Wasps in the genus *Trichogramma* are probably the most commonly mass-produced and released parasitoids of insects, primarily because of their ability to parasitize a wide range of Lepidoptera. *Trichogramma* spp. have been used for more than 100 yr as biological control agents of lepidopteran pests ( [@CIT0027], [@CIT0042], [@CIT0025], [@CIT0007], [@CIT0045]). During this time, *Trichogramma* spp. have been released against at least 28 pest species on 20 crops, including cereals, vegetables, and trees, in more than 50 countries and on more than 32 million ha each year ([@CIT0014], [@CIT0022], [@CIT0039], [@CIT0038], [@CIT0047]). There are more than 200 species of *Trichogramma*, but only 19 have been mass-reared and used effectively for biological control ([@CIT0007]). Substantial production of *Trichogramma* spp. has occurred in France, the Netherlands, Switzerland, Germany, the United States, Canada, Mexico, China, and Russia, with lesser facilities in South and Central America, Australia, southern and eastern Europe, South Africa, India, and southern Asia ([@CIT0039], [@CIT0009]).

The most common host eggs for rearing different *Trichogramma* spp. are from the Mediterranean flour moth, *Ephestia kuehniella* (Zeller), often referred to as *Anagasta kuehniella* (Zeller); rice moth, *Corcyra cephalonica* (Stainton) (Lepidoptera: Pyralidae); and the Angoumois grain moth, *Sitotroga cerealella* Olivier (Lepidoptera: Gelechiidae) ([@CIT0029]). The suitability of these hosts for rearing *Trichogramma* spp. depends on many factors, including characteristics of the host eggs, ease of mass rearing, storage capability, high parasitoid production ([@CIT0040]), and cost ([@CIT0018]). Mass rearing of hosts to produce *Trichogramma* spp. tends to be expensive and labor-intensive ([@CIT0045]). However, the Mediterranean flour moth can be reared on simple and inexpensive diets, such as wheat flour and many other foods ([@CIT0041], [@CIT0034], [@CIT0028], [@CIT0013], [@CIT0006], [@CIT0024]). In an attempt to reduce the cost of host production, larvae of the rice moth have been reared on relatively inexpensive cereals, such as finger millet, *Eleusine coracana* L. Gaertn; soft white wheat, *Triticum aestivum* L.; short-grained white rice, *Oryza sativa* L.; and durra sorghum, *Sorghum bicolor* L. ([@CIT0030]). Parasitism of *C. cephalonica* eggs by *T. chilonis* did not vary due to larval diet but significantly more parasitoids survived to emerge and lived longer when the eggs were produced by moths derived from larvae fed finger millet. Since there is no yolk in the eggs of many parasitoid wasps, such as *Trichogramma* spp., the host eggs must provide all the essential nutrients for wasp embryonic and larval development ([@CIT0012]). Also, the host must have a high rate of reproduction with a short life cycle ([@CIT0011], [@CIT0029]).

The challenge for mass-producing parasitoids, such as *Trichogramma brassicae* Bezdenko, is to maintain wasp quality while reducing production resources and associated costs ([@CIT0021]). In a tritrophic system involving an artificial diet, insect host, and parasitoid, the first step is to formulate an affordable larval diet and design an efficient rearing system for the host. The host stage that is utilized for production must be acceptable to the parasitoid for oviposition, sustain larval development, and yield effective adults. Required characteristics of the parasitoid females include adequate size, fecundity, longevity, and level of parasitism ([@CIT0046]). Postproduction quality control guidelines based on these characteristics have been developed for at least 13 hymenopteran parasitoids, including three *Trichogramma* spp. ([@CIT0020]). These guidelines and field evaluations of target pest populations help to assure that the parasitoids are effective when released ([@CIT0005], [@CIT0032], [@CIT0023], [@CIT0015], [@CIT0042]). The objective of this research was to determine if increasingly less complex and expensive *E. kuehniella* larval diets could be used to produce viable adults that oviposit a high number of eggs that are acceptable for parasitism by *T. brassicae* females and produce high-quality parasitoids.

