SJAR Spanish Journal of Agricultural Research Span. j. agric. res. 2171-9292 1695-971X Consejo Superior de Investigaciones Científicas España 10.5424/sjar/2024224-20852 sjar/2024224-20852 RESEARCH ARTICLE Phenology, biological and cultural control of the new almond pest Eurytoma amygdali (Hymenoptera, Eurytomidae) in Spain Fenología, control biológico y cultural de la nueva plaga del almendro, Eurytoma amygdali (Hymenoptera, Eurytomidae) en España Phenology and control measures of almond wasp https://orcid.org/0000-0003-3793-3851 Rodrigo Eugenia Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Supervision Visualization Writing – original draft Writing – review & editing 1 https://orcid.org/0000-0003-3203-0127 Benedito Vicent Investigation Writing – review & editing 2 https://orcid.org/0000-0001-8905-6083 Xamaní Pilar Data curation Methodology 3 https://orcid.org/0000-0002-1803-5581 Baena Manuel Investigation 4 https://orcid.org/0000-0003-1545-6011 Ferragut Francisco Formal analysis Investigation Writing – original draft Writing – review & editing 1 Instituto Agroforestal del Mediterráneo, Universitat Politècnica de Valencia Camino de Vera s/n 46022 Valencia Spain Instituto de Ingeniería del Agua y Medio Ambiente Universitat Politècnica de València Camino de Vera s/n 46022 Valencia Spain Departamento Ecosistemas Agroforestales, Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural, Universitat Politècnica de València Camino de Vera s/n 46022 Valencia Spain 4Departamento de Biología y Geología, I.E.S. Alhaken II, Manuel Fuentes “Bocanegra” 14005 Córdoba Spain * Correspondence should be addressed to Eugenia Rodrigo: erodrigo@eaf.upv.es 30 12 2024 30 12 2024 22 4 20852 Received 15 10 2023 Accepted 25 07 2024 Published 28 11 2024 © 2024 CSIC 2024 CSIC https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License. Abstract <italic toggle="yes">Aim of study:</italic>:

Eurytoma amygdali Enderlein (Hymenoptera, Eurytomidae), the almond wasp, is a new pest detected in Spain. Although the insect is present in Europe, in Spain no damage was detected until 2016, increasing substantially in the following years. The phenology of almond wasp and the natural enemies complex were investigated and the results of the parasitism rate and cultural control measures were analyzed.

 <italic toggle="yes">Area of study</italic>:

Five Spanish provinces, important regions for almond production where E. amygdali causes serious harvest losses.

 <italic toggle="yes">Material and methods</italic>:

Samples of different almond varieties were taken from ecological almond plots in the Spanish provinces of Valencia, Alicante, Albacete, Murcia and Cuenca.

 <italic toggle="yes">Main results</italic>:

The first almond wasp eggs were observed at the beginning of May and completely developed larvae, early in July. Wasp flight occurred from mid-March to late May. Two natural enemies were associated with the pest, Pyemotes amygdali Çobanoğlu & Doğanlar (Acari, Pyemotidae), and Opilo domesticus (Sturm) (Coleoptera, Cleridae). Pyemotes amygdali parasitized wasp larvae, adult females and males, and Opilio domesticus fed on wasp larvae. O. domesticus was very scarce, unlike P. amygdali. In the plots with parasitized wasps, the parasitism rate ranged from 0.6% to 44% for P. amygdali.

Our results suggest that sanitation measure was an important cultural pest control practice, because in the plantations where mummified almonds were removed, 96% of edible almonds were harvested, compared to 18% if mummified almonds were not removed from trees.

 <italic toggle="yes">Research highlights</italic>:

Cultural control could be an important measure to control the almond wasp, because it allows biological conservation control, by preserving natural enemies present in the environment and avoid the negative effect of insecticides.

 Resumen <italic toggle="yes">Objetivo del estudio</italic>:

Eurytoma amygdali Enderlein (Hymenoptera, Eurytomidae), la avispilla del almendro, es una nueva plaga detectada en España. Aunque el insecto está presente en Europa, en España no se detectaron daños hasta 2016, aumentando de forma sustancial en los años siguientes. En este estudio se ha investigado la fenología de la avispilla del almendro y el complejo de enemigos naturales, así como la tasa de parasitismo y el efecto de medidas culturales de control.

 <italic toggle="yes">Area de estudio</italic>:

Cinco provincias españolas que son importantes zonas productoras de almendras y donde E. amygdali ha provocado graves pérdidas de cosecha.

 <italic toggle="yes">Materiales y métodos</italic>:

Se tomaron muestras en parcelas de cultivo de almendro ecológico en las provincias españolas de Valencia, Alicante, Albacete, Murcia y Cuenca.

