**Self-Selection of Agricultural By-Products and Food Ingredients by** *Tenebrio molitor* **(Coleoptera: Tenebrionidae) and Impact on Food Utilization and Nutrient Intake**

#### **Juan A. Morales-Ramos 1,\*, M. Guadalupe Rojas 1, Hans C. Kelstrup <sup>2</sup> and Virginia Emery <sup>2</sup>**


Received: 15 October 2020; Accepted: 20 November 2020; Published: 24 November 2020 -

**Simple Summary:** Insects have been considered as an alternative to fishmeal in animal feed formulations. Current methods for mass producing them remain expensive and, although cost is not the current market driver for insect products, they remain off the main stream. One way to reduce production costs is to lower the cost of insect diets. This could be accomplished by using agricultural by-products as ingredients to formulate insect diets. In this study 20 ingredients were tested as dietary components for the yellow mealworm. Ingredients included dry potato and cabbage; the bran of wheat and rice; by-product meals from vegetable oil production; spent distiller's grains from brewery and ethanol production; and hulls of different grains. A method called self-selection was used to approach the optimal proportion of these ingredients in mealworm diets by measuring their relative consumption. Nine combinations of eight ingredients were presented to groups of mealworms while carefully measuring the relative consumption of each ingredient. Results showed that the most suitable ingredients for mealworm production were dry cabbage and potato, the bran of wheat and rice, the meals of canola and sunflower, and distilled grains from corn and barley. This information will be used to formulate and evaluate diet formulations for the yellow mealworm in future research.

**Abstract:** Nutrient self-selection was used to determine optimal intake ratios of macro-nutrients by *Tenebrio molitor* L. larvae. Self-selection experiments consisted of 9 combinations (treatments) of 8 ingredients, from a total of 20 choices, radially distributed in a multiple-choice arena presented to groups of 100 *T. molitor* larvae (12th–13th instar). Larvae freely selected and feed on the pelletized ingredients for a period of 21 days at 27 ◦C, 75% RH, and dark conditions. Consumption (g) of each ingredient, larval live weight gained (mg), and frass production were recorded and used to calculate food assimilation and efficiency of conversion of ingested food. The macro-nutrient intake ratios were 0.06 ± 0.03, 0.23 ± 0.01, and 0.71 ± 0.03 for lipid, protein, and carbohydrate, respectively on the best performing treatments. The intake of neutral detergent fiber negatively impacted food assimilation, food conversion and biomass gain. Food assimilation, food conversion, and biomass gain were significantly impacted by the intake of carbohydrate in a positive way. Cabbage, potato, wheat bran, rice bran (whole and defatted), corn dry distillers' grain, spent brewery dry grain, canola meal and sunflower meal were considered suitable as *T. molitor* diets ingredients based on their relative consumption percentages (over 10%) within treatment.

**Keywords:** insects as feed and food; nutrition; food assimilation; food conversion; insect dietetics; insect rearing; macro-nutrients

#### **1. Introduction**

In recent years, the yellow mealworm, *Tenebrio molitor* L. (Coleoptera: Tenebrionidae) has been considered as a potential source of animal protein in feeds for fish [1–6] and livestock [7–14]. An increasing number of companies have been founded every year since 2013 that focus on insect mass production as animal feed [15]. One of the most important aspects of mass production involves the formulation of inexpensive yet effective diets that maximize biomass productivity over time. Recent research has focused on the potential use of agricultural by-products as insect food to reduce production costs of insect biomass [16–19]. However, current studies on *T. molitor* have focused on the effects of single ingredients on biological and food utilization parameters. No attempts have been made to evaluate combinations of multiple by-products as ingredients with the aim to develop diets for *T. molitor*. Self-selection studies incorporating by-products have been used to develop complete diets for the house cricket, *Acheta domesticus* L. [20].

Developing adequate diet formulations for insects using multiple undefined (oligidic) ingredients is a complex procedure and can take multiple years of research, particularly in insect species with long life cycles such as *T. molitor*. Optimal diets can be obtained from multiple oligidic ingredients by allowing insects to select the optimal ratios of each ingredient in a multiple-choice experimental setting. This method is known as self-selection and was first proposed by Waldbauer and Friedman (1991) [21]. The objectives of this study were to (1) determine the ingredients with the highest potential for formulation of insect diets using the self-selection method, (2) establish the optimal macro-nutrient intake ratios of *T. molitor* based on the self-selected intake of 20 ingredients, and (3) explore the impact of intake of macro-nutrients, neutral detergent fiber (NDF), phytosterol, and minerals including Fe, Mg, Ca, Zn, Cu, and Mn on the biomass gain, food assimilation and efficiency of conversion of ingested food (ECI).

#### **2. Materials and Methods**

#### *2.1. Experimental Design*

The colony stock, the rearing procedures and rearing hardware used in this study were as described by Morales-Ramos et al. [22]. Larvae of *T. molitor* used in the experiments were separated by size from the stock colony using sifters of standard numbers 10 and 12, which selected larvae with head capsule width measuring between 1.4 to 2 mm. According to estimates by Morales-Ramos et al. [23] these head capsule measurements correspond to larvae between 3 and 5 instars prior to pupation, which could include instars 11 to 14. However, because the stock used in this study has been selected for larger size, the experimental group of larvae could have included earlier instars.

Experimental units consisted of groups of 100 larvae maintained in multiple-choice arenas designed to provide equal access to 8 different food choices. Groups of larvae from each experimental unit were weighed at the beginning and end of the experiment and their weight was recorded for each of the units of each treatment. The multiple-choice arenas consisted of breathable round plastic dishes (120 × 25 mm, Pioneer Plastics 53C, Pioneer Plastics, North Dixon, KY, USA) modified by the addition of 8 sample plastic vials 20 mL (72 mm height × 25 mm diameter) (Product # 73400, Kartell s. p. a., Noviglio, Milan, Italy). The sample vials were cut to a height of 35 mm to fit inside the dishes and assembled perpendicularly to the dish in a radial pattern (45◦ apart) with equal distance to the center of the dish (Figure 1A). A 5 mm diameter opening was drilled into one side of each of the vials pointing perpendicularly to the center of the dish, to allow larvae to enter the vials (Figure 1B, a). A depression (90 × 2 mm) was constructed at the center of the dish with screened bottom (0.5 mm screen openings) to allow the collection of frass in a second dish located under the arena (Figure 1).

**Figure 1.** Multiple-choice arenas for self-selection experiments: (**A**) top and bottom views; (**B**) assembled arena with cover and bottom dish; (**C**) experimental unit with food choices and larvae, (a) opening into modified vial as food-choice compartment.

#### *2.2. Food-Choice Treatments*

Food choices consisted of 20 food products and agricultural by-products, which included dry white cabbage, potato flour, alfalfa pellets, wheat bran, rice bran (whole and solvent defatted), spelt screenings; meals from canola, soybean, olive, sunflower, cotton, and kelp; hulls from rice, oat, and peanut, and coffee chaff; and dry distilled grains from corn, wheat, and barley from ethanol production and brewery. Nine treatments of eight different combinations of these food choices were selected for the study (Table 1). The criterium of selection for the combination treatments was based on the relative content of each of the macro-nutrients (protein, lipid, and carbohydrate) and dietary fiber as neutral detergent fiber (NDF). Combinations should contain at least one food choice with high content of each of the macro-nutrients and dietary fiber to allow the mealworm larvae to select a complete diet. For instance, diet 1 contains canola meal as high protein ingredient, potato flour as high carbohydrate, corn distilled grain and dry cabbage for lipids and hulls from peanuts and rice as high fiber ingredients (Table 1).


**Table 1.** Combination of eight food choices presented to *Tenebrio molitor* larvae groups in nine self-selection treatments.

Food ingredients were ground into a fine powder using a high-speed food processor. Powdered food ingredients were individually mixed with reverse osmosis (RO) water at 50% to 70% ratio to obtain a consistency of dough. The food ingredients were then formed into sticks using a cut 10 mL syringe. These sticks were dried in a vacuum oven at 50 ◦C for a period of 48 h. This procedure resulted in stable dry sticks of each of the food ingredients listed in Table 1 with dimensions that allowed them to be introduced in the compartments of the multiple-choice arenas (Figure 1C).

Food combination treatments consisted of 10 repetitions each (=10 experimental units totaling 1000 larvae). Food ingredients were randomly distributed in the arena compartments to minimize the proximity effects among the different ingredients. At the beginning of the experiment, a measured amount of each of the corresponding food ingredients was added to the corresponding arena compartment in each of the experimental units. The initial amount of each food ingredient provided was recorded for each of the experimental units from each of the combination treatments. Experimental units were maintained in environmental chambers at 27 ◦C, 75% RH (relative humidity) and dark conditions for a period of three weeks. Experimental units were monitored daily to observe the consumption of each of the ingredients. Food ingredients that were depleted by consumption, were replenished with a measured amount of the corresponding food ingredient, which was recorded for each of the experimental units.

#### *2.3. Data Collection and Analysis*

At the end of a three-week period, larvae from each experimental unit were counted and weighed alive as a group. The remaining food was collected separately by ingredient, separated from frass, dried in a vacuum oven, and weighed. Frass was separated from food by sifting the remains using a standard No. 35 sieve (0.5 mm openings). The frass was collected, dried, and weighed using the same drying procedure. To collect the remaining food, all the vials in the arena were capped, the arena was inverted, and the contents of each vial were emptied, one by one, into a standard number 35 sieve by removing the cap to separate food from frass. The consumption of each ingredient (Ii) was calculated as total weight added of ingredient 'i'—remaining weight of ingredient 'i', were i = 1 to 8. The total food consumption (FC) was calculated as the sum of the consumption of all the eight ingredients. Assimilated food (AF) was calculated as AF = FC − frass weight. The percent consumption of each ingredient was calculated as (Ii/FC) × 100. The weight of live mealworm biomass gained (LWG) was calculated as ending group weight—initial group weight. Mortality was extremely low (0.32 ± 0.21%) and dead larvae were cannibalized by surviving larvae (no cadavers were found), therefore, the ending live biomass measure per group included the loss of biomass due to mortality. Because the initial biomass dry weight could not be directly determined, the dry weight biomass gained (DWG) was calculated as LWG × the proportion of dry matter of mealworm larvae. The proportion of dry weight of mealworm larvae was previously determined from 25 groups of 10 larvae, which were weighed live, then frozen at −25 ◦C, dried in a vacuum oven at 50 ◦C, and weighed dry. The dry weight proportion of *T. molitor* late instar larvae was 0.38. The efficiency of conversion of ingested food (ECI) was calculated based on Waldbauer [24] as ECI = (DWG/FC) × 100 for each experimental unit.

Nutrient intake by *T. molitor* larvae was estimated from the self-selected consumption of the choice ingredients using the nutrient matrix calculation described by Morales-Ramos et al. [20,25]. The macro nutrient (lipid, protein and carbohydrate) content of the ingredients used in this study was obtained from data published in multiple sources [26–37]. The nutrient intake data was used to calculate the 3-way ratios of macro nutrient intake as described by Morales-Ramos et al. [25] and calculated as protein intake ratio = Pi/MNi, lipid intake ratio = Li/MNi, and carbohydrate intake ratio = Ci/MNi, where Pi, Li, and Ci are intakes of protein lipid and carbohydrate, respectively and MNi is the total intake of all three macronutrients and the sum of all three ratios is always = 1. The intake of other nutrients including neutral detergent fiber (NDF) and minerals including iron, magnesium, manganese, calcium, and zinc was also estimated.

Data consisting of live biomass gained, total food consumption, percent food assimilation, and efficiency of conversion of ingested food were compared among treatments using general linear mixed model (GLMM) and the Tukey–Kramer HSD (honestly significant difference) test for least square means of JMP software version 14.1 [38]. The effect of nutrient intake on food assimilation and efficiency of food conversion (ECI) was analyzed using multiple regression. The stepwise followed by backwards elimination methods were used to determine the optimal number of independent variables required in the model to explain food assimilation and ECI using the *Cp* statistic as the criterion to include or exclude variables [38–40].

