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United States Department of Agriculture

Agricultural Research Service

Research Project: Mass Production of Biological Control Agents

Location: Biological Control of Pests Research Unit

2011 Annual Report

1a.Objectives (from AD-416)
The long-term objective of this project is to develop an improved understanding of the biology, nutrition, and behavior of natural enemies of pest arthropods. Our primary target pest will be the two-spotted spider mite, Tetranychus urticae. Some of the most important natural enemies of this mite are phytoseiid predatory mites and coccinellids. We aim to implement, improve and increase the mass rearing capabilities of these organisms by developing artificial diets and novel rearing technology to enable economical biological control of this pest by augmentation of its natural enemies and to develop novel techniques of quality control and quality assurance. Our work will focus on the following: (1) Determine nutritional, behavioral, and physical conditions necessary for in vivo and in vitro insect rearing and use the results to implement mass production systems of biological control agents. (2) Determine the impact of mass rearing procedures on survival, reproduction, and gene expression. (3) Assess optimal conditioning of insects for release.

1b.Approach (from AD-416)
Research activities will focus on a specific predator, Stethorus punctillum and a generalistic predator, Coleomegilla maculata. Studying the nutritional requirements of a generalist versus a specific predator will provide insights on diet development of these 2 organisms. The generalist predator is expected to be an easier target for artificial diet development and success will have greater impact since C. maculate is an important predator of several economically damaging pest species.

3.Progress Report
The first year of the project resulted in a successful establishment of new species of beneficial organisms. Two colonies of the spotted lady bug, Coleomegilla (C.) maculata, of different origins were established using different rearing methods. Five species of stink bugs (Hetereptera: Pentatomidae) were established including Nezara (N.) viridula, Euschistus servus, Acrosternum hilare, and Podisus maculiventris. Work on artificial diets for these organisms yielded promising formulations for C. maculata and N. viridula and significant advances were achieved on developing new rearing methods for all the above species. In the area of lab rearing of C. maculata, extensive literature research on artificial diets and chemical ecology were conducted. Based on the literature and field observations, detailed chemical analyses of prey of the major nutrient groups were conducted. Beetles fed on the diet safely and completed development. Predators collected from the wild can be easily and quickly adapted to laboratory culture on artificial diet because the new diet formulation closely resembles natural prey. Larvae and adult C. maculata collected from the field readily feed on the diet and are able to develop and reproduce. This new discovery opens the door for development of a complete artificial-based rearing system, eliminating the need for greenhouses. On the search for alternative, novel sources of protein that can be incorporated into an artificial diet for C. maculata, preliminary experiments with proteins from marine organisms (microalgae, crustaceans) are yielding promising results. A commonly used protein source for human consumption known as Spirulina algae (in powdered form), in combination with brine shrimp (used as commercial food for tropical fish), provides suitable protein sources that allow efficient development and reproduction of C. maculata in lieu of any insect protein. A new method of refining artificial diets involving nutrient self-selection was tested in Tenebrio molitor (yellow mealworm) with success. In a multiple choice experiment, larvae of the yellow mealworm selectively chose optimal ratios of 2 nutrients. In the area of bio-ecological interactions of plant allelochemicals, spider mites, and its lady beetle predator, Stethorus punctillum, experiments showed that plant chemicals such as linamarin appear to block protein absorption by spider mites. This resulted in negative effects on development time, growth, and body size of the predator.

1. Biology studies by Phytoseiulus (P.) persimilis. Previous year studies on temperature dependent development rate and fecundity of spider mites, Phytoseiulus persimilis, showed that the most favorable temperatures were between 25 and 27ºC. New studies were done at 8, 10, and 15ºC to more closely determine the optimal temperature for short term storage of P. persimilis using an encapsulated artificial diet. The previous study showed that temperature affected each of the biological parameters (development time, survival, fecundity) in different ways. Evaluation of stored mites showed that the optimal temperature is 15ºC for an 8 day period. The life and fertility table analysis allowed us to analyze all of these parameters, producing a single statistic that measures population growth. We concluded that optimal short term storage temperature for P. persimilis can fluctuate between 15 and 17ºC. These results provide critical information about the optimal conditions for mass storage of P. persimilis. This information will be especially valuable for biocontrol producers to reduce losses due to overproduction of mites.

