Efficacy of the parasitoid, Trichogrammatoidea bactrae Nagaraja (Hymenoptera: Trichogrammatidae) on the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) egg masses

The Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) is one of the major insect pests, causing a significant damage on different cultivated agricultural crops. Developing an alternative non-chemical tool, an effective and environmentally friendly method to suppress pest's infestation is essentially needed. Therefore, biological control by releasing the egg parasitoids could be the most promising tool for integrated pest management. This study was designed to evaluate the efficacy of the egg parasitoid, Trichogrammatoidea bactrae Nagaraja (Hymenoptera: Trichogrammatidae) as a bio-control agent against S. littoralis egg masses with different physical characteristics (number of egg layer and degree of scale density) in a no-choice and choice tests, under laboratory conditions. Also, the parasitoids’ fitness in terms of parasitism percentage, developmental period, adults’ emergence percentage, female offspring percentage, and longevity were investigated. The results revealed that T. bactrae wasps had a great ability to parasitize S. littoralis egg masses, but with different rates, related to their layers and scales’ thickness in both tests. The highest parasitism percentage was observed on one-layer eggs, followed by two layers. However, 3-layer eggs were the least preferable one. High numbers of adult emergencies (> 80%) were observed in all tested egg masses, except in the case of 3 layers with high scales. Furthermore, female-biased sex ratios were noticed at all examined eggs, with only the exception of high-scaly eggs with a single layer that recorded the lowest rate (≤ 45%). Besides, the survival of adult female parasitoids was not significantly affected in both tests. T. bactrae could be used as a bio-control agent against S. littoralis egg masses with different physical characteristics based on the achieved results.


Background
The Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), is one of the major phytophagous insect pests in Egypt as well as in many other countries (Pineda et al. 2004), causing significant damages on different cultivated agricultural crops such as cotton, corn, clover, soybeans, peanuts, flowers, fruits, and vegetables (Kandil et al. 2003). Management of this polyphagous pest is still dependent upon chemical insecticides (Dahi et al. 2016). However, chemical control has not provided a long-term solution for this pest problem because of the high costs, environmental pollution, and hazards to human's health. Besides, insecticide-resistance insect strains, drastic effects on natural enemies, resurgence, and outbreaks of secondary pests are well known (El-Heneidy et al. 2015). Developing an alternative non-chemical tool, an effective and environmentally friendly method to suppress pest infestation is essentially needed. Therefore, the biological control by releasing the egg parasitoids could be the most promising tool for integrated pest management. In this context, the potential of different Trichogramma species against different Spodoptera spp. have been studied by several researchers worldwide (Toonders and Sánchez 1987;Beserra et al. 2002 andBrotodjojo and Walter 2006;Bueno et al. 2010;Díaz et al. 2012;Dequech et al. 2013;Puneeth and Vijayan 2013;Figueiredo et al. 2015;Saljoqi et al. 2015;Jaraleño-Teniente et al. 2020). However, the efficiency of these parasitoids showed variation in their range related to the species of Trichogramma. Moreover, this variability may be due to the egg masses' layers deposited by Spodoptera females covering with the dense scales, which can alter the parasitism behavior (Toonders and Sánchez 1987;. Up to date, there is no detailed information on the performance of the Trichogrammatoidea species towards any Spodoptera spp. This study is an attempt to assess the efficacy of egg parasitoid, Trichogrammatoidea bactrae Nagaraja as a bio-control agent against S. littoralis eggs with different physical characteristics (number of egg layers and degree of scale density) in a no-choice and choice tests, under laboratory conditions. As far as we know, this study is the first to evaluate this parasitoid species against the target pest.

Methods
Cultures of the cotton leafworm, S. littoralis and the egg parasitoid, T. bactrae were reared under standard rearing conditions of (25 ± 2°C and > 60% RH with 14 L: 10D cycle) at the mass rearing unit of Trichogramma, Plant Protection Research Institute, Agricultural Research Center (ARC), Assiut Governorate, Egypt.

