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PLOS ONE logoLink to PLOS ONE
. 2021 Feb 11;16(2):e0245029. doi: 10.1371/journal.pone.0245029

Experimental warming influences species abundances in a Drosophila host community through direct effects on species performance rather than altered competition and parasitism

Mélanie Thierry 1,2,*, Nicholas A Pardikes 2, Chia-Hua Lue 2, Owen T Lewis 3, Jan Hrček 1,2
Editor: Lucas D B Faria4
PMCID: PMC7877627  PMID: 33571220

Abstract

Global warming is expected to have direct effects on species through their sensitivity to temperature, and also via their biotic interactions, with cascading indirect effects on species, communities, and entire ecosystems. To predict the community-level consequences of global climate change we need to understand the relative roles of both the direct and indirect effects of warming. We used a laboratory experiment to investigate how warming affects a tropical community of three species of Drosophila hosts interacting with two species of parasitoids over a single generation. Our experimental design allowed us to distinguish between the direct effects of temperature on host species performance, and indirect effects through altered biotic interactions (competition among hosts and parasitism by parasitoid wasps). Although experimental warming significantly decreased parasitism for all host-parasitoid pairs, the effects of parasitism and competition on host abundances and host frequencies did not vary across temperatures. Instead, effects on host relative abundances were species-specific, with one host species dominating the community at warmer temperatures, irrespective of parasitism and competition treatments. Our results show that temperature shaped a Drosophila host community directly through differences in species’ thermal performance, and not via its influences on biotic interactions.

Introduction

It is becoming evident that many species are declining as the climate changes [1, 2], and increasing numbers of extinctions are expected as a result in the coming decades [3]. Animals are directly impacted by warming temperatures through changes in their fecundity, mortality, metabolic rates, body growth rate, and phenology [47]. Species in the tropics are likely to be more sensitive to global warming because they are closer to their upper thermal limits [3, 8], and the predicted increase in temperatures by a few degrees would exceed their thermal maxima. Ectotherms, such as insects, have particularly narrow thermal limits and are facing severe declines in abundances with rising temperature [9]. Warming temperatures directly affect physiology and demography depending on species’ thermal tolerances (i.e., their ability to survive exposure to extreme temperatures) and their thermal performance (i.e., their fitness-related traits over a range of temperatures). Both thermal tolerance and thermal performance are expected to influence population sizes and community structure with ongoing global warming [5].

However, ecological communities are not defined solely by the species that compose them, but also by the way those species interact with one another, via both trophic and non-trophic interactions [10, 11]. Trophic interactions, such as predation, herbivory, or parasitism have strong effects on community composition and evenness [12, 13]. Non-trophic interactions such as competition and pollination are also ubiquitous and can alter community composition in many ways (e.g. if some species are competitively excluded, or if species coexistence is enhanced) [1416]. Trophic and non-trophic interactions act together to structure ecological communities [1719], and a theoretical understanding is emerging of how these different types of interactions shape the structure and dynamics of more complex ecological networks [20]. However, empirical evidence on the combined effects of trophic and non-trophic interactions on the structure of terrestrial species-rich communities remain sparse. Moreover, global warming may modify such mechanisms structuring ecological communities, since warming temperatures are expected to have direct effects on both component species and their interactions [21, 22]. Temperature can alter resource-consumer interactions via its effects on metabolic processes such as growth and reproduction, and change in behaviors [2325]. The main mechanisms behind species interactions response to climate change are the differences in effects among interacting species, such as asymmetrical responses in their phenology [26], growth rate [27], and body mass [28]. Furthermore, changes in the outcome of species interaction with warming temperatures can have cascading effects on individual fitness, populations and communities [25, 29, 30]. Despite calls for more investigations of how species interactions respond to global climate change [31, 32], most such studies focus either on aquatic systems [21, 33], on a single interaction type [34], or on a small number of species [35]. We urgently need more data to predict how environmental changes modify different types of interactions (both trophic and non-trophic) in more complex ecological networks [36, 37].

Insect host-parasitoid communities are excellent model systems to investigate how species and their interactions respond to warming temperatures [14]. Parasitoids are insects which develop in or on the bodies of arthropod hosts, killing the host as they mature, and playing an important role in regulating host populations in both natural and agricultural ecosystems [38]. As ectotherms, many parasitoid traits involved in species interactions are sensitive to changes in temperature [39, 40]. Empirical studies suggest that global warming could weaken top-down control by parasitoids by increasing parasitoid mortality, by decreasing parasitoid virulence and/or increasing host immune response, and by increasing host-parasitoid asynchrony, thus increasing the frequency of pest outbreaks [4143]. However, most studies of host-parasitoid interactions are limited to a pair of interacting species, and it is unclear how host-parasitoid communities respond to warming temperatures when more complex systems are considered [14, 44]. Community level responses to global warming may depend on how species interact, and the effect of species interactions on community structure might change depending on environmental conditions. For instance, parasitoids can mediate host coexistence, but the outcome may depend on temperature [45]. Furthermore, competitive interactions among hosts can affect the responses of species and communities to environmental changes [30], but such responses may differ for intraspecific and interspecific competition [46]. Thus, to help forecast the impacts of global warming on host-parasitoid communities, it will be critical to examine the combined responses of species and their interactions under simulated warming conditions [47].

In this study, we use a laboratory experiment with intra vs. inter specific competition between hosts and parasitism in a fully factorial design to investigate how temperature affects host communities directly through difference in species responses, and indirectly through effects on parasitism and competition with other host species. We used host abundances and their relative frequencies to describe the host community. We also measured host body mass as a proxy for host fitness under the different treatments, and because an increase in temperature generally produces smaller individuals, which could influence the outcome of competition [28]. We focus on a set of three Drosophila species which are members of a natural Drosophila-parasitoid community in Australian tropical rainforests [48]. We test the predictions that elevated temperature will affect the relative abundance of the hosts directly through the thermal performance of individual species, and indirectly through effects on their interactions with other species. Elevated temperatures could alter the competitive abilities of the hosts (linked to species’ thermal performance) and the extent to which they are parasitized (linked to effects of temperature on parasitoid attack rates and virulence) [39], with consequences for the relative abundance of hosts in the community [14]. An interactive effect of trophic and non-trophic interactions on host relative abundances is expected due to a trade-off between resistance to parasitoids and larval competitive abilities [49]. This study aims to disentangle the direct and indirect effects of warming on structuring our focal tropical Drosophila community, and provides an important step forward in our understanding of the potential mechanisms driving tropical insect community responses to global warming.

Materials and methods

Study system

The experiment was established from cultures of Drosophila species and their associated parasitoids collected from two tropical rainforest locations in North Queensland, Australia: Paluma (S18° 59.031’ E146° 14.096’) and Kirrama Ranges (S18° 12.134’ E145° 53.102’) (<100 m above sea level). Drosophila and parasitoid cultures were established from 2017 to 2018, identified using both morphology and DNA barcoding, and shipped to the Czech Republic under permit no. PWS2016-AU-002018 from Australian Government, Department of the Environment. Three host species (Drosophila birchii, D. pseudoananassae and D. sulfurigaster, together accounting for ~ 48% of the host abundances sampled at the study sites [48]) and two of their natural larval parasitoid species Asobara sp.1 (Hymenoptera: Braconidae; Smithsonian National Museum of Natural History (NMNH) reference vouchers USNMENT01557096 [BOLD sequence accession: DROP042-21] and USNMENT01557097 [BOLD sequence accession: DROP043-21] and Leptopilina sp.1 (Hymenoptera: Figitidae; NMNH reference vouchers USNMENT01557104 [BOLD sequence accession: DROP050-21] and USNMENT01557117 [BOLD sequence accession: DROP053-21]) able to parasitize all three host species were used in this experiment. The parasitoid species are new undescribed species unambiguously identified by the above vouchers and sequences in order for this paper to be linked to them once they will be formally described. Data on thermal performance of the three host species have been previously measured by MacLean, Overgaard, and collaborators [50, 51] (Table 1). All cultures were maintained at 23°C on a 12:12 hour light and dark cycle at Biology Centre, Czech Academy of Sciences. Drosophila isofemale lines were maintained on standard Drosophila medium (corn flour, yeast, sugar, agar and methyl-4-hydroxybenzoate) for approximately 15 to 30 non-overlapping generations. To ensure genetic variation, five lines from each host species were combined to establish mass-bred lines immediately before the start of the experiment. Isofemale lines of parasitoid lines were maintained for approximately 10 to 20 non-overlapping generations prior to the start of the experiment by providing them every week with 2-day-old larvae of Drosophila melanogaster. This host species is not present naturally at the field locations where hosts and parasitoids originated, and was not used in the experiment, thus avoiding bias of host preferences. Single parasitoid isofemale lines were used.

