.25 µl BioMix (Bioline), 0.1 µl (20 µM) of forward

and 0.1 µl (20 µM) reverse primer, 5.55 µl distilled water and 1.0

µl sample DNA. The PCR reaction was performed at 94

◦

C for

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Heyworth et al. Symbionts Aid Recovery From Heat-Shock

2 min, followed by 35 cycles of: 94

◦

C for 30 s, 55

◦

C for 30 s, and

◦

C for 1 min. It concluded with 6 min at 72

◦

C and then cooled

the sample to 4

◦

C indeﬁnitely. PCR products were run on a 1%

agarose gel and t h e presence of a band conﬁrmed the presence of

the symbiont.

Five aphid lines were used in the experiment, two uninfected

with facultative symbionts (200 and 218) and the remainder

infected singly with one of three facultative symbionts, Regiella,

Fukatsuia, and Hamiltonella (Supplementary Table S1). Regiella

was injected into aphid genotype 200, while the other two

symbionts were injected singly into genotype 218. These ﬁve

aphid lines comprise three pairwise comparisons between

uninfected and infected aphids, with the uninfected line of 218

used in two comparisons. This design aimed to compare host

ﬁtness and symbiont densities within each pair across the same

aphid genetic background and thus we conducted no analyses

across multiple pai rs. These speciﬁc isolates of symbiont were

chosen because preliminary results indicated that they were likely

to provide heat shock protection.

To produce these infections of Hamiltonella, Regiella, and

Fukatsuia we used hemolymph injections from infected donor

aphids (Supplementary Table S1). Hemolymph was extracted

from donor aphids under a microscope using glass needles

and re-injected into the appropriate aphid line and surviving

aphids raised to adulthood. Glass needles were pulled from Kwik-

Fil

borosilicate glass capillaries (1B100-4, World Precision

Instruments, 1 mm diameter) using a P-97 Flaming/Brown

micropipette Puller (Sutter Instrument Co.). The oﬀspring of

the surviving aphids were tested for the successful establishment

of the new infection when they were adults, and these aphid

lines were retested regularly to ensure the maintenance of the

new aphid-symbiont combinations. All injected lines had been

maintained in the laboratory for at least a year before being used

for experiment al assays.

Heat Shock Protocol

This assay was designed to understand symbiont dynamics after

aphids have been exposed to heat shock. Aphids were exposed

to either a single peak of high temperatures or were maintained

at a steady control temperature. The “heat shock” temperature

chosen was 38.5

◦

C, which was based on a series of pilot studies

(data not shown). Our aim was to ﬁnd a temperature that had

a strong negative eﬀect on ﬁtness, but at whi ch approximately

half the i ndividuals in an aphid population still survived. The

aim was to explore the phenotypes of the symbionts and not to

model natural situations directly. The temperature experienced

by aphids near the ground is often considerably higher than

meteorological records and depends, for example, on aspect and

slope of the site, but it is likely that aphids are exposed to similarly

high temperatures in Northern England on hot summer d ays

(

Bennie et al., 2008; Suggitt et al., 2011).

To produce age-controlled populations of each of the ﬁve

lines, groups of young adults were placed into petri dishes (9 cm

diameter) that contained a single broad bean (Vicia faba var.

Sutton Dwarf) leaf, placed in 2% agar. V. faba is a host plant that

almost all pe a aphids perform on (

Ferrari et al., 2008). The adults

were left to reproduce for 24 h at 20

◦

C, and the oﬀspring were

subsequently put onto 2 week old V. faba plants in groups of 50

and enclosed in a vented, transparent cage. On the following day

(aphid age 24–48 h) the populations were moved into cabinets

where they were either exposed to heat st ress or 20

◦

C as a

control. Temperature cabinets were rigorously checked before

and during the experiments to ensure even distribution of heat

and the same relative humidity (50%) in both cabinets. Plants

containing aphids were also placed in a randomized block pattern

within the cabinet to remove any potential eﬀects of uneven

heat distribution.

While the control treatment was left at 20

◦

C, the temperature

in the heat treatment was increased from 20 to 38.5

◦

C ste adily

over the course of 2 h, held at 38.5

◦

C for 4 h, and then

decreased back to 20

◦

C over a further 2 h. Surviving aphids

from bot h treatments were moved onto fresh 2-week-old plants

on the day after heat shock to mitigate any temperature

eﬀects on the plant itself. These plants were moved into a

diﬀerent controlled-temperature room (20

◦

C), where aphids

from both heat treatments were kept together until being

collected for analysis.