Materials and Methods {#s1}
=====================

Rearing *Ephestia kuehniella* {#s2}
-----------------------------

*Ephestia kuehniella* eggs were obtained from a colony that had been maintained for several years at the USDA, Agricultural Research Service, Center for Medical, Agricultural and Veterinary Entomology in Gainesville, FL using modified rearing methods for stored-product insects ([@CIT0010]). The 24-h-old eggs were placed in each of four 13.5 cm long × 7.5 cm wide × 9.5 cm high standard plastic rearing containers with 400 eggs and 400 g of diet in each container. The infested containers were covered with plastic mesh lids and held in a reach-in growth chamber (Percival Model I-36VL) at 26 ± 1°C, 50 ± 5% RH, and 16:8 (L:D) h. After the larvae pupated and adults emerged in the containers, 200--400 1-d-old moths were transferred to a 15 cm diameter × 13.7 cm high polypropylene funnel covered with plastic mesh over the mouth and held in the growth chamber for mating and oviposition. The first generation eggs traveled down the neck of the funnel and were collected on wax paper. After 24 h, the eggs were poured onto filter paper to separate them from the moth scales and used to establish two new colonies with the different larval diets.

The colonies were maintained on either a standard or reduced diet, and eventually a minimal larval diet ([Table 1](#T1){ref-type="table"}). The respective diets contained 10.98, 12.20, and 12.58% crude protein; 5.80, 2.29, and 1.73% lipid; and 83.77, 89.23, and 90.63% dry matter. Protein was measured by the Kjeldahl method (AOAC 920.87), lipid by the Soxhlet method (AOAC 920.39), and dry matter by the standard method (AOAC 942.05) ([@CIT0017]). The standard diet was prepared by mixing the dry and wet ingredients separately before combining them, and then the honey and glycerin were premixed and added ([@CIT0010]). The finished diet was covered with paper toweling and air-dried in the laboratory for 4 d, ground with a hammer mill, and stored for 5 d in a freezer at −18°C. A second colony was maintained on a previously used and less complex larval diet ([@CIT0008]). The reduced diet was prepared by mixing glycerin thoroughly with wheat flour and yeast. After three generations, there were no significant differences in some of the rearing parameters for the first two colonies, so a third colony was added and maintained on a minimal diet consisting of only whole wheat flour. *Ephestia kuehniella* is a major pest in flour mills ([@CIT0043]). Fresh diets were prepared for each generation and placed in the standard plastic rearing containers. Initially, separate colonies were established for the standard and reduced diets by sprinkling 100 eggs on each diet in three larval rearing containers per colony. Eggs for the minimal diet colony were derived from the third generation of the colony reared on the reduced larval diet, 100 eggs being added to each of three larval rearing containers. Larvae from the reduced diet eggs would undergo less change in diet ingredients than larvae from standard diet eggs. Thus, the standard and reduced diet colonies were reared for seven generations and the minimal diet colony was limited to four generations.

###### 

The ingredients and percentage by weight of the standard, reduced, or minimal larval diet for rearing *Ephestia kuehniella* larvae^*^a^*^

  Ingredients^*b*^       Grams   \%
  ---------------------- ------- -------
  **Standard diet**              
   Laboratory rat chow   930     9.77
   Rolled oats           382     3.98
   Toasted wheat germ    240     2.50
   Brewer's yeast        487     5.07
   White cornmeal        2,497   25.99
   Whole wheat flour     2,220   23.11
   Honey                 1,500   15.62
   Glycerol              1,350   14.06
  **Reduced diet**               
   Whole wheat flour     348     87
   Glycerol              40      10
   Brewer's yeast        12      3
  **Minimal diet**               
   Whole wheat flour     400     100

^*a*^The standard diet was developed to maintain research colonies ([@CIT0010]), as was the reduced diet ([@CIT0008]). The whole wheat flour diet was considered minimal but adequate because *E. kuehniella* is a major pest in flour mills ([@CIT0043]).