Table 1. Host species thermal tolerance upper limit (CTmax) and thermal performances: Optimal temperature (Topt) and thermal breadth (Tbreath defined here as the range where performance is above 80% of optimal) for overall species fitness (product of fecundity, developmental success and developmental speed) and fecundity measured as egg-laying capacity ± SD.

Data are from [51].

Host species D. birchii D. pseudoananassae D. sulfurigaster
CTmax 38.51 ± 0.32 39.02 ± 0.32 36.55 ± 0.11
Fitness Topt 25.33 ± 1.05 24.00 ± 0.45 24.72 ± 0.73
Fitness Tbreath 4.27 ± 0.57 5.15 ± 0.36 4.51 ± 0.31
Fecundity Topt 26.18 ± 0.62 24.62 ± 1.52 24.84 ± 0.72
Fecundity Tbreath 5.37 ± 1.16 9.31 ± 1.11 5.26 ± 0.44

Experimental design

To disentangle the effects of warming temperatures on host species and their interactions, we manipulated the presence of parasitoids and interspecific competition between host species in a fully factorial design (Fig 1) at ambient and elevated temperatures. We aimed to study the independent and combined effects of parasitism and host competition when both forms of antagonistic interaction occur at strong (but realistic) levels. As the focus of the experiment was to compare the direct and indirect effects of warming temperatures on host communities, competitive interactions between parasitoids were not assessed nor manipulated, but potentially present in all treatments with parasitoids. Parasitoid preferences were not quantified, but the two parasitoid species used were able to parasitize all three hosts species during trials.

Fig 1. Schematic representation of the steps of the protocol and the experimental treatments.

Fig 1

Orange, pink, and blue nodes represent the three host species, and white and grey nodes represent the two parasitoid species. Solid arrows show possible trophic interactions, and dashed arrows show possible competitive interactions in each treatment. The type of competition between host species (intraspecific/interspecific) and presence or absence of parasitoids in the cages were manipulated in a fully factorial design: a) intraspecific competition, b) interspecific competition, c) intraspecific competition with parasitism, and d) all interactions.

Transparent plastic boxes (47cm x 30cm x 27.5cm) with three ventilation holes (15 cm in diameter) covered with insect-proof nylon mesh served as the experimental units (S1 Fig). Each box contained three 90 mm high and 28 mm diameter glass vials containing 2.5 mL of Drosophila food medium. Interactions were manipulated by establishing vials containing a single host (Fig 1A and 1C) or multiple host species (Fig 1B and 1D), and by including (Fig 1C and 1D) or excluding (Fig 1A and 1B) parasitoids. A total of 60 three-day-old virgin adult hosts, with 1:1 sex ratio, were placed in each vial to allow mating and oviposition (i.e., a total of 180 adults per box) and removed after 48 hours. In the multi-host treatment, the 60 hosts were split evenly across the three species (i.e., 20 adults for each species). The density of adult hosts was selected based on preliminary observations to achieve a high level of resource competition (i.e., the density at which strong intraspecific competition was observed for all host species; S1 Table) while keeping the number of adults for each of the three host species and the total number of adult hosts consistent across treatments and species. The treatment allowed competition both at the adult stage for oviposition space, and at the larval stage of their offspring for food resources [52, 53], but we did not aim to identify which was the primary source of competition. All results relate to the host offspring (their abundances and frequencies).

For treatments that included parasitoids (Fig 1C and 1D), ten parasitoids (3–7 days old, 1:1 sex ratio) from each species (n = 2, i.e., 20 parasitoids per box), corresponding to 9% of the total number of adult hosts, were placed in a box immediately after the hosts were removed (at 48h) and remained in each box for 72 hours, creating high but realistic parasitoid pressure (within the range of parasitism rate observed in this system in nature: 8–42% [48]). Vials were removed from the boxes simultaneously with the parasitoids (72 hours after parasitoid introduction), and individually sealed. Each treatment combination was replicated once across four time-blocks, and each treatment and replicate were therefore represented by three vials. The duration of the experiment corresponded to a single generation of both the hosts and the parasitoids (i.e., about 30 days for the species with the longest developmental time to emerge).

The experimental temperatures were chosen to simulate current mean yearly temperature at the two study sites [48]: 23.2 ± 0.4°C (65.9 ± 2.8% humidity), and projected temperatures representing a plausible future scenario under climate change: 26.7 ± 1.0°C (65.1 ± 2.8% humidity). The simulated difference was therefore 3.5°C (projected change in global mean surface temperature for the late 21st century is 3.7°C for the IPCC RCP8.5 baseline scenario [54]). Vials were placed at their corresponding temperature treatment from the first day the adult hosts were introduced for mating and oviposition to the last emergence (up to 40 days). All four blocks included both ambient and warming temperature treatments.

To calculate parasitism rates for each host-parasitoid species pair, pupae from the three vials of each box were randomly sampled 12 days after the initiation of the experiment. All sampled pupae were transferred into 96-well PCR plates (on average 169 ± 30 SD pupae sampled per box) and kept at their corresponding temperature treatment until adult insects emerged (up to 40 days for the slowest-developing parasitoid species). Sampled pupae were identified to their corresponding host species on the basis of pupal morphology (S2 Fig), and the outcome was recorded as either a host, a parasitoid, an empty pupal case, or an unhatched pupa. We assumed that any pupae which were empty at the time of sampling resulted in adult hosts because this period was too short for parasitoids to complete development and emerge. We calculated parasitism rates from the pupae sampled in plates only. Parasitism rates for each host-parasitoid pair were calculated as the proportion of each parasitoid species that emerged from the total number of sampled pupae of each host species.

All hosts that emerged (from both vials and sampling plates) were used to quantify the following aspects of host community structure: abundances of each host species, and their relative frequencies (i.e., the fraction of all host individuals belonging to each host species). All hosts and parasitoids that emerged from vials before and after subsampling for parasitism rates were collected, identified, and stored in 95% ethanol until four consecutive days of no adult emergences. Individual dry body mass of hosts was measured with 1 μg accuracy using a Sartorius Cubis ™ micro-balance. Only fully-eclosed and intact individuals were included in body mass measurements.

Statistical analysis

All vials with fewer than ten total emergences or pupae were removed from analyses of host abundances, frequencies, and parasitism rates (S2 Table, deleted observation due to D. sulfurigaster), as these outcomes were associated with low success during the mating process and not with experimental treatments (results with the whole dataset can be found in S3 Table). We used 3-day-old hosts and allowed them to mate and lay eggs for 48 hours. Drosophila sulfurigaster females generally take 4 days to mature compared to 3–4 days for D. birchii females and 3 days for D. pseudoananassae, which could explain the low abundances sometimes observed for D. sulfurigaster compared with the two other host species.

Data were analyzed with generalized linear models (GLMs). After testing for overdispersion of the residuals, abundance data were modeled using a negative binomial error distribution, host body mass using a gaussian error distribution, and frequencies of host species and parasitism rates using a quasibinomial error distribution. Parasitism (two levels), type of competition (two levels), host species (three levels), parasitoid species (two levels), and temperature (two levels) were included as categorical predictor variables within each model. Blocks were included in the models as a fixed effect. Each two-way interaction was tested and kept in our models if judged to be statistically significant on the basis of backward selection using Likelihood-ratio tests. Interaction between temperature and parasitism, temperature and competition, and parasitism and competition were systematically kept in our models as the experiment was designed to test for the significance of these interactions. The three-way interaction between temperature, parasitism, and competition was tested for host abundances, host frequencies, and host body mass, but was not significant. Significance of the effects was tested using type III analysis of deviance with F-tests.

