Acta agriculturae Slovenica, 121/3, 1–8, Ljubljana 2025 doi:10.14720/aas.2025.121.3.21435 Original research article / izvirni znanstveni članek Efectiveness of four release doses of two parasitoids species, Trichogram- ma (Hymenoptera: Trichogrammatidae) in some biological aspects of the lesser date moth Batrachedra amydraula Meyricke, 1916 (Cosmopteri- dae: Batrachedridae) on date palm trees in Basrah /Iraq. Ghazwan FAISAL AL-SAEDI 1, 2 Received December 16, 2024, accepted April 11, 2025 Delo je prispelo 16. december 2024, sprejeto 11. april 2025 1 Date Palm Research Center, University of Basrah / Iraq. 2 corresponding author: ghazwan.khalaf@uobasrah.edu.iq Efectiveness of four release doses of two parasitoids spe- cies, Trichogramma (Hymenoptera: Trichogrammatidae) in some biological aspects of the lesser date moth Batrachedra amydraula Meyricke, 1916 (Cosmopteridae: Batrachedridae) on date palm trees in Basrah /Iraq. Abstract: Date palms are infested with various destructive insect pests, which cause major economic losses. One of these pests is the smaller date moth Batrachedra amydraula Mey- ricke, 1916. The study aimed to examine the effectiveness of two different Trichogramma egg parasitoid (Trichogramma brassi- cae Bezdenko, 1968 and T. evanescens Westwood, 1833) in the biological control of the lesser date moth B. amydraula. Each parasitoid species was examined at four different release doses (250, 500, 750, and 1000 parasitoid/tree). The findings revealed that all species of Trichogramma had a substantial effect, par- ticularly at high doses, on the high parasitism of B. amydraula eggs, reaching 86.75 % for a dose of 1000 T. evanescens para- sitoids/tree, with a significant difference reached 83.49 % at a dose of 1000 T. brassicae parasitoids/tree. The maximum rate of emergence of T. evanescens adults was at a dose of 1000 para- sitoids/tree, which was much higher than T. brassicae reached 64.20 %. The response of absolute doses of Trichogramma fe- males to B. amydraula spawning sites was evaluated, and the parasite’s egg sites were determined according to tree height. The best parasitism rate was recorded for both Trichogramma species on trees whose height ranged from 1.5 m-2 m. Key words: natural enemies, lesser date moth, Tricho- gramma, date palm, B. amydraula. Učinkovitost štirih velikosti izpustitve parazitoidnih vrst iz rodu, Trichogramma (Hymenoptera: Trichogrammatidae) na nekatere biološke parametre manjšega dateljevega molja Batrachedra amydraula Meyricke, 1916 (Cosmopteridae: Ba- trachedridae) na palmah v Basri, Irak Izvleček: Dateljevec napadajo različni škodljivci, ki pov- zročajo velike ekonomske izgube, med njimi je mali dateljev molj (Batrachedra amydraula Meyricke, 1916). Namen razi- skave je bil preučiti učinkovitost dveh razčičnih parazitoidov jajčec iz rodu Trichogramma (Trichogramma brassicae Bezden- ko, 1968 and T. evanescens Westwood, 1833) za biološki nad- zor malega dateljevega molja. Vsaka parazitoidna vrsta je bila preučevana v štirih različno velikih izpustih (250, 500, 750 in 1000 parasitoidov na drevo). Rezultati so pokazali, da imata obe vrsti iz rodu Trichogramma precejšen učinek na jajčeca molja, še posebej ob večjih izpustih, kar je doseglo 86,75 % za izpust 1000 parazitoidov vrste T. evanescens na drevo, a značilno raz- liko 83,49 % pri izpustu 1000 parazitoidov vrste T. brassicae na drevo. Največje število odraslih osebkov vrste T. evanescens je bilo pri pri izpustu 1000 parazitoidov na drevo, ki je bilo veliko večje kot pri izpustu vrste T. brassicae , ki je doseglo le 64,20 %. Ovrednoten je bil odziv velikosti izpustov samic iz rodu Tricho- gramma na mesta razmnoževanja molja, kjer je bilo ugotovlje- no število parazitiranih jajčec glede na višino drevesa. Največji obseg parazitizma je bil zabeležen za obe vrsti parazitoidov na drevesih, ki so bila visoka med 1,5 m-2 m. Ključne besede: naravni sovržniki, mali dateljev molj, Trichogramma, dateljevec, B. amydraula. Acta agriculturae Slovenica, 121/3 – 20252 G. FAISAL AL-SAEDI 1 INTRODUCTION Date palms (Phoenix dactylifera L) are considered