^*b*^Purina Rodent Laboratory Chow (<http://multipurina.ca/en/rodents/products>), Quaker Old Fashioned Oats (<http://www.quakeroats.com>), Kretschmer Toasted Wheat Germ (<http://www.kretschmer.com/wheatgerm-products>), MP Biomedicals Brewer's Yeast (<http://www.mpbio.com>), white cornmeal (<http://www.quakeroats.com>), organic whole wheat pastry flour (<http://www.bobsredmill.com>), honey (<http://www.publix.com>), Glycerol, Anhydrous, J.T.Baker (<https://www.fishersci.com>). Glycerin from Bulk Apothecary (\$1.46 per lb) was used for the cost calculations (<https://www.bulkapothecary.com/>).

Life History of *Ephestia kuehniella* {#s3}
-------------------------------------

The life history parameters determined for each of the three *E. kuehniella* colonies were as follows: immature development time (days), female pupal mass (mg), adult emergence (%), female longevity (days), eggs oviposited per female (n), mass of eggs (µg), egg length (mm), egg width (mm), and egg hatch (%). The three containers for each new larval diet colony were held in the same growth chamber as the original colony. Immature development time was measured by recording days from egg hatch to adult emergence. The mass of 10 randomly selected female pupae per container from each colony and generation was determined with an electronic balance (Mettler Toledo, AL204 Analytical Balance). Adult emergence was recorded every morning. Female and male longevity was from emergence to the last day the moth survived. To measure fecundity, 20 pairs of 1-d-old male and female pupae were transferred randomly from each colony into 20 separate 115 ml funnels (Thermo Scientific Nalgene Polypropylene Powder Funnel) with the mouth of each covered with plastic mesh. A 100 mm × 15 mm Petri dish bottom lined with wax paper was positioned under the neck of each funnel to collect the eggs. The wax paper supporting the eggs was removed and replaced daily and the less than 24-h-old eggs were poured onto filter paper and counted using a stereomicroscope (Leica Microsystems, Inc. Buffalo Grove, IL). Egg quality was assessed based on the mass, size, and percent hatch of eggs oviposited by females from the three colonies. Six samples of 100 eggs from each container and colony were weighed individually with an electronic balance (Mettler XP6 Microbalance). To measure egg length and width, a small wet brush was used to stick 20 randomly sampled eggs per colony and generation onto a piece of cellophane tape that was transferred to a microscope slide. Images were obtained with a dissecting microscope (MZ125, Leica Microsystems) fitted with a camera (KY-F70B, JVC Americas Corp., NJ) and stored using commercial software (Auto-Montage v.5.02.0096, Synoptics Ltd., United Kingdom). The percentage of eggs that hatched was recorded for 20 randomly sampled eggs from each container and colony. The *E. kuehniella* life history data for percent adult emergence and percent egg hatch was arc-sin transformed for statistical analysis and means were compared using analysis of variance (PROC GLM, SAS 9.4) and Tukey-HSD.

Rearing of *Trichogramma brassicae* {#s4}
-----------------------------------

A *T. brassicae* colony was established at the University of Florida using several shipments of parasitized *E. kuehniella* eggs provided by the Beneficial Insectary, Inc. (Redding, CA). This stock colony was maintained in the growth chamber for about 1 mo by exposing the wasps to *E. kuehniella* eggs from the standard diet colony. The host eggs were attached to 5 cm × 1 cm pieces of filter paper using 20% honey solution. Each standard egg card supporting 200--400 eggs was placed into an individual 13 mm × 100 mm glass tube and exposed to 20--40 newly emerged wasps for 6--7 h. After 4 d, the eggs became black as the parasitoids developed. The emerged host larvae were removed daily to prevent them from feeding on the remaining eggs.

Life History of *Trichogramma brassicae* {#s5}
----------------------------------------

Eggs from the second *E. kuehniella* generation reared on the standard and reduced diets were used to initiate the *T. brassicae* life history study. Individual *T. brassicae* females were exposed to 24-h-old *E. kuehniella* eggs derived from each of the two larval diet colonies to obtain data from subsequent generations. Female wasps were placed into individual 10 cm high × 1.5 cm diameter glass tubes each containing a standard egg card with 70 host eggs. The tubes were closed with cotton plugs and placed in the growth chamber. After a 24-h exposure to *T. brassicae*, the host egg cards were removed and placed into separate fresh tubes. Each *T. brassicae* female was provided with a second egg card to determine the percent parasitism of 140 eggs. The number of parasitized eggs and male and female wasps that emerged were recorded from each colony, including the minimal diet colony beginning in the fourth *E. kuehniella* generation. The size of the wasps was determined by measuring the length of the left hind tibia of 10 randomly selected females newly emerged from eggs derived from each colony. Images were obtained with the dissecting microscope used to measure the *E. kuehniella* eggs. The *T. brassicae* life history data for percent emergence and percent females was arc-sin transformed for statistical analysis and means were compared using analysis of variance (PROC GLM, SAS 9.4) and Tukey-HSD.