Post-hoc multiple comparisons were performed using the emmeans package, and P-values were adjusted using the Tukey method. Model assumptions were verified with the DHARMa package. All analyses were performed using R 3.5.2 [55] with the packages stats, MASS [56], car [57], performance [58], DHARMa [59], and emmeans [60].

Results

In total, 7627 individuals (7063 hosts and 564 parasitoids) were reared across all treatments and replicates (238.3 ± 13.3 SD on average per box). Across all treatments and replicates, a total of 2717 pupae were sampled to estimate parasitism rates, of which 2227 (82%) produced an adult host or parasitoid. Mean host abundances, host body mass, and parasitism rates are presented for each treatment in S4 Table. We focused on the effects of temperature, parasitism, competition and their interactions on host abundances, host frequencies, and host body mass (Table 2).

Table 2. Table showing the effect of temperature (23°C or 27°C), parasitism (present or absent), competition between host species (intraspecific or interspecific), host species (n = 3), parasitoid species (n = 2), interactions between terms, and block (n = 4) on host abundances, host frequencies, host body mass, and parasitism rate.

Df Host abundances Host frequencies Host body mass Parasitism rate
Temperature 1 1.41 (ns) 0.47 (ns) 1.88 (ns) 4.89 *
Parasitism 1 21.80 *** 0.03 (ns) 2.98 (ns) - -
Competition 1 0.15 (ns) 0.06 (ns) 10.76 ** 1.14 (ns)
Host species 2 27.07 *** 64.7 *** 426.64 *** 2.47 (ns)
Parasitoid species 1 - - - - - - 2.29 (ns)
Temperature x Parasitism 1 0.26 (ns) 0.05 (ns) 0.60 (ns) - -
Temperature x Competition 1 0.00 (ns) 0.07 (ns) 1.32 (ns) 0.04 (ns)
Temperature x Host species 2 7.90 *** 24.12 *** - - - -
Parasitism x Competition 1 1.58 (ns) 0.00 (ns) 4.49 * - -
Competition x Host species 2 - - - - 27.80 *** - -
Host x parasitoid species 2 - - - - - - 20.23 ***
Block 3 1.02 (ns) 0.47 (ns) 4.53 ** 1.49 (ns)
-
Df error 68 68 65 70
0.87 0.05 0.93 0.10

Degrees of freedom (Df) for each F-ratio are given for each factor and for the error. F values are presented with the significance of the effect:

(***) P < 0.001,

(**) P < 0.01,

(*) P < 0.05, (ns) P > 0.05.

Direct effect of warming on the host community

The effect of temperature on host relative abundances varied significantly across host species (Table 2 and Fig 2). At 23°C, D. birchii and D. pseudoananassae had similar relative abundances across treatments (mean frequency of D. birchii = 0.426 ± 0.05; mean frequency of D. pseudoananassae = 0.471 ± 0.05 for all treatments combined at 23°C). At 27°C, Drosophila pseudoananassae relative abundances increased by 12.8% (Post Hoc odd ratio (OR) = 0.336, P < 0.0001) while D. birchii relative abundances decreased by 56.1% (Post Hoc OR = 3.190, P < 0.0001) (mean frequency of D. birchii = 0.187 ± 0.02; mean frequency of D. pseudoananassae = 0.743 ± 0.02 for all treatments combined at 27°C). The change in frequency of D. sulfurigaster with temperature was not significant (at 23°C: 0.178 ± 0.03, at 27°C: 0.118 ± 0.02; Post Hoc OR = 1.361, P = 0.440). Elevated temperature had no effect on host body mass (F1,65 = 1.88, P = 0.175, S3 Fig).

Fig 2. Effect of experimental treatments on host frequencies.

Fig 2

Experimental warming changed the frequencies of hosts for all treatments. See Fig 1 for detailed description of the treatments. The small points represent the values from each block, the large points represent the grand mean, and the bars represent standard errors of the means.

Effect of biotic interactions on the host community

Parasitism significantly reduced mean abundances of all three host species by 50 ± 0.22 (SEM) hosts on average across species (β = -0.339, F1,68 = 21.80, P < 0.0001; Fig 3A), and the negative effect of parasitism was consistent across host species (Table 2). Competition type did not significantly impact host abundances or relative host frequencies. Effects of competition on host body mass depended both on host identity (F2,65 = 27.80, P < 0.0001), and on presence or absence of parasitoids (F1,65 = 4.87, P = 0.038). D. pseudoananassae was the host species that varied the most in body mass with treatments (S3 Fig). Its body mass decreased with interspecific competition in the absence of parasitoids but increased with interspecific competition with presence of parasitoids. Changes in body mass for the other two host species were less pronounced.

Fig 3. Effect of experimental treatments on host community and host-parasitoid interactions.

Fig 3

(a) Host abundances (■: D. birchii, ♦: D. pseudoananassae, ▲: D. sulfurigaster) were significantly reduced by parasitism across treatments. (b) Parasitism rates were reduced at higher temperature (□: Asobara sp.—D. pseudoananassae, ˄: Asobara sp.—D. birchii, +: Asobara sp.—D. sulfurigaster, × Leptopilina sp.—D. pseudoananassae, ◊: Leptopilina sp.—D. birchii, ˅: Leptopilina sp.—D. sulfurigaster). See Fig 1 for detailed description of the treatments. The small points represent the values from each block, the large points represent the grand mean, and the bars represent standard errors of the means. Significance of treatment effects is indicated as follows: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (ns) P > 0.05.

Indirect effect of warming on host community structure through parasitism and interspecific competition

Experimental warming significantly decreased parasitism rates for all host-parasitoid pairs (β = -0.29, F1,70 = 4.89, P = 0.030, Table 2 and Fig 3B). However, the effects of parasitism and competition did not vary with temperature in affecting any of our measures of community structure (P > 0.05, Table 2).

Discussion

Our results revealed that experimental warming directly affected Drosophila host community structure through differences in thermal performance among species, and decreased parasitism rates, without effects on host competition. However, warming did not impact the effect of parasitism on host community structure over the timescale investigated. The type of competition (intraspecific or interspecific) among hosts did not change host community structure.

Our results suggests that ongoing rises in global temperatures could directly alter arthropod host community structure through differences in thermal performance across species, as has been shown for communities of fish [61], plants [62], and insects [63]. Changes in host frequencies in warmer temperatures was primarily due to a dramatic increase in the relative abundance of a single host species, D. pseudoananassae, the species with the largest thermal performance breath [51], and our main conclusions should thus not be impacted by the low abundances sometimes observed for D. sulfurigaster due to mating problems. This increase occurred across all combinations of parasitism and competition treatments, and without a change in Drosophila body mass, suggesting a direct effect of temperature on host fecundity due to the preferred temperature of the adults for egg-laying and/or offspring egg-to-adult viability related to their thermal preference [64]. In our system, D. pseudoananassae distribution is limited to low elevation sites [48], and this species has a higher thermal tolerance and a bigger thermal breadth than either of the other two species considered in this study [51]. In nature, Drosophila species distributions are driven by differences in innate thermal tolerance limits, with low phenotypic plasticity for thermal tolerance limits in both widespread and tropical species [65]. This suggests that warming temperatures, in the context of global climate change, will have a strong effect on community composition through direct effect on fitness.

Our data also revealed a significant decrease in parasitism rates with warming. Reviews suggest that parasitism rates would decrease under global warming scenarios due to an increase in parasitoid mortality, and host-parasitoid spatial and temporal asynchrony [14, 44]. However, the presence of parasitoids significantly decreased abundances of the three host species independently of the temperature regime, suggesting that warming treatments did not decrease attack rate, but decreased successful parasitism rate [66, 67]. The decrease in parasitism rates at higher temperatures could also result from improved host immune response, decreasing the vulnerability of hosts to parasitoid attacks [68]. Therefore, host immune function responses to temperature should be considered alongside host thermal performance and tolerance to predict the effects of increasing temperatures on host communities [14]. This experiment was performed over a single generation, so long-term consequences of decreased parasitism rates with elevated temperatures for host-parasitoid dynamic cannot be assessed, but a decrease in parasitism rates could lead to the release of hosts from top-down control. However, in the case of a simple linear tritrophic interaction, the results of Flores-Mejia et al. [69] suggest that parasitoid top-down control might be less sensitive to temperature than previously thought. Nevertheless, with warming temperatures, stronger host and parasitoid genotype congruence has been observed, which could decrease parasitoid diet breadth and thus decrease parasitism rates [70]. In our experiment, the role of parasitoids in lowering insect abundance was not reduced under experimental warming. However, parasitism rates were reduced, suggesting that an indirect effect of warming temperatures on the structure of the host community, mediated by parasitoids, might emerge over multiple generations.

