Effectiveness of Torymus sinensis Kamijo Releases in Controlling the Chestnut Gall Wasp, Dryocosmus kuriphilus Yasumatsu, in Trás-os-Montes, Portugal

Abstract

The chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu) is an invasive pest that attacks species of the genus Castanea, inducing gall formation on buds and leaves, which can significantly reduce tree growth, fruiting, and overall chestnut production. Native to China, D. kuriphilus has become a serious threat to chestnut orchards worldwide. Torymus sinensis Kamijo, a parasitoid also originating from China, is highly specific to D. kuriphilus and is currently considered the most effective biological control agent against this pest. This study aimed to evaluate the establishment of T. sinensis as well as its effectiveness in controlling D. kuriphilus at release sites between 2020 and 2023. Releases of T. sinensis were conducted in the municipality of Bragança with a sex ratio of 120 females to 70 males. The parasitoids were randomly released across three chestnut trees with infestation levels ranging from moderate (26–50% of the canopy affected by galls) to very severe (>80% of the canopy affected). At each release site, 250 galls were collected annually, and 10% of these galls were dissected to calculate parasitism rates by T. sinensis. Results revealed a positive correlation between the monitoring year and the parasitism rate. Following the releases, parasitism rates increased gradually, reaching values between 15% and 40%. T. sinensis successfully established itself in chestnut orchards and parasitized D. kuriphilus, despite normal population fluctuations being observed across years and orchards.

1. Introduction

The parasitoid Torymus sinensis Kamijo, 1982 (Hymenoptera: Torymidae), is recognized as a specific biological control agent for the chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu. This pest induces the formation of galls on buds, leaves, and petioles, leading to tree weakening and significantly impacting tree production. Losses can reach up to 80% by the fourth year of infestation [1]. Native to China, D. kuriphilus has become a serious threat to chestnut orchards worldwide.

T. sinensis is a univoltine species, with females laying an average of 70 eggs during their lifespan of approximately 30 days. Fertilized eggs develop into diploid females, while unfertilized eggs produce haploid males [2,3]. In early spring, T. sinensis females oviposit within newly formed D. kuriphilus galls, typically laying one egg per host larva. The parasitoid larva hatches shortly thereafter, consuming its host by late spring before entering dormancy. Pupation occurs in late winter, and adults emerge in early spring of the following year, coinciding with chestnut tree sprouting and the formation of new D. kuriphilus galls [2,3].

The first introduction of T. sinensis occurred in Japan in 1975, and it was subsequently released in all regions where D. kuriphilus had become established. These include the USA (late 1970s), Italy (2002), France (2011), Croatia (2014), Hungary (2014), Bosnia and Herzegovina (2016), Spain (2015), Portugal (2015), and Slovenia (2015) [2,4,5,6,7,8]. In Europe, T. sinensis was first introduced in Italy in 2002, marking the beginning of its use as a biological control agent on the continent [6].

The release of T. sinensis has proven to be the most effective strategy for controlling D. kuriphilus. Its success depends on a carefully planned release process, which requires synchronization of the pest and parasitoid life cycles. This process also relies on comprehensive knowledge of D. kuriphilus biology, chestnut tree phenology, and the influence of climatic conditions on both pest development and crop growth [9].

Monitoring efforts in countries where T. sinensis has been released reveals considerable variability in parasitism rates. For example, in Campania, Italy, parasitism rates ranged from 0.5% to 164% between 2009 and 2016 [10]. Similarly, monitoring conducted in Bosnia and Herzegovina in 2017 showed rates between 44.83% and 74% [4].

In Portugal, the national action plan for the control of D. kuriphilus, coordinated by the Direção Geral de Agricultura e Veterinária (DGAV), outlines guidelines for biological control, including monitoring programs for both D. kuriphilus and T. sinensis, as well as protocols for the release of T. sinensis [1].

The objective of this study was to evaluate the parasitism rates of T. sinensis during the release year and their evolution over the subsequent four years.

2. Materials and Methods

2.1. Torymus sinensis Releases

Over four years (2020–2023), a total of 851 T. sinensis releases were conducted in chestnut orchards across 36 civil parishes in the municipality of Bragança. The release sites were spaced at least 500 m apart and selected based on the absence of prior releases and the presence of moderate (26–50% of the canopy of three trees with galls) to severe (>80% of the canopy of three trees with galls) infestations. Each release involved a sex ratio of 120 females to 70 males, distributed in 11 tubes (3 cm in diameter and 12 cm in length).