Results {#s6}
=======

Life History of *Ephestia kuehniella* {#s7}
-------------------------------------

Immature *E. kuehniella* reared on the standard larval diet developed significantly faster than those fed the reduced or minimal diets ([Table 2](#T2){ref-type="table"}). The development time for immatures reared on the minimal diet increased significantly during generations 6--7. The mass of female *E. kuehniella* pupae from larvae reared on the standard diet was significantly greater than the mass of pupae from the reduced diet in generations 2--3, and for both of the other two diets in generations 4--7 ([Table 2](#T2){ref-type="table"}). Additionally, for generations 4--7, female pupae from the reduced diet had a greater mass than those produced on the minimal diet. The mass of female pupae from the standard diet increased significantly in the fourth generation from 26.4 ± 0.3 to 29.0 ± 0.2 mg. The percent emergence of adult *E. kuehniella* from larvae reared on the standard, reduced, or minimal diet decreased significantly in that order for all generations. By generation 7, females from the standard diet lived for significantly fewer days than females from the reduced and minimal diets. The standard diet produced significantly more eggs per female than the reduced diet, except for the first generation. The lowest number of eggs per female was produced by the minimal diet colony. The number of eggs peaked in generation 4 before decreasing significantly by generation 7 for females from all three diets.

###### 

Life history of *Ephestia kuehniella* larvae, pupae, and adults produced using three larval diets

  Diet        Generation^*a*^                                                                                                                               
  ----------- ---------------------------------- ---------------- ----------------- ---------------- ---------------- ------------------ ------------------ ------
              Immature development time (days)                                                                                                              
  Standard    39.3 ± 0.1bAB                      39.0 ± 0.3bABC   39.8 ± 0.1bA      39.5 ± 0.2cAB    38.9 ± 0.3cBC    38.2 ± 0.0cC       39.3 ± 0.3cAB      6.9
  Reduced     41.7 ± 0.0aABC                     40.5 ± 0.4aC     42.0 ± 0.4aAB     42.8 ± 0.1bA     42.8 ± 0.3bA     41.6 ± 0.1bABC     41.1 ± 0.4bBC      9.5
  Minimal     ---                                ---              ---               51.1 ± 0.6aB     49.9 ± 0.1aB     53.5 ± 0.2aA       53.3 ± 0.1aA       28.7
  *F*-value   455.9                              11.1             39.8              250.1            471.2            6217.3             812.9              
              Female pupal mass (mg)                                                                                                                        
