The adverse impact of transforming neem oil (Azadirachta indica) into a nano formulation for application as a botanical pesticidal agent against the desert locust Schistocerca gregaria

Introduction

The desert locust crisis impacts numerous countries and regions. Typically residing in 30 countries, desert locusts have the potential to expand their range to as many as 60 during periods of population surges. These insects are among the oldest migratory pests globally, inflicting extensive damage to crops. Outbreaks of the desert locust species Schistocerca gregaria are not a recent occurrence. Among various locust species, S. gregaria is particularly harmful. Although locusts belong to the grasshopper family, they exhibit behavioral changes that enable them to form dense, mobile swarms and migrate over considerable distances. An invasion of desert locusts represents a serious threat to food security, as these insects can consume an amount of food equivalent to their body weight each day. For example, a swarm occupying a single square kilometer can contain a range of 40 - 80 million adult locusts, and a small swarm of 1 km² can consume as much food in a single day as would be required to feed 35,000 individuals (FAO, 2020). Desert locusts undergo an incomplete metamorphosis characterized by three developmental stages: egg, hopper (nymph), and adult. Locusts are classified as solitarious when they exist in low population densities and as gregarious when found in high densities. The desert locust, S. gregaria, exhibits significant plasticity in its behavioral, coloration, morphological, and physiological characteristics in response to factors such as crowding, temperature, and other external conditions. As the population of solitarious hoppers increases, their behavior shifts, leading to aggregation and the formation of groups, particularly when vegetation begins to desiccate. Additionally, their coloration alters, resulting in the appearance of dark spots (FAO, 2001).

In recent times, insecticides have faced considerable criticism due to their environmental hazards, the development of resistance among insect populations, and their impact on non-target organisms. The understanding of the necessity to minimize the use of chemical pesticides and safeguard the ecological environment is steadily advancing. In the management strategies for desert locusts, we are also diligently seeking new pesticides that pose minimal risks to non-target organisms, operators, residents, livestock, and the surrounding environment (Bayoumi et al., 2022; Van Huis et al., 2007; Lomer et al., 2001). As a result, the pursuit of new environmentally friendly solutions has become a significant undertaking (Nassar et al., 2018). Researchers prioritize strategies aimed at preventing desert locust populations from escalating to harmful levels. The application of oils for locust control offers the benefits of being environmentally friendly and ecologically safe, thereby ensuring sustainable management. Plants are regarded as one of the most abundant sources of natural bioinsecticides, which can be utilized as pest control agents. This includes the use of botanical toxicants, repellents, synergists, growth regulators, and antifeedants to combat various insect pests (Disha et al., 2020; El-Shazly et al., 2019; El-Sonbati et al., 2014).

Azadirachtin, recognized as the primary active ingredient in neem oil, is predominantly found in the seeds and has been shown to significantly enhance mortality rates, interfere with metamorphosis, and reduce overall activity levels in locusts and grasshoppers. Azadirachtin, for instance, has the capability to diminish food intake, disrupt the success of moulting from the fifth instar to the adult stage, induce the occurrence of over-aged nymphs, inhibit the growth of desert locusts, and influence their reproductive and developmental processes. Neem oil has the potential to reduce the reproductive capacity and egg-hatching success of desert locusts, while also influencing their growth and development (Bashir & El Shafie, 2014).  It acts swiftly, decomposes rapidly, and exhibits low toxicity to mammals (Lomer et al., 2001). So, using neem oil has the advantages of environmental friendliness, ecological security, hence controlling locusts continuously. The present study is an attempt through semi-field conditions to evaluate the efficacy of azadirachtin in both forms bulk and nano against S. gregaria.

Materials and methods

Mass rearing of insects

The insects were obtained from the stock culture maintained for several generations at Locust and Grasshopper Research Department, Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt. Insects were reared under crowded conditions in the laboratory according to (Ouedraogo et al. 2002) in specific closed cages, fed on brunches of Egyptian clover, Trifolium alexandrinum, leaves in winter and Sesban, Sesbania sesban, in summer. The locust's cages were kept at 30±2°C and 30-50% relative humidity.

Preparation of nano capsules

Polyethylene glycol (PEG) loaded nanoemulsion was formulated utilizing an oil-in-water (o/w) emulsification technique, based on a modified approach outlined in references (Zhang et al., 2008; Mohammad et al. 2022). The PEG-neem nanocapsules were synthesized through the miniemulsion polymerization method. Neem oil (obtained from Luna Company, Egypt) was emulsified with distilled water in a 1:1 (v/v) ratio using Tween 80, with stirring conducted for 10 minutes. The resulting emulsion was then introduced into a 3% PEG solution at a 1:1 (v/v) ratio while maintaining drop wised in continuous mechanical stirring at ambient temperature. The emulsion underwent sonication for 60 minutes using an ultrasonic cleaner, model WUC-DO3H, operating at 290W and 60 Hz, followed by an additional 3 minutes of sonication with a high-energy ultrasonic probe (model VCX750, 750W, 20 kHz). The suspension of the loaded nanocapsules was allowed to equilibrate overnight, resulting in the formation of nanocapsules dispersed in an aqueous solution.

Transmission Electron Microscopy (TEM)

The morphological shape of prepared nano-formulation was carried out using TEM (Jeol, JEM-2100). The nano-capsule suspension was diluted with distilled water and deposited onto a carbon coated copper grid and particle sizes were determined by TEM; the diameter of the particles is shown using the scale bar of the photo (Fig.1).

Gas chromatography/mass spectrometry (GC/MS)

The analysis of crude and PEG nano-capsules via GC-MS was conducted utilizing a gas chromatography-mass spectrometry instrument located at the Department of Medicinal and Aromatic Plants Research, National Research Center. The equipment employed was a TRACE GC Ultra Gas Chromatograph (THERMO Scientific Corp., USA), which was coupled with a thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer). The GC-MS system featured a TG-5MS column (30 m x 0.32 mm i.d., 0.25 μm film thickness). The analyses were performed with helium as the carrier gas at a flow rate of 1.0 mL/min and a split ratio of 1:10, following a temperature program that commenced at 60°C for 1 minute, then increased at a rate of 3.0°C/min to 240°C, where it was maintained for an additional minute. Both the injector and detector were set to 240°C. Samples were diluted (1:10 hexane, v/v) and 1 μL of the mixtures was injected consistently. Mass spectra were acquired through electron ionization (EI) at 70 eV, covering a spectral range of m/z 40-450.

