Document Type : Original Article

Author

Department of Insect Population Toxicology, Central Agricultural Pesticides Laboratory, Agriculture Research Center, Dokki, Giza, Egypt

10.22034/jpbb.2022.363668.1030

Abstract

Introduction: Fecundity (egg laying) and fertility (egg hatching) are important components of insect reproduction. Gossypol has been reported to be an active anti-reproductive inhibitor by reducing or completely impairing fecundity and fertility in various animal species.
Methods: Tested sublethal dose (LC25) of gossypol effects on the fourth instar larvae reproduction parameters and detoxifying enzyme activities of the cotton leaf worm, Spodoptera littoralis (Lepidoptera: Noctuidae) compared with control was evaluated.
Results: Gossypol showed toxicity against the second and the fourth instars larvae of S. littoralis after 72 hours of treatment; the LC50 values were 162.56 and 296.30 mg (ai)/Kg of artificial diet, respectively. Gossypol significantly suppressed oviposition and adult emergence, as well as prolonged the pre-oviposition period. According to the results, the highest fecundity inhibition more than 43% was observed in mating of treated females (TF) with treated males (TM) in addition to the highest fertility inhibition of 33%. Gossypol significantly reduced the activities of the detoxification enzymes including glutathione S-transferase (GST), lactic dehydrogenase (LDH), and protein content.
Conclusion: Overall, the results showed that the exposure of larvae by the sublethal dose (LC25) of gossypol negatively affected the egg laying and egg hatching as well as reduced the activities of the detoxification enzymes in the cotton leaf worm. These findings may pioneer new approaches to control this pest.

Graphical Abstract

Suppressive Effects of Gossypol on the Spodoptera littoralis (Lepidoptera: Noctuidae) Reproduction and Enzymatic Properties

Highlights

‎► Gossypol at sublethal dose (LC25) showed toxicity to Spodoptera littoralis. ► ‎Gossypol cause adverse effects reproduction in S. littoralis, altering essential ‎parameters for its survival. ► Gossypol induced higher activities of detoxifying ‎enzymes.  ‎

Keywords

Main Subjects

  1. Introduction

The most hazardous larval stage with an insatiable and polyphagous appetite of the cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae). It has the power to completely seriously harm more than 100 different varieties of commercially important crops. The physiological stress brought on by severe infestations may also make the host plants more vulnerable to other diseases and pests [1, 2]. Due to the high resistance of S. littoralis to commonly used synthetic chemical insecticides, it has caused severe damage to crops in Egypt and other countries [3], and thus has been classified as a “super pest” [1, 2]. Chemical insecticides have been used extensively and excessively to protect crops for decades, which has put the environment and human health at danger as well as raised insect resistance and residue problems [4]. The development of Integrated Pest Management (IPM) programs, such as the use of plant allelochemicals, has become increasingly significant as an alternative to the discovery of novel, generally safe, and natural products effective against pest species [5].

Plant allelochemicals, a class of the secondary metabolites made by plants, have been suggested as a means of controlling insects since they play a key role in plant-insect interactions and insect defense such as gossypol [6, 7].

Gossypol is a polyphenol isolated from the seed, stem, and roots of the cotton plant (Gossypium sp.), which involve in the defense system against pathogens and insect herbivores [8]. The pure substance, in the form of a crystalline, yellowish powder that is toxic to various organisms and also has a negative effect on insect development [9, 10, 11, 12]. Studies conducted in China, Africa, and Brazil have shown that gossypol provokes infertility in various animal species [13], including man [14]. Targeting the reproduction ability of insect pests is crucial in their control insect. Despite different evaluations of gossypol, as far as we know there is no/less information on its use as an inhibitor of fecundity and fertility in insects via larvae-exposure. Thus, the present study was performed to evaluate gossypol at sublethal dose (LC25) for its inhibitory effects on the reproductive characteristics and the activity of detoxifying enzymes in S. littoralis.