very important economic trees in different countries (Abass and Maziel, 2019). A large number of insect pests attacks palm trees in all their parts, which sometimes cause them serious and significant damage represented by the weakness of the palm tree, which leads to a re- duction in its date productivity in terms of quality and quantity (Al-Jubouri , 2007; Al-Saedi, 2023). These dam- ages also lead to the death of the palm tree. At the top of the palm tree, the pollen and fruits of the palm tree are exposed to many pests and insects that attack the flower pods before and after they open. They also attack the fruits in their various stages. One of these pests is less- er date moth (Batrachedra amydraula Meyricke, 1916) (Cosmopteridae: Batrachedridae), which usually cause fruit withering, pod breaking and falling. Several fac- tors such as pests, weather conditions, and diseases affect health of palm trees. The lesser date moth (B. amydraula Meyricke) in Iraq is a serious pest that infects date palm trees (Downson and Aten, 1962; Levi Zada et al. 2011; Haldhar et al. 2017). The harmful stage of the pest is the larvae that feed inside the fruits, and also the larvae form networks around the flowers and later infect the fruit. The use of chemical pesticides has affected the rela- tionship between date palm pests and natural enemies. Thus integrated pest management (IPM) programs have become more complex because of increasing the pests (Latifian, 2001; Latifian and Zaerae, 2009; Latifian, 2012). Parasitoid of the genus Trichogramma are among the most common groups of natural enemies because they are relatively easy to rear and are among the most im- portant egg parasitoid of the agricultural pest Lepidop- tera. Biological control has been used as an alternative to chemical control in many South American and Euro- pean countries, especially using egg parasitoids (Ivezic et al. 2022; Parra and Zucchim, 2004). The use of biological control methods to control B. amydraula is increasing- ly considered an essential management strategy within IPM. Parasitoids of the genus Trichogramma are among the most common groups of natural enemies because they are relatively easy to culture in the laboratory and are among the most important egg parasitoid of agricultural pests of the order Lepidoptera. Trichogramma parasitoids were used commercially as mass releases in more than 32 million hectares against more than 30 major insects worldwide (Wajnberg and Hassan 1994; Smith 1996; Ayvaz and Karaborklu, 2008; Pizzol et al., 2010; Pizzol, 2012). T. brassicae Bezdenko, 1968 was was widely used worldwide for the control of lepidopteran pests as well as in egg control of T. absoluta (Meyrick, 1917) in some countries (Tissoli and Parra, 2001; Goncalves-Gervasio, 2003; Garcia et al. 2005; Medeiros et al., 2006; Desneux et al., 2010; Molla et al., 2011; Vasconcelos, 2013). In ad- dition, Kuske et al. (2003) evaluated the effects of high releases of T. brassicae against the European corn borer Ostrinia nubilalis (Hübner, 1796) and was very success- ful in reducing the pest population and reducing yield losses. In Iran, T. brassicae is the most widespread species (Ebrahimi et al., 1998; Moezipour et al., 2008). Various factors can influence the effect of high releases of para- sitoids, for example, host selection by parasitoids may be influenced by characteristics of both the host species and the host plant attacked by the pest (Chau and Mack- auer, 2001; Desneux and Ramirez, 2009). Differences in parasitism depend on the host on which the parasitoids are reared and the effect of the size of the host born on natural parasitism rates of the host in the field, where female parasitoids usually accept host eggs of the same size or larger than the host (El-Wakeil, 2007; Nurindah et al., 1999). However Kolliker-Ott et al., 2003) reported no relationship between mass preference and perfor- mance of T. brassicae reared on the novel host Ephestia kuehniella Zeller, 1879 when parasitizing the target host of the European corn borer Ostrinia nubilalis. Otherwise, the