UV spectrophotometric examinations

The active ingredient contents of loaded nano-capsules were assessed through a refluxing process. A total of 1g of the loaded nano-emulsions were refluxed with 10 ml of methanol at 65°C for one hour to ensure complete extraction of the encapsulated oil. Following this, the samples were centrifuged at 10,000 x g for 10 minutes. The absorbance of the methanol solution containing the extracted neem oil was measured at 214 nm according to Jerbin et al. (2012), using a CHEM-7 UV spectrophotometer, with absolute methanol serving as the blank. The oil concentration was determined using a calibration curve derived from pure oil samples within a specific concentration range for neem oil. The encapsulation parameters were established following the methodology outlined by Kumar et al. (2014) as detailed below:

Bioassay tests

Five topical applications were conducted to investigate the effects of neem oil from A. indica, in both its nano and bulk forms, on S. gregaria. The treatments were administered to one-day-old fifth instar nymphs following their most recent fourth molt. The treatments included two doses of neem oil at concentrations of 5 ppm and 2.5 ppm. So, the insects were inoculated (using the micro pipette) with 10 and 5 µl per nymph for the two doses, respectively. These concentrations were based on the assumption that a drop of 5µl of the solution will reach the insect by using Ultra Low Volume (ULV) sprayer. So, we converted this volume to ppm and utilized it and its double as two concentrations. Additionally, a control group of untreated nymphs was included. The control and experimental nymphs were maintained individually in plastic cylinder containers (10 cm X 25 cm), with six replicates for each group, comprising five nymphs per replicate. As the capsule ingredients are not separately used to control the insect, they were used as built-in parts of the treatments, not in the untreated check. During the experiment, the nymphs were provided daily with branches of Egyptian clover, T. alexandrinum, leaves and were monitored until they either molted into adults or died. The duration of each nymphal instar and appearance of any malformations (insects shown damage in their legs, wings, and antennae after molting into adults) were meticulously recorded, and the entire procedure was conducted under semi-field conditions (not controlled but ambient temperature and humidity).

Biochemical activity

Preparation of enzyme extracts

The biochemical impacts of neem oil, whether in bulk or nano-formulations, were investigated, focusing on total protein and chitinase activity. The treatment involved the topical application of neem emulsions (both nano and bulk) to fifth instar nymphs of S. gregaria at concentrations of 2.5 and 5 ppm. Enzyme activity and total protein levels were assessed after 24, 48, and 72 hours. The nymphs were homogenized in sodium phosphate buffer (0.1M, pH 7.0) at a ratio of 0.1 g of body weight to 1.5 ml of buffer. The homogenates were then centrifuged at 10,000 xg for 15 minutes at 4°C, and the supernatant was utilized as the enzyme source. This supernatant was subsequently stored at -20˚C until further analysis.

Evaluation of total protein

Total protein concentration was determined using a colorimetric method with the Protein - Biuret kit from Biogiagnostic Company, following the procedure outlined by Gornall et al. (1949). The sample mixture consisted of 25 µl of the sample and 1 ml of the protein reagent (alkaline cupric sulfate). Similarly, the standard mixture included 25 µl of the standard and 1 ml of the protein reagent. All mixtures were thoroughly combined and incubated for 10 minutes at 37°C. The absorbance of the sample (ASample) and the standard (AStandard) was measured against a reagent blank at a wavelength of 550 nm. The color remained stable for one hour. The protein concentration was calculated using the following equation:

Protein Concentration (mg/g insect body weight) =

Determination of Chitinase activity

Chitinase activity was evaluated utilizing 3,5-dinitrosalicylic acid reagent to quantify the free aldehydic groups of hexosaminase released during chitin digestion, following the methodology outlined by Ishaaya & Casida (1974). The specific activity of chitinase is reported as µg of N-acetyl glucosamine released per minute per milligram of sample.

Statistical analysis

One-way analysis of variance (ANOVA), followed by Tukey’s HSD test (SPSS software) was used to analyze data. Statistically significant means were separated at P < 0.05.

The mortality was corrected according to Abbott's formula (Abbott, 1925)

Where: n = Number of insect population, T = treated, Co = control

Results

Transmission Electron Microscopy

The morphological characteristics and particle sizes of the synthesized neem nano-emulsion were analyzed using TEM, as depicted in Fig. 1. The particles of the nano-formulation exhibited an almost spherical morphology with a smooth surface texture. The preparation of PEG nano-capsules containing neem oil was conducted through a multi-step process utilizing a high-energy ultrasonic technique. The resulting emulsion of neem oil and PEG manifested in the form of core-shell capsules, where the inner core comprised the oil and the shell was constituted of PEG serving as the loading material. The average sizes of the cores ranged from 30 to 100 nm, while the dimensions of the core-shell capsules varied between 90 and 120 nm.

Fig. (1): Transmission electron microscopy of the prepared neem nanoemulsion loaded on PEG

GC/MS analysis of crude and Poly Ethylene Glycol (PEG) loaded emulsion

The chemical composition of neem oil, specifically in its crude and PEG-loaded emulsion forms, was analyzed using GC/MS, as depicted in fig. 2 and 3, and detailed in table 1. The spectral analysis revealed the presence of eight distinct peaks. The compounds within each emulsion were identified, showing percentages of 98.75% for the crude emulsion and 97.63% for the nano emulsion. Oleic acid, the predominant compound in both emulsions, accounted for 55.64% in the crude form and 63.37% in the PEG-loaded nanoemulsion. This was followed by palmitic acid, which constituted 19.02% and 20.56% in the respective emulsions. Notably, linoleic acid exhibited a significant reduction during the nano preparation process, with values of 14.90% and 0.99% for the crude and nano emulsions, respectively. Stearic acid was present at 5.98% in the crude emulsion and 8.14% in the nano-loaded emulsion. Additionally, hexadecadienoic acid was found at 2.10% and 1.84%, respectively, while the final peak, representing eicosanoic acid, showed values of 1.11% and 1.59%.

Determination of Encapsulation Efficiency (EE) and Loading Capacity (LC) of prepared loaded nano-formulations

The incorporation of neem oil into PEG nano-capsules has been studied. The results in table (2) showed that Encapsulation Efficiency (EE%) was 72.58±0.99%. Loading Capacity (LC%) was 40.328±0.55.

