Preference of Major Stored Product Insects in Fortiﬁed Rice with Basil

: As the world’s population grows, the needs for feeding it follow the same path. Considering these conditions, ensuring the quantity and quality of raw materials, such as cereals, seems imperative. Stored product insects are responsible for signiﬁcant losses in the post-harvest stages of agricultural products and the available chemical methods for their control are decreasing, due to their negative impact on the environment and humans. To this end, the evaluation of the efﬁcacy of non-chemical methods for the management of storage insects is crucial. In the present study, we conducted two trials of choice tests based on rice fortiﬁed with basil to determine its potential as a non-chemical repellent of stored product insects. In the ﬁrst trial, we evaluated the repellent activity of rice enriched with basil on adults of Sitophilus oryzae , Rhyzopertha dominica , Tribolium castaneum and Oryzaephilus surinamensis , as well as on T. castaneum larvae. In the second trial, the same procedure was followed with deltamethrin-treated rice fortiﬁed with basil. The results of the ﬁrst trial showed that for most of the insect species tested, the rice fortiﬁed with basil was moderately repellent, while for O. surinamensis , it was attractive. Surprisingly, in the second trial, the deltamethrin-treated rice fortiﬁed with basil showed a repellent effect on O. surinamensis adults and T. castaneum larvae, while no repellency was observed for the rest of insect species examined. Observation time was not signiﬁcant for any of the insect species, combinations and trials, with the exception of the rice fortiﬁed with basil vs. Blanc combination in O. surinamensis . Our ﬁndings suggest that the use of deltamethrin-treated rice fortiﬁed with basil was effective as a repellent for O. surinamensis adults and T. castaneum larvae.


Introduction
The expected increase in the world's population requires up to a 50% increase in food availability [1][2][3]. Cereals have been the main source of human nutrition for centuries [4]. At a time when more than 690 million people are undernourished, the need for grain quality and quantity insurance is considered imperative [3,5]. Stored product insects are primarily responsible for post-harvest losses occurring during storage [4]. Specifically, grain losses during the storage period in India are estimated at USD 1 billion [6,7], of which, USD 364 million are losses due to insect infestation [8]. However, this amount increases dramatically, reaching up to 20% of production, as far as developing countries are concerned [9].
Oryza sativa L., commonly known as rice, belongs to the Poaceae family and is one of the most nutritious and widely consumed staple foods [10]. Indeed, according to the Food and Agricultural Organization of the United Nations (FAO) in 2019, rice came third in the global ranking of the most produced agricultural grains, while the upward growth of rice production continued in 2021-2022, when 509.87 million metric tons of rice were consumed worldwide [11]. China is the largest producer and consumer of rice worldwide and its A commercial formulation of deltamethrin (K-Othrine 25 SC, 2.5% w/w active ingredient (a.i.), Bayer Hellas, Attiki, Greece) was used in the experiments. The label dose of deltamethrin (9.7 mg of grain/m 2 ) was utilized and applied in uninfested and insecticidefree fortified rice with basil. Insecticide spraying solutions were prepared by diluting the appropriate amount of the insecticide in 50 mL of distilled water. Afterwards, 1 mL of the spraying solution was sprayed on 110 g of rice fortified with basil, using a specialized airbrush (Badger 100, Kyoto BD-183 K Grapho-tech, Kyoto, Japan). Then, the grains were placed in a glass jar and shaken manually for 2 min to ensure the equal distribution of the insecticide in the entire grain mass.

Choice Tests 2.4.1. Test 1-Untreated Choice Tests
The tests were conducted inside plastic Petri dishes (90 mm diameter, 15 mm high) with 63.61 cm 2 bottom surface (Greiner Bio-One, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), which were divided into two equal parts. For the one-choice tests, 0.5 g of conventional rice (C) or fortified rice with basil (F) was placed in one part of the dish, whereas the other part was left empty and served as blank (B) (control area). For the two choice-tests, 0.5 g of fortified rice (F) was placed in one part of the dish and 0.5 g of conventional rice (C) was placed in the other part (control area). One unsexed individual of each insect species was released in the center of the dish, with different Petri dishes for each insect species. The insect presence on the treated and untreated area or in the middle of the Petri dish was recorded 5, 10, 15, 30, and 60 min after exposure. There were 20 dish replicates for each insect species and life stage.