  Standard    24.8 ± 0.2C                        25.9 ± 0.3aBC    26.4 ± 0.3aB      29.0 ± 0.2aA     29.0 ± 0.3aA     28.8 ± 0.4aA       28.9 ± 0.3aA       36.1
  Reduced     24.3 ± 0.2C                        24.2 ± 0.3 bC    25.4 ± 0.3bB      25.4 ± 0.3bB     26.6 ± 0.2bA     26.6 ± 0.3bA       26.2 ± 0.2bAB      15.8
  Minimal     ---                                ---              ---               24.1 ± 0.3cC     25.7 ± 0.2cA     24.2 ± 0.2cBC      25.1 ± 0.3cAB      7.8
  *F*-value   3.2                                17.7             6.2               80.4             41.4             51.5               50.9               
              Adult emergence (%)                                                                                                                           
  Standard    84.0 ± 1.9aAB                      82.5 ± 1.6aB     84.8 ± 1.7aAB     91.8 ± 1.5aA     86.0 ± 1.1aAB    83.0 ± 1.1aAB      87.3 ± 3.6aAB      2.7
  Reduced     73.3 ± 2.2bAB                      72.8 ± 1.7bB     73.8 ± 1.1bAB     79.0 ± 1.3bA     76.5 ± 0.7bAB    71.3 ± 0.5bB       73.5 ± 1.0bAB      3.9
  Minimal     ---                                ---              ---               68.5 ± 1.9cA     68.3 ± 2.1cA     65.8 ± 1.0cAB      61.5 ± 0.9cB       4.3
  *F*-value   14.0                               18.5             30.6              55.6             38.5             94.7               34.6               
              Female longevity (days)                                                                                                                       
  Standard    8.3 ± 0.2A                         8.0 ± 0.2AB      7.8 ± 0.2AB       7.3 ± 0.2bBC     6.9 ± 0.2bC      7.8 ± 0.1bAB       8.4 ± 0.2bA        9.1
  Reduced     8.7 ± 0.1AB                        8.6 ± 0.3AB      8.1 ± 0.3BC       8.1 ± 0.3abBC    7.5 ± 0.2bC      8.8 ± 0.3aAB       9.6 ± 0.3aA        6.2
  Minimal     ---                                ---              ---               8.9 ± 0.6aA      9.0 ± 0.4aA      8.6 ± 0.3aA        10.0 ± 0.3aA       2.2
  *F*-value   3.3                                3.3              0.6               4.0              16.3             5.0                9.1                
              Eggs per female (n)                                                                                                                           
  Standard    279.5 ± 8.3D                       295.9 ± 6.5aDC   321.2 ± 8.6aABC   351.5 ± 8.8aA    348.7 ± 8.9aAB   326.6 ± 10.0aABC   312.2 ± 10.1aBCD   8.9
  Reduced     264.5 ± 9.7AB                      265.1 ± 5.9bAB   277.3 ± 8.4bAB    287.2 ± 10.8bA   266.8 ± 6.8bAB   263.3 ± 5.2bAB     249.6 ± 10.6bB     1.9
  Minimal     ---                                ---              ---               240.9 ± 8.2cA    239.0 ± 9.0cA    225.8 ± 7.9cAB     210.9 ± 5.0cB      3.3
  *F*-value   1.4                                12.2             13.4              35.5             47.7             41.1               32.8               

