Evaluation of Artemia franciscana Cysts to Improve Diets for Mass Rearing Stethorus gilvifrons, a Predator of Tetranychus turkestani

Simple Summary The ladybird beetle Stethorus gilvifrons is a native predator of spider mites in the Mediterranean region that could be mass-reared and released to control spider mite populations on crop plants. The aim of this research was to test the hypothesis that brine shrimp cysts can improve diets for mass rearing of S. gilvifrons in the absence of prey, i.e., spider mites. The diet treatments included brine shrimp cysts alone (D1), brine shrimp cysts plus a vitamin B complex (D2), brine shrimp cysts plus date palm pollen (D3), or brine shrimp cysts plus date palm pollen and Mediterranean flour moth eggs (D4). The results revealed that only two diets, D3 and D4, supported predator development to the adult stage and reproduction. The predator reproductive rate and life table estimates indicated that D4 was superior. In conclusion, only a mixed diet of brine shrimp cysts, date palm pollen, and flour moth eggs is suitable for mass rearing S. gilvifrons. Abstract Stethorus gilvifrons is an acarophagous coccinellid distributed in the Mediterranean region and could potentially be mass-reared for the augmentative biological control of Tetranychus turkestani and related species on crop plants. The hypothesis that brine shrimp Artemia franciscana cysts can improve diets for rearing of S. gilvifrons was tested in laboratory experiments. The diet treatments included A. franciscana cysts (D1), A. franciscana cysts plus a vitamin B complex (D2), A. franciscana cysts plus date palm pollen (D3), and A. franciscana cysts plus date palm pollen and Ephestia kuehniella eggs (D4). The results indicated that D1 did not support immature development. D2 supported egg–larval development but not pupal–adult development. Both D3 and D4 supported development to the adult stage and reproduction. However, D4 was the most effective diet, determined by observations of S. gilvifrons oviposition behavior and fecundity. A life table analysis corroborated these results; an intrinsic rate of increase, net and gross reproductive rates, and mean generation time were best for S. gilvifrons fed D4 rather than D3. A mixed diet composed of A. franciscana cysts, date palm pollen, and E. kuehniella eggs can be used to mass rear S. gilvifrons.

Due to the rapid development of resistance of tetranychids to acaricides [13,14], nonchemical alternative methods of controlling T. turkestani are being pursued. Biological control using specialist predators in the tribe Stethorini (Coleoptera: Coccinellidae) could be a promising alternative if mass rearing and augmentative releases of one or more species could control tetranychids, such as T. turkestani, under semi-field conditions in greenhouses or high tunnels or in open agricultural fields [15][16][17].
One promising candidate for mass rearing and augmentative releases is Stethorus gilvifrons (Mulsant) (Coleoptera: Coccinellidae). Its proclivity for attacking and consuming tetranychids, including T. turkestani immature and adult stages, has been demonstrated in previous studies [18][19][20][21]. Preliminary research to develop cost-effective methods to mass rear S. gilvifrons has provided evidence, for the first time, that S. gilvifrons can complete development and reproduce on a diet devoid of natural prey, i.e., tetranychid mites. Factitious diets containing eggs of the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) and date palm Phoenix dactylifera L. (Arecales: Arecaceae) pollen supported the development and limited reproduction of S. gilvifrons in lieu of T. turkestani [22]. In a follow-up study, S. gilvifrons was successfully reared on an artificial diet based on vertebrate protein, hen's egg yolk, and E. kuehniella eggs [23]. The addition of 2,4-dihydroxybenzioc acid, a potential oviposition stimulant, to this diet significantly improved the reproductive output [23].
Since E. kuehniella eggs are expensive, this study was initiated to determine the suitability of a less expensive factitious food, i.e., decapsulated cysts (eggs) of brine shrimp Artemia franciscana Kellogg (Anostraca: Artemiidae) for S. gilvifrons. A. franciscana cysts are two-fold less expensive to produce than E. kuehniella eggs [24]. Several aphidophagous coccinellids can develop and produce some progeny on a diet of A. franciscana cysts. For example, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae) developed to the adult stage and the females produced some progeny when reared on a diet solely of A. franciscana decapsulated cysts [25]. Yet, progeny production improved significantly when C. maculata females were fed a diet containing a fatty acid (palmitic acid) and A. franciscana eggs, which had been pulverized into a fine powder [26]. Note that diets containing a mixture of plant products, e.g., bee pollen, and A. franciscana cysts are more nutritious than A. franciscana cysts alone for rearing another coccinellid, Adalia bipunctata (L.) [27].
In this study, the hypothesis that A. franciscana cysts can improve diets for S. gilvifrons was tested. The objectives were to test the diet treatments, including A. franciscana cysts (D1), A. franciscana cysts plus a vitamin B complex (D2), A. franciscana cysts plus date palm pollen (D3), and A. franciscana cysts plus date palm pollen and E. kuehniella eggs (D4) on development and reproduction of S. gilvifrons. A life table analysis was conducted to further define which diet mixture was most nutritious for S. gilvifrons.

