Diet suitability through biological parameters in Ostrinia furnacalis (Lepidoptera: Crambidae) clades

Introduction

Maize (Zea mays L.) is one of the most important food crops in the world, providing at least 30% of the dietary calories to more than 4.5 billion people in 94 developing countries. In parts of Africa and Mesoamerica, maize alone contributes over 20% of dietary calories (Erenstein et al., 2022; Lu et al., 2024).

Numerous lepidopteran pests consume maize crops, leading to substantial shortfalls in production. Among them, Ostrinia furnacalis (Lepidoptera: Crambidae) is considered one of the most injurious insect pests of the maize crop (Abbas et al., 2024; Alam et al., 2023; Hiruy & Getu, 2018). It has a wide distribution from eastern Asia to the western Pacific islands and causes a yield loss of 6–9 million tons annually in China (Li et al., 2022). The adults are active from early May to late October each year (Han et al., 2020). The adults of O. furnacalis  are categorized into three clades (I, II and III), and confirmed by their unique cytochrome c oxidase subunit I (COI) gene sequences, morphological and biological features. The COI gene sequences play a role in the expression of specific morphological and biological features of O. furnacalis. The COI sequences for clade I span from MN720652 to MN720668, the COI sequences for clade II span from MN720669 to MN720686 and the COI sequences for clade III span from MN720687 to MN720703. As a result of differentiation in the COI sequences, the three clades show differences in wing postmedial line, male sacculus construction, lifespan, male dynamics and host preferences. These differences create substantial difficulties in pest forecasting and control (Han et al., 2020). So, a more efficient approach for maize growers is to manage this species at the clade level rather than addressing the entire species. The clades of O. furnacalis provide a remarkable model for studying their life history through the quantification of biological characters and fitness parameters under various diets, as a means for controlling this pest.

Mass rearing is a technique employed to produce large numbers of insects fed on natural or artificial diets (Guo et al., 2019) for multifaceted study purposes, including insect biology, insecticide testing and the production of biological control agents (Rahayu & Trisyono, 2018). Artificial diets are widely used to rear several insect and biological control agents efficiently, yielding insects with superior fitness over those reared on natural diets (Rahayu & Trisyono, 2018; Yang et al., 2024). Several studies have been carried out on the benefit of using artificial diets in lepidopterans such as O. furnacalis (Rahayu & Trisyono, 2018), Helicoverpa armigera (da Silva et al., 2021), Plodia interpunctella (Jung et al., 2021) and Spodoptera frugiperda (Abbas et al., 2023; Ramos et al., 2022). In contrast, using a natural diet in the laboratory seems to be more laborious because it requires daily changes of the diet and produces a limited number of insects (He et al., 2023; Zhao et al., 2023). Various infections and diseases arise in insects during laboratory rearing. More recently, different antibiotics have been added to artificial diets to treat diseases in insects, contributing to the success of large-scale culturing (Thakur et al., 2016; Zahra, 2021). The existing literature suggests that insects possess a limited amount of energy. When insects are affected by disease, they use a significant portion of their energy in fighting the disease. This, in turn, leaves the insects with less energy for growth and development (Gao et al., 2020). So, when an antibiotic is added to an artificial diet, it prevents the insects from becoming diseased and preserves their energy for growth (Elnahal et al., 2022; Van Boeckel et al., 2015). For example, silkworms (Bombyx mori) are larger, with heavier cocoons, when reared on a diet with antibiotic added (Savithri, 2007), rifampicin and aureomycin has been added to an artificial diet to treat aphids (Lin et al., 2015) and penicillin is used to treat disease in Spodoptera exigua (Thakur et al., 2016). The growth of gut bacteria in Plutella xylostella increased in insects reared on an artificial diet supplemented with rifampicin (Gao et al., 2021). Another antibiotic, chloramphenicol, has also been added to artificial diet for the optimal growth of insects (Li et al., 2020). However, there is limited knowledge on the growth of O. furnacalis clades regarding the effects of antibiotics.

Therefore, this article aimed to investigate whether these three clades of O. furnacalis respond similarly or differently in terms of survival across all life stages when reared on a natural diet or on various artificial diets. Our hypothesis focused on analyzing the impact of artificial diet with antibiotic added, artificial diet alone and natural diet on the three clades of O. furnacalis during mass rearing. To address this hypothesis, we conducted a comprehensive analysis of the insects by considering various biological characteristics, including developmental time, weight of larvae, pupae and adults, larval head capsule width, pupation percentage, adult emergence rate and sex ratio. Our research aims to contribute knowledge by providing a comprehensive evaluation of biological characteristics throughout all life stages under the influence of different diets in laboratory settings. This investigation lays the foundation for more refined rearing techniques.

