Seed Traits and Curculio Weevil Infestation: A Study in Quercus mongolica

Abstract

Exploring host preference and resource partitioning among seed predator species is essential for understanding the coexistence mechanisms and guiding effective forest pest management. This study aimed to elucidate how seed traits influence infestation dynamics and species interactions, focusing on acorn weevils infesting Quercus mongolica. Species identification and clarification of their evolutionary relationships within the Curculio genus were performed through phylogenetic analyses of the mitochondrial cytochrome c oxidase subunit I gene sequences. The seed infestation patterns were assessed by comparing the infestation rates across various seed size classes. Furthermore, the correlations between the seed morphological traits (length, width, aspect ratio, and weight) and weevil abundance were analyzed. The phylogenetic results revealed well-supported monophyletic clades corresponding to Curculio arakawai and Curculio sikkimensis. This confirmed the clear genetic separation between these two distinct weevil species, thereby substantiating the divergence observed in weevil populations correlated with different seed hosts. The infestation patterns revealed the association of weevil species-specific preferences with seed size: C. arakawai predominantly infested larger acorn seeds, whereas C. sikkimensis predominantly infested smaller acorn seeds. C. sikkimensis favored smaller ones. Both species exhibited positive correlations between abundance and seed length and width in larger seeds; however, the seed weight displayed no significant effect. These results indicate niche differentiation mediated by seed size and morphology, which likely reduced interspecific competition and facilitated coexistence. This study elucidates species-specific host selection patterns in acorn weevils and highlights acorn traits as crucial factors shaping seed predator assemblages. The findings provide valuable insights for developing targeted pest management strategies and supporting sustainable oak forest regeneration.

1. Introduction

Seed predation markedly shapes the plant population dynamics and community assembly by directly influencing seed survival, dispersal, and recruitment, thus influencing ecosystem structure and function [1]. Oaks (Quercus spp.) are keystone species in temperate forests. They produce acorns, constituting an essential resource base for various seed predators, particularly weevils belonging to the family Curculionidae [2]. The heterogeneous nature of seed traits [3] helps examine how resource variation facilitates predator coexistence through niche differentiation [4].

Seed morphological traits, such as size, shape, and mass, affect the predator accessibility and nutritional quality of seeds, which in turn shape interspecific interactions and infestation dynamics [5]. Larger seeds improve the opportunities for larval development but may impose physical or chemical constraints, thereby selectively filtering associated insect communities [6]. From the perspective of the resource partitioning theory, the variation in seed morphology can reduce direct competition among sympatric predators by specializing in different seed size classes [7,8], thereby enabling coexistence in line with Chesson’s modern coexistence framework [9,10].

Interspecific competition among co-occurring species in the genus Curculio is a pivotal driver governing infestation patterns and community composition. For example, Curculio glandium T. Marsham, 1802 larvae exhibit intraspecific competition and cannibalism when crowded in single acorns [11], reflecting limited resource availability and competitive exclusion pressure. Such behaviors are not yet established in C. glandium. However, it is reasonable that C. glandium larvae sharing the same acorn are also involved in competitive interactions, consistent with the competitive exclusion principle [12]. Understanding these interactions is vital because they mediate the intensity of seed predation, thus influencing host reproductive success. Curculio weevils consume acorn tissues, thus displaying a parasitic relationship with oak trees and thereby affecting host reproductive success [13]. The infestation dynamics are closely related to seed traits and species-specific biological characteristics such as body size and developmental timing. According to Muñoz et al. (2014), two Curculio species reduce direct competition through resource partitioning by ovipositing in acorns of different sizes: C. glandium prefers smaller acorns, whereas Curculio elephas J.C. Fabricius, 1781 selects larger ones. This size-based resource segregation is likely a crucial mechanism facilitating their coexistence [14].

Molecular barcoding using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences is fundamental for accurate species delimitation and phylogenetic insight, especially given the morphological similarities in larval stages that can impede the precise assessment of species-specific ecological roles [13,15]. This molecular approach is crucial for understanding the diversity and interactions within assemblages such as Curculio species found on Quercus mongolica Fisch. ex Ledeb., 1850. For instance, species such as Curculio sikkimensis K.M. Heller, 1927 and Curculio hilgendorfi Harold, 1878 overlap spatially but exhibit potentially divergent seed size preferences, thereby revealing niche partitioning [16,17].

