Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?

Body size is commonly associated with biological features such as reproductive capacity, competition, and resource acquisition. Many studies have tried to understand how these isolated factors can affect the body pattern of individuals. However, little is known about how interactions among species in multitrophic communities determine the body shape of individuals exploiting the same resource. Here, we evaluate the effect of fruit infestation, parasitism rate, and seed biomass on size, allometric and asymmetric patterns of morphological structures of insects that exploit the same resource. To test it, we measured 750 individuals associated with the plant Senegalia tenuifolia (Fabaceae), previously collected over three consecutive years. Negative allometry was maintained for all species, suggesting that with increasing body size the body structure did not grow proportionally. Despite this, some variations in allometric slopes suggest that interactions in a multitrophic food web can shape the development of these species. Also, we observed a higher confidence interval at higher categories of infestation and parasitism rate, suggesting a great variability in the allometric scaling. We did not observe fluctuating asymmetry for any category or species, but we found some changes in morphological structures, depending on the variables tested. These findings show that both allometry and morphological trait measurements are the most indicated in studies focused on interactions and morphometry. Finally, we show that, except for the fluctuating asymmetry, each species and morphological structure respond differently to interactions, even if the individuals play the same functional role within the food web.

Introduction

During their development, organisms are exposed to numerous stresses, such as severe weather conditions [1], pollution [2], deficiency of nutrients [3], competition [4], predation and parasitism [5, 6]. These stresses can directly affect the body size of organisms [7], which in turn can influence changes in their fertility, survival, and dispersion [8-10]. When organisms suffer stresses above their tolerance, they need to compensate for the loss of energy, reducing the amount of energy allocated for the growth, maintenance, and development of the morphological structures, which produces distortions in development, body size and symmetry [11-16]. However, some organisms can withstand stress through “buffering” mechanisms, producing pre-determined phenotypic characteristics, which limit the variation of the phenotype and reduce the effects of stress on their development [17].

Two tools commonly used to evaluate possible phenotypic responses caused by stress are the allometry and fluctuating asymmetry (FA) [15, 18]. Static allometry is used to evaluate the mechanisms that influence variations in the growth of co-specific individuals in the same stage of life. The relationship between individuals’ development is observed considering body size and its organs, or between two organs (e.g. scaling relationship) [19-23]. FA refers to small random deviations from the symmetry of bilaterally symmetrical traits and it can be used as an indicator of the individual's ability to maintain a symmetrical development in the face of biotic and abiotic disorders [16, 24].

In recent years, with technological advances, there has been a significant increase in morphometric investigations, especially in the group of insects. This has directly reflected in a better understanding of the influence of abiotic and biotic factors on the morphological aspects of these individuals [16, 19, 23, 25–28]. Nevertheless, most studies have analyzed isolated factors influencing the body size of organisms [23, 27–33] and few considered multiple interactions affecting body size at the same time [34, 35]. Recently, we observed that interactions in a multi-trophic network can drive to changes in the body size of seed-feeding insects differently, in which abundant species have their body size more affected than less abundant ones [35]. Likewise, although there are several studies considering isolated factors influencing morphometric patterns (e.g. allometry and FA) [16, 20, 23, 25, 36], we are not aware of previous studies that have assessed how multiple interactions may or may not affect allometric patterns and FA of insects that share the same resource, especially in natural environments.

Thus, the present study evaluates the effect of different interactions on the morphology of three different insects, which exploit the same plant resource. To do that, we assessed the allometry and fluctuating asymmetry of the beetles Merobruchus terani Kingsolver, 1980, and Stator maculatopygus (Pic, 1930) (Chrysomelidae: Bruchinae) and the wasp Allorhogas vulgaris Zaldívar-Riverón & Martinez [37] (Hymenoptera: Braconidae: Doryctinae). These three species of seed-feeding insects are associated with the fruits of the plant Senegalia tenuifolia (L.) Britton & Rose (Fabaceae: Mimosoideae). Their immature stages live and feed inside the seed and when they reach adulthood, they can feed on nectar and pollen [38-40]. Moreover, because they spend much of their lives inside the seed, and their mobility is limited by their life history, these insects can experience high levels of competition and parasitism. Furthermore, their morphological characteristics are closely related to the features of their resource [27, 28, 33, 41, 42]. These biological aspects make this system an advantageous study model for understanding how interactions can affect insect morphometric patterns in a natural system.

We hypothesize that an increase in fruit infestation, parasitism rate, and a reduction in seed biomass cause: (I) a negative change in allometric slopes; (II) greater deviations in the fluctuating asymmetry of these species; (III) and reduction in the size of their morphological structures. To test these hypotheses, we selected the morphological traits that best explain the body size variation in some species of bruchine beetles [20, 23], namely pronotum length, left and right elytra and biomass; and for the wasp: right and left wing, right and left tibia length, and total body length of A. vulgaris [43, 44]. We evaluated the size, the allometric relationships and the fluctuating asymmetry of these morphological traits, according to different categories of seed infestation, parasitism rate and seed biomass to investigate the possible influence of these variables on the morphological structures of these species.

Materials and methods

Field location

We carried out this study at the municipalities of Lavras and Luminárias, in fragments of Brazilian Cerrado (savanna) in the south of Minas Gerais state, south-eastern Brazil. The study areas were divided into three main fragments called Ae, La, and Lu, which were 6 km apart from each other. Across these three areas, we established seven subareas which were at least 400 m from each other (Ae-1: 21°14′5.71′′S- 44°57′8.66′′W; Ae-2: 21°14′7.87′′S-44 58′0.06′′W; La-1: 21°-18′3.46′′S- 44°58′0.53′′W; Lu-1: 21 31′1.36′′S-44°53′1.78′′W; Lu-2: 21 31′5.13′′S-44°52′6.32′′W; Lu-3: 21 31′5.31′′S-44 52′3.84′′W; and Lu-4: 21°41'9.88''S- 44°96'7.18''W); see [45] for additional details of the study site.

The plant system, the assessment of fruits, seed biomass and insects

No permits or authorization were obtained as Brazilian authorities did not require permits for collection by public roads. Senegalia tenuifolia is locally known as unha-de-gato, and it is a pioneer species largely distributed in South America [46]. Senegalia tenuifolia is characterized as a scandent shrub, i.e., a shrub that is able to become a liana and reach a greater height (up to 8 m) [47]. The flowering period occurs from November to January, and the ripening occurs from January to August (Faria, L.D.B., personal communication). After this phase, fruits open and seeds begin to fall on the ground [45].

