Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) in Minho (Northern Portugal): Bioecology, Native Parasitoid Communities and Biological Control with Torymus sinensis Kamijo (Hymenoptera: Torymidae)

Abstract

The Asian chestnut gall wasp Dryocosmus kuriphilus Yasumatsu, native to China, was first detected in Europe in Italy in 2002. In Portugal it was declared in 2014, and it has since affected the Portuguese chestnut production. The introduction of its natural parasitoid Torymus sinensis Kamijo started through inoculative releases according to the National Action Plan for the Control of Dryocosmus kuriphilus, established by the Direção Geral de Alimentação e Veterinária (DGAV), in 2015. This research was carried out during 2018 and 2019, in five chestnut orchards in the Minho region (Northern Portugal). Between January and March 2018, at each location, three monthly harvests of 100 buds were carried out to determine the infestation level. Between April and June 2018, 240 galls were harvested every two weeks, with the purpose of studying the bioecology of D. kuriphilus. In each sampling site, 160 galls were placed in emergence boxes to follow adults of the D. kuriphilus population, and 80 galls were dissected with a binocular magnifying glass, to count and record the evolution of the larval stages of D. kuriphilus and of the parasitoids. The identification of T. sinensis and native parasitoids was carried out. The parasitism rates and Simpson diversity indices were determined. The percentage of buds infested by D. kuriphilus was high in all studied places, varying between 67% and 80%, regardless of the number of T. sinensis releases made and of the chestnut cultivars present in each orchard. The highest rate of parasitism by T. sinensis was 0.5044% and it was recorded in an orchard where there were two releases of T. sinensis in 2016 and 2017 (Arcos de Valdevez—Rio Cabrão—P1). Considering that the exotic parasitoid has already been released in the region since 2016, its establishment in all the chestnut orchards studied was confirmed, although with very low parasitism rates. The natural parasitism associated with the native parasitoid species of D. kuriphilus was confirmed in this work, recording values between 34% and 51% of the parasitized chestnut galls, with 11 species of native parasitoids having been identified: Eupelmus azureos, Eupelmus uruzonus, Eurytoma brunnivientris, Eurytoma pistaciae, Megastigmus dorsalis, Mesopolobus sp., Ormyrus pomaceus, Sycophila biguttata, Sycophila iracemae, Sycophila variegatta and Torymus flavipes.

1. Introduction

The chestnut tree is a deciduous tree belonging to the genus Castanea and the family Fagaceae [1,2]. According to Bounous et al. [3], the genus Castanea includes thirteen species, seven present in North America, five in East Asia and one in Europe, specifically, the European chestnut tree (Castanea sativa Mill.). These species have varied sizes, with maximum heights between 6 and 30 m [4]. For many years, the chestnut had a primary role in the diet of rural families, playing a critical subsistence function similar to that of the potato today and constituting a fundamental resource in the economy of the regions where the chestnut tree has adapted [5]. Consumed throughout many months of the year (October to July), whether cooked or converted to flour, chestnuts represented one of the primary food sources for those populations [6]. On the other hand, chestnut wood was valued and used as a building material, in the manufacture of furniture and a wide range of utensils for farming, basketry and cooperage, as well as firewood for heating [7].

In Portugal, the chestnut tree is very present in the region of Trás-os-Montes (Northeastern region) and Beiras (Central region), being also found in some smaller centers in the Northern Alentejo (Serra de S. Mamede and Marvão), Northern Algarve (Monchique) and Minho (Northwestern region) regions, and, in a dispersed way, in several points at the north of the Tagus River. It is also present, although in isolated spots, both on the island of Madeira (Curral das Freiras and Serra de Água) and in the Azores [8,9,10,11,12]. The edaphoclimatic conditions that characterize the different production areas provided the development of a high number of varieties (cultivars) [13]. To respond to market demands for high-quality products, whilst also seeking to promote the preservation of the national genetic heritage, demarcated regions with a Protected Designation of Origin (PDO) were created to promote regional chestnut varieties, such as Padrela, Terra Fria, Soutos da Lapa and Marvão. In the Minho region, the Amarelal variety is the one that best adapts to the soil and climate conditions within the national varieties. The hybrid varieties, Bouche de Betizac, Marigoule and Marsol, have also been introduced in recent years, being earlier and reaching larger calibers, although with a lower conservation power.