 <italic toggle="yes">Principales resultados</italic>:

Se observaron los primeros huevos de la avispilla a principios de mayo y larvas completamente desarolladas a principios de julio. El vuelo de la avispilla tuvo lugar desde mediados de marzo a finales de mayo. Se han encontrado dos enemigos naturales asociados a la plaga, Pyemotes amygdali Çobanoğlu & Doğanlar (Acari, Pyemotidae), y Opilio domesticus (Sturm) (Coleoptera, Cleridae). Pyemotes amygdali parasitó larvas, hembras y machos adultos de avispilla, y O. domesticus se alimentó de larvas de avispilla. Opilio domesticus fue muy escaso a diferencia de P. amygdali. En las parcelas con avispilla parasitada, la tasa de parasitismo por P. amygdali osciló entre 0.6% y 44%.

Nuestros resultados sugieren que el saneamiento fue un importante método de control cultural de la avispilla, ya que en las parcelas donde las almendras momificadas se eliminaron, el 96% de las almendras cosechadas fueron almendras comestibles, frente al 18% de almendras comestibles que se recogieron en las parcelas donde las almendras momificadas no se eliminaron.

 <italic toggle="yes">Aspectos destacados de la investigación</italic>:

El control cultural puede ser una importante medida de control de la avispilla del almendro ya que permite el control biológico por conservación, respetando a los enemigos naturales presentes en el medio ambiente y evitando el efecto negativo de los insecticidas.

 almond wasp biological cycle natural enemies sanitation harvest avispilla del almendro ciclo biológico enemigos naturales saneamiento cosecha Conselleria de Agricultura, Medio Ambiente, Cambio Climático y Desarrollo Rural (Generalitat Valenciana) S8456000 Introduction

The genus Eurytoma Illiger (Hymenoptera, Eurytomidae) displays a wide range of hosts and biology, with eight Palearctic species associated with stone fruit. Of these species, the almond wasp Eurytoma amygdali Enderlein (Hymenoptera, Eurytomidae) attacks only almond trees and is considered one of the most important pests of this crop. The insect is widely distributed throughout almond-growing areas in SE Europe, and has been recently detected in Romania, some countries of the former Soviet Union and in Middle Eastern countries like Syria, Turkey, Iran, Lebanon and Cyprus (Zerova & Furson, 1991; Noyes, 2019; Cioacă et al., 2022).

Previous studies conducted on E. amygdali biology indicate that it has one generation per year (Mentjelos & Atjemis, 1970; Plaut 1971b; 1972; Talhouk, 1977). In the Mediterranean basin where the pest was studied, adult flight and reproduction begin in March-April and end late in May (Plaut, 1971b; Talhouk, 1977; Katsoyannos et al., 1992). Females lay eggs in the endosperm of seeds when the endocarp is still soft. They can lay up to 7 eggs per seed, but only one larva survives (Mentjelos & Atjemis, 1970; Plaut, 1972; Talhouk, 1977). The larva enters diapause in June-July when it has consumed almost all the seed. The majority of larvae break their diapause after roughly 7 to 8 months, although a proportion of larvae can remain in diapause for 1, 2 years or more. Larvae pupate in January-February and wasps emerge in March-April (Mentjelos & Atjemis, 1970; Talhouk, 1977; Cakar, 1980). From June, infested fruits shrivel and remain firmly attached to the trees for a long time (Plaut, 1971b; 1972; Zerova & Fursov, 1991).

The natural enemy complex of E. amygdali is composed of the parasitoid hymenoptera Adontomerus amygdali Boucek, (Hymenoptera, Torymidae), Aprostocetus bucculentus (Kostjukov) (Hymenoptera, Eulophidae), Gugolzia bademia Doğanlar (Hymenoptera, Pteromalidae) Exeristes roborator (Fabricius) (Hymenoptera, Ichneumonidae), parasitoid mites Pyemotes amygdali Çobanoğlu and Doğanlar (Acari, Pyemotidae) and predatory beetles, Thanasimus spp Latreille (Coleoptera, Cleridae) (Çobanoğlu and Doğanlar, 2006; Doğanlar et al., 2006; Schäckermann et al., 2015; Abdul-Rassoul & Mohammed, 2017; Tolga & Yoldas, 2018).

Eurytoma amygdali is considered one of the most important almond pests, especially in the SE Mediterranean (OEPP/EPPO, 2004), because it can cause up to 80% harvest loss (Mentjelos & Atjemis, 1970; Talhouk, 1977; Cakar, 1980). Sanitation is used to control almond wasp and consists of collecting and removing infested fruit containing diapausing larvae from trees. Chemical control with insecticides is also applied. Systemic insecticides are applied after females oviposit inside fruit to kill any neonate larvae (Mentjelos & Atjemis, 1970; Plaut, 1971a; Katsoyannos et al., 1992). Tzanakakis et al. (1997), proposed using insecticides to control adult populations before oviposition occurs. The use of resistant cultivars has been suggested to control this pest (Katsoyannos et al., 1992) because some cultivars showed different infestation rates (Tzanakakis et al., 1997; Ibrahim et al., 2008; Saeidi, 2021).