Our results demonstrate that differences in thermal performance across host species may be a stronger determinant of how host communities respond to warming temperatures than shifts in the strength of biotic interactions in arthropod host communities. We used high, but realistic levels of competition and parasitism that would have allowed us to detect their effects on host species relative abundances if there were any. We did not find an interactive effect of parasitism and competition treatments on host abundances and frequencies. This result is in line with results from another laboratory experiment performed on the same system [71] showing that parasitism did not significantly affect host competitive coefficients. Furthermore, the type of competition between hosts did not significantly affect total host abundance, suggesting that the amount of food included was only able to support a certain number of hosts that did not vary with the type of competition. Aspects of our results contrast with those from a field transplant experiment on two species drawn from the same Australian Drosophila-parasitoid community [72]. Investigating fitness of D. birchii and D. bunnanda along an elevation gradient, the authors found an interacting effect between the abiotic environment and interspecific competition. However, the field experiment excluded parasitoids, and the elevational gradient studied is likely to include variations such as humidity as well as temperature, which might influence the outcome [73]. Our results also contrast with the conclusions from a systematic review on the mechanisms underpinning natural populations response to climate [47]. They found greater support for indirect effects of climate on populations through altered species interactions than direct effects. However, this review included drought in addition to temperature in the climatic variables, and the relative importance of biotic and abiotic mechanisms varied with trophic level. Moreover, the authors brought out a bias in the published studies toward temperate ecosystems and mammals, highlighting the need for more studies investigating the mechanisms driving tropical arthropod community responses to global climate changes.

Our study serves as example of the mechanisms that can be expected to drive community responses to global warming, but general conclusions on the potential impact of warming temperature on host-parasitoid networks will require replication with different species compositions and different systems. Especially, most host-parasitoid systems are tri-trophic (plants-arthropods-parasitoids), and climate warming is likely to impact host-parasitoid networks through bottom-up effects [74]. Few such experiments have been undertaken, despite the need to better disentangle direct and indirect effects of warming temperature on species communities. Ideally, future studies will also need to investigate the longer-term dynamics of such systems. Moreover, as temperatures continue to increase, species from diverse taxa are shifting their distribution worldwide to higher latitudes and elevations [75], changing their biotic environment with novel species interactions and different community assemblages [76]. Dispersal was not permitted in this study, but is likely to mediate some of the effects of warming temperature on species and their interactions [30, 77]. Understanding the mechanisms driving community responses to warming scenarios is particularly important for tropical communities, which face more severe impacts of climate warming than temperate communities [8], and contain most threatened species of global concern [78]. Here, we demonstrate that warming had a direct effect on our focal tropical Drosophila host community through differences in thermal performance, without affecting the relative strength of parasitism and competition.

Supporting information

S1 Fig. Transparent plastic boxes (47cm x 30cm x 27.5cm) with three ventilation holes (15 cm in diameter) covered with insect-proof nylon mesh used as experimental unit allowing parasitoids to attack one of the three experimental vials containing 2-days-old host larvae for 72h.

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S2 Fig. Drosophila birchii, D. pseudoananassae, and D. sulfurigaster pupae photography for morphological identification.

Not in scale (photo credit to Jinlin Chen).

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S3 Fig. Interactive effect of competition with host species, and with presence of parasitoids on mean host body mass (squares: D. birchii, diamonds: D. pseudoananassae, triangles: D. sulfurigaster).

See Fig 1 for detailed description of the treatments. The small points represent the values from each block and each host-parasitoid pair, the large points represent the grand mean, and the bars represent standard errors of the means. Blue: ambient temperature (23°C), red: warming treatment (27°C).

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S1 Table. Mean number of offspring per species with 10, 30, 60, 90 or 180 adult hosts (1:1 sex ratio) in a 5 mL host-media glass vial.

Choice of host number in the main experiment was based on these preliminary data to correspond to strong competition for all host species.

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S2 Table. Number of observations per temperature, treatments (Intraspecific competition: No interaction between host species; Interspecific competition: Direct competition between host species; Parasitism: Intraspecific competition with parasitism; All interactions: Interspecific competition with parasitism), and host species in the whole dataset, and with the reduced dataset used for analyses (excluding observations with fewer than 10 emerging insects or pupae).

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S3 Table. Table showing the effect of temperature (23°C or 27°C), parasitism (presence or absence), competition between host species (intraspecific or interspecific), host species (n = 3), interactions between terms, and block (n = 4), on host abundances, and host frequencies for the whole dataset (without any deleted observations due to D. sulfurigaster).

Degrees or freedom (Df) for each F-ratio are given for each factor and for the error. F values are presented with the significance of the effect: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (ns) P > 0.05.

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S4 Table. Summary table for mean (± SD) host abundances (Host ab.), individual host body mass (Host BM), total parasitism rate (PR), and parasitism rates of each parasitoid species (Asobara sp. and Leptopilina sp.) for each temperature (23 and 27°C), treatments (competition: Intra or inter, parasitism: Present or absent), and host species (D. birchii, D. pseudoananassae, D. sulfurigaster).

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Acknowledgments

We thank Inga Freiberga, Anna Jandová, Martin Libra and Joel Brown for their help during the collection of the data, Petr Šmilauer for his advices on the statistical analysis, and Jinlin Chen and J. Chris D. Terry for useful comments on the results. The drawings used for Fig 1 was made by Tereza Holicová.

Data Availability

All raw data used for this study are available from the Zenodo database: https://doi.org/10.5281/zenodo.4362189

Funding Statement

We acknowledge funding support of JH from the Grantová Agentura České Republiky no. 17-27184Y. MT was further supported by Jihočeská Univerzita v Českých Budějovicích (GA JU no. 038/2019/P), and OTL by the UK Natural Environment Research Council (NE/N010221/1).

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Decision Letter 0

Lucas D B Faria

15 Jan 2021

PONE-D-20-39886

Experimental warming influences species abundances in a Drosophila host community through direct effects on species performance rather than altered competition and parasitism

PLOS ONE

Dear Dr. THIERRY,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Dear authors, I have received the reviewers' revision of the manuscript, and overall they considered it an essential and novel experimental contribution to the subject species interaction and climate warming. Further, the results shed light on the mechanism describing direct and indirect effects on communities' structure and function under climate variance. Reviewers' primary concern, and mine, point out difficulties to follow the Methods section raising many questions to be clarified by the authors. Besides, provide details - i.e., which institution approved and protocol number if it was the case -  about species entrance in the country.

Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process by 27th February.

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Despite some issues in the Methods section, the manuscript can be accepted under minor revision by the authors.

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: This manuscript details the results of a laboratory experiment testing whether climate warming, species interactions (intraspecific competition, interspecific competition, and parasitism), or their interaction affect 3-species tropical Drosophila communities. The manuscript is mostly well-written (aside from parts of the Methods) and is skillfully couched in terms of pertinent larger questions and the literature. The experiment appears to have been carried out using sound methods, and analyzed and presented appropriately. The results should be interesting and useful to a broad audience interested in effects of climate warming on ecological communities. My main concern is that the methods – especially relating to the experimental design – are not explained as clearly as they could be. However, this should require a relatively minor revision. I have several other concerns below, all of which are minor as well. I feel confident that upon revision, this manuscript should represent a very useful contribution to understanding of ecology and global change.

Introduction:

• Lines 56-57: clarify: evidence for what exactly is lacking?

• In the part of the paragraph following that sentence, as well as in lines 77-82, it is a little difficult to follow the flow. Transitions may help.

• Last paragraph is a confusing. Clarify what is measured and why. First sentence makes it seem like you just focus on relative abundance, but the next sentence mentions body mass without fully explaining how it fits in. Clarify predictions – that sentence is confusing. Should also be more clear about what was done, i.e. overview of the experimental design.

• Lines 95-97: seems out of place at the end of the introduction.