Aphids were removed to measure symbiont density at two

time points. The ﬁrst was 24–26 h after the start of the peak heat

shock period (when the aphids were 3 days old), and the second

11 d ays post-heat shock (when the aphids were 14 days old, ∼6

days after an aphid would usually begin reproducing). These

two time points were chosen to investigate symbiont densities

immediately after stress, and to test if recovery by t h e onset

of reproduction was possible. Buchnera densities are known to

decrease as aphids age (

Simonet et al., 2016) and so this second

time point was chosen to be the potential highest density of

Buchnera during an aphid’s development.

The aphids were ﬂash frozen using dry ice and kept at

−80

◦

C until DNA extraction. In addition, one surviving apterous

individual from each group was placed on a petri dish with a V.

faba leaf (as above) to measure the number of oﬀspring produced.

These dishes were refreshed every 3–4 days to ensure healt hy V.

faba leaves. Oﬀspring counts continued until all aphids had died,

measuring total lifetime fecundity. There were 5–6 replicates

for fecundity counts and symbiont density for each of the ﬁve

aphid lines in each treatment (i.e., 10–12 replicates in total for

each line), these were performed in two temporal blocks with

approximately half the replicates of each treatment in each block.

qPCR Protocol

DNA was extracted from the aphids after samples were defrosted

at room temperature. Aphids were homogenized in a 200 µl

5% Chelex solution made in distilled water. Ten microlite rs of

proteinase K (10 mg/ml) was added per sample, and samples were

incubated for 6 h at 56

◦

C to facilitate digestion. They were then

“boiled” at 100

◦

C for 10 min before being centrifuged at 13,000

rpm for 3 min and the supernatant containing the DNA pipetted

into a clean 1.5 ml Eppendorf tube which was stored at −20

◦

until use. Five aphids per replicate were pooled to generate a

sample for the ﬁrst time point (24–26 h after heat shock) and one

aphid for the second time point (11 days later).

Samples were run in duplicate using SYBR



Green reagent on

a StepOnePlus

Real Time PCR machine (A pplied Biosystems).

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Heyworth et al. Symbionts Aid Recovery From Heat-Shock

Each reaction consisted of 10 µl FAST SYBR 2× mastermix

(Applied Biosystems), 1 µl forward primer (7 µM), 1 µl reverse

primer (7 µM), 6 µl nuclease-free water, and 2 µl DNA sample.

qPCR primers for Regiella, Fukatsuia, Hamiltonella, and the

aphid housekeeping gene elongation factor-1 alpha (EF-1α)

(Supplementary Table S2) were tested to ensure high eﬃciency

and similarity between primer sets. Melt curves were performed

on each plate to ensure the primers were speciﬁc to each target

and only bound once. Cycling conditions were 95

◦

C for 20 s,

followed by 40 cycles of 95

◦

C for 3 s and 60

◦

C for 30 s. The

melt curve involved a further 95

◦

C for 15 s, 60

◦

C for 1 min

and then a gradual increase to 95

◦

C over 15 min. Each 96-well

qPCR plate was a nalyzed using StepOne Software v2.2.2 (Applied

Biosystems) and Cycle threshold (Ct) values were obtained by

comparing each primer sample to a single standard curve of

known concentration and using identical t h reshold and baseline

levels for each primer target across plates. Standard curves

were created by amplifying positive control samples using PCR,

calculating DNA concentrations using a High Sensitivity D NA

Assay on a 2100 Bioanalyzer system (Agilent), and then serially

diluting the sample 1:10 with distilled water to create a 5-sample

curve comprising known concentrations decreasing from 10

pmol/ml. Samples with Ct values over 30 were classed as negative,

conﬁrmed by our negative controls. This corresponds to a copy

number of <52 per 2 µl sample for all primers, and is below the

threshold of detection. Where the diﬀerence in Ct values between

technical replicates was >1.5, the sample was either rerun or not

used in the analysis.

The standard curves were used to calculate the DNA

concentration of each sample, and this was converted into copy

numbers per 2 µl of DNA extract. To control for aphid size and

extraction quality, copy numbers for each sample were presented

relative to those of a housekeeping aphid gene as a control, giving

a ratio of symbiont copy numbers to aphid copy numbers.