Although the presence of E. amygdali is known in several countries in the Mediterranean Basin such as France, Greece and Turkey (Zerova & Fursov, 1991; Noyes, 2019), its presence and any damage it may cause have not been confirmed in Spain. Since 2016, 30% harvest losses have been observed, which increased to 70% the following year. Given this situation, between 2018 and 2019, E. amygdali was declared a pest in some Spanish Autonomous Communities like the Valencian Community (DOGV 2018. Resolution of 23 February 2018), the Murcia Region (BORM 2018 Order of 26 December 2018) and Castilla-La Mancha (DOCM 2019 Order 33/2019 of 25 February). These documents indicate that farmers are obliged to take control measures against this insect. As no almond wasp studies are available in Spain, the objectives of the present work were to study the phenology of E. amygdali, investigate the natural enemies complex and analyze parasitism rate and the effect of sanitation on the level of almond wasp infestation.

 Material and methods Study sites

In 2018, two organic almond tree plots, managed in the same way, were chosen for having presented severe almond wasp damage the year before (2017). Both of these plots are located in the Valencian Community (eastern Spain): one in the town of Biar (Alicante) (38° 37’ 14.74’’ N – 0° 47’ 55.80’’ W) and the other in Los Isidros district (Requena, Valencia) (39° 25’ 52.63’’ N – 1° 16’ 17.96’’ W). The trees in the two plots were the same age and had a similar crown size. In the Biar plot, almonds were of the “Guara” variety, while the variety in the Los Isidros plot was “Largueta”.

In 2019, it was decided to intensify the study, because no natural enemies were found from the 2018 harvest. Collaboration from farmers in the areas affected by almond wasp was requested so they would send us mummified almonds from the 2019 harvest. Samples were taken from ecological almond plots in the Spanish provinces of Valencia, Alicante, Albacete, Murcia and Cuenca. Table 1 shows the plots and the different almond varieties present in fields if they were known.

Spanish provinces where mummified fruits were collected from the 2019 harvest, geographical indications , total number of almonds opened and almond tree variety if known.
Province Town/District Lot a Plot a almonds b Variety/Varieties
Valencia c Venta Moro 14 41 47 Largueta, Guara, Vayro
14 165 119 Largueta
16 101 46 varied
61 46 139 Ferraduel, Largueta roja, Largueta, Constantí, Asperilla
Requena 130 389 118 Largueta, Largueta oja
120 435 149 Largueta, Largueta roja, Ferraduel
120 431 103 Asperilla, Largueta
128 46 30 Constantí, Vayro, Largueta roja, Largueta
Los Isidros 287 Unknown
Alicante c Villena 49 109 171 Guara
Biar 8 100 230 Guara
Castalla 22 97 103 Unknown
Albacete d Fuentealbilla 38 129 93 Unknown
Casas Ibáñez 29 36 100 Unknown
Hellín 31 196 169 Filippo Ceo
33 10 223 Guara y Filippo Ceo
36 3 31 Filippo Ceo y Guara
48 230 752 Guara
28 112 231 Filippo Ceo
31 169 134 Guara
Mahora 40 33 32 Unknown
Almansa 523 5005 112 Guara
523 5001 147 Guara
Tobarra 13 75 191 Guara and Ferragnes
Albacete 138 5086 84 Guara
137 27 82 Guara
Madrigueras 311 Unknown
55 Lauranne
Casas de Ves 152 Unknown
Cenizate 1 10027 101 Unknown
Golosalvo 13 208 185 Unknown
Cuenca d Granja Iniesta 15 65 100 Lauranne
Murcia e Yecla 99 Unknown
Jumilla 106 Unknown

Data from SIGPAC (lot and plot): the Geographical Information System of Spanish Agricultural Plots (source: SIGPAC, 2023) (https://sigpac.mapama.gob.es/fega/visor/).

Total number of almonds opened.

The province belongs to the Autonomous Communities of: Valenciac, Castilla-La Manchad and Murciae

Phenology and natural enemies of <italic toggle="yes">E. amygdali</italic> The 2018 trial. Phenology of <italic toggle="yes">E. amygdali</italic>

To determine the flight, egg and larval periods, the “Los Isidros and Biar” plots were periodically sampled from March to November 2018 (fortnightly from May to July, and monthly until November). On each sampling date, in the field, the growth and ripening of the fruits were monitored and the presence of adults in flight was reported. To detect the presence of flying almond wasps, on each sampling date, in the afternoon (from 12:00 to 14:00) the entire plot was walked through by passing along rows of almond trees, looking at the tree canopy, at eye level or slightly higher. Wasp presence was noted, but not the number of wasps observed. During each sampling, 100 fruits were picked at random and taken to the Crop Protection Laboratory at the School of Agricultural Engineering and Environment (ETSEAMN, Universitat Politècnica de València (UPV)). In the laboratory, almond fruits were opened up and the presence of eggs or larvae, the number of larvae per seed, the length of the larvae, and the location on the almond tissue (nucellus, endosperm, and embryo) were recorded.

To determine the emergence period and sex ratio, almonds were harvested. Harvest took place on 25 July 2018 in Biar and on 30 August 2018 in Los Isidros and were placed inside cardboard boxes measuring 15 x 15 x 31 cm. Two holes were made in rearing boxes, one covered with gauze to allow aeration and the other with a transparent glass tube to allow light to enter. Specifically, 90 fruits per tree from Los Isidros (a total of 56 trees) were placed inside the boxes. In Biar, all the collected fruits (about 5,000 from 80 trees) were kept into the boxes. A maximum of 60 almonds was placed in each box, and boxes were stored in a chamber under natural conditions with natural light, temperature between 17°C and 28°C and relative humidity between 70% and 80%. The boxes with almonds remained inside the chamber from the end of July/August 2018 to June 2019. From March 2019, glass tubes were examined every 2 days and the numbers of emerged males and females were recorded. When no wasp was observed inside glass tubes, all the boxes were opened to report natural enemies.

