Effect of entomopathogenic nematodes Steinernema species (steinernematidae: rhabditida) and Heterorhabditis bacteriophora (heterorhabditidae: rhabditida) on the digestive enzymes and midgut histology of the African migratory locust Locusta migratoria migratorioides (acrididae: orthoptera)

In some governorates of Egypt, the African migratory locust L. migratoria migratorioides is in a gregarious phase. Swarm development was successfully prevented by biological control agents. In this work, the two entomopathogenic nematode species, Steinernema sp. (SII)and Heterorhabditis bacteriophora (HP88) were investigated as natural enemies against the fifth nymph and adult African locusts at various concentrations (300, 600, 900, 1200, and 1500 Infected juveniles/100 g. soil). To ascertain fatal activity at 25 °C, the nematode-inoculated sand method was used. The two nematode species were semi-field administered against fifth nymphs and adult stages at (25 ± 2 °C) and 55–60% relative humidity with concentrations (3000, 6000, 9000, 12000, and 15000 Infected juveniles/kg. soil). Protease, amylase, invertase, and trehalase levels in the digestive enzymes of both fifth nymphs and adults fed with LC50 of both nematodes significantly decreased, but lipase and chitinase levels significantly increased. Adult locusts treated with the LC50 of S. sp. SII had basophilic epithelial cells, severe lumen hemorrhage, and highly aberrant proliferating cytoplasm, whereas the LC50 of H. bacteriophora HP88 displayed necrosis in an epithelial cell with vacuoles, loss of nucleus, and loss of goblet cells.


Introduction
Because they form large swarms that travel across nations, consume crops, and disrupt agriculture, locusts have long been dreaded (Lecoq and Cease 2022). A subspecies of the migrating locust is called Locusta migratoria migratorioides. An epidemic of L.migratoria migratorioides was reported in Angola in 2020, (Dcha 2020). It was present in Egypt's western desert and played a significant role in the large land reclamation initiatives of Sharq El-Owinat and Toshka (Soliman et al. 2019). The first line of defense against wild locust swarms, which can contain up to 8 million locusts, is chemical insecticides, although the majorities of them are non-specific and can affect both beneficial and harmless living things. The food chain has also been impacted by the repeated use of pesticides that are persistent and not biodegradable, which have damaged ecosystems in the water, air, and soil (Carriger et al. 2006;Gunstone et al. 2021). Acute or chronic illnesses can develop in people who have been exposed to pesticides, either directly or indirectly (Mostafalou and Abdollahi 2012). Additionally, it has an impact on both target and non-target creatures, including birds, fish, amphibians, humans, earthworms, predators, and pollinators (Gill and Garg 2014). However, the high costs of emergency response and rising understanding of the negative environmental effects of chemical control make biological control more appealing (Lomer et al. 2001;Zhang and Hunter 2017). Nematodes, protozoa, bacteria, fungi, and viruses are examples of biological agents (Rai et al. 2013;Deka et al. 2021). Except for Antarctica, entomopathogenic nematodes (EPNs) from the families Steinernematidae and Heterorhabditidae are widespread and employed to control soil-borne insect pests (Wright et al. 2005). Both Heterorhabditis bacteriophora (Heterorhabditidae) and Steinernema sp (Steinernematidae) may survive in a wide range of climatic situations, including grasslands, forests, and coasts, from hot regions to cold mountains (Bhat et al. 2020;van der Linden et al. 2022). The EPNs, S. sp. (SII), and H. bacteriophora (HP88) have been applied against many insect pests, such as red palm weevil Rhynchophorus ferrugineus , the sand flies Phlebotomus papatasi . The EPNs, Steinernema sp, and H. bacteriophora, have been used against a variety of insect pests, including the tea mosquito bug Helopeltis theory and bunch caterpillar, Andrea bipunctata (Amuri and Devi 2020), locusts and grasshopper (Ibrahim et al. 2018). The current work compares the effectiveness of two EPNs, S. sp. SII and H. bacteriophora HP88, against fifth nymphs and adults of the African migratory locust, L. migratoria migratorioides, in a lab setting at 25 o C and open air in semi-field conditions.