The S. tenuifolia fruits were previously collected in 2012, 2013, and 2014 in June, July, and August, during the plant’s ripening season. In this stage, plants can be recognized by their brownish color. Nevertheless, we only considered the collections of the months of July and August, as insects collected in June were not developed, making insect identification impossible. As S. tenuifolia is a liana, we could not distinguish the number of different plants at each site, so we opted to collect the fruits randomly in each subarea [33]. We collected 25 fruits from seven subareas for each month; thus, for each year, we had a sample size of 350 fruits except for 2013 (349 fruits sampled) totaling 1,049 fruits. Furthermore, there were different numbers of sampling sites for each area (see Table 1 for more details). All fruits were collected when still attached to the mother plant and taken to the laboratory, where each fruit was stored in individual PVC tubes covered with voile to enable air circulation. The fruits were stored for three months to allow the insects inside the seeds to complete their development and emerge as adults. After this, we opened the fruits and assessed the seed biomass by separating them into paper bags and drying at 40°C for 48 h. Subsequently, they were weighed in precision analytical balance in order to obtain the dry biomass.

Table 1

Sampling site areas with the amount of fruits gathered per subareas in each year.

Sampling sites and number of fruits per area in each year
Areas	2012	2013	2014
Ae	Ae1, Ae2	Ae1, Ae2	Ae1, Ae2
	100 fruits	99 fruits	100 fruits
La	La1	La1	La1
	50 fruits	50 fruits	50 fruits
Lu	Lu1, Lu2, Lu3, Lu4	Lu1, Lu2, Lu3, Lu4	Lu1, Lu2, Lu3, Lu4
	200 fruits	200 fruits	200 fruits

The emerged insects of each fruit were identified at the genus level and species level when possible, stored in 1.5-mL labeled plastic microtubes containing 70% ethanol and deposited in the Entomological Collection of the Laboratory of Ecology and Complexity at the Federal University of Lavras, Minas Gerais, Brazil. Subsequently, we used these specimens to measure their morphological structures. Building upon data previously collected, we gathered information on seed biomass of S. tenuifolia and on the abundance of the three seed-feeding species (M. terani Kingsolver, 1980, S. maculatopygus (Pic, 1930) and A. vulgaris Zaldívar-Riverón & Martínez 2018), and their parasitoids in each fruit [45, 48]. We chose these three species because they are the most abundant species consuming S. tenuifolia. The simplified food web is displayed in Fig 1.

Fig 1

The Senegalia tenuifolia and related trophic levels.

The resource plant represents the first trophic level, the second trophic level comprises the three main seed-feeding species, the third trophic level comprises the parasitoid species, and the fourth trophic level comprises the hyperparasitoid species. Solid lines indicate observed interaction and dashed lines indicate potential interaction. Potential interactions were based on information from previous studies. Figure obtained from previous studies [35].

Morphological measurements

Specimens of M. terani, S. maculatopygus, and A. vulgaris were placed on slides in the dorsoventral position for microscopy. The slides were photographed using a Leica M205A stereoscopic microscope coupled to a Leica DFC295 camera. Subsequently, measurements of the morphological structures were made based on the photos uploaded in Leica application suite. The structure measurements in the two coleopteran species were pronotum width, and both left and right elytra length and width [20, 23] (Fig 2A and 2B). For the hymenopteran species A. vulgaris, we measured total body size length (thorax + abdomen) [43, 49, 50], +R vein until the end of 3RSb vein, wing width measured from the junction between anterior wing length measured from the beginning of C+Sc, the parastigma and stigma to the end of vein 1A (Fig 2C) (adapted from [44]); and posterior tibia length was measured from the junction of the femur and the tibia to the junction of the tibia and tarsus [23, 35]. Wings and tibia from the wasp A. vulgaris individuals were dissected from their bodies to reduce measurement error; we measured both the right and left sides (Fig 2C). All measurements were performed three times.

Fig 2

Body traits of the seed-feeding beetles (A) Merobruchus terani and (B) Stator maculatopygus (Chrysomelidae: Bruchinae) and the wasp (C) Allorhogas vulgaris (Hymenoptera: Braconidae: Doryctinae) taken to estimate morphometry, allometry, and fluctuating asymmetry of their morphological structures, according to resource size, fruit infestation, and parasitism rate. The solid arrows indicate the length and the dashed arrows the width of (a) pronotum, (b) left elytra, (c) right elytra for the beetles, and (1) indicates the measurement of total body size length (thorax + abdomen); (2) the forewing length measured from the beginning of C+Sc+R vein until the end of 3RSb vein, forewing width measured from the junction between the parastigma (Pt) and stigma (Stg) to the end of vein 1A; and (3) posterior tibia length for the wasp.

Data analysis and categories

We tested the effect of fruit infestation, parasitism and resource size (seed biomass) on morphometric patterns of the three seed-feeding species. To do it, we calculated the fruit infestation rate (FIR) (1) and fruit parasitism rate (FPR) (2) by the following formulae:

Where HT is the total abundance of herbivorous, SA is the total number of seed per fruit, PT is the total number of parasitoids in the fruit, and HT is the total abundance of seed-feeding insects in the fruit (i.e., Merobruchus terani, Stator maculatopygus and Allorhogas vulgaris).

We defined three categories for both fruit infestation and parasitism rate: “Low”, from 0 to 0.30; “Medium”, from 0.31 to 0.60; and “High” from 0.61 to 1 (adapted from [43]). In addition, to have an approximately equal amount of samples by each category of seed biomass, we divided them into: “Small”; from 0.025 mg to 0.266 mg; “Medium” from 0.271 mg to 0.454 mg; and “Large” from 0.454 mg to 1. 993 mg.

Preliminary Spearman correlation analysis indicated that the elytra length and width of M. terani (correlation of 0.88) and S. maculatopyus (correlation of 0.77), as well as the anterior wing length and width of A. vulgaris (correlation of 0.89) were highly correlated. Thus, the morphometric analyses were conducted using only the length of these morphological structures.