Materials and methods

Results

Head capsule width relative to each larval instar stage and weight among the clades

The regression and correlation analysis revealed that the three clades were very similar in terms of head capsule size relative to instars and weight of O. furnacalis larvae, with correlation values close to 1 for these parameters. Thus, there was a solid relationship between larval head capsule width, instar and weight of O. furnacalis larvae. Interestingly, for the relationship between head capsule width and larval instar, the third and fourth instars of clade III had significantly wider head capsule widths for all diets (1,4 mm in logarithm, y = 0,1996x + 0,2,855, R² = 0,9,934). Whereas clades II and I had fewer wide head capsule widths (clade II, 1,3 mm in logarithm, y = 0,1984x + 0,2,886, R² = 0.9979; 1,2 mm in logarithm, y = 0,1956x + 0,2,887, R² = 0,9,974) with all diets (Fig. 1). In terms of the relationship between head capsule width and the weight of O. furnacalis larvae, clade III had wider head capsule widths correlated with heavier weights (y = 676608 × ⁶⁴³²⁶, R² = 0,9,963, P < 0.0001), compared with clade II (y = 65608 × ⁶²²³³, R² = 0,9,948, P < 0.0001) and clade I (y = 66890 × ⁶²²³³, R² = 0,9,936, P < 0.0001) (Figure 1 and 2).

Pupation and sex ratio of O. furnacalis (%)

At the clade level, clade III had a higher number of pupae proceed to the adult stage on natural, artificial and antibiotic diets (74.8%, 81.1% and 89.5%, respectively; df = 49, F = 48.451, P <˂ 0.005) than clade I (64.2%, 71.3% and 78.3%, respectively; df = 49, F = 3.21, P <˂ 0.3173) or clade II (72.5%, 79.7% and 81.7%, respectively; df = 49, F = 0.053, P <˂ 0.8132) (Fig. 3). Also, clade III had a higher sex ratio (df = 49, $$$ {x}_{\mathrm{tab}}^2 $$$=0.65,0.64,0.63, x² <˂ 0.0010) compared with clade I (df = 49, $$$ {x}_{\mathrm{tab}}^2 $$$=0.55,0.54.0.53, x² <˂ 0.0603) or clade II (df = 49, $$$ {x}_{\mathrm{tab}}^2 $$$=0.51,0.50,0.49, x² <˂ 0.0040) on natural, artificial and antibiotic diets. Clade III showed a higher pupation rate and sex ratio on the antibiotic diet (Table 4).

Table 4. Adult emergence and Sex ratio (%) in three Clades under Natural, Artificial and Antibiotic Diet

	Adult emergence (%)
	Natural Diet			Artificial Diet			Antibiotic Diet
	Clade 1	Clade 2	Clade 3	Clade 1	Clade 2	Clade 3	Clade 1	Clade 2	Clade 3
Larvae to Adult (number)	64.2 ± 0.11a	72.5 ± 0.11b	74.8 ± 0.08c	71.3 ± 0.11a	79.7 ± 0.11b	81.1 ± 0.11c	78.3 ± 0.11a	81.7 ± 0.11b	89.5 ± 0.05c
Sex Ratio (%)
Male	43.2 ± 0.20a	44.2 ± 0.14b	49.8 ± 0.14c	48.3 ± 0.14a	52.2 ± 0.17b	56.6 ± 0.14c	34.9 ± 0.08a	39.5 ± 0.05b	42.1 ± 0.05c
Female	41.7 ± 0.20a	43.7 ± 0.14b	44.1 ± 0.14c	42.4 ± 0.14a	42.7 ± 0.17b	43.3 ± 0.14c	59.0 ± 0.08a	58.5 ± 0.05b	57.9 ± 0.05c
Clades (Average)	149.1	160.4	168.7	162	174.6	181	172.2	179.7	189.5
Diet (Average)	159.4			172.5			180.46

• Values (Means ± SE, followed by same letter for each row were not significantly different.

Discussion

Biological performance studies on clades of O. furnacalis under different diets are crucial for a comprehensive understanding of their survival and dispersal. This knowledge is particularly relevant to agroecosystems, where understanding variations in the developmental periods and population dynamics of the clades of O. furnacalis is essential for effective management. Although many previous studies have evaluated the development of O. furnacalis on different host plants and artificial diets (Jie et al., 2022; Rahayu & Trisyono, 2018; Xin-Hua et al., 2023), there is limited information on the biological performance of the clades of O. furnacalis (Han et al., 2020).