Locust rearing
Adult locusts, L. migratoria migratorioides (♂ and ♀), were cultured in a laboratory using samples taken from field-grown maize in the Abu Rawash town of Giza, Egypt. Locusts were raised for more than two generations in the gregarious phase in wooden cages (40 × 40 × 30 cm) at a density of (30-60) adults/cage. For the locusts to lay the eggs, each cage had a third of the sand. Instead of using artificial light, locusts were exposed to sunlight for more accurate findings (Hill and Taylor 1933). Every day, cages were cleaned. Depending on the season, fresh alfalfa (Medicago sativa) or corn (Zea mays) leaves were fed to the locusts.

Entomopathogenic nematode species
S. sp. SII and H. bacteriophora HP88, two EPNs, were acquired from the Department of Parasitology and Animal Diseases at the National Research Centre in Dokki, Egypt, and were raised for numerous generations in the Plant Protection Institute at the Agricultural Research Center. Infectious juveniles (IJs) of the two EPNs were housed in the last instar larvae of the larger wax moth Galleria mellonella.

Bioassay test
At the Department of Pest Physiology, Plant Protection Institute, ARC, Egypt, studies were conducted using doses of 300, 600, 900, 1200, and 1500 IJs/100 g. soil for two tested EPN species at 25 o C 60% for 7 days against fifth nymphs and adults of L. migratoria migratorioides. Nematode-inoculated Sand as a method: 100 g of sterilized, air-dried sandy soil (60 sand, 20 clay, and 20% silt) was combined with the aforementioned amounts of two applied EPNs in aerated plastic jars (8 × 10 × 10 cm), (Van Sambeek and Wiesner 1999). The soil's moisture content was preserved at 10% (v/w) by adding distilled water. Freshly cleaned maize leaves were fed to the locusts. Each nematode species' IJs were exposed to three adults or five nymphs /aerated plastic jars. A sevenday check was carried out to determine the fatality rate. Two nematode species, five concentrations, one species of locust, two instars, and three vessels/concentrations were employed in total for the testing. Nymphs and adults were kept under control with only water. The test was repeated in three trials over time using the two nematode species.

Semi-field experiment
The experiment's goal was to investigate the effects of EPNs SII and HP88 infection on fifth-and adult-stage African locusts in settings that were more like nature. To prevent external infestations and prevent the experimental locusts from escaping, all containers in this study were covered with netting material. In this study, 50 fifth instar nymphs or 30 adults / plastic containers (20 × 15 × 15 cm) containing 1 kg of moistened, sterile sand. 3000, 6000, 9000, 12000, and 15000 (IJs/ kg. soil) were the two EPN concentrations. Three replicates, fifty 5 th nymphs or thirty adults, and either a concentration or control were employed. The experiment was conducted three times using the two nematode species. At a temperature of 25° C 2, a soil moisture content of 20%, and a relative humidity of 55-60%, all containers were placed near the field-grown corn in the Plant Protection Institute, Giza, Egypt, in September. After 7 days of treatment, the mortality rates of both the 5 th nymphs and the adults of the L. migratoria migratorioides were recorded (Nouh 2022).

Reproduction of EPNs in L. migratoria migratorioides
The bioassay experiment produced dead locust cadavers, which were collected and stored in white traps (White 1927;Sobhy et al. 2020). A single adult or nymph locust was placed into each trap, which was subsequently incubated at 25 °C. The nearby water was used to introduce the newly emerged worms. These were taken out of the water and placed in storage bottles. It took up to 10 to 14 days for all of the EPNs to be harvested. Statistics were run on the nematode emergence data that were collected.

Digestive enzyme activities assayed for 5 th nymphs and adult locusts
Fifth nymphs and adults of locusts were used as controls, and after being exposed for 72 h in semi-field plastic containers, they were given the LC 50 doses of SII and HP88. After the locusts' wings and legs were removed, they were homogenized to create a sample solution and test the digestive enzymes' [protease, lipase, amylase, invertase, trehalase, and chitinase] activities by the procedures outlined by (Kreema et al. 2021), with a slight modification to the lipase activity outlined by (Singab et al. 2022). Each replication was taken from 30 locusts, both fifth nymphs and adults, and three replicates were analyzed.

Histopathological effects of the midgut of the adult locust
As controls, ten adult locusts were employed, and the same number of them received the LC 50 concentrations of both species' EPNs. According to (Nasiruddin and Mordue 1993), insects were dissected in Ringer solution (pH 6.8), and the midgut was isolated from the rest of the gut, fixed in Bonn's fluid, and embedded in paraffin.