Methods:

• Line 103: cultures were established in both 2017 and 2018? clarify.

• Lines 105-106: clarify “together accounting for ~48% of the host communities as the study sites.” Those species comprised 48% of fly individuals across the sites? Or some other meaning?

• Lines 110-112: remember to remove this note

• Why was care taken to ensure genetic diversity in the Drosophila but not in the parasitoids?

• Line 131: 15 cm diameter holes would take up half the box – should read 15 mm?

• Line 136: I take it that the adult flies were then removed after 48 hours and before the parasitoids were added – but this should be stated to clarify.

• Lines 148-149: sentence seems out of place – could be moved to earlier in the paragraph

• Figure 1 should be altered to more accurately represent experimental design. E.g. if adult flies and parasitoids were not present at the same time, don’t show them together. Also, no larvae are shown. Perhaps just show larvae instead of adult flies.

• Lines 151-152: what does this mean – that the experiment was ended when both flies and parasitoids emerged? How long was this? But next sentence (last sentence of paragraph) seems to imply the experiment was only one day – do you mean that each block was started a day apart but then ran for many days?

• First Experimental design paragraph is confusing as methods are presented out of order. It is not easy to understand what was done. Also there is at least one important hole in the methods: how long after introducing the adult flies were the parasitoids introduced?

• Lines 163-166: clarify how this projected temperature falls among climate warming scenarios, e.g. mid-range for 2100?

• What was the experimental design of the temperature treatments, i.e. were two of the time blocks warm and two normal?

• Lines 183-185: Move this justification for looking at body mass to the introduction.

• Line 188: Since D. sulfu. represented all the “failures,” and there were many, this should be pointed out in the text, not just in the supplemental info. Relatedly, might this impact the conclusions? Maybe address this in the discussion.

• Lines 198: remove “ANOVA.” Likelihood ratio tests and the “anova()” command in R have nothing to do with ANOVA (confusingly).

• Include overview of the fly species’ thermal performances rather than just citing the papers.

Results:

• Table 1 & Table S3: Remove “ANOVA,” since you ran GLMs, not ANOVAs.

• Include line numbers in second half of ms.

• “Direct effect of warming” paragraph: also mention the third species. 4th sentence: clarify rel. abundances changed from which treatment to which treatment?

• Figures are blurry – provide high definition versions.

• Fig. 3: include the shapes/species in the legend(s), rather than in the caption.

• Last part of the final sentence in the Results sounds more like discussion.

Discussion:

• First paragraph: first sentence sounds a bit general – include study species/system. Include more in this paragraph: especially, competition.

• Towards the end of second paragraph: a bit confusing that you say D. pseudoananassae did not have the highest thermal tolerance in the paper but did in this experiment. Why mention the first part about the other study?

• The claim that climate change will affect tropical communities more severely needs to be backed up with a citation.

• Last sentence should be moved to earlier in the discussion.

References:

Need to be edited.

Reviewer #2: Thierry et al. performed a community-level experiment to understand the direct and indirect effects of warming on host species. They found that warming directly affect the abundance of host species and decrease parasitism rates for host-parasitoid pairs. Besides, the top-down control was still maintained by the presence of parasitoids decreasing host abundance regardless of the temperature. They conclude that the warming had a direct effect on host community structure through differences in host thermal performance but not via biotic interactions (competition and parasitism).

The manuscript is well written and makes novel contributions to a topic underexplored in the literature because it access the impact of warming on a community-wide system incorporating direct and indirect effects. Although the experiment was conducted over a single host generation, the results shed a light on how the direct and indirect effects could play a role in structuring a host community under warming conditions.

Detailed comments as follows:

Abstract:

line 23: Would it be more clear to use global climate change instead of global change?

line 30: it may be more clear if authors specify what metric was used when saying ‘host communities’. E.g host species abundance and frequency

Introduction: overall, really well written and provide a good context for the predictions stated at the end of this section.

Lines 88 - 91: If possible, I would rephrase the prediction for clarity. e.g We tested the predictions that the elevated temperature will affect the relative abundance of the host directly through the thermal performance of the species, and indirectly through effects on their interactions such as hosts competitive abilities and parasitoids virulence.

Throughout the manuscript, you use the terms: attack rates, parasitoid virulence, parasitism (yes or not) and parasitism rates (host-parasitoid species pair). I think the manuscript would benefit from explaining or deleting some of these terms. For example: I think it would be clearer if you could specify in the manuscript what do you consider as the parasitoid virulence metric? Is it the parasitism rates, the ‘yes or no parasitism’, both or none? In the Line 92 the word ‘parasitism’ refers to: ‘yes or no parasitism’ or to ‘parasitism rates (host-parasitoid species pair)’?. Please, could you rephrase the sentence in lines 92 – 93 (…while the effects on parasitism will be linked to effects of temperature on parasitoid attack rate and virulence.) for clarity?

Methods:

Line 116: what do you mean by revive genetic variation? Would it be to ensure or to rescue?

Lines 133 – 135: the single species and multiple species treatments were only for hosts right? The parasitoid-host species interaction was a sample from the single and multiple host species treatment, right? In these lines I would add ‘single-host species’ and ‘multiple-host species’ for clarity.

Line 160: In figure 1 legend we read ‘c)parasitism only’. However this treatment also includes host intraspecific competition according to what it is showing in Figure1, right? I would then change this label.

In Figure1: I think it would be more informative if you could add a dashed line (or indicate somehow) the intraspecific competition in letter a and c. Besides, in the mixed-species treatment do you think that intraspecific competition could also have happened together with the interspecific competition? If yes, do you think intraspecific could have been stronger than interspecific competition in these mixed-species treatments (figure 1 letter b and d)?

In Line 171 ‘the sampled pupae were identified to their corresponding host species’, how was it done? Do the Drosophila species have visually different pupal case? Was it how you link a parasitoid to a host species to calculate parasitism rates?

Line 176: I would make it more clear here that the parasitism rates was calculated for each host-parasitoid pair.

Line 182 we read ‘second generation started’, does it mean the F1 offspring? Are you counting the Parental generation as first?

Line 186: you mean fully-ecloded?

Results:

Line 213: do you mean host frequencies instead of host proportions?

In Figure 3 legend: in letter ‘b’ we read: (b) Parasitism was reduced at higher temperature. Do you mean here parasitism rates? In your manuscript I separate parasitism as ’yes or no’ and parasitism rates as ‘the host-parasitoid pair.’

In Figure 2 legend: do you refer to ‘proportion’ as the host frequency?

Discussion:

Below I apologize for not indicating the line number that I’m referring to. It happened because the number of the lines is not appearing in my pdf version.

In the third paragraph of the discussion you have: ‘However, presence of parasitoids significantly decreased... parasitoid virulence’. It would be more clear if there is a following up sentence to better explain this sentence and the decrease in virulence. For example, why parasitoids would decrease virulence with increasing temperature?

The idea that warming decreased parasitism rates, but did not impact the effect of the presence of parasitoid in decreasing host abundance, could be better explored in the discussion. For example, although parasitoid preference for host species was not quantified, Lavandero and Tylianakis et al 2013 shows that genetically similar parasitoids become more likely to attack genetically similar hosts in warmer sites. Therefore, there could be a shift in the parasitoid preference for a host species or even for a narrower subset of host species that would decrease parasitism rates (host-parasitoid pair) due to parasitoid competition under warming conditions.

In the fourth paragraph, fourth line we read: ‘…allowed us to detect their effects on host species relative abundances if they were any.’ Should the word ‘they’ be ‘there’?

In the last paragraph for the discussion we read: ‘The role of parasitoids as essential… after several generations’. What do you mean by suggesting that the indirect effects of the temperature on the host community through parasitism could be observed after several generations?

**********

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Reviewer #1: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Feb 11;16(2):e0245029. doi: 10.1371/journal.pone.0245029.r002

Author response to Decision Letter 0


23 Jan 2021

Dear Editor,

Thank you for considering our manuscript for publication in PLOS ONE and for the opportunity to submit a revised version. We are glad that the reviews were positive and found our paper to be interesting and novel. We appreciate the constructive criticisms made by the reviewers and the Academic Editor, and have made appropriate changes in response as detailed point by point below. We hope that the revision will allow the manuscript to be published in PLOS ONE.