Statistical Analysis

Data were analyzed using the R software v. 3.4.1 (

R Core Team,

2018). Since our core question was to test whether the three

symbiont species can provide heat shock protect ion, but not to

compare th e extent of this protection, the data were analyzed

separately for each symbiont species. Thus, in each analysis the

infected line was compared with the same uninfected aphid

genotype, within and across heat treatments. The d a ta for the

uninfected line 218 was therefore used twice, paired wit h line 218

infected with Hamiltonella or Fukatsuia. Similarly, we analyzed

the two time points separately because symbiont densities

change during aphid development, which would complicate the

interpretation of th e analysis.

Lifetime fecundity of the set of Regiella lines was analyzed

using a general linear model assuming a normal error

distribution. The number of oﬀspring was the response

variable, and temporal block, facultative symbiont presence,

heat treatment and the interaction between symbiont presence

and heat treatment were the explanatory variables. The sets

of Fukatsuia and Hamiltonella lines were analyzed with

a non-parametric Kruskal-Wallis test, because the model

assumptions of parametric models were not met. This was

followed by Wilcoxon tests to identify diﬀerences between

speciﬁc treatments.

The densities of the symbionts were also analyzed with a

general linear model assuming a normal error distribution.

This was split into six analyses, separate for the lines relating

to each symbiont species at each time point, to simplify the

interpretation. For Buchnera densities, t he explanatory variables

were again temporal block, f acultative symbiont presence and

heat treatment as well as the interaction between the latter factors.

For the densities of the facultative symbionts, only block and

heat treatment were explanatory variables. In most cases model

assumptions were met without transforming the data, only the

Buchnera densities at the ﬁrst time point in the Regiella lines were

log-transformed. For Regiella densities at the ﬁrst time point and

Buchnera densities in the set of Regiella lines at the second time

point, Kruskal Wallis and Wilcoxon tests were used as described

for t h e fecundity data.

For all general linear models, post-hoc tests were only

performed when the factor or interaction was signiﬁcant in

the main analysis. This was conducted using the R package

“phia” (

De Rosario-Martinez, 2015), with Holm’s correction for

multiple comparisons. All data are available as Supplementary

Material (Data Sheets 2–4).

RESULTS

Effects of Facultative Symbionts on

Fecundity After Heat Shock

We exposed aphids to a short spike of high temperature and

measured facultative and obligate symbiont densities and aphid

ﬁtness after 1 and 11 days. As expected, heat shock decreased

the number of oﬀspring produced in an aphid’s lifetime in all

three sets of lines [Regiella F

(1, 19)

= 103.23, P < 0.001; Fukatsuia:

W = 109, P = 0.03, Hamiltonella: W = 136.5, P = 0.001;

Figure 1]. H owever, the extent of this decrease was modiﬁed by

the presence of Regiella and Fukatsuia [Regiella, symbiont × heat

treatment: F

(1, 19)

= 5.78, P = 0.03; Fukatsuia: heat treatment in

uninfected lines W = 36, P = 0.004 and in infected lines W =

13, P = 0.48]. Fukatsuia provided the greatest protection from

heat as there was no diﬀerence in the fecundity of the infected

lines in the control and heat shock treatment, whereas there

was a greater reduction in fecundity in the uninfected aphids

than in the infected aphi ds for the Regiella lines. In contrast,

there was a similar decrease in fecundity for both uninfected and

infected Hamiltonella lines following heat shock (uninfected: W

= 36, P = 0.004; infected: W = 34.5, P = 0.008; Figure 1). At

benign temperatures, two of the symbionts also aﬀected lifetime

fecundity: the presence of Hamiltonella increased lifetime

fecundity (W = 4, P = 0.03), whereas Fukatsuia decreased

it (W = 34, P = 0.013), and there was no diﬀerence for

Regiella (Figure 1).