However, the results reveal that a natural diet of fresh maize leaves significantly affects the biological parameters of developmental time and weight for each clade of O. furnacalis, leading to prolonged developmental times and lower weights of larvae. This suggests that these leaves are less suitable for the insects of each clade. These variations are associated with low food quality, the nutritional constitution of different plant tissues and the relationship between protein and carbohydrates that are unavailable to the insects (Bisht et al., 2018; Wang et al., 2019). For some lepidopteran species, a prolongation in developmental time indicates compensatory action, where larvae subjected to a poorer nutritional source tend to compensate for food stress by lengthening the immature stage and not reaching the ideal stage for proper weight gain (Silva et al., 2018). Previous research has shown that an antibiotic diet outperforms then other diets in terms of development because it is a high-caliber diet that requires less time for growth (Mohd Yusof et al., 2021).

The antibiotic diet used in this study led to heavier weights in all life stages of O. furnacalis, whereas the artificial and natural diets did not yield weights comparable with the antibiotic diet. Numerous scientists have said that the use of antibiotics inhibits the growth of invading microorganisms, leading to favorable conditions for insect development and weight gain (Kim et al., 2022; Morales-Ramos et al., 2022). It is possible to gradually eliminate a few microbes in some insects when feeding the insects with antibiotics over several generations. Clade III outperformed the other clades with a shorter development time and higher weight than Clades I and II. Previous studies have indicated that the inclusion of antibiotics in artificial diets can suppress the growth of invading microorganisms, contributing to favorable insect development (Li et al., 2020).

We established strong relationships of head capsule width with each instars stage and their weights. Our results tell that clade III as compared to clade I and II O. furnacalis had most wider head capsule width ranged between (1.4–1.7 mm in logarithm) with significant increase in 4^th–5^th instar in antibiotic diet. Because it saves the energy level of insects, that helps them to grow well. These results aligning with previous research on different species such as Lobesia botrana (Delbac et al., 2010) and S. frugiperda (Tiwari et al., 2023). Over results also align with the another findings specific on O. furnacalis, that also indicated a correlation of larval instars with larval head capsule widths and weight (Rahayu & Trisyono, 2018; Tiwari et al., 2023). Our findings also matched with another research that showed that size increment ratio of larval head capsule width in each molt generally constant from 1.3 to 1.7 mm in logarithm (Gullan & Cranston, 2014).

The number of pupations, adult emergence and sex ratio produced from the larvae fed with the antibiotic, artificial and natural diets were not closely related, indicating that these diets did not affect O. furnacalis Clades in the same way. In our findings, natural and artificial diets supported the larvae in developing into larger female adults, while the antibiotic diet supported the development of more male adults and exhibited a better sex ratio than artificial and natural diets. Similar results were found in this study (Gopalakrishnan & Kalia, 2022). Based on our findings, the biological and fitness parameters of different Clades of O. furnacalis reared on the antibiotic diet were rated as good, while the artificial diet exhibited moderate fitness. In contrast, the natural diet proved to be a poor diet. Regarding Clades, Clade III exhibited higher fitness as compared with Clade I and II.

The significance of incorporating antibiotics into artificial diets act as a growth booster for the mass rearing of three Clades of O. furnacalis in the laboratory (Galarza et al., 2021; Phuong et al., 2016). Antibiotics function as growth promoters by addressing bacterial infections (Wang et al., 2021) and potentially amplifying the overall growth and development (Galarza et al., 2021). This research holds the potential to significantly enhance the successful mass-rearing of three Clades of O. furnacalis in the laboratory. Despite the success presented in this study, further improvement of the existing antibiotic diet is needed.

Conclusion

The antibiotic diet used in this study showed a notable effectiveness in comparison to both artificial and the natural diets, by producing large number of O. furnacalis healthy insects. Besides diet, rearing conditions also played a crucial role in successful insect rearing. Consequently, the antibiotic diet, along with rearing conditions described in this study, can be employed for culture maintenance of all three O. furnacalis Clades. These investigations highlighted the reliability and effectiveness of the antibiotic diet for mass rearing of O. furnacalis. In relation to Clades, the biological parameters of all three Clades exhibited most significant results when subjected to the antibiotic diet. Notably, Clade III displayed markedly enhanced growth and development compared to Clade I and II. However, it remains crucial to ascertain whether these O. furnacalis Clades, reared in laboratory conditions, respond similarly to the natural conditions. Nevertheless, further comprehensive studies are essential to enhance and refine this antibiotic diet for future applications.

Funding information

We thank Jilin Province Major Science and Technology Project Mission Statement (20230302005NC), Jilin Province Science and Technology Development Plan Project (20190303075SF) and Program for Innovative Research Team of Baicheng Normal university (IRTBCNU) for their financial support towards this research.