effectiveness of Trichogramma release may depend not only on the biological characteristics of the parasit- oid species or strains used, but also on their interactions with the pest plant (Tabone et al. 2010; Andrade et al. 2011; Yuan et al. 2012). Trichogramma is primarily used as a biocontrol agent through releases but the effect of Trichogramma generations growing within a crop can play an important role in the success of biological control programs (Mills, 2010). Torres et al., 2001) showed that the female parasitoid preferentially lays eggs in the upper part of the plant, followed by the middle and lower parts respectively. In the current study, the efficiency of four doses of these parasitoids in controlling B. amydraula Meyrick, 1916[ was examined. The main goal of the study was to determine how T. brassicae and T. evanescens Westwood, 1833 impact the distribution of B. amydraula eggs on date palm trees and study the effect of tree height on the efficiency of parasitism. 2 MATERIALS AND METHODS 2.1 HOST PRODUCTION Insects were collected from the University of Bas- rah, Date Palm Research Center (Basrah, Iraq) where the parasitoids, Trichgramma brassicae and T. evanescens were produced by the Mediterranean flour moth, Ephe- stia kuehniella (Lepidoptera: Pyralidae) in large quanti- ties on flour. The Mediterranean flour moth was reared Acta agriculturae Slovenica, 121/3 – 2025 3 Effectiveness of four release doses of two parasitoids species, Trichogramma ... the lesser date moth ... date palm trees in Basrah /Iraq. in the laboratory on flour inside plastic cages (50 x 50 x 50 cm). All cage walls were covered with a piece of terry- cloth. Inoculated females of E. kuehniella were obtained by mating a male with a virgin female. Then, after one day the mating, E. kuehniella females were used in the experiments, and then the insects were placed in plastic cones, the base of which were covered with a terrycloth, through which the eggs are collected. Then the fertilized eggs were taken and placed in plastic containers (40 x 25 x 25 cm) filled with 2 kg of flour and were infested with E. kuehniella eggs. The moths begin to appear after they spread on the flour and moths continue to emerge for two months. 2.2 BREEDING THE PARASITE The parasitoids, Trichogramma brassicae and T. evanescens (Hymenoptera: Trichogrammatidae) were produced in large quantities on the eggs of the Medi- terranean flour moth, Ephestia kuehniella. Eggs of the Mediterranean flour moth E. kuehniella were pasted onto cardboard measuring 8 x 4 cm. Each card contained 250, 500, 750, and 1000 eggs. T. brassicae and T. evanescens are reared in glass containers with a height of 21 cm and a diameter of 10cm covered with muslin cloth by using rubber bands that are kept in place until use. Six cards of freshly collected eggs (1 day old) were placed in each glass jar along with 2 cards containing parasitic eggs that would hatch within 24 hours. E. kuehniella and Trich- gramma species were reared at a temperature of 25 ± 2 °C, 70 % relative humidity (El-Dakroury et al., 2002). 2.3 THE EFFECT OF THE HOST LOCATION ON TRICHOGRAMMA PARASITISM AND ON B. AMYDRAULA EGGS This study was conducted in Abu Al-Khasib area in Basrah Governorate, southern Iraq, in 2019. Halawi and Al-Sayer varieties were used. The parasitoids T. brassicae and T. evanescens were released in 2019 at the beginning of March. Four cards with doses of (250, 500, 750, and 1000 parasitoids/tree) were hung on palm branches at different heights (1.5-2 m), (2.5-3 m), and (3.5-4 m). The sample size was 10 branches (replicates)/ palm tree. 2.4 STUDY PARAMETERS Presence of live larvae of B. amydraula on fruits whether they are fallen or not with the presence of silk tissue was recorded. Samples were collected and placed in plastic bags for the purpose of examination in the laboratory. The parameters were: incidence and severity of the moth infection, the number of moth adults, the sex ratio, and the moth mortality (which should be cor- rected according to Henderson-Tilton`s formula,1955). The examination was conducted at two-week intervals from the beginning of March 2022 until the beginning of June 2023. 