Facultative Symbiont Densities After Heat

Shock

We measured the densities of the three facultative symbionts

at two time points after exposure to heat, 24–26 h and 11

Frontiers in Ecology and Evolution | www.frontiersin.org 4 March 2020 | Volume 8 | Article 56

Heyworth et al. Symbionts Aid Recovery From Heat-Shock

FIGURE 1 | The effect of heat shock and facultative symbiont presence on the

number of offspring produced by pea aphids. Aphids from genotype 200 that

were uninfected or carrying Regiella are compared in the ﬁrst panel. Aphids

from genotype 218 that were uninfected or carrying Fukatsuia or Hamiltonella

are compared separately in the second and third panels, respectively. In all

comparisons, there was a signiﬁcant effect of heat shock compared to

controls, but no overall effect of symbiont presence. Means and standard

errors are shown. Within each panel separately, upper case letters denote

signiﬁcant differences between aphids with different symbionts within the

control treatment and lower case letters denote signiﬁcant differences in the

heat shock treatment. The asterisks show signiﬁcant differences between heat

treatments for aphids of the same symbiont status (**P < 0.01, ***P < 0.001).

days post-heat shock (Figure 2). Compared to non-heat shocked

controls the densities of two of the symbionts, Fukatsuia and

Hamiltonella, were lower on the day after heat shock [Fukatsuia:

(1, 8)

= 65.05, P < 0.001, Hamiltonella: F

(1, 8)

= 6.64, P =

0.03], whereas densities of Regiella are unaﬀected (W = 28, p

= 0.13; but note that this is signiﬁcant in a less conservative

parametric test). By the second time point, taken when the

aphids were young adults, there was no diﬀerence between

population densities in heat stressed or control aphids for any

of the three facultative symbionts [Fukatsuia: F

(1, 8)

= 0.35, P =

0.57, Hamiltonella: F

(1, 7)

= 0.95, P = 0.36, Regiella: F

(1, 6)

= 0.01,

P = 0.91; Figure 2], suggesting that heat did not have long-term

eﬀects on faculta tive symbiont populations.

Obligate Symbiont Densities Under Heat

Shock

Compared to the control treatment densities of Buchnera were

decreased on the day after he at shock in each of the three pairs

of lines, regardless of facultative symbiont infection [Regiella

lines: F

(1, 19)

= 80.36, P < 0.001, Fukatsuia lines: F

(1, 18)

70.71, P < 0.001, Hamiltonella lines: F

(1, 18)

= 56.07, P <

0.001; Figure 3]. Regardless of treatment, Buchnera densities

were higher in the lines harboring Fukatsuia [F

(1, 18)

= 33.86, P <

0.001] or Hamiltonella [F

(1, 18)

= 24.52, P < 0.001] compared to

uninfected lines, an eﬀect that was not seen in the Regiella lines

(1, 19)

= 0.15, P = 0.71]. For the Fukatsuia and Hamiltonella

lines there was also a signiﬁcant interaction between symbiont

presence and heat treatment [Fukatsuia: F

(1, 18)

= 9.57, P =

0.006; Hamiltonella: F

(1, 18)

= 4.80, P = 0.04]: in both cases,

FIGURE 2 | Densities of facultative symbionts in pea aphids after heat shock

or in the control treatment (A) 24–26 h after the onset of the heat shock and

(B) 11 days after heat shock. Densities are shown as the copy number of the

gyrB gene of the facultative symbiont relative to copy number of the aphid

gene EF1-α. Means and standard errors are shown. Asterisks denote

differences between heat treatments for aphids carrying a given symbiont

(*P < 0.05, ***P < 0.001).

Buchnera densities in the control treatment were higher in lines

with facultative symbionts compared to uninfected aphids but

there was no diﬀerence between these lines after heat shock. The

interaction was not signiﬁcant for the Regiella lines [F

(1, 19)

2.58, P = 0.13] where the extent of the loss of Buchnera did not

diﬀer between infected and uninfected lines.

At the later time point, when aphids were young adults, heat

shock again reduced Buchnera densities on average [Regiella

lines: W = 105, P = 0.004; Fukatsuia lines: F

(1, 19)

= 6.85, P =

0.02; Hamiltonella lines: F

(1, 17)

= 24.84, P < 0.001]. Fukatsuia

presence on average also signiﬁcantly increased the density of

Buchnera regardless of treatment which was due to high Buchnera

densities in the heat shocked aphids [F

(1, 19)

= 7.89 , P = 0.01]; a

diﬀerence that was not found for Regiella (W = 46, P = 0.37)

or Hamiltonella presence [F

(1, 17)

= 0.17, P = 0.69]. Importantly,

there was a signiﬁcant interaction between symbiont infection

and temperature in the Fukatsuia lines [F

(1, 19)

= 7.55, P = 0.01]

Frontiers in Ecology and Evolution | www.frontiersin.org 5 March 2020 | Volume 8 | Article 56