The release process was conducted on days without rain or wind to minimize interference with parasitoid activity. Parasitoids were randomly distributed among three heavily infested chestnut trees. The tubes were opened, and insects were gently stimulated to exit onto the leaves, ensuring their dispersal into the orchard.

2.2. Monitoring the Effectiveness of T. sinensis

The effectiveness of T. sinensis releases was monitored during winter (January) each year from 2021 to 2024. Monitoring was conducted at 25% of the release sites for each year (45 sites in 2020, 56 in 2021, 52 in 2022, and 50 in 2023), as well as at previously monitored sites from earlier years (5 sites in 2020, 15 in 2021, 29 in 2022, and 37 in 2023).

At each monitored site, a total of 250 galls were collected from five to six randomly selected chestnut trees [1]. The samples were taken from branches located at various heights (1.5–3 m above the ground) to ensure representative sampling.

The collected galls were transported to the laboratory, where they were cleaned with a brush and stored at room temperature in emergence boxes (Figure 1) until adult insects emerged. These boxes were constructed from cardboard (32 cm × 27 cm × 10 cm) and equipped with two collection tubes (3 cm in diameter and 12 cm in length) for emerging insects. After all insects had emerged, the boxes were opened, and adults were collected and identified using a binocular microscope and identification keys.

Subsequently, 10% of the collected galls were dissected to count the number of larval chambers. The parasitism rate was calculated using Equation (1).

$$\mathrm{Parasitism} \mathrm{rate}={\displaystyle \frac{Nº T. sinensis}{250 galls\times nº of chambers}}\times 100$$

(1)

2.3. Statistical Analysis

The success of T. sinensis releases was evaluated by calculating the median number of T. sinensis individuals captured, as well as the average parasitism rates and their standard errors for each civil parish. Linear regression analyses and ANOVA tests were performed to assess the relationship between the number of T. sinensis released and the number subsequently captured, as well as the relationship between the year of release and parasitism rates. The homogeneity of variance test (Levene’s test) was also carried out on the data analyzed.

To carry out the ANOVA test to assess the relationship between the number of parasitoids released and those collected, it was necessary to group the data into three categories: Low (100–599), Medium (600–999), and High (1000–4000).

All statistical analyses and calculations were conducted using Microsoft Excel (Microsoft Office LTSC Professional Plus 2021), PAST, version 0.45 (http://www.uio.no/õhammer/past accessed on 15 March 2025), and JASP, version 0.19.3 (https://jasp-stats.org/download/ accessed on 15 March 2025).

3. Results

The number of releases conducted in each chestnut orchard and the average parasitism rates of T. sinensis are presented in

Table 1. Released, collected, and parasitism rate of T. sinensis in 36 chestnut orchards located in the municipality of Bragança, Portugal.

Parish	Torymus sinensis Kamijo Released (a)							Torymus sinensis Kamijo (b)
	Number of Sites Sampled	2020	2021	2022	2023	Released		Collected	Median (c)	Parasitism Rates ±SE (%) (d)
Baçal	3	190	380			570		482	241	12.75	±	9.02
Carragosa	4	380	380	570		1330		610	45	7.37	±	4.25
Castro de Avelãs	4	190	190	190	190	760		599	178	14.95	±	7.475
Coelhoso	5	190	190	190	380	950		1065	256.5	24.13	±	12.06
Donai	8	380	570	190	380	1520		863	189.5	12.23	±	6.11
Espinhosela	15	760	760	380	950	2850		1934	257.5	12.3	±	6.15
França	2		190	190		380		295	147.5	14.7	±	10.39
Gondesende	6	380	190	190	380	1140		856	98	13.18	±	6.59
Gostei	1		190			190		223	223	22.3
Grijo de Parada	2			380		380		661	661	33
Mós	4	190	190	190	190	760		468	57.5	11.68	±	5.84
Nogueira	1		190			190		251	251	25.1
Outeiro	3		190	380		570		784	392	24.15	±	17.08
Pâramio	16	380	570	570	1520	3040		2332	168	10.55	±	5.28
Pinela	11	380	950	190	570	2090		2630	486.5	27.83	±	13.91
Quintanilha	1			190		190		257	257	25.6
Quitela de Lampaças	4	190	190	190	190	760		1211	274.5	26.5	±	13.25
Rabal	2		190	190		380		374	187	18.7	±	13.22
Rebordãos	4	190	190	190	190	760		367	90	9.33	±	4.66
S. Comba de Rossas	7	190	380	190	570	1330		1500	242	16.73	±	8.36
S. Pedro de Sarracenos	4	190	190	190	190	760		353	86	10.1	±	5.05
Salsas	15	760	570	950	570	2850		2143	370	16.45	±	8.23
Samil	4	190	190	190	190	760		629	142.5	15.9	±	7.95
Sendas	10	570	380	380	570	1900		836	75	7.55	±	3.78
Serapicos	15	380	950	950	570	2850		1980	353	12.08	±	6.04
Sortes	5	190	380	380	190	1140		996	224.5	22.29	±	11.14
U.F. de Aveleda e Rio de Onor	2		190	190		380		404	202	20.2	±	14.28
U.F. de Izeda, Calvelhe e Paradinha Nova	3	190		380		570		861	430.5	24.95	±	17.64
U.F. de Castrelos e Carrazedo	4	190	190	190	190	760		431	73	10.75	±	5.38
U.F. de Parada e Failde	16	950	950	570	570	3040		2545	664.5	17.28	±	8.64
U.F. de Rebordainhos e Pombares	7	190	190	190	190	760		903	126	22.98	±	11.49
U.F. de Rio Frio e Milhão	1			190		190		223	223	22.2
U.F. de Sé, Santa Maria e Meixedo	4	190	190	190	190	760		575	88.5	14.38	±	7.19
U.F. de Sao Julião de Palácios e Deilão	3	190		380		570		790	395	22.35	±	15.80
Vilarinho	1				190	190		174	174	17.4
Zoio	6	380	190		380	950		1120	76.5	14.3	±	7.15

