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, 1991Zerova MD, Fursov VN, 1991. The Palaearctic species of Eurytoma (Hymenoptera: Eurytomidae) developing in stone fruits (Rosaceae: Prunoideae). B Entomol Res81: 209-219. 10.1017/S0007485300051294; Noyes, 2019Noyes JS, 2019. Universal Chalcidoidea Database. World Wide Web electronic publication. http://www.nhm.ac.uk/chalcidoids (accessed June 2023).; Cioacă et al., 2022Cioacă L, Ciceoi R, Stănică F, 2022. Almond seed wasp, Eurytoma amygdali, a new fruit pest in the romanian fauna. Scientific Papers. Series B, Horticulture66: 34-39.).
Previous studies conducted on E. amygdali biology indicate that it has one generation per year (Mentjelos & Atjemis, 1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934; Plaut 1971bPlaut HN, 1971b. On the biology of adult of almond wasp, Eurytoma-amygdali End (Hym, Eurytomidae), in Israel. B Entomol Res61: 275-281. 10.1017/S0007485300057795; 1972Plaut HN, 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res61: 681-687. 10.1017/S0007485300047519; Talhouk, 1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x). In the Mediterranean basin where the pest was studied, adult flight and reproduction begin in March-April and end late in May (Plaut, 1971bPlaut HN, 1971b. On the biology of adult of almond wasp, Eurytoma-amygdali End (Hym, Eurytomidae), in Israel. B Entomol Res61: 275-281. 10.1017/S0007485300057795; Talhouk, 1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x; Katsoyannos et al., 1992Katsoyannos 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 Appl62: 9-16. 10.1111/j.1570-7458.1992.tb00635.x). 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, 1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934; Plaut, 1972Plaut HN, 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res61: 681-687. 10.1017/S0007485300047519; Talhouk, 1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x). 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, 1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934; Talhouk, 1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x; Cakar, 1980Cakar L, 1980. Eurytoma amygdali End. (Hym., Chalcidoidea, Eurytomidae) a pest of almond in Macedonia. Zastita Bilja, 31, 263-272. (in Bosnian with French abstract)). From June, infested fruits shrivel and remain firmly attached to the trees for a long time (Plaut, 1971bPlaut HN, 1971b. On the biology of adult of almond wasp, Eurytoma-amygdali End (Hym, Eurytomidae), in Israel. B Entomol Res61: 275-281. 10.1017/S0007485300057795; 1972Plaut HN, 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res61: 681-687. 10.1017/S0007485300047519; Zerova & Fursov, 1991Zerova MD, Fursov VN, 1991. The Palaearctic species of Eurytoma (Hymenoptera: Eurytomidae) developing in stone fruits (Rosaceae: Prunoideae). B Entomol Res81: 209-219. 10.1017/S0007485300051294).
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, 2006Çobanoğlu 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 East39: 101-106. 10.1080/09397140.2006.10638189; Doğanlar et al., 2006Doğanlar 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 Sci2: 282-286.; Schäckermann et al., 2015Schäckermann 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 Entomol40: 12-21. 10.1111/een.12244; Abdul-Rassoul & Mohammed, 2017Abdul-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 Mus14: 301-306. 10.26842/binhm.7.2017.14.4.0301; Tolga & Yoldas, 2018Tolga 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.).