Insecticidal activity of neem oil nano and bulk formulations on the 5^th nymphal instar of S. gregaria

The findings are displayed in Table (3), Fig. (3), they illustrate the impact of neem A. indica nanoemulsion and crude oil on the fifth nymphal instar of S. gregaria. After the topical application treatment, the mortality rates for the nano-emulsions at concentrations of 5 and 2.5 ppm were recorded at 73.33% and 56.11%, respectively. Contrary to expectations, the mortality percentages for the crude oil treatments at both 5 and 2.5 ppm were significantly higher, achieving 100% mortality, in stark contrast to the 0.0% observed in the control group.

Table 1: Gas chromatogram analysis of neem crude oil and PEG loaded nano emulsion

Table 2: Values of Encapsulation Efficiency (EE%) and loaded capacity (LC%) of the prepared neem oil nano-emulsion

Fig. (2): Upper graph: Gas chromatogram/mass spectrometry analysis of neem crude emulsion. Lower graph: Gas chromatogram/mass spectrometry analysis of neem PEG-loaded nano emulsion.

Effect of Nano and bulk formulations of Neem Oil on Different Biological Aspects of S. gregaria-the 5^th nymphal instar

A notable increase in the number of over-aged nymphs was recorded during the treatment period for nymphs exposed to 2.5 ppm nanoemulsion, amounting to 21.25 days. While no significant differences were observed between the 5 ppm nanoemulsion and the control group, the durations were 18.8% and 18.39 %, respectively. Furthermore, the data presented in Table (3) and Fig. (3 and 4) indicated a highly significant decrease in the rate of adult emergence, with 100% of the control nymphs successfully developing into adults, in contrast to 0% emergence in both the 5 ppm and 2.5 ppm crude oil treatments. In the case of nanoemulsions at 5 ppm and 2.5 ppm, the adult emergence rates were recorded at 26.67% and 43.89%, respectively. It is important to note that both doses of nanoemulsion (5 ppm and 2.5 ppm) resulted in 100% malformation, as illustrated in Fig (4), where the insects exhibited damage to their legs, wings, and antennae after molting into adults. Conversely, no such deformities were observed in the control group.

The research indicated that nano-emulsion of neem oil produced morphogenesis effects at all administered concentrations of 5 and 2.5 ppm, disrupting the morphogenesis of newly molted nymphs of S. gregaria when applied to the fifth-instar nymphs (Fig. 4). This treatment led to varying degrees of malformations, including significantly or slightly curled bodies, as well as deformities in the antennae and legs. These abnormalities arose from the nymphs' inability to molt into the subsequent nymphal instar or their transition into malformed adult forms. The malformations observed included nymphs that could not completely shed their last nymphal exuviae, which adhered to their bodies, resulting in curled legs and coiled, underdeveloped short wings, along with individuals displaying characteristics of an intermediate nymphal-adult stage. Daily observations revealed a marked antifeedant effect of neem in both crude oil and nano-emulsion treatments on S. gregaria nymphs, irrespective of the doses used. The growth inhibition attributed to neem was evident in both treatment types. The application of both crude and nano treatments on the fifth-instar nymphs of S. gregaria resulted in a reduction in body mass compared to the control group. Additionally, the amount of fecal waste produced by the desert locusts serves as a reliable measure of their food intake. Consequently, the daily production of fecal pellets was diminished and appeared dry and small in S. gregaria fifth-instar nymphs treated with crude oil (5 and 2.5 ppm) and subsequently with nano-emulsion (5 and 2.5 ppm). In contrast, the control insects produced a typical quantity of fecal matter with a standard shape and size. In this study, it was noted that the topical application of A. indica crude oil at doses of 5 and 2.5 ppm to the nymphs of S. gregaria influenced their body coloration prior to death, with the cuticle color changing from yellow with black patches to a uniformly black appearance in all deceased specimens (Fig. 5).

Table 3: Percentage mortality, duration, adult emerge and adult malformation of Schistocerca gregaria against two doses of neem oil and its nano and bulk forms

Fig. (3): Mortality%, Nymphal duration days, Adult emergence% and Adult malformation% of Schistocerca gregaria against two doses nano and bulk forms of neem oil.

Fig. (4): Molting failure of treated Schistocerca gregaria 5^th nymphal instars with nano-emulsion of neem at 5 and 2.5 ppm. A: Normal adult. B: Curled and twisted wings. C: The old cuticle is linked to the developed adults. D: Nymphs were unable to transition into adult and died without completing the molting process.

Physiological effects (biochemical analysis)

The information presented in Tables 3 and 4 illustrates the impact of neem nanoemulsion and crude oil on the total protein and chitinase activity of S. gregaria nymphs after 24, 48, and 72 hours. The parameters examined were influenced by various treatments and dosages of neem nanoemulsions in comparison to the control group. Table (4) indicates that a 5 ppm dose (contact treatment) significantly affected both total protein and chitinase activity. A notable reduction in total protein was observed 24 hours post-treatment with the nanoemulsion when compared to the control. Conversely, the crude oil treatment did not alter total protein levels during the same timeframe; however, after 72 hours, a significant decrease was noted in total protein levels when compared to both the control and the nano treatment (F value 7.44). Additionally, chitinase enzyme activity exhibited a significant decline after 24 hours in the nano treatment relative to the control, while a significant increase was recorded in chitinase activity following the crude oil treatment during the same period (F value 3.40).

Table (5) presents the effects of a 2.5 ppm dose (contact treatment) on total protein and chitinase activity. A significant decrease in total protein was observed 24 hours post-contact treatment with crude oil, whereas the nano treatment did not affect total protein levels in comparison to the control (F value 3.72). Furthermore, chitinase activity significantly decreased after 24 hours of nano treatment, while crude oil treatment led to a significant increase in chitinase activity during the same period when compared to the control nymphs (F value 21.80).

Fig. (5): Body color of treated Schistocerca gregaria 5^th nymphal instars with crude oil of neem with 5 and 2.5 ppm. A: Normal nymphs. B: treated nymphs.