Heyworth et al. Symbionts Aid Recovery From Heat-Shock

FIGURE 3 | The effect of heat shock and facultative symbiont presence on

densities of the primary symbiont Buchnera in pea aphids. In each panel

Buchnera densities are shown 1 or 11 days after heat shock. Uninfected

aphids are compared to aphids carrying (A) Regiella, (B) Fukatsuia, and (C)

Hamiltonella. The uninfected replicates are the same for the Fukatsuia and

Hamiltonella lines. Means and standard errors are shown. In all cases, there

was a signiﬁcant overall difference between the heat shock and control

treatments. There was also a signiﬁcant overall effect of symbiont presence for

the Fukatsuia lines at both time points, and Hamiltonella lines 1 day after heat

shock. These main effects are not illustrated on the ﬁgure. post-hoc results

from signiﬁcant interactions are denoted by upper case letters for signiﬁcant

differences between aphids with different symbionts within the control

treatment, and lower-case letters denote s igniﬁcant differences in the heat

shock treatment. The asterisks show signiﬁcant post-hoc differences between

heat treatments for aphids of the same symbiont status

(**P < 0.01, ***P < 0.001).

and an equivalent eﬀect in the Regiella lines (heat t reatment in

uninfected lines: W = 36, P = 0. 004 and in infe c ted lines: W

= 17, P = 0.42) that was not seen for the Hamiltonella lines

(1, 17)

= 1.95, P = 0.18]: Buchnera densities after heat shock

were signiﬁcantly reduced in uninfected aphids but not when

Fukatsuia or Regiella were present.

DISCUSSION

Our results show that diﬀerent aphid symbionts can protect

the aphid from heat and help the obligate symbiont to recover

after heat shock. Infection with Regiella and Fukatsuia was

closely linked to Buchnera recovery after heat shock and led

to increased production of oﬀspring compared t o uninfe ct ed

controls where as there was no such protection in aphids infected

with Hamiltonella. This pattern diﬀers from other studies (

Russell

and Moran, 2006; Doremus and Oliver, 2017), which found

that Hamiltonella but not Regiella or Fukatsuia provided heat

protection. As diﬀerent lines of insects and symbionts were

used in these studies, it is likely that these protective eﬀects

are dependent on symbiont, host genotype or their interaction

and are thus not a universal feature of symbiont infection. The

prevalence of he at protection may be overestimated here since

we chose genotypes based on preliminary results.

Buchnera densities a re closely linked to aphid ﬁtness.

Disrupting the obligate symbiosis by removing Buchnera leads

to large reductions in oﬀspring production and often host death

(Koga et al., 2003; Akman Gündüz and Douglas, 2009) Overly

high densities of Buchnera can also lead to a reduction in

ﬁtness (Chong and Moran, 2016), meaning that the relationship

between density of t h e symbiont and number of oﬀspring

produced is not dire ct ly proportional. However, removal of

Buchnera, via antibiotics or heat, as also shown in our results,

generally leads to aphid sterility in the absence of facultative

symbionts (Dunb ar et al., 2007; Koga et al., 2007).

A key question that we addressed was whether the facultative

symbionts protect Buchnera from the eﬀects of heat, and if so

whether Buchnera is dire c tly protected or its recovery facilitated.

The densities of both Buchnera and the facultative symbionts

were reduced 24 h after heat shock. In aphids carrying Fukatsuia

or Regiella, these densities returned to levels that were similar to

those in non-heat shocked controls, thus demonstrating a clear

role of the facultative symbionts in the recovery of Buchnera.

The most parsimonious interpretation of the observed pattern

is that th e facultative symbionts do not provide immediate

protection, although it is possible t hat the decline in Buchnera

DNA density occurs due to diﬀerent processes in aphids with

and without facultative symbionts. It is conceivable that Buchnera

is only truly killed by heat inside the latter and growth

is merely arrested in the former and thus some immediate

protection occurs.

The patterns of obligate and facultative symbiont densities

obser ved here suggest that a diﬀerent mechanism underlies the

heat protection provided by Regiella and Fukatsuia compared

to that provided by Serratia (

Montllor et al., 2002; Burke G.

et al., 2010). After heat shock, Serratia in pea aphids lyse and

this coincides with metabolomic changes (Burke G. et al., 2010).