Eurytoma amygdali is considered one of the most important almond pests, especially in the SE Mediterranean (OEPP/EPPO, 2004OEPP/EPPO2004, Bulletin OEPP/EPPO Bulletin34, 427–438. Stone fruits. 10.1111/j.1365-2338.2004.00750.x), because it can cause up to 80% harvest loss (Mentjelos & Atjemis, 1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934; Talhouk, 1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x; Cakar, 1980Cakar L, 1980. Eurytoma amygdali End. (Hym., Chalcidoidea, Eurytomidae) a pest of almond in Macedonia. Zastita Bilja, 31, 263-272. (in Bosnian with French abstract)). 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, 1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934; Plaut, 1971aPlaut 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 Sci2: 113-114. 10.1002/ps.2780020305; Katsoyannos et al., 1992Katsoyannos 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 Appl62: 9-16. 10.1111/j.1570-7458.1992.tb00635.x). Tzanakakis et al. (1997Tzanakakis 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 Entomol90: 1635-1640. 10.1093/jee/90.6.1635), 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., 1992Katsoyannos 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 Appl62: 9-16. 10.1111/j.1570-7458.1992.tb00635.x) because some cultivars showed different infestation rates (Tzanakakis et al., 1997Tzanakakis 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 Entomol90: 1635-1640. 10.1093/jee/90.6.1635; Ibrahim et al., 2008Ibrahim 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). JJAS4(1): 139-151.; Saeidi, 2021Saeidi Z, 2021. Resistance of different almond cultivars/genotypes to almond fruit wasp, Eurytoma amygdali (Hymenoptera: Eurytomidae). J Crop Prot10(3): 535-545.).
Although the presence of E. amygdali is known in several countries in the Mediterranean Basin such as France, Greece and Turkey (Zerova & Fursov, 1991Zerova MD, Fursov VN, 1991. The Palaearctic species of Eurytoma (Hymenoptera: Eurytomidae) developing in stone fruits (Rosaceae: Prunoideae). B Entomol Res81: 209-219. 10.1017/S0007485300051294; Noyes, 2019Noyes JS, 2019. Universal Chalcidoidea Database. World Wide Web electronic publication. http://www.nhm.ac.uk/chalcidoids (accessed June 2023).), 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.
| Province | Town/District | Lota | Plota | almondsb | Variety/Varieties |
|---|---|---|---|---|---|
| Valenciac | 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 | |||
| Alicantec | Villena | 49 | 109 | 171 | Guara |
| Biar | 8 | 100 | 230 | Guara | |
| Castalla | 22 | 97 | 103 | Unknown | |
| Albaceted | 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 | |
| Cuencad | Granja Iniesta | 15 | 65 | 100 | Lauranne |
| Murciae | 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/).
Phenology and natural enemies of E. amygdali
⌅The 2018 trial. Phenology of E. amygdali
⌅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 E. amygdali
⌅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, 1998Gerstmeier 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.; Çobanoğlu & Doğanlar, 2006Çobanoğlu 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 East39: 101-106. 10.1080/09397140.2006.10638189; Bahillo de la Puebla et al., 2021Bahillo 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 E23: 3-98).
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.
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 E. amygdali
⌅The 2018 trial. Phenology of E. amygdali
⌅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.
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.
The 2019 trial. Natural enemies of E. amygdali
⌅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).
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.
Discussion
⌅In our studied area, E. amygdali showed an annual generation in accordance with previous studies (Mentjelos and Atjemis, 1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934; Plaut, 1971bPlaut HN, 1971b. On the biology of adult of almond wasp, Eurytoma-amygdali End (Hym, Eurytomidae), in Israel. B Entomol Res61: 275-281. 10.1017/S0007485300057795; 1972Plaut HN, 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res61: 681-687. 10.1017/S0007485300047519). 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 (1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934) and with Plaut (1972Plaut HN, 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res61: 681-687. 10.1017/S0007485300047519).
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 (1972Plaut HN, 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res61: 681-687. 10.1017/S0007485300047519) and Talhouk (1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x) reported.