Table 4: Effects of 5 ppm A. indica nano and bulk emulsions on certain biochemical aspects of the 5^th instar nymphs of Schistocerca gregaria

Discussion

TEM analysis

This study demonstrated that the inner cores of active ingratiate (neem oil) were developed as a single core of smaller particles within each capsule. To achieve the reduced size of the capsules, both a cleaner sonicator and a high-energy ultrasonic probe were employed. It was indicated that the utilization of the high-energy ultrasonic probe is a crucial step in producing small nano-capsules. Our findings were consistent with those reported by Bayoumi et al., (2022). However, the authors noted that relying solely on mechanical stirring is inadequate for producing uniformly sized nano-capsules, and extending the sonication time, either prior to or following the addition of oil to the loading material, resulted in smaller capsules. In this study, the capsule sizes were larger than those reported by Youssef et al. (2018), who prepared peppermint/alginate nano-capsules and found that the diameter of peppermint oil nano-capsules ranged from 70 to 100 nm. The application of sonication after cross-linking with calcium chloride contributed to a reduction in the diameter of the nano-capsules without the use of a solvent. A similar observation was made by Lertsutthiwong et al. (2008), who reported that the size of the prepared nano-capsules was influenced by the use of high-energy sonication, sonication duration, and increased dilution with distilled water.

Mortal potency of the neem formulations against nymphs of S. gregaria

The research conducted by Linton et al. (1997) demonstrated that the application of azadirachtin to S. gregaria during the 5^th instar stage significantly increased mortality rates among these insects, with the death rates in the treated group being markedly higher than those in the control group. Specifically, mortality rates for the 5^th instar S. gregaria were recorded at 56% and 64% for dosages of 7 µg and 5 µg of azadirachtin, respectively, in contrast to 5.3% mortality in the control group. Notably, 40% of the insects treated with 7 µg and 5 µg of azadirachtin perished during the ecdysis process, while only 5.3% of the control group experienced similar outcomes. Furthermore, Hamadah et al. (2013) observed that all insects in the 4^th and 5^th nymphal instars of S. gregaria succumbed to treatment with high doses of Neemazal from A. indica, with mortality rates reaching 60% and 70% at lower concentration levels, respectively. Azadirachtin has also been shown to exert toxic effects on the 4^th nymphal instars of S. gregaria, with potential mortality rates of 100% after 72 hours of treatment with clover leaves emerged in a concentration of 25%, while a lower concentration of 5% resulted in 65% mortality (Al-Maroug et al. 2022). The oils derived from A. indica seeds exhibited mortality rates ranging from 65% to 100% in S. gregaria (Nicol et al., 1991; Schmutterer & Frekes, 1990). The observed partial or complete mortality in S. gregaria can be linked to feeding inhibition, which leads to prolonged starvation and eventual death (Hamadah et al., 2013). Additionally, the mortality of desert locusts due to plant extracts may disrupt metamorphosis in the final instar nymphs of S. gregaria, potentially as a result of hormonal regulation disturbances. It has been suggested that the prevention of metamorphic ecdysis and subsequent death may be achieved by modulating the ecdysteroid peak or influencing the release of eclosion hormone (Al-Sharook et al., 1990).

Table 5: Effects of 2.5 ppm A. indica nano and bulk emulsions on certain biochemical aspects of the 5^th instar nymphs of  Schistocerca gregaria

Neem oil has been shown to disrupt the growth and developmental processes in desert locusts. Specifically, instars treated with azadirachtin, referred to as over-aged nymphs, exhibit an extended instar duration, allowing them to survive for several weeks longer than the typical duration. Furthermore, over-aged nymphs of L. migratoria and S. gregaria that were treated with azadirachtin demonstrate activity in both the corpus allatum and juvenile hormone.

Neem oil has been shown to disrupt the growth and developmental processes in desert locusts. Specifically, instars treated with azadirachtin, referred to as over-aged nymphs, exhibit an extended instar duration, allowing them to survive for several weeks longer than the typical duration. Furthermore, over-aged nymphs of L. migratoria and S. gregaria that were treated with azadirachtin demonstrate activity in both the corpus allatum and juvenile hormone.

A study by Linton et al. (1997) indicated that 20 days post-treatment, all insects in the control groups had reached adulthood, while only 12% of those treated with 5µg of azadirachtin achieved adulthood. In contrast, a higher dosage of 7mg resulted in the production of over-aged nymphs in S. gregaria. Neemazal has been observed to inhibit the development of both penultimate and final instar nymphs, significantly prolonging the nymphal duration in penultimate instars, particularly at concentration levels of 3.7, 1.8, 0.9, and 0.4%, with durations of 19.0±2.8, 17.7±3.2, 13.2±2.8, and 15.5±3.9 days, compared to 9.6±2.6 days for control nymphs. A similar inhibitory effect was noted for the last instar nymphs, especially at concentrations of 1.8% and 0.9%, which resulted in durations of 15.8±4.0 and 15.9±3.9 days (Hamadah et al., 2013), respectively, versus 12.3±1.2 days for control nymphs. The application of Neemazal on penultimate instar nymphs of S. gregaria resulted in delayed development, characterized by an extended developmental duration and a regression in the developmental rate relative to the concentration level (Nicol et al., 1991). This prolonged developmental duration serves as a significant indicator of inhibited growth in desert locusts following treatment with A. indica seed oil. Additionally, azadirachtin notably extended the duration of fourth instar nymphs of S. gregaria, with the most significant prolongation observed at concentrations of 10% and 15%, yielding durations of 16.7 and 17.6 days, respectively vs. 10.2 days of control (Al-Maroug et al., 2022).

In alignment with the findings of the current study on S. gregaria, the application of neem in its nano form on the 5^th instar nymphs resulted in a delay in development when compared to the control group. There exists a quantitative relationship between juvenile hormone and ecdysteroids (Schmutterer & Frekes, 1990). The temporal alteration of this balance is crucial for the successful development of the insect; thus, a postponement of the ecdysteroid peak prior to molting, as noted by Bashir and El Shafie (2014) following azadirachtin injection, may induce 'solitarisation effects.' This disruption caused by azadirachtin within the hormonal system is also linked to malformations, molting disturbances, and developmental delays. Azadirachtin has been shown to affect the success of molting between the 5^th instar and adult stages, inhibiting the growth of desert locusts. Neem oil similarly regulates growth and development in desert locusts. The treatment or ingestion of azadirachtin can result in diminished feeding levels in insects, which is essential for achieving the necessary body mass prior to ecdysis. Commonly observed abnormalities in insects treated with azadirachtin include mortality during the molting process, where the insect fails to shed the old cuticle. More than 45% of molts in Azadirachtin-treated insects resulted in death during ecdysis, whereas no mortality was recorded in the control group. Research indicates that Azadirachtin treatment significantly affects the loss of molts in insects, likely due to their inability to ingest sufficient air volumes to rupture the old cuticle and facilitate the expansion of the new one during ecdysis (Linton et al. 1997; Hamadah et al., 2013). The same phenomenon was observed in the treated 5^th instar nymphs of S. gregaria in the present study. Neemazal, an extract derived from neem, disrupts the metamorphosis process in S. gregaria by generating nymphal-adult intermediates, which may interfere with the normal ecdysone or ecdysteroid levels required for proper metamorphosis. Azadirachtin has been documented to inhibit ecdysteroid levels. Furthermore, azadirachtin treatment impedes neurosecretion of prothoracicotropic hormone, leading to physiological disruptions (Hamadah et al., 2013).