 The 2019 trial. Natural enemies of <italic toggle="yes">E. amygdali</italic>

The mummified fruits collected in 2019 from five Spanish provinces were taken to the ETSEAMN (Table 1) and placed inside cardboard boxes (31 x 15 x 15 cm) in a chamber under natural conditions (natural light, temperature between 17°C and 28°C and relative humidity between 70% and 80%). As previously indicated, cardboard box interiors were dark with only an opening for airing and another hole covered with a transparent glass tube. These fruits were left inside the chamber from August 2019 to June 2020. From June, fruits were opened and the number of fruits with a wasp exit hole, the fruits with dead wasps inside, the fruits with natural enemies and those that failed for unknown reasons were reported. Identification of natural enemies was carried out by specialist taxonomists based on morphological characters using identification keys (Gerstmeier, 1998; Çobanoğlu & Doğanlar, 2006; Bahillo de la Puebla et al., 2021).

 Parasitism rate

The parasitism rate per plot was calculated as the percentage of almonds with a parasitized wasp in relation to almonds with an exit hole, almonds with a dead wasp inside and almonds with a parasitized wasp.

almonds with a parasitized wasp ( almonds with an exit hole + almonds with a dead wasp inside + almonds with a parasitized wasp ) * 100 Infestation level The 2018 trial

At harvest time (25 July 2018 in Biar and 30 August 2018 in Los Isidros), 10 fruits were collected per tree in Los Isidros. In Biar the number of collected fruits was half of the fruit of each tree. All the fruits were taken to the laboratory, where they were opened and it was noted whether they were healthy or unhealthy seeds. Of the unhealthy seeds, it was also noted whether they had almond wasps inside or failed for other reasons. It is noteworthy that after the 2017 harvest, all the fruits left on the almond trees in Los Isidros plot were removed, as well as all those from the surrounding plots.

 The 2019 trial

The level of infestation in Los Isidros plot was very low in 2018, so it was decided to repeat the experiment in 2019 only in Biar on the same plot from the previous year and where all the almond fruits were manually collected. In 2019 in Biar, almonds were mechanically collected using a trunk shaker with an umbrella hitched to an orchard tractor. After harvesting, on 26 August 2019, all fruits that did not fall after the application of the trunk shaker (58,09 ± 6,01 almonds) were collected manually, in the same way as the previous year. They were placed inside separate bags and taken to the laboratory, where they were opened and examined, following the same criteria applied the year before.

 Results Phenology and natural enemies of <italic toggle="yes">E. amygdali</italic> The 2018 trial. Phenology of <italic toggle="yes">E. amygdali</italic>

During field sampling, in Biar, the first adults were detected on 17 April 2018 and the last flying wasps were seen on 15 May 2018. In Los Isidros, flight started from 24 April 2018 and ended on 11 May 2018.

Inside the kernels, the first eggs were observed at the beginning of May (6 May in Biar). When seed tissue was still soft and transparent, up to four larvae were found per seed (15 May 2018 in Biar and Los Isidros). When the seed reaches its final size, it begins to dry and harden, the number of larvae decreased to one larva/kernel, sometimes with two larvae/kernel, and very rarely with three larvae/kernel. If one fruit housed two kernels, there was one larva in each kernel.

Larva length varied over time (Figure 1). By mid-May, larvae were 0.46 ± 0.015 mm long and were distributed all over the seed tissue (nucellus, endosperm and embryo), but were not easily detected, except for well-chitinised mandibles. At the end of June and the beginning of July, larval size increased up to approximately 8 mm (the longest size) in both studied areas. In July, the completely developed larva had consumed all the seed content and remained inside the seed coat.

Mean size (± Standard Error) of <italic toggle="yes">Eurytoma amygdali</italic> larvae on the different sampling dates in 2018 for the two study plots (Los Isidros and Biar) in the Valencian Commmunity (eastern Spain).

Figure 2 shows the emergence of adult almond wasps from the harvested almond fruits which kept in a chamber under natural conditions. Almond wasps emerged abundantly between 14 and 18 March 2019, with males being more abundant. Flight ended 2.5 months later (on 29 May 2019). In total, 272 females and 192 males emerged from almonds, and no predators or parasitoids were found.

Emergence of adult males/females <italic toggle="yes">Eurytoma amygdali</italic> wasps from the mummified fruits placed inside a climate chamber from the end of July 2018 to June 2019. The 2019 trial. Natural enemies of <italic toggle="yes">E. amygdali</italic>

From the 2019 harvest almonds left inside the chamber, 5,032 almonds were removed from boxes and opened. Of all these almonds, 46.2% had an exit opening and the kernel had been consumed, 18.9% had a dead wasp inside, 21.2% failed for unknown reasons, 11.4% contained a live larva in diapause, 1.9% had a mite-parasitized wasp inside, 0.16% contained one larva, adult or exuvial clerid Coleoptera, 0.2% had larval and adult Cerambycidae, and 0.04% had adult Buprestidae.