The larva enters diapause when it has consumed all the seed content (Plaut, 1972Plaut HN, 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res61: 681-687. 10.1017/S0007485300047519). The effect of temperature on diapause termination has been recently studied (Tzanakakis & Veerman, 1994Tzanakakis ME, Veerman A, 1994. Effect of temperature on the termination of diapause in the univoltine almond seed wasp Eurytoma amygdali. Entomol Exp Appl70: 27-39. 10.1111/j.1570-7458.1994.tb01756.x; Margaritopoulos & Tzanakakis, 2006Margaritopoulos JT, Tzanakakis ME, 2006. Diapause completion in the almond seed wasp, Eurytoma amygdali (Hymenoptera: Eurytomidae) following early low temperature treatment. Eur J Entomol103: 733–742. 10.14411/eje.2006.097). This process begins at the end of May in Israel, which is approximately one month earlier than in our studied area. Similar to Talhouk (1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x), 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, 1971bPlaut HN, 1971b. On the biology of adult of almond wasp, Eurytoma-amygdali End (Hym, Eurytomidae), in Israel. B Entomol Res61: 275-281. 10.1017/S0007485300057795; Talhouk, 1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x; Katsoyannos et al., 1992Katsoyannos 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 Appl62: 9-16. 10.1111/j.1570-7458.1992.tb00635.x; Yiğit et al., 2020Yiğit 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 A4: 9-12. 10.46460/ijiea.737755).
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 (1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934), 23% for Talhouk (1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x) or 1% for Plaut (1972Plaut HN, 1972. On the biology of the immatures stages of the almond wasp, Eurytoma amygadali End (Hym. Eurytomidae) in Israel. B Entomol Res61: 681-687. 10.1017/S0007485300047519). 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, 1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934; Talhouk, 1977Talhouk 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 Entomol83: 145-154. 10.1111/j.1439-0418.1977.tb02384.x; Cakar, 1980Cakar L, 1980. Eurytoma amygdali End. (Hym., Chalcidoidea, Eurytomidae) a pest of almond in Macedonia. Zastita Bilja, 31, 263-272. (in Bosnian with French abstract)).
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, 2006Çobanoğlu 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 East39: 101-106. 10.1080/09397140.2006.10638189). 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 (2006Çobanoğlu 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 East39: 101-106. 10.1080/09397140.2006.10638189) and Doğanlar et al. (2006Doğanlar 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 Sci2: 282-286.). The study by Doğanlar et al. (2006Doğanlar 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 Sci2: 282-286.), 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. (2006Doğanlar 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 Sci2: 282-286.), who obtained parasitism rates between 7.56% and 44.53%. According to Çobanoğlu & Doğanlar (2006Çobanoğlu 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 East39: 101-106. 10.1080/09397140.2006.10638189), 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. (2021Bahillo 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 E23: 3-98), 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, 1929Correa 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 Coimbra1, 1-25.; Español, 1959Español F, 1959. Los Cléridos (Cleridae) de Cataluña e Islas Baleares (Col., Cleroidea). Publicaciones del Instituto de Biología Aplicada Barcelona 30, 105-146.). 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, 2000Bahillo de la Puebla P, López Colón JI, 2000. El género Opilo Latreille, 1802 en la Península Ibérica (Coleóptera, Cleridae)Boln Asoc esp Ent24: 213-227.; Bahillo de la Puebla et al., 2021Bahillo 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 E23: 3-98). Doğanlar et al. (2006Doğanlar 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 Sci2: 282-286.) 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, 1954Gauss R, 1954. Der Ameisenbuntkafer Thanasimus (Clerus) formicarius Latr. als Borkenkaferfeind. In. Die Grosse Borkenkaferkalamitat in Südwest-Deutschland J944-5J. G.Wellenstein [Ed.]. Ulm.), or 2 years in Scandinavia (Schroeder 1999Schroeder 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 Entomol1: 127-135. 10.1046/j.1461-9563.1999.00018.x). 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., 2019Haustein 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 Entomol116: 362–371. 10.14411/eje.2019.038). 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, 1970Mentjelos J, Atjemis A, 1970. Studies on the Biology and Control of Eurytoma amygdali in Greece. J Econ Entomol63: 1934-1936. 10.1093/jee/63.6.1934; Plaut 1971aPlaut 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 Sci2: 113-114. 10.1002/ps.2780020305; Katsoyannos et al., 1992Katsoyannos 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 Appl62: 9-16. 10.1111/j.1570-7458.1992.tb00635.x), and insecticides applied to the tree canopy against adults (Tzanakakis et al., 1997Tzanakakis 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 Entomol90: 1635-1640. 10.1093/jee/90.6.1635). 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.