Journal Pre-proof

Protection of cowpea seeds and toxicity against cowpea weevils by the essential oils
from Lippia alba (verbenaceae) and Schinus terebinthifolius (anacardiaceae)

Rafael Torre, Elisabeth Alves Duarte Pereira de Medeiros, Camila da Silva Barbosa
Pereira, Ana Clara Ramos Menezes, Igor Sampaio Fontes, Larissa Vitória Ribeiro
Pereira, Diego Henrique Fernandes Paiva, Andre Marques dos Santos, Pedro Corrêa
Damasceno-Junior, Marco Andre Alves de Souza

PII: S0261-2194(24)00098-X
DOI: https://doi.org/10.1016/j.cropro.2024.106670
Reference: JCRP 106670

To appear in: Crop Protection

Received Date: 30 January 2024

Revised Date: 15 March 2024
Accepted Date: 20 March 2024

Please cite this article as: Torre, R., Medeiros, E.A.D.P.d., Pereira, C.d.S.B., Menezes, A.C.R., Fontes,
I.S., Pereira, Larissa.Vitó.Ribeiro., Paiva, D.H.F., Santos, A.M.d., Damasceno-Junior, Pedro.Corrê.,
Souza, M.A.A.d., Protection of cowpea seeds and toxicity against cowpea weevils by the essential oils
from Lippia alba (verbenaceae) and Schinus terebinthifolius (anacardiaceae), Crop Protection (2024),
doi: https://doi.org/10.1016/j.cropro.2024.106670.

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© 2024 Published by Elsevier Ltd.

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 1 Protection of cowpea seeds and toxicity against cowpea weevils by the essential oils

2 from Lippia alba (Verbenaceae) and Schinus terebinthifolius (Anacardiaceae)

4 Rafael Torre1, Elisabeth Alves Duarte Pereira de Medeiros1, Camila da Silva Barbosa Pereira1,

5 Ana Clara Ramos Menezes1, Igor Sampaio Fontes1, Larissa Vitória Ribeiro Pereira2, Diego

6 Henrique Fernandes Paiva2, Andre Marques dos Santos2, Pedro Corrêa Damasceno-Junior3,

7 Marco Andre Alves de Souza1*

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9 1 – Laboratory of Aromatic and Medicinal Plants, Department of Biochemistry, Federal Rural

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10 University of Rio de Janeiro, Brazil.

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11 Rafael Torre; ORCID: 0000-0002-8248-7080 -p
12 Elisabeth Alves Duarte Pereira de Medeiros; ORCID: 0000-0002-3500-2730
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13 Camila da Silva Barbosa Pereira; ORCID: 0000-0002-8283-7845
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14 Ana Clara Ramos Menezes; ORCID: 0009-0000-4931-288X

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15 Igor Sampaio Fontes; ORCID: 0009-0003-1313-5756

16 Marco Andre Alves de Souza*; ORCID: 0000-0003-2173-3513

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17
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18 2 – Laboratory of Plant Biochemistry, Department of Biochemistry, Federal Rural University of

19 Rio de Janeiro, Brazil.

20 Andre Marques dos Santos; ORCID: 0000-0002-2397-3775

21 Larissa Vitória Ribeiro Pereira; ORCID: 0009-0008-2228-5576

22 Diego Henrique Fernandes Paiva; ORCID: 0009-0006-9412-1381

23

24 3 – Department of Agrotechnology and Sustainability, Institute of Agronomy, Federal Rural

25 University of Rio de Janeiro, Brazil.

26 Pedro Corrêa Damasceno Junior; ORCID: 0000-0001-8879-4850

27

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28 *Corresponding author: Marco Andre Alves de Souza. Address: Rodovia Br 465, km 7,

29 Universidade Federal Rural do Rio de Janeiro, Pavilhão de Química, Sala 26. CEP: 23.897-

30 00023089, Seropédica, Rio de Janeiro, Brazil. Phone: +55 (21) 2681-4600. e-mail:

31 decoerej@ufrrj.br

32

33 ABSTRACT

34 Alternative methods have been widely studied to mitigate problems arising from the use of

35 pesticides in agriculture. Among them is the use of essential oils to control stored grain pests. In

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36 this context, the aim of this study was to evaluate the effect of fumigation of cowpeas with 12

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37 essential oils having different chemical profiles, derived from the germplasm collection of

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38 Schinus terebinthifolius and Lippia alba at Rio de Janeiro Federal Rural University. These oils

39
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were applied at various concentrations (0.1 to 1.0 mg/mL of air) against cowpea weevils
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40 (Callosobruchus maculatus). The chemotypes evaluated included citral, citral/limonene,
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41 carvone/limonene, and linalool from the essential oils of L. alba, as well as α-pinene, sabinene,

42 α-phellandrene/α-pinene, β-pinene/α-pinene, δ-carene/α-pinene, limonene, α-

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43 phellandrene/sabinene, and myrcene from the essential oils of S. terebinthifolius. Various

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44 aspects of the insects’ reproductive cycle were evaluated, including mortality, egg laying,
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45 emergence of new adults, emergence rate, and larval hatching, along with grain mass loss. The

46 results indicated life cycle inhibition of all evaluated oils, with the essential oil from the UFRRJ

47 ECB028 (linalool CT) genotype having the lowest LC50 (0.10 mg/mL of air), completely halting

48 the insects’ life cycle and totally preventing seed mass loss. Overall, the results pointed to

49 certain genotypes as promising for the development of technologies aimed at seed protection.

50 Keywords: Vigna unguiculata, Callosobruchus maculatus, bushy matgrassis, Brazilian

51 verbena, pink-pepper, Brazilian pepper tree.

52 1 INTRODUCTION

53 The cowpea (Vigna unguiculata (L.) Walp), originating from Africa, is a nutrient-rich species

54 now cultivated and consumed in all continents, making it a significant commodity (Ketema et

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55 al., 2020). However, the storage of cowpeas requires protective measures, since the cowpea

56 weevil (Callosobruchus maculatus Fabricius, 1775. Coleoptera: Bruchidae) stands out as the

57 primary post-harvest pest, attacking the grains during storage (Kalpna et al., 2022).

58 The cowpea weevil is a cosmopolitan pest originating from Africa that has spread worldwide,

59 capable of infesting various legumes besides the cowpea (Kébé et al., 2017; Tuda et al., 2014).

60 During the larval stage, the endosperm of seeds is consumed by the larvae, reducing their mass

61 and impairing their nutritional and sanitary quality by facilitating the infestation of opportunistic

62 fungi (Farrell et al., 2002; Rajendran, 2020). High reproductive success rates, rapid

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63 development, a short life cycle, and excellent adaptation to diverse climatic conditions have

64 made the cowpea weevil a challenging pest to control, potentially causing losses of up to 80%

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of production within a few months (Kpoviessi et al., 2019).
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66 Currently, the fumigation of stored grains with metallic phosphides, generating phosphine gas,
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67 is the most widely used method for chemical protection of stored grains (Rajendran, 2020).

68 Phosphine (PH3) gas is toxic to pests but is also harmful to mammals (World Health
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69 Organization et al., 1988; Tuet et al., 2020). Inhalation of this gas produces immediate effects
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70 such as nausea, respiratory problems, headaches, and gastrointestinal and pulmonary disorders.

71
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Therefore, the average maximum phosphine exposure level for adults during working hours is

72 set at 0.42 mg/m3 in most developed countries (Davis, 2003; Saini and Kaushik, 2021).