2.5 STATISTICAL ANALYSIS The rate of parasitism results, adult emergence, sex ratio and adult longevity were analyzed statistically ac- cording to a randomized complete block design (CRBD) using one-way analysis of variance (ANOVA) and the results were compared by calculating the smallest dif- ference. If significant differences were detected, com- parisons were made using and significance of means was determined based on Duncan’s test at P ≤0.05. Statistical analysis was performed using SPSS Statistics for Win- dows version 21 (Al-Rawi and Khalafallah, 1980). 3 RESULTS The results of releasing four doses of the parasite Trichogramma brassicae and T. evanescens to control the lesser date moth B. amydraula showed that there is a significance in the rate of parasitism of T. evanescens reached 86.75 %, 67.11 %, 55.32 % and 42.89 % at 1000, 750, 500 and 250 parasitoids/ tree respectively. These val- ues were slightly higher than the rate of parasitism of T. brassicae which was recorded 83.49 %, 58.67 %, 47.33 % and 33.79 % at 1000, 750, 500 and 250 parasitoids/ tree respectively (Fig.1). Figure 1: Parasitism according to the four doses of Tricho- gramma brassicae and T. evanescens on B. amydraula eggs. Acta agriculturae Slovenica, 121/3 – 20254 G. FAISAL AL-SAEDI However, there was no significance in the rate of emergence of adults of the T. evanescens parasite at the doses of 1000,750,500 and 250 parasitoids/tree which were 64.2, 63.59, 63.45 and 61.83  % respectively. The results of adult emergence of T. brassicae parasite were slightly lower than T. evanescens after releasing 1000, 750, 500 and 250 parasitoids/tree, which were 59.4 %, 60 %, 58.85 % and 58.34 %, respectively (Figure 2). The mortality rate of T. evanescens parasite reached 35.8 %, 36.05 %, 36.55 % and 38.17 % at doses of in the figure 3 they were 250, 500,750 and 1000 parasitoids/ tree, with a no significant in the figure 3 difference from the T. brassicae parasite, which recorded 40.6  %, 40  %, 41.15 % and 41.66 %, respectively, and this shows that the mortality rate of T. evanescens was lower than that of T. brassicae, but the difference between the mortality rates of these two parasitoid was significant when 1000 para- sitoids/tree were released. The results showed that the fe- male percentage of T. evanescens was slightly close to that of T. brassicae and was 61.8 %, 60.27 %, 59 % and 58.66 % for the doses 1000, 750, 500 and 250 parasitoids/tree for T. evanescens and was 61.6 %, 60.04 %, 58.83 % and 58.46 % for the doses 1000, 750, 500 and 250 parasitoids/ tree for T. brassicae. However, the differences between the two parasitoid were all no significant in the figure 4 at the doses 1000 and 750 parasitoids/tree (Figure 4). 3.1 DISTRIBUTION OF THE PARASITE AND ITS PARASITISM ON THE PLANT CANOPY The parasitism rate by the most preferred height of trees showed the behavior of the parasite T. evanes- cens on palm trees, a great parasitism on height 1.5-2 m and reached 58.95 %, 52.84 %, 50.42 % and 48.30 % for doses 1000, 750, 500 and 250 parasitoids/tree, com- pared to the parasite T. brassicae which reached 50.51 %, 42.70 %, 40 % and 39.99 % for doses 1000, 750, 500 and 250 parasitoids/tree. As for the height of trees 2.5-3 m, the parasitism rate was recorded lower than the previ- ously mentioned height, with a significant difference, reaching 39.43 %, 28.26 %, 27.73 % and 20.04 % for the doses of 1000, 750, 500 and 250 parasitoids/tree for the T. evanescens parasite, and it reached 39.16 %, 36.92 %, 36 % and 31.79 % for the doses of 1000, 750, 500 and 250 parasitoids/tree for the T. brassicae parasite. While a sig- nificant difference was found for the parasitism of T. eva- nescens on tree height 3.5-4 m which reached 10.71 %, 18.90 %, 21.85 % and 22.66 % for doses of 1000, 750, 500 and 250 parasitoids/tree, as well as for the parasitism of T. brassicae which reached 10.33 %, 20.38 %, 24 % and 28.22 % for doses of 1000, 750, 500 and 250 parasitoids/ tree (Figure 5). Figure 2: Adult emergence according to the four doses of Trich- ogramma brassicae and T. evanescens on B. amydraula eggs. Figure 3: Mortality according to the four doses of Trichogram- ma brassicae and T. evanescens on B. amydraula eggs. Figure 4: Female ratio according to the four doses of Tricho- gramma brassicae and T. evanescens on B. amydraula eggs. Acta agriculturae Slovenica, 121/3 – 2025 5 Effectiveness of four release doses of two parasitoids species, Trichogramma ... the lesser date moth ... date palm trees in Basrah /Iraq. 4 DISCUSSION The present study showed good efficiency of Tricho- gramma brassicae and T. evanescens in controlling the damage levels of the lesser date moth B. amydraula. Overall, T. evanescens showed the highest egg parasitism and thus appears to be more effective when compared with T. brassicae in biocontrol activity against the lesser date moth B. amydraula. Our results were comparable to those of Mohammad et al. (2011), who noticed that the lesser date moth infestation levels in Anbar Governorate were significantly reduced after two weeks of releasing 100, 200, and 300 T. evanescens or T. principium per palm tree, with higher efficiency for the parasite T. evanescens. Both parasitoid were effective in reducing the infestation of this pest on date palm fruits during both the Hababok and Jamri stages for all release rates. On the other hand, study by Alrubeai et al. (2014) demonstrated the effec- tiveness of the egg parasitoid T. evanescens in reducing the lesser date moth during the 2011 and 2012 seasons and showed that releasing the egg parasitoid at a rate of 500 and 1000 parasitoids/palm tree achieved a reduction of 55.06 % and 67.45 %, respectively, in the infestation rate and was a good option for controlling the lesser date moth as an alternative method of chemical control. Our research was examined two species of Trichogramma par- asitoid at different heights of palm trees. In comparison, our results showed that the parasitism efficacy was higher than that reported by Alrubeai et al. (2014) who conduct- ed it in central Iraq and focused on one height and one caltivar of palm trees using one species of Trichogramma parasitoid. In another study, Gameel et al. (2014) showed the effectiveness of releasing the egg parasitoid T. evane- scens on the eggs of the lesser date moth B. amydraula at a rate of ten releases in envelopes (20,000 egg parasitoid/ acre) which led to a significant reduction in the infesta- tion after successive releases of the parasitoid to reach a high level (75.06 %) in the 2013 season, overall, the re- duction rate ranged from 72.49 % to 75.97 % with an av- erage of 73.74 % across all trials. Mohammad et al. (2011) found that the highest control efficiency obtained by T. evanescens parasitizing the lesser date moth was 70.8 % when used at a rate of three capsules per palm tree (cap- sules containing about 100 parasitized pupae). In another study, Alrubeai et al. (2014) found that releasing the egg parasite T. evanescens at a rate of 500 and 1000 parasit- oids/palm tree achieved a reduction in the infection rate of 55.06 % and 67.45 %, respectively. The emergence rate of T. evanescens adults was ac- ceptable and slightly higher than that of T. brassicae. Oth- erwise, the size of the host eggs reared on different hosts appears to influence the biological characteristics of Trichogramma parasitoid (Greenberg et al. 1998, Broto- djojo and Walter, 2006). This may be explained by the fact that Trichogramma reared on smaller hosts are less robust and perform less well than those reared on larger host eggs (Liu et al. 1998). On the other hand, studies on other plants have shown that different species of Tricho- gramma differ in their preference for Tuta absoluta eggs, for example Zouba et al. (2013) found that T. absoluta eggs were small in size and that the rate of egg parasit- ism by T. bourarachae 65.64 % was significantly higher compared to T. cacoeciae 57.05  %. Furthermore, stud- ies indicate that larger host eggs can positively influence factors such as the percentage of parasitism, the