Dryocosmus kuriphilus Yasumatsu is considered one of the most serious pests of the genus Castanea worldwide, as the galls allow the development of D. kuriphilus inside individual chambers, interrupt the growth of new branches and significantly reduce fruit set, reducing the fruit production of chestnut (C. sativa) by 50–80% [14,15]. Native from China, over the last few decades, this pest has been identified in different countries. In 1958 was first observed in Korea [16]. On the American continent its presence was confirmed in 1974 in chestnut trees imported from China [14]. In 1975 its presence was reported in Japan [16]. The gall wasp was accidentally introduced into mainland Europe via Northwest Italy in 2002 through infected plant material from China, rapidly expanding to several western European countries [17,18]. In 2005, it was identified in France and Slovenia, and in 2009 in Switzerland and Hungary [19]. In Portugal, the first pest outbreaks were detected in the municipality of Barcelos in June 2014 (Minho region, Northwestern Portugal) [20]. By that year, the insect had already been reported in 75 parishes in the region. In August 2014, the first outbreaks were confirmed on the island of Madeira. Despite the measures taken, the situation worsened in 2015, with the pest being detected in Trás-os-Montes, namely in the three main chestnut-producing areas (Terra Fria, Padrela and Soutos da Lapa), as well as in the Center region, in the municipalities of Trancoso, Aguiar da Beira, Anadia and Fundão [21]. This rapid expansion across the European continent suggests multiple human-made introductions, as it highly exceeds the estimated dispersal rate for this insect, which is estimated at around 8 km·yr⁻¹ [22]. Since 2003, D. kuriphilus has been included in the European and Mediterranean Plant Protection Organization (EPPO) A2 list, which aggregates all quarantine organisms and on which all countries apply specific regulations for their monitoring and control [23].

The first approach to minimizing the impact of the chestnut gall wasp was the classic biological control, implemented in Portugal since 2015 through releases of the exotic parasitoid Torimus sinensis Kamijo in the locations where the presence of the pest was confirmed at high levels, a condition considered necessary for the adaptation and establishment of the parasitoid. The national action plan for the control of D. kuriphilus, under the coordination of the DGAV, was established to define the prospection actions, to control the chestnut-gall wasp in the national territory and to prevent the spread of the pest in Portugal. According to this plan all orchards were monitored, recording the evolution of chestnut phenology and the beginning of gall formation to determine the best moment to release T. sinensis. Thus, in each orchard the releases were carried out in the phenological state D (appearance of veins and development of leaves). The natural limitation exerted by a diverse set of native auxiliary species, although playing an important role, does not appear to be sufficient (in the light of current knowledge) for effectively controlling this pest. On the other hand, using chemical means has not proved to be a valid option, and the lack of other biochemical and genetic methods is still not feasible in the short term.

The life cycle of D. kuriphilus starts in early summer (June–July) with the emergence of adults, which lay eggs exclusively into the chestnut tree buds during the summer and their larvae overwinter inside the buds [20]. Adult longevity is less than 10 days, during which they can lay more than 100 eggs in groups of three to five per bud [20]. At the begging of the spring, when the chestnut tree begins its vegetative activity, the D. kuriphilus larvae induce the formation of insect galls and feed for approximately 30 days until they achieve pupation, which will last until the beginning of summer, when the adult insect emerges. Thus, the occurrence of D. kuriphilus and their galls is shaped by the distribution of their host plant, their ecological interactions include oviposition, gall formation, parasitism and feeding. Parasitoid T. sinensis is an univoltine species, with one generation per year, and haplodiploid, giving birth to haploid males from unfertilized eggs and diploid females from fertilized ones [24]. Females of T. sinensis lay their eggs in early spring (March–April) inside the newly formed galls (in the body of the host D. kuriphilus, or in the wall of the larval chamber). Usually, one egg is placed per host larvae, but under natural conditions, several eggs per larva have been observed in a single chamber. However, only one parasitoid larva completes its development due to cannibalism among the larvae of T. sinensis. After emerging, the larva feeds from their host larva at the end of spring and enter into a dormant state until late winter, when pupation starts. Synchronous with the developing of chestnut trees and the formation of galls caused by D. kuriphilus, adults of T. sinensis emerge in early spring. The life duration of T. sinensis depends mainly on the ambient temperature and the type and quantity of food available, and adults can reach 40 or more days [25]. The length of the egg deposition season for T. sinensis is normally set according to its lifespan [26]. Figure 1 shows the schematic life cycle of D. kuriphilus and T. sinensis.