  1. Materials and Methods

2.1. Insects

The Spodoptera littoralis (Boisduval) strain used in this study obtained from an original colony that was kept in the Insect Population Toxicology Department of the Central Agricultural Pesticides Laboratory at the Agriculture Research Center in Giza, Egypt. This colony was raised under controlled conditions of 25±1 °C, 65±5% RH, with a photoperiod of 16:8h (L: D) without being exposed to any insecticides. The early 2nd and 4th instars larvae were transferred into Petri dishes and given an artificial rearing diet for used in the experiments.

2.2. Feeding assays

Gossypol (purchased from Sigma-Aldrich, St. Louis, USA) was incorporated into the diet at six concentrations, which were 25, 50, 100, 200, 300, and 400 mg (ai)/Kg diet. Control diets (without gossypol) were also included. Thirty newly molted active (<24 h old) 2nd and 4th-instars larvae were selected and starved for 4 h to induce a higher feeding rate before releasing them into the artificial diet containing gossypol. The larvae were given an artificial meal, and after 72 h their death was observed, it is considered as fatal if it exhibits severe shrinkage compared to the control larvae or if it is unable to move properly when moved with a soft brush. Three replicates of each treatment were used in the tests, which were carried out under the identical conditions as previously mentioned. Using Finney (1971) [15] approach to probability analysis, the values of LC25 and LC50 were determined.

2.3. Sublethal effects on fecundity and fertility

Newly molted active (<24 hours old) fourth-instar larvae were fed for 24 h on an artificial diet containing 72.90 mg (ai)/Kg diet of gossypol used as a sub-lethal concentration based on the results of the preliminary bioassays in this study. Females and males’ survivors were used to evaluate the sub-lethal effect of gossypol on the various reproductive aspects. Three mating combinations were conducted: 

Treated females mated with treated males (TF × TM)

Untreated females mated with treated males (UF × TM)

Treated females mated with untreated males (TF × UM)

Untreated females mated with untreated males (control) (UF × UM)

Five replicates were conducted for each combination. Checked a rearing cage daily until oviposition were initially observed then collected daily on filter paper, which served as an oviposition substrate and was replaced with new filter paper daily. Eggs were counted daily with binocular until female death; female was not replaced if they died before the male. Fecundity (total number of eggs/female) and fertility (percentage egg hatchability), pre-oviposition and oviposition periods were determined. During the experiment, every day replaced artificial diet and the 10% sucrose solution to kept fresh and ensure the normal growth of larvae and adults.

2.4. Biochemical studies

2.4.1. Enzyme source preparation

To prepare the crude homogenates that serve as enzyme sources, S. littoralis larvae in their fourth instar fed on an artificial diet treated with gossypol at a sub-lethal concentration of 72.90 mg (ai)/Kg diet were employed, with untreated larvae serving as a control. Whole larva except the hind mid gut were dissected, and then homogenized in 5 volumes (w/v) of saline solution (0.9%). Homogenization was performed in sterilized Eppendorf tube (1.5 mL) using a manual homogenizer at approximately 4 °C. Homogenate were centrifuged 5000 rpm at 4 °C for 30 min to separate the supernatant. The supernatant sample from each individual was collected to use for the enzyme assay of glutathione S-transferase (GST), lactic dehydrogenase (LDH), and determination of protein content.

2.4.2. Determination of enzyme activity

Activity of two enzymes different groups GST and LDH were analyzed with colorimetric methods using specific substrates for each enzyme. The assay was performed in triplicate. Using 4-chloro-1,3-dinitrobenzene (CDNB) as substrate was used to measure the estimated GST -EC 2.5.1.18 activity [16]. Estimation of LDH -EC 1.1.1.27 activity was measured using bioMèrieux kits, France, as recommended by the German Society of Clinical Chemistry (1972) [17]. The control group was not treated with the enzyme extract. Protein content was measured by the method of Bradford (1976) [18].

