2 Materials and
methods
2.1 Study organisms
The laboratory culture of the host P. interpunctella and larval
hosts parasitised by V. canescens larvae were kept in temperature
controlled incubators at University of Leeds, UK, under a constant
temperature of 28℃ and 16:8 light: dark cycles (Jones et al., 2015,
Mugabo et al., 2019). Adult V. canescens were kept at ambient
temperature, with a sugar water solution for maintenance. Larval hosts
were provided with a wheat bran diet (same with Mugabo et al., 2019). No
artificial source of moisture was provided inside the incubator for the
laboratory culture to date (Figure S1).
2.2 Experimental
procedure
We conducted a life history experiment where hosts were kept
individually for their entire life cycles. Eggs were collected for 24
hours from approximately 50 randomly-selected newly emerged host adults
from the laboratory culture (Jones et al., 2015). The eggs were then
transferred into 25 well (1cm x 1cm) clear plastic plates (Sterilin
Limited, Thermo Fisher Scientific, UK), with a single egg plus 0.3g food
added into each cell, which is sufficient for complete host development
(see Mugabo et al., 2019). A piece of 2-ply tissue paper and a nylon
mesh were placed between the well-plate and lid to provide ventilation,
while also preventing host larvae from moving between cells or escaping.
We prepared 1360 eggs for the experiment, and assigned these eggs in
different treatment combinations. We first placed half of the eggs
(i.e., 680 eggs), in an incubator with artificially manipulated
humidity, with the other half placed in an incubator with no humidity
manipulation, both under a constant 28 ℃ with 16:8 light: dark cycle.
Artificial humidity was created by placing ~1000 ml of a
saturated sodium chloride solution in the humidity treatment incubators
(Solomon, 1951). Pilot data showed that at a constant temperature of 28
℃ or 38 ℃ the solution can increase the relative air humidity (RH) from
30 ± 5% in the incubators with no solution to 60 ± 5% (Fig S1 -S4).
Larval development was checked daily to identify when they were at
either the 4th instar or 5th instar.
To avoid changes in the microclimate during monitoring of host
development inside the 25-well plates, which may affect their
development rates, 200 extra eggs were set up in the same way and
monitored to estimate the overall developmental status of the
experimental cohort. When host larvae had reached their early fourth
instar, half of them were parasitised in each humidity treatment (i.e.,
340 eggs per humidity treatment; hereafter ‘H-P’ treatment) and
half were unparasitized in each humidity treatment (hereafter ‘H’
treatment). To parasitize hosts in the ‘H-P’ treatment, we took each
host out of its cell, and placed it in a petri dish under normal
laboratory conditions. A newly emerged parasitoid wasp from the stock
culture was placed with a host until it was parasitised, which was
confirmed when V. canescens performed a unique cocking motion
(Rogers, 1972). Each parasitized host was put back into its original
cell and original treatment, to continue development.
170 parasitized hosts and 170 unparasitized hosts that were kept in a
humid or non-humid environment, were then exposed to heat waves in their
fourth instar (immediately after parasitizing), and the rest were kept
in their original incubators until they were exposed to heat waves in
their fifth instar (~5th day after
parasitizing). Heat waves were generated by transferring parasitised and
unparasitized larvae into incubators at a constant 38℃ incubators
(either with or without artificially increased humidity) for different
durations of 6 hours or 72 hours. Control parasitised and unparasitized
larvae were kept at 28ºC (i.e. no heatwave). Larvae exposed to the
heatwaves were returned to 28ºC and corresponding humidity conditions
and monitored daily until host or parasitoid adults emerged and died
naturally. Collectively, the treatments comprised unparasitized hosts
and hosts parasitised in the 4th instar, which were
kept in a humid or non-humid environment, and exposed to heatwaves of
durations of 0, 6, or 72 hours in their fourth or fifth instar. There
were 50 individual larvae for the 0-hour treatment, 60 larvae for the
6-hour heat waves, and 60 larvae for 72-hour heat waves, in each
combination of humidity × parasitism × larval stage.
We measured the following traits in unparasitized hosts: 1) host
emergence 2) juvenile development time (i.e., from egg to adult
emergence) 3) mid femur length (which is correlated with body size,
Jones et al., 2015, Mugabo et al., 2019). For parasitized hosts, we
measured 1) relative emergence success —whether a host (i.e.
parasitoids were killed by heat stress; where encapsulation was not
considered), wasp (i.e. parasitoids survived and killed the host), or
neither emerged (i.e., both were killed) 2) wasp juvenile development
time (i.e., from parasitism to wasp emergence) 3) hind tibia length (as
a measure of adult body size, Harvey et al., 2001).