Frontiers in Ecology and Evolution | www.frontiersin.org 6 March 2020 | Volume 8 | Article 56

Heyworth et al. Symbionts Aid Recovery From Heat-Shock

At the same time Buchnera densities are maintained at similar

levels to those at benign temperatures (Burke G. et al. , 2010). In

our experiments, in aphids with Regiella or Fukatsuia, Buchnera

densities initially decrease. This demonstrates that the protection

provided by the facultative symbionts is not instant and suggests

that the protection is probably not due to a constitutively

activated aphid stress response, but it is still possible that the

facultative symbionts prime a stress response by the aphid that

helps recovery later on.

The provision of heat-protective compounds, through either

lysis or release from a live cell, is also consistent with our

obser vations.

Burke G. et al. (2010) explored which metabolites

are aﬀected by heat treatment when Serratia lyse. They found

three metabolic changes linked to the presence of the protective

Serratia after heat stress, one of which is a decrease in

concentration of the antioxidant indole-3-lactate, as well as two

other unidentiﬁed metabolites (

Burke G. et al., 2010). The initial

decline of Fukatsuia in our study suggest that lysis is likely

in this sy mbiont (and possibly in Regiella where the decline

is signiﬁcant using less conservative statistics). However, the

protective compounds released during this process appear to act

later than in Serratia (Burke G. et al., 2010) as Buchnera also

declines initially. In some cases, facultative symbionts can replace

the function of an obligate symbiont (Koga et al., 2003, 2007)

and this could be a way of protecting the host from the eﬀects of

heat. In our system, bot h Buchnera and the facultative symbionts

recover demonstrating that this functional replacement is not a

likely mechanism here.

Some strains of Buchnera are more resistant to heat than

others (

Dunbar et al., 2007; Moran and Yun, 2015) and it

is possible that the two aphid genotypes here carry diﬀerent

Buchnera genotypes. Aphids in laboratory populations often

carry Buchnera strains with the ibpA

mutant allele that are

more sensitive to heat but have higher ﬁtness (Burke, G. R. et al.,

2010). We did not sequence ibpA in the genotypes used here

since we were interested in the eﬀects of facultative symbionts

and the Buchnera strain is the same within all our comparisons.

It is worth noting that the Fukatsuia and Hamiltonella lines

both had t h e same aphid and Buchnera genotype but only

Fukatsuia protected, suggesting that the Buchnera strain does

not bias our conclusions. However, in natural populations the

absence of facultative symbiont infections is correlated with a

higher incidence of this mutation (

Burke, G. R. et a l., 2010).

Aphids thus have two mechanisms which protect Buchnera from

heat: the absence of the heat-sensitive ibpA

mutant allele

and the presence of protective facultative symbionts. Facultative

symbionts appear to confer low ﬁtness in the presence of ibpA

and are thus likely selected against in aphids with this mutation

(

Burke, G. R. et al., 2010). It thus suggests that in natural

populations facultative symbionts may only be able to rescue

the a phid Buchnera from heat in aph ids that carry the heat-

tolerant ibpA

allele a nd this may explain the scarcity of ibpA

in natural populations.

As well as the protective eﬀect of Fukatsuia and Regiella,

we observed interesting changes in symbiont densities at

benign temperatures. Fukatsuia decreased fecundity, as shown

previously (

Heyworth and Ferrari, 2015; Doremus and Oliver,

2017

); the densities of Buchnera conﬁrm that this is not due

to suppression of the obligate symbiont (Koga et al., 2003),

which has been observed for a costly infection by Rickettsia in

pea aphids (Sakurai et al., 2005). Surprisingly, infection with

Fukatsuia or Hamiltonella leads to an increase of Buchnera

population levels in younger aphids. This may beneﬁt both the

aphid and Buchnera when facultative symbionts are present,

because additional nutrients may be required. It is possible that

either the aphid upre gulates Buchnera densities or that this strain

of Buchnera responds to the presence of facultative symbionts by

increasing its growth rate. In either case, the density of Buchnera

cells in infected aphids is comparable to uninfected aphids once

the aphids are adult.

The ability of facultative symbionts to protect obligate

nutritional symbionts from heat stress has implications for

the frequencies and spread of both the microbes and the

insects themselves. Many ph ytophagous insects rely on obligate

symbionts to provide essential nutrition, but these are often

vulnerable to ecologically stressful situations (

Bennett and

Moran, 2015; Kikuchi et al., 2016) due to severe genome

reduction during coevolution with their hosts (McCutcheon

and Moran, 2011). This genome reduction probably led to the

heat sensitivity that some facultative symbionts ameliorate, an

example of a symbiosis rescuing another symbiosis. It seems

improbable that this rescue resulted from close coevolution

due to the relatively transient nature of facultative symbiosis

infections (Smith et al., 2015).

