Location: Biological Control of Pests Research
2020 Annual Report
Objectives
Objective 1: Discover new biological control agents for invasive insect pests, especially invasive hemipterans, such as the bagrada bug and the kudzu bug.
Objective 2: Develop practical, mass rearing methods for agriculturally important insects, especially insect pests needed for the production of their natural enemies (such as stink bugs), insect biological control agents (such as predatory pentatomids and coccinellids), and insects potentially important as a food supply for animals and humans (such as mealworms and crickets).
Sub-objective 2A: Develop a reliable method for continuous production of the green stink bug Nezara viridula.
Sub-objective 2B: Study new methods to produce extracts from the yellow mealworm Tenebrio molitor and the house cricket Acheta domesticus and incorporate them into artificial diets for the predators Podisus maculiventris and Coleomegilla maculata.
Sub-objective 2C: Evaluate agricultural by-products as sources of food for the production of Tenebrio molitor and Acheta domesticus.
Objective 3: Develop effective biological control strategies for insect pests of crops grown under cover (e.g. high tunnels and greenhouses).
Approach
Climate matching software will be used to determine the most likely locations of natural enemy adapted populations across native ranges of M. cribraria and B. hilaris. The USDA-ARS European Biological Control Laboratory near Montpellier, France, will play a key role regarding parasitoid introductions of B. hilaris from many regions of Asia and Africa. Scientists at the university in Japan will make additional collections across the geographic range of the host within Japan. Scientists at USDA-ARS-IIRU, Newark, DE, will provide his host specificity expertise and make his Asian contacts available for the Kudzu Bug project.
Development of artificial diets for N. viridula will be approached by detailed chemical analyses of plant foods suitable for development and reproduction of N. viridula approximating their nutritional requirements. Artificial diets will be formulated to replicate the concentration and ratios of major nutritional components of broccoli, green lima beans and raw peanuts, which have been used to rear N. viridula. Diets will be compared to natural food sources broccoli, green snow peas, and raw peanuts plus a nutrient supplement previously developed (unpublished). Rearing conditions other than diet, such as optimal rearing density and adult reproductive curves, will also be studied.
Extracts of T. molitor larvae and pupae and A. domesticus nymphs will be produced by freeze-drying them at -25ºC and by spray drying of homogenized insects. Dry samples will be ground to particles of at least 30 µm. Extracts produced from dried T. molitor pupae will be used to produce artificial diet formulations for C. maculata. The diet formulations will be compared on their suitability to produce quality predators using life table analysis of C. maculata. The formulations will also be compared to a control consisting of natural food. The same procedure will be used for artificial diet formulations for P. maculiventris.
Four different types of agricultural by products will be tested as viable options to formulate diets for T. molitor and A. domesticus. 1) Peanut shells, 2) corn cobs, 3) discarded cabbage, and 4) residual from corn fermentation for ethanol production. Each by-product will be chemically analyzed to determine the content of protein, lipid, and carbohydrate. Diets will be formulated by mixing ingredients with wheat bran at different ratios depending of their nutrient content based on the chemical analyses. Diets will be evaluated by determining and comparing immature survival, development time and the efficiency of food conversion for each of the two species of insects.
Release and evaluation techniques for lady beetles as predators of strawberry will be develop and evaluated. This study will involve (A) testing the effectiveness of augmentative releases of lady beetles to control aphids in high tunnels, (B) testing the predation potential of larvae in the presence of aphid-tending ants in the laboratory and in high tunnels, and (C) testing the assertion that molecules in wax filaments on the cuticle of S. creperus larvae subdue ant aggression.
Progress Report
This is the final report for this project which will be replaced by new project pending completion of research review process.
Two key natural enemies of the kudzu bug were imported from Japan for host specificity testing against eggs of native stink bug species. The parasitoid wasp Ooencyrtus nezarae could attack most stink bugs tested, while Paratelenomus saccharalis was specific to kudzu bug eggs. Three species of egg parasitoids of the bagrada bug were imported from Pakistan and colonized for host specificity testing. Host specificity tests continued from 2017 to 2018.