Allometry was evaluated according to categories of fruit infestation, parasitism rate and seed biomass, estimating the rate of variation of the pronotum and elytra (mean of the right and left sides) of M. terani and S. maculatopygus in relation to the individual body weight, for A. vulgaris, we estimated the rate of variation of the wing and tibia (mean of the right and left sides) in relation to the total body size of each individual. In this analysis, we used ‘major axis’ regressions (i.e., geometric mean), which are considered more appropriate when both x and y variables are measured with error [23, 51]. The values of slope and confidence intervals from the analyses were used in a single plot to illustrate the allometric relationships among species and all categories.

We evaluated the FA patterns of species according to categories of fruit infestation, parasitism rate, and seed biomass. We selected the following morphological structures: elytra length (right and left) of M. terani and S. maculatopygus and wing and tibia length (right and left) of A. vulgaris. We used generalized linear mixed models (GLMM) analysis with restricted likelihood (REML), which produces unbiased estimates for the values of fluctuating asymmetry (i.e., fixed effects), considering possible measurement errors among individuals (i.e., random effects) [52]. The fixed effects evaluate directional asymmetry (DA), while the random effects evaluate fluctuating asymmetry (FA). All models considered random terms for the intercept, which estimates an average value among the three individual measurements. Besides, the structure of the random effect for the slope evaluates FA, estimating the same growth rate between the sides (M1), or different growth rates between the sides (M2). We also considered variations in the slope between the sides associated with the categories of infestation rate (M3) and parasitism rate (M4). We selected the best model through the likelihood ratio test (see supporting information S1 Table). Considering the best model for the structure of random effects (RE), we also evaluated the variation in the size of the structures among the categories of fruit infestation, parasitism rate, and seed biomass. Significance levels between groups were tested by post-hoc Tukey, preventing overestimated inferences with the ‘lsmeans’ package [53]. All analyses were performed using R software v.3.4.1 [54]. Allometry analyses were performed using the ‘lmodel2’ package [55]. REML analyses were conducted with the ‘lme4’ package [56] and ‘lmerTest’ package [57]. All relevant data are within the paper and its Supporting Information files.

Results

We measured 750 seed-feeding insects, 534 of these were M. terani individuals, 146 were A. vulgaris individuals, and 70 were S. maculatopygus individuals, distributed according to the categories displayed in Table 2. The number of insects present in the highest categories of fruit infestation and parasitism was lower concerning the medium and low categories for all species, except for A. vulgaris, which was more abundant in fruits with higher infestation rate. We did not find individuals of M. terani or S. maculatopygus in fruits with a high infestation rate, and their abundances were lower in high parasitism rate (Table 2).

Table 2

Total abundance of the three seed-feeding insects, Merobruchus terani, Stator maculatopygus and Allorhogas vulgaris, associated to the plant Senegalia tenuifolia by category of fruit infestation, parasitism and seed biomass.

CATEGORIES	Merobruchus terani	Stator maculatopygus	Allorhogas vulgaris
Low fruit infestation (LFI)	323	45	34
Medium fruit infestation (MFI)	191	4	51
High fruit infestation (HFI)	0	0	61
Low parasitism rate (LPR)	432	43	114
Medium parasitism rate (MPR)	71	4	28
High parasitism rate (HPR)	15	3	0
Small seed (SS)	183	8	61
Medium seed (MS)	181	11	62
Large seed (LS)	154	31	23
Total abundance	534	70	146

Regarding the allometric analysis, we found slope values less than one, which suggests negative allometric patterns for all species (Figs 3, 4 and 5). This means that the structures measured (pronotum and elytra length in the beetles, and wing and tibia length in the wasp) increased proportionally less in their length concerning their body size. Despite that, we observed small variations of allometric slopes and confidence intervals among the categories and species, indicating some kind of effect on their morphological pattern (See supplementary material, S2–S4 Tables).

Fig 3

Negative allometry depicted by the slopes and their confidence intervals (CI) for the pronotum and elytron allometry (pronotum length and elytron length in relation to body weight) among infestation, parasitism rate, and seedbiomass categories for Merobruchus terani individuals (CI of 95%).

Low (0–0.30%), medium (0.31–0.60%), and high (0.61–1%) infestation and parasitism rate; and among small, medium, and large seeds.

Fig 4

Negative allometry depicted by the slopes and their confidence intervals (CI) for the pronotum and elytron allometry (pronotum length and elytron length in relation to body weight) among infestation, parasitism rate, and seed biomass categories for Stator maculatopygus individuals (CI of 95%).

Low infestation categories (0%-0.30%) and low parasitism categories (0%-30%), and between small, medium, and large seeds.

Fig 5

Negative allometry depicted by the slopes and their confidence intervals (CI) for the tibia and wing allometry (tibia length and wing length in relation to body size) among infestation, parasitism rate, and seed biomass categories for Allorhogas vulgaris individuals (CI of 95%).

Low (0–0.30%), medium (0.31–0.60%), and high (0.61–1%) infestation and parasitism rate (low and medium); and among small, medium, and large seeds.

Fruit infestation positively influenced the pronotum allometry (increased allometric scale) of M. terani and both wing and tibia allometry of A. vulgaris. The slope variation between the pronotum and body weight (pronotum allometry) in M. terani individuals was 0.08 in LFI (low fruit infestation), 0.16 in MFI (medium fruit infestation) (Fig 3). To A. vulgaris, the slope was 0.45 in LFI, 0.55 in MFI and 0.71 in HFI (high fruit infestation) between its wing and total body size (wing allometry), and 0.45 in LFI, 0.55 in MFI and 0.76 in HFI between its tibia and total body size (tibia allometry) (Fig 5). On the other hand, we found a weak negative effect (decreased in allometric scale) of the fruit infestation on the elytra allometry of M terani, with a variation of allometric slope between elytra and body weight of 0.15 in LFI; and 0.12 in MFI (Fig 3).

The rate of parasitism negatively influenced the pronotum and elytra allometry of M. terani individuals and the wing and tibia allometry of A. vulgaris individuals. The slope variations between pronotum and body weight of M. terani (pronotum allometry) were: 0.07 in LP (low parasitism), 0.03 in MP (medium parasitism); and 0.03 in HP (high parasitism); and between elytra and body weight of M. terani (elytra allometry) were 0.16 in LP; 0.25 in MP; and 0.06 in HP (Fig 3). The slope variations of the wing and total body size of A. vulgaris (wing allometry) were 0.60 in LP and 0.46 in MP, and between the tibia and total body size of A. vulgaris (tibia allometry) were LP: 0.57; MP: 0.49 (Fig 5).