#### **3. Results**

The means of consumption of each of the ingredients within each of the combination treatments are presented in Table 2. The relative consumption of each ingredient within combination treatments is illustrated as percentages in Figure 2. The ingredients that were consumed in higher proportion were dry potato in treatment 8 (41.01%); crude rice bran in treatments 3, 4, 5, 6, and 7 (40.47%, 34.87%, 30.37%, 32.27% and 33.18%, respectively); wheat bran in treatments 1 and 9 (30.49% and 37.1%, respectively); and corn dry distiller's grain with solubles (DDGS)in treatment 2 (34.63%). The least consumed ingredients were rice hulls in treatments 1 and 2 (0.31% and 0.13%, respectively); coffee chaff in treatment 8 (1.48%); peanut hulls in treatments 3, 6 and 7 (1.66%, 2.77%, and 3.19%, respectively); soybean meal in treatment 9 (2.06%); olive meal in treatment 4 (2.27%); and sunflower meal in treatment 5 (2.58%) (Figure 2). In general, highly consumed ingredients had a high carbohydrate content. The ingredients consumed in low percentages generally contained high amounts of fiber at the expense of other nutrients, such as rice hulls, coffee chaff and peanut hulls or have a combination of high fiber and high protein contents like meals of olive, soybean and sunflower.


*Insects* **2020** , *11*, 827

**Figure 2.** Proportional consumption of food ingredients by *T. molitor* larvae in nine treatments of different combinations of eight ingredients.

Despite the great diversity observed in the relative consumption of ingredients between treatments, the intake ratios of macro nutrients (lipid + protein + carbohydrate = 1) tended to converge close to a set of ranges between 0.03 to 0.16 of lipid, 0.21 to 0.25 of protein and 0.62 to 0.74 of carbohydrate (Table 3, Figure 3) with overall means of 0.1 ± 0.04, 0.24 ± 0.04, and 0.66 ± 0.06 for lipid, protein, and carbohydrate, respectively. The only exception was treatment 2 which showed significantly higher protein (0.36) (*F* = 379.1; df 8, 81; *p* < 0.0001) and lower carbohydrate (0.55) (*F* = 460.8; df 8, 81; *p* < 0.0001) intake ratio than all the other treatments (Table 3) outlying visibly in the graph of Figure 3. However, the rest of the treatments showed some significant differences among them in the macro nutrient intake ratios (*F* = 304, 379.1, and 460.8 for lipid, protein and carbohydrate, respectively; df 8, 81; *p* < 0.0001) that were less obvious in Figure 3 (Table 3).


**Table 3.** Macro nutrient intake ratios of *T. molitor* larvae in nine self-selection treatments with different combinations of eight food ingredients.

Mean ± standard deviation. Means with the same letter are not significantly different after Tukey–Kramer HSD test at α = 0.05.

**Figure 3.** Ternary plot of self-selected macro-nutrient intake ratios (L = lipid, P = protein, C = carbohydrate) in nine combination treatments of eight ingredients.

These differences in the intake ratios of macro nutrients resulted in significant differences in group live biomass gain (*F* = 10.15; df = 8, 81; *p* < 0.0001), overall dry-weight food consumption (*F* = 10.91; df = 8, 81; *p* < 0.0001), food assimilation (*F* = 29.13; df = 8, 81; *p* < 0.0001), and ECI (*F* = 28.41; df = 8, 81; *p* < 0.0001) among choice treatments (Figure 4). The highest live biomass gain was observed in treatment 5 (7.3 ± 0.28 g), followed by treatments 1 (6.91 ± 0.41 g) and 7 (6.83 ± 0.74 g). The highest assimilation was observed in treatment 8 (55.25 ± 2.03%) followed by treatment 5 (50.86 ± 1.86%). The highest ECI was observed in treatment 5 (9.87 ± 0.45%) followed by treatment 8 (9.48 ± 0.64%). In general, the best performing treatments were 5, 8, and 1 (Figure 4). Treatment 2 was the worst performer among choice treatments, showing the lowest live biomass gain (5.45 ± 0.49 g), the lowest food assimilation (39.19 ± 2.11%), and the lowest ECI (7.18 ± 0.5%) (Figure 4). The low performance of larvae groups of treatment 2 may be associated with the significant deviations in macronutrient intake ratios observed in this treatment (Figure 3). The optimal macro-nutrient ratios for *T. molitor* may be closer to those observed in average for treatments 1, 5, and 8, which were 0.06 ± 0.03, 0.23 ± 0.01, and 0.71 ± 0.03 for lipid, protein, and carbohydrate, respectively.

**Figure 4.** Circles represent means and brackets represent standard deviation of (**A**) dry-weight food consumption, (**B**) live biomass gain, (**C**) percent food assimilation, and (**D**) efficiency of conversion of ingested food (ECI) by groups of 100 *T. molitor* larvae in nine self-selection treatments of eight ingredients. Means with the same letter are not significantly different at α = 0.05 after Tukey–Kramer HSD test.

Live biomass gain was significantly impacted by efficiency of food conversion (ECI) (*R*<sup>2</sup> = 0.53; *F* = 100.74; df = 1, 88; *p* < 0.0001) and food assimilation (*R*<sup>2</sup> = 0.13; *F* = 13.35; df = 1, 88; *p* = 0.0004) in a positive way. Consumption of some ingredients have significant effects on biomass gain, food assimilation and ECI. For instance, consumption of potato had a significant positive effect on food assimilation (β = 0.01; *R*<sup>2</sup> = 0.57; *F* = 116.64; df = 1, 88; *p* < 0.0001), but consumption of corn DDGS had the opposite effect on food assimilation (<sup>β</sup> = <sup>−</sup>0.007; *R*<sup>2</sup> = 0.21; *F* = 23.0; df = 1, 88; *p* < 0.0001).

Ingredients that had a mean consumption percentage of at least 10% in any given choice treatment were considered relevant ingredients (RI). Relevant ingredients included potato, cabbage, wheat bran, crude rice bran, defatted rice bran, corn DDGS, spent brewery DG, canola meal, and sunflower meal. Multiple regression analysis indicated that the consumption of all the relevant ingredients had a significant positive effect on live biomass gain (*R*<sup>2</sup> = 0.7; *F* = 20.75; df = 9, 80; *p* < 0.0001). Only consumption of potato, cabbage, rice bran whole, and spent brewery DG had a significant positive effect on food assimilation (partial *F* Ratios = 49.47, 12.17, 6.62, and 8.55; df = 9, 80; *p* < 0.0001, = 0.0008, = 0.0119, and = 0.0045, respectively). Significant negative effects on food assimilation were observed with consumption of canola and sunflower meals (partial *F* Ratios = 6.39 and 4.49; df 9, 80; *p* = 0.0135 and 0.0371, respectively). The resulting optimized model for assimilation (after stepwise) agreed with the full model analysis including the 6 variables that showed significant effects on food assimilation (*R*<sup>2</sup> = 0.75; *p* = 42.6; df = 6, 83; *p* < 0.0001). In the full model (9 independent variables) the efficiency of food conversion (ECI) was only affected significantly by the consumption of potato, and this effect was positive (partial *F* Ratio = 13.31; df 9, 80; *p* = 0.0005). However, when this model was analyzed with the stepwise method, an optimized 3-variable model resulted that included potato, rice bran, and canola meal all affecting ECI significantly and positively (*R*<sup>2</sup> = 0.64; *p* = 49.9; df = 3, 88; *p* < 0.0001). Significant quadratic effects on live biomass gain were observed from consumption of potato (β<sup>1</sup> <sup>=</sup> 0.146, <sup>β</sup><sup>2</sup> <sup>=</sup> <sup>−</sup>0.023; *R*<sup>2</sup> = 0.18; *F* = 9.47; df = 2, 87; *p* = 0.0002), corn DDGS (β<sup>1</sup> = 0.077, <sup>β</sup><sup>2</sup> <sup>=</sup> <sup>−</sup>034; *<sup>R</sup>*<sup>2</sup> <sup>=</sup> 0.38; *<sup>F</sup>* <sup>=</sup> 26.2; df <sup>=</sup> 2, 87; *<sup>p</sup>* <sup>&</sup>lt; 0.0001) and spent brewery DG (β<sup>1</sup> <sup>=</sup> <sup>−</sup>0.105, <sup>β</sup><sup>2</sup> <sup>=</sup> <sup>−</sup>0.024; *R*<sup>2</sup> = 0.27; *F* = 16.02; df 2, 87; *p* < 0.0001). Biomass gain was maximized at an intermediate level of consumption of these three ingredients.

Intake ratios of some nutrients had a significant impact on food assimilation and efficiency of food conversion (ECI). The optimal multiple regression models obtained after stepwise and backwards elimination procedures consisted of only 2 dependent variables explaining food assimilation and 4 variables explaining ECI. Models are valid only within the ranges observed for these variables, presented in Table 4. Food assimilation was impacted significantly by carbohydrate and neural detergent fiber (*R*<sup>2</sup> = 0.73; *F* = 117.93; df = 2, 87; *p* < 0.0001) (Table 5). These two variables also impacted ECI in addition to the minerals Mg and Mn (*R*<sup>2</sup> = 0.73; *F* = 57.48; df = 4, 85; *p* < 0.0001) (Table 6).


**Table 4.** Summarized estimated nutrient intake means and ranges in 90 self-selection observations from 9 different 8-choice combination treatments (in mg/100 mg).


**Table 5.** Model from stepwise on percent assimilation.


Model: R<sup>2</sup> = 0.73; F = 57.48; df 4, 85; *p* < 0.0001.

#### **4. Discussion**

It is apparent by the results presented in this study that *T. molitor* larvae tend to balance their intake of macro nutrients by selecting among a variety of ingredients when feeding. This agrees with previous studies confirming the ability of *T. molitor* to self-select for optimal macro-nutrient intake ratios [41–44]. The intake ratios of macro nutrients by *T. molitor* larvae converged within a narrow range of values among eight of the nine combination treatments of different food ingredients. Treatment 2 was the exception showing excess intake of protein and reduced intake of carbohydrate. Deviation of macro-nutrient intake ratios observed in treatment 2 coincided with a low performance of growth and food utilization of the larvae grown in this treatment. The reason for the deviations in macro-nutrient intake ratios observed in treatment 2 may have been the absence of an additional ingredient with low protein content besides defatted rice bran. There was an unusually high consumption of corn DDGS (34.66 ± 4.13%) and spent brewery DG (29.75 ± 1.46%) in this treatment resulting in a combined mean consumption of 64.41% of these two ingredients from the mean total food consumption in treatment 2. In the other three treatments where these two ingredients were present together (treatments 5, 6, and 7), their combined consumption did not exceed 26% of the total food consumed. Additionally, consumption of corn DDGS and spent brewery DG did not exceed 21.5% when presented alone within the food choices (treatments 1, 3, 4, and 8). The high consumption of these two distilled grain ingredients in treatment 2 is itself an anomaly and may have been driven by the need for lipid intake, which was extremely low (lower than 3.6%) in the rest of the ingredients presented in treatment 2: two defatted ingredients (canola meal and rice bran defatted), alfalfa pellets, the hulls of peanut and rice, and coffee chaff [26,27,33,34]. The lipid content of corn DDGS and spent brewery DG is reported to be higher than 8% [26,27,30,34,35,37].

The optimal macro nutrient ratios for *T. molitor* may be those observed in the best performing treatments (1, 5, and 8): 0.06 ± 0.03 (max 0.12 min 0.03), 0.23 ± 0.01 (max 0.25 min 0.2), and 0.71 ± 0.03 (max 0.75 min 0.65) for lipid, protein, and carbohydrate, respectively. Rho and Lee (2016) [45] determined that an equal ratio of protein and carbohydrate was the best for *T. molitor* based on adult fecundity and longevity. However, this study is not comparable with ours because both studies were done on different life stages and measured different life cycle parameters.