73 Pesticides have a residual effect that persists in the environment longer than natural products,

74 leading to water, soil and air contamination. Consequently, it is harmful human and animal

75 health (Saini and Kaushik, 2021). Furthermore, irresponsible use contributes to the development

76 of resistant populations of agricultural pests (Sağlam et al., 2015).

77 The use of essential oils from aromatic plants has been gaining traction as an environmentally

78 friendly and low-risk alternative. In addition to these advantages, the complexity of the

79 chemical profile of essential oils reduces the likelihood of the emergence of resistant insect

80 populations (Basaid et al., 2021; Isman, 2020; Lucia and Guzmán, 2021).

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 81 There are few studies investigating the biotechnological potential of essential oils from native

82 aromatic species for the control of agricultural pests of interest. In this context, exploring the

83 chemical diversity of natural products and prospecting for biological activities is an interesting

84 path that contributes to the valorization of these native species. Therefore, this study assessed

85 the toxicity of essential oils from different genotypes of Lippia alba (Mill.) N.E.Br. ex Britton

86 & P. Wilson (Verbenaceae) and Schinus terebinthifolius Raddi (Anacardiaceae), originating

87 from the aromatic species collection of Rio de Janeiro Federal Rural University (UFRRJ),

88 against the pest C. maculatus.

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89 2 MATERIAL AND METHODS

90 2.1 Material

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Seeds of Vigna unguiculata, belonging to the commercial class White, subclass Fradinho,
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92 cultivar BRS Itaim, were obtained from a local market and sterilized at -20 °C for 24 hours.
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93 Insects of the species Callosobruchus maculatus (Fabricius, 1775) were provided by the

94 Agricultural Sciences Center of Federal University of Ceará. Since 2012, a colony has been
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95 maintained by the Laboratory of Aromatic and Medicinal Plants at UFRRJ. The plant material,
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96 used as a source for essential oil distillation, was obtained from genotypes in the germplasm
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97 collection of the Department of Agrotechnologies and Sustainability at the Agronomy Institute

98 of UFRRJ. These genotypes are duly registered in the National System for Management of

99 Genetic Heritage and Associated Traditional Knowledge (SisGen) under the code AF533EB for

100 the Schinus terebinthifolius (Brazilian pepper tree) and code AFF48EE for the Lippia alba

101 collection (Brazilian verbena or bushy matgrassis).

102 2.2 Source material and essential oil distillation

103 The essential oils were obtained through hydrodistillation of dried and crushed S.

104 terebinthifolius fruits (genotypes: UFRRJ ARO050, UFRRJ ARO079, UFRRJ ARO011,

105 UFRRJ ARO025, UFRRJ ARO032, UFRRJ ARO078, UFRRJ ARO022) and dried whole

106 leaves of L. alba (genotypes: UFRRJ ECB021/022, UFRRJ ECB037/029/016, UFRRJ

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107 ECB003/008, UFRRJ ECB028) for a period of 2 hours using a modified Clevenger apparatus.

108 Each of the essential oils represented a different chemical group (chemotype, CT) characteristic

109 of their respective collections (Table 1 and Figure S1). Some L. alba essential oils, used in the

110 biological assays, are a mixture of essential oils from genotypes with similar chemical profiles

111 (see Table 1) because they produced smaller amounts of essential oil.

112 2.3 Chemical analysis and identification

113 Chemical analysis was carried out according to a previously published protocol/program (Alves

114 et al., 2019) and is presented below in summary form. To separate, detect and quantify the

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115 constituents, 1 µl of each essential oil sample (10 µl/ml) was injected into a Hewlett-Packard

116 5890 Series II gas chromatograph (Palo Alto, USA) equipped with a flame ionization detector

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(GC-FID), in split/splitless injector mode (split ratio of 1:20) and a fused silica capillary column
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118 (5% Phenyl 95% dimethylpolysiloxane), with size of 30 m × 0.25 mm (i.d.) × 0.25 μm (film
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119 thickness). To separate and identify the substances, 1.0 µl of essential oil sample (10 µl ml-1)

120 was injected into a QP-2010 Plus (Shimadzu, Japan) gas chromatograph coupled to a mass
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121 spectrometer (GC-MS) and the programming were the same as described for the GC-FID and
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122 the previously published article from our group (Alves et al., 2019). The identification of

123
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volatile compounds in the essential oil was based on linear retention indices (LRI) and mass

124 spectra of the samples according to the literature (Adams, 2007; Alves et al., 2019; van Den

125 Dool and Dec. Kratz, 1963).

126 2.4 Toxicity assay

127 2.4.1 Procedure

128 The method involved static fumigation, where the volatiles from the essential oil reached

129 equilibrium with the atmosphere inside the experimental unit. The experimental unit comprised

130 a 50 mL Falcon tube containing 30 whole cowpea grains, pre-sterilized at -20 °C for 24 hours in

131 a freezer, along with ten recently emerged C. maculatus insects, aged up to two days, sexed at a

132 1:1 ratio. Each Falcon tube was sealed with a lid containing a 2 cm diameter filter paper disk

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133 glued on the inside, with or without the essential oil. Eight essential oils obtained from S.

134 terebinthifolius genotypes and four from L. alba were evaluated. The concentrations of the

135 essential oils varied depending on the quantity available for the assays. See Table 1 for the

136 essential oils tested and the respective concentrations used in the assays. Treatments were

137 produced by weighing a quantity of essential oil directly onto the filter paper attached to the

138 inside of the Falcon tube lid using an analytical balance. Treatments and the controls (without

139 essential oil) were prepared with 6 replicates (n=6) and carried out in two experiments. The

140 experimental units were incubated in a climate-controlled chamber at 28 °C (±2 °C) and relative

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141 humidity of 65±10% throughout the experiment. The parameters of the evaluations conducted

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142 are displayed in Table 1.

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143 2.4.2 Observed variables -p
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144 Mortality – After 48 hours of incubation, insects were removed from the tubes for mortality
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145 counting and subsequent disposal. Insects stimulated with dissecting forceps that did not move

146 were considered dead. The filter paper lids were then replaced with woven nylon screens.
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147 Egg Laying (Oviposition) – Between 6 and 9 days of incubation, seeds were removed from the
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148 experimental units for egg-laying counting (oviposition).

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149 Hatching – Using a stereomicroscope, the color of eggs deposited on the seeds was assessed on

150 the ninth day of incubation. Those showing a whitish color were considered hatched (Figure

151 S2).

152 Fertility Rate – Considering oviposition and hatching, the fertility rate was calculated as the

153 ratio of the number of eggs with larval hatching to oviposition, multiplied by 100.

154 Emergence – After 20 days of incubation, daily observation of the experimental units was

155 conducted to count the daily emergence of new adult insects and the total emergence over a 10-

156 day period after the onset of emergence.

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157 Emergence Rates – The emergence rate was calculated as the ratio of total emergence to the

158 total number of eggs, multiplied by 100.

159 2.4.3 Seed Weight Loss (%)

160 This was determined based on the dry matter of the seeds, considering that the insects' action on

161 the seeds results in a change in seed moisture content before and after the experiment.