Regarding the seed biomass categories, we found a slight negative variation in the elytra allometry (variation between elytra and body weight) of M. Terani with slope values of 0.13 in SS (small seed), 0.12 in MS (medium seed) and 0.11 in LS (large seed). No variations were found for M terani pronotum allometry, since the slope was 0.09 in all categories of seed biomass (SS, MS, and LS) (Fig 3). On the other hand, we found a positive variation in wing allometry of A. vulgaris individuals, with slopes of 0.60 in SS, 0.50 in MS, and 0.70 in LS, as well as their tibia allometry with slopes of 0.64 in SS, 0.46 in MS, and 0.61 in LS (Fig 5). For S. maculatopygus individuals, we found higher allometric values in MS category, both to their elytra (slope values of 0.03 in SS, 0.10 in MS, and 0.03 in LS) and pronotum (slope values of 0.03 in SS, 0.10 in MS, and 0.08 in LS) (Fig 4). Furthermore, a closer inspection on confidence intervals indicated isometry of A. vulgaris at high rates of infestation, parasitism, and large seeds, once confidence intervals were ≥ 1 in these situations (Fig 5).

We did not observe fluctuating asymmetry for any morphological structure tested, as the results obtained by the selecting models showed no significant differences among the models tested for all species (see supplementary material S5 Table). Thus, in all cases, we considered the simplest model (M1), which does not distinguish random effects for the slope between the right and left sides (i.e., absence of FA). Additionally, we verified a significant increase in the elytra length of M. terani with the increase of seed biomass and fruit infestation (Fig 6). The effect of seed biomass was significantly dependent on the category, but not on the side, which suggests that there is neither fluctuating (FA) nor directional asymmetry (DA).We also observed a significant effect of wing size of A. vulgaris in relation to seed biomass, regardless of the side: smaller seeds caused a greater increase in the wing size of A. vulgaris (see supplementary material, S7 Table) (Fig 7). We did not observe any relation of seed biomass, parasitism rate and infestation rate in the elytra length of S. maculatopygus (see supplementary material, S8 Table), elytra length of M. terani (see supplementary material S6 Table), or in the tibia length of A. vulgaris (see supplementary material S7 Table).

Fig 6

Fluctuating asymmetry and the effect of seed biomass and infestation rate on the elytra length of Merubruchus. terani.

The effect was estimated using mixed linear models adjusted by the REML method; the result compares the right and left sides of the elytra. The Y- axis represents the elytra length of the M. terani and the X-axis represents the seed biomass according to the side evaluated and infestation rate: BL (large seed, left elytron), BR (large seed, right elytron), ML (medium seed, left elytron), MR (medium seed, right elytron), SL (small seed, left elytron), and SR (small seed, right elytron). Different letters indicate statistically different means between categories.

Fig 7

Fluctuating asymmetry and the effect of seed biomass on the wing length of Allorhogas vulgaris.

The effect was estimated using mixed linear models adjusted by the REML method; the result compares the right and left sides of the wing. The Y-axis represents the wing length of A. vulgaris and the X-axis represents the seed biomass: BL (large seed, left wing), BR (large seed, right wing), ML (medium seed, left wing), MR (medium seed, right wing), SL (small seed, left wing), SR (small seed, right wing). The purple color represents the left side and the blue color represents the right side. Different letters indicate statistically different means among categories.

Discussion

Through the measurement of various morphological traits of the three main seed-feeding insects associated with fruits of S. tenuifolia, we have demonstrated the potential influence of multiple trophic interactions on the body patterns (especially in allometry and morphometry) of seed-feeding insects that share the same resource. We tested this by using different categories of fruit infestation, parasitism rate, and resource size. In general, we found a negative allometric pattern for all species with some variations in allometric slopes, suggesting that interactions in a multitrophic food web can indeed shape the development of these insects’ bodies. Moreover, we observed a substantial increase in confidence intervals at high levels of infestation and parasitism, which suggests that, at high intensity, stresses can lead to an increase in variations of the three seed-feeding insects' bodies, changing their allometric scaling, but not their allometric pattern (slope values < than 1). We did not observe fluctuating asymmetry for any of the species studied; however, we observed a significant increase in the elytra of M. terani at high infestation and a reduction in wing length of A. vulgaris in large seeds.

In the group of insects, both body size and locomotion structures (e.g. wing and tibia) are essential for dispersion, competitive ability, and escape from predators [10]. The scale between individuals’ body sizes and their morphological structures can be changed due to strong environmental and genetic pressures during their development [58, 59]. However, to avoid abrupt changes affecting the performance of individuals, there is a certain flexibility in the growth of body size and its morphological structures [60]. We observed this flexibility in the allometric pattern of all the seed-feeding insect species studied that despite maintained a negative pattern regardless the categories tested, produced greater variations in morphological traits at high levels of fruit infestation and parasitism.

The fruit infestation, parasitism rate, and biomass of the resource did not change the morphological allometric pattern of the three seed-feeding insect species. However, as we found a great variation in allometric slopes, it seems these variables (e.g. fruit infestation, fruit parasitism and seed biomass) can influence the insect's development, both in its allometry and abundance. Therefore, since these variables negatively changed (decreased) some allometric scaling of these three seed-feeding species, our first hypothesis was partially corroborated. The fruit infestation and seed biomass caused a positive (increased allometric slopes) variation in the allometry of the pronotum of M. terani and the allometry of wings and tibia of A. vulgaris. Studies have shown that the amount of resource available during the larval phase can affect the relationship between body size and morphological structures of some individuals [5, 58, 60–63]. In one study, for instance, they observed that insects that did not have enough food during their larval stage had their body size reduced and wings proportionally larger [60]. This happened because the initial lack of food prevented the body size of individuals from growing, whereas the wings continued to grow exponentially, increasing their wing allometric coefficient. Also, a continuous growth in appendages of holometabolous insects was observed, even after their body size had stopped growing [64]. Thus, the increase of fruit infestation, and consequently, the decrease in resource availability would be affecting the body weight of these individuals, without necessarily leading to a decrease in their morphological structures, increasing the allometric slope of their morphological structures.