Yours sincerely,

Mélanie Thierry, Nicholas A. Pardikes, Chia-Hua Lue, Owen T. Lewis & Jan Hrček

Academic editor’s comments:

Dear authors, I have received the reviewers' revision of the manuscript, and overall they considered it an essential and novel experimental contribution to the subject species interaction and climate warming. Further, the results shed light on the mechanism describing direct and indirect effects on communities' structure and function under climate variance. Reviewers' primary concern, and mine, point out difficulties to follow the Methods section raising many questions to be clarified by the authors. Besides, provide details - i.e., which institution approved and protocol number if it was the case - about species entrance in the country.

* We appreciate the positive comments on our manuscript. We have rewritten the Methods section and modify Figure 1 to clarify the experimental protocol, and have added details on the permit [line 111 in the revised manuscript].

We have added that the species were “shipped to the Czech Republic under permit no. PWS2016-AU-002018 from Australian Government, Department of the Environment” [lines 111-112 in the revised manuscript].

Additional Editor Comments:

Despite some issues in the Methods section, the manuscript can be accepted under minor revision by the authors.

Reviewers' comments:

Reviewer #1:

This manuscript details the results of a laboratory experiment testing whether climate warming, species interactions (intraspecific competition, interspecific competition, and parasitism), or their interaction affect 3-species tropical Drosophila communities. The manuscript is mostly well-written (aside from parts of the Methods) and is skillfully couched in terms of pertinent larger questions and the literature. The experiment appears to have been carried out using sound methods, and analyzed and presented appropriately. The results should be interesting and useful to a broad audience interested in effects of climate warming on ecological communities. My main concern is that the methods – especially relating to the experimental design – are not explained as clearly as they could be. However, this should require a relatively minor revision. I have several other concerns below, all of which are minor as well. I feel confident that upon revision, this manuscript should represent a very useful contribution to understanding of ecology and global change.

* We appreciate the reviewer’s interest in our manuscript. As stated above, we have rewritten the Methods section and edited Figure 1 to clarify the experimental design.

Introduction:

• Lines 56-57: clarify: evidence for what exactly is lacking?

* We have clarified the sentence [lines 56-58 in the revised manuscript].

• In the part of the paragraph following that sentence, as well as in lines 77-82, it is a little difficult to follow the flow. Transitions may help.

* We added transitions to help the flow of the paragraphs [lines 57 and 79-82 in the revised manuscript].

• Last paragraph is a confusing. Clarify what is measured and why. First sentence makes it seem like you just focus on relative abundance, but the next sentence mentions body mass without fully explaining how it fits in. Clarify predictions – that sentence is confusing. Should also be more clear about what was done, i.e. overview of the experimental design.

* We clarified in this paragraph what was measured (“host abundances and their relative frequencies”), why (“to describe the host community”), our predictions [lines 94-99 in the revised manuscript], and an outline of the experimental design (“a laboratory experiment with intra vs. inter specific competition between hosts and parasitism in a fully factorial design”).

• Lines 95-97: seems out of place at the end of the introduction.

* We rewrite this sentence [lines 101-104 in the revised manuscript].

Methods:

• Line 103: cultures were established in both 2017 and 2018? clarify.

* We changed it to “from 2017 to 2018” for clarity [line 110 in the revised manuscript].

• Lines 105-106: clarify “together accounting for ~48% of the host communities as the study sites.” Those species comprised 48% of fly individuals across the sites? Or some other meaning?

* We specified that the number refers to host abundances sampled [line 113 in the revised manuscript].

• Lines 110-112: remember to remove this note

* We have provided a final version of the species identity text [lines 114-119 in the revised manuscript].

• Why was care taken to ensure genetic diversity in the Drosophila but not in the parasitoids?

* Including wide genetic diversity of the hosts exceeds standard practice for ecological laboratory experiments, but was not possible for parasitoids: Unfortunately, we did not have enough isofemale lines of parasitoids in culture to combine them in the same way that we did for the Drosophila.

• Line 131: 15 cm diameter holes would take up half the box – should read 15 mm?

* 15 cm is the correct size, which takes about 1/3 of 3 of the sides. We have provided a picture of the experimental boxes in Supplementary Figure S1 for visualization.

• Line 136: I take it that the adult flies were then removed after 48 hours and before the parasitoids were added – but this should be stated to clarify.

* Yes, this is correct. We have now stated this more clearly in the manuscript and added the steps of the protocol in Figure 1.

• Lines 148-149: sentence seems out of place – could be moved to earlier in the paragraph

* We moved it to the beginning of the experimental design [lines 141-143 in the revised manuscript].

• Figure 1 should be altered to more accurately represent experimental design. E.g. if adult flies and parasitoids were not present at the same time, don’t show them together. Also, no larvae are shown. Perhaps just show larvae instead of adult flies.

* The figure has been edited as suggested.

• Lines 151-152: what does this mean – that the experiment was ended when both flies and parasitoids emerged? How long was this? But next sentence (last sentence of paragraph) seems to imply the experiment was only one day – do you mean that each block was started a day apart but then ran for many days?

* Yes, each block started a day apart and ran until everything emerged (about 30 days maximum for the species with the longest developmental time). We deleted the last sentence of the paragraph to avoid this confusion.

• First Experimental design paragraph is confusing as methods are presented out of order. It is not easy to understand what was done. Also there is at least one important hole in the methods: how long after introducing the adult flies were the parasitoids introduced?

* We added the time when the parasitoids were introduced (48h after the hosts, when we removed the adult hosts) [lines 165-166 in the revised manuscript]. We also modified Figure 1 to show the different steps of the protocol.

• Lines 163-166: clarify how this projected temperature falls among climate warming scenarios, e.g. mid-range for 2100?

* We added the projected increase in temperature for the late 21st century according to the IPCC RCP8.5 baseline scenario [lines 184-185 in the revised manuscript].

• What was the experimental design of the temperature treatments, i.e. were two of the time blocks warm and two normal?

* We specified that all four blocks included both temperature treatments [line 187 in the revised manuscript].

• Lines 183-185: Move this justification for looking at body mass to the introduction.

* We have moved it to the Introduction as suggested [lines 91-93 in the revised manuscript].

• Line 188: Since D. sulfu. represented all the “failures,” and there were many, this should be pointed out in the text, not just in the supplemental info. Relatedly, might this impact the conclusions? Maybe address this in the discussion.

* We added a discussion of this issue in the Methods section [lines 210-214 in the revised manuscript] and mentioned it in the Discussion section [lines 294-296 in the revised manuscript].

• Lines 198: remove “ANOVA.” Likelihood ratio tests and the “anova()” command in R have nothing to do with ANOVA (confusingly).

* Removed

• Include overview of the fly species’ thermal performances rather than just citing the papers.

* We have added in a new Table 1 containing host species thermal tolerance upper limit and thermal performances (optimal temperature and thermal breadth for overall species fitness and fecundity) measured by MacLean, Overgaard, and collaborators.

Results:

• Table 1 & Table S3: Remove “ANOVA,” since you ran GLMs, not ANOVAs.

* Removed

• Include line numbers in second half of ms.

* We have added the lines numbers for the whole manuscript.

• “Direct effect of warming” paragraph: also mention the third species. 4th sentence: clarify rel. abundances changed from which treatment to which treatment?

* We added the third species in the text [lines 250-251 in the revised manuscript], and stated that the relative abundances were for all four treatments combined at 23°C versus 27°C.

• Figures are blurry – provide high definition versions.

* All figures have been updated with a higher resolution.

• Fig. 3: include the shapes/species in the legend(s), rather than in the caption.

* The shapes of panel (a) represent host species whereas the shapes of panel (b) represent each host-parasitoid pair. The figure would be too small to add a legend for each panel. Therefore, we considered that adding the legend for the shapes in the figure rather than in the caption would be confusing.

• Last part of the final sentence in the Results sounds more like discussion.

* This sentence has been removed.

Discussion:

• First paragraph: first sentence sounds a bit general – include study species/system. Include more in this paragraph: especially, competition.

* We completed the first paragraph with adding that results are on Drosophila host community, that experimental warming had no effects on host competition, and that the type of competition (intraspecific or interspecific) among hosts did not change host community structure [lines 285-289 in the revised manuscript].