2.5. Statistical analysis

Statistics were used for data analysis as mean ± SE. SAS software was used to run a one-way analysis of variance (ANOVA) to see if there were statistically significant differences between the individual means. The Tukey's test was used to examine mean values at the 0.05 level or lower of probability. 

  1. Results

3.1. Toxicity of gossypol

Toxicity of gossypol against the second and the fourth-instars larvae of S. littoralis was determined via the artificial diet incorporation method. Log-probit regression analysis of concentration-mortality data showed that, after 72 h of treatment, for the artificial diet, the LC50 and LC25 values for 2nd instar larvae were 162.56 and 52.77 mg (ai)/Kg diet, respectively and for the 4th instar larvae were 296.30 and 72.90 mg (ai)/Kg diet, respectively (Table 1). Bioassays to examine sublethal dose (LC25) effect of gossypol exposure was conducted at a LC25 value of 72.90mg (ai)/Kg diet for the 4th instar larvae.

 

 

Table 1. Toxicity of gossypol to 2nd- and 4th-instars larvae of Spodoptera littoralis

Larval stage

Slope (±SE)

LC50 (95% CL) a

mg (ai)/Kg diet

LC25 (95% CL) a

mg (ai)/Kg diet

χ2 (df = 4) b

2nd instar larvae

1.37 (0.004)

162.56 (227.61-116.23)

52.77 (95.13-29.27)

2.56

4th instar larvae

1.10 (0.003)

296.30 (438.23-200.34)

72.90 (138.09-38.48)

5.46

a95% confidence limits, b Chi-square value (χ2), and degrees of freedom (df) were calculated by probit analysis with SPSS.

 

3.2. Sublethal effects on adult emergence and longevity

Emerging adults of the control and the gossypol treatment showed different numbers (F= 257.78; p= 0.05) as greater numbers of adults were produced in the control (Table 2). In the same experiment, longevity adults of the control and the gossypol treatment showed different numbers for both sexes, however, the greater numbers of females were produced (Table 2). There were significant differences between the males and females (F= 8.670–0.859; p< 0.01), males were more susceptible than females.

Table 2. Adult emergence and longevity of Spodoptera littoralis after the exposure of the fourth-instar larvae to sublethal dose (LC25) of gossypol

Treatment

Adult emergence (%)

Adult longevity (day)

Gossypol

73.43 ± 4.57b

15.07 ± 3.77ab

10.17 ± 1.08b

Control

97.00 ± 1.89a

      16.43 ± 2.74a

15.00 ± 7.00a

Means in the same column with the same letter are not significantly different from one another. Data are five replicates of mean ±SE.

 

3.3. Sublethal effects on pre-oviposition and oviposition period 

Adult female of S. littoralis when larva fed on gossypol had a shorter oviposition period 4.71 days than adult female in control 13.67 days. The pre-oviposition period ranged from 3.54 days in gossypol treatment to 2.41 days in control (Table 3). Compared with the control, there was almost complete suppression of oviposition in the gossypol-treated as well as a prolonged pre-oviposition period (F= 792.88–1.987; p< 0.01).

Table 3. Pre-oviposition and oviposition period in the female of Spodoptera littoralis after the exposure of the fourth-instar larvae to sublethal dose (LC25) of gossypol

Treatment

Pre-oviposition period (day)

Oviposition period (day)

Gossypol

3.54 ± 1.70b

4.71 ± 0.157b

Control

2.41 ± 0.227a

13.67 ± 0.146a

Means in the same column with the same letter are not significantly different from one another. Data are five replicates of mean ±SE.