Description of Nymphal Performance Affected by neem

Neem formulations effected malformation

The utilization of nano formulations in varying doses has prompted morphogenesis changes in S. gregaria, leading to different levels of malformations characterized by nymphal-adult intermediate stages. This investigation indicates that azadirachtin induces morphogenesis effects in S. gregaria following treatment of fourth instar nymphs, resulting in the above-mentioned malformations. These abnormalities arise when nymphs fail to molt into the subsequent nymphal instar or when the molting process culminates in malformed adult stages. This study documents that the nymphal morphogenesis of S. gregaria is disrupted by azadirachtin treatment administered to penultimate instar nymphs. Observed deformities include the inability of nymphs to shed the last nymphal exuviae, which remain attached to their bodies, resulting in an adult-like form with curled legs and incompletely developed short wings, alongside the emergence of nymphal-adult intermediate stages (Al-Maroug et al., 2022). Previous research done by Nassar et al. (2018) demonstrated various impacts on the transformation of immature adult forms due to botanical extracts, with some exhibiting no effects or contradictory outcomes, contingent upon the activity of the plant species and the susceptibility of the insect species. Consequently, azadirachtin has been shown to influence both the hormonal system of S. gregaria and juvenile hormone levels (Al-Maroug et al., 2022). Our findings corroborate Josephrajkumar et al. (1999) who reported morphogenesis defects in adult S. gregaria following neem oil treatment of final instar nymphs. The performance of S. gregaria may be compromised by changes in ecdysteroid titers, which can lead to alterations in lysosomal enzyme activity and subsequent morphological abnormalities.

Neem formulations effected feeding

The antifeedant characteristics of azadirachtin are well-documented. This compound has been shown to decrease food intake and hinder the growth of desert locusts (Linton et al. 1997). The effects of A. indica are linked to its saponin constituents, which may influence cell membranes, thereby impairing digestion and nutrient absorption (Bogumil & Wieslaw, 2006, De Geyter et al., 2012). Azadirachtin interferes with gut functionality, resulting in a flaccid, partially filled gut, diminished protein digestion efficiency, and lowered feeding rates. Injections of azadirachtin produce a significant antifeedant response in 5^th instar nymphs of S. gregaria, as indicated by the average daily production of fecal pellets (Linton et al., 1997). Consistent with these findings, insects treated with both doses of bulk and nano formulations exhibited a pronounced antifeedant effect on S. gregaria, evidenced by a decrease in daily fecal pellet output, which was notably smaller and drier in comparison to the control group.

Neem formulations effected nymphs color

Indent imbalances in the levels of ecdysteroids and juvenile hormones are evident in the effects associated with elevated juvenile hormone titers. These effects include the presence of green hemolymph, brown and green cuticles, a lack of black pigmentation, and supernumerary molts in locusts, as well as cuticular melanization that results in black spots linked to low ecdysteroid levels and the absence of juvenile hormone (Schmutterer & Frekes , 1990, Nicol & Schmutterer, 1991, Gelbic & Ne´mec, 2001). The role of the bright yellow and black coloration observed in gregarious late-instar nymphs of S. gregaria remains ambiguous. However, it has been proposed that this coloration serves as warning signals, as these gregarious nymphs, who inhabit desert environments, consume plants that contain alkaloids and other toxic compounds (Cullen et al., 2017). During the nymphal stage of S. gregaria, the shift in coloration, which is influenced by population density and the consumption of native toxic host plants, leads to the development of warning coloration, known as aposematism (Sword et al. 2000). Numerous studies have indicated that juvenile hormone promotes green body coloration in the nymphal stages of various acridid species (Pener & Simpson, 2009). In regions with ample vegetation, S. gregaria exhibits green phenotypes and solitary behavior. Yet, as vegetation diminishes due to arid conditions, these locusts undergo a phase change to a gregarious form, displaying yellow with black patterns (FAO, 2001). Despland & Simpson (2000) indicated that food quality significantly impacts mortality and development in desert locusts. High-quality food sources may enhance population growth by hastening the emergence of later instar hoppers, reducing mortality rates, and enabling the production of two generations within a single season. Based on these findings and our data, we deduce that the color change to black in fifth-instar nymphs occurred following treatment with neem in bulk form, likely due to the cessation of feeding and an imbalance in ecdysteroid and juvenile hormone levels.

The active components responsible for the insecticidal properties of botanical insecticides are typically lipophilic, allowing them to permeate the cell membranes of insects and disrupt their digestive and neurological systems (Da Cruz et al. 2020). Liu et al., (2013) reported that compounds such as terpenoids, alkaloids, and steroids, which are synthesized by plants, contribute to this insecticidal activity. Research has identified key chemical compounds, including α-terpinyl acetate, α-terpineol, 4-terpineol acetate, and linalool, as significant agents that can effectively kill and repel insects. Analysis using gas chromatography-mass spectrometry (GC/MS) revealed a substantial reduction in linoleic acid due to the nano formulation process, suggesting that an extended ultrasonic treatment of three minutes negatively affected the stabilization of linoleic acid. Furthermore, Youssef & Abdelmegeed (2021) noted that the preparation of a Mentha piperita alginate nanoemulsion did not significantly alter the oil compounds, with the exception of the absence of eucalyptol, as only two minutes of ultrasonic treatment was employed in their methodology. This may account for the enhanced efficacy of bulk oil in nano formulations as an insecticidal product.