Diet and rearing methods for the Southern green stink bug were developed. Stinkbugs developed faster in the new diets than in the conventional food mix consisting of raw peanuts and fresh vegetables, which include cabbage, broccoli, and green beans. Adults fed with the new diet formulation produced more eggs than adults fed the conventional food mix. The green stink bug was able to complete development and reproduce in the new diet formulation in total absence of fresh vegetables. The new diet was introduced to the main colony in late 2018 increasing survival and allowing significant reduction in space and labor. Colonies of the Southern green stink bugs have been maintained for more than 10 generations using these diets in the absence of fresh vegetables. These diets could potentially be modified to rear other species of stink bugs providing new ways to study them for developing new methods of control. Full evaluation of these diets using life table analysis is pending. Diet evaluation experiments have been designed and hardware consisting of experimental rearing cages have been constructed. Experiments were scheduled to begin in early March 2020 but were cancelled due to the facilities lock down imposed to prevent the spread of Covid-19.
Morphological changes in the reproductive system associated with reproductive age were characterized in the Bagrada bug and the Southern green stink bug. This method could be useful in determining reproductive age of individuals from the field or from unknown age. Necrotic processes observed in the ovaries of green stink bugs were linked to the presence of microsporidia parasites, which can be a problem in cultured colonies of stink bugs.
Nutritional studies completed during the previous project plan culminated on the development of an effective artificial diet for the pink spotted lady beetle. This diet formulation was later enhanced with the addition of 7% dry mealworm pupae powder. The best method for producing mealworm pupae powder was determined in subsequent studies. Mealworm pupae were dried by four different methods including freeze dry, vacuum oven dry, air dry, and a combination of blanching at 95°C for 1 minute and then vacuum oven drying. The best method was blanching the mealworm pupae before drying them in a vacuum oven to prevent degradation by enzymatic reactions. Other insect powders were also effective and even superior to the mealworm powders to enhance lady beetle artificial diets. Diets that incorporate house fly powder were superior than diets that contained mealworm powder by reducing development time and immature mortality and increasing adult fecundity and egg viability. A colony of the pink spotted lady beetle was established to be maintained entirely with artificial diet for multiple generations. By the time this report was completed, this lady beetle colony had been successfully maintained for more than 4 years (approximately 30 generations) exclusively on artificial diet in the total absence of natural prey. A greenhouse evaluation study of diet-reared lady beetles planned to test their effectiveness controlling spider mite infestations in greenhouse bean plants during spring and summer of 2019 has been postponed duet government shot down in 2019 and Covid-19 pandemic in 2020.
A new concept for the development of insect diets based on self-selection and statistical analysis was conceived, tested, and executed during the 5-year plan in two species of commercially produced insect species, the house cricket and the yellow mealworm. The self-selection method consists of presenting multiple food ingredients to groups of insects and carefully measuring the consumption of each of the ingredients. The relative consumption of each ingredient is then used as a base to formulate prototype diets. This method was used to formulate 7 cricket diets and 8 mealworm diets using agricultural by-products as the ingredients. Evaluation of the cricket diets was completed in December 2018 and peer reviewed publication was produced. The mealworm diets were evaluated between August 2019 to July 2020. These two studies culminated with the development of three cricket diets and three mealworm diets that outperformed the reference and control diets with a significant reduction in costs. It is estimated that the new diets provide at least twice the profits in biomass gain compared with feeding costs as the reference diets. Other improvements in the rearing system of crickets and mealworms included a new inverted water dispenser for the house cricket. This water system is designed to force crickets to drink water in an inverted position, preventing them from contaminating the water by defecation. A patent was submitted for this technology. A new system to estimate early instar larval densities for the yellow mealworm was developed based on frass production within a 5-week period. Adult frass was separated from the frass of young larvae by using a sift of standard number 50. The amount of frass produced per batch was correlated with the actual number of young larvae collected using linear regression. The model was then used to estimate the number of young larvae based on the weight of their frass produced within a 5-week period. This system provided control of larval densities in the production trays and resulted in a 2-fold increase in mealworm biomass production while reducing the rearing space requirements to half. Also, the stock mealworm colony has been under selection for larger pupal size for a period of 8 years. These efforts culminated in a genetically distinctive colony line that produce significantly larger larvae and pupae and display a significantly higher food conversion efficiency than commercial mealworm colonies in the U.S.