162 Therefore, 30 cowpea seeds from each experimental unit were weighed before and after the

163 assays, and the weight was recorded. To determine the dry matter of the cowpea seeds used in

164 the assays, a sample of 30 seeds (n=3) from the same batch was weighed before and after drying

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165 in an oven at 102 °C for 72 hours, and the dry matter found was estimated for the experimental

166 units. The seeds from the respective experimental units underwent the same drying procedure at

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the end of the experiment to calculate dry matter. Attention! The cowpea seeds placed in the
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168 tubes were not subjected to drying at 102°C; samplings from the batch were used for this
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169 estimation. The difference in dry matter before and after the experiment was the basis for

170 determining the percentage of seed weight loss (%WL) at the end of the assays. Equation:
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171 %WL=100-(100A)/(BD/C), where: A=dry matter of experimental unit seeds at the end of the
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172 test; B= mass of experimental unit seeds at the beginning of the test; C= mass of the seeds

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sampled from the same batch before drying; D=seed sample dry matter from the same batch

174 after drying.

175 2.5 Statistical analysis

176 The data were organized and subjected to analysis of variance (one-way ANOVA) in a

177 completely randomized design, and the means of the treatments were compared using the Tukey

178 test (α=0.05). Insect mortality data were subjected to median lethal concentration (or LC50)

179 analysis to estimate the lethal concentration for 50% of the population using GraphPad Prism

180 9.0 software (GraphPad Software, USA) and non-linear regression and dose-response functions.

181 For the biological assay, the assumptions of normality and homogeneity of variance were

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182 checked using GraphPad Prism v.9 software, no data transformation was necessary and the data

183 were unbalanced, with a small number of data points lost or discarded.

184 3 RESULTS

185 The main characteristic distinguishing the essential oils from different genotypes was their

186 chemical composition, as presented in Tables 2 and 3. Based on the chemical variations of the

187 major substances and with the aid of multivariate analysis (Figure S1), essential oils containing

188 the following major substances, here referred to as chemotypes (CT), were selected: sabinene

189 CT (UFRRJ ARO050), α-phellandrene/α-pinene CT (UFRRJ ARO079), α-pinene CT (UFRRJ

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190 ARO011), β-pinene/α-pinene CT (UFRRJ ARO025), δ-carene/α-pinene CT (UFRRJ ARO032),

191 α-phellandrene/sabinene CT (UFRRJ ARO078), myrcene CT (UFRRJ ARO022), limonene CT

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192 (UFRRJ ARO094), citral/limonene CT
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193 ECB037/029/016), carvone/limonene CT (UFRRJ ECB003/008), and linalool CT (UFRRJ
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194 ECB028).

195 3.1 Mortality and LC50

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196 After 48 hours of exposure to the essential oil volatiles, dead insects were counted. All of them
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197 showed some level of toxicity to the cowpea weevil. No mortality or significant difference was
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198 observed between the control and treatments for the genotypes UFRRJ ECB021/022

199 (citral/limonene CT; F=1.507; df=5, 29; P=0.2182) and UFRRJ ECB037/029/016 (citral CT;

200 F=1.0, df=5, 30; P=0.4346) (Table 4). In assays with essential oils obtained from the genotypes

201 UFRRJ ARO094 (limonene CT), UFRRJ ARO022 (myrcene CT), and UFRRJ ECB003/008

202 (carvone/limonene CT), total insect mortality was not observed in the treatment with the highest

203 concentration of essential oil (respectively, 65.00, 81.67 and 73.33% mortality) (Table 4). The

204 most interesting mortality results were found at the concentration of 0.25 mg/mL of air for the

205 treatment with the essential oil of the genotype UFRRJ ECB028 (linalool CT; Mortality=100%)

206 and at the concentration of 0.50 mg/mL of air in treatments with the essential oils of the

207 genotypes UFRRJ ARO011 (α-pinene CT), UFRRJ ARO050 (sabinene CT), UFRRJ ARO025

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208 (β-pinene/α-pinene CT), UFRRJ ARO079 (α-phellandrene/α-pinene CT), UFRRJ ARO032 (δ-

209 carene/α-pinene CT), and UFRRJ ARO078 (α-phellandrene/sabinene CT) (respectively, 98.33,

210 93.33, 88.33, 90.0, 91.67 and 83.33% mortality) (Table 4).

211 The mortality data were subjected to nonlinear regression analysis, and the best fit to the data

212 was the sigmoidal distribution, considering one of the factors as the mortality for 50% (LC50) of

213 the population. The results indicated that the substances from the genotypes L. alba UFRRJ-

214 ECB-028 (linalool CT) and S. terebinthifolius UFRRJ ARO025 (β-pinene/α-pinene CT)

215 presented LC50 values of 0.102 and 0.287 mg/mL of air, respectively, the lowest such values

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216 within their collections (Table 5). It was not calculated the LC50 in the treatment with the

217 essential oil of the genotype UFRRJ ARO022 (myrcene CT), because the data did not meet the

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important statistical assumptions necessary for analysis.
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219 3.2 Egg laying (oviposition)
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220 Egg laying was counted between the sixth and ninth day of incubation, and a significant

221 reduction in the average egg laying was observed. Egg laying values were observed in insects
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222 exposed to 0.50 mg/mL of air of essential oils from the genotypes UFRRJ ARO025 (β-
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223 pinene/α-pinene CT), UFRRJ-ARO 079 (α-phellandrene/α-pinene CT), UFRRJ ARO094

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224 (limonene CT), UFRRJ ARO078 (α-phellandrene/sabinene CT), UFRRJ ECB003/008

225 (carvone/limonene CT) and results were 24.85, 25.52, 22.33, 21.25 and 23.69%, respectively.

226 When exposed to 0.25 mg/mL of essential oil from the genotype UFRRJ ARO050 (sabinene

227 CT) results was 31.84%. Among the essential oils evaluated, the genotype UFRRJ ECB028

228 (linalool CT) stood out, promoting approximately 70% inhibition in egg laying in the treatment

229 with 0.10 mg/mL of air (Egg laying=29.96%; F=65.07; df=5, 30; P<0.0001) (Table 6).

230 3.3 Emergence

231 Table 7 presents the consolidated values of new adult emergence. Overall, the emergence of a

232 new generation of adult insects was inhibited by all tested essential oils. A decrease in

233 emergence was observed from the concentration of 0.25 mg/mL of air for S. terebinthifolius

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234 oils. All tested essential oils from S. terebinthifolius, at concentrations equal to or higher than

235 0.50 mg/mL of air inhibited the emergence of new adults by more than 50% (Table 7).

236 Treatments with essential oils from the L. alba showed the best results, with almost total

237 inhibition of new adult emergence when insects were exposed to the concentration of 0.25

238 mg/mL of air from the essential oils of the genotypes UFRRJ ECB021/022 (citral/limonene CT;

239 Emergence=4.37%; F=117.5; df=5, 29; P<0.0001) and UFRRJ ECB037/029/016 (citral CT;

240 Emergence=3.33%; F=220.1; df=5, 30; P<0.0001). Notably, essential oils UFRRJ ECB003/008

241 (carvone/limonene CT; F=202; df=5, 30; P<0.0001) and UFRRJ ECB028 (linalool CT;

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242 F=579.0; df=5, 30; P<0.0001) inhibited 100% of new adult emergence from the concentration

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243 of 0.10 mg/mL of air (Table 7).