^*a*^For each life history parameter, means (±SE) in columns followed by different lowercase letters and in rows followed by different capital letters are significantly different (*P* \< 0.05).

The mass of *E. kuehniella* eggs from the standard diet was significantly greater than eggs from the minimal diet but not the reduced diet in generations 4--7 ([Table 3](#T3){ref-type="table"}). The reduced diet egg mass was not different than the mass of minimal diet eggs. Eggs from the standard diet were not significantly longer than eggs from the reduced diet after generations 4 but were longer than eggs from the minimal diet. The width of eggs was equivalent for the three diets. The percentage of egg hatch did not differ significantly from standard and reduced diets for all generations; however, it was greater for the standard diet than the minimal diet in generations 4--7. Also for these generations, there was no significant difference in the percent hatch of eggs from the reduced and minimal diets. Egg mass, length, and hatch did not vary significantly within diets for all generations, but egg width was variable for the standard and reduced diets.

###### 

Characteristics of *Ephestia kuehniella* eggs produced by females from larvae reared on three diets

  Diet        Generation^*a*^                                                                                                     
  ----------- ------------------- -------------- -------------- ---------------- --------------- --------------- ---------------- ------
              Mass of eggs (µg)                                                                                                   
  Standard    26.9 ± 0.2A         26.9 ± 0.3A    27.3 ± 0.2A    27.5 ± 0.1aA     27.5 ± 0.1aA    27.1 ± 0.2aA    27.3 ± 0.2aA     1.5
  Reduced     26.4 ± 0.4A         26.4 ± 0.3A    27.0 ± 0.2A    26.9 ± 0.2 abA   26.9 ± 0.3abA   26.5 ± 0.1abA   26.8 ± 0.1 abA   1.1
  Minimal     ---                 ---            ---            26.5 ± 0.2bA     26.3 ± 0.1bA    26.1 ± 0.2bA    26.5 ± 0.2bA     1.3
  *F*-value   1.5                 1.5            1.7            6.3              9.0             8.6             8.5              
              Egg length (mm)                                                                                                     
  Standard    ---                 0.544A         0.544A         0.544aA          0.543aA         0.543aA         0.542aA          0.06
  Reduced     ---                 0.535A         0.538A         0.538abA         0.537abA        0.539abA        0.538aA          0.09
  Minimal     ---                 ---            ---            0.527bA          0.528bA         0.530bA         0.529bA          0.2
  *F*-value   ---                 1.98           1.18           4.65             4.36            3.59            5.67             
              Egg width (mm)                                                                                                      
  Standard    ---                 0.303B         0.304B         0.319A           0.312AB         0.310AB         0.315A           6.4
  Reduced     ---                 0.299C         0.308B         0.318A           0.310AB         0.300BC         0.307BC          10.0
  Minimal     ---                 ---            ---            0.312A           0.312A          0.308A          0.308A           1.5
  *F*-value   ---                 0.26           2.47           3.14             0.19            2.66            3.03             
              Egg hatch (%)                                                                                                       
  Standard    94.7 ± 1.8aA        93.7 ± 2.5aA   95.3 ± 1.6aA   96.3 ± 1.4aA     99.0 ± 1.0aA    97.0 ± 1.3aA    97.3 ± 1.2aA     1.2
  Reduced     92.0 ± 0.9aA        92.7 ± 2.2aA   93.0 ± 1.6aA   94.3 ± 1.2 abA   96.3 ± 1.7abA   95.0 ± 1.3abA   94.7 ± 1.4abA    0.98
  Minimal     ---                 ---            ---            91.7 ± 0.6bA     91.3 ± 2.4bA    92.0 ± 1.3bA    91.0 ± 1.3bA     0.08
  *F*-value   1.70                0.09           1.05           4.33             4.76            3.65            5.66             

^*a*^For each life history parameter, means (±SE) in columns followed by different lowercase letters and in rows followed by different capital letters are significantly different (*P* \< 0.05).

Life History of *Trichogramma brassicae* {#s8}
----------------------------------------

The number of *E. kuehniella* eggs from the standard diet that were parasitized by *T. brassicae* females increased significantly in generation 5 and was greater than the other two diets in generation 7 ([Table 4](#T4){ref-type="table"}; [Fig. 1](#F1){ref-type="fig"}). There was no change in the number of parasitized eggs for the reduced and minimal diets during generations 4--7. After generation 4, wasps emerged from about 95% of the parasitized eggs regardless of *E. kuehniella* larval diet. Female wasps from the standard diet eggs had a significantly longer average tibia length of 0.150 ± 0.002 mm (mean ± SE) in generation 7 compared with wasps from reduced diet eggs (0.145 ± 0.001 mm) and minimal diet eggs (0.145 ± 0.002 mm). The sex ratio of wasps from the standard diet eggs was not statistically different from wasps produced by the reduced diet eggs but the proportion of females was greater than for the minimal diet eggs. The proportion of female wasps that emerged from eggs produced by the standard, reduced, and minimal diets remained consistent within diets across generations.

###### 

Parasitism, emergence, and sex ratio of *Trichogramma brassicae* from eggs of *Ephestia kuehniella* produced by females from larvae reared on three diets

