Evaluation of artificial diets for rearing Spodoptera frugiperda (Smith) (Lep: Noctuidae) intended biocontrollers search

doi:10.21203/rs.3.rs-2321302/v1

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Evaluation of artificial diets for rearing Spodoptera frugiperda (Smith) (Lep: Noctuidae) intended biocontrollers search

https://doi.org/10.21203/rs.3.rs-2321302/v1

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To develop a methodology for laboratory rearing of S. frugiperda for the production of bio-controllers, which allow biological control of this important pest. Four artificial diets were evaluated, two of them with grain flour (bean and chickpea) and agar; two prepared with cassava and lentil starch.

Methodology: The tests were carried out in three consecutive generations; in the first one the efficiency of the diets with agar was estimated, and in the second one diets without agar and the best diet of the first trial. A final test included all artificial diets. Larval size, pupal weight and length, adult longevity and fertility, and duration of each developmental stage were determined.

Results: Larval development in bean and wheat germ had no significant differences (p>0.05) (L1=2.57 mm; L2=5.83 mm; L3=9.95 mm; L4=14.7 mm; L5=21.4 mm; L6=28.01 mm and L1=2.55; L2=5.79; L3=10.16; L4=14.62 and L6=27.56 mm respectively) with the reference diet. Pupae weights ranged from 0.21g to 0.18g. The average weights in each of the cases were, 0.18 g, 0.19g, and 0.16g approximately for the diets containing bean, wheat germ, and chickpea respectively; and showed significant differences (p>0.05) with the control (0.21g.).

Conclusion: The bean diet did not show significant differences (p>0.05) with the control for the insect development time (53, 39 days, 48.53 days, and 43.61 days for the artificial diet and 36.37 days, 38.42 days and 41.56 days for the reference diet).

Breeding

diet

insect

Lepidoptera

rearing

pest

The codling moth Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) is a polyphagous species native to the tropics, distributed from northern Argentina and Chile to the southern United States (Andrews and Quezada, 1989). In its larval stage, this insect feeds on tender shoots and especially on the buds of plants of different crops, including cotton, sorghum, soybean, corn, and rice (Alonso-Alvarez, 1991). In Colombia, it is estimated to cause economic losses ranging from 13% and 18% in rice and sugarcane plantations, respectively (ICA, 2010). Although S. frugiperda causes direct damage to the plantation, it also affects the economy of farmers (CATIE, 1990), raising production costs since chemical control is the most widely used to combat this insect, and the use of pesticides unbalances the ecosystem, destroys beneficial organisms and stimulates resistance in the pest. Therefore, it is necessary to rethink the management of agricultural systems looking for new solutions such as biocontrol (Navarro and Doreste, 1945). In the specific case of the department of Norte de Santander, there is still no tradition in the use of beneficial insects due to the absence of studies that explore the biotechnological potential of natural enemies, the ideal conditions for breeding and development of both the pest and the controller, thus limiting the large-scale production of biological inputs and their subsequent transfer to the farmer. Consequently, it is pertinent to develop studies on the multiplication of S. frugiperda (host) of excellent quality as a first step for the study and selection of natural enemies (host) with great specificity, to generate clean alternatives for the management of their populations in crops of economic importance in this region of the country.

2.1. Location of the experiment and biological material

The experiment was carried out in the Entomology laboratory of the Agricultural and Environmental Research Center of the Francisco de Paula Santander University, located in the municipality of Los Patios, Department of Norte de Santander, Colombia. S. frugiperda eggs were acquired from the colony previously established under controlled conditions.

2.2Artificialdietsand feeding of larvae

Five modified diets (Abbasi, et al.; 2007; Alfazary et al., 2012 Arévalo and Polanía, 2009; Morales et al., 2010), were included in three experiments, which corresponded to three formulations that included agar as a gelling agent, while two others lacked this ingredient, and a natural diet based on corn slices (Table 1).

Once the individuals emerged, neonate larvae (< 24 hours) were individualized in ventilated glass containers of 6 cm in diameter and 4 cm deep, and previously sterilized, inside which 1 cm² of artificial diet was incorporated. The same treatment was carried out for each of the diets evaluated (Lastra and Gómez, 2006).