S. littoralis rearing
Initial culture of S. littoralis egg batches used in the study was obtained from the experimental farm of Plant Protection Department, Faculty of Agriculture, Assiut University. The insect rearing technique was conducted according to the methodology described by Dahi (1997). Newly hatched larvae were reared on fresh caster bean leaves (Ricinus communis L.) and supplied daily until pupation. The pupae were collected and transferred to new wooden boxes (35 × 35 × 35 cm 3 ), covered with a layer of fine wood dust to keep moisture until adult moth emergence. Adults were fed on 10% sugar solution hanged inside the cage. The inner walls of the cage were covered with sheets of (A 4 ) white copy paper, and branches of oleander (Nerium oleander L.) as the ovipositional site. Newly laid egg masses were collected daily to start the experiments, and the others were used to maintain the insect colony.

T. bactrae rearing
The parasitoid used in this study was imported for the first time from Australia in 1992 by Dr. A. H. El-Heneidy (ARC, Egypt) and was established under Egyptian environmental conditions (Mohamed and El-Heneidy 2020). This parasitoid species was mass-reared on eggs of the Angoumois grain moth Sitotroga cerealella (Oliver) (Lepidoptera: Gelechiidae) for 4 successive generations, under standard rearing conditions.

Experimental design
The deposited egg masses (≤ 24 h) (approximately 150 ± 50 eggs) according to their physical characteristics (no. of egg layers and degree of scale density) were divided into 9 treatments as one layer of eggs with no scales, with low scale density, with mid scale density, and with high scale density; two layers of eggs with low scale density, with mid scale density, and with high scale density; and three layers of eggs with low scale density and with high scale density. The degrees of scales were divided according to Brotodjojo and Walter (2006). Thirty newly emerged T. bactrae wasps without any previous oviposition experience were exposed to each type of the egg masses in plastic jars (500 ml) for 24 h to avoid super-parasitism in 2 types of tests separately. Nochoice test, by offering only one type of egg masses individually, and for choice test, by offering all tested egg masses simultaneously to the adult parasitoids. After 24 h of exposure to the parasitoid, parasitized egg cards from each treatment were collected and maintained in new plastic jars (250 ml) under standard rearing conditions. The egg masses were replaced by new ones for 3 days. The length of the experiment was decided depending on Doyon and Boivin (2005) that more than 70% of parasitization occurred within the first 3 days after adult emergence. For each treatment (egg masses), 30 replicates were used.

Biological parameters
The fitness of the egg parasitoid, T. bactrae on S. littoralis egg masses with different physical characteristics was assessed by measuring the following biological variables in both no-choice and choice tests as parasitism percentage (no. of parasitized eggs (blackened eggs)/total no. of eggs exposed). The total number of eggs on each egg mass was determined by counting (no. of hatched larvae + unhatched eggs (dead larvae) + blackened parasitized eggs) (Dequech et al. 2013). Besides, developmental period (days) (the average time till adult emergence), adult emergence rate (no. of all emerged adults/no. of parasitized eggs), and females' ratio by examining dead adults under a stereomicroscope (no. of emerged adult females/total individuals) were determined. The longevity of adults was recorded daily by keeping 50 emerged adults individually from each examined egg masses into glass vials (2 cm diameter × 4 cm height) till mortality. For parasitoid nutrition, few droplets of 10% sugar solution were provided daily until the wasps died.

Statistical analysis
Obtained data were subjected to one-way analysis of variance (ANOVA). Data were arcsine √proportions transformed before analysis to meet normality. Means were separated by t test at P ≤ 0.05 level. All calculations and graphs were used by Microsoft Excel® software according to Fowler et al. (1998).