  Diet        Generation^*a*^                                                                                                                 
  ----------- --------------------------------- ----------------- ------------------ ------------------ ------------------- ----------------- ------
              Parasitized eggs per female (n)                                                                                                 
  Standard    90.70 ± 6.25D                     94.65 ± 6.01CD    106.25 ± 7.15BCD   127.15 ± 7.13aAB   121.30 ± 7.00aABC   136.20 ± 8.99aA   6.57
  Reduced     77.85 ± 6.27B                     79.95 ± 5.83B     105.25 ± 7.92A     112.05 ± 7.17abA   108.10 ± 7.61aA     101.30 ± 9.03bA   57.4
  Minimal     ---                               ---               92.35 ± 7.48A      91.40 ± 8.11bA     84.60 ± 6.98bA      96.20 ± 5.67bA    0.46
  *F*-value   2.10                              3.08              1.06               5.75               13.67               7.32              
              Emergence (%)                                                                                                                   
  Standard    92.06 ± 1.27aC                    92.94 ± 1.06aCB   97.33 ± 0.43aA     95.31 ± 0.62ABC    95.77 ± 0.56AB      95.21 ± 0.66ABC   5.43
  Reduced     80.98 ± 1.46bB                    78.26 ± 1.29bB    93.72 ± 0.79bA     94.20 ± 0.49A      93.77 ± 0.67A       95.08 ± 1.05A     57.4
  Minimal     ---                               ---               92.12 ± 1.88bA     95.12 ± 1.23A      92.05 ± 2.05A       94.45 ± 0.61A     1.05
  *F*-value   34.12                             0.56              8.68               2.40               2.96                0.60              
              Sex ratio (% female)                                                                                                            
  Standard    0.66 ± 0.03AB                     0.66 ± 0.03AB     0.79 ± 0.03aA      0.71 ± 0.04AB      0.68 ± 0.05AB       0.65 ± 0.04aAB    2.53
  Reduced     0.69 ± 0.02A                      0.70 ± 0.03A      0.70 ± 0.03abA     0.61 ± 0.03A       0.63 ± 0.03A        0.60 ± 0.03abA    2.14
  Minimal     ---                               ---               0.62 ± 0.04bA      0.66 ± 0.05A       0.57 ± 0.06A        0.50 ± 0.04bA     2.08
  *F*-value   0.53                              0.75              7.50               2.12               1.35                3.92              

^*a*^The eggs were used to initiate the indicated *E. kuehniella* generation. For each life history parameter, means (±SE) in columns followed by different lowercase letters and in rows followed by different capital letters are significantly different (*P* \< 0.05).

![Mean (±SE) number of *Ephestia kuehniella* eggs parasitized in each generation by *Trichogramma brassicae* females. The eggs were from moths reared as larvae on a standard, reduced, or minimal diet.](iez076f0001){#F1}

Discussion {#s9}
==========

*Ephestia kuehniella* were reared successfully for multiple generations on a standard, reduced, or minimal larval diet; however, the immatures developed fastest on the standard diet, the female pupae had the greatest mass, and more adults emerged. Previously, larvae reared on durum wheat flour compared to soft wheat flour also had a shorter development time and the adults produced more progeny ([@CIT0024]). This was attributed to a higher protein content and quantity of water-soluble and lipo-soluble vitamins in the durum wheat flour-based diet. Larvae of the closely related almond moth, *Cadra cautella* (Walker), also exhibited superior development on high-protein diets ([@CIT0019], [@CIT0001]). In our study, *E. kuehniella* females from the standard diet had a shorter life span, but oviposited more eggs that had a somewhat greater mass and length, and a higher percent hatch. It has been determined that larval feeding is prolonged if diets have lower protein and carbohydrate content, and the adults produce fewer eggs ([@CIT0002]). We determined that the number, mass, and length of eggs, and percent hatch, were intermediate for the reduced diet and lowest for the minimal diet. A high level of fecundity of *E. kuehniella* moths also was observed if larvae were fed a nutritionally rich diet containing 43.5% maize meal, 43.5% whole meal wheat flour, 10% glycerin, and 3.0% yeast ([@CIT0048]). An *E. kuehniella* larval diet enriched with yellow maize also supported greater fecundity ([@CIT0028]). In our study, significantly increasing the lipid in the larval diet was correlated with a higher rate of reproduction in *E. kuehniella* and their eggs were larger and more viable.

The *T. brassicae* oviposited more eggs per female in the largest eggs that were from *E. kuehniella* reared on the standard larval diet. *Trichogramma* spp. assess host eggs based not only on size but also shape, color, age, chorion thickness, nutritional content, and previous parasitism ([@CIT0037], [@CIT0003], [@CIT0036], [@CIT0031], [@CIT0035]). In our study, wasps emerged from about 95% of the parasitized eggs produced by all three larval diets but the standard diet eggs yielded a higher proportion of females. It is known that more and larger *Trichogramma* spp. wasps emerge from larger more nutritious host eggs and a greater proportion of them are females ([@CIT0004], [@CIT0026], [@CIT0033], [@CIT0012]). Thus, the mean *T. brassicae* female:male sex ratio of 69:31 could have been due to oviposition of more fertilized female eggs in the largest *E. kuehniella* eggs from the standard diet. Also, the size and number of progeny could have been affected by different nutritional or environmental conditions of the larvae or pupae. A sex ratio of 56:44 has been reported for commercial *T. brassicae* ([@CIT0016]).