Rearing colonies of one ant species, two lady beetle species, and two aphids were established in the laboratory to study the impact of ant predation on the effectiveness of lady beetles as biocontrol agents against aphids. Strawberry (Camarosa and Chandler cultivars) plants were planted in pots in four, identical high tunnels, 24 feet long, and 18 feet wide. Lady beetle releases were done during summer and fall seasons of 2017. Factors affecting success or failure of releases have been reviewed and discussed in a peer-reviewed publication. Adults of the pink spotted lady beetle were released for aphid control on strawberry plants in two of four “old” high tunnels during summer 2018. Predatory mites were also released in two of four high tunnels for two-spotted spider mite control. Aphid and spider mite populations were reduced within two weeks of the releases. Another lady beetle, Stethorus (S.) punctillum, was released as an alternative to curb populations of the carmine mite on strawberry plants. Lady beetle adults and progeny were effective in reducing the population of the carmine mite by 95% within three weeks after release. The effectiveness of S. punctillum to control carmine mite populations on crop plants has not been previously reported and constitute a promising way to control this pest. An invasive aphid Aphis (A.) ruborum and an aphid parasitoid Aphelinus varipes were discovered on cultivated strawberry in high tunnel greenhouses in Washington County, Stoneville, Mississippi, USA. The occurrence of A. ruborum in Mississippi represents a new state record. The host-parasitoid association of Aphis ruborum and the parasitoid Aphelinus varipes has not been reported anywhere in the world.
Starting this fiscal year, there were limitations on Objective 3 due to the critical technician vacancy. These limitations reduced experimental expectations for fiscal year 2020 for this objective. Further, with Covid-19 introduction to the U.S. and later spread to the state of Mississippi, facilities were closed in early April 2020 forcing the cancellation of all field experiments planned for this objective.
Covid-19 also impacted Sub-objectives 2A and 2B. Activities in the NBCL were limited to colony maintenance and only critical personnel was allowed in the building starting in mid-March. This forced the cancellation of the lab experiment planned for these two sub-objectives. However, all the colonies associated with these two subobjectives (Nezara viridula and Coleomegilla maculata) have been successfully maintained and remain in optimal health allowing the continuation of these studies later.
Experiments were already in progress for Subobjective 2C when the NBCL was forced to close due to Covid-19. These studies continued to be attended by critical personnel. These studies consisted of the evaluation of eight mealworm diets formulated using agricultural by-products. Ingredients used in the diet formulations included wheat bran, rice bran, defatted rice bran, canola meal, dry corn distilled grain, spent brewery barley, dry potato, oat hulls, and peanut hulls. Growth and development of mealworm larvae as well as efficiency of food conversion and assimilation were evaluated and compared among the different diets and a control consisting of wheat bran only and a reference diet. All diet formulations performed better than the control and reference diet except for diet 5. Diet 2 was the best resulting in the highest biomass productivity and food conversion efficiency. Also, larvae fed with diet 2 completed development faster than larvae fed with all other formulations.
Accomplishments
1. Identifying oviposition stimulants for the pink spotted ladybeetle (Coleomegilla maculata) from plant-derived natural products. ARS researchers in Stoneville, Mississippi and Peoria, Illinois, discovered that several bioflavonoids (plant secondary metabolites) stimulated oviposition in ladybird beetles. More precisely, powdered formulations of quercetin, taxifolin, naringenin, genistein, and catechin hydrate stimulated oviposition behavior in the pink spotted ladybeetle. For example, in 12-day bioassays involving multiple females (20 individuals/cage), oviposition was common in cages with quercetin, but rare in cages without quercetin. The stimulation of oviposition in the presence of bioflavonoids has the potential benefits of: (1) reducing cannibalism, by restricting egg-laying to a location away from feeding sites, and (2) increasing egg harvesting from a specific location rather than random locations in rearing cages. The possible end result will be more efficient mass production of ladybeetles for augmentative biological control of plant pests on small fruits and vegetables in commercial greenhouses and high tunnels.