According to studies in other countries, the recent introduction of the parasitoid T. sinensis is seen as the most promising alternative for chestnut gall wasp control [16,18,27,28,29]. At the same time, it is essential to study the chestnut ecosystem in the Minho region to identify and monitor the complementary action of the native parasitoids on the natural limitation of D. kuriphilus populations. Thus, the objectives defined for this research were to study the bioecology of D. kuriphilus, characterizing the different stages of development in the production conditions of the Minho region. In addition, the objectives were to evaluate the adaptation and establishment of the exotic parasitoid T. sinensis in the natural environment of chestnut orchards in the study area of the five orchards studied (Arcos de Valdevez and Ponte de Lima) where it has been released in a controlled manner since 2015, and to identify the native parasitoids associated with the chestnut ecosystem in the Minho region. Although not considered specific, this may play a complementary and essential role in the natural limitation of D. kuriphilus, evaluating their diversity, abundance and parasitism rate.

2. Materials and Methods

2.1. Location of the Orchards under Study

This research was carried out from 2018 to 2019 in five chestnut orchards in Alto Minho, four in the municipality of Arcos de Valdevez and one in the municipality of Ponte de Lima. These orchards were selected to conduct the biological treatment in the Alto Minho region, with their chestnut trees being seriously infected by the wasp (presence of galls in 51–80% of shoots). The chestnut orchards selected are characterized in

Table 1. Identification and characterization of the studied orchards (P1 to P5) in Arcos de Valdevez (AVV) and Ponte de Lima (PTL), with one (1T), two (2T) or no treatment (NT) of T. sinensis.

Orchard	Area (ha)	Location (Parish, Municipality)	Varieties	Nr. of Releases T. sinensis	Year of the Releases
P1AVV2T	1.22	Rio Cabrão (Arcos de Valdevez)	Amarelal	2	2016 2017
P2AVV2T	0.29	Távora S. Maria (Arcos de Valdevez)	Marigoule	2	2016 2017
P3AVV1T	0.35	Rio de Moinhos (Arcos de Valdevez)	Amarelal	1	2017
			Marsol
P4AVVNT	1.10	S. Paio (Arcos de Valdevez)	Amarelal	0
			Bouche Betizac		-----
			Marigoule
P5PTL2T	1.83	Gondufe (Ponte de Lima)	Marigoule	2	2016 2017

.

2.2. Quantification of D. kuriphilus Populations

2.2.1. Framework

Between January and March 2018, 100 chestnut buds were harvested monthly in each chestnut orchard. The material collected was packaged, adequately identified and transported to the laboratory. In the laboratory, the buds were dissected using a binocular magnifying glass (Model SZ-ET/Olympus brand). The number of D. kuriphilus larvae per bud was recorded, and the percentage of infested buds was determined. From the bud burst, between April and June 2018, 240 spring galls were harvested fortnightly in each orchard in a total of 5 harvests per orchard. The material identified and transported to the laboratory was cleaned to remove leaves and branches. Each sample was divided into two sub-samples: one composed of 80 galls to be subjected to dissection with a binocular magnifying glass (to detect parasitized larvae/pupae of D. kuriphilus and larvae of parasitoids inside the gall), and another sample composed of the remaining 160 galls, which were placed in cardboard boxes (20 cm × 36 cm × 13 cm) and properly labelled for weekly recording of the emergence of D. kuriphilus and parasitoids until May 2019. The cardboard boxes were previously identified and adequately insulated with opaque tape on the inside of the box to prevent insects from escaping. The boxes were placed at room temperature in the laboratory and without direct light. The adults emerging from the galls were captured in a transparent tube (Falcon Tube, 50 mL) and screwed into an exit hole in the correctly identified box (orchard, date of gall harvest). The quantification of D. kuriphilus adults was recorded weekly, systematically replacing the Falcon tubes in each box, and the insects were transferred to Eppendorf tubes containing 70% alcohol and stored in the laboratory for subsequent quantification and identification.