On the other hand, the infestation categories and seed biomass negatively influenced (decreased) the elytra allometry slopes of M. terani. This result is similar to the one obtained in another study, in which it was observed that different morphological structures in the same individual can present different plasticity in face of biotic and abiotic factors [59]. Therefore, as the same factor (e. g. fruit infestation and seed biomass) can have a different effect on different morphological traits, we strongly recommend measuring more than one morphometric structure in the same individual, especially in morphometric studies.

The parasitism rate negatively changed the allometric pattern of both the pronotum and elytra of M. terani, and the wing and tibia of A. vulgaris. This may have occurred because the body size and morphological structure growth in holometabolous insects occur at different times. First, there is an increase in body size, which is proportional to the amount of food available during the larval phase. After that, individuals stop eating and enter the pupal stage, when their morphological structures start the development process [65]. Insects exposed to the presence of parasitoids can change their foraging habitat inside fruits [66, 67]. In one study, it was found that both insect hosts and their parasitoids can detect the presence of each other by vibrations emitted when they are foraging [66]. The parasitoids can detect their host by the vibration emitted while they are feeding on the seeds, and the host can detect the presence of parasitoids by the vibration they emit when they insert their ovipositor inside the fruit. Consequently, to survive, these hosts stop eating or accelerate their development [68, 69]. Thus, although the morphological structures grow independently from body size, both the lack of food and the acceleration of their development could have caused these changes in the allometric scale of these seed-feeding insect species.

Furthermore, we were unable to observe the effect of the infestation and parasitism rate on the allometry of the structures of S. maculatopygus, as they were absent in the medium and high categories, showing somehow their low tolerance to stresses caused by fruit infestation and parasitism, something already suggested in other studies [23, 33, 35]. Besides, the values of the elytra and pronotum allometry of S. maculatopygus were close to zero, indicating that the allometry of S. maculatopygus is not a very sensitive parameter to changes in the amount of the resource, which can be reinforced by the results of other studies, in which the body size of S. maculatopygus was not related to the size of the resource [23, 35].

In addition to allometry pattern, another approach used in morphometric studies is FA. Some studies consider FA to be a good indicator of instability in the development of individuals under stressful situations [1, 12, 70–72]. However, other studies failed to find FA responses under stressful situations caused by larval density [73] and lack of food [74, 75]. Likewise, we did not find differences between the right and left sides (e.g., FA) of the three seed-feeding insects, regardless of the categories tested (e.g., infestation rate, parasitism rate, and seed biomass), rejecting our hypothesis that greater stresses would cause greater deviations in the symmetry of the three seed-feeding insect’ species. Some studies have suggested that body size is more sensitive to environmental disturbances than FA, since body size of individuals can present a greater plasticity than FA [75-77]. Therefore, due to the great plasticity of body size, there is a dampening of the impact caused by stressful factors in the development of organisms, thus maintaining their shape, symmetry, and, consequently, performance.

Our results confirm that both body size and morphological structure size are more plastic (higher variability) than changes in their symmetry (FA), since we verified some significant relationships between the measured body structures and the categories (e.g., competition and size of the resource), regardless of the side evaluated. The seed biomass showed a positive relationship with the elytra size of M. terani, and a negative one with the wing size of A. vulgaris. Besides, we found that, at high infestation rate, M. terani elytra’s size is bigger. The increase in the M. terani elytra was also observed in another study, in which it was verified that increase of competition generates both bigger elytra and body size for this species [23]. By contrast, the results obtained by A. vulgaris indicate that larger fruits cause a decrease in their wings. However, this result may reflect the habit of this species to feed on the ends of the seed, allowing the exploitation of the resource by more than one individual [78]. In addition to this, we observed in the laboratory that the seeds preyed upon by these individuals are almost completely consumed, which would explain this negative relationship between the seed biomass and the body size of A. vulgaris [35].

We did not find significant influences of fruit infestation and parasitism on the morphological traits of any of the three species, refuting our hypothesis that higher levels of infestation and parasitism would cause a decrease in the morphological structures of these species. Also, the hypothesis that a decrease in the amount of resource would decrease the growth of morphological structures was only corroborated for M. terani. These results show that although fruit infestation and parasitism are limiting factors on the abundance of these individuals at higher rates, it is not necessarily reflected in changes in their symmetry, but instead can cause changes in the size of morphological structures, indicating that this approach would be most interesting in studies focusing on interaction and morphometry.

In conclusion, we have demonstrated for the first time the influences of fruit infestation, parasitism rate, and resource size on the body shape of three seed-feeding insect species that share the same resource. We did that by analyzing their allometry, symmetry, and morphometry. We found that although these species have a similar functional role (consuming seeds) in the food web, their bodies have different ways to respond to these interactions. Also, although their body scaling did not change its pattern, keeping slope values <1, the relationship between body size and morphological structures varies substantially at high levels of infestation and fruit infestation (higher CI values), demonstrating that this tool can be effectively used in evaluations of this type. Last, we suggest more studies evaluating the relationship between FA and stresses caused by interactions within a food web, since it was not clear if there was no effect on FA, or if body parts changed first.

Model testing the existence or not of fluctuating asymmetry with different combinations of random effects for the intercept and slope, by testing mixed models with restricted likelihood (REML).

(DOCX)

Click here for additional data file.

Allometric coefficient with slope value, confidence interval according to the categories and morphological structures of Merobruchus terani.

(DOCX)

Click here for additional data file.

Allometric coefficient with slope value, confidence interval according to the categories and morphological structures of Stator maculatopygus.

(DOCX)

Click here for additional data file.

Allometric coefficient with slope value, confidence interval according to the categories and morphological structures of Allorhogasvulgaris.

(DOCX)

Click here for additional data file.

Result comparing the fluctuating asymmetry models of Merobruchus terani and Stator. maculatopygus elytra and Allorhogas vulgaris’ wing and tibia.

All models have the same structure with different random factors. In which, M1 is the model with no difference in sides; M2 with different slopes for each side; M3 with different slopes for each side in infestation categories; M4 different slopes for each side in parasitism categories.

(DOCX)

Click here for additional data file.

Fluctuating asymmetry between left and right sides for elytra length of Merobruchus terani, according to categories of seed biomass, fruit infestation and parasitism rate.