Table 1

Adjusted composition for the ingredients of the artificial diets evaluated for feeding the larval stage of *S. frugiperda.*
Ingredients	Bean diet (Osores, 1982)	Chickpea diet (Arévalo and Polanía, 2009).	Wheat germ diet (Morales et al, 2010)	Lentil Diet (Alfazary et al., 2012).	Starch diet (Abbasi et al., 2007).
	Adjusted quantities of ingredients
Distilled water	500ml	500ml	500ml	590ml	260ml
Bean flour	50 g.
H. of chickpea		75g.			62 g
Wheat germ	35 g.		100g.
Lentil				180g.
Rice				25g.
Starch					50g
Agar	5g.	7g.	6g.
Yeast	15g.	12g.	12g.	18.5g.	3.5g
Multivitamin			1 tablet		1 tablet
Ambramycin			0.04g.
Ascorbic Acid	0.75g.	3g.	1.17g.	3g.	1.17g
Sorbic acid	0.66g.	1g.	0.75g.	4g.	0.75g
Methyl paraben	2 g in 5ml H₂O	2 g in 5ml H₂O	2 g in 5ml H₂O	2.5 g in 5ml H₂O	2g H₂O
Formaldehyde	1.5 ml	1ml	1ml	1ml	1 ml

2.3 Evaluation of developmental parameters in the larval stage of S. frugiperda.

The trials were set up as follows: Trial 1: Bean Chickpea Corn Germ (diets with Agar) Trial 2: Bean Lentil Corn Starch (diets without agar) Trial 3: Bean Chickpea Germ Lentil Corn Starch (all diets). During the entire larval stage and every two days, the feed was removed and larval size was determined as a fundamental parameter to estimate the quality of the diet (Arévalo 2009). The trials were organized in a completely randomized design using 30 larvae per treatment and every 48 hours the larval size and molting time of the cephalic capsule were determined to determine the duration between instars. Data will be subjected to analysis of variance and Tukey mean comparison test (P = 0.05).

2.4 Evaluation of developmental parameters in the pupal stage of S. frugiperda.

Once the larval stage was completed, and 24 hours after pupal formation, the individuals were sexed and individualized in containers with ventilated lids similar to those used in the larval stage; inside the containers, an absorbent towel disk and sterile cotton balls moistened daily with distilled water were placed. In each trial, larvae fed consecutively on each of the substrates of interest were used (Arévalo and Polanía, 2009; Abbasi, 2007). The trial was organized in a completely randomized design using the surviving pupae from the biological parameters determination trial. Pupal length and weight were determined every 48 hours in all treatments. The data were subjected to an analysis of variance and Tukey mean comparison test (P = 0.05).

2.5. Evaluation of developmental parameters in the adult stage of S. frugiperda.

For the evaluation of the developmental parameters of the adult stage of S. frugiperda, pairs of pupae were placed in rearing cages consisting of cylindrical plastic containers 14 cm in diameter at the base and 25 cm in height, lined on the inside with bond paper, an absorbent paper was used at the bottom of the container, a disc of bond paper with holes was used as a lid, and a muslin cloth was placed over the lid to guarantee ventilation of the insects and allow free circulation of air inside the cage. To maintain humidity inside the container, cotton balls moistened with sterile distilled water were placed daily until the emergence of adults. Individuals were fed with a 10% sugar solution. The rearing units were checked daily to determine the number of spawns. Postings were removed from the bond paper by cutting them with scissors and counted daily. (Lastra and Gómez, 2006). The trials were organized in a completely randomized design. The number of egg laying and longevity of adults were determined every 48 hours in all treatments until the death of the last individual. Data were subjected to an analysis of variance and Tukey mean comparison test (P = 0.05).

2.6. Determination of the life cycle of S. frugiperda fed on artificial diets under laboratory conditions.

The duration of the developmental stages of S. frugiperda was determined in the different treatments evaluated and in the three trials carried out. The data were subjected to an analysis of variance and Tukey mean comparison test (P = 0.05).

2.7. Economic analysis

The cost of each artificial diet was determined considering the commercial value of the ingredients and the consumption of each larva until pupal formation and survival. (Murúa et al., 2003). Considering the biological and economic aspects, the best artificial diet for the maintenance of S. frugiperda larvae was selected.

2.8. Statistical analysis

A completely randomized experimental design was used, with 5 experimental diets and

30 replicates for the larval stage. All data were subjected to ANOVA analysis of variance. The groups of averages were compared with Tukey's test (p = 0.05). All statistical analyses were developed using S.A.S. 9.0.