Ingredients that were considered relevant based on relative consumption percentage (over 10%) included potato, cabbage, wheat bran, crude rice bran, defatted rice bran, corn DDGS, spent brewery DG, canola meal, and sunflower meal. Multiple regression analyses of consumption of relevant ingredients versus live biomass gain showed significant positive effects. These results can be interpreted as evidence that such ingredients are suitable for inclusion in diets for *T. molitor*, especially when biomass production is one of the main priorities. However, consumption of relevant ingredients did not always have positive effects on food assimilation. For instance, canola and sunflower meals had significant negative effects on assimilation. Food assimilation is not necessarily critical for biomass production when the

food provided has a low cost, as in this case where agricultural by-products are used. Analysis of nutrient intake ratios showed that intake of fiber negatively affects food assimilation. This may explain the negative effects of canola and sunflower meals on assimilation, since both meals have a relatively high fiber content. Food conversion efficiency (ECI) was impacted positively by the consumption of potato, rice bran and canola meal. Because both food assimilation and ECI significantly impacted biomass gain in a positive way, we may consider the ingredients that impact both parameters in a positive way as highly suitable for inclusion in insect diets. Potato, rice bran, cabbage, spent brewery DG, and canola meal seem to be highly suitable as ingredients in *T. molitor* diets, but defatted rice bran, corn DDGS, and sunflower meal are promising if provided in the correct proportions. Wheat bran, potato, and cabbage have been used and are currently used regularly in *T. molitor* diets for mass production [46]. The rest of the ingredients are not currently used in commercial production, but some studies have assessed their potential, such as on spent brewery DG [32].

Macro-nutrient intake ratios were an important factor affecting live biomass gain, food assimilation and ECI. Macro-nutrient ratios were optimal for *T. molitor* within ranges of 0.06 ± 0.03, 0.23 ± 0.01, and 0.71 ± 0.03 for lipid, protein, and carbohydrate, respectively. Nutrient intake analyses showed that the intake of carbohydrate significantly and positively impacted live biomass gain, food assimilation and ECI. The intake of protein did not impact these three parameters within the ranges observed in this study. It appears that protein intake was strongly regulated by self-selection in most treatments, with the only exception of treatment 2. Other studies have reported that high protein intake reduce development time and pupal size [42] and increased adult longevity and fecundity [43]. In this study the impact of high intake of protein on biomass productivity and food utilization was negative. High intake levels of fiber also had a negative impact on food assimilation and ECI. Li et al. (2015) [47] reported that the optimal intake levels of crude fiber for *T. molitor* is within a range of 5 to 10%. In this study we did not compare intakes of crude fiber, but the self-selected percentages of ND fiber were between 22.52 ± 0.62% in treatment 5 and 34.94 ± 0.94% in treatment 9.

#### **5. Conclusions**

The macro-nutrient intake ratios resulting from ingredient self-selection by *T. molitor* fell within narrow margins: Lipid intake was between 0.12 and 0.03, protein between 0.25 and 0.2, and carbohydrate between 0.75 and 0.65. Deviations from these ranges of macro nutrient intake ratios resulted in a diminished performance in larval growth and food utilization.

The relevant ingredients, based on their relative consumption by *T. molitor* larvae included potato, cabbage, wheat bran, crude rice bran, defatted rice bran, corn DDGS, spent brewery DG, canola meal, and sunflower meal. Consumption of relevant ingredients significantly affected live biomass production in a positive way in *T. molitor* larvae.

Both food assimilation and efficiency of conversion of ingested food were positively impacted by ingestion of carbohydrate and negatively impacted by ingestion of fiber. Ingredients that enhanced both of these parameters had relatively high carbohydrate and low fiber content such as potato. However, levels of carbohydrate and fiber should not depart from the self-selected ranges observed, because excessive or deficient intake of those nutrients can have a detrimental impact on growth and food utilization in *T. molitor* larvae.

**Author Contributions:** Conceptualization, J.A.M.-R., M.G.R. and H.C.K.; methodology, J.A.M.-R., M.G.R. and H.C.K.; software, J.A.M.-R.; validation, J.A.M.-R. and H.C.K.; formal analysis, J.A.M.-R.; investigation, J.A.M.-R. and M.G.R.; resources, J.A.M.-R., M.G.R., H.C.K. and V.E.; data curation, J.A.M.-R.; writing—J.A.M.-R. and M.G.R.; writing—review and editing, J.A.M.-R., M.G.R., H.C.K. and V.E.; visualization, J.A.M.-R.; supervision, J.A.M.-R.; project administration, M.G.R. and V.E.; funding acquisition, V.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded in part by USDD-DARPA, SBIR Grant No. SB172-002, D2-2236.

**Acknowledgments:** We thank Damian Tweedy for his assistance in mixing and forming ingredient pellets.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Disclaimers:** Mention of companies or product brand names does not constitute an endorsement by the U. S. Department of Agriculture or the U. S. Government. The views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government.

#### **References**


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## *Article Enterobacter* **sp. AA26 as a Protein Source in the Larval Diet of** *Drosophila suzukii*

**Katerina Nikolouli 1,2,\*,†, Fabiana Sassù 1,2,3,†, Spyridon Ntougias 4, Christian Stauffer 2, Carlos Cáceres <sup>1</sup> and Kostas Bourtzis <sup>1</sup>**


**Simple Summary:** *Drosophila suzukii* has caused considerable damages to a variety of soft fruit crops. The sterile insect technique (SIT) is one of the most promising candidates that has recently attracted significant research efforts. A SIT package requires highly productive and cost-efficient mass rearing of the insects to produce a sufficient amount of males that will be sterilized and consequently released in the target area. Operational costs of mass-rearing facilities can be remarkably high, mainly due to the larval diet used for rearing. Gut symbiotic bacteria have been shown to enhance the productivity and development of fruit flies when used as supplements or protein source of their larval diet. In this study, we evaluated whether *Enterobacter* sp. AA26 could replace inactive brewer's yeast as a protein source in *D. suzukii* larval diet and effects on the biological quality of the flies are discussed.

**Abstract:** The Spotted-Wing Drosophila fly, *Drosophila suzukii*, is an invasive pest species infesting major agricultural soft fruits. *Drosophila suzukii* management is currently based on insecticide applications that bear major concerns regarding their efficiency, safety and environmental sustainability. The sterile insect technique (SIT) is an efficient and friendly to the environment pest control method that has been suggested for the *D. suzukii* population control. Successful SIT applications require massrearing of the strain to produce competitive and of high biological quality males that will be sterilized and consequently released in the wild. Recent studies have suggested that insect gut symbionts can be used as a protein source for *Ceratitis capitata* larval diet and replace the expensive brewer's yeast. In this study, we exploited *Enterobacter* sp. AA26 as partial and full replacement of inactive brewer's yeast in the *D. suzukii* larval diet and assessed several fitness parameters. *Enterobacter* sp. AA26 dry biomass proved to be an inadequate nutritional source in the absence of brewer's yeast and resulted in significant decrease in pupal weight, survival under food and water starvation, fecundity, and adult recovery.

**Keywords:** spotted-wing drosophila; symbiotic bacteria; gut microbiota; pest-management; mass-rearing; insect fitness

#### **1. Introduction**

The gut of insects is the receptacle of a rich diversity of symbiotic bacteria, which can influence nutrient assimilation, host physiology, biology and ecology, including sexual and social behavior, and fitness [1–5]. The study of these microorganisms has brought to light mechanisms of symbiotic interactions that have proved to be a source of knowledge to manipulate insect behavior and develop microbe-based pest control techniques

**Citation:** Nikolouli, K.; Sassù, F.; Ntougias, S.; Stauffer, C.; Cáceres, C.; Bourtzis, K. *Enterobacter* sp. AA26 as a Protein Source in the Larval Diet of *Drosophila suzukii*. *Insects* **2021**, *12*, 923. https://doi.org/10.3390/ insects12100923

Academic Editors: Man P. Huynh, Kent S. Shelby and Thomas A. Coudron

Received: 30 August 2021 Accepted: 2 October 2021 Published: 9 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

i.e., repellents, attract-and-kill, and mass trapping [6,7]. Recently gut microbiota have been exploited to increase the efficacy of the sterile insect technique (SIT) [8,9]. The SIT is an environment-friendly approach for population management with appreciable economic and social benefits that has been effectively applied worldwide on different insect pests [10,11]. SIT highly relies on efficient mass-rearing methods allowing the continuous production of large numbers of high-quality insects [12]. Insects (preferentially males) are then sterilized by irradiation and released with an overflow ratio into a target area, where they are expected to compete with wild males and mate with wild females [12]. Successful matings between sterile males and wild females will not produce viable offspring that eventually cause the intended population to decline in the next generations [10].

One of the most significant parts of a SIT programme is the larval diet used during insect mass-rearing. The artificial diet is one of the most critical and expensive components for the production of high-quality sterile insects and its development is guided by the availability of ingredients, nutritional value, ease of storage, labour and cost [13,14]. The diet used for mass-reared insects should supply proper and sufficient nutritional factors allowing for an efficient maturation into the adult stage and high adult fitness [13,15]. In addition, the components of the mass-rearing diet should be cost-effective without any side effects on insect fitness and overall colony productivity [13,16]. In the long run, diet expenses and insect performance should be well-balanced to achieve highly competitive insects at a reduced cost.

Several studies have focused on exploiting insect gut symbiotic bacteria as probiotics or alternative protein sources to enhance the SIT of fruit flies [17–28]. In the case of *Ceratitis capitata* (Wiedemann) (Diptera: Tephritidae), enrichment of the larval diet with live bacteria of the *Enterobacteriacae* family shortened the immature development stages, extended survival and improved the male mating competitiveness [9,17,21–24]. Increased fecundity was also observed when *Bactrocera oleae* (Rossi) (Diptera: Tephritidae) was fed with *Enterobacteriacae* microbiota [27,29]. Similarly, bacteria such as *Candidatus* Erwinia dacicola [30] and *Pseudomonas putida*, which are common in the wild population of *Bactrocera oleae* (Rossi) (Diptera: Tephritidae), were found beneficial for larval development and female fecundity, respectively [27,29].

In all the above studies the beneficial probiotic effect was shown by inoculating the diet with live bacteria while the use of inactive bacteria is limited [18,28]. Kyritsis and colleagues [28] assessed whether the use of inactive *Enterobacter* sp. AA26 biomass could be used in the larval diet of *C. capitata* as an alternative protein source to replace the costly brewer's yeast. Incorporating dead *Enterobacter* sp. AA26 benefited substantially the biological quality and productivity of reared *C. capitata* and paved the way to explore the use of inactive bacteria as the main protein source in the larval diet.

*Drosophila suzukii* (Diptera: Drosophilidae) is one of the most damaging insect pests of soft skinned fruits in North America and Europe in the last decade, but also a newly introduced detrimental insect in South America and Africa [31–34]. The also-called spotted wing Drosophila (SWD) fly can damage a wide range of economically important soft-skin fruits that are easily perforated by the females sclerotized ovipositor to lay their eggs [35,36]. Eggs hatch and larvae feed on the fruit pulp which causes the total rot of the fruit and an economic impact that accounts for millions of revenue losses each year [37]. *Drosophila suzukii* infestations are primarily controlled by the application of various insecticides, but the rising concerns on their poor effectiveness and possible impacts on the health of farmers and consumers have initiated the development of eco-friendly pest management tactics such as netting and tunneling, attract-and-kill baits deployment, alternative oviposition sites, and parasitoid releases [38,39]. Researchers have recently begun to evaluate SIT as a strategy to control *D. suzukii* populations in confined areas [40–42]. Currently, speciesspecific protocols of irradiation, mass-rearing, packaging, and quality control are fully or partially available for the implementation of this technique to control *D. suzukii* populations [42–44]. Nevertheless, the use of probiotics in *D. suzukii*'s larval or adult diet can

help to further improve some of these protocols i.e., increase the mass-rearing productivity and/or boost the quality of the released sterile flies.