Results are displayed in comparison to the right elytra.

(DOCX)

Click here for additional data file.

Fluctuating asymmetry between left and right sides for wing and tibia length of Allorhogas. vulgaris, according to categories of seed biomass, fruit infestation and parasitism rate.

Results are displayed in comparison to the right side of these structures.

(DOCX)

Click here for additional data file.

Fluctuating asymmetry between left and right sides for elytra length of Stator maculatopygus according to categories of seed biomass, fruit infestation and parasitism rate.

Results are displayed in comparison to the right elytra.

(DOCX)

Click here for additional data file.

26 Aug 2020

PONE-D-20-22527

Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?

PLOS ONE

Dear Dr. Tamires Camila Camila Talamonte de Oliveira

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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Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Colleagues, one of our reviewers ma de a series of important and detailed suggestions in the manuscript. I strongly encourgae you to follow, correct or answer the criticism of this reviewer in a second round. I agree with the reviewer in most of the comments.

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'TCTO thanks the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq) (141129/2018-2) for financial support.  LDBF thanks CAPES, CNPq (306196/2018-2), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support.'

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Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #1: Please find below my comments concerning the manuscript by Oliveira et al., entitled: “Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?” (PONE-D-20-22527). The authors present an interesting study regarding to multiple biotic factors that can shape the growth and development patterns of three species with similar ecological functions. This is a seminal work in this specific topic that highlight important aspects the importance of investigating several drivers factors at the same time. The text is well written, the analyses are appropriate, and I think the findings will be interesting for a broader audience. I think the study is sound and I have only some comments or recommendations.

Minor comments

L 25. What are these categories?

L 30-32. This sentence seems inconsistent and / or words are missing. Please, rewrite.

L 42. “create” could be respond through?

L 46. Are there any recent references?

L 67-69. Didn't you say that above? Line 64?

L 92: “and for the wasp: right and left wing, right and left tibia length and total body

length of A. vulgaris.“

L 92. Are there any references considering these measures for wasps?

Methods

L 108. I missed at least one sentence describing basic information about the plant such as growth form, average height, etc.

L 113-115. Were these fruits collected from the same individuals? If not, how many different individuals? Were the annual collections carried out in the same individuals? Please, provide these important details.

L 113. “seven” or eight?

L 180. Why the length and not the width?

L 238-247. The legend of Figures 4 and 5 looks similar, both in relation to S. maculatopygus.

L 262. Isn't it Figure 3? Please, see my comment above and check this figures citation on MS.

Discussion.

L 338-400. Please, detail how the great variation in allometric slopes can influence in the abundance of individuals.

L 378-379. Did the authors test this?

L 388. Maia et al. (2017).

L 411. Space before “By contrast …”

Reviewer #2: Review of manuscript #PONE-D-20-22527 “Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?” by Oliveira et al.

General comments and assessment:

The manuscript (henceforth MS) assesses if multitrophic interactions might shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects in a plant species from Brazilian Cerrado. The study is innovative and brings a great advance to the understanding of how ecological interactions can influence individual performance. I really liked to read this work and I think this is a great contribution.

I can point out two major issues in the MS: there are many typos and English grammar issues. The MS is well structured, but some parts are hard to follow, or totally not understandable. The MS needs to be proofread by a native. I could provide many small issues over the MS, but it would be good to recheck the spelling and grammar.

The second point it is the lack of details in some parts of the methodology, which I highlight bellow. The main issue that I pointed out, it is that the authors did not mention how many plants they used, or how many fruits per plants they collected, which can bias the analyses, eg. spatial correlation, low variability of samples.

More specific comments:

Abstract

In my opinion the abstract needs some improvement and clarity to really reflect what was found in MS. I found some typos and issues in English grammar. Also the structure is not good, since nothing about methodology is mentioned. Further, the conclusions do not seem to follow the results. So I recommend to reword some parts and recheck English typos.

L6, and 8 – Separate city name from country with comma.

L17 – HAVE TRIED… - present perfect, not continuous.

L21-22 – You tested the effects of specific variables on morphological structures. In L22-23, you tested the effects of these specific variables on allometric patterns, fluct. asymmetry, and MORPHOLOGICAL STRUCTURE. Doesn’t morphological structure encompass these two other results (allometry and asymmetry)? What I mean it is that you used only morphology before, but later you used morphology, asymmetry, and allometry. So it would be good to standardize and be clear which parameter is different from each other and which can be encompassed by the other. In your conclusion you used only morphology; so I expect that this term encompasses the other two. If I wrong, please be clear in your statements.

L24 – Methodology is missing. I recommend you to add some words about methodology here. Maybe you can add a few words before the sentence “we tested (L. 22)…” telling time and specie used. Like: “For that, analyzing PLANT SPECIES (FAMILY) over four consecutive years, we tested how these FRUIT-RELATED TRAITS affect…

L24-27 – What is a “negative allometric pattern…”? Do you mean that the relationship between the variables fit in your models showed a negative relationship with allometric parameters? If so reword the sentence. It’s hard to follow what you want to say.

L27-28 – Again, what is the relationship between variables and

L29 – FOUND

L30-32 – When you say “besides” you give an idea of addition to “allometry”. However, later you mentioned “the most” a superlative, comparative. This is contradictory. Or both parameter, allometry and morphological, are good to evaluate, or one is better than other.

L31- “allometry, …”

L32-34 – According to your results in abstract, I can’t see that each species respond differently. You wrote that you didn’t find fluctuation asymmetry for categories of species. Only that. So I can’t say only reading the abstract that each species respond differently.

Introduction

L39-40 – change semicolons to commas. Not common in English.

L43 – change “can create” to “influence”

L44 – change “destinated” to “allocated”

L51 – add “FA” abbreviation here, when you say for the first time “fluctuating asymmetry”. Remove “fluctuating asymmetry” from L55 and L71. Use only the abbreviation instead.

L65 – For me it is a little weird mentioning your own study in a third person “Oliveira et al. …”. I would use: Recently, we observed … (Oliveira et al. 2020).

L66 – change “;” to “,”.

L69 – If you provided an abbreviation for a term (FA), please use it over the MS (there are many other parts with the term and not the abbreviation), and not the term itself.