Parasitism percentage
The T. bactrae wasps were able to parasitize the S. littoralis egg masses, but with different rates (Fig. 1) according to their different layers and degrees of scales' thickness, in both no-choice and choice tests (Table 1). Regardless the degree of scales, the highest parasitism percentage was significantly (P < 0.001) recorded in one layer eggs (66.67, 60.53%), followed by two layers (58.23, 48.53%), while the least preferable one was three layers egg masses (29.03, 22.67%) in both tests, respectively. In addition, the thick scaly eggs with three layers had significantly (P = 0004, 0.007) the least parasitism rate (< 20%) in all egg masses in both tests, respectively. When the parasitoids had no choice on egg masses, the rates of parasitism were more prominent than when those were given choice among different eggs. The percentage of parasitism was highly significantly differed among all tested egg masses (ANOVA, F 8,168 = 33.421, 73.069; P < 0.001) in no-choice and choice tests, respectively.

Developmental period
The average time required to emerge the parasitoid was non-significantly changed among the egg masses with different physical characteristics (ANOVA, F 8,72 = 0.337; P = 0.949) in the case of no-choice test (Fig. 2). In contrast, when the parasitoids were allowed to choose between all examined eggs at a time, the developmental period varied significantly (ANOVA, F 8,72 = 2.625; P = 0.013). These differences were associated only with the degree of scales covering the eggs, not the layers.

Adults' emergence percentage
High numbers of adult emergency were observed in all egg masses in both tests ( Table 2). The percentage of emergence was significantly changed (F 8,126 = 5.385; P < 0.001) as a result of the scales' thickness only in the case of no-choice test. On the other hand, both of the layer's number and the degree of scales affected significantly on the emergence rate of the parasitoid in choice test (F 8,126 = 17.218; P < 0.001). The lowest emergence was recorded on high-scaly three-layer eggs in both tests.

Females' percentage
The overall sex ratio of emerged adults was femalebiased, but with different rates, regardless of the number of egg layers as well as the scale density (Table 3). The only exception was noticed in the case of one-layer eggs covering with thick scales; the percentage of females was decreased dramatically in both tests and recorded the lowest rate (≤ 45%). The female percentage was highly significantly influenced by the different physical characteristics of eggs in no-choice and choice tests (ANOVA, F 8,92 = 17.946, 13.779; P < 0.001), respectively.

Longevity of adults
The survival of fed female parasitoid at 25°C was non-significantly affected by the egg layers and the scale density in both tests (ANOVA, F 8,125 = 0.385, 1.455; P = 0.927, 0.185), respectively (Fig. 3).