Stethorus gilvifrons Colony
The initial population of S. gilvifrons was originally collected from a sugarcane field (Amir-Kabir Agro-Industry Co., Ahvaz, Iran) around Khuzestan Province in Ahvaz (coordinates, 31 • 19'13"N, 48 • 40'09"E), Iran. Preadults and adults of S. gilvifrons were cultured in separate containers at controlled environmental conditions (25 ± 1 • C, 65% ± 5% RH, and 16-h photophase). The S. gilvifrons colony was maintained for several consecutive generations on factitious diets, including mixtures of A. fransciscana cysts, date palm pollen, and E. kuehniella eggs. The identity of S. gilvifrons was confirmed by H. Hodek for a previous study [22]. Voucher specimens were deposited in the insect collection of Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Preparation of Factitious Diets
The initial colony of E. kuehniella was purchased from the Golestan Mooud Insectary Company, Ahvaz, Iran and reared continuously based on the method described previously [28]. Eggs were collected daily and stored in a refrigerator at 4 • C for less than two weeks before use. Date palm pollen sheaths were purchased from a rural supplier in Ahvaz City at a cost of 3.0-3.5 USD per kg (equivalent to four sheaths). Approximately 6 g of pollen grains were contained in each kg of sheath. Pollen grains were removed from the sheaths and stored in a freezer at −18 • C before use. A vitamin B complex (BECOVIT ® ) was purchased from a drugstore in Ahvaz.
Capsulated A. franciscana cysts were originally prepared by the Aquatics Department, Faculty of Veterinary, Shahid Chamran University of Ahvaz. To decapsulate the cysts, 1 g of A. franciscana cysts was weighed and added to 1-L distilled water, and then, 10 g of sodium chloride was added to achieve a 1% salinity. Next, 3 mL of 5% sodium hypochlorite was added to solution, and the cysts were ventilated using an oxygen pump. The decapsulated A. franciscana cysts were leached and screened-out of the solution using a mesh screen (60 µ, 250 mesh), then stored in a hydrated state in a refrigerator until experimentation.
Decapsulated A. franciscana cysts were removed from the distilled water before the final preparation for experiments. The factitious diets tested in this study were as follows: A. franciscana cysts (0.5 g), A. franciscana cysts + vitamin B complex (0.5 g + 1 mL), A. franciscana cysts + date palm pollen (0.5 g + 0.04 g), and A. franciscana cysts + date palm pollen + E. kuehniella eggs (0.5 g + 0.04 g + 0.5 g). A ceramic mortar and pestle were used to mix the diet components, except E. kuehniella eggs, until well-blended. Then, the E. kuehniella eggs were gently mixed with the A. franciscana cysts/date palm pollen blend. Finally, the diet compositions were transferred to microtubes and stored in a freezer at −18 • C until experimentation.