L73-76 – I think this is not your main aim. If you see your abstract, that one is a good and appropriate aim. Provide a general view of your study and not a specific, as you did. You evaluated the effects of different interactions on the morphology of three different insects, which exploit the same plant resource. So this is bigger than what you have provided as aim. Next, you can provide all this information about the system.

L76: remove space between “Hymenoptera” and “:”

L84 – change “interesting” to “advantageous”

L86 – here you can clearly see that there is no relationship between your current aim and what you hypothesized. What is the relationship between “increase in competition and parasitism rate” and your current aim in the Introduction? The answer is none. So, because of that, you need to reword your aim.

L86-89 – For (I), you didn’t mention a specific relationship (positive, negative, or neutral) as for (II), which was “greater”, and (III), which was “reduction”. Why that? I would expect a reduction in allometric patterns.

L90 – change “explained” to “explain”

L91 – change “bruchine beetles for the Coleopteran [19,22]: pronotum” to “bruchine beetles [19,22], namely pronotum”

L92-93 – change “for the wasp right and left wing right and left tibia length and total body

length of A. vulgaris” to “for the wasp, right and left wing, right and left tibia length, and total body length of A. vulgaris”

L93-94 – You mentioned “allometric relationships and FA”, but in other parts you wrote three variables: allometry, FA, and morphology. Please, be consistent with what you are really testing and standardize you MS. See my comments in Abstract about this. Mention one term which encompasses all three, or the three terms.

L94-95 – does “seed infestation” refer to “competition” (L86)? Again, please standardize your MS and do not change the terms over the MS.

Methodology

L103-106 – the degree symbol is not correct. Remove ‘0’ before degree numbers with two digits (044).

L106 – Reference is not correctly formatted. “Tuller et al. (2015)”

L108 – Inform some characteristic of the plant species studied before saying how you assessed beetles and fruits. Is it a shrub, tree,…? What was the mean size of each plant individual? We need to know some plant traits that make your MS replicable.

L111-112 – It is not clear why you removed these data. Low abundance in the same plant, same fruit, or in general? Not clear. Because if you have low abundance in the same fruit/plant is not a reason for removing the data.

L113 – remove “Therefore”, you are not concluding something.

L113-122 – In Table 1, there is Lu-4, but not in the text. If you excluded data from 2011, why adding this information in the Table 1? Since you used 2012-2014, you can only focus on these years, and you do not need to mention Ae1 subarea, since it appeared only in 2011. Also you informed La2 in the text, but not in the Table. You need to rewrite all this part of your methodology, including 2.1 subtopic and Table 1. Remove 2011 data, and reword Ae2 and Ae3 to Ae1 and Ae2. Make further modifications accordingly to these suggestions.

L113 – How many plants did you use? How many plants per subarea? Were the same plants over months and years? How many fruits per plant?

L118 – Is it possible to give some information about fruit ripening at the moment of collection?

Table 1 – Depending on the information provided in the text, you don’t need to provide a table, since it will repeat the same information.

L1234 – remove “they were”

L128 – How did you assess seed biomass? Did you dry seeds or not? When did you do that? After how many days, weeks, months? Did you open the fruit to collect seeds or not? Please provide informations about it.

L129-130 – You have already provided full name of species with authors. Please mention only genus abbreviation and epithet.

L131 – Didn’t understand. Did you use Tuller et al. 2015 to collect these data? I can’t follow this message. It seems that you used biomass and abundance data from Tuller, and not from your study. Maybe you collected the data accoding to Tuller methodology. Please clarify that.

L131 – Wrong citation format for PlosOne.

L134 – Fig. 1 - This is not a plant species food web, but a plant-insect food web. S… tenuifolia is part of the food web. Do not need to write gray scale unless you will explain something from it.

L139 – change to … “previous studies [citation, according to PlosOne rules]”.

L146 – change “structures measures” to “structure measurements”

L147 – change to “width, and both”

L147 – change to Fig. 2AB

L149 – add space between “)[“

L151 – change to Fig. 2C

L151 – Format the citation according to Journal rules.

L155 - change to Fig. 2D

L 159 – remove space between “Hymenoptera” and “:”

L169 – change to “tested”, and to “and resource size”

L169 – Fruit infestation is competition? Be clear. Earlier you mentioned competition and infestation. Standardize your terms.

L169-170 – You mentioned the supplementary here as more details about the effects of some variables on morphometric patterns. However, these two sup. files are only formulas to calculate fruit infestation and parasitism. Also, these formulas can easily be added in the MS by: FIR=HT/SA or FIR=HT.SA-1, where FIR is the fruit infestation rate by seed-feeding insects, HT is the total abundance of herbivorous, SA is the total number of seed per fruit; and FPR=PT.(HT+PT)-1, where FPR is the fruit parasitism rate by both coleopteran and hymenopteran parasitoid present in the fruit, PT is the number total of parasitoids in the fruit, and HT is the abundance total of seed-feeding insects in the fruit (Merobruchus terani, Stator maculatopygus and Allorhogas vulgaris). If you keep the formulas in sup., please add formulas according to the order you mention the variables in the MS, i.e., first infestation, then parasitism formula.

L171-173 – change to “We defined three categories for both fruit infestation and parasitism rate (adapted from FORMATED CITATION): “Low”, from 0 to 0.30; “Medium”, from 0.31 to 0.60, and “High” from 0.61 to 1.

L175-176 – use “from” and “to” to say range of seed size, and not hyphen.

L174(169) – you used the term seed size, but you are actually using biomass. So standardize your text and say exactly what you are measuring, which seems to be biomass and not size.

L175-176 – Use only three decimals.

L178 and others – Inform which correlation, Spearman, Pearson, Kendall...

L187 – change “;” to “,”

L191 – change to “We evaluated the fluctuating asymmetry patterns of the species”

L194 – change to “We used mixed models analysis with restricted likelihood (REML),”

L196 – add comma after “i.e.”. Standardize to “fixed effects” or “fixed terms”

L198 – This paragraph is other part of mixed analysis. So, join this paragraph with the previous one.

Results

L221 – change to “lower”

L222 – Table 2 with space. Provide full name of plant species, and insects as well. Provide range for all variables or none. Change to “Total abundance” only. We already know that it is per species. Suggest LFI, MFI, HFI; LPR, MPR, HPR.

L227 – Change to “Figs.”