number of parasitoid emerging per egg, lifespan, and sex ratio (Waage and Ming, 1984; Bai et al. 1992; Greenberg et al. 1998). In fact, the rate of emergence of adult Trichogram- ma can also vary with the size and quality of the host egg, the number of parasitoid developing in each egg, the period of development in the host egg and the tem- perature (Doyon and Boivin, 2005; Pratissoli et al., 2005). In this study, we obtained acceptable levels of control of the lesser date moth B. amydraula, despite reducing the release doses. The results showed an inverse relationship between the release dose and the number of parasitoids of B. amydraula eggs, as expected the highest parasitism rate was recorded for B. amydraula eggs, so these results suggest that releasing 400 parasitoids of both T. brassicae and T. evanescens per tree could be the most appropriate dose in this study. Release doses of Trichogramma were also selected in a study by Cabello et al. (2009) where it was shown that releasing 75 adults per square meter every 3 or 4 days was very effective in reducing pest numbers. Previ- ous studies, including Alsaedi (2021), have shown that the recommended doses of T. brassicae and T. embryoph- agum (Hartig, 1838)at a rate of (50, 100 and 200 adults/ tree) depending on the pest infestation levels during the first weeks of the date fruit growing season can effectively reduce the number of B. amydraula. This study showed that parasitism of T. brassicae and T. evanescens was as- sociated with the number of eggs laid by B. amydraula on different parts of the plant canopy and tree height, and that both species tended to parasitize eggs on shorter host trees. This suggests that selection by B. amydraula on this site provides a better place for egg laying by the order Lepidoptera. Or it may be because the wind affects the parasite, pushing it from the top to the bottom to fall on shorter trees. When looking at the different plant structures, the level of bleaching by B. amydraula was higher in the upper part of the short trees than in the other parts. In this regard, a similar pattern of parasitism on host plant parts was discussed by Romeis et al. (1998) for other Trichogramma species on maize, cotton and pea, these results indicated that Trichogramma adjusts Acta agriculturae Slovenica, 121/3 – 20256 G. FAISAL AL-SAEDI its parasitism to host distribution. It is known that one important plant characteristic that has a significant effect on parasitoid performance is the presence of non-glan- dular or glandular appendages (trichomes) (Oliveira et al. 2017). This is especially true for small parasitoid such as Trichogramma species that can be trapped by trichome secretions or have reduced walking speed on such surfac- es (Romeis et al., 1998; Kennedy, 2003). Trichogramma is primarily used as a biological control agent by release but the effect of Trichogramma generations growing within the crop can play an important role in the success of bio- logical control programs (Mills, 2010). Torres et al., 2001) showed that the female parasitoid preferentially laid eggs in the upper part of the plant, followed by the middle and lower parts respectively. This was in contrast to the find- ing in this study that the parasitoid preferred lower trees and may be due to wind speeds in relation to taller trees. 5 CONCLUSIONS Both Trichogramma brassicae and T. evanescens demonstrated significant effectiveness in parasitizing Ba- trachedra amydraula eggs, particularly at higher release doses. The highest parasitism rate was observed with T. evanescens at a dose of 1000 parasitoids per tree, indicat- ing that increasing the release dose enhances biological control efficiency. T. evanescens exhibited a higher adult emergence rate compared to T. brassicae at the highest release dose, suggesting a difference in their development and survival rates. The percentage of female parasitoids did not differ significantly between the two Trichogram- ma species, indicating that they have similar reproductive capacity. 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