2. Genetic studies on Lygus spp. Continuous colonies of two arthropod species are still maintained in the Stoneville Research Quarantine Facility (SRQF). A stable homozygous red-eyed strain of Lygus lineolaris was established to supplement insect genetics efforts. We have established a thriving colony of lady beetles, Coleomegilla (C.) maculata. Efforts to culture the bean plataspid, Megacopta cribraria, have been underway but are presenting substantial difficulties. Ribonucleic acid (RNA) interference microinjection experiments produced a polygalacturonase knockdown manuscript, in addition to a publication documenting Inhibitor of Apoptosis (IAP) knockdown. Mass reared insects are an essential precursor to molecular genetic studies, especially de novo sequencing as will be required to study the changes in gene expression of biological control agents such as C. maculata in response to artificial diet. The variety of mass rearing and molecular genetic experience gained over the past years provided our team with critical skills for future research projects as defined by our research project.

3. Factitious food of Coleomegilla (C.) maculata. Currently, preliminary experiments with protein from marine organisms (microalgae, crustaceans) are yielding promising results. A commonly used protein source for human consumption, known as Spirulina algae (in powdered form) in combination with brine shrimp (used as commercial food for tropical fish), provide suitable protein sources that allow efficient development and reproduction of C. maculata in lieu of any insect protein. We reared C. maculata for five consecutive generations on brine shrimp as the only food source. The development and reproduction of predators reared on brine shrimp is comparable to that of predators reared on a common insect prey, lepidopteran (Ephestia moth) eggs. Both Spirulina microalgae and brine shrimp are promising sources of protein that could be incorporated into an artificial diet for C. maculata and other generalist predators. This discovery provides further information on the suitability of brine shrimp as an alternative source of protein for incorporation into an artificial diet. The costs associated with commercial production of brine shrimp is less than that of producing insect eggs. Therefore, isolation of protein molecules, (or the development of cell lines) from brine shrimp should be encouraged in hopes of providing cheap, non-insect protein for an artificial diet suitable for commercial use by the biocontrol industry.

4. In vivo rearing of nematodes on Tenebrion (T.) molitor. Studies on the nutrition of the mealworm, T. molitor, used as hosts for production of beneficial nematodes resulted in development of 6 diet formulations that significantly improved immature survival, development time, food utilization efficiency, and reproductive potential. Two of these diet formulations increased T. molitor susceptibility to infection by 2 species of entomopathogenic nematodes and resulted in higher nematode yields when T. molitor larvae were used. Studies on the effect of adult density on progeny production yielded optimal adult densities for maximum larvae production. Age dependent fecundity studies of T. molitor provided new insight on the optimal adult age to replace the reproductive colony. New egg collection methods for Coleomegilla (C.) maculata, were developed with use of water absorbing polymers. A new method was developed to provide water for rearing stink bugs and eliminate water contamination and disease transfer among individuals using the polymers. New cages designed for stink bug rearing facilitate watering and reduce labor and bacterial infection. The new diet formulations increase the reproductive rate of T. molitor, and by increasing susceptibility increase the number of entomopathogenic nematode produced per larvae. Instead of using different diet formulations for these purposes, a single formulation accomplishes both host and parasite production). The optimal density of T. molitor adults for reproduction, and the cutout date for replacement of reproductive adults can reduce the number of adults required to maintain T. molitor continuous production. This enables producers to direct fewer resources for colony maintenance and more for production. New methods of egg collection for C. maculata could significantly reduce labor on mass producing this predator. Similarly, improved cage designs for rearing stink bugs could reduce labor and improve insect survival.

Review Publications
Riddick, E.W., