Hole Size Matters: An Indirect Method for Measuring Gall Wasp Community Emergence?

© 2024 Academia Sinica, Taiwan Open Access Hole Size Matters: An Indirect Method for Measuring Gall Wasp Community Emergence? Ricardo Clark-Tapia1, Victor Aguirre-Hidalgo1, Juli Pujade-Villar2, Cecilia Alfonso-Corrado1, and Felipe Tafoya3,* 1Instituto de Estudios Ambientales, Universidad de la Sierra Juárez, Avenida Universidad S/N, Ixtlán de Juárez, Oaxaca 68725, México. E-mail: [email protected] (Clark-Tapia ), [email protected] (Aguirre-Hidalgo), [email protected] (Alfonso-Corrado) 2Universitat de Barcelona. Departament de Biologia Animal Avda. Diagonal, 645. 08026 Barcelona, Spain. E-mail: [email protected] (Pujade-Villar) 3Tecnológico Nacional de México, Instituto Tecnológico El Llano Aguascalientes, Km. 18 Carretera Aguascalientes -S.L.P., El Llano Aguascalientes, México. C.P. 20330. *Correspondence: E-mail: [email protected] (Tafoya) Received 21 November 2023 / Accepted 26 June 2024 / Published 11 November 2024 Communicated by Y. Miles Zhang Quantifying the abundance and diversity of small insects, especially those with specific environmental associations or hidden habitats, can be challenging using direct methods. Galls are specialized microhabitats that host a wide range of interactions between species. However, assessing their abundance and associated community diversity takes time and effort. In this study, we propose an indirect approach using the size of gall emergence holes to estimate the abundance of gall wasps (Cynipini) and their associated wasp species. We examined eight different types of gall structures collected in the temperate forest of Mexico. We identified every gall-emerging individual, classifying them as gall inducer, inquiline, or parasitoid. Kruskal-Wallis tests, correlation analysis and a mixed linear model (LMM) were used to evaluate differences between mesosoma size and gall hole size for each emerged species in each gall type. Our results showed that mesosoma and hole size significantly differed between Cynipini wasps and their associated wasp species. LMM showed a significant relationship between the size of the mesosoma and the diameter of the emergence hole among the analyzed wasp species. Upon validating the method, a low emergence rate and low abundance of the studied Cynipini wasps were observed, attributed to negative interactions and inadequate development of the wasps. We emphasize the potential of gall emergence hole size as an indicator of species abundance and emergence rates within Cynipini gall complexes. Furthermore, strategies are discussed to improve their effectiveness and reliability in future studies to increase our understanding of the ecological dynamics and evolutionary processes of gallforming wasps. Key words: Cynipini, Diameter, Mesosoma, Negative interactions, Wasp BACKGROUND The estimation of the population size or abundance of insect species, especially those that are difficult to see due to their small size, is a critical ecological indicator (Sileshi 2007; Schowalter 2022). This estimate is essential for understanding the significant fluctuations between different habitats over short periods due to changing environmental conditions or negative interactions with another organisms (e.g., van der Sluijs 2020; Kehoe et al. 2021). Furthermore, understanding insect richness is critical to developing effective management and conservation strategies, especially amid widespread insect declines, in which smaller insects are highly susceptible and vulnerable to environmental changes (Sileshi 2007; Schowalter 2022). However, quantifying insect abundance and diversity is often a complex and challenging task (Sileshi Citation: Clark-Tapia R, Aguirre-Hidalgo V, Pujade-Villar J, Alfonso-Corrado C, Tafoya F. 2024. Hole size matters: An indirect method for measuring gall wasp community emergence? Zool Stud 63:35. doi:10.6620/ZS.2024.63-35. Zoological Studies 63:35 (2024) doi:10.6620/ZS.2024.63-35 1 © 2024 Academia Sinica, Taiwan 2007; Clark-Tapia et al. 2022). Direct methods for estimating insect abundance, such as live insect capture, can be complicated due to difficulties in capturing and handling insects in areas where direct assessment is complex, costly, and unspecific, which may be detrimental to insect populations themselves (Stork 2018), mainly when these populations are fragile or at risk. Diverse indirect methods have been developed to address this challenge. These methods often entail assessing the marks caused by the insects on their host plants (Dyck et al. 2021). Such approaches include inspecting excreta (Majer 1983; Nichols et al. 2007), plant damage (Schooler and McEvoy 2006) or employing cumulative recording data to obtain valuable measures of range change (Thomas 2005). These methods enable the estimation of insect abundance and diversity without relying on direct observations, which can significantly assist in biodiversity management and species conservation efforts (Nichols et al. 2007; ClarkTapia et al. 2022). Moreover, indirect methods allow data collection on an extensive time and space scale, which is crucial for understanding biodiversity change trends (Samways et al. 2010 2020). Nevertheless, it is essential to highlight that these indirect methods are particularly valuable for studying species that are challenging to quantify or have specific survival requirements, such as gall wasps of the Cynipidae family. In North America, over 80% of gall wasps are from this family, mainly grouped in the tribe Cynipini; all representatives of this tribe only utilize the oaks (Fagaceae: Quercus) as their primary hosts (Price 2005; Russo 2007), inducing galls or cecidia in response to oviposition (Redfern 2011; Hearn et al. 2019). These structures can serve as new microhabitats, giving rise to the development of ecological niches (Stone and Schönrogge 2003; Gilbert 2009; Pujade-Villar 2013). The recording and observation of gall appearance and structure have proven effective in studying the diversity of Cynipini and their relationship with host plants (Stone et al. 2002; Ito and Hijii 2004; Lobato-Vila et al. 2022; Martínez-Romero et al. 2022). Gall structure classification is also an effective tool in studies regarding biodiversity patterns and conservation strategies at the ecosystem level (Price et al. 2004; Clark-Tapia et al. 2022; Ward et al. 2022). However, it is essential to note that the appearance of the galls does not provide information about species abundance and diversity. Although only one gall-inducing wasp emerges from each larval chamber of a given type of gall (single or multiple larval chambers), multiple species associations can also emerge. These associations include parasitoid species, primarily from hymenopteran families such as Chalcididae, Braconidae, Ichneumonidae, Eulophidae, Eupelmidae, Eurytomidae, Pteromalidae, Ormyridae, and Torymidae, as well as inquiline species, which are generally other cynipids belonging to the tribe Synergini (Stone et al. 2002; Melika 2006; Russo 2007; LobatoVila and Pujade-Villar 2021). In this context, the size of exit holes in galls has been used as indirect evidence of multitrophic interactions (Russo 2007; Stone et al. 2008; Cooper and Rieske 2010). This study aimed to propose an indirect method to quantify the emergence percentage and abundance of the Cynipini gall wasp and study multitrophic interactions using the size of the insects’ emergence hole. To achieve this goal, the methodological proposal was first validated by collecting, monitoring, and measuring emergence holes and interactions in various types of galls. Subsequently, the emergence percentages and abundance of gall wasps were estimated. This will contribute to the general understanding of insect ecology and open new perspectives for research in this field. MATERIALS AND METHODS Gall sampling To conduct this study, we collected eight types of Cynipini galls in two temperate forests of Mexico; six came from Sierra Fría, Aguascalientes, and two from Sierra Juárez, Oaxaca, hosted in five oak species (see Table 1 for the host species and location of each sampling site). Selected cynipids induce galls, particularly on leaves and stems, exhibiting different shapes and sizes (Figs. 1–8, Table 1). Sampling was carried out in each forest by selecting an area with the presence of the host corresponding to each type of gall. In each sampling area, three transects were established, each with a length of 100 meters and separated by 500 meters. Subsequently, a sample of each gall type was collected from the sampled trees without evidence of emergence or damage. It is important to note that the number of galls collected per tree varied depending on the abundance of each type, and not all trees had galls. Therefore, we aimed to obtain at least 50 samples of each gall type (see Table 1 for inducer and associate gall species, gall size, sample size, and sampling sites). We visited each sampling site during the fieldwork to establish transects for gall collection (see Table 1). In Aguascalientes, the collection period was extended from October 15 to November 10, while in Oaxaca it occurred from November 15 to December 20, 2022, at the beginning of the gall wasp emergence period. page 2 of 15Zoological Studies 63:35 (2024) © 2024 Academia Sinica, Taiwan Fig. 1. Dimensions of the emergence holes of the galls induced by Erythres hastata ŏ (a), and associated genera: Synergus sp. ♀ inquiline (b), Ormyrus sp. ♀ parasitoid (c), Synergus sp. ♂ inquiline (d), and Ormyrus sp. ♂ parasitoid (f), at 2:1 scale for enhanced visualization and 1:1 real scale estimated using the average gall size. Fig. 2. Dimensions of the emergence holes of the galls induced by Cynips sp. ŏ (a), and associated parasitoids genera: Torymus sp1. ♀ (b), Ormyrus sp. ♀ (c), Torymus sp.2 ♀ (d), and Ormyrus sp. ♂ (f), at a 1:1 real scale estimated using the average gall size. page 3 of 15Zoological Studies 63:35 (2024) © 2024 Academia Sinica, Taiwan Fig. 3. Dimensions of the emergence holes of the galls induced by Druon sp. ŏ (a), and associated parasitoid genera: Torymus sp.2 ♀ (b), Ormyrus sp. ♀ (c), and Ormyrus sp. ♂ (d), at 2:1 scale for enhanced visualization and 1:1 real scale estimated using the average gall size. Fig. 4. Dimensions of the emergence holes of the galls induced by Andricus sp. ŏ (a), and associated parasitoid genera: Ormyrus sp. ♀ (b), Torymus sp2. ♀ (c), Ormyrus sp. ♂ (d), and Eupelmus sp. ♀ (e), at a 1:1 real scale estimated using the average gall size. page 4 of 15Zoological Studies 63:35 (2024) © 2024 Academia Sinica, Taiwan Fig. 5. Dimensions of the emergence holes of the galls induced by Kinseyella quercusobtusata ŏ (a), and associated parasitoid genera: Torymus sp1. ♀ (b), Sycophila sp. ♂ (c) and Torymus sp1. ♂ (d), at 2:1 scale for enhanced visualization and 1:1 real scale estimated using the average gall size. Fig. 6. Dimensions of the emergence holes of the galls induced by Disholcaspis sp. ŏ (a), and associated parasitoid genera: Ormyrus sp. ♀ (b), Torymus sp. ♀ (c), Eurytoma sp. ♂ (d), Ormyrus sp. ♂ (e), at 2:1 scale for enhanced visualization and 1:1 real scale estimated using the average gall size. page 5 of 15Zoological Studies 63:35 (2024) © 2024 Academia Sinica, Taiwan Fig. 7. Dimensions of the emergence holes of the galls induced by Atrusca sp. ŏ (a), the associated family Vespidae ♀ (b), and associated parasitoid genera: Torymus sp3. ♀ (c), and Eulophidae (d), at 2:1 Scale for enhanced visualization and 1:1 real scale estimated using the average gall size. Fig. 8. Dimensions of the emergence holes of the galls induced by Zapatella gabriellae ŏ (a), and associated parasitoid genera: Torymus sp. ♀ (b), Torymus sp. ♂ (c) and Synergus sp. ♂ (d), at a 1:1 real scale estimated using the average gall size. page 6 of 15Zoological Studies 63:35 (2024) © 2024 Academia Sinica, Taiwan Table 1. Characteristics of galls induced by Cynipini Tribe and associated species. Abbreviations for genera: E = Erytres, K = Kinseyella and Z = Zapatella Cynipini and associated species Sexual generation type Species code Gall size (mm, mean ± SD) Species host and location Sample size Sampling sites E. hastata Synergus sp. Ormyrus sp. Synergus sp. Ormyrus sp. Asexual (ŏ) Female (♀) Female (♀) Male (♂) Male (♂) Eh Sy_f Oy_f Sy_m Oy_m 12.3 ± 0.8 Quercus laurina Humb. and Bonpl. stems 158 Aguascalientes 22°05'56''N 102°42'18''W Cynips sp. Torymus sp. Ormyrus sp. Torymus sp. Ormyrus sp. Asexual (ŏ) Female (♀) Female (♀) Male (♂) Male (♂) Cy Tm_f Oy_f Tm_m Oy_m 5.4 ± 1.41 Q. resinosa Liemb. leaves 58 Aguascalientes 22°10'16''N 102°31'15''W Druon sp. Ormyrus sp. Torymus sp. Ormyrus sp. Asexual (ŏ) Female (♀) Tm_f Male (♂) Dn Oy_f Tm_m Oy_m 7.3 ± 2.9 Q. chihuahuensis Trel. leaves 56 Aguascalientes 22°09'46''N 102°31'28''W Andricus sp. Ormyrus sp. Torymus sp.2 Ormyrus sp. Eupelmus sp. Asexual (ŏ) Female (♀) Female (♀) Male (♂) Female (♀) Ad Oy_f Tm_f2 Oy_m Eu_f 2.98 ± 0.09 Q. chihuahuensis Trel. leaves 166 Aguascalientes 22°09'46''N 102°31'28''W K.quercusobtusata Torymus sp. Sycophila sp. Torymus sp. Asexual (ŏ) Female (♀) Male (♂) Male (♂) Kq Tm_f Sh_m Tm_m 25.2 ± 4.4 Q. resinosa leaves 112 Aguascalientes 22°10'16''N 102°31'15''W Disholcaspis sp. Ormyrus sp. Torymus sp. Eurytoma sp. Ormyrus sp. Asexual (ŏ) Female (♀) Female (♀) Male (♂) Male (♂) At_1 Oy_f Tm_f Ey_m Oy_m 7.6 ± 1.8 Q. chihuahuensis stems 123 Aguascalientes 22°09'46''N 102°31'28''W Atrusca sp. Torymus sp.3 Eulophidae sp. Vespidae Asexual (ŏ) Female (♀) Male (♂) Female (♀) At_2 Tm_f Elp Vp 30 ± 4.1 Q. obtusata Humb. and Bonpl. leaves 50 Oaxaca 17°18'55''N 96°28'56''W Z. gabriellae Torymus sp. Torymus sp. Synergus sp. Asexual (ŏ) Female (♀) Male (♂) Male (♂) Zg Tm_f Tm_m Sy_m 9.6 ± 1.3 Q. crassifolia Humb. and Bonpl. stems 53 Oaxaca 17°08'22''N 96°36'44''W Authors of the species of gall wasps: E. hastata Kinsey; K.quercusobtusata Pujade-Villar and Melika; Z. gabriellae Cuesta-Porta and Pujade-Villar. page 7 of 15Zoological Studies 63:35 (2024) © 2024 Academia Sinica, Taiwan Method validation Collection, monitoring, and measurement During sampling, mature galls were collected and identified in the field by their coloration and previous experience. These galls were placed in cloth bags and transported to the laboratory. Once in the laboratory, all galls were visually examined using a stereoscope to ensure no emergences or damage occurred. The selected galls were placed individually in sheer organza bags, then placed in a box and stored in the laboratory. Of all the analyzed galls, only those induced by Druon sp. showed multiple chambers (Fig. 3). In this gall type, the filaments or hairs covering the structure were removed to obtain accurate measurements of the emergence holes. After they were placed in the organza bags, the galls were monitored weekly for three months to detect any wasp emergence, except for Druon sp. The galls of this species were checked every day because the simultaneous emergence of wasps was recorded. In cases of multiple emergences, the species was matched to the emergence based on the size and shape of the hole. In this gall, each emergence hole was marked with an indelible marker. All emerged wasps (Cynipini, inquilines or parasitoids) were stored in Eppendorf tubes with 96% ethyl alcohol, and subsequent measurements were taken of the emergency hole and the mesosoma of the wasp using a VE-910 eyepiece micrometer previously calibrated with acetate sheets with millimeter marks as a reference. This process was conducted considering that the mesosoma’s width in wasps is a reliable indicator of size (e.g., Ohl and Thiele 2007). Emergence Analysis For each gall type, we first identified emerging species (cynipids, parasitoids, inquilines) to the genus level, except for species in Eulophidae and Vespidae. Subsequently, the exit hole’s diameter was measured twice, in perpendicular directions, using a previously calibrated micrometer with an ocular. The diameter values obtained from the two measurements were averaged to obtain a representative value of the hole emergence diameter. To establish an association between the size of the exit hole and the mesosoma size of emerging wasps, we compared the diameter of the emergence hole with the width of the mesosoma. This comparison was based on the premise that the mesosoma, being more rigid than the metasoma, provides a reliable measure of size. Therefore, if the width of the mesosoma allowed it to pass through the hole, we inferred that the metasoma would too. This assessment was primarily based on the width of the mesosoma, which has previously been established as a dependable indicator of size in wasps (e.g., Ohl and Thiele 2007). Data analysis Validation method Kruskal-Wallis and Mann-Whitney analyses were used to compare diameter and mesosoma differences between species and to perform species-specific pairwise comparisons. These analyses were performed in R v.4.10 (R Core Team 2021) using the nparcom package (Konietschke et al. 2015) and visualized in the ggplot2 (Wickham et al. 2016) and GridExtra v.2.3 (Auguie and Antonov 2017) packages. Holm’s correction was also applied to the p-value to control type I errors. The relationship between diameter and mesosoma was examined, focusing mainly on analyzing the gallinducing wasp due to the limited number of individuals of other species. This association was evaluated using Pearson correlation and linear regression through the nparcom package (Konietschke et al. 2015) in R. Additionally, a mixed linear model (LMM) analysis was conducted to investigate the relationship between the diameter size of emergence holes (diameter) and the pronotum length (mesosoma) in relation to insect species (species). The diameter and mesosoma were log-transformed to achieve a normal distribution. The model was fitted using the lmer() function from the lme4 package v. 1.1-27 (Bates and Sarkar 2007) in R. The model was specified as follows: Diameter = β0 + β1 * Mesosoma + β2 * Species + bi + ε where: • Diameter represents the size of the emergence hole. • Mesosoma represents the length of the mesosoma. • Species is a categorical variable indicating the insect species. • β0 is the intercept of the model. • β1 and β2 are the coefficients associated with the explanatory variables Mesosoma and Species, respectively. • bi is the random effect of each species, considered as a normally distributed random variable with a mean of zero and variance σ2 Species. • ε represents the error term. A random effect for the Species variable was included to capture individual differences between species in the size of the emergence hole diameter. page 8 of 15Zoological Studies 63:35 (2024) © 2024 Academia Sinica, Taiwan This allowed modeling differences between species as random effects, facilitating the assessment of individual differences between species and their impact on the size of the emergence hole diameter. The restricted maximum likelihood (REML) method was used for