Previous studies have characterized bacterial species frequently occurring within the gut of *D. suzukii*, in some cases as an attempt to improve the effectiveness of bait traps used for its population management [45,46], and in others to understand its peculiar specialization in ripe and ripening fruits compared to other drosophilid species [47–50]. As a result, bacterial families such as *Enterobacteriaceae, Acetobacteraceae, Lactobacillaceae*, and *Enterococcaceae,* which are generally observed in association with different wild or laboratory-reared species of drosophilid [51], were also found in *D. suzukii* [52,53]. Several studies have identified *Enterobacteriaceae* to be abundant in *Drosophila melanogaster* [51,54,55] and *D. suzukii* [47,49,50,52,53,56]. Martinez- Sañudo et al. observed that *Enterobacteriaceae* in *D. suzukii* were more abundant and diverse in newly colonized areas compared to flies adapted in the new habitat [53]. Interestingly, *Enterobacter* sp. has been identified in few *D. suzukii* studies with varying diversity and abundance [52,53,57].

Distinct microbial populations have been correlated with positive impacts on fly longevity and development [49,58]. Bing et al. showed that *D. suzukii* utilizes microbes as a source of protein when reared on fruit-based diets [49]. During undernutrition *D. melanogaster* can use a wide range of microbes which serve as a protein source [58]. However, it is not yet clear how all fly-associated microbes contribute to nutrition.

A comparative study between *Enterobacter* sp. AA26 and Torula yeast (a yeast type that is different than brewer's yeast) showed that *Enterobacter* sp. AA26 was equivalent to yeast in terms of nutritional value [59]. The biomass of the strain AA26 provided all the essential and non-essential amino acids and vitamins required for the development of *C. capitata* larvae. Considering the abundance of *Enterobacteriaceae* species in fruit flies, including *D. suzukii*, the beneficial effects of *Enterobacter* sp. AA26 on the mass-reared *C. capitata*, the high protein content of *Enterobacter* sp. AA26 and the role of microbes as protein sources for *Drosophila* flies, we evaluated *Enterobacter* sp. AA26 as a potential protein source that could totally or partially replace inactive brewer's yeast in the *D. suzukii* larval diet. Assessing the potential beneficial effect of the same bacteria species in different insect species is of critical importance because if results are positive, they will justify economically the insect production in mass-rearing facilities. We assessed the effect of *Enterobacter* sp. AA26 on several fitness parameters, including pupal weight and recovery, adult emergence rate, sex ratio, flight ability, adult survival under stress and female fecundity. These fitness parameters are considered production and quality indicators of flies used for SIT programmes and ensure that insects of poor quality that can lead to a lack of effective control and higher programme costs are excluded [60].

#### **2. Materials and Methods**

#### *2.1. Drosophila Suzukii Rearing Colony*

All experiments were performed using the *D. suzukii* colony maintained at the Insect Pest Control Laboratory (IPCL) of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Seibersdorf, Austria. The colony was obtained from the Agricultural Entomology Unit of the Edmund Mach Foundation in San Michele All'Adige, Trento, Italy. Adult flies were maintained in metal-framed and mesh-covered cages of 45 × 45 × 45 cm under controlled laboratory temperature, RH and light conditions (22 ± 5 ◦C, 65 ± 5% RH, and 14:10 h light:dark (L:D) photoperiod).

#### *2.2. Enterobacter* sp. AA26 *Biomass Production and Larval Diet Preparation*

Inactive *Enterobacter* sp. AA26 biomass was produced as described in Kyritsis et al. [28]. Briefly, a 1 L laboratory-scale bioreactor was used to grow *Enterobacter* sp. AA26 under aseptic conditions. The bioreactor was fed with Luria-Bertani (LB) broth and operated under the fill and draw mode. Adequate aeration of the bioreactor was achieved through an air pump and continuous agitation. The bacterial biomass was collected by centrifugation at 4000× *g* for 10 min and stored at −80 ◦C until use.

The biomass was dried at 60 ◦C for 24 h and subsequently grinded in a Planetary Ball Mill PM100 for 3.5 min, until a fine powder was obtained. The rearing larval diet, consisting of 28% wheat bran, 7% inactive brewer's yeast (*Saccharomyces cerevisiae*), 13% sugar, 0.45% sodium benzoate, 0.45% nipagin, and 51% water, was used as a standard control diet. All solid components (apart from nipagin) were weighted and mixed into water to a total volume of 1 L. The solution was constantly stirred and brought to boil. It was left simmering for 10 min and then cooled slightly for an additional 10 min. Nipagin was diluted in 10 mL water, added to the media and mixed well. To evaluate the effect of yeast replacement with *Enterobacter* sp. AA26, we used 1) a full replacement diet (hereafter as "total") containing 7% *Enterobacter* sp. AA26 biomass instead of inactive brewer's yeast, and a partial replacement diet with 3.5% inactive brewer's yeast and 3.5% *Enterobacter* sp. AA26 (hereafter as "partial"). We decided to use the 1:1 *Enterobacter* sp. AA26:yeast ratio based on the similar study performed in medfly [28].

#### *2.3. Drosophila Suzukii Egg Collection*

Eggs used for the experiments were obtained following the wax-rearing procedure developed at the IPCL [44]. Eggs were collected from the mass-rearing colony of the IPCL and placed on moist filter paper to avoid desiccation. All eggs were collected within a period of 4 h to prevent sample variation. Twenty-four hours after the collection, eggs were transferred on a wet black net in a Petri dish (size: 70 × 15 mm (D × H)) containing about 150 g of diet. Four replicates of 500 eggs each were collected per treatment (i.e., *n* = 2000 eggs per treatment). These eggs were used to evaluate pupal weight and recovery, adult emergence rate, sex ratio, flight ability, adult survival under stress and female fecundity. The number of replicates used in each experiment are shown below in the respective M&M section.

#### *2.4. Effect of Enterobacter* sp. AA26 *on Pupae and Adult Recovery*

To assess the pupae and adult recovery, we measured number of pupae, adult emergence and sex ratio. Ten days after the egg collection, all pupae were removed from the diet and counted. All pupae that recovered from the same larval treatment (4 petri dishes per treatment) were placed on moist paper and left in boxes until adult emergence in a room with regulated temperature, humidity and light. Adult emergence was recorded daily using CO2 anesthesia. The sex ratio was determined as proportion of males per total number of adults. Adult emergence and sex ratio data were also collected from the flight ability and survival under stress experiments and all data coming from the different assays were combined for statistical analysis.

#### *2.5. Effect of Enterobacter* sp. AA26 *on Pupal Weight*

To determine the pupal weight, dark-brown pupae 24 h before emergence were selected from each treatment. Until the day of the experiment pupae were maintained in a room with regulated temperature, humidity and light to avoid any bias in the weight data. Pupal weight was determined by weighing independently pools of 10 pupae. Each single pool represented one sample. For each treatment (partial, total, and control), we performed 12 replicates (*n* = 120 pupae per treatment). All data were combined for statistical comparisons.

#### *2.6. Effect of Enterobacter* sp. AA26 *on Survival*

The adult survival was tested under food and water deprivation. Two days before emergence, 100 pupae from each treatment were randomly collected and individually placed into a 96-well microtiter plate sealed with plastic film. Pupae were checked twice a day to record the time of emergence, the time of death, and the sex of each fly. The film on the top of each well was delicately perforated to allow air exchange. Plates were kept in the dark to reduce the mobility at standard laboratory conditions. One plate was set up for each treatment. Each single adult represented one sample.

#### *2.7. Effect of Enterobacter* sp. AA26 *on Flight Ability*

The flight ability test was performed following the standard Quality Control Procedures applied to evaluate the sterile insects used in SIT applications. Pupae from each treatment were placed within a ring of paper centered in the bottom of the Petri dish to allow newly emerged flies a place to rest. A black plexiglass tube was placed over the Petri dish and was lightly coated inside with unscented talcum powder to prevent the flies from walking out. Flies that emerged were periodically removed from the vicinity of the tubes to minimize fly-back or fall-back into the tubes. For each treatment four replicates were conducted each consisting of 30 pupae (*n* = 120 pupae per treatment).

#### *2.8. Effect of Enterobacter* sp. AA26 *on Female Fecundity*

Newly emerged adults were selected from each treatment, sexed and separated into "triplets" made of one female and two males to ensure female insemination. Triplets were then transferred into 200 cm3 volume rectangular plexiglass cages and were provided with water and standard adult diet under the standard laboratory conditions. Twenty replicates were performed for each of the treatments and the control group. After 72 h, males were discarded, and females were individually placed into a Petri dish provided with raspberryjuice agar substrate as egg oviposition site. Oviposition was allowed for 48 h without any interruption, after which the females were discarded, and the number of laid eggs was counted. Each single female represented a replicate. Females that did not lay any eggs or laid fewer than 10 eggs were not included in the analysis.

#### *2.9. Statistical Analyses*

All statistical analyses were performed using R version 4.0.5 [61].

Pupal weight: Pupal weight data are continuous variables and therefore assume a normal distribution. A linear model was applied for their analysis.

Pupae and adults recovery: The number of pupae and adults recovered per treatment are count data and were analyzed with GLM with negative binomial distribution and a log link function. Negative binomial was applied due to overdispersion detected in the Poisson and the Quasi-Poisson GLM models [62,63]. Analysis of deviance was performed with an F-test [64].

Sex ratio (proportion males): Sex ratio proportional data assume a binomial distribution and were analyzed with a GLM-binomial family and a logit link function [65]. Analysis of deviance was performed with a Chi-squared test [64].

Adult survival: The survivorship curves were calculated using a Kaplan–Meier approach (survfit package) [66]. The package survival was used for modeling the survival data [67].

Flight ability: Rate of fliers are proportional data and assume a binomial distribution [65]. Data were analyzed with a GLM-binomial family and a logit link function. Analysis of deviance was performed with a Chi-squared test [64].

Fecundity: Fecundity data are count data and were analyzed with a generalized linear model (GLM) with negative binomial distribution and a log link function. Negative binomial was applied due to overdispersion detected in the Poisson model [62,63]. Analysis of deviance was performed with an F-test [64].

Residuals of the models were checked for normality and homogeneity of variance. Goodness-of-fit of the models was visually inspected with half-normal plots with simulation envelopes [68]. In addition, analysis of variance was performed for each model to check assess differences in the fit statistics [65]. Overdispersion of the generalized linear models was checked with the DHARMA package [69]. DHARMA tests if the simulated dispersion is equal to the observed dispersion and supports the visual inspection of the residuals. Lsmeans package were used for the pair-wise comparisons of the fitted model estimates [70]. In all cases, the mean ± standard error is reported. In all boxplots, both the mean and the median values are depicted. Significant differences between treatment groups are indicated in the boxplots with asterisks (\*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05, ns: *p* > 0.05; confidence level used: 0.95, alpha = 0.05). Non-significant differences are not shown in the boxplots.

#### **3. Results**

#### *3.1. Effect of Enterobacter* sp. AA26 *on Pupal Weight*

Brewer's yeast replacement with *Enterobacter* sp. AA26 had a significant effect on the average weight of the pupae (linear model: F = 26.45, df = 2, 33, *<sup>p</sup>* = 1.395 × <sup>10</sup><sup>−</sup>7) (average pupal weight: total: 0.0146 g ± 0.0007; partial: 0.0162 g ± 0.0006; control: 0.0166 g ± 0.0008). Pairwise comparisons among the tested diets (total, partial, control) indicated that the pupal weight was significantly lower in the total replacement when compared either with the partial or the control treatments (t-value = 5.351, *p* ≤ 0.0001; and t-value = 6.942, *p* ≤ 0.0001, respectively), while, between the partial and the control treatments, the difference was not significant (t-value = 1.591, *p* = 0.1212) (Figure 1).