Discussion
There is an urgent need for minimizing the dependence of chemical pesticides in the management of insect pests. Therefore, in view of the problems based on the chemical, alternative environmentally friendly methods are essentially needed. Most of the researches focused on different Trichogramma species against different Spodoptera species and none of them studied T. bactrae and its association with S. littoralis. It is important to bear in mind that understanding and knowing the factors affecting the efficacy of T. bactrae wasps against S. littoralis eggs with different physical characteristics is crucial to improve the control program against this pest in the field. Host preference is an important parameter for biological control programs as more than one pest species of different physical characteristics may occur in the field (Goulart et al. 2011). Meanwhile, host recognition and acceptance are mainly driven by physical and chemical cues, which are mostly detected by the sensilla on the antennae and the ovipositor; these assess the suitability of a host for parasitization (Schmidt 1994). Parasitism capacity is an important indicator for the parasitoid efficiency, reproduction, and flourishing as a bio-control agent (Nurindah and Cribb 1997). Based on the present results, the higher parasitism rates of T. bactrae were observed on the single-layer eggs, maybe due to the parasitoid was able to access and examine all the eggs deposited in the mass, followed by the twolayers. However, three-layer eggs had the least rate in both no-choice and choice tests, because the parasitoid was only able to arrive and parasitize the outer, the edge, and/or the exposed eggs, but unable to parasitize the lower layer. Based on our observations, scales with low and mid thickness degree covering the eggs seemed to be as a chemical stimulus and attracted the female parasitoid of T. bactrae to parasitize, but with different rates related to the overlapping layers. The findings are mostly in accordance with those reported by other researchers on different Trichogramma species as mentioned by  that the parasitism percentage on S. frugiperda egg masses with one, two, and three layers were (66.24, 45.20, and 40.10%), respectively. Besides,  recorded 63.2% parasitism by T. atopovirilia on single-layer eggs of the same pest. As a result of releasing T. pretiosum parasitoid against this pest on maize fields, the rates increased according to the number of releases (1-3) (69.8, 79.2, and 68.75%), respectively (Figueiredo et al. 2015). On the other hand, T. pretiosum and T. minutum wasps were parasitized on (44.8 and 51.6%) of S. exigua eggs, respectively (Greenberg et al. 1998). On S. litura eggs, the parasitism rate by T. chilonis reached 80.31% (Puneeth and Vijayan 2013). Obtained results on the high-scaly eggs seemed to be less attractive or unpreferable host and had a negative impact on overall behavior. This may be because of different females of Trichogramma species reabsorbed their oocytes during unsuitable conditions or hosts' deprivation, causing a reduction in parasitism percentage and the period of fertility (Hougardy et al. 2005). The parasitism rates on S. frugiperda egg layers with thick scales were very low (≤ 10%) as reported by Toonders and Sánchez (1987) and Beserra et al. (2002 in Brazil and Mexico. This may be due to the females avoided parasitism on high-scaly eggs that prevents their progeny, searching for new preferable ones. Another aspect as reported by Noldus (1989), S. frugiperda eggs are rarely attacked by T. pretiosum because this parasitoid does not respond to its semiochemicals and thus led to low parasitism.  Occasionally, T. bactrae parasitoid tried to remove the scales by moving on the egg mass and passing the front legs over the antennae, also rubbing the hind legs with each other's, cleaning the scales adhered to the body. Similar behavior was observed by the egg parasitoid, Telenomus remus Nixon females on S. frugiperda egg masses (Carneiro and Fernandes 2012).
Ultimately, the variability in the number of parasitized eggs noticed on the previous and the present studies may be related to the different Spodoptera species, also to the geographical origin, and the parasitoid strain. Therefore, an adaptation of a specific parasitoid strain from a specific region against the target pest must be taken into consideration for the success of the biological control program.
Afterwards, high adult emergence rates were recorded in all examined egg masses, with exception of high-scaly three-layer eggs. This finding was in similar line with that of Bueno et al. (2010) who recorded the emergence rate of T. pretiosum from S. frugiperda eggs was higher than 88% in all tested temperatures (18-32°C). Another important parameter in the biological control programs is sex ratio. Higher rates of females in their progeny are vital in mass production and responsible for the direct suppression of the target pest when released in the field (Bueno et al. 2009). With the following mating, the female wasps store their sperms in the spermatheca, and through parasitization, it can decide the sex ratio of their progeny by controlling the entry of sperm to the egg (Suzuki et al. 1984). At the present experimental conditions, the overall sex ratio of emerged adults among all examined egg masses was female-biased. Contrary to this prediction, on high-scaly eggs with a single layer, the percentage of females decreased significantly (< 45%) in both no-choice and choice tests. This may be due to the presence of densely scales covered the eggs, making them as unsuitable or unattractive hosts for the decision to deposit fertilized eggs and thus decreased the number  of produced females in the progeny. In addition, as reported by Houseweart et al. (1983), the sperm depletion or decrease in the females' spermatheca led to a decrease in the number of fertilized eggs, so it forced to parasitize but produced unfertilized eggs (males). Certainly, this laboratory study is necessary to improve the biocontrol program against this pest. Moreover, further field studies of the efficacy of this parasitoid toward different Spodoptera species are needed.

Conclusions
The results proved that T. bactrae is an important parasitoid species with a great ability to parasitize S. littoralis egg masses related to their layers and the degree of scales' thickness, in choice and no-choice tests. The most preferable egg mass to this parasitoid was one layer, followed by two layers. Highest numbers of adult emergency were observed in all examined egg masses, except in the case of the 3 layers with high-scaly eggs. T. bactrae could be used as a bio-control agent against S. littoralis egg masses with their different physical characteristics.