2.3 Data analysis
Data consisted of multiple trait measurements in unparasitized and
parasitized hosts as responses, and three experimental treatments i.e.,
humidity, larval stage, duration of heatwaves were predictors. Humidity
and larval stage were categorical variables, each with two levels, and
duration of heat waves was treated as a continuous variable on a log
(x+1)-transformed scale. The number of wasps that emerged from host
larvae (i.e., without heat waves) as controls for those exposed during
the 5th instar were less than the minimum requirements
of statistical tests (i.e. < 3), which may bias overall
estimates if treated as an independent group. Therefore, we increased
the number of observations in this group by shuffling all no-heatwave
controls within each humidity treatment and split equally in numbers
into two larval stages, using ‘sample_n()’ function in the ‘tidyverse’
package (Wickham et al., 2019).
Statistical analyses were conducted using R (v 4.3.2, RCoreTeam, 2022).
We used a generalised linear model (GLM) with a binomial error
distribution to investigate if humidity, larval stage, and duration of
heat waves affected the adult emergence of unparasitized hosts. In
parasitized hosts, we performed a multinomial logit regression model to
investigate if the relative success of hosts and parasitoids was
affected by the experimental manipulations, using ‘nnet’ package (Ripley
et al., 2016). Probability of success was measured by three possible
outcomes of each parasitized host: 1) parasitoid emergence – where the
host was killed by the successful emergence of a parasitoid; 2) host
adult emergence – where a parasitoid failed to kill the host; 3) none
– where both the host and the parasitoid failed to emerge (i.e., both
were killed). Humidity, larval stage, duration of heat waves, and their
interactions were included as predictors. To compare how the survival of
parasitoids was changed due to experimental manipulations, we set up the
outcome of ‘parasitoid emergence’ as the reference group, indicating
that any changes in the outcomes in parasitized larvae (i.e., from
parasitoid emergence to host emergence; or from parasitoid emergence to
no emergence) were the results of experimental treatments (where
encapsulation was not considered). The multinomial logit coefficients of
‘host adult emergence’ and ‘none’ relative to ‘parasitoid emergence’
were estimated and compared using two-tailed Wald z-tests (Kwak and
Clayton-Matthews, 2002).
Host and parasitoid larval development were assumed to be
gamma-distributed under the constant temperature (Li et al., 2022), so
the GLMs were fitted with a log-link Gamma error distribution for hosts
and parasitoids that emerged to the adult stage. Body size of
unparasitized adult hosts and parasitoids were analysed using GLMs with
a log-link gaussian error distribution. Humidity, larval stage, duration
of heat waves, and their interactions were included as independent
variables in these models. Model residuals were checked using a
simulation-based approach by running n=250 simulations in ‘DHARMa’
package (Hartig and Hartig, 2017) and tested for normality using
Kolmogorov-Smirnov test.
Finally, we used a piecewise structural equational model (’piecewiseSEM’
package, Lefcheck, 2016) to investigate the direct and indirect effect
of humidity, larval stage, and duration of heat waves on hosts, and how
they subsequently affected the phenotype of parasitoids (body size). The
advantage of this method is that piecewise SEMs can switch from global
to local estimation, allowing for the fitting of equations with a range
of distributions (Shipley, 2000). As the emergence of parasitoids
resulted in the death of the host, we quantified the host’s contribution
to the parasitoid using host larval survival time (i.e., the date of
parasitoid emergence minus the date of host egg laying). The hind tibia
length of parasitoids was used as a measure of parasitoid performance to
avoid the correlation between two endogenous variables (i.e., host
survived time and parasitoids’ sizes). As such, our piecewise SEM
included two underlying structured equations which specified a) the
effect of larval stage and duration of heat waves on host survival time,
and b) the effect of host larval survival time, duration of heat waves,
and host larval stage on the hind tibia of parasitoids. Both equations
were fitted by a GLM with a log-link gaussian distribution (Gamma GLMs
were not supported), with model assumptions checked by the ‘DHARMa’
package (Hartig and Hartig, 2017). To examine if the effect of heat
waves on hosts and parasitoids were modified by humidity, we performed a
multigroup analysis incorporating humidity as a grouping variable.
Whilst constraining both paths may result in a saturated model (i.e.,
having 0 degrees of freedom to evaluate model fit), the process of
automatically adjusting coefficients in piecewise SEMs allows us to
examine if any of the paths varied among groups (Lefcheck, 2016). Thus,
any significant interactions indicated whether heat waves interacted
with humidity, and any constrained effects implied the path to the hosts
or parasitoids did not differ in humidity. Standardized coefficients
were calculated for non-categorical variables.