2.2.2. Quantification of Parasitoids and Global Rate of Parasitism

The hatching parasitoids in each orchard were monitored by sampling spring galls following the same methodology described for D. kuriphilus. Likewise, from the observation of spring galls dissected biweekly, values related to the population density of parasitoids inside the galls were recorded and determined according to the methodology already described. Only the first three harvests (240 galls) were considered, to exclude possible counting errors and to ensure a correct interpretation of the parasitism situation by observing the chambers (structures where D. kuriphilus develops, and an indicator of the attack intensity of the pest) after the emergence of D. kuriphilus adults and native parasitoids. To determine the percentage of parasitized galls and to assess the parasitism rates, spring galls harvested in 2018 in the five chestnut orchards were dissected under a binocular magnifying glass, and each chamber was evaluated as parasitized or not according to its condition, i.e., the presence of a black spot covering approximately half of the inner wall of the chamber, or the host being found (D. kuriphilus), as proposed by Santos et al. [30]. Global parasitism rates were calculated by relating the number of parasitized chambers to the total number of chambers in the sample, using Equation (1):

$$\mathrm{Parasitism}\mathrm{rate}=\left(\frac{\mathrm{no}.\mathrm{of}\mathrm{parasitized}\mathrm{chambers}}{\mathrm{Total}\mathrm{nr}.\mathrm{of}\mathrm{chambers}}\right)$$

(1)

2.2.3. Identification of the Parasitoids

The parasitoids were morphologically identified at the genus and species level using taxonomic keys and by comparison with reference specimens previously identified and deposited at the Entomology laboratory (ESA/IPB/CIMO, Bragança, Portugal) [30,31].

2.2.4. Indicators of the Parasitoids Population Structure

The diversity of the populations studied was analyzed by determining species abundance, the relative frequency and Simpson’s diversity index. Abundance represents the number of individuals of each species, and relative frequency represents the percentage relative to the number of times each species was identified in the studied locations. The diversity of native parasitoids was evaluated using the Simpson diversity index using Equation (2) [32]:

$$\mathrm{C}=1-\frac{{{\displaystyle \sum }}_{\mathrm{i}=1}^{\mathrm{S}}{\mathrm{n}}_1\left(\mathrm{n}-1\right)}{\mathrm{N}\left(\mathrm{N}-1\right)}$$

(2)

where,

• n—number of individuals of a species;
• N—total number of organisms.

Biological diversity is more significant the closer to 1 the value of the Simpson diversity index is.

2.2.5. Quantification of Torimus Sinensis Populations and Effective Parasitism Rate

Field releases of T. sinensis were carried out in 2016 and 2017 using 190 insects, 70 males and 120 females, in 4 of the 5 orchards studied in the present work [33]. To study the establishment of T. sinensis in the Minho region, field samples were collected in the five chestnut orchards of Minho. Monitoring, quantifying and identifying T. sinensis was carried out following the methodology previously described (Section 2.2.2) for natural parasitoids until February 2019. Parasitism rates were calculated by relating the number of T. sinensis parasitoids that emerged with the total number of chambers in the sample, according to Nieves-Aldrey et al., and is presented in Equation (3) [34]:

$$\mathrm{Parasitism}\mathrm{rate}=\left(\frac{\mathrm{no}.\mathrm{of}T. sinensis}{\mathrm{no}.\mathrm{of}\mathrm{galls}\times \mathrm{MCG}}\right)\times 100$$

(3)

where,

• no. of T. sinensis—number of T. sinensis parasitoids per sample;
• no. of galls—number of galls collected per site;
• MCG—average number of chambers per gall.

2.2.6. Assessment of the Existence of Statistically Significant Differences between Populations of D. kuriphilus

To determine the occurrence of statistically significant differences between the populations of D. kuriphilus from the different orchards monitored in the present study, an analysis of the variance of the means of occurrences between the different groups was used. For this purpose, an analysis of variance (one-way ANOVA) was performed using the IBM^® SPSS^® Statistics software—Version 27.0.1.0—64-bit edition (Armonk, NY, USA).

3. Results

3.1. Bioecology of D. kuriphilus

The dissection of winter buds, carried out between January and March 2018, showed the presence of D. kuriphilus in all chestnut orchards (Figure 2 and

Table 2. D. kuriphilus infestation evaluated by the percentage of infested buds and average number of D. kuriphilus larvae per bud ± standard deviation in orchards P1 to P5 of Arcos de Valdevez (AVV) and Ponte de Lima (PTL), with one (1T), two (2T) or no treatment (NT) of T. sinensis in 2018.

Location	No. of Buds	% of Infested Buds	Average No. of Larvae/Buds ± Standard Deviation
P1AVV2T	200	78.0%	2.54 ± 2.03
P2AVV2T	200	80.0%	2.66 ± 2.02
P3AVV1T	200	74.0%	2.90 ± 2.30
P4AVVNT	200	77.5%	2.92 ± 2.33
P5PTL2T	300	67.0%	2.04 ± 1.93

). The buds infested with D. kuriphilus ranged from 67% in the P5PTL2T orchard to 80% in the P2AVV2T orchard. Regarding the mean number of D. kuriphilus larvae per bud, the values varied between 2.04 ± 1.93 in the P5PTL2T orchard and 2.92 ± 2.33 in the P4AVVNT orchard.