• Towards the end of second paragraph: a bit confusing that you say D. pseudoananassae did not have the highest thermal tolerance in the paper but did in this experiment. Why mention the first part about the other study?

* In our study, we did not measure host species thermal tolerance but only recorded their abundances and frequencies at ambient and elevated temperatures. We deleted the part mentioning thermal performance optimum to avoid confusion in the Discussion.

• The claim that climate change will affect tropical communities more severely needs to be backed up with a citation.

* We have added the reference [line 359 in the revised manuscript].

• Last sentence should be moved to earlier in the discussion.

* The last sentence was moved as suggested [lines 320-323 in the revised manuscript].

References: Need to be edited.

* References have been edited through our reference manager Mendeley.

Reviewer #2:

Thierry et al. performed a community-level experiment to understand the direct and indirect effects of warming on host species. They found that warming directly affect the abundance of host species and decrease parasitism rates for host-parasitoid pairs. Besides, the top-down control was still maintained by the presence of parasitoids decreasing host abundance regardless of the temperature. They conclude that the warming had a direct effect on host community structure through differences in host thermal performance but not via biotic interactions (competition and parasitism).

The manuscript is well written and makes novel contributions to a topic underexplored in the literature because it access the impact of warming on a community-wide system incorporating direct and indirect effects. Although the experiment was conducted over a single host generation, the results shed a light on how the direct and indirect effects could play a role in structuring a host community under warming conditions.

Detailed comments as follows:

Abstract:

line 23: Would it be more clear to use global climate change instead of global change?

* We have changed it as suggested [line 23 in the revised manuscript].

line 30: it may be more clear if authors specify what metric was used when saying ‘host communities’. E.g host species abundance and frequency

* We have specified that effects were measured on host abundances and frequencies [line 30 in the revised manuscript].

Introduction: overall, really well written and provide a good context for the predictions stated at the end of this section.

Lines 88 - 91: If possible, I would rephrase the prediction for clarity. e.g We tested the predictions that the elevated temperature will affect the relative abundance of the host directly through the thermal performance of the species, and indirectly through effects on their interactions such as hosts competitive abilities and parasitoids virulence.

* The prediction was rephrased following this suggestion [lines 94-96 in the revised manuscript].

Throughout the manuscript, you use the terms: attack rates, parasitoid virulence, parasitism (yes or not) and parasitism rates (host-parasitoid species pair). I think the manuscript would benefit from explaining or deleting some of these terms. For example: I think it would be clearer if you could specify in the manuscript what do you consider as the parasitoid virulence metric? Is it the parasitism rates, the ‘yes or no parasitism’, both or none?

* Parasitism rate reflects both attack rate and successful parasitism rate, and relate to parasitoid virulence. We only measured parasitism rates, but we could hypothesize that the decrease in parasitism rates with warming temperature was due to a decrease in successful parasitism rate and not attack rate by looking at the effect of parasitism treatment (presence or absence) on host abundances at both temperatures. We have now made sure that the terms used were clear and consistent through the manuscript.

In the Line 92 the word ‘parasitism’ refers to: ‘yes or no parasitism’ or to ‘parasitism rates (host-parasitoid species pair)’?. Please, could you rephrase the sentence in lines 92 – 93 (…while the effects on parasitism will be linked to effects of temperature on parasitoid attack rate and virulence.) for clarity?

* The sentence was rewritten for clarity [lines 96-99 in the revised manuscript].

Methods:

Line 116: what do you mean by revive genetic variation? Would it be to ensure or to rescue?

* We have changed revive to ensure as suggested [line 126 in the revised manuscript].

Lines 133 – 135: the single species and multiple species treatments were only for hosts right? The parasitoid-host species interaction was a sample from the single and multiple host species treatment, right? In these lines I would add ‘single-host species’ and ‘multiple-host species’ for clarity.

* We have modified the sentence as suggested [lines 151-152 in the revised manuscript].

Line 160: In figure 1 legend we read ‘c)parasitism only’. However this treatment also includes host intraspecific competition according to what it is showing in Figure1, right? I would then change this label.

* We have changed the legend according to suggestion.

In Figure1: I think it would be more informative if you could add a dashed line (or indicate somehow) the intraspecific competition in letter a and c.

* We did not add a dashed line for intraspecific competition but changed the legend of panel (c) to add intraspecific competition as suggested above.

Besides, in the mixed-species treatment do you think that intraspecific competition could also have happened together with the interspecific competition? If yes, do you think intraspecific could have been stronger than interspecific competition in these mixed-species treatments (figure 1 letter b and d)?

* We have added a discussion about the absence of effect of the type of competition between hosts on host abundances in the Discussion section [lines 331-333 in the revised manuscript].

In Line 171 ‘the sampled pupae were identified to their corresponding host species’, how was it done? Do the Drosophila species have visually different pupal case? Was it how you link a parasitoid to a host species to calculate parasitism rates?

* Yes, we chose host species that could be tell apart from their pupae. We have now specified that pupae were identify morphologically [lines 192-193 in the revised manuscript], and have added a new Supplementary Figure S3 showing photography of the pupae for the three host species.

Line 176: I would make it more clear here that the parasitism rates was calculated for each host-parasitoid pair.

* We have clarified this [lines196-197 in the revised manuscript].

Line 182 we read ‘second generation started’, does it mean the F1 offspring? Are you counting the Parental generation as first?

* We have removed this part to avoid confusion and replace it by “until four consecutive days of no adult emergences” [line 203 in the revised manuscript]. We previously considered F0 as the parental generation and F1 as the offspring.

Line 186: you mean fully-ecloded?

* We believe “eclosed” is the correct spelling.

Results:

Line 213: do you mean host frequencies instead of host proportions?

* Yes, we have modified this according to the suggestion [line 237 in the revised manuscript].

In Figure 3 legend: in letter ‘b’ we read: (b) Parasitism was reduced at higher temperature. Do you mean here parasitism rates? In your manuscript I separate parasitism as ’yes or no’ and parasitism rates as ‘the host-parasitoid pair.’

* Yes, we have changed the legend as suggested (“parasitism rates” instead of “parasitism”) and have made sure that the terms were clear throughout the revised manuscript.

In Figure 2 legend: do you refer to ‘proportion’ as the host frequency?

* Yes, we have changed the legend as suggested.

Discussion:

Below I apologize for not indicating the line number that I’m referring to. It happened because the number of the lines is not appearing in my pdf version.

* Apologies. Line numbers have now been added for the whole manuscript.

In the third paragraph of the discussion you have: ‘However, presence of parasitoids significantly decreased... parasitoid virulence’. It would be more clear if there is a following up sentence to better explain this sentence and the decrease in virulence. For example, why parasitoids would decrease virulence with increasing temperature?

* We have changed “parasitoid virulence” for “successful parasitism rates” [lines 309-310 in the revised manuscript], and reorganized parts of this paragraph for clarity.

The idea that warming decreased parasitism rates, but did not impact the effect of the presence of parasitoid in decreasing host abundance, could be better explored in the discussion. For example, although parasitoid preference for host species was not quantified, Lavandero and Tylianakis et al 2013 shows that genetically similar parasitoids become more likely to attack genetically similar hosts in warmer sites. Therefore, there could be a shift in the parasitoid preference for a host species or even for a narrower subset of host species that would decrease parasitism rates (host-parasitoid pair) due to parasitoid competition under warming conditions.

* Thank you for this suggestion. We have reorganized the discussion of this result and now discuss the suggested reference [lines 318-320 in the revised manuscript].

In the fourth paragraph, fourth line we read: ‘…allowed us to detect their effects on host species relative abundances if they were any.’ Should the word ‘they’ be ‘there’?

* Yes, corrected

In the last paragraph for the discussion we read: ‘The role of parasitoids as essential… after several generations’. What do you mean by suggesting that the indirect effects of the temperature on the host community through parasitism could be observed after several generations?

* We have moved this part of the Discussion to a previous paragraph where we explained the idea in more detail [lines 320-323 in the revised manuscript].