 

3.4. Sublethal effects on fecundity (egg-laying) and fertility (egg-hatching)

Gossypol caused a significant decrease in the number of eggs laid by female S. littoralis as well as the hatching rate in different types of mating combinations over the control (Table 4). The highest decrease was observed in mating of females (TF) and males (TM) (F= 5630.7–420.2; P< 0.050.000) followed by mating of females (UF) and males (TM) (F= 3121.2–161.2; P > 0.05–0.305), while the lowest decrease was observed in mating of females (TF) and males (UM) (F= 618.9–99.34; P > 0.05–0.305). The mating of both exposed females TF and TM showed the highest egg laying inhibition of 43.08%, and the hatching rate was significantly reduced by 66.01% compared with 98.43% in the control, followed by mating of UF and TM was numbers of eggs laid (32.08%) and hatching rate (77.00%). Less reduction was observed in the numbers of eggs laid (11.95%) and hatching rate (88.55%) in type of mating combination of TF and UM.

 

Table 4. Fecundity (egg-laying) and fertility (egg-hatchability) inhibition rates in the different mating combinations of Spodoptera littoralis after the exposure of the fourth-instar larvae to sublethal dose (LC25) of gossypol

Mating combination

Fecundity inhibition

Fertility inhibition

Untreated females (UF) × Untreated males (UM)

318 ± 3.00a

     98.43 ± 4.55a

    Treated females (TF) × Treated males (TM)

181 ± 7.00d

66.01 ± 2.954d

     Untreated females (UF) × Treated males (TM)

216 ± 7.00c

      77.00 ± 4.00c

     Treated females (TF) × Untreated males (UM)

280 ± 4.00b

85.55 ± 0.461b

Means in the same column with the same letter are not significantly different from one another (P = 0.05). Data are five replicates of mean ±SE.

 

3.5. Enzyme activity

Measurement of glutathione S-transferase (GST), and lactic dehydrogenase (LDH) activities revealed significant differences between treated and untreated larvae of S. littoralis (Table 5). The GST activity was 44.83 nmol/min/mg protein in untreated larvae which is approximately 2-fold its value in treated larvae with sublethal dose (LC25) of gossypol. Furthermore, the obtained results showed a significant decrease LDH leakage in treated larvae by 57.48% of control value. Moreover, the protein content of the gossypol-treated larvae was significantly lower than that of untreated larvae (Table 5).

 

Table 5. In vivo effect of gossypol at LC25 on the activity of glutathione S-transferase, lactic dehydrogenase and protein content of Spodoptera littoralis larvae

Treatment

a GST

LDH

Protein content

Activity

% of Control

Activity

% of Control

Activity

% of Control

Gossypol

24.37 ± 0.670b

-45.64

181.67 ± 4.83b

-42.52

13.40 ± 0.383b

-12.02

Control

44.83 ± 1.657a

 

316.06 ± 3.63a

 

15.40 ± 2.493a

 

a Glutathione S-transferase activity is expressed as nmole CDNB conjugated formed/min.mg protein. b Lactic dehydrogenase activity is expressed as U/L supernatant. c Protein content is expressed as mg/g tissue. Means in the same column with the same letter are not significantly different from one another.

 

  1. Discussion

In this study, results confirmed that gossypol exhibits active toxicity against the second and fourth instar larvae of Spodoptera littoralis. Previous studies have shown that gossypol has significant toxicity against larvae of Heliothes versine (Fabricius) [9], Helicoverpa armigera (Hübner) [19], Spodoptera littoralis (Boisduval) [11], and adults of Aphis gossypii (Glover) [6].

The results from the present study focused on the inhibitory effect of gossypol on fecundity and fertility of S. littoralis. Gossypol at sublethal dose (LC25) considerably influenced the reproduction parameters of this pest. The fecundity and fertility of S. littoralis exposed to the gossypol decreased in the different types of crosses such as TF × TM, TF × UM and UF × TM over the control (UM × UF). But in all of the test, the mating cross TF × TM is more affected than other types such as TF × UM and UF × TM. These findings are supported by the findings of Ismail (2020a) [11] who reported that feeding S. littoralis diets containing gossypol dramatically affected the species' longevity, fertility, and fecundity over the course of two subsequent generations. Guo et al. (2013); Du et al. (2004) [10, 20] who indicated that the gossypol dramatically sped up development time and reduced the reproduction of Aphis gossypii (Homoptera: Aphididae) [20], and Bemisia tabaci (Hemiptera: Aleyrodidae) [10]. Ismail (2020b) [12] found that the Thrips tabaci (Thysanoptera: Thripidae) and Pectinophora gossypiella (Lepidoptera: Noctuidae) populations were significantly and negatively affected when cotton thrips and pink bollworm fed on cotton plants contained high levels gossypol. 