Four arthropod species, which differed from almond wasp, were detected and identified. Opilo domesticus (Sturm) (Coleoptera, Cleridae) (Figures S1 and S2 [suppl.]) and Pyemotes amygdali Çobanoğlu and Doğanlar (Acari, Pyemotidae) (Figure S3 [suppl.]) were identified as predator and parasitoid respectively. Trichoferus fasciculatus (Faldermann) (Coleoptera, Cerambycidae) and Anthaxia (Haplanthaxia) millefolii (Fabricius), are not natural enemies and were eating the almond shell.

Pyemotes amygdali was observed in all the studied provinces, 41% of the plots contained parasitized wasps, and in these plots, the percentage of parasitism ranged from 0.26% to 44.44% (Table 2).

Mean percentage of parasitism due to <italic toggle="yes">Pyemotes amygdali</italic> in the different plots of the same sampled town/district calculated from mummified almonds for the 2019 harvest.
Province Town/District Percentage of parasitism
Valencia Venta del Moro 0.82
Requena 8.3
Los Isidros 0.76
Alicante Villena 0
Biar 0
Castalla 3.53
Albacete Fuentealbilla 4.29
Casas Ibáñez 44.44
Hellín 0.26
Mahora 0
Almansa 0
Tobarra 0
Albacete 0
Madrigueras 13.235
Casas de Ves 0
Cenizate 0
Golosalvo 0
Murcia Yecla 1.33
Jumilla 3.08
Cuenca Graja de Iniesta 16.42

Only four O. domesticus specimens were found. Clerids were inside almonds in August 2019 when almonds were harvested from Requena, Almansa and Venta del Moro plots. We found an adult insect when opening a mummified almond in July 2020. The other specimens were larvae, which were reared individually in Petri dishes with alive E. amygdali larvae. The petri dishes with clerid larvae remained in the chamber under natural conditions until they became adults in July 2021, approximately 2 years later.

 Infestation level

In 2018, 560 almonds were opened in Los Isidros, of which 6.8% had a live wasp inside and 74% were edible. In Biar, 4,791 almonds were opened, of which 71.7% contained a live wasp, and only 17.5% were edible.

In 2019, 8,842 almonds were collected and opened in Biar, of which 19.7% contained a live wasp, and 71% were edible almonds.

In Biar, the plot was the same in both study years. These differences in harvest for both years are shown in Table 3, which also indicates that the percentage of edible almonds from the 2019 harvest was 95.8%, compared to only 17.5% the previous year.

Percentage of fruits infested by almond wasp and edible nuts for the 2018 and 2019 harvests at Biar.
Year Number of harvested almond trees Total almond fruits harvested Percentage of fruits containing a live wasp Percentage of edible nuts
2018 75 4,791 71.1 17.5
2019 152 61,162 2.9 95.8
Discussion

In our studied area, E. amygdali showed an annual generation in accordance with previous studies (Mentjelos and Atjemis, 1970; Plaut, 1971b; 1972). The first eggs inside seeds were detected at the beginning of May. Newly hatched larvae measured around 0.46 mm long and were observed in the three seed parts (nucellus, endosperm and embryo), which agrees with Mentjelos & Atjemis (1970) and with Plaut (1972).

According to our results, larva grew and fed on seed content from May to the end of June and beginning of July, when maximum size was reached., Although initially up to three larvae per kernel were detected, finally only one per kernel developed, exactly as Plaut (1972) and Talhouk (1977) reported.

The larva enters diapause when it has consumed all the seed content (Plaut, 1972). The effect of temperature on diapause termination has been recently studied (Tzanakakis & Veerman, 1994; Margaritopoulos & Tzanakakis, 2006). This process begins at the end of May in Israel, which is approximately one month earlier than in our studied area. Similar to Talhouk (1977), at the beginning of July the larva was fully developed, had consumed the seed contents and remained inside the intact seed coat.

While making direct field observations in 2018, we observed adult wasps flying from mid-April to mid-May. In 2019, from the infested almonds placed inside boxes, emergence of adults peaked at mid-March, and ended at the end of May, a period that lasted approximately two months. According to our results, males were emerged earlier than females as a result of the protandry described for this species in the previous studies (Plaut, 1971b; Talhouk, 1977; Katsoyannos et al., 1992; Yiğit et al., 2020).

According to our results, 11% of fruits contained a live larva in diapause. The percentages of larvae in diapause reported by previous studies vary, with 42% for Mentjelos and Atjemis (1970), 23% for Talhouk (1977) or 1% for Plaut (1972). For the three years that our study lasted (from 2018 to 2020), we observed adult almond wasps emerging two years after oviposition. Our results are consistent with other studies showing that the greater majority of the larvae break their diapause after seven to eight months while only a small percentage can remain in diapause for up to three winters (Mentjelos & Atjemis, 1970; Talhouk, 1977; Cakar, 1980).