The observations of the chestnut buds and galls and the monitoring of the emergence of adults of D. kuriphilus allowed us to determine the evolution of the different stages of development (Figure 3).

Table 2 also shows the relative values of the infested buds, and the average number of D. kuriphilus larvae per bud and the respective standard deviations, which showed an attack level between 67% (P5PTL2T) and 80% (P2AVV2T).

Based on the results of the 2018 spring gall dissection (

Table 3. Results of the dissection of spring galls harvested in 2018 in the five chestnut orchards (P1 to P5) located in Arcos de Valdevez (AVV) and Ponte de Lima (PTL), with one (1T), two (2T) or no treatment (NT) of T. sinensis.

Location	No. of Dissected Galls	No. of D. kuriphilus Chambers	Average No. of Chambers Per Gall ± Standard Deviation
P1AVV2T	240	1514	6.31 ± 2.46
P2AVV2T	240	1544	6.43 ± 2.49
P3AVV1T	240	1541	6.42 ± 3.22
P4AVVNT	240	1676	6.98 ± 3.65
P5PTL2T	240	1487	6.20 ± 2.90

), the number of chambers with D. kuriphilus was lower in the Ponte de Lima chestnut orchard (P5PTL2T), with a higher number of chambers (1676 chambers) being recorded of D. kuriphilus in the Arcos de Valdevez orchard where there was no T. sinensis release (P4AVVNT), oscillating between and in the Arcos de Valdevez orchard without any T. sinensis treatment (P4AVVNT).

Regarding the emergence of D. kuriphilus from spring galls, a total of 4027 adult insects were recorded from 1 June 1 to 10 August 2018. The peak of emergences occurred on 29 June in the P2AVV2T orchard (Arcos de Valdevez), and on 13 July in the P5PTL2T orchard in Ponte de Lima. In the remaining orchards, peaks of emergence were recorded in the interval between the dates above and it is concluded that the pest was present in all orchards with higher populations until June 20, the date from which populations began to decline (Figure 4 and Figure 5). For the statistical comparison of the populations, an analysis of variance (one-way ANOVA) was carried out with a comparison of the variance of the means of the different groups. The results obtained point to p values greater than 0.05, indicating that there were no statistically significant differences between the groups studied.

3.2. Quantification of Parasitoids and Rate of Natural Parasitism Associated with D. kuriphilus

The results of the evaluation of the presence of parasitoids in the spring galls collected in 2018, after their dissection, confirmed the presence of parasitoids and the natural parasitism of D. kuriphilus (Figure 6). It was found that the percentage of parasitized galls varied between 34% and 51% in the orchards of Arcos de Valdevez—P3AVV1T (one release of T. sinensis in 2017) and P2AVV2T (two releases of T. sinensis in 2016 and 2017), respectively (

Table 4. Absolute and relative values of spring galls parasitized by T. sinensis and autochthonous parasitoids in five chestnut orchards in Minho (P1 to P5) located in Arcos de Valdevez (AVV) and Ponte de Lima (PTL), with one (1T), two (2T) or no treatment (NT) of T. sinensis (NT) in 2018.

Location	Total No. of Galls	No. of Parasitized Galls	% of Parasitized Galls
P1AVV2T	240	89	37%
P2AVV2T	240	123	51%
P3AVV1T	240	82	34%
P4AVVNT	240	103	43%
P5PTL2T	240	100	42%

).

The parasitism rate (

Table 5. Total number of chambers, number of parasitized chambers and parasitism rate in 5 chestnut orchards in Minho (P1 to P5) located in Arcos de Valdevez (AVV) and Ponte de Lima (PL), with one (1T), two (2T) or no treatment (NT) of T. sinensis in 2018.

Location	Total Chambers	No. of Paratized Chambers	% Parasitized Chambers
P1AVV-2T	1514	114	7.5%
P2AVV-2T	1544	150	9.7%
P3AVV-1T	1541	104	6.7%
P4AVV-NT	1676	133	7.9%
P5AVV-2T	1487	118	7.9%

) varied between 6.7% (P3AVV1T) and 9.7% (P2AVV27), considering the ratio between the number of chambers with the presence of parasitoids and the total number of chambers.