Statistics
The lethal activity of EPNs species was analyzed using (SPSS) version 27 software (SPSS Inc 2020), to determine the LC 50 , LC 90 , lower bound, and upper bound (95% confidence limits). P < 0.05 denoted a significant difference between groups when analyzing the reproduction rate of the tested EPNs species and the impact of EPNs on biochemical analyses.

Lethal activity
In Abu Rawash, Giza, Egypt, the destruction of greenery by locusts is represented in (Fig. 1a). The LC 50 values for the fifth nymphs and adults, when SII was delivered via the nematode-inoculated sand approach, were (1201.953, 1279.473 IJs/100 g. soil), respectively, according to ( Table 1). The LC 50 values for the treatment with HP88 utilizing the nematodeinoculated sand method were (1960.94, 2071.085 IJs/100 g. soil) for the fifth nymphs and adults, respectively (Table 1), (Fig. 1b, c). The semi-field application of the EPN, SII indicated LC 50 values for fifth nymphs and adults of (6695.01, 8123.9 IJs/kg soil), whereas the treatment with HP88 revealed LC 50 values for fifth nymphs and adults of (10119.7, 12285.4 IJs/kg soil), respectively ( Table 2). The treated deformed adults were shown in (Fig. 2a-c) as the fifth nymphs with the highest concentrations of SII and HP88 (15000 IJs/ kg).

Reproduction of EPNs in L. migratoria migratorioides
The results of the reproductive experiment showed that every examined EPN species was able to enter and reproduce inside the hemocoel of adult locusts that were in their fifth instar (Figs. 3 and 4). For fifth-stage nymphs, there were significant variations in the production of IJs between the two strains (F = 13.96; d f = 2; P = 0.001), and for adults (F = 9.24; d f = 2; P = 0.004). The highest progeny output among the examined EPNs was noted for SII (78333 881.61, 200333 Fig. 1 a Severe damage to the green leaves of some plant leaves was affected by the African migratory locust, Locusta migratoria migratorioides, in Abu Rawash, Giza, Egypt. b Died fifth nymph of L. migra-toria migratorioides treated with SII and HP88. c Died adult's ♀ and ♂ of L. migratoria migratorioides treated with SII and HP88 1 3 1201.47) for 5 th nymph, adults, respectively. HP88 (32666 333.1, 95000 1154.7) was the next best progeny producer for 5 th nymph, and adults, respectively. Additionally, for the two EPN isolates studied, the study found a good connection between mean reproduction rates and nematode concentrations (r = 0.998 in SII, r = 0.999 in HP88) for the 5 th instar, (r = 0.989 in SII, r = 0.955 in HP88) for the adult stage.

Histopathological observations of the adult L. migratoria migratorioides treated with SII and HP88
In the present study, the histological characteristics of the midgut were explored. The basic cell type of the midgut in adult locusts is the goblet cell, which is located between the columnar epithelial cells and degenerative cells at the basement membrane (Fig. 5a). Numerous villi were observed, which were well supplied with blood vessels. Villi had apical microvilli that projected into the lumen. Goblet cells, interstitial glands, and columnar cells constitute the main bulk of the epithelium of the midgut (Fig. 5b). Large numbers of goblet cells were found, while the interstitial cells occupied the spaces between the goblet cells. In addition, longitudinal and circular muscles were present. After injection of SII, the epicuticle showed severe corrugation, and the epidermal layer was completely split. The epithelial cells showed basophilic, the lumen showed severe hemorrhage, and the cytoplasm showed severely abnormally proliferated (Fig. 6a, b). Injection with HP88 induced a thinning and corrugated cuticular surface with distortion Fig. 2 a Malformed and dwarf adult L. migratoria migratorioides treated as 5 th instar nymph, with SII. b Malformed adult L. migratoria migratorioides treated as 5 th instar nymph with HP88. c Control adult L. migratoria migratorioides Fig. 3 Progeny production by 5 th nymphal L. migratoria migratorioides exposed to different concentrations of infective juveniles (IJs) of Entomopathogenic nematodes (EPNs): SII and HP88. In a bar column with different alphabet letters were significantly different at P = 0.001 of subcuticular layers. The musculature region showed a disorganized appearance. These changes included necrotic epithelial cells with vacuoles, loss of nuclei, and loss of goblet cells (Fig. 7a-c).