As we and others (Montllor et a l., 2002; Burke G. et al.,

2010

) have shown, carrying certain isolates of facultative

symbionts can protect obligate symbionts from a single, short

exposure to heat, but it remains to be investigated whether

this protection is also eﬀective under long-term or regular

exposure to extreme temperatures. The temperature-dependent

ﬁtness eﬀe ct s are likely to alter the frequencies of facultative

symbionts in natural populations, but will do so in concert

with other abiotic and biotic factors, including the frequencies

of heat-tolerant Buchnera strains. The symbionts’ ability to

aﬀect interactions between host and natural enemies is also

well-documented (Hr

cek et al., 2016). These interactions can

be aﬀected by a change in temperature, through an eﬀect of

temperature on the natural enemy itself (

Roux et al., 2010;

Nguyen et al., 2013) or on the interaction between host, symbiont

and natural enemy (Guay et al., 2009; Jeﬀs and Lewis, 2013;

Heyworth and Ferrari, 2016). In addition, both vertical and

horizontal transmission frequencies of symbionts can be aﬀected

by temperature (Anbutsu et al., 2008; Osaka et al., 2008; Liu et al.,

2019). A combination of temperature-dependent ﬁtness eﬀects

and transmission dynamics is therefore a likely reason for the lack

of a clear correlation between symbiont mediated beneﬁts seen

in laboratory experiments and symbiont frequencies observed

in the ﬁeld (Oliver et al. , 2014), and probably contributes

to the geographic variation in the composition of facultative

symbiont communities (Montllor et al., 2002; Tsuchida et al.,

2002; Sepúlveda et al., 2017

). There are, however, examples where

the patterns based on laboratory experiments are observed: the

frequencies of the aphid heat-protective symbiont Serratia are

high in host populations in the warmer climes of Southern

Frontiers in Ecology and Evolution | www.frontiersin.org 7 March 2020 | Volume 8 | Article 56

Heyworth et al. Symbionts Aid Recovery From Heat-Shock

USA (Chen and Purcell, 1997; Montllor et al., 2002) a nd more

generally in arid compared to temperate regions (Henry et al.,

2013). Similarly, land temperature correlates with symbiont

prevalence in midges (Morag et al., 2012).

Facultative symbionts alter insect ﬁtness under stressful

conditions and can aﬀect not just the host, but also species

that the host interacts with directly and indirectly (

McLean and

Godfray, 2016; Doremus et al., 2018). In extreme cases, hosting

a defensive symbiont can lead to cascading extinctions and

the collapse of entire communities (Sanders et al., 2016). Our

work highlights how the host and its symbiont community is

aﬀected by temperature and that this temperature-dependency

might result in changes of community interactions under

climate change.

DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the

article/Supplementary Material.

AUTHOR CONTRIBUTIONS

EH, MS, and JF conceived the ideas and designed methodology,

collected the data, wrote the manuscript, and gave ﬁnal approval

for publicati on. EH and JF analyzed the data .

FUNDING

EH was supported by a studentship from th e Biotechnology

and Biological Sciences Research Council (BBSRC, award

BB/F016751/1) and JF and MS by award BB/J00524X/1

from BBSRC.

ACKNOWLEDGMENTS

We would like to thank Alison Fenwick, Chris Lancaster, and

Paul Scott for setting up and monitoring the cabinets for the heat

shock treatment, Sally James in the Technology Facility at the

University of York for support with the qPCR , and Sa lly Raines

for helping with aphid culture and helpful discussions.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found

online at: https://www.frontiersin.org/articles/10.3389/fevo.

2020.00056/full#supplementary-material

Data Sheet 2 | Densities of the obligate sy mbiont Buchnera aphidicola in the two

heat treatments in the presence and absence of facultative symbionts.

Data Sheet 3 | Densities of the facultative s ymbionts in the two heat treatments.

Data Sheet 4 | Lifetime fecundity of pea aphids in the two heat treatments in the

presence and absence of facultative symbionts.

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Conﬂict of Interest: The authors declare that t he research was conducted in the

absence of any commercial or ﬁnancial relationships that could be construed as a

potential conﬂict of interest.

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