The target pest may become more vulnerable to the impacts of pesticides as a result of the modifications in detoxification enzyme activity described above [21, 22]. These findings showed the treatment larvae of S. littoralis with sublethal dose (LC25) of gossypol resulted in a significant reduction of the GST activity. These outcomes are consistent with those of Lee et al. (1982) [23] who demonstrated a comparable suppression of the GST activity following gossypol administration. These results are in line with the findings of Lee et al.  (1982) [23] who have shown a similar inhibition of GST activity after treatment with gossypol. The changes in activity of lactic dehydrogenase (LDH) is indicative of cellular and bio-membrane change, these implies that the compound induced oxidative stress. Oxidative damage or stress usually refers to the impairment of the function of cellular components, reactive oxygen species (ROS) can worsen the harm already done to cells and biomembranes (such as enzymes, nucleic acids, membranes, and proteins). ROS include superoxide radicals (O-2), hydroxyl free radicals (OH), and hydrogen peroxide (H2O2) [24]. In this study, the LDH activity decrease when treated with sub-lethal concentration of gossypol. The decrease in the LDH activity of can result in a reduced elimination of ROS which in turn can denature different biomolecules of the insect body. The denaturation of biomolecules can stop all the cellular processes, so leading to the death of the insect [25]. The reduction in the LDH activity of S. littoralis after treatment with gossypol is in line with the findings of Lee et al.  (1982) [23] who observed similar inhibition of the LDH activity and the degree of enzyme inactivation is both gossypol and enzyme-concentration-dependent.

Total protein level of larvae after treatment with sub-lethal concentration of gossypol was significantly decreased. This decline in protein content is probably due to gossypol with the hormones regulating protein synthesis. These results are supported by Ismail (2020c); Gall and Behmer (2014) [22, 26] reported that the decrease in protein level might be due to the inhibition of metabolizing enzymes or an imbalance between the rate of protein synthesis and the rate of biodegradation or may be due to the dissociation of protein into amino acids, dissociation of protein into amino acids, resulting in the reduction of energy storage of the insect. Likewise, Ismail (2020c) [22] reported that the decreased of protein induced by the chemical compounds in larvae of S. littoralis. This study found that even at low concentrations of gossypol, the reproduction performance of S. littoralis is impacted, as well as the pest's potential to impair many essential processes. Based on this data, the acquired results could be considered a promising approach for gossypol-based treatment of S. littoralis.

  1. Conclusion

According to the findings, gossypol can be used to protect plants from the significant polyphagous pest Spodoptera littoralis. The sensitivity of biochemical parameters in Gossypol-treated sets was high, resulting in the reduced fecundity and fertility in S. littoralis females. The results shed light on the potential role of gossypol in overcoming insecticide resistance, paving the way for gossypol to be used in integrated pest management strategies to maintain the sustainable pest control.

List of abbreviations

GST: glutathione S-transferase

LDH: lactic dehydrogenase

UF × UM: Untreated females mated with untreated males

UF × TM: Untreated females mated with treated males

TF × UM: Treated females mated with untreated males

TF × TM: Treated females mated with treated males

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

All data generated or analyzed during this study are included in this published article.

Competing interests 

The authors declare that they have no competing interests.

Funding  

No funds, grants, or other support was received.

Authors' contributions

S.M.I. subject selection, study design, carried out the experiments, paper writing, collecting, interpretation of the data, and performing statistical analysis. The author read and approved the final manuscript.

Acknowledgements 

Not applicable.

ORCID

Seham Mansour Ismail : https://www.orcid.org/0000-0002-4885-7383

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