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244 3.4 Hatching and fertility rates -p
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245 The egg hatching was counted by observing whether they were translucent or white (Figure
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246 S2), determining the fertility rate (Table 8). There was no interesting effect of S. terebinthifolius

247 oils on the fertility rate (UFRRJ ARO032: 99.48 – 90.14% and UFRRJ ARO094: 76.38 –
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248 92.74%), meaning that, in absolute terms, the number of hatched eggs in relation to the total
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249 egg laying in the treatments remained the same as the control (Figure 1). On the other hand,
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250 treatments in which insects were exposed to essential oils from the genotypes UFRRJ

251 ECB021/022 (citral/limonene CT) and UFRRJ ECB037/029/016 (citral CT) drastically inhibited

252 fertility, by inhibiting the hatching of almost all eggs from the concentration of 0.25 mg/mL of

253 air (Hatching=7.39; F=91.20; df=5, 29; P<0.0001 and Hatching=4.51; F=225.8; df=5, 30;

254 P<0.0001) and 100% inhibition from 0.10 mg/mL of air when exposed to the essential oils of

255 ECB-003/008 and ECB-028 (Table 9).

256 3.5 Emergence Rates

257 The emergence rate expresses the ratio between new adults and the total egg laying, and overall

258 the results were similar to those observed for the fertility rate, with negligible inhibitory effects

259 for S. terebinthifolius essential oils. On the other hand, essential oils from the genotypes UFRRJ

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260 ECB003/008 (carvone/limonene CT; F=515.1; df=5, 30; P<0.0001) and UFRRJ ECB028

261 (linalool CT; ; F=930.7; df=5, 30; P<0.0001) produced an emergence rate equal to zero at the

262 concentration of 0.10 mg/mL of air, and for the genotypes UFRRJ ECB-021/022

263 (citral/limonene CT; F=168.8; df=5, 30; P<0.0001) and UFRRJ ECB-037/039/016 (citral CT;

264 F=205.1; df=5, 30; P<0.0001), the emergence rate was close to zero at the concentration of 0.25

265 mg/mL of air (Table 10).

266 3.6 Seed weight loss (%)

267 Considering the difference in dry mass before and after a reproductive cycle, it was possible to

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268 calculate the percentage of seed mass loss, and therefore assess the degree of protection

269 conferred by essential oils to the seeds. The results pointed to a decrease in cowpea weight loss

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when insects were exposed to different S. terebinthifolius and L. alba essential oils for 48 hours
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271 at various concentrations (Table 11). For S. terebinthifolius essential oils, protection of nearly
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272 60% was observed at the concentration of 0.50 mg/mL of air (UFRRJ ARO032: weight loss

273 =19.48%; F=26.66; df=5, 28; P<0,0001 and UFRRJ ARO094: weight loss =13.15%; F=37.01;
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274 df=4, 23; P<0,0001). For essential oils from the genotypes UFRRJ ECB003/008
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275 (carvone/limonene CT; F=386.3; df=5, 30; P<0.0001) and UFRRJ ECB028 (linalool CT;

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F=1471.0; df=5, 30; P<0.0001), close to 100% protection was observed at the concentration of

277 0.10 mg/mL of air, and for the genotypes UFRRJ ECB-021/022 (citral/limonene CT; F=137.0;

278 df=5, 29; P<0.0001) and UFRRJ ECB-037/039/016 (citral CT; F=168.8; df=5, 30; P<0.0001) at

279 the concentration of 0.25 mg/mL of air (Table 11).

280 4 DISCUSSION

281 Different essential oils from S. terebinthifolius and L. alba have been characterized in the

282 aromatic plant collections at UFRRJ (Figure S1, Tables 2 and 3). One way to valorize these

283 essential oils has been to explore technological applications, focusing, for example, on the

284 agricultural sector for animal and plant health, as undertaken by various authors (Adorjan and

285 Buchbauer, 2010; Alonso-Gato et al., 2021; Chaubey and Chaubey, 2019; Ellse and Wall,

286 2014).

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287 Some researchers have investigated the chemical composition of essential oils to identify the

288 active principle responsible for toxicity, and with some success they have identified one or more

289 substances (Ma et al., 2020; Paventi et al., 2020). On the other hand, other authors have

290 evaluated and confirmed the synergistic effect of substances in essential oils (Chen et al., 2021;

291 Gaire et al., 2020; Liang et al., 2020). In any case, the key is to identify a specific oil that

292 exhibits activity and can be a suitable natural product for the development of a technological

293 application, as demonstrated by Isman and colleagues (Isman, 2020; Isman et al., 2011).

294 In our study, mortality was the initial variable observed, and we found that regardless of the

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295 essential oil, the majority exhibited some level of toxicity, reducing the life expectancy of the

296 insects to varying extents (Table 4). Various articles have highlighted the toxic effects of S.

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terebinthifolius essential oils (Marcela S. Alves et al., 2015; Fouad et al., 2023; Vicenço et al.,

298 2020) and L. alba essential oils (de Albuquerque Lima et al., 2021; Deka et al., 2021) on pests.
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299 The study conducted by Oliveira et al. (2017) highlighted the toxicity of S. terebinthifolius

300 essential oil against the pest insect C. maculatus. Significant results were observed, including
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301 effects on mortality, with a lethal concentration for 95% of individuals (LC95) of 38 μL/L of air,
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302 as well as a reduction in egg laying and the emergence of new adult insects, consistent with

303
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those found in the present study. Alves et al. (2015), in their work, also investigated the effects

304 of L. alba essential oil, citral chemotype, on C. maculatus. When applying this essential oil at a

305 concentration of 0.4 µL/mL of air, they observed a mortality of 66.7%, an inhibition of 85.1% in

306 egg laying, and a complete inhibition (100.0%) in the emergence of new adults. Additionally, in

307 the same study, Alves and colleagues reported that S. terebinthifolius essential oil caused a

308 mortality of 33.3%, an inhibition of 82.4% in egg laying, and an inhibition of 96.9% in the

309 emergence of new adults.

310 S. terebinthifolius essential oil also demonstrated toxicity against other agricultural pest insects.

311 According to Abdelgaleil et al. (2016), the fumigant effect of this essential oil exhibited an LC50

312 of 28.16 mg/mL of air against Sitophilus oryzae (Coleoptera: Curculionidae), commonly known

313 as the rice weevil. Additionally, Bezerra et al. (2023) reported mortality rates of 83.33% in

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314 nymphs and 75.75% in adults of Aphis craccivora Koch (Hemiptera: Aphididae), commonly

315 referred to as the black bean aphid. These same authors also observed, under greenhouse

316 conditions, mortality rates of 73.52% in nymphs and 62.85% in adults.

317 Several studies have observed the toxic effects of L. alba essential oil on insects. For instance,

318 Benelli et al. (2018) reported that L. alba essential oil demonstrated an LC50 of 59.6 μL/L

319 against 4th instar larvae of Culex quinquefasciatus (Diptera: Culicidae) and an LD50 of 110

320 μg/adult in Musca domestica (Diptera: Muscidae). According to Peixoto et al. (2015), essential

321 oils from different genotypes of L. alba presented LC50 values against Sitophilus zeamais

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(Coleoptera: Curculionidae) ranging from 15.2 to 16.7 μL/mL and from 54.6 to 70.8 μL/mL,

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322

323 respectively, for carvone and citral chemotypes. The same authors also evaluated the effect

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against the insect Tribolium castaneum (Coleoptera: Tenebrionidae), observing a range of LC50

325 values from 19.5 to 28.7 and from 67.2 to 107.8 μL/mL, respectively, for carvone and citral
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326 chemotypes. Additionally, Shukla et al. (2011) found that L. alba essential oil, at a
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327 concentration of 0.1 µL/mL, exhibited 100% mortality and ovicidal, larvicidal, and pupicidal
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328 activities of 49.1%, 22.8 to 50.9%, and 21.87%, respectively.