3.3. Evaluation of developmental parameters in the larval stage of S. frugiperda.

The evaluations of larval sizes in the first trial established that there was no significant difference for first-instar larvae, whose length in all cases did not exceed 2.66 mm. The bean, chickpea, and wheat germ diets, which measured 2.62 mm, 2.54 mm, and 2.63 mm, respectively, showed no significant difference from the control.

However, these results did not show a clear trend for instars two, three, four, and six for the control. Nevertheless, the records of the last instar established that the larvae fed with the bean and wheat germ diets reached an average length of 25.29 mm and 25.62 mm, respectively; this value did not differ from the control treatment, which reached a value of 25.86 mm. Larvae fed with chickpeas reached an approximate size of 22 mm, differing significantly from those fed with corn (Table II).

When evaluating the second group of diets for feeding S. frugiperda included those whose gelling compound was starch or lentil and an additional treatment that consisted of the best diet of the first trial (bean) and the control treatment (corn slices), it was established that all treatments differed from the control treatment. It was established that all treatments differed from the control treatment. The larvae of the control presented a length per instar of L1 = 2mm; L2 = 6.56mm; L3 = 11.4mm; L4 = 16.98mm; L5 = 21.87mm; and L6 = 28.27mm approximately; these values were closely followed by the growth of the larvae fed with starch and beans, which did not differ from each other and presented significant differences with the reference diet; and in turn these three with the diet containing lentils. It is important to note that larvae fed on a natural diet, of bean and starch, reached the pupal stage through six larval instars, while those fed a lentil-based diet only reached the fifth instar where the entire population died (Table 2).

Table 2

The average length of the larval stage of *s. frugiperda* fed with artificial diets under laboratory conditions. averages with the same letters do not show significant differences, according to Tukey's test (p = 0.05).
Test	Diet	L1	L2	L3	L4	L5	L6
Test	Diet	(mm)	(mm)	(mm)	(mm)	(mm)	(mm)
1	Beans	2.62 a*	5.54 ab	8.82 ab	12.15 c	17.55 a	25.29 a
	Chickpea	2.54 a	5.03 b	8.19 b	11.8 c	17.34 a	22 b
	Germ	2.63 a	5.83 a	8.9 a	13.55 b	18.48 a	25.62 a
	Corn	2.66 a	6.1 a	10.02 a	15.49 a	20.8 b	25.86 a
2	Beans	2.69 a	5.43 b	8.64 b	12.99 b	19.68 b	25.98 b
	Lentil	2.22 b	4.0 c	5.12 c	7.53 c	10.3 c	------
	Starch	2.27 b	5.08 b	8.02 b	12.46 b	17.21 b	24.99 b
	Corn	2.7 a	6.56 a	11.4 a	16.98 a	21.87 a	28.27 a
3	Beans	2.57 ab	5.83 a	9.95 ab	14.7 a	21.46 ab	28.01 a
	Chickpea	2.28 bc	5.67 a	9.02 ab	14.42 a	20.5 bc	27.28 a
	Germ	2.55 ab	5.79 a	9.92 ab	14.62 a	20.71 bc	27.56 a
	Lentil	2.09 c	4.11 b	4.9 c	------	------	------
	Starch	2.27 bc	4.7 b	8.43 b	12.67 b	19.15 c	25.21 b
	Corn	2.72 a	6.25 a	10.16 a	14.92 a	22.17 a	29.08 a

The trial in which the diets that included agar and those that included other types of gelling agents such as starch and lentil were tested. It allowed us to establish that the length of the larvae fed with the bean-based diet reached sizes of L1 = 2.57 mm; L2 = 5.83 mm; L3 = 9.95 mm; L4 = 14.7 mm; L5 = 21.4 mm; L6 = 28.01 mm approximately, values that did not present significant statistical difference with the larvae of S. frugiperda fed with the reference diet, which exhibited no significant statistical difference with the larvae of S. frugiperda larvae fed with the reference diet, which exhibited sizes of L1 = 2.72; L2 = 6.25; L3 = 10.16; L4 = 14.92; L5 = 22.17; L6 = 29.08 mm in each of the indicated instars. Although the diet with wheat germ did not present significant differences with the control in instars L1, L2, L3, L4, and L6 with sizes of 2.55 mm; 5.79 mm; 10.16 mm;14.62 mm and 27.56 mm approximately, it is important to point out that instar five presented a variation with the value reached by the control for this time of development (Table II).