a—Number of Torymus sinensis Kamijo released in n0 year; b—Number of Torymus sinensis Kamijo collected; c—Median of Torymus sinensis collected in each year; d—Data refer to 250 galls sampled in each sites. Parasitism rate is calculated: ${\displaystyle \frac{nº T. sinensis}{250 galls\times nº of chambers}}\times 100$.

.

Over the four years of the study, a total of 851 releases were performed, introducing 161,690 T. sinensis individuals into the municipality of Bragança. This included 102,120 mated females (Table 1). During the release year (n₀, corresponding to 2020, 2021, 2022, and 2023), 38,570 T. sinensis individuals were introduced across all sites, with 32,725 individuals recovered during monitoring in the same release year. In total, 50,750 galls were collected across all monitoring events.

The recovery rate of parasitoids was positively associated with the number of releases conducted. Linear regression analysis (Figure 2) revealed a positive correlation (R² = 0.8068, F = 136.152, p = 0.080254) between the number of T. sinensis released and the number recovered. The R² value indicated that 80.68% of the variation in the dependent variable (Y), the number of T. sinensis recovered, was explained by the regression model, suggesting a relatively good fit to the data and a strong explanatory power.

However, the p-value associated with the F-test was 0.080254 (p > 0.05), indicating that the model was not statistically significant. This suggests that while there is a clear trend in the data, the results may have been influenced by other factors not captured by the regression model or the variability in the data itself.

The one-way ANOVA test revealed a significant difference between the groups (F(2,33) = 19.11, p < 0.001). The collected averages were higher in the High group (M = 1602.1; SD = 743.2) than in Medium (M = 701.9; SD = 316.3) and Low (M = 444.5; SD = 246.5) groups. Tukey’s post hoc tests confirmed that High differed from Medium and Low (p < 0.05) and that Medium differed from Low (p < 0.05). (more information in Supplementary Materials).

Average parasitism rates in the release year (n₀, corresponding to 2020, 2021, 2022, and 2023) varied significantly across civil parishes, ranging from 7.37% in Carragosa to 33% in Grijó de Parada (Table 1).

Monitoring of releases (Figure 3) revealed parasitism rates that fluctuated between 0.5% (Carregosa) and 31.5% (Coelhoso) in 2020 (A), 0.7% (Parâmio) and 49.4% (Quintela de Lampaças) in 2021 (B), 3.1% (Sendas) and 41.1% (Pinela) in 2022 (C), and 0.1% (Mós) and 59.8% (Rebordainhos) in 2023 (D). These results indicate variability in parasitism rates over the years and among locations, reflecting potential differences in environmental conditions, release success, or local population dynamics of D. kuriphilus and T. sinensis.

For chestnut orchards monitored continuously over four years, parasitism rates exhibited year-to-year variations, generally showing an upward trend. However, declines were observed in Calvelhe and Pinela. Notably, the sampling in Coelhoso revealed a decrease in parasitism rates from 2021 to 2022, and by 2023, no D. kuriphilus galls were found in this orchard (Figure 4).