3.3. Identification and Population Dynamics of Native Parasitoids

The weekly evolution of native parasitoid emergences from spring galls in 2018 (Figure 7) shows that the adult emergence period ran from 16 May to 22 August 2018, with two peaks, the first on 6 June, and the second, although less pronounced, between the weeks of 4 and 11 July 2018.

For the set of five chestnut orchards submitted to different treatments (releases) of T. sinensis, 430 specimens of parasitoids were identified. This identification revealed the existence of eleven species (

Table 6. Identification of native parasitoids in orchards located in Arcos de Valdevez (AVV) and Ponte de Lima (PTL), with one (1T), two (2T) or no treatment (NT) of T. sinensis in 2018.

Parasitoid Native Species	Chestnut Orchards
	P1AVV-2T	P2AVV-2T	P3AVV1T	P4AVV-NT	P5PTL-2T
Eupelmus azureos (Ratzeburg, 1844)	A	A	P	P	P
Eupelmus uruzonus (Dalman, 1820)	P	P	P	P	P
Eurytoma brunnivientris (Ratzeburg, 1852)	P	P	P	P	P
Eurytoma pistaciae (Rondani, 1877)	A	A	A	P	A
Megastigmus dorsalis (Fabricius, 1798)	P	P	P	P	P
Mesopolobus sp. (Westwood, 1833)	P	P	P	P	P
Ormyrus pomaceus (Geoffroy, 1785)	P	P	P	P	P
Sycophila biguttata (Swederus, 1795)	A	P	A	P	A
Sycophila iracemae (Nieves Aldrey, 1984)	P	P	P	P	P
Sycophila variegatta (Curtis, 1831)	P	P	P	P	P
Torymus flavipes (Walker, 1833)	P	P	P	P	P
No. of species	8	8	9	11	9

P—present; A—absent.

), belonging to seven genera and five families: Eupelmidae, Pteromalidae, Ormyridae, Eurytomidae and Torymidae (

Table 7. Systematic position of parasitoid species identified in chestnut orchards located in Arcos de Valdevez and Ponte de Lima in 2018.

Species	Family
E. azureos	Eupelmidae
E. urozonus
Mesopolobus sp.	Pteromalidae
O. pomaceus	Ormyridae
E. brunnivientris	Eurytomidae
E. pistaciae
S. biguttata
S. iracemae
S. variegatta
M. dorsalis	Torymidae
T. flavipes

). All families belonged to the superfamily Chalcidoidea.

3.4. Abundance and Specific Frequency of Native Parasitoids

The most abundant species were S. iracemae, T. flavipes, Mesopolus sp., O. pomaceus and S. variegada, with abundances of 189, 57, 44, 39 and 34, respectively. All these species were found in all chestnut orchards, so they presented relative frequency values of 100%. Species E. azureos was identified in three orchards (P3AVV1T, P4AVVNT and P5PTL2T), and the species S. biguttata was identified only in two orchards (P2AVV2T and P4AVVNT), presenting values of abundance and relative frequency of 60% and 5%, respectively. In turn, the least abundant and least frequent species was E. pistaciae, with values of 2% and 20%, respectively (

Table 8. Abundance and relative frequency of native species of parasitoids identified in 2018 in chestnut orchards located in Arcos de Valdevez and Ponte de Lima, with different treatments of T. sinensis.

Parasitoid Native Species	No. of Insects	M	%
E. azureos	5	3	60%
E. uruzonus	14	5	100%
E. brunnivientris	17	5	100%
E. pistaciae	2	1	20%
M. dorsalis	24	5	100%
Mesopolobus sp.	44	5	100%
O. pomaceus	39	5	100%
S. biguttata	4	2	40%
S. iracemae	190	5	100%
S. variegatta	34	5	100%
T. flavipes	57	5	100%

No.—number of individuals; M—abundance (number of counts in which each species appeared); %—relative frequency (frequency of appearance in total samples).

), present only in the P4AVVNT chestnut trees.

Table 9. Number of adults (NoA) of native parasitoids emerging in orchards in 2018, and the Simpson Diversity Index (SDI) calculated for the five orchards of Minho (P1 to P5), located in Arcos de Valdevez (AVV) and Ponte de Lima (PTL), with one (1T), two (2T) or no treatment (NT) of T. sinensis in 2018.