3.4 Evaluation of developmental parameters in the pupal stage of S. frugiperda.

Pupae size.

In the first trial conducted on pupae obtained from the feeding of larval feeding on each of the substrates, the size of pupae offered diets with beans and germ obtained average sizes of 15.54 mm and 15.69 mm, respectively, which showed no significant differences from the control whose pupae exhibited an average size of 15.82 mm (Fig. 1A).

On the other hand, in the evaluation of the pupal size of individuals fed with a diet without agar, and using another gelling agent, it was determined that the diets that included bean and starch reached an average size of 15.34 mm and 15.50 mm, respectively, and did not differ from the size of the pupae of the control treatment, which had an average size of 16.13 mm. No pupae size was recorded for pupae fed with lentils since larval individuals fed with this diet did not reach this stage of development (Fig. 1B).

When evaluating the five diets in the third trial, it was determined that all diets that included agar showed a good performance in terms of pupae length, obtaining measurements of 16.39 mm; 16.10 mm; 16.33 mm, for the diets with bean, chickpea, and germ, respectively, and did not differ significantly with the pupae from larvae fed with corn slices, whose average size was approximately 16.24 mm. The starch treatment with a size of 14.31 mm was significantly different from the control (Fig. 1C).

Weight of pupae.

It was determined that the results were variable. It was determined from the first trial that pupae weights ranged from 0.21g to 0.18g. The average weights in each of the cases were approximately 0.18 g., 0.19g, and 0.16g. for the diets containing beans, wheat germ, and chickpea, respectively; in all cases, the weights showed significant differences with the control, which reached an average weight of 0.21g. (Fig. 2A).

When evaluating the weight of pupae fed with agar-free diets, it was possible to establish that those containing bean and starch in their formulation did not show significant differences between them (0.16 g.); however, they differed from the control, whose individuals registered a weight of approximately 0.20 g. (Fig. 2B).

In the trial in which all diets were included, pupae weights of approximately 16.38g., 16.10g., and 16.33g. were obtained for the bean, chickpea, and wheat germ diets, respectively, which did not differ statistically significantly from the control, which recorded a weight value for this stage of development of approximately 16.2g. (Fig. 2C).

3.5 Evaluation of developmental parameters in the adult stage of S. frugiperda.

The average number of brood egg laying/brood pens in each of the treatments of the first trial was 20.3; 12.3 for the bean and corn diets, respectively, values that differed significantly. However, the treatments with the lowest number of brood egg laying/brood pens were the chickpea and wheat germ diets with a total number of egg-laying of approximately 4.3 and 9.0. In the second trial, values of approximately 22.3 egg laying/cage were recorded for the bean diet, a value that presented a statistically significant difference concerning the reference diet (12.3 egg laying). Individuals fed the starch diet obtained approximately 4 egg laying/cage. The lentil diet did not record any egg-laying because the larvae did not reach the pupal stage (Table 3). In contrast to the third

Table 3

Determination of adult longevity and number of egg-laying of the adult stage of *S. frugiperda* fed under laboratory conditions. Averages with the same letters do not show significant differences, according to Tukey's test (p = 0.05).
Treatment	Diet	Longevity	Total Positions
1	Beans	11.47 a*	20.3
	Chickpea	11.85 a	4.3
	Germ	9.52 b	9.0
	Corn	9.22 b	12.3
2	Beans	9.76 a	22.3
	Lentil	--------	------
	Starch	9.16 a	4.0
	Corn	9.43 a	12.3
3	Beans	9.12 ab	21.7
	Chickpea	10.9 a	6.0
	Germ	9.5 ab	12.7
	Lentil	--------	-------
	Starch	7.4 b	4.3
	Corn	8.67 b

It was determined that the diet that included beans had an important effect on the number of egg-laying/cage, with an average of 21.7 egg-laying per brood unit, which was the best treatment, followed by the diets with wheat germ and the reference diet, obtaining 12.7 egg laying/cage in each one. From rearing units with individuals fed with starch, only 4.3 egg laying were recovered approximately, while in the case of the lentil diet it was not possible to determine the fertility of the individuals since the larvae did not reach the pupal stage (Table 3).