Experimental Setup
The experiment compared the following diet treatments: A. franciscana cysts (D1), A. franciscana cysts plus a vitamin B complex (D2), A. franciscana cysts plus date palm pollen (D3), and A. franciscana cysts plus date palm pollen and Ephestia kuehniella eggs (D4). The developmental time, survival, adult body weight, adult periods (pre-and postoviposition), fecundity, sex ratio, and egg hatch of S. gilvifrons fed different diets were assessed. Using a completely randomized design, first instars of S. gilvifrons (<24 h) from the stock colony were transferred to Petri dishes (6-cm diameter, 1.6-cm length) in a cohort of 3 (n = 25) and supplied ad libitum with factitious diet at the base of each Petri dish on a metal net wadding on cotton strands. At least 30 mg of each factitious diet was always present in all Petri dish arenas. The diets were refreshed every two days; the old diet and feces were discarded. Newly emerged adults were sexed and weighed using an N-202 precise digital balance. Female and male adults from the same larval diet were paired and provided one of the diets in the individual Petri dishes (n = 15 for each treatment). Metal net wadding was utilized as an oviposition substrate in the factitious diet bioassays. Under the metal net wadding, cotton strands were used. Oviposition substrates were checked daily for eggs to determine the preoviposition period. The oviposition rate (daily fecundity) was monitored during the entire lifespan of the females. All eggs were transferred to new Petri dishes until adult emergence, and the number of male and female S. gilvifrons adults was recorded to determine sex ratio (proportion of females). The longevity of males and females was also determined. All experiments were conducted under controlled conditions (30 ± 1 • C, 65% ± 5% RH, and 16-h photophase).

Statistical Analysis
The datasets were first verified for normal distribution by the Kolmogorov-Smirnov test. Data on the immature survival and developmental time were analyzed using one-way analysis of variance (one-way ANOVA). Data on the adult body weight, preoviposition period, oviposition period, postoviposition period, adult longevity, total fecundity, sex ratio, and egg hatchability of S. gilvifrons reared on different diets were analyzed using a Student's t-test. Data on the immature survival rate and egg hatch rate were arcsinetransformed prior to analysis. Means were separated using Tukey's HSD test after the one-way ANOVA at the p ≤ 0.05 significance level [29].
As described in a previous study [23], an age-stage, two-sex life table procedure [30] was used to analyze the data in response to the development rate among the individuals and stages of development in the diet treatments. Population growth parameters such as the net reproductive rate (R o ), gross reproductive rate (GRR), intrinsic rate of natural increase (r), finite rate of increase (λ), mean generation time (T), and doubling time (DT) were calculated using the TWOSEX-MSChart program [31]. The bootstrap technique was used to determine the standard errors of the population growth parameters and multiple comparisons were made by the paired bootstrap test with 100,000 samples.

Immature Development and Survival
A diet of purely A. franciscana cysts (D1) did not support the development of S. gilvifrons. Thus, this data was not included in the statistical analysis ( Table 1). The survival rate of all immature stages combined did not differ significantly amongst the other three diets (F = 1.42, df = 2, 8; p = 0.313). The three diets in the analysis were A. franciscana cysts plus a vitamin B complex (D2), A. franciscana cysts plus date palm pollen (D3), and A. franciscana cysts plus date palm pollen and E. kuehniella eggs (D4). The development time was significantly shorter for males reared on D2 than on D3 or D4 (males: F = 3.98, df = 2, 18; p = 0.039) but not for females (F = 1.55, df = 2, 22; p = 0.234; Table 1). Note that none of the S. gilvifrons immatures reared on D2 survived to the pupal stage. The adult body weights of S. gilvifrons males and females did not differ significantly between the two diets D3 and D4 (male: t = 0.159; df = 44, p = 0.874; female: t = 0.187, df = 44, p = 0.853; Table 1).

Life Table Assessment
The diets had significant effects on several life table parameters of S. gilvifrons ( Table 3). The intrinsic rate of a natural increase (r) was greater with D4 than D3; the finite rate of increase (λ) was not affected significantly. The net reproductive rate (R o ) and gross reproductive rate (GRR) were significantly greater with D4 than D3. The mean generation time (T) was significantly less with D4 than D3, but the doubling time was unaffected by the diets.