Test 2-Treated Choice Tests
The same experimental procedure described for Test 1 was also followed in Test 2, in which we evaluated the insect response to deltamethrin-treated rice. For this reason, 0.5 g of deltamethrin-treated conventional rice or deltamethrin-treated fortified rice with basil (CD and FD, respectively) (please refer to Section 2.3 for deltamethrin application) was placed in one part of the dish (treated area), whereas in the other part, 0.5 g of conventional rice or rice fortified with basil was placed (untreated area). More specifically, we tested four different combinations: (i) deltamethrin-treated fortified rice with basil vs. conventional rice (FD vs. C), (ii) deltamethrin-treated conventional rice vs. basil fortified-rice (CD vs. F), (iii) deltamethrin-treated fortified rice with basil vs. basil fortified-rice (FD vs. F), (iv) deltamethrin-treated conventional rice vs. conventional rice (CD vs. C). Each treatment was replicated 20 times for each insect species and life stage.

Statistical Analysis
Prior to analysis, all data were tested for normalization and homogeneity using the Shapiro-Wilk test. Since the required assumptions for parametric tests were not met, nonparametric analyses were performed. For both choice tests (Test 1 and 2), for each treatment and insect species tested, the data were analyzed separately with a generalized linear model framework, with observation time as the response variable. To determine the effect of each treatment on insect choice, the average number of insects present in each part of the Petri dish for each observation time was subjected to Wilcoxon signed-rank test (p < 0.05). A Friedman's test was used to detect differences between treatments (p < 0.05), when there was a number of insects that chose to stand in the middle of the Petri dish. All data were analyzed using SPSS 29.0 (IBM Corporation, Armonk, NY, USA).

Test 1-Untreated Choice Tests
For all insect species examined, the observation time did not play a significant role in the insects' choice, with the exception of O. surinamensis and the combination of F vs. B (Table 1). Most S. oryzae adults (60%) opted for F in the combination F vs. B; however, no significant differences were detected either in this combination or in the rest of the combinations tested (F vs. C and C vs. B) ( Figure 1). Regarding R. domica adults, a percentage of 13-15% remained in the middle of the Petri dish for all the combinations examined (F vs. C, F vs. B, and C vs. B). This specific percentage of R. dominica individuals differed significantly compared to C, F, and B in the combinations F vs. C, F vs. B, C vs. B, respectively ( Figure 2). Overall, twice as many indi- Regarding R. domica adults, a percentage of 13-15% remained in the middle of the Petri dish for all the combinations examined (F vs. C, F vs. B, and C vs. B). This specific percentage of R. dominica individuals differed significantly compared to C, F, and B in the combinations F vs. C, F vs. B, C vs. B, respectively ( Figure 2). Overall, twice as many individuals of R. dominica selected C compared to F, 1.2 more individuals selected F compared to B, and 1.5 more individuals were detected in the B compared to C. Nevertheless, no significant difference was recorded in the mean number of R. dominica that chose one of the two options (F or C, F or B, and C or B) in all the combinations evaluated ( Figure 2). Regarding R. domica adults, a percentage of 13-15% remained in the middle of the Petri dish for all the combinations examined (F vs. C, F vs. B, and C vs. B). This specific percentage of R. dominica individuals differed significantly compared to C, F, and B in the combinations F vs. C, F vs. B, C vs. B, respectively ( Figure 2). Overall, twice as many individuals of R. dominica selected C compared to F, 1.2 more individuals selected F compared to B, and 1.5 more individuals were detected in the B compared to C. Nevertheless, no significant difference was recorded in the mean number of R. dominica that chose one of the two options (F or C, F or B, and C or B) in all the combinations evaluated ( Figure 2).  The percentage (%) of insects that chose to stand in the line in the middle of Petri dish, represented as "Line". For each combination, means followed by the same lowercase letter are not significantly different, according to chi-square test at 0.05; where no letters exist, no significant differences were noted. Chi-square parameters for the F vs. C combination were: x 2 = 8.4, df = 2, p = 0.015, for the F vs. B were: x 2 = 8.3, df = 2, p = 0.016, for the B vs. C were: x 2 = 10.00, df = 2, p < 0.001.
The percentage of T. castaneum adults that remained on the line in the center of the Petri dish fluctuated at smaller levels (6-7%) in relation to the respective R. dominica figures ( Figure 3). In the combination, F vs. C, 55% of the insects chose C, while the percentage of individuals that chose F was 67% in the combination F vs. B. Finally, in the combination C vs. B, the number of T. castaneum adults was equally distributed in the two dish parts ( Figure 3). In the case of O. surinamensis adults, the preference for F was stronger compared to C and B, while in the combination C vs. B, no statistically significant differences were detected ( Figure 4). In the F vs. C and F vs. B combinations, the highest number of T. castaneum larvae chose for F (58 and 59%, respectively), while in the C vs. B combination 54% of the larvae chose C and 46% B ( Figure 5). However, no statistical differences were detected for any of the combinations examined ( Figure 5). nation C vs. B, the number of T. castaneum adults was equally distributed in the two dish parts ( Figure 3). In the case of O. surinamensis adults, the preference for F was stronger compared to C and B, while in the combination C vs. B, no statistically significant differences were detected ( Figure 4). In the F vs. C and F vs. B combinations, the highest number of T. castaneum larvae chose for F (58 and 59%, respectively), while in the C vs. B combination 54% of the larvae chose C and 46% B ( Figure 5). However, no statistical differences were detected for any of the combinations examined ( Figure 5).     For each combination, means with asterisks (*) indicate signifi differences between the two choices, according to Wilcoxon singed-rank test at p < 0.05 (in all df = 4). Where no asterisk (*) exists, no significant differences were found. Wilcoxon's singedtest parameters for F vs. C were: z = −2.0, df = 1, p = 0.046, for F vs. B were: z = −2.0, df = 1, p = 0.