2. Implementation of density management new techniques into mealworm production system resulting in a 4-month reduction in development time and an increase of 1-fold in biomass production. Current mass production of T. molitor in commercial farms does not control adult and larvae density resulting in substantial inefficiencies in reproduction and biomass production, which make insect protein expensive. Previous studies conducted in the NBCL, Stoneville showed that adult density impacts fecundity and larval density impacts food utilization efficiency, development time, and survival. However, methods to determine densities of newly hatched larvae were lacking. ARS researchers in Stoneville, Mississippi, developed a new method to estimate young larval densities by correlating waste production with larvae numbers. Controlling larval densities in rearing trays increased production and reduced the space requirements for mealworm mass production by shortening the development time while increasing the growth rate of larvae. This new technology can result in substantial cost reductions of mealworm production and a subsequent price reduction of mealworms for animal feed and food ingredients for human consumption.
3. Enhanced artificial diets for the pink spotted lady beetle. Commercial production of insect predators requires the development of effective artificial diets in order to reduce production costs. In the past, artificial diets for insect predators have been ineffective in producing quality predators due to nutrient deficiencies resulting from the lack of insect components in the diets. ARS researchers in Stoneville, Mississippi, developed new artificial diets for the pink spotted lady beetle using insect components derived from commercially available insect powders. Powders produced from yellow mealworm, house cricket, and house fly were incorporated to experimental artificial diet formulations and tested for their efficacy at producing quality lady beetles. Diets incorporating powders of the house fly produced lady beetles, which were of higher quality than the control diet and previously developed artificial diets containing mealworm powder. Also, insect powders derived from insects that were processed, such as oven roasted or blench and dry, were of higher quality than powders derived from row insects. These new diet formulations can be produced in high quantity at a reasonable price and constitute a promising solution to the costly mass production techniques currently used to produce lady beetles and other insect predators. If adopted by the biological control industry, this diet formulation could reduce the price of biological control agents. Reducing the cost of producing biological control agents could encourage the use of this method to control important pests and reduce the need for insecticide applications, which could benefit the environment and make agriculture more sustainable.
4. Agricultural by-products to develop diets for cricket and mealworm production as animal feed. Massive quantities of agricultural by-products are produced annually in the U.S. Most of these products, such as, distilled grains for ethanol production, rice bran, and extracted grain meals from vegetable oil production, are not fit for human consumption. However, some insects can feed on these products and convert them into useful animal protein. Insects produce high quality protein and could be an important alternative to fish meal in the formulation of feeds for different farm animals and aquaculture. The current costs of insect production at present are not competitive with fish meal due to inefficiencies of the rearing technology and high costs of insect diets. Using agricultural by-products to produce insect diets could reduce the cost of insect production. ARS researchers in Stoneville, Mississippi, developed diets for the house cricket and the yellow mealworm using 80 to 90% agricultural by-products. These diets produced insects of better quality than existing commercial diets at a much lower price. These diet formulations could allow the reduction of insect mass production by converting unusable agricultural by-products into valuable animal protein. The cost reduction of insect production could make it competitive with fish meal, which is an unsustainable source of animal protein required for the production of feeds for livestock and aquaculture. These new diets will benefit the existing insect production industry, which is one of the fastest growing industries in the U.S. Other industries, such as ethanol, vegetable oil, etc., will also benefit by the utilization of their by-products, which could become a commodity.