The variation in acorn traits, particularly seed size, is believed to strongly influence the feeding preferences and infestation patterns of different Curculio species. Such species-specific selectivity allows for sympatric Curculio weevils to coexist by minimizing direct resource overlap through behavioral adaptations and niche differentiation [16]. The limited resources within individual acorns further constrain the number of larvae that can develop successfully, resulting in density-dependent competition and shaping the larval survival and population dynamics [11]. Thus, the seed size serves as a key regulator of seed predator exploitation patterns and species coexistence, especially under resource-limited conditions [18]. Despite this, systematic studies integrating molecular identification with analyses of weevil infestation patterns and seed trait selection remain scarce. In this study, mitochondrial COI barcoding was employed for precise larval identification and combined with analyses of acorn morphological traits and weevil population data to examine resource partitioning and competitive interactions among sympatric Curculio species. The objectives were to (1) reveal species-specific preferences for acorn size and the corresponding resource differentiation; (2) clarify the effects of the seed morphological characteristics (length, width, shape, and mass) on weevil abundance and infestation dynamics; and (3) assess the density-dependent competition among larvae within individual acorns. It was hypothesized that coexisting Curculio species would exhibit significant preferences for different acorn size classes, facilitating resource partitioning and reducing interspecific competition; that different species would be associated with particular acorn size ranges, resulting in distinct patterns of larval competition and infestation; and that, among the seed traits, acorn width would be the most essential predictor of weevil abundance, reflecting morphological matching between predator and resource. This study may provide new insights into the mechanisms structuring seed predator communities in temperate forests and deepen our understanding of the ecological and evolutionary dynamics underlying oak–weevil interactions.

2. Materials and Methods

2.1. Site Description

The experimental sampling site was located in a Mongolian oak (Q. mongolica) forest in Jilin City (42.79° N, 127.22° E). This site is situated within a temperate coniferous and broad-leaved mixed forest region and is characterized by a subhumid continental monsoon climate. The site has a mean annual temperature of 3.9 °C, with the lowest average temperatures ranging from −18 °C to −20 °C in January and the highest average temperatures between 21 °C and 23 °C in July. The annual precipitation averages 650–750 mm. The soil type is classified as Cambisol (World Reference Base for Soil Resources), with a pH range of 5.5–7.0.

2.2. Plot Setup and Seed Collection

Two transects, spaced 100 m apart, were established within a 2-hectare natural Q. mongolica forest located on south-facing slopes with inclinations of less than 15°. Trees exhibiting similar diameter at breast height (DBH ± 2.5 cm) but markedly differing seed sizes were selected along each transect at intervals of 50–60 m. Five trees with large seeds (mean length 21.07 mm and width 17.20 mm) and five with small seeds (mean length 14.74 mm and width 13.22 mm) were chosen. Fallen seeds were collected using nylon mesh nets (37.4-µm mesh size) placed on the ground before larval emergence and were subsequently stored at 4 °C in the laboratory for further use.

2.3. Q. mongolica Seed Traits

A total of 2000 seeds were collected, with 200 seeds per tree from both large and small fruiting Mongolian oak trees. The seed length and width were measured using a 573-NTD13 digital caliper (Mitutoyo, Kawasaki, Japan) with an accuracy of 0.01 mm. The weight of each seed was determined using an LA-204/A analytical balance (Mettler-Toledo, Shanghai, China) with an accuracy of 0.0001 g.

The seed length is typically measured from the base (proximal end) to the apex (distal end) after removing the acorn cupule (cup). The seed width (diameter) is measured as the diameter of the widest part of the acorn. The seed aspect ratio (calculated as seed_length divided by seed_width) is used to determine the seed shape. A ratio close to 1.0 indicates that the length and diameter are approximately equal, classifying the seed as spherical. When the seed diameter (width) is significantly greater than the length (resulting in a ratio less than 1.0), the seed is classified as oblate. Conversely, if the seed length is significantly greater than the diameter (width) (yielding a ratio greater than 1.0), the seed is classified as prolate [19].

2.4. Quantitative Assessment of Infestation Parameters

Acorn weevils typically infest acorns by laying their eggs inside the developing seeds, where the larvae subsequently hatch and feed. The extent of this infestation and an understanding of host preference were assessed through dissection examinations on 1000 large-sized and 1000 small-sized acorns to determine the percentage of infested seeds. Combined with the relevant information recorded in Section 2.2, the precise number of weevil larvae found inside each dissected seed was then recorded. The specimens were individually placed into centrifuge tubes containing 95% ethanol for labeling and preservation. The seed-boring insects were identified based on their morphological traits, and their species and numbers were recorded.