During the first study year (2018), we did not find that any natural enemies emerged from infested almonds. We intensified the study in 2019 by extending the sampling area to five Spanish provinces affected by the almond wasp. Of the approximately 5,000 opened almonds in June-July 2020, most had been consumed by almond wasp (65.1%) or contained a larva in diapause (11.4%), 21.4% were failed almonds or attacked by other insects, and natural enemies were detected in 2.1% of almonds.

Two natural enemies of almond wasps were found: s P. amygdali and the predatory clerid O. domesticus. Pyemotes amygdali is a gregarious ectoparasitoid that was described as a new species in 2006 (Çobanoğlu & Doğanlar, 2006). This ectoparasitoid mite sucks the hemolymph of its host by attacking the intersegmental membrane and paralysing the insect. The female opisthosoma substantially increases in size when eggs develop inside her (phisogastric females), and eggs hatch within the female. This mite, that parasitized the larvae, pupae and adults of E. amygdali have been reported by Çobanoğlu & Doğanlar (2006) and Doğanlar et al. (2006). The study by Doğanlar et al. (2006), was conducted in three provinces in Turkey (Adana, Nizip and Hatay), and in eight localities. Pyemotes amygdali was detected only in three localities of the Hatay province, while we found it in the five Spanish provinces that we sampled. This finding indicates that this mite appears where the almond wasp is present. The presence of large phisogastric females makes these mites clearly visible on the parasitized insect. We found groups of mites parasitizing larvae, male adults and female adults of almond wasp. In the plots with parasitized wasps, the parasitism rate in our study ranged among plots from 0.56% to 44.4%, which coincides with the study by Doğanlar et al. (2006), who obtained parasitism rates between 7.56% and 44.53%. According to Çobanoğlu & Doğanlar (2006), this Pyemotidae species could easily reproduces on the host, and may be a good candidate for biological control of almond wasp.

We found O. domesticus, preying on E. amygdali larvae. According to Bahillo de la Puebla et al. (2021), 37 clerid species are cited in the Iberian Peninsula and Balearic Islands. They present relatively non-specific feeding habits, and both adults and larvae actively prey on xilophagus insects. Adult O. domesticus has been detected in the wood of conifers and frondose species, where it consumes mainly anobiidae and some cerambycidae species (Correa de Barros, 1929; Español, 1959). This insect has been reported to appear on the branches of some Quercus, beech (Fagus sylvatica L.), fig wood (Ficus carica L.), carob (Ceratonia siliqua L.) and cypress (Cupressus L.) species (Bahillo de la Puebla & López-Colón, 2000; Bahillo de la Puebla et al., 2021). Doğanlar et al. (2006) cited Thanasimus spp (Coleoptera, Cleridae) as a predator of E. amygdali. Therefore, it is the first time that O. domesticus is cited to prey on a hymenoptera species and inside an almond fruit. Information about the life cycle of O. domesticus and other species of the same family is scarce. One of the most widely studied genera is Thanasimus, predator of bark beetles (Coleoptera, Curculionidae), which are serious forest pests. Adult T. formicarius lives for 4-10 months and its life cycle completed in 1 year (Gauss, 1954), or 2 years in Scandinavia (Schroeder 1999). Korynetes caeruleus (de Geer 1775), predator of Anobium punctatum (de Geer 1774) (Coleoptera: Ptinidae), takes 2 years to develop from eggs to adults at 21°C and 75% RH, with a 4-month cold period at 40°C (Haustein et al., 2019). Approximately 2 years had elapsed from the time we observed O. domesticus larve until adults emerged, which roughly coincides with previous results.

The methods applied to control almond wasp include cultural measures, which consist in collecting and removing any mummified fruits attached to almond tree branches, and chemical control. Systemic insecticides have been used against recently hatched larvae found inside fruit (Mentjelos & Atjemis, 1970; Plaut 1971a; Katsoyannos et al., 1992), and insecticides applied to the tree canopy against adults (Tzanakakis et al., 1997). In our study area, in almond crops, we can generally state that, to date, not many insecticide applications are carried out. Insecticides were applied in spring to control aphids and copper products to control diseases (leaf curl disease, shot hole disease and red leaf blotch disease). Moreover, in organic almond cultivation, the applied products are non residual contact insecticides; e.g., narrow-range oil. This situation changed because of the damage that the insect was causing and severe crop loss. Between 2018 and 2019, E. amygdali was declared a pest in the Valencian Community and other Spanish Autonomous Communities, and farmers were obliged to take control measures against this insect. With the new situation, farmers carried out weekly insecticide applications in March and April, and have also used more persistent and more toxic active ingredients to date. Presently, the allowed active materials to be applied to almond crops are paraffin oil, lambda-cyhalotrin, pyrethrins (authorised for ecological crops) and spirotetramat (https://www.mapa.gob.es/es/agricultura/temas/sanidad-vegetal/productos-fitosanitarios/fitos.asp).