Discussion
The results of the current study showed that the EPNs S. sp. (SII) and H. bacteriophora (HP88) were effective against the Fig. 4 Progeny production by adult L. migratoria migratorioides exposed to different concentrations of infective juveniles (IJs) of Entomopathogenic nematodes (EPNs): SII and HP88. In a bar column with different alphabet letters were significantly different at P = 0.004  traits (EPN species/stain, application methods, behavior, and type of bacteria hosted), and host traits (species, development, immune system, and molecules emitted by the host), additionally, the abiotic environment (temperature, humidity, UV radiation, soil characteristics, and chemicals) and the biotic environment (rhizosphere characteristics, molecules emitted by damaged roots, and natural enemies) are divided into two categories (Mráček et al. 2005;Labaude and Griffin 2018). The symbiotic bacterium Xenorhabdus nematophila, which is connected to the intravascular structure, was found in significant numbers of Steinernema sp. IJs. The insecticidal toxins it produced after being released into the host insect's hemolymph traveled to the connective tissues around the midgut, muscle fibers, and tracheae, where they caused harm and served as a source of nutrients for bacterial and nematode development. In addition, a significant portion of the H. bacteriophora IJs included the symbiotic bacterium Photorhabdus luminescens linked to the intestinal lumen. They moved to the region between the extracellular midgut epithelium after being discharged into the hemolymph of the host insect. There, they produced insecticidal toxins that damaged the insect tissues and turned them into a nutritional soup for bacteria and nematodes (Hinchliffe et al. 2010;Da Silva et al. 2020;Santhoshkumar et al. 2021). According to (Chapman and Chapman 1998), digestive enzymes are primarily generated and secreted by the midgut epithelium of insect alimentary ducts from the brush edge of microvilli on the apical membrane. Amylase and protease, two digesting enzymes, were also found in the salivary gland complex (Li et al. 2017). It depends on feeding habits, the quality, and quantity of the food consumed, as well as the specific midgut habitats, how the digestive enzymes in the gut are made is complex (and frequently species-specific) (Holtof et al. 2019). The digestive enzymes lipase, amylase, and protease, which metabolize sugars, lipids, cellulose, and proteins in the insect midgut, are crucial for the production of energy and the metabolism of insect nutrition (Gökkuş et al. 2016;Bonelli et al. 2020). The digestive enzymes in this study's locusts were protease, lipase, amylase, invertase, trehalase, and chitinase since they consumed maize leaves. Different levels of the evaluated digestive enzymes were found in treated locusts compared to control ones after treatment with the LC 50 of both EPNs. Nymphal and adult-treated locusts showed a considerable reduction in protease activity. This is supported by numerous kinds of research on pests in general (Wang et al. 2012;Ibrahim, et al. 2015Ibrahim, et al. , 2018. The hydrolysis of triacylglycerol into its free fatty acids and glycerol backbone is one of the many reactions that lipase is in charge of (Yao et al. 2021). Because X. nematophila or P. luminescens bacteria secrete various secreted enzymes, such as hemolysis, lipases, and proteases that contribute to pathogenicity or nutrient acquisition for the bacterium and its nematode host, lipase activity was dramatically boosted (Richards and Goodrich-Blair 2010). Due to the toxic effects of the EPNs, S. sp. (SII), the H. bacteriophora (HP88), and their associated bacteria, the treatment of 5 th nymph and adult locusts considerably reduced the levels of amylase, invertase, and trehalase when compared to the control (Muhammad et al. 2022). This was accepted (Ibrahim, et al. 2015;Tang et al. 2018). Chitin, a polymer of N-acetyl-D-glucosamine, is mostly produced by fungi, arthropods, and nematodes and makes up a significant portion of the insect cuticle. It supports the cuticles of the skin and trachea, the peritrophic matrices lining the gut epithelium, and insect development and morphogenesis in insects (Kramer and Muthukrishnan 1997;Merzendorfer and Zimoch 2003;Subbanna et al. 2018;Da Silva et al. 2020 andHenriques et al. 2020). Due to the presence of X. nematophila or P. luminescens bacteria, which create chitinase for their growth rate, the amount of chitinase was greatly enhanced in this study (Chen et al. 1996).
Dedication To the soul of Professor Monzer Mohammed Abd El-Rahman.