Test 2-Treated Choice Tests
Observation time did not affect the selection of any of insect species and treated c binations tested (Table 2). For the majority of the combinations tested, deltameth treated rice, either fortified with basil or conventional, collected the highest percentag S. oryzae adults. More specifically, 59% chose the FD in the FD vs. C combination, chose the CD in the F vs. CD combination, and 59% chose the CD in the CD vs. C com nation ( Figure 6). An exception was the combination of FD vs. F, where F collected 53

Test 2-Treated Choice Tests
Observation time did not affect the selection of any of insect species and treated combinations tested (Table 2). For the majority of the combinations tested, deltamethrintreated rice, either fortified with basil or conventional, collected the highest percentage of S. oryzae adults. More specifically, 59% chose the FD in the FD vs. C combination, 55% chose the CD in the F vs. CD combination, and 59% chose the CD in the CD vs. C combination ( Figure 6). An exception was the combination of FD vs. F, where F collected 53% of S. oryzae individuals compared to FD, which collected 47%. However, no differences were detected in this combination or in the others evaluated ( Figure 6). In the case of R. dominica, statistically significant differences were presented only in the combination CD vs. C, where CD collected two times more adults compared to C (Figure 7). The other combinations collected a similar mean number of R. dominica adults in both options (Figure 7).   Adults of T. castaneum showed a completely different preference for the combinations examined compared to the previous two insect species (Figures 6-8). In this case, for most of the combinations examined, untreated rice (without deltamethrin), either F or C, collected the highest number of individuals, except for the F vs. CD combination (Figure 8). Among the treatments, a significantly higher number of T. castaneum adults were detected in C (66%) compared to CD (34%) (Figure 8). For each combination, means with asterisks (*) indicate significant differences between the two choices, according to Wilcoxon singed-rank test at p < 0.05 (in all cases df = 4). Where no asterisk (*) exists, no significant differences were found. Wilcoxon's singed-rank test parameters for CD vs. C were z = −2.0, df = 1, p = 0.043.
Adults of T. castaneum showed a completely different preference for the combinations examined compared to the previous two insect species (Figures 6-8). In this case, for most of the combinations examined, untreated rice (without deltamethrin), either F or C, collected the highest number of individuals, except for the F vs. CD combination (Figure 8). Among the treatments, a significantly higher number of T. castaneum adults were detected in C (66%) compared to CD (34%) (Figure 8).
The repellent effect of deltamethrin was strongly demonstrated in the case of O. surinamensis. In particular, only 1/5 of O. surinamensis adults chose FD in the combination, FD vs. C, while correspondingly, in the rest of the combinations the higher percentage of individuals chose F or C compared to FD or CD (Figure 9). Nevertheless, significant Sustainability 2023, 15, 11379 9 of 15 differences between the two options were shown in three out of four combinations tested (Figure 9). A similar behavior to O. surinamensis was also observed in the larvae of T. castaneum (Figures 9 and 10). In all combinations evaluated, the percentage of larvae that chose the deltamethrin-treated rice (FD or CD) was significantly lower than the percentage that chose the untreated rice (F or C) ( Figure 10). In addition, the highest percentage of preference was 66% and observed in C both in the combination, FD vs. C, and in CD vs. C ( Figure 10).