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329 Among the analyzed essential oils in this study, the genotype UFRRJ ECB 028 (linalool CT)

330
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exhibited the strongest toxic effect, with a LC50 of 0.1017 mg/mL of air (Table 5). This same

331 essential oil induced 100% mortality of the insects at a concentration of 0.25 mg/mL of air

332 (Table 4). It is important to note that, considering the static fumigation method, only the

333 volatiles in equilibrium within the experimental unit exerted a toxic effect. The insect does not

334 come into direct contact with the essential oil. Essential oils from different species that had

335 linalool as the major component also yielded similar fumigation results against insect pests

336 (Kheloul et al., 2020; Kim and Lee, 2014; López et al., 2008; Peixoto et al., 2015).

337 But what would be the explanation for the toxic effect of linalool? Monoterpenes derived from

338 plants have been extensively studied for their insecticidal activity, with several studies

339 demonstrating their impact on different pest insects (Abdelgaleil et al., 2021; Karabörklü and

340 Ayvaz, 2023). However, the precise mechanisms of action remain incompletely elucidated.

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341 Review articles have highlighted that monoterpenes, including linalool, may exert their toxicity

342 through a variety of pathways, encompassing neurotoxic effects and inhibition of detoxification

343 enzymes. Abdelgaleil et al. (2021) reviewed the mode of action of linalool in different pest

344 insects, noting its inhibitory effect on acetylcholinesterase (AChE) activity. Karabörklü and

345 Ayvaz (2023) also included the inhibition of glutathione-S-transferase (GST) and Na+/K+–

346 ATPase in their list. In summary, evidence suggests that monoterpenes such as linalool can

347 exert insecticidal activity through a variety of targets, including nervous and enzymatic systems.

348 Egg laying was affected by all tested essential oils (Table 6), even those where mortality was

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349 not high. For example, genotypes UFRRJ ECB021/022 (citral / limonene CT) and UFRRJ

350 ECB037/029/016 (citral CT) caused a low but significant reduction in egg laying, but almost no

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mortality was observed (Tables 4 and 6). In this case, it appears that toxicity exclusively

352 influences embryo formation in the egg, decreasing hatching and consequently fertility and the
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353 emergence of new adults (Tables 7, 8, and 9).
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354 There was a substantial reduction in hatching, and consequently a decrease in fertility and insect
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355 emergence rates, under the influence of L. alba essential oils (Figure 1, Tables 8 and 10).
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356 However, the essential oils of S. terebinthifolius UFRRJ ARO032 and UFRRJ ARO094, which

357
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barely reduced hatching, also promoted some reduction in the emergence of new adults (Figure

358 1 and Table 7). What could be the explanation? In this case, overall the treatments did not differ

359 from the control for fertility and emergence rates (Tables 8 and 10). Therefore, the decrease in

360 the emergence of a new generation of adult insects was a consequence of the reduction in total

361 egg laying (Figure 1 and Table 6). Probably the production and maturation of eggs, considering

362 all the energy flow required for this event, were hindered, preventing egg laying. If egg laying is

363 lower, certainly, the progeny will be lower as well.

364 These results suggest that the volatile substances in essential oils have various biochemical

365 targets and act on different reproductive stages. The effects of essential oils on egg laying are a

366 consequence of the depletion of diverted energy resources, such as those directed towards the

367 xenobiotic pathway, as reported in the literature (Alves et al., 2023, 2019). Additionally, various

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368 authors have indicated that the sequestration of energy resources crucial for oogenesis is the

369 reason for the reduction in oviposition, as well as the emergence of new adults (El-Minshawy et

370 al., 2018; Santos et al., 2021; Wang and Horng, 2004).

371 Among all the results presented, what stands out the most is the effect of essential oils and their

372 potential application related to seed protection. In practical terms, how the use of essential oil

373 protects the seed from the action of the bruchid can be easily assessed by the seed mass loss.

374 The results indicated excellent protection provided by the essential oils of L. alba, especially the

375 genotypes UFRRJ ECB 0003/008 (carvone/limonene CT) and UFRRJ ECB 028 (linalool CT),

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which starting from 0.10 mg/mL of air protected the seeds 100% against the weevils’ action,

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376

377 since no mass loss was observed after incubation of the seed with the insect (Table 11). Similar

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results have been obtained with other essential oils versus grain storage pests such as C.

379 maculatus and Acanthoscelides obtectus (Rodríguez-González et al., 2019; Viteri Jumbo et al.,
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380 2018).
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381 6 CONCLUSION
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382 In general, the results are promising, by opening perspectives for the use of essential oils in
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383 grain protection. Our findings demonstrated that essential oils from L. alba genotypes were

384
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more efficient in seed protection, especially the genotypes UFRRJ ECB 0003/008

385 (carvone/limonene CT) and UFRRJ ECB 028 (linalool CT). The reduction in hatching was the

386 primary factor that contributed to seed protection, by decreasing the larval stages that consume

387 the seeds, thus reducing their mass, sanitary value, and nutritional content. These results offer

388 interesting prospects for the development of technologies aimed at pesticide-free seed

389 protection. Also, the results presented contribute, in a broader sense, to integrated pest

390 management in agriculture. It represents an additional effective technological possibility for

391 ensuring the health of seeds and grains, protecting them from pests and diseases, while

392 simultaneously contributing to the reduction of synthetic pesticide use and promoting more

393 sustainable practices.

394 ACKNOWLEDGEMENTS

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395 We gratefully acknowledge support from the Postgraduate Program in Chemistry (PPFQ-IQ-

396 UFRRJ), Rio de Janeiro State Research Foundation (FAPERJ) and Office to Coordinate

397 Improvement of Higher Level Personnel (CAPES).

398 DECLARATIONS

399 Ethics approval – Not applicable.

400 Competing interests – The authors declare they have no known competing financial interests

401 or personal relationships that could have appeared to influence the work reported in this paper.

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402 Authors' contributions – Rafael Torre, Elisabeth Medeiros, Camila Pereira: conceptualization,

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403 formal analysis, writing - original draft; Ana Menezes, Igor Fontes, Larissa Pereira, Diego

404
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Paiva: investigation; Andre Santos, Pedro Damasceno-Junior, Marco Souza: supervision,
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405 writing - review & editing; Marco Souza: project administration, funding acquisition.
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406 Funding – This study was financed in part by Rio de Janeiro State Research Foundation
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407 (FAPERJ) - Finance Codes: E-26/211374/2021 and E-26/211.547/2021; and by the Office to

408 Coordinate Improvement of Higher Level Personnel (CAPES), Finance Code 001.
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587 https://doi.org/10.1371/journal.pone.0207618

588 Wang, M.H., Horng, S. Bin, 2004. Egg dumping and life history strategy of Callosobruchus

589 maculatus. Physiol. Entomol. 29, 26–31. https://doi.org/10.1111/J.0307-6962.2004.0357.X

590

591

592 FIGURE CAPTIONS

593

23
594 Figure 1. Absolute values of egg laying, hatched eggs (green column), and non-hatched
595 eggs (red column) of C. maculatus under the influence of volatile compounds from
596 essential oils of the UFRRJ ARO032 (a) and UFRRJ ARO094 (b) collections of S.
597 terebinthifolius, and the UFRRJ ECB021/022 (c), UFRRJ ECB016/029/037 (d), UFRRJ
598 ECB028 (e), and UFRRJ ECB003/008 (f) L. alba collections.