**Figure 1.** Effect of *Enterobacter* sp. AA26 on pupal weight. Data were analyzed with a linear model to define if the different treatments had a significant effect on the average pupal weight. Boxplots span the interquartile range and whiskers indicate the highest and lowest observations. The line and the dot inside each box represent the median and the mean, respectively. Significant differences between treatment groups are indicated with asterisks (\*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05, ns: *p* > 0.05; confidence level used: 0.95, alpha = 0.05).

#### *3.2. Effect of Enterobacter* sp. AA26 *on Developmental Parameters* 3.2.1. Pupae Recovery

The mean number of pupae recovered per treatment grown in the total replacement diet was 261 ± 20.15 compared to 318 ± 31.2 and 352 ± 51.5 of the partial and control treatments, respectively (Figure 2a). Analysis of deviance indicated that treatment is not a

significant predictor for pupal recovery (GLM negative binomial model: F = 2.0914, df = 2, 9, *p =* 0.123) based on the GLM-negative binomial model. A marginal significant difference was detected at the pairwise comparison of the total and partial treatments (z = 2.026, *p* = 0.0427).

**Figure 2.** Effect of *Enterobacter* sp. AA26 on pupal and adult recovery. A GLM (negative binomial family) was used to analyze the impact of the various treatments on the recovery of pupae and adults; (**a**). Number of pupae recovered per treatment; (**b**). Number of adults recovered per treatment. Boxplots span the interquartile range and whiskers indicate the highest and lowest observations. The line and the dot inside each box represent the median and the mean, respectively. Significant differences between treatment groups are indicated with asterisks (\*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤0.05, ns: *p* > 0.05; confidence level used: 0.95, alpha = 0.05).

#### 3.2.2. Adult Recovery and Sex Ratio

Analysis of deviance indicated that treatment is a significant predictor for the number of adults recovered (GLM negative binomial model: F = 5.9898, df = 2, 9, *p* = 0.002504). Total replacement of brewer's yeast with *Enterobacter* sp. AA26 significantly decreased the adult recovery compared to both the partial replacement and the control treatment (z = 2.610, *p* = 0.0090 and z = 3.416, *p* = 0.0006, respectively) (Figure 2b). We did not observe any significant adult recovery decrease between the partial replacement and the control treatment (z = 0.808, *p* = 0.4190) (Figure 2b).

The partial and total replacement of *Enterobacter* sp. AA26 did not affect the sex ratio of the emerged adults (GLM binomial model: analysis of deviance: χ<sup>2</sup> = 4.8549, df = 9, *p* = 0.218) (Figure 3).

#### *3.3. Effect of Enterobacter* sp. AA26 *on Adult Survival under Food and Water Starvation*

The presence of *Enterobacter* sp. AA26 had a negative impact on the *D. suzukii* adult survival under food and water starvation (♀-rank test: <sup>χ</sup><sup>2</sup> = 19, df = 2, *<sup>p</sup>* = 7 <sup>×</sup> <sup>10</sup>−5; ♂logrank test: <sup>χ</sup><sup>2</sup> = 40.5, df = 2, *<sup>p</sup>* = 2 × <sup>10</sup>−9). Males developed in the total replacement diet had significantly shorter survival times compared to the ones developed in the partial and the control diets (log-rank test: χ<sup>2</sup> = 5.7, df = 1, *p* = 0.02; log-rank test: χ<sup>2</sup> = 15.8, df = 1, *<sup>p</sup>* = 7 × <sup>10</sup><sup>−</sup>5, respectively). In the case of females, the survival probability was significantly shorter when grown in the total replacement diet compared to the control one (log-rank test: <sup>χ</sup><sup>2</sup> = 18.9, df = 1, *<sup>p</sup>* = 1 × <sup>10</sup><sup>−</sup>5), but no significant difference was detected between the partial and the control diets (log-rank test: χ<sup>2</sup> = 0.4, df = 1, *p* = 0.5) (Figure 4).

**Figure 3.** Sex ratio of males developed in the partial and total replacement of *Enterobacter* sp. AA26 diet and control diet. Sex ratio was determined as the percentage of males per total number of adults. A GLM (binomial family) analysis was performed to determine the effect of *Enterobacter* sp. AA26 diet replacement. Boxplots span the interquartile range and whiskers indicate the highest and lowest observations. The line and the dot inside each box represent the median and the mean, respectively. Significant differences between treatment groups are indicated with asterisks (\*\*\* *p* ≤0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05, ns: *p* > 0.05; confidence level used: 0.95, alpha = 0.05).

**Figure 4.** Effect of *Enterobacter* sp. AA26 on female (**left**) and male (**right**) survival under starvation. Significant differences were measured with a log-rank test. The x-axis represents time in hours.

#### *3.4. Effect of Enterobacter* sp. AA26 *on Flight Ability*

The addition of *Enterobacter* sp. AA26 in the *D. suzukii* larval diet did not affect the adult flight ability (GLM binomial model-analysis of deviance: χ<sup>2</sup> = 14.829, df = 9, *p* = 0.1942). The rate of fliers was calculated based on the emerged pupae and data showed that there was no significant difference among the three treatments. The rate of fliers was 76.3% ± 1.28, 77.7% ± 0.80, and 85% ± 0.80 for the total, partial and control treatments, respectively (Figure 5).

**Figure 5.** Flight ability of *D. suzukii* adults. The rate of fliers was calculated based on the emerged pupae. A GLM (binomial family) analysis was performed to determine the effect of *Enterobacter* sp. AA26 diet replacement. Boxplots span the interquartile range and whiskers indicate the highest and lowest observations. The line and the dot inside each box represent the median and the mean, respectively. Significant differences between treatment groups are indicated with asterisks (\*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤0.05, ns: *p* > 0.05; confidence level used: 0.95, alpha = 0.05).

#### *3.5. Effect of Enterobacter* sp. AA26 *on Fecundity*

Analysis of deviance indicated that treatment is a significant predictor for the egg production of *D. suzukii* females (GLM negative binomial model: F = 15.263, df = 2, 37, *p =* 2.352 × <sup>10</sup>−7). The mean number of eggs per female grown in the total replacement diet (22.1 ± 3.88) was significantly lower compared to the partial (35.9 ± 6.76) and the control diets (44.7 ± 6.01) (total-partial: z = 3.299, *p* = 0.0010; total-control: z = 5.516, *p* < 0.0001). The model did not detect a significant difference between the partial and the control treatments (z = 1.848, *p* = 0.0646) (Figure 6).

**Figure 6.** Effect of *Enterobacter* sp. AA26 on *D. suzukii* female fecundity. The number of eggs per female are shown in the y-axis for each of the treatment groups and control group. Analysis of fecundity were performed using a GLM (negative binomial family). Boxplots span the interquartile range and whiskers indicate the highest and lowest observations. The line and the dot inside each box represent the median and the mean, respectively. Significant differences between treatment groups are indicated with asterisks (\*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05, ns: *p* > 0.05; confidence level used: 0.95, alpha = 0.05).

#### **4. Discussion**

In our study, results indicate that *Enterobacter* sp. AA26 dry biomass is not adequate to fully replace inactive brewer's yeast as a protein source in *D. suzukii* larval diet. In particular, complete replacement of brewer's yeast resulted in significant decrease in pupal weight, survival under food and water starvation, fecundity, and adult recovery. In addition, neither the partial nor the complete replacement of yeast with *Enterobacter* sp. AA26 had any significant impact on flight ability, sex ratio, and pupal recovery.

Core aspects of insect physiology are influenced or regulated by gut microbial communities by promoting digestive activities, boosting immune responses and restricting pathogen colonization [71]. Several studies have characterized the diverse microbial communities harboring the gut of natural *Drosophila* populations, with *Enterobacteriaceae* being one of the predominant families [51,72,73]. Studies on *D. suzukii* also confirmed the presence of *Enterobacteriaceae* in their digestive tract [52,53]. *Enterobacter* belongs to the *Enterobacteriaceae* family and is considered one of the most dominant genera of the gut of several insect species [74–77]. Due to their pivotal role in host physiology and biology, *Enterobacter* spp. could be exploited for the control of pest species. Recent studies have explored the possibility of using *Enterobacter* spp. as probiotic supplement in larval diets of mass-rearing insects for large scale operational SIT programs [22–24,28]. *Enterobacter* sp. AA26 was isolated from the gut of *C. capitata* males and females and assessed for its probiotic effects on several fitness parameters [23,24]. Results in *C. capitata* indicated that addition of *Enterobacter* sp. AA26 increased pupae and adult production and decreased rearing duration for several developmental stages. On the other hand, pupal weight, sex

ratio, male mating competitiveness, flight ability or life span under food and water deprivation were not affected. *Enterobacter* sp. AA26 dry biomass was also tested as a potential protein source that could fully replace the brewer's yeast in the *C. capitata* larval diet [28]. *Enterobacter* sp. AA26 proved to be an adequate nutritional source for *C. capitata* larvae since the immature stages' mortality decreased, the pupae development was accelerated, the pupal weight increased and the adult survival under food and water deprivation was elongated.

*Drosophila suzukii* is a continuously expanding threat and a SIT-based approach has been proposed as a promising control option. Mass-rearing protocols and the larval diet are important aspects that will determine the production of insects of high biological quality that will be released in the target area. *Drosophila suzukii* diet is currently based on brewer's yeast as a protein source. Previous studies have pointed out the critical role of yeast for *D. suzukii* as a source of dietary protein that is absent from fruits [78–81]. Yeast has been proved to have an essential nutritional role in *D. suzukii* larval development, survivorship, eclosion rate, and adult body mass [82–84]. The mass-rearing protocol of *D. suzukii* is currently based on a wheat bran larval diet that includes 7% inactive brewer's yeast [44]. Information collected from SIT facilities, currently rearing *C. capitata,* indicate that yeast-related expenses can be very high and, in some cases, they can be as high as 12% of the whole production cost [28]. Therefore, yeast replacement with a cheaper protein source could significantly decrease the cost of a mass-rearing facility. However, this replacement should not compromise the production of high biological quality insects. The yeast substitute should be of equal nutritional value, if not higher.

Following the promising results of a similar study in *C. capitata*, we tested whether *Enterobacter* sp. AA26 dry biomass could be a reliable alternative protein source. The total replacement of brewer's yeast had detrimental effects on female fecundity, pupal weight, and adult survivorship and recovery, thus indicating that *Enterobacter* sp. AA26 cannot fulfill the protein requirements of *D. suzukii* when yeast is absent from the diet. Interestingly, the partial yeast replacement did not present severe effects (apart from the adult recovery rate), suggesting that halving the yeast quantity is still sufficient to produce fit adults. In contrast, Kyritsis et al. suggested that *Enterobacter* sp. AA26 dry biomass can be used as an adequate replacement of brewer's yeast without any negative impact on the biological quality and the productivity rate of *C. capitata* [28]. The different physiology and biology of the two insect species might be one of the reasons why *Enterobacter* sp. AA26 failed to "act" as a suitable protein source. In addition, *Enterobacter* sp. AA26 is a *C. capitata* gut isolate, and although *Enterobacteriaceae* prevail in *D. suzukii* gut and *Enterobacter* sp. has been detected in a few *D. suzukii* studies, no data is available whether this specific AA26 strain is a member of the *D. suzukii* gut microbiota. Strain inconsistency or even bacterial competition could explain why *Enterobacter* sp. AA26 was an unfavorable protein source for *D. suzukii*. A recent study has shown that an infection of *E. ludwigii* in *D. melanogaster* affected the development of the flies and caused age-dependent neurodegeneration, thus indicating that specific *Enterobacter* sp. can negatively impact core aspects of the biology and behavior of the flies [85]. Further studies are required to identify promising microbe candidates with high nutritional value for *D. suzukii*.