Figure 7.
Average choice rate (%) of Rhyzopertha dominica adults for each of the three following combinations: Fortified with deltamethrin vs. Conventional rice (FD vs. C), Fortified vs. Conventional with deltamethrin rice (F vs. CD), Fortified with deltamethrin vs. Fortified rice (FD vs. F), and Conventional with deltamethrin vs. Conventional rice (CD vs. C). For each combination, means with asterisks (*) indicate significant differences between the two choices, according to Wilcoxon singedrank test at p < 0.05 (in all cases df = 4). Where no asterisk (*) exists, no significant differences were found. Wilcoxon's singed-rank test parameters for CD vs. C were z = −2.0, df = 1, p = 0.043.
Adults of T. castaneum showed a completely different preference for the combinations examined compared to the previous two insect species (Figures 6-8). In this case, for most of the combinations examined, untreated rice (without deltamethrin), either F or C, collected the highest number of individuals, except for the F vs. CD combination (Figure 8). Among the treatments, a significantly higher number of T. castaneum adults were detected in C (66%) compared to CD (34%) (Figure 8).  Conventional with deltamethrin vs. Conventional rice (CD vs. C). For each combination, means with asterisks (*) indicate significant differences between the two choices, according to Wilcoxon singedrank test at p < 0.05 (in all cases df = 4). Where no asterisk (*) exists, no significant differences were found. Wilcoxon's singed-rank test parameters for CD vs. C were z = −2.0, df = 1, p = 0.041.
The repellent effect of deltamethrin was strongly demonstrated in the case of O. surinamensis. In particular, only 1/5 of O. surinamensis adults chose FD in the combination, FD vs. C, while correspondingly, in the rest of the combinations the higher percentage of individuals chose F or C compared to FD or CD (Figure 9). Nevertheless, significant differences between the two options were shown in three out of four combinations tested (Figure 9). A similar behavior to O. surinamensis was also observed in the larvae of T. castaneum (Figures 9 and 10). In all combinations evaluated, the percentage of larvae that chose the deltamethrin-treated rice (FD or CD) was significantly lower than the percentage that chose the untreated rice (F or C) ( Figure 10). In addition, the highest percentage of preference was 66% and observed in C both in the combination, FD vs. C, and in CD vs. C ( Figure  10).

Discussion
There are several published works that confirm the repellency of Ocimum sp against stored product insects [33,34,36,40,[43][44][45][46][47][48]. For example, Al-Ghamdi and W [35] evaluated the efficacy of O. basilicum and the screwpine, Pandanus tectorius Par (Pandanales: Pandanaceae), powders against T. castaneum with satisfactory results. ever, all these papers involved essential oils, extracts, or dried plant materials. Ag et al. [41] in an experiment conducted on fortified rice with basil and spearmint foun the level of infestation of R. dominica, S. oryzae, and O. surinamensis was reduced com to conventional rice. This is the first paper that evaluates the repellent effect of bas fortifying material on rice either alone or in combination with deltamethrin.
For most of the insect species tested in the present study fortification with bas a moderate repellency effect. Indeed, S. oryzae, R. dominica, and T. castaneum adults sh 51 and 40, 58 and 39, and 55 and 26% repellency in fortified rice with basil in F vs. F vs. B combinations, respectively. The findings of our research are in line with th other works concerning the repellent action of both O. basilicum [36] and other spec the genus, Ocimum [33,43]. Al-Ghamdi and Wong [36]