f
r oo
-p
re
lP
na
ur
Jo

24
599 Table 1. Genotype codes, source material for essential oil production, their respective chemotypes, assay concentrations, and observed variables
600 in the assays.
Concentration b Mortality, Oviposition, Hatching, Fertility,
Genotype Chemotype (CT) (mg/mL of air) Emergence, Emergence Rate and Weight Loss
UFRRJ ARO050 sabinene X -
UFRRJ ARO079 α-phellandrene / α-pinene X -

f
oo
0.10; 0.25; 0.50; 0.75
UFRRJ ARO011 α-pinene X -
and 1.0

r
β-pinene / α-pinene

-p
UFRRJ ARO025 X -
δ-carene / α-pinene

re
UFRRJ ARO032 X X

lP
UFRRJ ARO078 α-phellandrene / sabinene X -
0.10; 0.50 and 1.0
UFRRJ ARO022 myrcene X -

na
0.10; 0.25; 0.50 and X X

ur
UFRRJ ARO094 limonene
0.75
Jo
UFRRJ ECB021/ 022 a citral / limonene X X
UFRRJ ECB037/ 029/ 016 a citral 0.10; 0.25; 0.35; 0.50 X X
UFRRJ ECB003/ 008 a carvone/ limonene and 0.75 X X
URRJ ECB028 a linalool X X

601 a – The essential oils from these codes were mixed in equal mass proportion for fumigation assays due to their similar composition (Figure S1).
602 ARO – Code for the S. terebinthifolius genotypes and ECB – Code for L. Alba genotypes. b – Each of the tested essential oils was accompanied
603 by a control treatment, which consisted of the absence of essential oil on the filter paper.

25
604 Table 2. Chemical composition of essential oils from S. terebinthifolius fruits.
UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ
Compound RIC a
ARO011 ARO025 ARO022 ARO079 ARO032 ARO050 ARO094 ARO078
myrcene 996 0.9 0.4 66.2 1.6 1.6 2.2 1.0 2.1
α-pinene 942 48.5 28.8 0.9 19.4 24.5 7.4 4.6 11.3
limonene 1031 0.0 0.0 0.0 0.0 0.0 0.0 62.7 0.0
β-pinene 984 10.6 47.5 0.5 0.5 0.5 0.6 0.1 0.7
sabinene 979 1.9 0.0 0.1 0.3 0.4 37.2 0.5 21.1
δ-3-carene 1016 0.1 0.0 0.0 0.1 34.8 0.0 0.0 0.0
α-phellandrene 1010 1.0 0.4 0.6 31.4 0.0 8.8 9.5 22.1
sylvestrene 1031 4.2 4.4 1.9 15.7 14.1 10.4 0.0 9.2
germacrene D 1492 6.9 5.2 17.6 5.6 7.1 2.8 13.9 5.1
p-cymene 1029 0.4 0.3 0.0 7.8 3.7 4.6 0.0 3.5

f
4-terpineol 1183 1.1 0.6 0.1 0.1 0.1 7.8 0.1 2.6

oo
β-caryophyllene 1429 5.0 2.0 6.2 4.4 3.2 2.4 0.5 2.4
elemol 1561 6.6 0.0 0.5 1.9 0.0 0.5 0.2 4.4

r
elemol acetate 1681 0.0 0.0 0.0 3.4 0.0 0.0 0.2 0.2
γ-terpinene 1063
caryophyllene oxide 1594
0.5
0.4
0.2
0.7
-p
0.0
0.6
0.0
0.8
0.0
0.7
3.4
1.1
0.0
0.4
1.3
0.7
re
γ-eudesmol 1635 4.0 0.3 0.3 0.1 0.2 0.2 0.6 2.2
α-zingiberene
lP

1498 0.3 0.8 0.0 0.4 0.9 0.4 0.1 1.0

α-terpinene 1022 0.3 0.1 0.0 0.1 0.0 1.9 0.0 0.8
TotAl 92.6 91.5 95.4 93.4 91.8 91.6 94.4 90.8
na

605 a – Retention index calculated (van Den Dool and Dec. Kratz, 1963).
ur

606
Jo

607

608

609

610

611

612

613

614

615

26
616 Table 3. Chemical composition of essential oils from L. alba leaves.
UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ
Compound RIC a
ECB021 ECB022 ECB037 ECB029 ECB016 ECB003 ECB008 ECB028
sabinene 972 0.5 0.4 0.3 0.2 0.2 1.7 1.6 0.9
myrcene 991 0.3 0.2 4.3 2.2 2.9 0.4 0.4 0.3
p-cymene 1023 2.4 2.0 0.6 0.3 0.4 0.0 0.0 0.0
limonene 1028 7.6 6.7 0.4 0.2 0.2 23.1 20.7 0.0
eucalyptol 1030 0.0 0.0 0.2 0.1 0.0 0.0 0.0 6.2
(E)-β-ocimene 1047 0.3 0.3 0.9 0.5 0.8 0.5 0.5 0.9
γ-terpinene 1057 2.7 2.4 0.2 0.0 0.1 0.0 0.2 0.0
cis-sabinene hydrate 1066 0.0 0.2 0.0 0.2 0.0 0.8 0.7 0.0
linalool 1101 1.3 0.9 1.4 1.1 1.2 1.8 1.7 63.1
pinocarvone 1161 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2

f
myrtenal 1195 0.0 0.0 0.5 0.7 0.6 0.0 0.0 0.0

oo
cis-p-1(7),8-dien-2-ol 1210 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.6
carvone 1243 0.0 0.0 0.0 0.0 0.0 59.2 56.6 0.0

r
neral 1243 29.6 29.6 29.8 32.1 30.4 0.0 0.0 1.0
geranial
geranyl acetate
1274
1385
42.4
0.1
43.4
0.1
-p
43.1
0.3
45.5
0.2
44.3
0.0
0.0
0.0
0.4
0.6
1.4
0.0
re
β-caryophyllene 1418 0.3 0.3 3.5 3.6 4.3 0.0 0.4 3.7
α-humulene
lP

1451 0.2 0.2 0.5 0.6 0.5 0.0 0.2 0.9

germacrene D 1480 2.9 3.0 1.5 0.2 1.2 4.1 4.7 4.5
elemol 1551 3.2 3.1 0.0 0.0 0.0 3.9 4.7 0.0
na

germacrene B 1555 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8
caryophyllene oxide 1582 0.0 0.0 4.7 7.1 6.0 0.0 0.0 0.0
ur

Total 93.8 92.9 92.1 94.7 93.0 95.5 93.3 89.5

617 a – Retention index calculated (van Den Dool and Dec. Kratz, 1963).
Jo

618

27
Table 4. Mortality (%) of C. maculatus under the influence of volatiles from essential oils of S. terebinthifolius and L. alba collections.