Previous studies on fruit flies have clearly indicated that insect fitness is correlated to both the type and dose of the amino acids provided by the diet [86,87]. A study by Azis et al. 2019, compared the amino acid and vitamin content of *Enterobacter* sp. AA26 and Torula yeast [59]. *Enterobacter* sp. AA26 proved to be a sufficient source of all the essential nutrients required by *C. capitata*. Both essential and non-essential amino acids and vitamins were included in adequate amount in *Enterobacter* sp. AA26 biomass thus making it a strong candidate for the replacement of Torula yeast. Although glutamic acid and proline represented a lower fraction of the protein content compared to *Enterobacter* sp. AA26, the overall performance of the diet was equal to the Torula one for *C. capitata* larvae [59]. However, one cannot rule out the possibility that this lower percentage of glutamic acid and proline could affect the *D. suzukii* development and fitness. At the present study, Torula yeast has not been used and therefore it is not safe to extrapolate any conclusions related to the inactive brewer's yeast used for *D. suzukii*, since the two yeast types have different nutritional properties [88].

The outcomes of our study suggest that the "lack of performance" of *Enterobacter* sp. AA26 might not be due to the protein content per se but due to the protein quality. The nutritive value of the yeast's protein content is quite high and studies on the proteinogenic composition of yeast extracts have shown that the protein proportion of brewer's yeast can be more than 60% and the proportion of free amino acids more than 30%, thus indicating a rich protein source [88,89]. Apart from the essential amino acids, the content of minerals, vitamins and lipids play a role in the nutrient composition of brewer's yeast. Significant differences between brewer's yeast and *Enterobacter* sp. AA26 in the bioavailability of nutrients, as well as in the interactions among individual nutrients could be a reason explaining the reduced performance of *Enterobacter* sp. AA26. To the best of our knowledge there is not a corresponding study that compares *Enterobacter* sp. AA26 with inactive brewer's yeast, and thus our insight regarding their differences in terms of nutritional value is limited. A future study could shed light on this aspect and reveal why *Enterobacter* sp. AA26 is not sufficient as a protein source for the development of *D. suzukii*.

#### **5. Conclusions**

In the context of an SIT application, improvement of the mass-rearing protocols is always in the center of research efforts. Our findings clearly highlighted the importance of yeast as a diet component for *D. suzukii*. The *C. capitata* gut isolate, *Enterobacter* sp. AA26, cannot replace nutritionally the inactive brewer's yeast in the *D. suzukii* larval diet. However, one should not exclude the possibility that bacterial isolates coming from the *D. suzukii* gut could have a beneficial effect on the fly productivity or be an adequate protein source that would eventually lead in yeast replacement. Future studies should focus on dissecting the gut bacterial diversity of *D. suzukii* and employ the most abundant inhabitants as candidates for the yeast replacement. In addition, cost estimation studies should also be performed for the promising candidates to elucidate their potential as core larval diet components in mass-rearing facilities.

**Author Contributions:** Conceptualization, K.N., C.C. and K.B.; Data curation, K.N. and F.S.; Formal analysis, K.N. and F.S.; Funding acquisition, C.S.; Investigation, K.N. and F.S.; Methodology, K.N.; Resources, S.N.; Supervision, C.S., C.C. and K.B.; Validation, K.N. and F.S.; Writing—original draft, K.N. and F.S.; Writing—review & editing, K.N., F.S., S.N., C.S., C.C. and K.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture. This research was also funded by the Austrian Science Fund (FWF): I 2604-B25.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the authors.

**Acknowledgments:** The authors would like to thank Rui Pereira and Marc Vreysen for their support throughout this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Effect of Cold Storage on the Quality of** *Psyttalia incisi* **(Hymenoptera: Braconidae), a Larval Parasitoid of** *Bactrocera dorsalis* **(Diptera: Tephritidae)**

**Jia Lin 1,2,3, Deqing Yang 1,2,3, Xuxing Hao 1,2,3, Pumo Cai 1,2,3,4, Yaqing Guo 1,2,3, Shuang Shi 1,2,3, Changming Liu 1,2,3 and Qinge Ji 1,2,3,\***


**Simple Summary:** Biological control programs primarily rely on the mass-release of high-quality bioagents in order to successfully suppress pests. However, producing such bioagents on a large scale and within a short timeframe or in a single step is extremely difficult. Therefore, it is important to consider methods that could increase the shelf life and help to synchronize the release schedule of bioagents reared in different batches. In the present study, we determined the effects of various cold storage protocols on the emergence and quality of *Psyttalia incisi*, a larval parasitoid of *Bactrocera dorsalis*. Our results indicated that there were no negative impacts on the emergence parameters and adult quality when late-age *P. incisi* pupae were stored at 13 ◦C for 10 or 15 d. This information is valuable in facilitating the mass-rearing of *P. incisi* and helping to improve the efficiency of biological control programs using *P. incisi* against *B. dorsalis.*

**Abstract:** *Psyttalia incisi* (Silvestri) is the dominant parasitoid against *Bactrocera dorsalis* (Hendel) in fruit-producing regions of southern China. Prior to a large-scale release, it is important to generate a sufficient stockpile of *P. incisi* whilst considering how best to maintain their quality and performance; cold storage is an ideal method to achieve these aims. In this study, the impacts of temperature and storage duration on the developmental parameters of *P. incisi* pupae at different age intervals were assessed. Then, four of the cold storage protocols were chosen for further evaluating their impacts on the quality parameters of post-storage adults. Results showed that the emergence rate of *P. incisi* was significantly affected by storage temperature, storage duration, and pupal age interval and their interactions. However, when late-age *P. incisi* pupae developed at a temperature of 13 ◦C for 10 or 15 d, no undesirable impacts on dry weight, flight ability, longevity, reproduction parameters of post-storage adults, emergence rate, or the female proportion of progeny were recorded. Our findings demonstrate that cold storage has the potential for enhancing the flexibility and effectiveness of the large-scale production and application of *P. incisi.*

**Keywords:** *Psyttalia incisi*; oriental fruit fly; cold storage; emergence rate; quality; reproduction

#### **1. Introduction**

*Batrocera dorsalis* (Hendel) (Diptera: Tephritidae) is a notorious pest of economic importance, largely due to its traits of polyphagia, superior dispersal ability, outstanding climate adaptability, and high fecundity [1]. Over the last two decades, this fly has spread into many tropical and subtropical regions due to both human transportation and adult fly

**Citation:** Lin, J.; Yang, D.; Hao, X.; Cai, P.; Guo, Y.; Shi, S.; Liu, C.; Ji, Q. Effect of Cold Storage on the Quality of *Psyttalia incisi* (Hymenoptera: Braconidae), a Larval Parasitoid of *Bactrocera dorsalis* (Diptera: Tephritidae). *Insects* **2021**, *12*, 558. https://doi.org/10.3390/insects 12060558

Academic Editor: Man P. Huynh

Received: 11 May 2021 Accepted: 4 June 2021 Published: 16 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

migration, causing considerable damage to commercial fruits and horticultural products as well as the associated import and export trade [2,3]. At present, the primary strategy for suppressing this pest involves spraying chemical insecticides, either alone or in combination with food-based lures [4]. However, there are numerous problems associated with this strategy, including insecticide resistance, environmental depredation, and effects on food safety, which has led to pressure for an alternative strategy to be developed [5,6]. Biological control, which has the advantages of being long-lasting and environmentally friendly, has recently gained much attention and is regarded as the prime alternative tactic against *B. dorsalis* populations [7].

*Psyttalis incisi* (Silvestri) (Hymenoptera: Braconidae: Opiinae) is a solitary opiine endoparasitoid, whose preferential host is the early larval instar of *B. dorsalis* [8]. It has been reported that *P. incisi* exist naturally and occupy a dominant proportion (77.6%) of the parasitic wasp population against *B. dorsalis* in fields of Zhangzhou City, Fujian Province, China, and contributed a limited reduction in *B. dorsalis* populations [9]. Hence, *P. incisi* is a highly suitable bioagent for biological control programs against *B. dorsalis* in that region of China. However, in order for such programs to be effective, millions of parasitoids need to be first produced and then transported into the affected areas. In order to improve the ease at which this can be achieved, it is important to consider methods by which the shelf life of *P. incisi* can be increased. This will help to ensure a sufficient stockpile of parasitoids and allow releases to be appropriately timed given their physiological characteristics.

Exposing bioagents to low temperatures can extend their developmental duration and is particularly valuable for the inundative biological control program which entailed a large number of biocontrol agents [10]. However, keeping parasitoids at a sub-ambient temperature can result in cold injuries and excessive consumption of energy reserves, resulting in undesirable effects on the quality and quantity of post-storage parasitoids [11]. Hence, to minimize losses in the performance of parasitoids after undergoing storage, packaging, and shipment, recent research has focused on optimizing the tradeoffs between quality and the cold storage protocol [12–14]. For braconid parasitoids against tephritid pests, the impacts of cold storage have been evaluated in several species, such as *Psyttalia humilis* (Silvestri), *Psyttalia ponerophaga* (Silvestri), *Fopius arisanus* (Sonan), and *Diachasmimorpha longicaudata* (Ashmead) (Hymenoptera: Braconidae); these works have provided a lot of valuable information for facilitating the practical application of biological control programs [15–17]. However, to date, cold storage has never been investigated as an auxiliary approach for the large-scale rearing and release of *P. incisi*. Owing to the vital role of *P. incisi* in the management of *B. doralis*, it is crucial to optimize the flexibility and effectiveness of large-scale production and mass-release programs of *P. incisi* through cold storage techniques.

In the present study, we first determined the effects of various storage temperatures (4, 7, 10, and 13 ◦C) and exposure durations (10, 15, 20, and 25 days) on the emergence rate of *P. incisi* at different pupal age intervals within parasitized *B. dorsalis* puparia. Further experiments were then carried out to examine the dry weight, flight ability, longevity, and reproduction parameters of parental *P. incisi* (G1) along with emergence rates, and the female proportion of progeny (G2).

#### **2. Materials and Methods**

#### *2.1. Insect Colonies*

*Bactrocera dorsalis* and *P. incisi* were initially collected from orchards in and around Fujian institutes of Tropical Crops (117◦30 58.95 E, 24◦37 31.37 N, and 26 m altitude), in Zhangzhou City, Fujian Province, China in 2004. These orchards are composed of 'Pearl' guava (*Psidium guajava* L.), carambola (*Averrhoa carambola* L.), and wax apple (*Syzygium samarangense*), the annual average temperature of this area was 21 ◦C and annual average rainfall was 1603 mm [9]. *Bactrocera dorsalis* and *P. incisi* were then identified in the laboratory [18], and these vouchers were deposited in the UN (China) Center for Fruit Fly Prevention and Treatment, Fujian Agriculture and Forestry University. *Bactrocera dorsalis*

were permitted to oviposit in a plastic bottle that was neatly pierced with holes; eggs were collected and transferred to a tray containing a mill feed diet for larval development, prepared in accordance with Chang et al. [19]. Puparia were collected from the bottom of the tray and transferred into a gauze cage (30 × <sup>30</sup> × 30 cm3) until emergence. Fly adults were provided with a diet of yeast extract and sugar (1:3, wt:wt) and water. For the rearing of *P. incisi*, excessive numbers of second-instar *B. dorsalis* larvae were transferred to an oviposition plate (diameter: 9 cm, high: 0.5 cm) which was covered with 80 mesh net to prevent larvae from escaping. Then, two oviposition plates were provided to 500 pairs of *P. incisi* adults within a cage for 24 h to avoid super-parasitism. Honey and water were provided for *P. incisi* adults. *Bactrocera dorsalis* and *P. incisi* used in the experiments were reared under the controlled conditions of 25 ± 1 ◦C, 65 ± 5% relative humidity (RH), and a 12:12 h (L:D) photoperiod.