parameter estimation and statistical inference. The significance of the model coefficients was evaluated using hypothesis tests, and the p-value associated with each coefficient was calculated. Abundance estimation Finally, with all the collected galls, the proposed emergence method was evaluated. To do this, the percentage of emergence for each gall wasp (GC) was estimated, along with two causes of emergence failure: 1) due to the presence of negative interactions (Pa) and 2) inadequate development of the larvae, considered empty galls (Vn). Kruskal-Wallis and Mann-Whitney analyses were used to evaluate differences among GC, Pa and Vn for each gall type. Also, the abundance of each species was estimated by counting the number of emerged adults of each species. The tests were executed and visualized in R. RESULTS Validation method In inducer gall wasps, only females were identified (asexual generation), while in associated wasps, individuals of both sexes were observed. Figures 1 to 8 show the gall types induced by each Cynipini wasp and the identified negative interactions, illustrating the size proportions of the species within each gall. The most frequent negative interactions involved parasitoids of the genera Torymus and Ormyrus, which were present in 88% and 63% of the analyzed gall species, respectively. Other interactions with wasps, such as Synergus (inquilines), Eurytoma, Eupelmus, and Sycophila (parasitoids), as well as Vespidae (predators emerging from the larval chamber), had a frequency of occurrence lower than 25%. Significant differences were found in the diameter of the emergence hole between Cynipini wasps and associated species (Fig. 9, Table S1). The diameter of the emergence hole of the analyzed galls of inducer wasps was significantly larger than that of all associated species. Furthermore, significant differences were observed between the associated species, especially between the sexes, when present in the samples. Additionally, the mesosoma size of the inducer species was significantly larger (Hc = 42.43; p < 0.001) than that of the associated wasp species, except for E. hastata, which showed no significant differences compared to the females of Synergus sp. Furthermore, among the associated gall species, significant differences (Hc = 32.15; p < 0.01) were observed between females and males in the size of the mesosoma of parasitoid wasps (Fig. 9, Table 1S). Association analysis revealed a close relationship between mesosoma size and the diameter of the hole in several gall wasps. Specifically, a significant association was found in the species Druon sp., K. quercusobtusata, Z. gabriellae, and Andricus sp., although this last species had a weak association (Table 2). On the other hand, E. hastata, Cynips sp., Disholcaspis sp., and Atrusca sp. did not have statistically significant associations. In some cases, a negative association between mesosome size and hole diameter was observed in wasp species (inducers, inquilines, or parasitoids). Smaller holes led to emergence failures, as the wasps became trapped in the hole or inside the gall. On the other hand, linear mixed models showed a significant relationship between the mesosoma body and the size diameter among the analyzed wasp species. On average, across all species examined, an increase in mesosoma size correlated with an increase in hole diameter, with an estimated average coefficient of 0.527 (p < 0.05). However, it is worth noting that the magnitude and direction of this association varied among species, with some significant coefficients indicating differences in emergence hole diameter between specific species. Except for E. hastata, all models had positive coefficients for the mesosoma, indicating an increase in mesosoma size associated with an increase in diameter (Fig. 10). Abundance estimation According to the proposed method, it was found that most gall wasp species had low emergence percentages. However, two species, Druon sp. and Z. gabriellae, had emergence percentages greater than 30%, with Druon sp. having the highest percentage at 48.5%; the remaining Cynipini species showed emergence rates below 30% (Fig. 11). Specifically, in K. quercusobtusata, Disholcaspis sp., and Atrusca sp., their low emergence percentages were attributed to negative interactions (predation), which had percentages of 69.6%, 54.2%, and 55.7%, respectively. On the contrary, the non-appearance in Andricus sp. and E. hastata was due to inadequate development of the wasp (Fig. 11). No significant differences were observed between the analyzed Cynipini gall wasps, presence of negative interactions or species association, and empty percentages. The estimated abundances of all species page 9 of 15Zoological Studies 63:35 (2024)