The relative costs of the standard, reduced, and minimal *E. kuehniella* larval diets were based on ingredient prices in Karaj, Iran during May 2019. These prices are unrealistic for mass rearing but suitable for comparing the costs of the three experimental diets. Glycerol was considerably more expensive than all the other ingredients, so a less expensive alternative was substituted for the cost comparisons ([Table 1](#T1){ref-type="table"}). This resulted in costs of \$0.957, \$0.692, and \$0.661 for 100 g of the standard, reduced, and minimal larval diet, respectively. Larvae reared on 100 g of these diets produced 44, 37, and 31 females (50% females, [Table 2](#T2){ref-type="table"}) that oviposited a mean of 312, 250, and 211 eggs per female, respectively, yielding 13,728, 9,212, and 6,541 eggs at a cost of 0.070, 0.075, and 0.101 per 1,000 eggs (cost 100 g diet/total eggs produced by the females × 1,000). When 140 eggs from the three separate diets were exposed to *T. brassicae* females to determine their acceptance for oviposition, more of the standard diet eggs were parasitized, followed by the reduced and minimal diets ([Table 5](#T5){ref-type="table"}). After adjusting for differences in parasitism, parasitoid emergence, yield of parasitoids, and cost of host eggs, the standard diet was less expensive than the reduced and minimal diets per 1,000 parasitoids. Moreover, parasitoids from the standard diet were significantly larger and potentially would be more effective when released in the field.

###### 

Cost of 1,000 *Trichogramma brassicae* produced from *Ephestia kuehniella* eggs oviposited by females from a standard, reduced, or minimal larval diet

  Diet       Eggs parasitized/female (no.)^*a*^   Parasitoid emergence from eggs (%)   Parasitoids/140 host eggs (no.)   Parasitoids/1,000 host eggs (no.)   Cost/1,000 host eggs (\$)   Cost/1,000 parasitoids (\$)
  ---------- ------------------------------------ ------------------------------------ --------------------------------- ----------------------------------- --------------------------- -----------------------------
  Standard   136                                  95.21                                129.49                            924.93                              0.070                       0.075
  Reduced    101                                  95.08                                96.03                             685.93                              0.075                       0.109
  Minimal    96                                   94.45                                90.67                             647.64                              0.101                       0.156

^*a*^Each parasitoid female was provided with 140 host eggs over 48 h to determine the total number of eggs a female could parasitized ([Table 4](#T4){ref-type="table"}).

The objective of this research was to reduce the number of ingredients in *E. kuehniella* larval diet, and thereby cost, without decreasing the quantity or quality of eggs produced by females from larvae fed the less complex diets. The eggs had to be accepted by *T. brassicae* females for oviposition and yield high-quality wasps. Unfortunately, decreasing the dietary ingredients prolonged *E. kuehniella* larval development, lowered pupal mass, reduced adult emergence, and yielded fewer eggs per female that had less mass and were smaller. Fewer of the eggs from the simpler diets were parasitized by *T. brassicae* females than eggs from the standard diet and the diminished eggs yielded smaller wasps. Conversely, the standard *E. kuehniella* larval diet resulted in the production of more and larger eggs, and a greater number of them were parasitized by *T. brassicae* females than eggs from the reduced and minimal diets. The standard larval diet ultimately was less expensive to use because the resulting *T. brassicae* had a significantly higher rate of reproduction than wasps from the other host eggs.

The authors thank Jim Nation, Dan Hahn, and Lyle Bus, University of Florida Entomology and Nematology Department, along with Patrick De Clercq, University of Ghent Department of Plants and Crops, for guidance and Alex Gannon, University of Florida Doctor of Plant Medicine Program for technical support. Sinthya Penn, Beneficial Insectary, provided the *T. brassicae* used in this study. Partial funding was provided by a grant from the USDA, National Institute of Food and Agriculture, Crop Protection and Pest Management Competitive Grants Program (Award number 2017-70006-27149).