#### *2.2. Effects of Storage Temperature, Storage Duration and Pupal Age Interval on the Emergence Parameters of P. incisi*

Newly-formed *B. dorsalis* puparia were collected daily, with parasitized and unparasitized puparia distinguished by several characteristics as described in Wang and Messing [20] and Danne et al. [15]. Briefly, for parasitized puparia, the major characteristics included oviposition scars on the cuticle, a gap inside the fly pupa caused by consumption of the fly body by a parasitoid larva, and being relatively smaller and browner. For unparasitized puparia, the appendages of the fly could be clearly observed through the cuticle under a microscope.

Based on a preliminary experiment that identified the immature stage of *P. incisi* (through dissecting puparia), parasitized *B. dorsalis* puparia were incubated at 25 ◦C for 3, 6, and 9 days for *P. incisi* to develop into prepupae, middle-age pupae (some appendages, the shape of abdomen and thorax are observable but without any color; eye and ocelli are rufous), and late-age pupae (body is tawny, eye and ocelli are dark, mouth is dark brown), respectively. The three developmental stages were subsequently stored in four incubators (PRX-25013, Safu, Ningbo, China) set at constant low temperatures (4, 7, 10, and 13 ◦C, respectively) for four durations (10, 15, 20, and 25 d), all at 75% RH. A control group comprised parasitized puparia that developed in an incubator set at 25 ◦C and 75% RH until emergence. For each treatment and control, Petri dishes contained 30 parasitized puparia each were prepared. To ensure a ventilated condition to avoid the outbreak of pathogens, gauze was used to cover the dishes. Dishes were randomly assigned to the various treatment groups and inspections of each incubator were performed daily (for 30 s) to observe whether *P. incisi* adults had emerged during the cold exposure period. After cold exposure, treatments were held under control conditions and the number and sex of emerged *P. incisi* adults were recorded daily. All unemerged puparia of each treatment were dissected 5 days after the last *P. incisi* emerged or 15 days after cold treatment protocol (for those treatments without *P. incisi* emerged). A total of nine replicates were performed for this experiment.

#### *2.3. Effects of Pupal Cold Storage on Quality of P. incisi Adults*

To determine the effects of cold exposure on the G1 quality and G2 emergence parameters, four pupal cold storage protocols that did not have significant negative impacts on the emergence rate of *P. incisi* were selected for further experiments based on the results of Experiment 2.2. After the cold storage protocol, this series of bioassays were conducted under controlled conditions of 25 ± 1 ◦C and 65 ± 5% RH.

The four cold storage protocols were as follows:

Cold storage 1 (CS1): late-age *P. incisi* pupa (9-day-old parasitized *B. dorsalis* puparia) stored at 13 ◦C for 10 days;

Cold storage 2 (CS2): middle-age *P. incisi* pupa (6-day-old parasitized *B. dorsalis* puparia) stored at 13 ◦C for 10 days;

Cold storage 3 (CS3): late-age *P. incisi* pupa (9-day-old parasitized *B. dorsalis* puparia) stored at 13 ◦C for 15 days;

Cold storage 4 (CS4): middle-age *P. incisi* pupa (6-day-old parasitized *B. dorsalis* puparia) stored at 13 ◦C for 15 days.

#### 2.3.1. Dry Weight

Parasitized puparia were transferred to a plastic bowl within a gauze cage. Parasitoids that had emerged within 24 h without foraging any food were used in this bioassay. For each treatment, 1 group of 50 females and 1 group of 50 males were respectively placed inside a total of 6 centrifuge tubes. All adults were kept at –20 ◦C for 20 min and were then dried in an oven at 60 ◦C for 48 h. Subsequently, the gross dry weight of a cohort of 50 females or males from each group was measured using a semimicro balance (CP225D, accuracy of 0.01 mg, Sartorius, Göttingen, Germany). Nine replicates were conducted in this experiment.

#### 2.3.2. Flight Capacity

Fifty newly-emerged (<24 h) females and 50 newly-emerged males were caught using 5 mL centrifuge tubes. These centrifuge tubes were held at 4 ◦C for 10 s to immobilize the adult wasps. Subsequently, two tubes each of females and males were placed into a hollow black cylinder that had been placed in the center of a cage and the lids of these tubes were opened and no longer closed to allow them to escape. Talc powder was uniformly daubed around the interior of the black cylinder to ensure that adults could not climb out. To provide illumination, a 30 W fluorescent light was directed at the top of the cage from a distance of 20 cm. The collection of fliers was performed every 12 h. The experiment was performed until all parasitoid wasps were dead. The flight capacity was calculated as (the total number of *P. incisi* flew out the hollow black cylinder / the total number of *P. incisi* used) × 100%. For each treatment, nine replicates of this experiment were conducted.

#### 2.3.3. Longevity and Reproductive Parameters

Pairs of *P. incisi* adults that had emerged within 24 h were confined in individual centrifuge tubes to observe mating behavior; this ensured that all adults used in this experiment were mated. Subsequently, each pair of *P. incisi* adults were transferred into a plastic jar (diameter: 15 cm, high: 10 cm) and the top was wrapped with gauze for ventilation. Honey and water were provided: honey was daubed on the gauze and water was absorbed in a sponge inside the plastic jar. Excessive numbers of second-instar *B. dorsalis* larvae were transferred to a small oviposition plate (diameter: 3.5 cm, high: 0.5 cm) that was covered with 80 mesh net, and then a small oviposition plate was provided for *P. incisi* adults to parasitize for 24 h. The larvae were refreshed daily until the female had died. After parasitism, larvae were reared on artificial diets until pupation. Larvae and pupae that died during development were removed for further observation and dissection under a microscope to determine whether they had been parasitized. The number of both sexes of the progeny of each treatment and control were documented daily. The pre-oviposition period, oviposition period, post-oviposition period, longevity of G1, and emergence rate as well as the female proportion of G2 were recorded for each treatment and control. A total of 15 replicates were performed for each treatment.

#### *2.4. Statistical Analysis*

All data were analyzed after checking that the data were normally distributed and there was homogeneity of variances (SPSS Inc., Chicago, IL, USA). Percentage data were arcsine square-root-transformed for further statistical analysis; however, untransformed data are presented in tables. The effects of storage temperature, storage duration, pupal age interval, and their interactions on emergence and proportion of female G1 were analyzed by univariate three-way ANOVA (generalized linear model, GLM). One-way ANOVA was conducted to analyze the effects of the pupal cold storage protocol on dry weight, flight ability, longevity, and reproduction parameters of G1 *P. incisi* adults and the emergence rate and female proportion of G2. Differences between treatments and control were assessed using a one-way ANOVA with Tukey's honestly significant difference (HSD) test (*p* < 0.05) for multiple mean comparisons.

#### **3. Results**

#### *3.1. Effects of PupalCold Storage on the Emergence Parameters of P. incisi* 3.1.1. Emergence Rate

A significant difference was observed in the emergence rate of *P. incisi* among storage temperature (*F*3, 432 = 1286.705, *p* < 0.001), storage duration (*F*3, 432 = 426.532, *p* < 0.001), pupal age interval (*F*2, 432 = 196.2910, *p* < 0.001), storage temperature × storage duration (*F*9, 432 = 12.342, *p* < 0.001), storage temperature ×pupal age interval (*F*6, 432 = 68.522, *p* < 0.001), storage duration × pupal age interval (*F*6, 432 = 6.573, *p* < 0.001), and their interactions (*F*18, 432 = 3.881, *p* < 0.001).

Overall, the emergence rate of *P. incisi* pupae of the same age decreased as temperature decreased and storage duration increased. Regardless of pupal age interval and storage duration, *P. incisi* pupae stored at 4, 7, and 10 ◦C exhibited significantly lower emergence rates compared to the control group. However, no significant differences were observed for middle-age *P. incisi* pupae stored at 13 ◦C for 10 days (*p* = 1.000) and 15 days (*p* = 0.343) as well as late-age *P. incisi* pupae stored at 13 ◦C for 10 days (*p* = 1.000) and 15 days (*p* = 0.779) in comparison with the control group. Surprisingly, after being subjected to the same cold storage protocol of 4, 7, and 10 ◦C, middle-age *P. incisi* pupae presented the highest emergence rate compared to the prepupae and late-age pupae (Table 1).

**Table 1.** Emergence rate of *P. incisi* after being subjected to different pupal cold storage treatments (*n* = 9).


Note: Data are presented as mean ± SE. Different lowercase letters indicate significant differences at the 0.05 level by Tukey's test. Control means *P. incisi* pupae developed at 25 ◦C. <sup>E</sup> means that part of *P. incisi* have emerged during the cold storage protocol.

#### 3.1.2. Female Proportion

The proportion of females was not affected by storage temperature (*F*3, 253 = 0.469, *p* = 0.704), storage duration (*F*3, 253 = 0.698, *p* = 0.554), pupal age interval (*F*2, 253 = 0.027, *p* = 0.973), storage temperature × storage duration (*F*6, 253 = 0.490, *p* = 0.816), storage

temperature × pupal age interval (*F*5, 253 = 0.093, *p* = 0.993), storage duration × pupal age interval (*F*6, 253 = 0.087, *p* = 0.998), or their interactions (*F*3, 253 = 0.069, *p* = 0.976). There was no significant difference between the control group and all treatments (Table 2).

**Table 2.** Proportion of emerging *P. incisi* females after being subjected to different pupal cold storage treatments (*n* = 9).


Note: Data are presented as mean ± SE. Different lowercase letters indicate significant differences at the 0.05 level by Tukey's test. Control means *P. incisi* pupae developed at 25 ◦C. <sup>E</sup> means that part of *P. incisi* have emerged during the cold storage protocol. "-" means that the emergence rate was less than 5% after pupal cold storage, and therefore excluded from the analysis.

#### *3.2. Effects of Pupal Cold Storage on the Quality of P. incisi Adults* 3.2.1. Dry Weight

Pupal cold storage lead to significant impacts on the dry weight of both sexes of post-storage *P. incisi* adults according to one-way ANOVA (female: *F*4, 40 = 36.795, *p* < 0.001; male: *F*4, 40 = 22.323, *p* < 0.001). Furthermore, based on the result of Tukey's HSD test, except for the cold storage 4 (CS4) protocol (female: *p* < 0.05; male: *p* < 0.05), all other pupal cold storage treatments did not differ from the control for the dry weight of both sex adults (Table 3).

#### 3.2.2. Flight Capacity

The flight capacity of post-storage *P. incisi* adults was significantly affected by the cold storage protocol (female: *F*4, 40 = 3.520, *p* < 0.05; male: *F*4, 40 = 2.926, *p* < 0.05). CS4 lead to the lowest flight capacity of both sex adults and was significantly inferior to the control (female: *p* < 0.05; male: *p* < 0.05) (Table 3).


**Table 3.** The dry weight of G1 *P. incisi* post-storage females (a) and males (b) that had been subjected to different pupal cold storage treatments (*n* = 9).

#### 3.2.3. Longevity

For female parasitoids, pupal cold storage had significant effects on longevity (female: *F*4, 70 = 3.795, *p* < 0.01), and a remarkable reduction in female longevity was observed for CS4 (*p* < 0.05). However, the longevity of males was not significantly influenced by different treatments (*F*4, 70 = 1.669, *p* = 0.167) (Figures 1 and 2).