We do not have any data about the effect of authorised insecticides in Spain against the natural enemies that we found: P. amygdali and O. domesticus. Nevertheless, the secondary effects of these insecticides on a wide range of enemies are known. According to IOBC-WPRS Pesticide Side Effect Database (https://www.iobc-wprs.org/restricted_member/toolbox.cfm), the toxicity level of products lies between 1 (harmless) and 4 (harmful). The most widely used insecticide is lambda-cyhalotrin. This insecticide has a high toxicity level against a wide range of natural enemies. It has toxicity level 4 to Adalia bibunctata (L) (Coleoptera, Coccinellidae), level 3 to Episyrphus balteatus (De Geer) (Diptera, Syrphidae) both natural enemies of aphids, and level 4 to Opius concolor Szepligeti (Hymenoptera, Braconidae), parasitoid of olive fruit fly.

We conclude that sanitation could be an important measure for almond wasp control, as 90% of the mummified almonds contain larvae inside, that will become the population infesting almonds the following year By collecting mummified almonds, the side effects of insecticides (such as lambda-cyhalothrin) are avoided, allowing conservation biological control by P. amygdali and O. domesticus and other natural enemies present in the fields and in the natural environment.

 Supplementary material

(Figures S1, S2 and S3): accompanies the paper on SJAR’s website.

 Acknowledgements

We wish to thank Victoria Beltrán (Estación Experimental Agraria de Elche, Generalitat Valenciana) and Juan Salvador Samper (Mañán Sociedad Cooperativa) for their technical assistance and advice. Juan A. Berenguer and Narciso Martínez Gómez for providing the trial field at Biar and Los Isidros and to the technicians of the Servicio de Producción Ecológica Innovación y Tecnología (Generalitat Valenciana) for insecticide applications. Special thanks to the technicians of agricultural cooperatives and farmers for send us mummified almonds from the 2019 harvest. We want to dedicate this paper to Dr. Rafael Laborda Cenjor for his important contribution and who unfortunately passed away in 2021.

 Competing interests

The authors declare that no competing interests exist.

 Authors’ contributions

Eugenia Rodrigo: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing. Vicent Benedito: Investigation, Writing – review & editing. Pilar Xamaní: Data curation, Methodology. Manuel Baena: Investigation. Francisco Ferragut: Formal analysis, Investigation, Writing – original draft, Writing – review & editing.