3.6 Determination of the life cycle of S. frugiperda feeding on artificial diets under laboratory conditions.

According to the results of the evaluation of the diets with gelling agents other than agar (Table 4), it was established that there were no significant differences with the treatment and that the average duration of the adult stage in each case was 9.76 days; 9.16 days and 9.43 days for the diets with beans, starch, and corn, respectively. For the lentil diet, no results were recorded since the population declined without reaching at least the end of the larval stage. The longevity of the adult individuals in the trial in which all diets were included allowed comparing and establishing that the longest-lived individuals were those fed with the chickpea diet, whose duration was approximately 10.9 days; however, there was no significant difference with the insects fed with bean and wheat germ diets with a duration of the adult stage of approximately 9.12 days and 9.5 days, respectively. In contrast, the shortest longevities were recorded for insect populations fed starch and corn diets with a duration of 7.4 days and 8.67 days, respectively. The total development time from the feeding of the larvae with different artificial diets allowed for determining that the diet that used beans as a protein source was the one that registered a total life cycle time from the moment of offering the food the closest to the reference diet, not only in the time it takes the insect to develop but also in other biological parameters such as larval size and pupal size and weight, as well as longevity of the adult stage and number of egg-laying/cage. The total development times for beans were 53, 39 days, 48.53 days, and 43.61 days for the first, second, and third generations, while for corn they were 36.37 days, 38.42 days, and 41.56 days, respectively.