#### 3.2.4. G1 Reproductive Performance and G2 Emergence Parameters

Pre-oviposition period (*F*4, 70 = 0.353, *p* = 0.841), post-oviposition period (*F*4, 70 = 2.128, *p* = 0.086), G2 emergence rate (*F*4, 70 = 0.412, *p* = 0.799), and G2 female proportion (*F*4, 70 = 1.864, *p* = 0.127) were not significantly affected by pupal cold storage. However, significant effects were observed for total offspring per female (*F*4, 70 = 15.245, *p* < 0.001), daily offspring (*F*4, 70 = 3.625, *p* < 0.05), and oviposition period (*F*4, 70 = 3.893, *p* < 0.001). The highest total offspring produced by a female was in the control group, and there was a significant difference between the control in comparison to females subjected to CS2 (*p* < 0.05) and CS4 (*p* < 0.01). Furthermore, CS4 resulted in significantly lower daily offspring (*p* < 0.05) and a shorter oviposition period (*p* < 0.05) than the control (Tables 4 and 5).

**Figure 1.** Longevity of G1 *P. incisi* post-storage females (**a**) and males (**b**) that had been subjected to different pupal cold storage treatments. CS1: cold storage 1, late-age pupae stored at 13 ◦C for 10 d; CS2: cold storage 2, middle-age pupae stored at 13 ◦C for 10 d; CS3: cold storage 3, late-age late pupae stored at 13 ◦C for 15 d; CS4: cold storage 4, middle-age pupae stored at 13 ◦C for 15 d. Control means *P. incisi* pupae developed at 25 ◦C. Bars topped with the same letter do not differ significantly (*p* > 0.05) according to Tukey's HSD test (one-way ANOVA) (*n* = 15).

Note: CS1: Cold storage 1, late-age pupae stored at 13 ◦C for 10 d; CS2: cold storage 2, middle-age pupae stored at 13 ◦C for 10 d; CS3: cold storage 3, late-age late pupae stored at 13 ◦C for 15 d; CS4: cold storage 4, middle-age pupae stored at 13 ◦C for 15 d. Control means *P. incisi* pupae developed at 25 ◦C. Bars topped by the same letter do not differ significantly (*p* > 0.05) according to Tukey's HSD test (one-way ANOVA).

**Figure 2.** Survival curves of *P. incisi* post-storage female (**a**) and male (**b**) that had been subjected to different pupal cold storage treatments. CS1: cold storage 1, late-age pupae stored at 13 ◦C for 10 d; CS2: cold storage 2, middle-age pupae stored at 13 ◦C for 10 d; CS3: cold storage 3, late-age late pupae stored at 13 ◦C for 15 d; CS4: cold storage 4, middle-age pupae stored at 13 ◦C for 15 d. Control means *P. incisi* pupae developed at 25 ◦C (*n* = 15).

**Table 4.** Reproductive parameters of G1 *P. incisi* post-storage adults (*n* = 15).


Note: Data are presented as mean ± SE. Different lowercase letters indicate significant differences at the 0.05 level by Tukey's test. CS1: cold storage 1, late-age pupae stored at 13 ◦C for 10 d; CS2: cold storage 2, middle-age pupae stored at 13 ◦C for 10 d; CS3: cold storage 3, late-age late pupae stored at 13 ◦C for 15 d; CS4: cold storage 4, middle-age pupae stored at 13 ◦C for 15 d. Control means *P. incisi* pupae developed at 25 ◦C.

**Table 5.** Emergence rate and female proportion of progeny (G2) (*n* = 15).


Note: Data are presented as mean ± SE. Different lowercase letters indicate significant differences at the 0.05 level by Tukey's test. CS1: cold storage 1, late-age pupae stored at 13 ◦C for 10 d; CS2: cold storage 2, middle-age pupae stored at 13 ◦C for 10 d; CS3: cold storage 3, late-age late pupae stored at 13 ◦C for 15 d; CS4: cold storage 4, middle-age pupae stored at 13 ◦C for 15 d. Control means *P. incisi* pupae developed at 25 ◦C.

#### **4. Discussion**

The utilization of opiine parasitoids, especially *F. arisanu*, has successfully alleviated the serious damage of *B. dorsalis* to fruit products in Hawaii, which inspired other regions to adopt a biological control program for managing this pest [21]. However, today, *F. arisanu* has only been recorded in Zhanjiang City, Guangdong Province in China [22], which means that *F. arisanu* may not be able to be the dominant bioagent against *B. dorsalis* in some regions of China. Furthermore, considering the various ecological conditions in different regions of mainland China, it is indispensable to dig local parasitoid resources to develop the most suitable local biological control programs against *B. dorsalis*. Previous research in Zhangzhou City, Fujian Province, China, indicated that there were four parasitic wasp species against *B. dorsalis*, including *P. incisi, Pachycrepoideus vindemmiae* (Rondani), *Pachycrepoideus vindemmiae* (Rondani), and *Spalangia endius* (Walker) (Hymenoptera: Pteromalidae), of which P. *incisi* occupied a dominant proportion (77.6%) of this parasitic wasp population [9]. Therefore, P. *incisi* is a highly suitable bioagent for biological control programs against *B. dorsalis* in that region of China.

The present study is the first, to our knowledge, that has aimed to optimize the cold storage protocol of *P. incisi* to improve the efficiency and flexibility of biological control programs against *B. dorsalis*. Our results demonstrate that storage temperature, storage duration, pupal age interval, storage temperature × storage duration, storage temperature ×pupal age interval, and storage duration × pupal age interval and their interactions have significant impacts on the viability of immature *P. incisi*. However, when late-age *P. incisi* pupae were subjected to CS1 and CS3, the emergence parameters of both G1 and G2 progeny, and G1 quality parameters (including flight ability, dry weight, longevity, and reproduction parameters) did not differ significantly from the control treatment.

The essence of cold storage is to utilize a sub-optimum temperature to prolong the developmental time of a bioagent whilst maintaining its quality and effectiveness against a pest [23]. Cold storage protocols are extremely valuable for inundative biological control, given that they require large-scale production and the release of huge numbers of bioagents. However, modulating the development of bioagents via cold storage can pose lethal and sub-lethal effects to their survivorship [24,25]. In the present study, a reduction in the storage temperature or an extension of the storage period resulted in a decrease in the emergence rate of *P. incisi*. This is consistent with previous research on the cold storage of two other braconid parasitoids that are used against *B. dorsalis*, namely *F. arisanus* and *D. longicaudata*; these studies found that the viability of parasitized pupae was gradually reduced as the temperature decreased or the storage period was extended [16,17]. In addition, we interestingly found that middle-age *P. incisi* pupae exhibited superior performance in the emergence rate in comparison to prepupae and late-age pupae subjected to the same cold storage protocols of 4, 7, or 10 ◦C. Similarly, a study on *Encarsia formosa* (Gahan) (Hymenoptera: Aphelinidae) indicated that both early- and late-stage pupae exhibited inferior tolerance to low temperatures in comparison with mid-stage pupae, with the mid-stage pupae exhibiting higher survival rates [26].

Although cold storage clearly has advantages in enhancing the efficiency and flexibility of mass-rearing and release programs, the undesirable effects on the quality of post-storage insects are of concern. A significant amount of research has emphasized that subjecting bioagents to cold conditions can result in cold stress and excessive consumption of energy reserves, thereby influencing their quality parameters and their successful application under field conditions [11,27]. Our results demonstrated that the cold storage of *P. incisi* pupae was deleterious to the dry weight of post-storage adults. Lins et al. reported that the body mass loss was directly proportional to the extension in the storage period when prepupae of *Praon volucre* (Haliday) (Hymenoptera: Braconidae) were stored at 5 ◦C [28]. These weight losses during cold storage may chiefly result from the immature parasitoid wasps consuming a body of energy and lipid reserves to ensure survival during the period of low temperature. Excessive lipid loss is fatal to parasitoid adults as they cannot synthesize lipids by themselves, and thereby perform a trade-off between survivorship and fecundity, in turn affecting their lifespan [29–31]. In our study, the longevity of female *P. incisi* that emerged from middle-age pupae stored at 13 ◦C for 15 days was significantly shorter. This is in accordance with results found in *D. longicaudata* [17], which revealed that females that emerged from parasitized *B. doralis* pupae stored below 8 ◦C had a shorter lifespan than the control, regardless of the storage period. Likewise, the cold storage of other braconid wasps, such as *Aphidius ervi* (Haliday), *Aphidius picipes* (Nees), and *Bracon hebetor* (Say) (Hymenoptera: Braconidae), during the pupal stage, indicated a similar tendency in the longevity of post-storage adults [32–34].

Dispersibility, one of the most important quality parameters for parasitoids when considering the various, complex, and harsh field conditions they need to withstand whilst host-seeking, hiding from predators, and searching for resting places, is particularly vulnerable to the effects of low-temperature storage. In fact, the neuro-muscular dysfunction that can be induced by cold storage directly affects the bioagent's ability to disperse, and is a major obstacle to the practical application of post-storage insects in the field [10,35]. In concordance with this, our results demonstrated that subjecting middle-age *P. incisi* pupae to 15 days of cold storage at 13 ◦C resulted in the emerged adults displaying inferior flight ability. A study on *E. formosa* and *Encarsia eremicus* (Rose) (Hymenoptera: Aphelinidae) indicated that increasing the cold exposure period of pupae resulted in a reduction in the flight capacity of the emerged adults [26]. Similarly, *P. volucre* pre-pupae developed at a sub-ambient temperature of 5 ◦C exhibited lower flight capacity than the control group [28].

In addition to dispersibility, the reproductive system of bioagents is extremely susceptible to sub-optimum conditions. In our study, for middle-age *P. insici* pupae subjected to cold storage at 13 ◦C for 10 or 15 days, the number of progeny produced by post-storage adults was remarkably decreased. This is consistent with previous research on braconid parasitoids, whereby a reduction in fecundity was strongly associated with low temperature and storage period [28,33,36]. Furthermore, the reproductive parameters of *P. incisi* that originated from the Zhangzhou region showed a significant difference to the Hawaii *P. incisi* strain [37]. Liang et al. [38] indicated that there was a certain difference between two geographic *P. incisi* populations by using random amplified polymorphic DNA analysis, this may account for the remarkable difference in reproductive parameters between these two strains.

The viability of mass-release biocontrol programs is largely determined by the ability to generate sufficient reserves of the bioagent in a relatively short timeframe. Furthermore, flexibility is also required in both the rearing and release schedules to deal with unforeseen factors such as adverse weather and transportation delays. As such, the cold storage technique can help mitigate these problems [39] and is increasingly undertaken as a support approach in classical biological control programs [11]. Nonetheless, the adverse effects induced by sub-ambient temperature remarkably reduce the performance of post-storage bioagents. This consequently leads to challenges in balancing the logistics of the release program with field performance. In order to minimize the losses in quantity and fitness, recent studies have primarily concentrated on optimizing the cold storage protocol of the bioagent. As such, our present study aims to provide a foundation for optimizing the cold storage technique of *P. incisi* and our results demonstrate that *P. incisi* pupae subjected to CS1 and CS3 does not result in significant adverse effects on the emergence rate and quality of post-storage adults. Such information is vital for the mass production and release of *P. incisi* as a dominant biological control agent against *B. dorsalis* in that region of China. In addition, in this study, we used microscopic examination to distinguish parasitized puparia and unparasitized puparia to facilitate scientific research. However, for the mass storage of *P. incisi* pupae for release, we still propose using physical approaches, such as utilizing the suitable mesh of net that prevents flies from escaping, while without restricting the emerged *P. incisi*. Further studies will be carried out to assess the potential control efficacy of post-storage *P. insici* against the *B. dorsalis* population under field conditions.

**Author Contributions:** Methodology, J.L., C.L., and Q.J.; performed the experiments, J.L., D.Y., X.H., Y.G., and S.S.; writing—original draft preparation, J.L. and P.C.; writing—review and editing, J.L. and P.C., supervision, C.L. and Q.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Industry University Research Project of Fujian Science and Technology Department (2019N5003) and the National Key R&D Program of China (2017YFD0202000).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available from the corresponding author on reasonable request.

**Acknowledgments:** We extend our sincerest appreciation to Shumei Wang for giving us critical suggestions regarding this research.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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