 Funding
Funding agencies/institutions: Project / Grant
Conselleria de Agricultura, Medio Ambiente, Cambio Climático y Desarrollo Rural (Generalitat Valenciana) S8456000
References Abdul-Rassoul MS , Mohammed SM , 2017. First record of Adontomerus amygdali (Boucek, 1958) (Hymenoptera, Torymidae): a parasitoid of the almond fruit wasp, Eurytoma amygdali Enderlein, 1907 (Hymenoptera, Eurytomidae) in Erbil Provence, IRAQ. Bull Iraq nat His Mus 14: 301-306. 10.26842/binhm.7.2017.14.4.0301 Bahillo de la Puebla P , López Colón JI , 2000. El género Opilo Latreille, 1802 en la Península Ibérica (Coleóptera, Cleridae) Boln Asoc esp Ent 24: 213-227. Bahillo de la Puebla P , López Colón JI , Prieto Piloña F , 2021. La familia Cleridae Latreille, 1802 (Coleoptera) en la Península Ibérica e Islas Baleares. A E 23: 3-98 Cakar L , 1980. Eurytoma amygdali End. (Hym., Chalcidoidea, Eurytomidae) a pest of almond in Macedonia. Zastita Bilja, 31, 263-272. (in Bosnian with French abstract) Cioacă L , Ciceoi R , Stănică F , 2022. Almond seed wasp, Eurytoma amygdali, a new fruit pest in the romanian fauna. Scientific Papers. Series B, Horticulture 66: 34-39. Çobanoğlu S , Doğanlar M , 2006. A new Pyemotes (Acari: Pyemotidae) reared from larvae of the Almond Seed Wasp, Eurytoma amygdali (Hymenoptera: Eurytomidae) from Hatay, Turkey. Zool Middle East 39: 101-106. 10.1080/09397140.2006.10638189 Correa De Barros JM , 1929. Notas para o estudo das espécies da Fam. Cleridae existentes em Portugal. Arquivos da secçáo de Biología e Parasitologia do Museo da universidade da Coimbra 1, 1-25. Doğanlar O , Emin Yıldırım A , Doğanlar M , 2006. Natural Enemy Complex of Eurytoma Amygdali Enderlein, 1907 (Hymenoptera, Eurytomidae) in Eastern Mediterranean Region of Turkey; Notes on Their Interaction and Effectiveness. Res J Agric & Biol Sci 2: 282-286. Español F , 1959. Los Cléridos (Cleridae) de Cataluña e Islas Baleares (Col., Cleroidea). Publicaciones del Instituto de Biología Aplicada Barcelona 30, 105-146. Gauss R , 1954. Der Ameisenbuntkafer Thanasimus (Clerus) formicarius Latr. als Borkenkaferfeind. In. Die Grosse Borkenkaferkalamitat in Südwest-Deutschland J944-5J. G. Wellenstein [Ed.]. Ulm. Gerstmeier R , 1998. Checkered Beetles: Illustrated Key to the Cleridae and Thanerocleridae of the Western Palaearctic Buntkäfer: Illlustrierter Schlüssel zu den Cleridae und Thanerocleridae der West-Paläarktis. Magraf publishers, Weikersheim, Baden-Württemberg, 241 pp, 8 col plates, 304 text figs, maps. Haustein T , Busweiler S , Haustein V , von Laar C , Plarre R , 2019. Laboratory breeding of Korynetes caeruleus (Coleoptera: Cleridae) for the biological control of Anobium punctatum (Coleoptera: Ptinidae). Eur J Entomol 116: 362–371. 10.14411/eje.2019.038 Ibrahim MY , Al-Fouzoo T , Al-Naser Z , 2008. Biological and ecological studies of almond fruit wasp, Eurytoma amygdali End. (Hymenoptera: Eurytomidae) at Homs Governorate (Syria). JJAS 4(1): 139-151. Katsoyannos BI , Kouloussis NA , Bassiliou A , 1992. Monitoring populations of the almond seed wasp, Eurytoma amygdali, with sex pheromone traps and other means, and optimal timing of chemical control. Entomol Exp Appl 62: 9-16. 10.1111/j.1570-7458.1992.tb00635.x Margaritopoulos JT , Tzanakakis ME , 2006. Diapause completion in the almond seed wasp, Eurytoma amygdali (Hymenoptera: Eurytomidae) following early low temperature treatment. Eur J Entomol 103: 733–742. 10.14411/eje.2006.097 Mentjelos J , Atjemis A , 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol 63: 1934-1936. 10.1093/jee/63.6.1934 Noyes JS , 2019. Universal Chalcidoidea Database. World Wide Web electronic publication. http://www.nhm.ac.uk/chalcidoids (accessed June 2023). OEPP/EPPO 2004, Bulletin OEPP/EPPO Bulletin 34, 427–438. Stone fruits. 10.1111/j.1365-2338.2004.00750.x Plaut HN , 1971a. Behaviour of phosphamidon within the young almond fruit, and action of translocated phosphamidon and monocrotophos on the egg and the young larva of almond wasp Eurytoma amygdali . Pestic Sci 2: 113-114. 10.1002/ps.2780020305 Plaut HN , 1971b. On the biology of adult of almond wasp, Eurytoma-amygdali End (Hym, Eurytomidae), in Israel. B Entomol Res 61: 275-281. 10.1017/S0007485300057795 Plaut HN , 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res 61: 681-687. 10.1017/S0007485300047519 Saeidi Z , 2021. Resistance of different almond cultivars/genotypes to almond fruit wasp, Eurytoma amygdali (Hymenoptera: Eurytomidae). J Crop Prot 10(3): 535-545. Schäckermann J , Pufal G , Mandelik Y , Klein AM , 2015. Agro-ecosystem services and dis-services in almond orchards are differentially influenced by the surrounding landscape. Ecol Entomol 40: 12-21. 10.1111/een.12244 Schroeder LM , 1999. Prolonged development time of the bark beetle predator Thanasimus formicarius (Col.: Cleridae) in relation to its prey species Tomicus piniperda (L.) and lps typographus (L.) (Col.: Scolytidae). Agr Forest Entomol 1: 127-135. 10.1046/j.1461-9563.1999.00018.x Talhouk AS , 1977. Contributions to the knowledge of almond pests in East Mediterranean countries V. The fruit-feeding, Eurytoma amygdali End., and Anarsia lineatella Z. Z Angew Entomol 83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x Tolga M , Yoldaş A , Zeynep Y , 2018. Investigation on the damage rate of Eurytoma amygdali Enderlin (Hymenoptera: Eurytomidae) with first record of its parasitoid, Exeristes roborator (Fabricus) in almond orchards of Mugla and Manisa. Türkiye VII. Bitki Koruma Kongresi (Uluslararas1 Kat1hmh), 14-17 Kas1m 2018, Mugla, Türkiye. Tzanakakis ME , Papadopoulos NT , Katsoyannos BI , Drakos GN , Manolakis E , 1997. Premature fruit drop caused by Eurytoma amygdali (Hymenoptera: Eurytomidae) on three almond varieties. J Econ Entomol 90: 1635-1640. 10.1093/jee/90.6.1635 Tzanakakis ME , Veerman A , 1994. Effect of temperature on the termination of diapause in the univoltine almond seed wasp Eurytoma amygdali . Entomol Exp Appl 70: 27-39. 10.1111/j.1570-7458.1994.tb01756.x Yiğit T , Özgen İ , Canbay A , Koç İ , 2020. Infestation rate of almond seed wasp (Eurytoma amygdali Enderlein, Hymenoptera: Eurytomidae) on important commercial almond varieties in Malatya province (Turkey). I J I E A 4: 9-12. 10.46460/ijiea.737755 Zerova MD , Fursov VN , 1991. The Palaearctic species of Eurytoma (Hymenoptera: Eurytomidae) developing in stone fruits (Rosaceae: Prunoideae). B Entomol Res 81: 209-219. 10.1017/S0007485300051294