Table 4

Determination of the life cycle of *S. frugiperda* fed artificial diets under laboratory conditions. Averages with the same letters do not show significant differences, according to Tukey's test (p = 0.05)
Test	Diet	L1 (Days)	L2 (Days)	L3 (Days)	L4 (Days)	L5 (Days)	L6 (Days)	Larval duration (Days)	Pre-Pupae (Days)	Pupae (Days)	Adult (Days)	Total cycle (Days)
1	Beans	6.65 ± 1.61 (5–11) b	2.54 ± 0.91 (2–4) b	4.27 ± 1.42 (2–8) a	3.77 ± 1.51 (2–7) b	3.59 ± 1.40 (2–6)b	6.54 ± 2.06 (2–10)b	27.63 ± 3.52 (21–33)b	2.72 ± 1,16 (2–6)a	10.57 ± 1.54 (9–15) a	11.47 ± 1.98 (9–15) a	52.39
	Chickpea	8.52 ± 1.66 (7–11) a	3.64 ± 1.77 (2–6) a	4.66 ± 2,69 (2–12) a	5.53 ± 2.85 (2–12) a	7.46 ± 4.14 (2–17) a	9.18 ± 5.12 (2–18) a	38.99 ± 4.04 (31–47) a	2.72 ± 1.01 (2–4) a	10.83 ± 0.98 (10–12) a	11.85 ± 1.83 (10–14) a	64.39
	Germ	4.77 ± 1. 28 (3–7) c	2.08 ± 0.40 (2–4) b	2.26 ± 0.69 (2–4) b	2.43 ± 1.04 (2–6) c	2.52 ± 0.90 (2–4) b	3.6 ± 1.31 (2–6) c	17.66 ± 3.22 (15–27) c	2.6 ± 0.94 (2–4) a	10.21 ± 1.23 (8–12) a	9.52 ± 1.50 (8–12) b	39.99
	Corn	4.7 ± 1.20 (3–7) c	2.08 ± 0.42 (2–4) b	2.18 ± 0.42 (2–4) a	1.18 ± 0.59 (2–4) c	2.27 ± 0.70 (2–4) b	2.36 ± 0.79 (2–4) c	14.77 ± 2.15 (13–19) c	2.38 ± 0.80 (2–4) a	10.05 ± 0.94 (8–12) a	9.22 ± 1.07 (8–11) b	36.37
2	Beans	6.17 ± 1.00 (5–7) b	2.59 ± 1.08 (2–6) a	2.88 ± 1.01 (2–4) b	3 ± 1.18 (2–6) b	4.91 ± 2.57 (2–10) ab	5.81 ± 1.22 (4–8) a	25.36 ± 2.95 (19–31) a	3.13 ± 1.32 (2–6) b	10.76 ± 0.94 (10–12) a	9.76 ± 1.04 (8–12) a	48.53
	Lein	7.35 ± 0.79 (7–9) a	2.66 ± 0.98 (2–4) a	7.2 ± 6.05 (4–4) a	6 ± 4.69 (2–14) a	6.66 ± 1.15 (6–8) a	-	-	-	-	-	-
	Starch	7.08 ± 0.91 (5–9) a	2.83 ± 1.31 (2–6) a	2.91 ± 1.32 (2–6) b	3 ± 1,32 (2–6) b	3.58 ± 1.56 (2–8) bc	6.26 ± 2.65 (2–12) a	25.66 ± 4.35 (19–33) a	4 ± 1.51 (2–8) a	10.88 ± 0.83 (10–12) a	9.16 ± 1.42 (7–11) a	49.65
	Corn	4.66 ± 1.30 (3–7) c	2.14 ± 0.53 (2–4) a	2.16 ± 0.55 (2–4) c	2.33 ± 0.76 (2–4) b	2.08 ± 0.42 (2–4) c	2.69 ± 1.29 (2–6) b	16.06 ± 2.24 (13–21) b	3 ± 1.02 (2–4) b	9.93 ± 0.94 (10–12) a	9.43 ± 1.04 (8–12) a	38.42
3	Beans	5.5 ± 1.04 (5–9) ab	2.59 ± 0.93 (2–4) c	2.52 ± 0.89 (2–4) c	2.89 ± 1.15 (2–6) b	2.37 ± 0.79 (2–4) b	4.74 ± 1.13 (2–6) b	20.48 ± 1.63 (19–25) b	2.74 ± 1.13 (2–6) b	11.27 ± 1.15 (9–13) a	9.12 ± 1.18 (7–11) ab	43.61
	Chickpea	5.92 ± 1.18 (5–9) ab	2.43 ± 1.04 (2–6) c	2.36 ± 0.79 (2–4) c	2.36 ± 1.00 (2–6) b	3 ± 1.02 (2–4) b	5.36 ± 1.29 (2–8) b	21.36 ± 3.19 (15–31) b	2.91 ± 1.02 (2–4) ab	12 ± 1.70 (10–16) a	10.9 ± 3.70 (7–17) a	47.17
	Germ	5.07 ± 0.37 (5–7) bc	2.14 ± 0.52 (2–4) c	2.34 ± 0.77 (2–4) c	3,1 ± 1,14 (2–6) b	2,62 ± 0.94 (2–4) b	4,5 ± 1.04 (2–6) a	19.71 ± 1.82 (17–23) b	2.88 ± 1.01 (2–4) ab	11.68 ± 1.62 (8–14) a	9.5 ± 1.44 (7–12) a	43.77
	Lein	6.11 ± 2.03 (5–11) ab	9 ± 0 (9–9) a	16 ± 1.41 (15–17) a	-	-	-	-	-	-	-	-
	Starch	7 ± 1.95 (3–13) a	4.2 ± 1.82 (2–10) b	4.32 ± 1.92 (2–8) b	5.18 ± 3.47 (2–14) a	5.69 ± 3.90 (2–14) a	8.67 ± 2.99 (4–16) a	31.5 ± 7.82 (23–85) a	3.82 ± 1.08 (2–6) a	9.4 ± 2.07 (7–12) b	7.4 ± 1.67 (5–9) b	52.12
	Corn	4.63 ± 0.79 (3–5) c	2.67 ± 1.24 (2–6) c	2.87 ± 1.18 (2–6) c	2.26 ± 0.69 (2–4) b	2.18 ± 0.59 (2–4) c	4.09 ± 1.44 (2–6) b	18.73 ± 2.49 (13–23) b	2.56 ± 0.92 (2–4) b	11.6 ± 1.55 (8–14) a	8.67 ± 1.11 (7–10) ab	41.56

3.7 Economic analysis

For the calculation of the costs of the artificial diets (Table 5), the amount of food consumed by the larvae until pupa formation was taken into account. The lowest cost was presented with the wheat germ diet, followed by beans.

Table 5

Calculation of the production cost of a tray of artificial diet capacity for feeding the larval stage of *S. frugiperda* under laboratory conditions.
$/ Tray	No. Portions/tray	$/Portion	No. Total portions	Total/treatment
$1,64	283,54	$ 0,0058	349,95	$2,020
$1,61	310,31	$ 0,0052	*
$1,62	303,72	$ 0,0053	330	$1,762
$0,88	153,18	$ 0,0058	450	$2,599
*life cycle not completed

However, when analyzing the data on the development parameters of the diets evaluated, it was found that the bean diet showed a similar performance when compared to the reference diet. Therefore, it is necessary to consider that the studies should be continued and with a greater number of generations evaluated, to establish which is the best diet for maintenance, while having a balance between biological aspects and production costs.