Attachment

Submitted filename: Exp2018_Response to reviewers.pdf

Decision Letter 1

Lucas D B Faria

27 Jan 2021

PONE-D-20-39886R1

Experimental warming influences species abundances in a Drosophila host community through direct effects on species performance rather than altered competition and parasitism

PLOS ONE

Dear Dr. THIERRY,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 13 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Lucas D. B. Faria

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Dear THIERRY, M. and authors

I have gone through the revised manuscript. Comments raised by the reviewers were responded by the authors ok. However, I still have doubts about figures (2, 3 and S7) and statistical tests.

Line 227 points out the existence of Tukey's post hoc test, however, I could not find through the text or at the figures any reference to it. Please confirm it and add the information or remove it from the text.

Figure 2, 3 and S7 labels ascribe (The small points represent the values from each block and each host-parasitoid pair, the large points represent the grand mean, and the bars represent standard errors of the means). However, I could no find a difference between point sizes. Please confirm it and add the information or remove it from the text.

Figure 3(b) label legends species as follow (□: Asobara sp. - D. pseudoananassae, ˄: Asobara sp. - D. birchii, +: Asobara sp. - D. sulfurigaster, × Leptopilina sp. - D. pseudoananassae, ◊: Leptopilina sp. - D. birchii, ˅: Leptopilina sp. - D. sulfurigaster). However, figure did not shows it. Please add it to the figure.

Tables 2 and S5 labels are quite similar (difference consist only "...for the whole dataset". Please explain the difference between them - by means of the whole dataset - in the S5 label so readers could follow the authors' idea easily. Further and regarding to F-values legend [F-values are presented with the significance of the effect: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (.) P < 0.1, (ns) P > 0.05"] - I understand that p-values greater than 0.05 are non-significative, so "(.) P < 0.1" does not make sense to me, and I am asking to avoid it, even though R model summary gives it. Then change it at the tables (.) to (ns).

Kind regards

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Feb 11;16(2):e0245029. doi: 10.1371/journal.pone.0245029.r004

Author response to Decision Letter 1


27 Jan 2021

Dear Editor,

Thank you for considering our manuscript for publication in PLOS ONE and for the opportunity to submit a revised version. We are glad that you consider our responses to the reviewers sufficient. We appreciate the constructive criticisms you have raised regarding our figures and statistical tests, and have made appropriate changes in response as detailed point by point below. We hope that this new revision will allow the manuscript to be published in PLOS ONE.

Yours sincerely,

Mélanie Thierry, Nicholas A. Pardikes, Chia-Hua Lue, Owen T. Lewis & Jan Hrček

Additional Editor Comments:

Dear THIERRY, M. and authors

I have gone through the revised manuscript. Comments raised by the reviewers were responded by the authors ok. However, I still have doubts about figures (2, 3 and S7) and statistical tests.

* Thank you for raising those mistakes and oversight. We have corrected the results, figures and the tables as suggested.

Line 227 points out the existence of Tukey's post hoc test, however, I could not find through the text or at the figures any reference to it. Please confirm it and add the information or remove it from the text.

* We corrected the methods, which now reads “Post-hoc multiple comparisons were performed using the emmeans package, and P-values were adjusted using the Tukey method.” [lines 227-228 of the new revised manuscript]. We have added the Post Hoc test results throughout the Result section [lines 348-352 of the new revised manuscript].

Figure 2, 3 and S7 labels ascribe (The small points represent the values from each block and each host-parasitoid pair, the large points represent the grand mean, and the bars represent standard errors of the means). However, I could no find a difference between point sizes. Please confirm it and add the information or remove it from the text.

* Apologies, the small points were lost with the format conversion of the figures. We uploaded revised versions of the figures with both point sizes.

Figure 3(b) label legends species as follow (□: Asobara sp. - D. pseudoananassae, ˄: Asobara sp. - D. birchii, +: Asobara sp. - D. sulfurigaster, × Leptopilina sp. - D. pseudoananassae, ◊: Leptopilina sp. - D. birchii, ˅: Leptopilina sp. - D. sulfurigaster). However, figure did not shows it. Please add it to the figure.

* This was the same problem than for the small points raised previously. The revised version of Figure 3 now shows those symbols.

Tables 2 and S5 labels are quite similar (difference consist only "...for the whole dataset". Please explain the difference between them - by means of the whole dataset - in the S5 label so readers could follow the authors' idea easily.

* We have added to the Table S5 label “(without any deleted observations due to D. sulfurigaster)”.

Further and regarding to F-values legend [F-values are presented with the significance of the effect: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (.) P < 0.1, (ns) P > 0.05"] - I understand that p-values greater than 0.05 are non-significative, so "(.) P < 0.1" does not make sense to me, and I am asking to avoid it, even though R model summary gives it. Then change it at the tables (.) to (ns).

* We changed the P-value significance as suggested.

Attachment

Submitted filename: Exp2018_PlosOne_Response to reviewers_v2.docx

Decision Letter 2

Lucas D B Faria

29 Jan 2021

Experimental warming influences species abundances in a Drosophila host community through direct effects on species performance rather than altered competition and parasitism

PONE-D-20-39886R2

Dear Dr. THIERRY,

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Additional Editor Comments (optional):

I have gone through the revised manuscript (R2) and I am satisfied with the changes. So I am pleased to say that the manuscript is accepted to be published in Plos One Journal.

Congratulations and kind regards

Reviewers' comments:

Acceptance letter

Lucas D B Faria

1 Feb 2021

PONE-D-20-39886R2

Experimental warming influences species abundances in a Drosophila host community through direct effects on species performance rather than altered competition and parasitism

Dear Dr. Thierry:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. Lucas D. B. Faria

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Transparent plastic boxes (47cm x 30cm x 27.5cm) with three ventilation holes (15 cm in diameter) covered with insect-proof nylon mesh used as experimental unit allowing parasitoids to attack one of the three experimental vials containing 2-days-old host larvae for 72h.

    (PDF)

    S2 Fig. Drosophila birchii, D. pseudoananassae, and D. sulfurigaster pupae photography for morphological identification.

    Not in scale (photo credit to Jinlin Chen).

    (PDF)

    S3 Fig. Interactive effect of competition with host species, and with presence of parasitoids on mean host body mass (squares: D. birchii, diamonds: D. pseudoananassae, triangles: D. sulfurigaster).

    See Fig 1 for detailed description of the treatments. The small points represent the values from each block and each host-parasitoid pair, the large points represent the grand mean, and the bars represent standard errors of the means. Blue: ambient temperature (23°C), red: warming treatment (27°C).

    (PDF)

    S1 Table. Mean number of offspring per species with 10, 30, 60, 90 or 180 adult hosts (1:1 sex ratio) in a 5 mL host-media glass vial.

    Choice of host number in the main experiment was based on these preliminary data to correspond to strong competition for all host species.

    (PDF)

    S2 Table. Number of observations per temperature, treatments (Intraspecific competition: No interaction between host species; Interspecific competition: Direct competition between host species; Parasitism: Intraspecific competition with parasitism; All interactions: Interspecific competition with parasitism), and host species in the whole dataset, and with the reduced dataset used for analyses (excluding observations with fewer than 10 emerging insects or pupae).

    (PDF)

    S3 Table. Table showing the effect of temperature (23°C or 27°C), parasitism (presence or absence), competition between host species (intraspecific or interspecific), host species (n = 3), interactions between terms, and block (n = 4), on host abundances, and host frequencies for the whole dataset (without any deleted observations due to D. sulfurigaster).

    Degrees or freedom (Df) for each F-ratio are given for each factor and for the error. F values are presented with the significance of the effect: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (ns) P > 0.05.

    (PDF)

    S4 Table. Summary table for mean (± SD) host abundances (Host ab.), individual host body mass (Host BM), total parasitism rate (PR), and parasitism rates of each parasitoid species (Asobara sp. and Leptopilina sp.) for each temperature (23 and 27°C), treatments (competition: Intra or inter, parasitism: Present or absent), and host species (D. birchii, D. pseudoananassae, D. sulfurigaster).

    (PDF)

    Attachment

    Submitted filename: Exp2018_Response to reviewers.pdf

    Attachment

    Submitted filename: Exp2018_PlosOne_Response to reviewers_v2.docx

    Data Availability Statement

    All raw data used for this study are available from the Zenodo database: https://doi.org/10.5281/zenodo.4362189


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