A linear regression analysis (Figure 5) between the year of monitoring and the parasitism rate revealed a positive correlation (R² = 0.1881; F = 7.647; p = 0.009). The R² value indicates that 18.81% of the variation in the parasitism rate is explained by the model. Although this value suggests limited explanatory power, it implies that other factors not included in the model may be influencing the parasitism rate.

The F-value is moderate, and the associated p-value of 0.009 is below the 0.05 significance threshold. This confirms that the relationship between the year and the parasitism rate is statistically significant at a 5% significance level, albeit with a limited ability to fully explain the observed variability.

There was no significant effect of year on the parasitism rate (ANOVA: F(3.30) = 2.45, p = 0.082). The average parasitism rates were 8.4% in 2020 and 25.3% in 2023, but the variation between years was not statistically significant. The assumption of equal variances was met (Levene p = 0.210) (more information in Supplementary Materials).

4. Discussion

The observed increase in T. sinensis populations over the years aligns with expectations, as this parasitoid requires time to establish and expand its population. Previous studies indicate that T. sinensis typically needs 5–7 years after the initial release or settlement to achieve high parasitism rates [11]. For example, in Japan, the first country to introduce T. sinensis, dispersal rates were estimated at less than 1 km/year during the initial years [11].

Fluctuations in natural parasitism rates over the years, along with the limited effectiveness of native parasitoids, support the importance of introducing T. sinensis as a biocontrol agent. Its efficacy has proven to be significant, delivering high levels of pest control within a few years post-introduction [11].

In the municipality of Bragança, during the release year (n₀) between 2020 and 2023, the average number of T. sinensis individuals collected ranged from 45 to 664 per civil parish. This compares favorably with Campania, Italy, where T. sinensis recovery ranged from 9 to 212 individuals [10].

Table 1 highlights considerable variability in parasitism rates across civil parishes. Similar variability has been observed in other countries and is often attributed to factors such as climatic conditions, agricultural practices, and the natural acclimatization of T. sinensis [10].

The relationship between the number of T. sinensis released and recovered (Figure 2) showed that higher recovery rates were generally associated with larger release efforts. Comparable trends were observed in Campania, Italy, where larger releases correlated with greater parasitoid recovery [10].

Parasitism rates of T. sinensis in some civil parishes of Bragança tended to increase over the four years (2020–2023), except in Calvelhe and Pinela, where rates declined. Notably, in Coelhoso, no Dryocosmus kuriphilus galls were found for monitoring in 2023, suggesting a significant reduction in pest presence. The effectiveness of T. sinensis has been consistently documented in other countries, particularly in Italy, where studies report positive trends in parasitism rates and long-distance movements of T. sinensis with parasitism rates ranged from 0.5% to 164% between 2009 and 2016 [4,5,10].

The linear regression analysis of parasitism rates and monitoring years (Figure 5) revealed a positive relationship between these variables. Following releases, parasitism rates increased gradually, ranging between 15% and 40%.

This study demonstrates that T. sinensis successfully colonized chestnut orchards in Bragança and parasitized D. kuriphilus. Despite normal population fluctuations observed across years and locations, the biocontrol agent showed its potential for managing the chestnut gall wasp effectively. Continued monitoring and release programs are recommended to further enhance T. sinensis establishment and long-term pest suppression.

5. Conclusions

The results of this study show that the releases of the parasitoid T. sinensis in chestnut orchards in the municipality of Bragança were successful in colonizing and controlling D. kuriphilus. The parasitism rate observed increased progressively over the four years of monitoring, reaching average values of between 15% and 40% in the different study sites, proving the ability of T. sinensis to establish and disperse in the conditions of the Trás-os-Montes region. Despite the spatial and temporal variability, influenced by differences in local climate, initial infestation status, and the density of releases, the general trend of increasing parasitism rates confirms the potential of this parasitoid as a biocontrol agent for this pest.

In addition, the positive correlation (R² = 0.81) between the number of specimens released and the number recovered reinforces the importance of a continuous and well-sized release effort to accelerate the consolidation of the T. sinensis population. The lack of statistical significance in some years or specific locations highlights the need for adjustments to the release strategy, namely in the choice of locations, the ideal time, and the density of releases, in order to optimize the effectiveness of biological control.

In practical terms, it is recommended to maintain this release program associated with a regular monitoring plan, allied to sustainable agronomic practices (maintaining plant diversity and reducing chemical pesticides), in order to boost the long-term suppression of D. kuriphilus in the Trás-os-Montes chestnut orchards. Future research should explore the interaction of T. sinensis with native parasitoids, as well as the impact of extreme climatic variables on the effectiveness of the biocontrol, in order to guarantee robust and resilient control of this invasive pest.