L230-232 – Why not comparing the slopes among categories using likelihood ratio test? It’s better than saying there is “some kind of effect”.

L248-249 – I can see a positive effect of infestation in elytra and pronotum on both beetles and pronotum and wing of the wasp. Why not for elytra and why not for the other beetle species?

L254-256 – How did you know that infestation affected negatively elytra?

L248-256 – In this case here, I’m quite sure that if CI does not overlap with slope=0 you have a significant influence of variable on insect parameter, and if it is above will have a positive and bellow a negative effect. Also these comments are to 257-265 and 266-278.

L287 – Add space after “(DA).”

L288 – remove “its”.

Discussion

L344 – format citation according to PlosOne rules.

L355-358 – This period is hard to follow. Maybe you should divide it or reword.

L356 – remove the dot after the species epithet.

L369 – format citation according to PLosOne rules. Also see L375.

L376-377 – can’t follow the sentence “of their development of the hosts”

L388 – check citation format.

L404 – what “this” refers to? Be clear.

L409 – remove comma after M. terani. Do not separate subject and verb.

L409-411 – Can’t understand these sentences. Reword it.

L412 – remove comma after species name.

L415 – What is “predated” – Do you mean “preyed upon by”?

L429 – remove “the” before “three”

L423 – incorrect use of food web. The food web is not of a plant species. The plant species is part of the food web. Reword it.

L433-434 – although their bodies scaling did not change ITS pattern - you are referring to scaling.

L434-435 – “they vary substantially in their allometric slopes at high levels of infestation and competition,”. I can’t know what is they, their,… very hard to follow.

L439 - change to “insects’ body”

Acknowledgements

If I’m not mistaken there is specific sentence for Brazilian foundation program CAPES.

FIGURES

In Fig. 1 there is the word FIGURAS at the top of the page. Change to FIGURES.

Fig 2-7 – Increase the size of number in x- and y-axis. They are very small.

Fig. 6 – You add posthoc test in your figure but you didn’t mention it in the analyzes. Did you use Tukey? Estimated marginal means? Also place the letters close to the SE bars.

Fig. 6-7 – Please do not use red color in MSs since it is not accessible for readers with reduced color vision. It would be grateful if you could change the color.

SUPPLEMENTARY

Table S1 – in M1 add space before ~

In M2 remove space after side in random effect

L26 - Change “biomass seed” to “seed biomass”

In legends of Figures and Tables provide full name of species. Abbreviate only if you mention earlier in the own legend.

**********

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Reviewer #1: No

Reviewer #2: Yes: Eduardo Soares Calixto

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26 Sep 2020

We have revised our manuscript following editor’s suggestion using a word processing program. Indeed, we agreed with most of the suggestions/comments pointed out and have answered point by point the comments raised by both reviewers. The MS English grammar has been revised, and all details about fruit collection were provided in the Materials and methods section. We hope this time expectations are met. Finally, we have organized the comments adding numbers to potential questions so we could answer each point.

The document with the specific answers were attached with the manuscript.

Submitted filename: PlosOne-Answers.docx

Click here for additional data file.

21 Oct 2020

PONE-D-20-22527R1

Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?

PLOS ONE

Dear Tamires Camila Talamonte de Oliveira

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 November 20. 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.

Please include the following items when submitting your revised manuscript:

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A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Kleber Del-Claro, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Dear Author, please do the needed corrections pointed by one reviewer and I will give the final decision without the need of a third round.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please find below my comments concerning the manuscript by Oliveira et al., entitled: “Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?” (PONE-D-20-22527). The authors presented an excellent review of the MS (point by point) which improved the quality of MS. In addition, there was a systematic review of English grammar. I am satisfied with the final version and would like to consider publishing this MS in PlosOne journal.

Reviewer #2: Review #2 of manuscript #PONE-D-20-22527 “Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?” by Oliveira et al.

Authors did a great job. The MS is very clear now. There still are some grammatical errors. I identified some, and I suggest again a careful check of English.

L112-113 – Do not use abbreviation for species name when starting a period. So provide full genus name.

L113, 209 and elsewhere – after “i.e.” add a comma

L220 – Add space between period and “The”.

L255 – I think there is a typo when informing the supplementary. After S2 there is a hyphen and comma, and then S4. Check it out.

L274-280 – Very long sentence. Please divide it in two at least.

L281 – Remove space after scale.

L289 – I believe after MP is a semicolon and not “:”

L296 – Change to “…seed). No variations…”

L301 – Remove “(medium seed)”, you have already mentioned it in 295. Then use the abbreviation.

L395 – I think “growths” is not correct. Do you mean: structure growth?

L406 – Change to [68,69]. Thus,… The dot is in a wrong place.

L415 – I did not understand this sentence. “to changes in the size of their resource,”. First I think it is “change”. And I don’t what is a change in size of a resource. Would it be amount of resource. Please check it out.

L419 – Use abbreviations constantly over the MS. Do not need to write the words again.

L426-429 – This sentence is not well written and clear. I suggest “Some studies have suggested that body size is more sensitive to environmental disturbances than FA, since body size of individuals can present a greater plasticity than FA [75-77].”. Please also check what you mentioned before has the same meaning now.

L453 – change to “are limiting factors”

L466-470. I found very repetitive theses sentences, and also you suggest/recommend studies in all of them. I recommend you to remove the last two sentences or reword them: “We recommend the use of insects’ body size and morphological structures in studies of food web. However, to clarify and better understand these approaches, more studies are necessary.”

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Reviewer #1: Yes: Diego Anjos

Reviewer #2: No

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21 Oct 2020

We thank the reviewers for the valuable comments on the manuscript. We have revised our manuscript following editor’s suggestion using a word processing program. Indeed, we agreed with most of the suggestions/comments pointed out and have changed point by point according to reviewer #2 corrections.

Submitted filename: Response to Reviwers.docx

Click here for additional data file.

23 Oct 2020

Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?

PONE-D-20-22527R2

Dear Dr. Tamires Camila Talamonte de Oliveira,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Kleber Del-Claro, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

27 Oct 2020

PONE-D-20-22527R2

Can multitrophic interactions shape morphometry, allometry, and fluctuating asymmetry of seed-feeding insects?

Dear Dr. Oliveira:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kleber Del-Claro

Academic Editor

PLOS ONE