Botanical formulations effected biochemical parameters

Proteins are essential components within cells, serving various functions including hormonal regulation (Sugumaran, 2010). They also contribute structurally, similar to carbohydrates and lipids. Furthermore, the synthesis of proteins is crucial for the maintenance of growth and reproductive processes in the body (Dimetry et al., 2019). Azadirachtin has been shown to influence pest development by interfering with the hormones that regulate moulting and metamorphosis (Amin et al., 2019). Mahmoud et al. (2014) indicated that the expression of proteins in adult S. gregaria treated with azadirachtin was reduced. Specifically, when adult locusts were administered a dose of 7.5 µg/g body weight, the total number of protein bands increased slightly from 10 in the control group to 11 in the treated group. However, when the dosage was raised to 15 µg/g body weight, the number of protein bands decreased from 10 to 8, and this number remained unchanged even with a further increase to 30 µg/g body weight. Additionally, protein expression can decline when insects are fed a diet containing azadirachtin or when larvae are injected with it. Reda et al. (2009) proposed that the reduction in hemolymph protein levels in S. gregaria may result from the detrimental effects of neem on the neurosecretory cells in the brain, which are responsible for protein secretion. This reduction may also stem from inadequate stimulation of neurosecretory activity, leading to a decrease in the protein levels in hemolymph necessary for oocyte development. Consistent with these findings, the current study demonstrated a decrease in total protein levels following neem treatments (both nano and bulk oil), particularly after 24 and 48 hours compared to the control group. It was noted that the reduction in hemolymph proteins in adult S. gregaria treated with Hexaflumuron, a chitin-synthesis inhibitor, may indicate a diminished capacity for protein synthesis. This decrease in hemolymph proteins can inhibit oogenesis and ovarian development, ultimately resulting in the sterility of the adult insect. Azadirachtin shows promise as a biopesticide for managing insect pests (Amin et al., 2019). The process known as apolysis involves the separation of epidermal cells from the old cuticle through the secretion of molting fluid and the formation of the ecdysal membrane, marking the initiation of ecdysis. This molting fluid is rich in proteases and chitinases, which are enzymes responsible for breaking down the primary components of the old endocuticle. In this study, a notable increase in chitinase activity was observed in nano and bulk formulations after 24, 48, and 72 hours following treatment. Additionally, Abdel-Aal (2006) reported that chlorfluazuron significantly enhanced chitinase activity in Spodoptera littoralis. These findings are consistent with previous research reported by Al-shannaf et al. (2012), they indicated that insect growth regulators, such as chlorfluazuron and pyriproxyfen, led to substantial increases in chitinase enzyme activity, with a reported increase of 130% in the larvae of the American bollworm, Helicoverpa armigera. The observed rise in chitinase activity may serve as a detoxification mechanism, which is considered as a main reason for the black color appeared on the treated nymphs and the individuals resulted from them.

Conclusion

The current research revealed the detrimental effects of topically administered neem A. indica on fifth-instar S. gregaria in semi-field settings. Prolonged sonication adversely impacted the key compound (Linoleic acid) within the nano formulation, leading to diminished pesticidal efficacy compared to the neem extract. Both formulations negatively influenced biological parameters, including mortality rates, failed molting, and deformities, in addition to affecting biochemical activities such as chitinase activity and total protein levels. Consequently, the nano formulation developed through this method does not seem to be an effective control agent for S. gregaria. Interestingly, bulk (crude oil)-treated individuals were shortly dead in the tested concentrations so that their durability was not compared with nano formulations.

Author's Contributions

Nadia Zekry Dimetry: investigation, methodology, supervision, formal analysis, visualization, draft preparation; Mahfouz Mohamed Mostafa Abd-Elgawad: conceptualization, formal analysis, visualization, final review and edit; Dalia Abdallah Youssef: investigation, methodology, formal analysis, visualization, draft preparation, final review and edit; Samira Mohamed Nabeel Abd-El Wahed investigation, methodology, formal analysis, visualization, draft preparation, final review and edit.

Author's Information

Funding

This research was funded by NRC In-House project No. 13050112 entitled: Pesticide alternatives against soilborne pathogens and pests attacking economically significant export crops.

Data Availability Statement

All data supporting the findings of this study are available within the paper.

Acknowledgments

We are grateful for the support of Prof. Dr. Elsayed A. Omer, Medicinal and Aromatic Plants Research Department, National Research Centre and his staff for their instructions and doing GC/MS analysis as a gift. The work was funded by NRC In-House project No. 13050112.

Ethics Approval

Insects were used in this study. All applicable international, national, and institutional guide lines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

Conflict of Interest

The authors declare no conflict of interest.

Generative AI statement

The authors declare that no Gen AI was used in the creation of this manuscript.

REFERENCES

Abbott, W. S. (1925) A method for computing the effectiveness of an insecticide. Journal of Economic Entomology, 18(2):265-267.

Abdel-Aal, A. E. (2006) Effect of chlorfluazuron, nuclear polyhydrosis virus (SLNPV) and Bacillus thuringiensis on some biological and enzymes activity of cotton leafworm, Spodoptera littoralis (Boisd). Bulletin of the Entomological Society of Egypt (ARE), Economic Series 32, 171-185.

Al-Maroug, N. A., A A, Nassar, M. I., Abdelatef, G. M., ELShazly, M. M., Abd ElFattah, E. A. E. & Abd El-Monem, D. H. (2022) Assessment of the commercial botanical insecticides against the desert locust, Schistocerca gregaria (Forsk.) (Orthoptera: Acrididae). Egyptian Academic Journal of Biological Sciences F. Toxicology and Pest Control, 14(2):121-132. https://doi.org/10.21608/eajbsf.2022.266750

Al-shannaf, H. M., Mead, H. M. & Sabry, A. H. (2012) Toxic and biochemical effects of some bioinsecticides and IGRs on american bollworm, Helicoverpa armigera (Hüb.) (Noctuidae: Lepidoptera) in cotton fields. Journal of Biofertilizers and Biopesticides, 3(2):1-6. http://dx.doi.org/10.4172/2155-6202.1000118

Al-Sharook, Z., Balan, K., Jiang, Y. & Rembold, H. (1991) Insect growth inhibitors from two tropical Meliaceae: effect of crude seed extracts on mosquito larvae. Journal of Applied Entomology, 111:425- 430. https://doi.org/10.1111/j.1439-0418.1991.tb00344.x

Amin, A. H., Bayoumi, A. E., Dimetry, N. Z. & Youssef, D. A. (2019) Efficiency of Nano-formulations of neem and peppermint oils on the bionomics and enzymatic activities of Agrotis ipsilon larvae (Lepidoptera: Noctuidae). International Journal of Natural Resource Ecology and Management, 4(5):102-111. https://doi: 10.11648/j.ijnrem.20190405.11  

Bashir, E. M. & El Shafie, H. A. (2014) Toxicity, antifeedant and growth regulating potential of three plant extracts against the desert locust Schistocerca gregaria Forskal (Orthoptera: Acrididae). American Journal of Experimental Agriculture, 4(8): 959-970.