According to the data collected in the three trials, it was evidenced that both the proteins provided by beans and wheat germ in each of the diets evaluated allow obtaining larvae of similar size to those fed with corn slices. It is important to measure the growth of an insect due to a food source since both body size and body weight is a quantitative trait that estimates the evolution and adaptation of an individual under laboratory conditions (Yokoyama and Miller, 1989). Bean grains are a source of important nutrients such as iron and zinc, but also of some amino acids essential for insect growth (Umaña, et al. 2013). The results obtained in this study are in agreement with those reported by Morales et al. (2010), who states that when they evaluated diets prepared with beans, they did not detect significant differences between this and the control and that mortality was not significant; the same is true of what was reported by Múrua (2003) who states that when using the diet of Osores et al. (1982), 95.5% of the larvae emerged to reach the adult stage and that only at the end of this stage does the death of the individuals occur. Therefore, the use of bean meal is recommended for incorporation in artificial diets for insect hatchlings, especially of S. frugiperda. The artificial diet containing wheat germ was shown to be efficient as it helps the optimal growth of the insect and therefore its amount should be optimal. In this study, 100 g of the wheat germ was used for the diet which also incorporated 12 g of brewer's yeast, in agreement with Cohen (2004) who points out that it can be added in this proportion in the artificial diet because of its high protein value (approximately 23%), additionally, it has a high amount of lipids and is rich in polyunsaturated acids, also essential and non-essential amino acids, a great amount of fiber, and vitamins except for vitamin A and ascorbic acid.

The results obtained for pupal size and weight in this study allowed determining the performance of each of the diets and their cumulative effect on the formation of the pupal stage, as an indicator of the good nutrition of an individual in its larval stage; therefore, it is assumed that greater weight, at a given moment, can ensure greater survival Morales et al, (2010). In the three trials carried out, it was determined that the diet prepared with beans obtained measurements that did not differ from the control in terms of size and weight, which is in agreement with Múrua (2003) who mentioned that individuals of S. frugiperda fed with the diet designed by Osores et al., (1992) were able to reach the pupal stage and have a satisfactory development that did not differ when compared with the reference diet. The results reported here do not coincide with those recorded by Alfazairy et al., (2012) who indicated that when feeding Helicoverpa armigera larvae with bean meal, the population evaluated when passing to the pupal stage presented mortality of 62.88% for the first generations, while from the fifth generation onwards these mortality levels were lower, it should be noted that in this research larval mortality did not exceed 3% and no deaths were recorded in the pupal stage for this diet.

From larvae fed with a wheat germ diet, pupae were obtained with similar characteristics in terms of size for the control, but in terms of weight there was a variation compared to those fed with corn, this can be explained by considering that size is not necessarily related to weight. The same behavior occurred with the data obtained from the population fed with chickpeas, which agreed with the records obtained by Morales et al. (2010), who reported that in three evaluated generations of S. frugiperda, the largest pupae weights 24 hours after their formation were greater than those of the control treatment (natural diet) and with Hamed and Nadeem (2008) who obtained pupae weights of 0.45 g in this same substrate. The same occurred with the weights of pupae from larvae fed with lentils, which in this study did not reach their formation and died, while Alfazairy et al. (2012) indicated that with this type of protein source the pupae recovered and reached a weight of 103 mg in comparison with the reference diet whose weights were below this value. Studies conducted by Abassi et al. (2007) on Helicoverpa armigera did not agree with the results obtained when evaluating the diet with starch, since they indicated that the weight of the pupae did not show significant differences for the diets prepared with agar, but did show significant differences in favor when compared with the control diet, indicating that pupation was 79%, in contrast in this study it was determined that the weight of the pupae from this food substrate had significant differences and lower values when compared with the natural reference diet. These divergent data with other authors may be because the studies conducted by these authors were carried out with insects for 5, 7, and even more, generations which were already adapted to the specific conditions of the laboratory, in the same way, insect strains behave differently in their adaptation process and phenotypic plasticity derived from a classical and evolutionary genetic adaptation mediated by the environment (Ghalambor et al., 2007).