Native Species	P1AVV-2T		P2AVV-2T		P3AVV-1T		P4AVV-NT		P5PTL-2T
	NoA	SDI	NoA	SDI	NoA	SDI	NoA	SDI	NoA	SDI
Eupelmus azureos	0	0.0000	0	0.0000	1	0.0000	2	0.0003	2	0.0003
Eupelmus uruzonus	3	0.0019	5	0.0028	1	0.0000	2	0.0003	3	0.0008
Eurytoma brunnivientris	1	0.0000	2	0.0003	7	0.0031	6	0.0045	1	0.0000
Eurytoma pistacina	0	0.0000	0	0.0000	0	0.0000	2	0.0003	0	0.0000
Megastigmus dorsalis	3	0.0019	4	0.0017	4	0.0009	5	0.0030	8	0.0072
Mesopolobus sp.	9	0.0226	4	0.0017	5	0.0015	10	0.0136	16	0.0306
Ormyrus pomaceus	1	0.0000	3	0.0008	20	0.0280	7	0.0063	8	0.0072
Sycophila biguttata	0	0.0000	3	0.0008	0	0.0000	1	0.0000	0	0.0000
Sycophila iracemae	18	0.0959	45	0.2773	63	0.2878	34	0.1689	29	0.1037
Sycophila variegatta	6	0.0094	9	0.0101	9	0.0053	6	0.0045	5	0.0026
Torymus flavipes	16	0.0752	10	0.0126	7	0.0031	7	0.0063	17	0.0347
Total	57	0.2068	85	0.3081	117	0.3296	82	0.208	89	0.187
SDI	0.793		0.692		0.670		0.792		0.813

shows the number of species and the absolute values of the species of native parasitoids identified in the orchards. It is possible to verify that the highest number of species occurred in the P4AVVNT orchard of Arcos de Valdevez (AVV), in which there were no releases of T. sinensis (NT). However, the global population of parasitoids was higher in the orchard where there was only one release of T. sinensis (1T), also located in Arcos de Valdevez (P3AVV1T). The assessment of biological diversity relative to native auxiliary fauna is represented by calculating the Simpson Diversity Index. The diversity of native parasitoids ranged between 0.670 (P3AVV-1T) and 0.813 (P5PTL-2T).

The relative abundance of native species of parasitoids in 2018, shown in Figure 8, evidenced the dominance of S. iracemae, regardless of the number of T. sinensis releases carried out in the different orchards.

3.5. Establishment of T. sinensis Populations in Minho

T. sinensis was present in chestnut galls throughout the year and overwintered in dead galls. Adults emerged in 2019 between February and March (Figure 9). The establishment of T. sinensis populations in the orchards with two parasitoid releases in 2016 and 2017 was assessed by recording the emergence of T. sinensis adults in 2019 from summer galls harvested in 2018. Figure 9 shows evidence that the highest number of hatched T. sinensis insects was recorded in the orchards of Arcos de Valdevez located in Rio Cabrão (P1) and Távora (P2). The parasitism rate varied between 0.0205% in the orchards of Arcos de Valdevez and Salvador not treated with T. sinensis (P4) and 0.5044 % in the orchard of Rio Cabrão (P1), with two treatments of T. sinensis.

4. Discussion

4.1. Bioecology of D. kuriphilus

The emergence of D. kuriphilus was recorded from 1 June to 10 August 2018. The peak of emergences occurred on 29 June in the P2AVV2T orchard (Arcos de Valdevez), and on 13 July in the P5PTL2T orchard in Ponte de Lima. In the remaining orchards, peaks of emergence were recorded in the interval between the above dates. Higher populations were observed until 20 June, from which date the populations began to decline. These populations evaluated by the dissection of winter buds carried out in five chestnut orchards located in Arcos de Valdevez and Ponte de Lima in 2018 confirm the high intensity of attack on the chestnut trees in all the orchards studied in the Minho region, further confirming the rapid spread of the chestnut gall wasp in Portugal, described since the pest appeared in 2014 and reported by the DGAV [35]. The percentage of buds infested by D. kuriphilus was high in all places studied, varying between 67% and 80%, regardless of the number of T. sinensis releases performed and the chestnut cultivars present in each orchard, as already mentioned by Lopes [36]. These values classify the attack intensity at level 3, the second most severe on the scale from 0 to 4 defined by the DGAV [37]. These results are in accordance with other authors [20,21,30,38,39], that have also conducted studies in Portugal. Recent research in Portugal, in different chestnut production conditions, had identical results. These authors developed a mathematical model that considered the separate evolution of D. kuriphilus and of T. sinensis, over a single season and concluded that for biological control to be efficient, it is necessary to implement, in each chestnut-producing region, a collective strategy based on the annual monitoring of infestation levels, as presented in this work.