5. Size increase in the yellow mealworm induced by selection. Although the production of insect protein holds promise as a substitute of fish meal in animal feed formulations, the market price of insect powders remains high due to the high costs of insect mass production under the current rearing systems. Current rearing procedures for insect mass production are primitive and have a great potential for improvement. Another aspect of insect biomass production that can be improved is the breeding of insect lines for more efficient growth and food conversion. ARS researchers in Stoneville, Mississippi, tested the possibility of obtaining improved lines of the yellow mealworm by selecting for a larger size. A continuous selection of larger mealworm pupae as reproductive stock for a period of 8 years has doubled the size of mealworm pupae in the experimental colony. This increase in size has not been accompanied by an increase in development time, which indicates that the growth rate of the mealworms has also increased. These results demonstrate the viability of selection strategy to improve the productivity of insect colonies and the potential that insect colonies have for further genetic improvement using conventional selection methods. Improved breeding stock could greatly increase the productivity and reduce costs of insect biomass production for animal feed.
6. Combined methods of dietary self-selection, nutrient matrix, and multiple regression analyses to develop oligidic insect diets. Insect diets that are formulated with no chemically defined ingredients such as grain flours, vegetables, and animal products are called oligidic. These types of diets are the least expensive to formulate and are the most stable and commercially viable. Because of their composition complexity, oligidic diets are the most difficult to develop requiring years of trial and error experiments followed by evaluations for insect performance. In previous work by ARS researchers in Stoneville, Mississippi, the use of self-selection techniques has been perfected to develop and optimize chemically-defined diets. Using analyses of nutrient composition and multiple regression has allowed the use of the self-selection technique to develop oligidic insect diets. This method works by using ingredient consumption data from multiple self-selection experiments with different combinations of ingredients. Consumption data of individual ingredients is used to calculate ingredient consumption ratios and nutrient intake ratios based on the known nutrient composition of the ingredients used in each experiment. Multiple regression analysis is used to correlate the intake of each nutrient with the biomass gain from start to finish of each experiment. This constitutes a holistic approach for insect diet development that yields an instant estimate of the optimal nutrient intake ratios for insect growth and development. This new method is applicable to insects that are at least partially omnivorous and can consume food from diverse sources. Many insect predator species are omnivorous, such as lady beetles and stink bug predators. This new method of insect diet development has the potential to revolutionize insect diet development and can benefit all fields where insect mass rearing is required, including insect research, sterile male release efforts for eradication of invasive pests, biological control industry and research, and insects as food and feed industry.
7. DNA barcoding technology identified an adventive aphid and a new association with an aphid parasitoid on strawberry. Aphids can cause major economic damage to many crops such as small fruits, i.e., cultivated strawberry and blackberry throughout the world. One goal of organic agriculture is to minimize the use of pesticides. A key to managing aphids without pesticides is to first identify the species and any associated natural enemies. ARS researchers in Stoneville, Mississippi, used morphology and DNA barcoding techniques to identify an adventive species (Aphis ruborum) new to eastern USA. This species is widely distributed in Europe, North Africa, and central Asia. DNA barcoding also identified at least one parasitic wasp species (Aphelinus varipes), previously introduced into the USA for classical biological control of another aphid species (Russian wheat aphid). This research is significant because it reports a new state record for Mississippi, USA and the eastern-most establishment of the aphid in the USA. Also, the association of the aphid with the parasitic wasp has never been reported before, anywhere in the world, to our knowledge.
Review Publications
Morales-Ramos, J.A., Rojas, M.G., Dossey, A.T., Berhow, M.A. 2020. Self-selection of food ingredients and agricultural by-products by the house cricket, Acheta domesticus (Orthoptera: Gryllidae): A holistic approach to develop optimized diets. PLoS One. 15(1):e0227400. https://doi.org/10.1371/journal.pone.0227400.
Ebrahimfar, J., Shishehbor, P., Rasekh, A., Riddick, E.W. 2020. Effect of factitious diets on development and reproduction of the ladybird beetle Stethorus gilvifrons, a predator of tetranychid mites. Biocontrol. 65:703-711. https://doi.org/10.1007/s10526-020-10033-y.