The statistical formulas used for calculations in this study were as follows:

Seed infestation proportion = (number of seeds that are infested/total seeds) × 100%

Proportion of seeds co-infested by multiple weevil species = (number of seeds
infested by multiple weevil species at the same time/total infested seeds) × 100%

Proportion of single-species infested seeds = (number of seeds infested by one weevil
species/total infested seeds) × 100%

Proportion of multi-species co-infested seeds = (number of seeds infested by multiple
weevil species at the same time/total infested seeds) × 100%

Proportion of a specific weevil species = (number of individuals of one specific weevil
species/total number of weevils) × 100%

2.5. Identification of Seed-Infesting Weevils

The total genomic DNA was extracted from weevil specimens using an Ezup Column Animal Genomic DNA Purification Kit (B51825-0100; Sangon Biotech, Shanghai, China) following the manufacturer’s protocols. The mitochondrial COI gene was amplified using the primers 2-COI-F (5′-GGATCACCTGATATAGCATTCCC-3′) and 2-COI-R (5′-CCTAAAAAATGTTGTGGGAAAAAGG-3′) (Yeasen Biotechnology, Shanghai, China). Polymerase chain reaction (PCR) amplification was performed in a 25-μL reaction mixture containing 1 μL of genomic DNA template (20–50 ng/μL), 1 μL each of 2-COI-F and 2-COI-R primers (10 μM), 12.5 μL of 2× Hieff PCR Master Mix (No Dye) (Yeasen Biotechnology, Shanghai, China), and 9.5 μL of nuclease-free ultrapure water. The thermal cycling protocol began with an initial denaturation at 95 °C for 5 min, followed by 35 cycles consisting of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 60 s, concluding with a final extension at 72 °C for 10 min. PCR products were separated using 1% agarose gel electrophoresis and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) following the manufacturer’s protocol. Purified amplicons were then quantified using the QuantiFluor-ST system (Promega, Madison, WI, USA). Equal molar amounts of each amplicon were pooled based on measurements taken with a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Bidirectional Sanger sequencing was conducted using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) with a BigDye Terminator v3.1 Cycle Sequencing Kit [20] Each sequencing reaction involved 20 ng of purified PCR product and 10 pmol of each primer, resulting in a final volume of 10 μL [21].

The morphological identification of weevil larvae Curculio sikkimensis and Curculio arakawai Matsumura, Kôno & H., 1928, Coleoptera: Curculionidae) was performed based on key diagnostic characteristics. C. sikkimensis larvae have a broader, reddish-brown cephalic capsule, whereas C. arakawai larvae have a narrower, yellowish-brown capsule. Both species exhibit subtle differences in the number and arrangement of setae on the pronotum, with C. sikkimensis larvae displaying a denser setal pattern on the terminal abdominal segments than C. arakawai. The mandible morphology also differs between the two: C. sikkimensis mandibles are broader with blunter inner teeth, whereas those of C. arakawai are relatively smaller with sharper inner teeth [22,23].

2.6. Phylogenetic Analysis

Mitochondrial DNA sequences from C. sikkimensis, C. arakawai, and related species were aligned and analyzed to understand their evolutionary relationships. The mitochondrial COI gene sequences were aligned using Multiple Sequence Comparison by Log-Expectation implemented in Molecular Evolutionary Genetics Analysis 11 (MEGA-11) [24]. The alignment was trimmed at both ends to remove ambiguous regions and gaps. This resulted in a dataset of equal sequence length for downstream analysis. The best-fit nucleotide substitution model was selected using MEGA’s Model Selection tool based on the Bayesian information criterion and comparing multiple candidate models. The general time-reversible model with gamma-distributed rate variation among sites and a proportion of invariant sites (GTR + G + I) was identified as optimal. Phylogenetic reconstruction was performed using the maximum likelihood method in MEGA. The tree topology was optimized using the nearest-neighbor interchange heuristic search algorithm. The node support was assessed using 1000 bootstrap replicates to evaluate the reliability of branching patterns. Sequences from closely related species belonging to the family Curculionidae were included as outgroups to root the tree and provide evolutionary context [21]. All sequence data used in the analysis are publicly available in the National Center for Biotechnology Information database with accession numbers PV688425, PV688427, PV688436, PV688438, PV688441, PV688444, PV688483, PV688507, PV688523, PV688526, PV688527, PV688539, PV688540, PV696121, and PV696122. The resulting phylogenetic tree was visualized and edited using MEGA 11 and FigTree v1.4.4 software for presentation purposes [25,26].