UFRRJ UFRRJ UFRRJ

Conc. UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ
ECB021/ ECB037/ ECB003/
(mg/mL) a ARO011 ARO050 ARO079 ARO025 ARO032 ARO094 ARO078 ARO022d ECB028
022 029/016 008
0.00 0.00b 0.00c 0.00b 2.00c 0.00b 0.00b 0.00b 0.00 4.00a 0.00a 0.00c 0.00c
0.10 0.00b 0.00c 0.00b 5.00c 0.00b 0.00b 3.33b 0.00 5.00a 0.00a 1.67c 41.67b
0.25 0.00b 21.67b 11.67b 40.00b 10.00b 0.00b - - 0.00a 1.67a 38.33b 100.0a

f
oo
0.35 - - - - - - - - 1.67a 0.00a 45.00ab 100.0a
0.50 98.33a 93.33a 90.00a 88.33a 91.67a 26.67b 83.33a 3.33 0.00a 0.00a 65.00ab 100.0a

r
0.75 100.0a 100.0a 96.67a 100.0a 95.00a 65.00a - - 0.00a 0.00a 73.33a 100.0a

-p
1.00 100.0a 100.0a 100.0a 98.33a 100.0a - 100.0a 81.67 - - - -

re
SS b 86471 75858 73938 60015 72155 14867 49467 143.8 13.89 28722 55347
-
(residual) (83.33) (1818) (4238) (6242) (1833) (74433) (1867) (553.3) (83.33) (9600) (5283)

lP
MS c 17294 15172 14788 12003 14431 3717 16489 28.76 2.778 5744 11069
-
(residual) (2.874) (60.56) (145.4) (221.3) (65.48) (323.2) (93.33) (19.08) (2.778) (320) (176.1)

na
F 6018 250.5 101.7 54.24 220.4 11.5 176.70 1.507 1.0 17.95 62.85
-
(DFn, DFd) (5, 29) (5, 30) (5, 29) (5, 28) (5, 28) (4, 23) (3, 20) (5, 29) (5, 30) (5, 30) (5, 30)

ur
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 - 0.2182 0.4346 <0.0001 <0.0001
a – Essential oils concentration (mg/mL of air). b – Sum of squares. c – Mean squares. d – The analysis of variance was not performed because
Jo
the data did not meet the important statistical assumptions necessary for analysis.

28
1 Table 5. Median lethal concentration (LC50) of the population of C. maculatus under
2 the influence of volatiles from essential oils of S. terebinthifolius and L. alba
3 collections.

LC50
Genotype Equation R2 b DF c SS d
(mg/mL of air)
UFRRJ ARO011 CT α-pinene Sigmoid 0.4423 0.999 31 83.33

UFRRJ ARO050 CT sabinene Sigmoid 0.3148 0.9766 32 1818

UFRRJ ARO079 CT α- phellandrene /α-pinene Sigmoid 0.3454 0.9458 31 4238

UFRRJ ARO025 CT β-pinene/α-pinene Sigmoid 0.2870 0.9057 30 6242

UFRRJ ARO032 CT δ-carene/α-pinene Sigmoid 0.3402 0.9744 30 1893

f
oo
UFRRJ ARO094 CT limonene Sigmoid 0.5235 0.6667 24 7433

UFRRJ ARO078 CT α- phellandrene /sabinene Sigmoid 0.3091 0.9636 20 1867

r
UFRRJ ARO022 CT myrcene a - - - - -

UFRRJ ECB003/008 CT carvone/limonene

-p Sigmoid 0.2800 0.7402 32 9954
re
UFRRJ ECB028 CT linalool Sigmoid 0.1017 0.9129 32 5283
lP

4 a – It was not calculated the LC50 in the treatment with the essential oil of the genotype UFRRJ ARO022
5 (myrcene CT), because the data did not meet the important statistical assumptions necessary for analysis.
6 b – coefficient of determination. c – Degrees of freedom. d – Sum of squares.
na
ur
Jo

29
7 Table 6. Egg laying (%) of C. maculatus under the influence of volatiles from essential oils of S. terebinthifolius and L. alba collections.

UFRRJ UFRRJ
Conc. UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ
ECB037/ ECB003
(mg/mL) a ARO011 ARO050 ARO079 ARO025 ARO032 ARO094 ARO078 ARO022 ECB021/022 ECB028
029/016 /008
0.00 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0ab 100.0a 100.0a 100.0a
0.10 108.8a 82.38a 80.48b 80.97a 112.50a 83.20ab 90.94a 83.61a 122.4a 95.92a 71.82b 29.96b
0.25 68.31b 31.84b 62.25c 51.25b 76.54ab 74.31b - - 75.18bc 56.38bc 43.14c 33.12b

f
oo
0.35 - - - - - - - - 78.92bc 68.37b 37.16c 32.49b
0.50 46.29bc 26.05b 25.52d 24.85bc 48.69bc 22.33c 21.25b 41.62b 68.23bc 57.40bc 23.69c 33.12b

r
0.75 46.23bc 18.39b 21.37d 19.51c 39.03c 27.27c - - 52.28c 32.40c 30.42c 38.61b

-p
1.00 38.51c 13.79b 21.96d 36.73bc 26.05c - 17.82b 22.90b - - - -

re
SS b 27029 39810 33733 28669 33496 26439 34879 22645 18169 19903 25542 22377
(residual) (4709) (5213) (1620) (5955) (8246) (4660) (3212) (4617) (12556) (7012) (4188) (2063)

lP
MS c 5406 7962 6747 5734 6699 6610 11626 7548 3634 3981 5108 4475
(residual) (162.4) (173.8) (55.87) (212.7) (294.5) (202.6) (160.6) (243) (433) (233.7) (139.6) (68.78)

na
F 33.29 45.82 120.8 26.96 22.75 32.62 72.39 31.07 8.39 17.03 36.59 65.07
(DFn, DFd) (5, 29) (5, 30) (5, 29) (5, 28) (5, 28) (4, 23) (3, 20) (3, 19) (5, 29) (5, 30) (5, 30) (5, 30)

ur
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
8 a – Essential oil concentration (mg/mL of air). b – Sum of squares. c – Mean squares.
Jo
9

30
10 Table 7. New adult emergence (%) of C. maculatus under the influence of volatiles from essential oils of S. terebinthifolius and L. alba
11 collections.

UFRRJ UFRRJ
Conc. UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ
ECB037/ ECB003
(mg/mL) a ARO011 ARO050 ARO079 ARO025 ARO032 ARO094 ARO078 ARO022 ECB021/022 ECB028
029/016 /008
0.00 100.0ab 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a
0.10 114.0a 91.40a 93.50a 77.40a 105.90a 87.4ab 100.8a 91.10a 69.02b 45.39b 0.00b 0.00b

f
oo
0.25 80.51b 35.08b 62.25b 35.89b 61.14b 70.41b - - 4.37c 3.33c 0.00b 0.00b
0.35 - - - - - - - - 4.73c 1.97c 0.00b 0.00b

r
-p
0.50 44.14c 30.83b 33.39c 19.85b 44.15bc 29.86c 36.08b 41.84b 2.79c 0.00c 0.00b 0.00b
0.75 52.30bc 18.28b 25.79cd 12.62b 40.55bc 30.21c - - 1.70c 0.61c 0.00b 0.00b

re
1.00 38.01c 20.79b 18.02d 27.45b 25.45c - 14.71b 27.73b - - - -

lP
SS b 29567 39967 35992 33750 30117 22760 35122 22285 50573 49564 50002 50000
(residual) (8522) (21733) (1260) (5289) (5069) (3093) (5757) (4785) (2496) (1351) (1485) (517.9)

na
MS c 5913 7993 7198 6750 6023 5690 11707 7428 10115 9913 10000 10000
(residual) (293.9) (724.4) (43.46) (188.9) (181) (134.5) (287.9) (251.9) (86.08) (45.04) (202) (17.26)

ur
F 20.12 11.03 165.6 35.73 33.27 42.32 40.67 29.49 117.5 220.1 202 579.
(DFn, DFd) (5. 29) (5. 30) (5. 29) (5. 28) (5. 28) (4. 23) (3.20) (3. 19) (5. 29) (5.30) (5. 30) (5. 30)
Jo
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
12 a – Essential oil concentration (mg/mL of air). b – Sum of squares. c – Mean squares.

31
13 Table 8. Fertility rate (%) of C. maculatus under the influence of volatiles from essential oils of S. terebinthifolius and L. alba collections.