Bayoumi, A. E., Amin, A. H., Dimetry, N. Z. & Youssef, D. A. (2022) Toxicity Evaluation of Two Neem Oil Nano- Formulations Using Swiss Male Albino Mice. Biomedical Journal of Science and Technical Research, 43(4):34763-34777. https://doi:10.26717/BJSTR.2022.43.006928    

Bogumil, S. G. L. & Wieslaw, O. (2006) Effect of low and high-saponin lines of alfalfa on pea aphid. Journal of Insect Physiology, 52(7), 737-743. https://doi.org/10.1016/j.jinsphys.2006.04.001

Cullen, D.A., Cease, A.J., Latchininsky, A. V., Ayali, A., Berry, K., Buhl, J., Keyser, R. De, Foquet, B., Hadrich, J.C., Matheson, T., Ott, S.R., Poot-Pech, M.A., Robinson, B.E., Smith, J.M., Song, H., Sword, G.A., VandenBroeck, J, Verdonck, R., Verlinden, H., Rogers, S.M., 2017. From molecules to management: mechanisms and consequences of locust phase polyphenism. In Advances in Insect Physiology 53:167-285. Academic Press. https://doi.org/10.1016/bs.aiip.2017.06.002

Da Cruz, R. C. D., da Silva Carvalho, K., Costa, R. J. O., da Silva, P. A., e Silva, S. L. D. C., Gualberto, S. A., de Gusmão N. B. & de Souza, I. A. (2020) Phytochemical and toxicological evaluation of a blend of essential oils of Croton species on Aedes aegypti and Mus musculus. South African Journal of Botany, 132, 188-195. https://doi.org/10.1016/j.sajb.2020.03.040

De Geyter, E., Swevers, L., Soin, T., Geelen, D. & Smagghe, G. (2012) Saponins do not affect the ec-dysteroid receptor complex but cause membrane permeation in insect culture cell lines. Journal of Insect Physiology, 58(1):18-23. https://doi.org/10.1016/j.jinsphys.2011.09.005

Despland, E. & Simpson, S. J. (2000) The role of food distribution and nutritional quality in behavioural phase change in the desert locust. Animal Behaviour 59(3): 643–652. https://doi.org/10.1006/anbe.1999.1335

Dimetry, N. Z., Amin, A. H., Bayoumi, A. E., Aly, M.A. & Youssef D. A. (2019) Comparative toxicity between neem and peppermint oils nano-formulations against Agrotis ipsilon (Hufm.) larvae (Lepidoptera: Noctuidae). Jounal of Botanical Research, Bilingual Publishing Co. 1:13-19.https://doi.org/10.30564/jrb.v1i1.590

Disha Varijakzhan, Chou-Min Chong, Kok-Song Lai & Swee-Hua Erin Lim (2020) Middle Eastern plant extracts: An alternative to modern medicine problems. Molecules, 25(5):1126. https://doi.org/10.3390/molecules 25051126.

El-Shazly, M. M., Ghazawy, N. A. R., Abdel Rahman, K. M., Elbassiony G. M. & Nassar, M. I. (2019) Tolerance of Heteracris littoralis (Orthoptera: Acrididae) to the toxic plant, Nerium oleander (Apocynacea). Journal of the Egyptian Society of Parasitology, 49(3):579-587. https://doi.org/10.21608/jesp.2019.68061  

El-Sonbati, A. Z., Diab, M. A., El-Bindary, A. A., Nozha, S.G. & Nassar, M. I. (2014) Evaluation and rearrangement of novel supramolecular 7-(2,3-Dihydro-1,3- benzothiazol-2-yl) quinolin-8-ol complexes and their biological effect. International Journal of Microbial and Biochemical Technology S4-001, 2-9. http://dx.doi.org/10.4172/1948-5948.S4-001.

FAO (2001) P.M. Symmons and K. Cressman Desert Locust Guidelines Biology and behaviour Second edition. https://www.fao.org/home/

FAO (2020) FAO Feature Story. Five things you should know about an age-old pest: the Desert Locust. https://www.fao.org/newsroom/story/Five-things-you-should-know-about-an-age-old-pest-the-Desert-Locust/en

Gelbic, I. & Ne´mec, V. (2001) Developmental changes caused by metyrapone and azadirachtin in Spodoptera littoralis (Boisd.) (Lep.,Noctuidae) and Galleria mellonella (L.) (Lep., Pyralidae). Journal of Applied Entomology 125(7):417–422. https://doi.org/10.1046/j.1439-0418.2001.00566.x

Gornall, A. G., Bardawill, C. J. and David, M. M. (1949) Determination of serum proteins by means of the biuret reaction. Journal of Biological Chemistry, 177(2):751-766.

Hamadah, Kh. Sh., Ghoneim, K. S., El-Hela, A. A., Amer, S. M. and Mohammad, A. A. (2013) Disturbed survival, growth and development of the desert locust Schistocerca gregaria by different extracts of Azadirachta indica (Meliaceae) and Nigella sativa (Ranunculaceae). Egyptian Academic Journal of Biological Sciences A. Entomology. 6(2):1-21.  https://doi.org/10.21608/eajbsa.2013.13246 

Ishaaya, I. & Casida, J. E. (1974) Dietary TH 6040 alters composition and enzyme activity of housefly larval cuticle. Pesticide Biochemistry and Physiology, 4(4):484-490. https://doi.org/10.1016/0048-3575(74)90073-X

Jerobin, J., Sureshkumar, R. S., Anjali, C.H., Mukherjee, A. & Chandrasekaran N. (2012) Biodegradable polymer based encapsulation of neem oil nanoemulsion for controlled release of Aza-A. Carbohydrate Polymers, 90:1750-1756.