2.7. Statistical Analysis

The data normality and variance homogeneity were examined using the Shapiro–Wilk test and Levene’s test, respectively, with R 4.3.2 using “stats” and “car.” Given the violation of assumptions, the infestation differences between large and small seeds were analyzed using the nonparametric Mann–Whitney U test. Statistical significance was established at p < 0.05.

The community-weighted means (CWM) of the seed morphological traits, such as seed length (seed_length), seed width (seed_width), seed weight (weight), and seed aspect ratio (length/width), were calculated by weighting the seed trait values in each sample by the relative abundance of the two weevil species C. sikkimensis and C. arakawai. The data were imported and processed in R version 4.3.2. The abundance values of C. sikkimensis and C. arakawai were transformed as log10(x + 1) to normalize the distributions. Similarly, the seed morphological traits, such as seed length, were log10-transformed to meet the assumptions of normality.

The CWM for seed length, width, weight, and aspect ratio in each sample was calculated as the abundance-weighted average of the trait values for the two weevil species, using their log-transformed relative abundance values as weights. Specifically, for sample j,

$$CW{M}_{seed\text{\_}trait}={\displaystyle \frac{p_{sikkimensis,j}\times t_{seed\text{\_}trait,sikkimensis}+p_{arakawai,j}\times t_{seed\text{\_}trait,arakawai}}{p_{sikkimensis,j}+p_{arakawai,j}}}$$

where p is the log-transformed abundance of species and t is the trait value.

Pearson correlation and linear regression analyses examined the relationships between species abundance and CWM for the seed length, width, weight, and aspect ratio. A p value < 0.05 indicated a statistically significant difference. These relationships were visualized using ggplot2 R4.4.1, incorporating scatter plots with linear model fits and annotated regression statistics [27,28].

3. Results

3.1. Weevil Identification

Well-supported monophyletic clades corresponding to C. sikkimensis and C. arakawai (bootstrap support > 70%) were recovered using phylogenetic analysis based on mitochondrial COI gene sequences. Both species formed distinct lineages, clearly separated from other closely related Curculio species such as C. hilgendorfi and Curculio pardus Chittenden, 1927. This genetic differentiation supported their taxonomic distinction and suggested a clear evolutionary divergence. The robust bootstrap values at major nodes indicated reliable phylogenetic relationships within this group (Figure 1).

3.2. Weevil Infestation

Seed size showed a trend to influence the infestation patterns of C. arakawai and C. sikkimensis, with higher overall infestation rates in large seeds compared with small seeds, although the difference was not statistically significant (Figure 2A; p > 0.05). Similarly, no significant difference was observed in the rate of single-seed infestation by both species between large and small seeds (Figure 2B; p > 0.05). However, species-specific preferences were evident in dual-species infestations. The proportion of C. arakawai in dual-species single-seed infestations was significantly higher in large seeds compared with small seeds (Figure 2C; p < 0.05). However, C. sikkimensis displayed the opposite pattern, with a significantly higher proportion in small seeds than large seeds (Figure 2D; p < 0.05). Furthermore, C. arakawai exhibited a significantly higher exclusive infestation rate in large seeds compared with small seeds (Figure 2E; p < 0.05), whereas C. sikkimensis showed no significant difference in seed sizes for exclusive infestations (Figure 2F; p > 0.05). Moreover, the abundance of C. arakawai was significantly higher in larger seeds compared with small seeds (Figure 2G; p < 0.05), whereas the abundance of C. sikkimensis was significantly higher in small seeds (Figure 2H; p < 0.05).

3.3. Seed Traits and Weevil Infestation Preference

The length of the large seeds showed a positive trend with abundance (Figure 3A; R² = 0.0287, p > 0.05), whereas the seeds width exhibited a significant positive correlation with abundance (Figure 3C; R² = 0.1384, p < 0.05). Seed aspect ratio had a nonsignificant positive trend (Figure 3E; R² = 0.0489, p > 0.05), and seed weight was not significantly correlated with abundance (Figure 3G; R² = 0.0038, p > 0.05). For the Arakawai variety, the seed length (Figure 3B; R² = 0.1424, p < 0.05) and seed width (Figure 3D; R² = 0.2031, p < 0.05) both showed significant positive correlations with abundance, whereas the seed aspect ratio (Figure 3F; R² = 0.0930, p < 0.05) and seed weight (Figure 3H; R² = 0.0030, p < 0.05) showed no significant correlations.