Discussion
There are several published works that confirm the repellency of Ocimum species against stored product insects [33,34,36,40,[43][44][45][46][47][48]. For example, Al-Ghamdi and Wong [35] evaluated the efficacy of O. basilicum and the screwpine, Pandanus tectorius Parkison (Pandanales: Pandanaceae), powders against T. castaneum with satisfactory results. However, all these papers involved essential oils, extracts, or dried plant materials. Agrafioti et al. [41] in an experiment conducted on fortified rice with basil and spearmint found that the level of infestation of R. dominica, S. oryzae, and O. surinamensis was reduced compared to conventional rice. This is the first paper that evaluates the repellent effect of basil as a fortifying material on rice either alone or in combination with deltamethrin.
For most of the insect species tested in the present study fortification with basil had a moderate repellency effect. Indeed, S. oryzae, R. dominica, and T. castaneum adults showed 51 and 40, 58 and 39, and 55 and 26% repellency in fortified rice with basil in F vs. C and F vs. B combinations, respectively. The findings of our research are in line with those of other works concerning the repellent action of both O. basilicum [36] and other species of the genus, Ocimum [33,43]. Al-Ghamdi and Wong [36] found that 1 h of exposure to plant powders of O. basilicum caused 52% and 44% repulsion of S. oryzae and T. castaneum adults, respectively. Similarly, 1,8 cineole, the main component of O. kenyense Ayob. ex A.J.Paton (Lamiales: Lamiaceae), showed a moderate repellant effect even when applied at the highest dose tested (10 µL/disc) against adults of T. castaneum (48% mean precent repellency value) and the larger grain borer, Prostephanus trunacatus Horn (Coleoptera: Bostrychidae) (50% mean percent repellency value) [33]. In the same research, 1,8 cineole evoked strong repellency against the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), and the granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae) [33]. Additionally, Bekele et al. [43] demonstrated a moderate repellant action of dry (10-65%) and ground (28-59%) leaves, and essential oil (35-39%) of O Ocimum sauve Willd (Lamiales: Lamiaceae) against R. dominica. For R. dominica and T. castaneum adults, a percentage of 6 to 15% stayed on the line in the middle of the Petri dish and essentially did not choose any of the two options. In the case of R. dominica adults, this could be attributed to the reduced mobility of this species compared to other stored product insect species [49], while with regard to T. castaneum adults, this could be due to the extremely difficult return of its large body to a prone position after being in a supine position.
On the other hand, O. surinamensis adults and T. castaneum larvae showed the lowest rates of repellence from rice fortified with basil with 31-33 and 41-42%, respectively. In fact, rice fortified with basil had an attractive effect on O. surinamensis, as 67 and 69% of individuals were attracted by its presence. Agrafioti et al. [41] showed that progeny production of O. surinamensis was enhanced when rice fortified with basil was used as a substrate compared to rice fortified with spearmint. Accordingly, in a field experiment conducted in India in a potato crop, yellow sticky traps baited with basil oil attracted more adults of the winged cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), than yellow sticky traps baited with lavender, geranium, and tea tree oil [50]. In addition, Moawad and Ebadah [51] observed that O. basilicum oil was the most attractive oil to 1st instar larvae of the Indianmeal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae), compared to the rose geranium, Pelargonium graveolens L'Hér. (Geraniales: Geraniaceae), and the clove, Syzygium aromaticum (L.) (Myrtales: Myrtaceae), oils. On the other hand, the fact that O. surinamensis was attracted to rice with basil may also be due to its ability to infest different types of dried fruit and herbs [52].
The combined action of plant derivatives with chemical or natural insecticides in order to enhance the insecticidal efficacy against stored product insects has been extensively studied [53][54][55][56][57][58][59][60][61][62]. For instance, Yang et al. [56] found that the combined application of the essential oil of Allium sativum L. (Asparagales: Amaryllidaceae) with diatomaceous earth against S. oryzae and the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), was significantly more effective than the single application of either the essential oil or the diatomaceous earth. The combination of basil with deltamethrin for the management of both agriculture- [63] and public-health-affecting insects [64] has shown satisfactory results. Silva et al. [63] evaluated the combined action of O. basilicum essential oil with deltamethrin for the control of the fall armyworm, Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae), and found that the dose of deltamethrin required to kill 50% of the population of S. frugiperda was reduced by 80% when applied in combination with O. basilicum oil compared to when applied alone. Similarly, in the work of Suwannayoud et al. [64], the mixture of Ocimum gratissimum essential oil with deltamethrin had a synergistic effect against the blow flies, Chrysomya megacephala (F.) (Diptera: Calliphoridae), Chrysomya rufifacies Macquart (Diptera: Calliphoridae), Lucilia cuprina Wiedemann (Diptera: Calliphoridae), and the house fly, Musca domestica L. (Diptera: Muscidae). Nonetheless, no research is available demonstrating the repellency of plant materials in combination with chemical insecticides with regard to stored product insects. Our results showed that O. surinamensis adults and T. castaneum larvae were attracted to rice fortified with basil in the first trial. However, when rice fortified with basil was combined with deltamethrin, a strong repellent effect was observed for O. surinamensis adults. Indeed, deltamethrin has been found to repel insects [65][66][67][68] through contact repellency or irritability, in which an insect is stimulated to leave the immediate toxic environment upon contact with the treated surface [69]. Still, in our study we are not able to determine which type of repellency took place at least in the stored product insects examined here.

Conclusions
Our results highlight the preference of major stored product insects in rice fortified with basil. In the untreated trials, most insect species showed moderate repellence from fortified rice with basil, with the exception of O. surinamensis, which was attracted to rice fortified with basil. On the contrary, O. surinamensis adults and T. castaneum larvae were repelled by the deltamethrin treated-rice that was fortified with basil. Integrated pest management in stored products is based on the use of more than one ecologically com-patible control method. Our data indicate that the combination of techniques, such as the fortification with other chemical compounds of low mammal toxicity and the application of deltamethrin could play an important role in stored product protection. The current work suggests that, under certain circumstances and for specific insect species, the use of rice fortified with basil and treated with deltamethrin is promising for stored rice protection. However, additional experimental work is required towards this direction.