4.2. Quantification of Parasitoids and Rate of Natural Parasitism Associated with D. kuriphilus

The natural parasitism associated with native parasitoid species of D. kuriphilus was confirmed in this work, recording values between 34% and 51% of parasitized chestnut galls. These values agree with another study by Santos et al. (2018), which was carried out between 2015 and 2017 [30]. According to these authors, the values of natural parasitism obtained in the chestnut orchards in Minho were consistently higher than those obtained in Trancoso, probably due to the milder weather conditions and the greater wealth of vegetation on the edges of the chestnut tree orchards. The results of the present study suggest the critical role those native parasitoids can play in the control of D. kuriphilus, providing greater protection for the chestnut crop against this serious chestnut pest. Regarding the number of chambers of D. kuriphilus recorded, and given the great laying capacity of the species, the effective natural parasitism rate was between 6.7% and 9.7%. These results confirm that, although native parasitoids play an essential role, their action was insufficient to contain the population of D. kuriphilus in the orchards studied. Native parasitoids may play an important role in biological control. They may support or hinder the success of introduced T. sinensis released for pest control purposes. In this work, the results of a one-year survey (2018) of D. kuriphilus populations and on parasitism rates by native indigenous parasitoids in five Portuguese chestnut orchards in the Minho region are given. A total of 11 species of native parasitoids were recorded, accounting for apparent differences in parasitism rates. This variability in parasitism observed in the different orchards may be related to climatic conditions, to the chestnut cultivars, to the number of T. sinensis releases and to the presence of areas with oaks around the orchards. In Italy, all native parasitoids recruited to ACGW are species associated with oak gall wasps. The adaptation of native parasitoids to the ACGW in our study is in accordance with other studies developed in Portugal [30,38], and with other researchers [40].

4.3. Identification and Population Dynamics of Native Parasitoids

In this study, eleven native parasitoids were identified in 2018 belonging to the families Eupelmidae, Eurytomidae, Ormyridae, Pteromalidae and Torymidae, all belonging to the superfamily Chalcidoidea. A study carried out in the northern region by Santos et al. identified nine species [30]. The results obtained in this study are also in agreement with others performed in countries such as Italy, Croatia, Japan and Korea, particularly concerning the identified insect families, all belonging to the superfamily Chalcidoidea [19,41,42,43]. These results agree with those of Bosio et al. [44], who considered them to be generalist parasitoids that parasitize gall-inducing insects on oaks and other hardwoods. The results referring to the emergence of parasitoids of different genera and species proved the richness and abundance of native species present in the five orchards studied in Minho, supported by the Simpson’s diversity index values obtained in all of the orchards. Although there were different population dynamics among the 11 identified species, adult insects emerged between 16 May and 22 August 2019, with a maximum peak on 6 June. The particularity of the presence of an adjacent area of oak trees is highlighted, which seems to have contributed to the diversity of the native parasitoids identified. In fact, in this chestnut orchard, the existence of a higher number of species was confirmed, suggesting the existence of the beneficial effects of the installation of oak species on the edges of chestnut orchards, as recommended by the Direção Regional de Agricultura e Pescas do Centro (DRAPC) in 2020, as a measure to promote biological diversity and the development of autochthonous parasitoids. Hence, it is essential to carry out specific studies to validate the existence of higher rates of parasitism in these circumstances.

5. Conclusions

D. kuriphilus population was high in all the orchards studied, independent of the number of T. sinensis releases performed and the chestnut cultivars present in each orchard. The exotic parasitoid T. sinensis was also present in all of the five orchards studied, and it was adapted to the new environments, providing good prospects for adapting to the new chestnut ecosystems in the Minho region, as demonstrated by other authors. It is concluded that the highest parasitism rate of T. sinensis (0.5044 %) occurred in the Souto de Arcos de Valdevez—Rio Cabrão (P1), where two releases of T. sinensis were carried out in 2016 and 2017. The lowest parasitism rate of 0.0205% was recorded in the orchard where there were no parasitoid releases (NT), located in Arcos de Valdevez—Salvador (P4). It is essential to consider that the origin of T. sinensis in the untreated chestnut orchard can be justified because, in another nearby orchard (700 m) not included in this study, a release of this parasitoid was carried out in 2017. The results obtained in this work suggest that the exotic parasitoid T. sinensis is adapting to the different ecosystems of the Minho region, and its action in the biological control of D. kuriphilus is expected to be confirmed over a more extended period in future works.