Josephrajkumar, A., Subrahmanyam, B. and Srinivasan, S. (1999) Plumbagin and azadirachtin deplete haemolymph ecdysteroid levels and alter the activity profiles of two lysosomal enzymes in the fat body of Helicoverba armigera (Lepidoptera: Noctuidae). European Journal of Entomology, 96(4):347- 353.

Kumar, S., Bhanjana, G., Sharma, A., Sidhu, M. C. and Dilbaghi, N. (2014) Synthesis, characterization and on field evaluation of pesticide loaded sodium alginate nanoparticles. Carbohydrate Polymers, 101:1061-1067. https://doi.org/10.1016/j.carbpol.2013.10.025

Lertsutthiwong, P., Noomun, K., Jongaroonngamsang, N., Rojsitthisak, P. and Nimmannit, U. (2008) Preparation of alginate nanocapsules containing turmeric oil, Carbohydrate Polymers, 74(2):209-214. https://doi.org/10.1016/j.carbpol.2008.02.009

Linton, Y. M., Nisbet, A. J. & Mordue, A. J. (1997) The effects of azadirachtin on the testes of the desert locust, Schistocerca gregaria Forskål. Journal of Insect Physiology, 43(11):1077-1084. https://doi.org/10.1016/S0022-1910(97)00060-7

Liu, X. C., Li, Y. P., Li, H. Q., Deng, Z. W., Zhou, L., Liu, Z. L. and Du, S. S. (2013) Identification of repellent and insecticidal constituents of the essential oil of Artemisia rupestris L. aerial parts against Liposcelis bostrychophila Badonnel. Molecules 18 (9):10733–10746. https://doi.org/10.3390/molecules180910733 

 Lomer, C. J., Bateman, R. P., Johnson, D. L., Langewald, J. and Thomas, M. (2001) Biological control of locusts and grasshoppers. Annual Review of Entomology, 46(1):667-702. https://doi.org/10.1146/annurev.ento.46.1.667 

Mahmoud, D. M., Elnaggar, T. F., Fattah H. M. A. & Kader, A. F. A. (2014) Effect of azadirachtin on the hemolymph protein of the adult desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). Egyptian Journal of Pure and Applied Science, 52(2):1-5.

Mohammad, A. A., Amer, H. M., El-Sawy, S. M., Youssef, D. A., Nour, S. A. & Soliman, G. M. (2022) Nematicidal activity of sweet annie and garden cress nano-formulations and their impact on the vegetative growth and fruit quality of tomato plants. Scientific Reports, 12(1):22302. https://doi.org/10.1038/s41598-022-26819-2

Nassar, M. I, Ghazawy, N. A., Torkey, H. M. & Rawy, S. M. (2018) Assessment of biorational botanical extracts on the Desert Locust Schistocerca gregaria Forskal (Orthoptera: Acrididae). Entomology and Applied Science Letters, 5(2):42-54.

Nicol, C. M. Y. & Schmutterer, H. (1991) Contact effects of seed oil from the neem tree Azadirachta indica (A. Juss.), on nymphs of the gregarious phase of the desert locust, Schistocerca gregaria (Forsk.). Journal of Applied Entomology, 111(2):197 –205. https://doi.org/10.1111/j.1439-0418.1991.tb00311.x

Pener, M. P. & Simpson, S. J. (2009) Locust phase polyphenism: an update. Advances in Insect Physiology, 36,1–272. https://doi.org/10.1016/S0065-2806(08)36001-9

Reda, F. A. Mona, I. M., El-Gammal, M. & Noura, M. M. (2009) Biochemical changes of the Chitin-synthesis inhibitor, Hexaflumuron (Consult) compound on the desert locust, Schistocerca gregaria (Forskal). Egyptian Academic Journal of Biological Sciences A, 1(1):59 – 68.

Ouedraogo, R. M., Kamp, A., Goettel, M. S., Brodeur, J. & Bidochka, M. J. (2002) Attenuation of fungal infection in thermo regulating Locusta migratoria is accompanied by changes in heamolymph proteins. Journal of Invertebrate Pathology, 81(1):19-24. https://doi.org/10.1016/S0022-2011(02)00117-9

Schmutterer, H. & Frekes, T. (1990) Beeinflussung von Metamorphose, Farbung und Verhalten der Wiisten-heuschrecke Schistocercu gregaria (Forsk.) under Afrikanischen Wanderheuschrecke Locusta migrutoria migratortorioides (R. & F.) durcli Niemsamcniil. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 97(4):431-438.

Sugumaran, M. (2010) Chemistry of cuticular sclerotization. Advances in Insect Physiology. 39:151-209. https://doi.org/10.1016/B978-0-12-381387-9.00005-1

Sword, G. A., Simpson, S. J., El Hadi, O. M. & Wilps, H. (2000) Density–dependent aposematism in the desert locust. Proceedings of The Royal Society B, London 267:63–68. https://doi.org/10.1098/rspb.2000.0967

Van Huis, A., Cressman, K. & Magor, J. I. (2007) Preventing desert locust plagues: Optimizing management interventions. Entomologia Experimentaliset Applicata, 122(3):191-214. https://doi.org/10.1111/j.1570-7458.2006.00517.x

Youssef, D. A., Bayoumi, A. E., Dimetry, N. Z., Amin, A. H. & Hoballah, E.M. (2018) Evaluating effect of peppermint oil (Mentha pipreta) and its nano-formulations on some enzymatic activities and bionomics of cotton leaf worm Spodoptera littoralis (Biosd.). Arab Universities Journal of Agricultural Sciences: Special Issue, 26 (2c), 1977-1991. https://doi: 10.21608/ajs.2018.31665

Youssef, D. A. & Abdelmegeed, S. M. (2021) Polymer-based encapsulation of peppermint oil (Mentha piperita) nano emulsion and its effects on life and some physiological activities of honeybees (Hymenoptera). Egyptian Pharmaceutical Journal, 20 (4):3 13-3 22.  https://doi.org/10.4103/epj.epj_49_21

Zhang, Y., Zhu, S., Yin, L., Qian, F., Tang, C. &Yin, C. (2008) Preparation, characterization and biocompatibility of poly (ethylene glycol)-poly (n-butyl cyanoacrylate) nano capsules with oil core via mini emulsion polymerization. European Polymer Journal, 44(6):1654–1661. https://doi.org/10.1016/j.eurpolymj.2008.03.019