Conc. (mg/mL) a UFRRJ ARO032 UFRRJ ARO094 UFRRJ ECB021/022 UFRRJ ECB037/029/016 UFRRJ ECB003/008 UFRRJ ECB028
0.00 99.48a 88.66a 87.54a 86.25a 91.90a 91.48a
0.10 98.47a 92.74a 51.83b 48.50b 1.07b 0.00b
0.25 99.22a 91.01a 6.47c 3.89c 3.02b 0.00b
0.35 - - 7.46c 5.42c 1.85b 0.00b

f
0.50 92.21ab 76.38b 2.79c 0.00c 0.00b 0.00b

oo
0.75 91.92ab 91.46a 5.77c 2.19c 0.00b 0.00b

r
1.00 90.14b - - - - -

-p
b
SS (residual) 499.1 (538.7) 1049 (1081) 32954 (2096) 37655 (1000) 41178 (367) 41841 (55.93)

re
c
MS (residual) 99.83 (19.24) 262.2 (46.99) 6591 (72.27) 7531 (33.33) 8236 (12.23) 8368 (1.864)

lP
F (DFn, DFd) 5.19 (5, 28) 5.58 (4, 23) 91.2 (5, 29) 225.9 (5, 30) 673.2 (5, 30) 4489 (5, 30)
P 0.0017 0.0027 <0.0001 <0.0001 <0.0001 <0.0001

na
14 a – Essential oils concentration (mg/mL of air). b – Sum of squares. c – Mean squares.

ur
Jo

32
15 Table 9. Larval hatching (%) of C. maculatus under the influence of volatiles from essential oils of S. terebinthifolius and L. alba collections.

Conc. (mg/mL) a UFRRJ ARO032 UFRRJ ARO094 UFRRJ ECB021/022 UFRRJ ECB037/029/016 UFRRJ ECB003/008 UFRRJ ECB028
0.00 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a
0.10 98.98a 104.6a 59.21b 56.23b 1.17b 0.00b
0.25 99.74a 102.7a 7.39c 4.51c 3.28b 0.00b
0.35 - - 8.53c 6.28c 2.02b 0.00b

f
0.50 92.68ab 86.14b 3.19c 0.00c 0.00b 0.00b

oo
0.75 92.40ab 103.2a 6.60c 2.54c 0.00b 0.00b

r
1.00 90.60b - - - - -

-p
b
SS (residual) 504.3 (544.3) 1335 (1375) 43005 (2735) 50617 (1345) 48761 (434.6) 50000 (66.86)

re
c
MS (residual) 100.9 (19.44) 333.8 (59.78) 8601 (94.31) 10123 (44.83) 9752 (14.49) 10000 (2.229)

lP
F (DFn, DFd) 5.187 (5, 28) 5.583 (4, 23) 91.20 (5, 29) 225.8 (5, 30) 673.2 (5, 30) 4487 (5, 30)
P 0.0017 0.0027 <0.0001 <0.0001 <0.0001 <0.0001

na
16 a – Essential oils concentration (mg/mL of air). b – Sum of squares. c – Mean squares.

ur
Jo

33
17 Table 10. New adults emergency rate (%) of C. maculatus under the influence of volatiles from essential oils of S. terebinthifolius and L. alba
18 collections.

UFRRJ UFRRJ
Conc. UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ UFRRJ
ECB037/ ECB003
(mg/mL) a ARO011 ARO050 ARO079 ARO025 ARO032 ARO094 ARO078 ARO022 ECB021/022 ECB028
029/016 /008
0.00 58.13b 52.98ab 63.41a 84.23a 88.48a 72.81b 54.47b 67.78a 69.82a 84.76a 86.43a 68.75a
0.10 48.50b 37.61b 65.60a 81.38a 85.85a 77.40ab 58.56ab 74.11a 39.94b 40.50b 0.00b 0.00b

f
oo
0.25 81.98a 76.65a 81.70a 60.88ab 78.05a 70.46b - - .4.16c 4.40c 0.00b 0.00b
0.35 - - - - - - - - 4.28c 2.29c 0.00b 0.00b

r
-p
0.50 45.92b 42.31ab 74.64a 62.99ab 80.53a 103.60a 99.28a 76.37a 2.51c 0.00c 0.00b 0.00b
0.75 51.98b 42.16ab 82.98a 52.09b 89.80a 82.30ab - - 3.16c 1.62c 0.00b 0.00b

re
1.00 46.96b 58.78ab 52.81a 63.45ab 98.33a - 51.00b 85.26a - - - -

lP
SS b 4944 6320 4334 5894 1429 4220 8894 938.9 21302 35269 37346 23634
(residual) (5189) (15029) (14869) (7429) (14387) (5895) (14449) (5270) (732.0) (1032) (435.0) (152.4)

na
MS c 988.9 1264 866.7 1179 285.8 1055 2965 313.0 4260 7054 7469 4727
(residual) (148.3) (417.2) (424.8) (218.5) (513.8) (256.3) (760.5) (277.4) (25.24) (34.39) (14.50) (5.079)

ur
F 6.669 3.030 2.040 5.394 0.5563 4.116 3.898 1.128 168.8 205.1 515.1 930.7
(DFn, DFd) (5, 35) (5, 36) (5, 35) (5, 34) (5, 28) (4, 23) (3, 20) (3, 19) (5, 29) (5, 30) (5, 30) (5, 30)
Jo
P 0.0002 0.022 0.0969 0.0009 0.7323 0.0117 0.0251 0.3626 <0.0001 <0.0001 <0.0001 <0.0001
19 a – Essential oils concentration (mg/mL of air). b – Sum of squares. c – Mean squares.

34
20 Table 11. Weight loss (%) of cowpea seeds due to the infestation of C. maculatus and as a function of the protection provided by the volatiles of
21 essential oils from S. terebinthifolius and L. alba collections.

Conc. (mg/mL) a UFRRJ ARO032 UFRRJ ARO094 UFRRJ ECB021/022 UFRRJ ECB037/029/016 UFRRJ ECB003/008 UFRRJ ECB028
0.00 37.43a 36.90a 38.52a 29.94a 32.27a 35.17a
0.10 39.38a 32.89ab 28.97b 15.27b 0.46b 1.77b
0.25 24.52b 26.10b 3.68c 2.31c 0.01b 1.56b

f
oo
0.35 - - 3.49c 2.32c 0.89b 1.22b
0.50 19.48bc 13.15c 1.88c 1.20c 0.85b 0.84b

r
-p
0.75 17.24bc 12.90c 1.32c 1.31c 0.84b 0.91b
1.00 12.62c - - - - -

re
b
SS (residual) 3325 (698.5) 2516 (374) 7391 (312.9) 4118 (143.6) 5017 (77.93) 5751 (23.45)

lP
c
MS (residual) 665 (24.95) 629.1 (17) 1478 (10.79) 823.6 (4.878) 1003 (2.598) 1150 (0.7818)
F (DFn, DFd) 26.66 (5, 28) 37.01 (4, 23) 137 (5, 29) 168.8 (5, 30) 386.3 (5, 30) 1471 (5, 30)

na
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
22 a – Essential oils concentration (mg/mL of air). b – Sum of squares. c – Mean squares.
ur
Jo

35
Jo
ur
na
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1 HIGHLIGHTS

2 Various chemotypes of L. alba and S. terebinthifolius essential oils were evaluated.

3 Weevils’ reproductive cycle and seed weight loss were assessed.

4 Essential oil from UFRRJ ECB028 genotype showed optimal results.

5 It completely halted the insects’ life cycle and achieved 100% seed mass loss prevention.

6 The UFRRJ ECB028 genotype is indicated for developmental of seed protection technology.

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

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