The longevity values obtained in this study did not suggest a clear trend compared to the control, since the diet containing beans and chickpeas obtained higher values in the first and third trials, while in the second trial it did not differ significantly from the control. Murúa (2003), reports that the average longevity of adults fed with bean meal was 22.37 days; the analysis of these data showed the existence of significant differences, being the longer duration compared to the reference diet.

The diets that did not contain agar did not show good behavior, since the longevity of the adult, although it did not have a clear behavior compared to the control, the number of egg-laying recovered from the treatment was well below the number of egg-laying obtained in the control, these results were in agreement with those described by Abassi et al (2007) who found that when feeding H. armigera the longevity was not significantly different from that of the control, however, the longevity of the males and females was not significantly affected, except for the second generation. On the other hand, other diets that did not contain agar such as lentils did not report adult longevity since they did not complete the larval stage of the evaluated population, however, Alfazairy et al, (2012) reported that the longevity of adults developed from larvae of S. littoralis reared for 9 consecutive generations on diets without agar were not significantly different and presented an average longevity of 0.2–2.4 - days more than the control. However, when analyzing the data on the development parameters of the diets evaluated and the cost/benefit ratio, it was found that the bean diet showed a similar performance when compared to the reference diet.

This study allowed us to determine that the bean meal diet can be implemented for the maintenance of S. frugiperda broodstock under laboratory conditions since the development parameters are close to those obtained when individuals were evaluated on a natural diet of corn slices. The cost/benefit ratio of implementing this diet is compensated by obtaining good quality insects for the multiplication of biocontrollers. However, it is necessary to continue the evaluations of agar-free starch diets as a potential substrate for feeding S. frugiperda to multiply its natural enemies, since it has been demonstrated that the behavior and development depend on the adaptation that the insect strain can acquire by feeding between five to seven consecutive generations, in contrast to this study that only evaluated the effect of feeding substrates on three consecutive generations.

Acknowledgments

To the Research and Extension Revolving Fund (FRIE) of the Francisco de Paula Santander University (UFPS), for providing the financial resources (FINU project contract No. 030-2011) for the execution of this research.

To the Research Group in Agricultural and Livestock Sciences (GICAP), for providing the physical and technological resources to carry out this scientific work.

To the student Erick Mejía and Luz América Tórres, for their valuable collaboration in the maintenance of the S. frugiperda colony in the Entomology Laboratory.

To the Chemical Technologist, Diana Natali Galvis Mogollon, assistant of the Laboratory of Animal Nutrition and Food Analysis, for her valuable support in the processing of the flours for the artificial diets.

Ethical Approval

Approved by the ethics committee of the faculty of agricultural sciences and the environment of Francisco de Paula Santander University

Competing interests

No competing interests

Authors' contributions

J.O. was in charge of the methodological process and analysis of results. J.V. was in charge of the revision, writing and editing of the document. All authors reviewed and accepted the manuscript.)

Funding

Francisco de Paula Santander University

Availability of data and materials

Data and materials are fully available upon direct request to the author's email address [email protected].

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No competing interests reported.

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Evaluation of artificial diets for rearing Spodoptera frugiperda (Smith) (Lep: Noctuidae) intended biocontrollers search

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Location of the experiment and biological material

2.3 Evaluation of developmental parameters in the larval stage of S. frugiperda.

2.4 Evaluation of developmental parameters in the pupal stage of S. frugiperda.

2.5. Evaluation of developmental parameters in the adult stage of S. frugiperda.

2.6. Determination of the life cycle of S. frugiperda fed on artificial diets under laboratory conditions.

2.7. Economic analysis

2.8. Statistical analysis

3. Results

3.3. Evaluation of developmental parameters in the larval stage of S. frugiperda.

3.4 Evaluation of developmental parameters in the pupal stage of S. frugiperda.

3.5 Evaluation of developmental parameters in the adult stage of S. frugiperda.

3.6 Determination of the life cycle of S. frugiperda feeding on artificial diets under laboratory conditions.

3.7 Economic analysis

4. Discussion

5. Conclusions

Declarations

Acknowledgments

Ethical Approval

Competing interests

Authors' contributions

Funding

Availability of data and materials

References

Additional Declarations

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