The length of the small seeds displayed a weak positive correlation with the abundance of C. sikkimensis (Figure 4A; R² = 0.0196, p > 0.05), whereas the seed width exhibited a significant positive correlation (Figure 4C; R² = 0.2785, p < 0.001). The seed aspect ratio followed a nonsignificant positive trend (Figure 4E; R² = 0.0196, p > 0.05), whereas the seed weight showed no significant correlation with abundance (Figure 4G; R² = 0.0029, p > 0.05). The seed width was positively correlated with the abundance of C. arakawai (Figure 4D; R² = 0.2998, p < 0.001), whereas the seed length (Figure 4B; R² = 0.0075, p > 0.05), seed aspect ratio (Figure 4F; R² = 0.0012, p > 0.05), and seed weight (Figure 4H; R² = 0.0007, p > 0.05) displayed no significant correlations.

4. Discussion

4.1. Acorn Size and Weevil Oviposition Preferences

The results of this study demonstrated that acorn size is a critical determinant shaping oviposition preference and larval development among Curculio species associated with Q. mongolica. Larger acorns are consistently selected by larger-bodied weevils, providing the optimal nutritional resources to support their reproduction and offspring growth [29]. These results clearly showed that size-based resource partitioning was a key coexistence mechanism among congeneric seed predators. Female weevils exhibited refined host selection behaviors and could assess multiple morphological seed traits such as length and width prior to oviposition [14,30]. This was consistent with the findings of studies on other seed predators in which visual and tactile cues governed host assessment. However, the present study further indicated that local interspecific differences in acorn trait preference might be more pronounced than previously recognized [31,32]. These preferences, which were shaped by ovipositor and body morphology, reduced the direct interspecific overlap and promoted niche differentiation, facilitating the stable coexistence of multiple Curculio species within the same oak population [33,34].

4.2. Multi-Species Infestation and Larval Competition

Size-based partitioning reduces niche overlap. However, the lack of oviposition marking or deterrence mechanisms in Curculio weevils leads to frequent multi-species and conspecific infestation within individual acorns, especially in larger seeds [35]. Consequently, larval competition is intensified, resulting in density-dependent effects on the survival and population dynamics [36,37]. The results of this study confirmed that offspring survival was not merely a function of oviposition success, but heavily moderated by intra- and interspecific larval interactions inside the developing seed, thus echoing established findings related to competitive exclusion and resource limitation in endoparasitic seed predators [38,39]. Variations in pest abundance and species dominance among acorn size classes highlight the dynamic nature of interspecific competition and suggest that community assembly in acorn weevils is driven not only by host trait selection, but also post-oviposition larval struggles [40]. The lack of oviposition avoidance underscores an important evolutionary tradeoff: while maximizing their oviposition chances, female weevils increase the risk of competitive mortality among their progeny [39,41].

4.3. Management Implications and Ecological Insights

Recognizing acorn size as a major driver of Curculio infestation holds practical significance for pest management. Targeted monitoring and intervention during periods when favored acorn size classes are abundant can enhance the effectiveness of control efforts [42]. Given substantial genetic and morphological variation within Q. mongolica populations, a clear potential exists to breed for acorn traits that reduce the susceptibility to weevil predation [32]. As Curculio larvae complete their development entirely within a single seed, the availability of space and nutrients imposes a strict upper limit on developmental success. Larger acorns may attract increased oviposition but also experience greater larval competition and multi-species infestation [3]. The observed positive correlation between larval body size and acorn weight, up to an asymptotic limit, supports optimal nutrition and fitness hypotheses, as larger larvae have better survival prospects for diapause and subsequent adult emergence [43]. The ability of female weevils to actively evaluate acorn morphology before egg laying, thus balancing the costs of egg deposition and the likelihood of successful offspring, demonstrates the evolutionary importance of host selection behavior in shaping oak–weevil dynamics [12]. Ultimately, the findings of this study suggest that acorn size and morphological variation underpin not only the ecological structure of seed predator communities, but also the effectiveness of management and breeding strategies for sustaining healthy oak forests [44].

5. Conclusions

Acorn size significantly influences the oviposition preference and larval development of C. sikkimensis and C. arakawai on Q. mongolica. Various species exhibit distinct size-based resource partitioning, facilitating their coexistence. Larger acorns provide more resources for bigger weevil species, but they also increase the risks of multi-species infestations and larval competition, ultimately impacting offspring survival. Female weevils evaluate host quality and adjust egg deposition to optimize progeny success. The lack of oviposition marking results in intensified interspecific competition. These insights support the development of targeted pest monitoring and control strategies and emphasize acorn traits as the key targets for breeding pest-resistant oak varieties, thereby promoting sustainable forest management.