Discussion
The failure of S. gilvifrons to complete development and reproduce when fed a diet of A. franciscana cysts with or without a vitamin B complex suggests that this diet lacked essential nutrients. However, S. gilvifrons was capable of developing to the adult stage and producing a limited number of progeny when fed a mixture of A. franciscana cysts and date palm pollen. This observation suggests that essential nutrients lacking in A. franciscana cysts are found in date palm pollen. In a recent study, S. gilvifrons completed development and produced more progeny when fed a mixture of E. kuehniella eggs plus date palm pollen rather than maize pollen or bee pollen [22]. Date palm pollen contains fatty acids, amino acids, flavonoids, saponins, sterols, and other components and has been reputed to improve fertility in humans [32]. Perhaps the content of essential nutrients, e.g., fatty acids, is greater in date palm pollen than in maize or bee pollen. More research is necessary to confirm this supposition.
The observation that a mixed diet of date palm pollen, A. franciscana cysts, and E. kueniella eggs was more nutritious than a diet with date palm pollen and A. franciscana cysts suggests that E. kuehniella provides essential nutrients not found in the other two components. The soluble protein content [25] and fatty acid content [33] are greater in E. kuehniella eggs than A. franciscana cysts, which could partially explain the results herein. Although a treatment composed of E. kueniella eggs only was not included in this study, a previous work indicated that a diet of E. kuehniella alone was less nutritious than natural prey, T. turkestani eggs, for S. gilvifrons [22].
Comparing the reproductive performance of S. gilvifrons fed optimal mixed diets in two closely rated studies versus this study suggests that the A. franciscana, E. kuehniella, and date palm pollen diet was superior. In other words, the total fecundity, net reproductive rate (R o ), and gross reproductive rate (GRR) were 59.72 eggs per female, 29.86, and 46.79, respectively, in this study. Yet, when fed a E. kuehniella, hen's egg yolk, and 2,4-DHBA diet, the S. gilvifrons total fecundity, R o , and GRR were 42.09 eggs per female, 16.30, and 32.38, respectively [23]. When fed a E. kuehniella and date palm pollen diet, the total fecundity, R o , and GRR were 40.62 eggs per female, 21.48, and 30.08, respectively [22]. A follow-up study comparing these three mixed diets in the same experimental design would confirm the superiority of the E. kuehniella, A. franciscana, and date palm pollen diet.
In terms of cost efficiency, eliminating or reducing the quantity of E. kuehniella eggs in a mixed diet for S. gilvifrons would represent significant savings. Therefore, more research to identify natural products or other factitious foods of high quality to support the reproduction of S. gilvifrons in a mass rearing system is encouraged. One possibility is the use of black soldier fly Hermetia illucens (L.) (Diptera: Stratiomyidae) larval hemolymph or larval powder as a replacement for E. kuehniella. H. illucens has a high protein (amino acid) content. Previous research suggested that larval hemolymph was a good substitute for vertebrate protein (hen's egg yolk) in an artificial diet for a phytoseiid mite, Amblyseius swirskii (Athias-Henriot), an important predator of tetranychid mites [34]. The utilization of H. illucens larval powder in diets for predatory insects or mites has not been reported in the literature.
In conclusion, this study indicated that A. franciscana cysts alone are not an adequate diet for S. gilvifrons, but mixing them with date palm pollen and E. kuehniella eggs does, in fact, support growth, development, and moderate reproduction. Future research should continue the search for inexpensive natural products or factitious foods that could replace E. kuehniella eggs in a mixed diet. Rearing over multiple generations without natural prey will be necessary to fully understand the effects of the factitious diet components on the health of S. gilvifrons and its subsequent capacity to recognize live prey, e.g., T. turkestani, on crop plants in greenhouses, high tunnels, or open fields upon augmentative release.

Institutional Review Board Statement: Not applicable.
Data Availability Statement: The authors will provide the pertinent data files on ResearchGate.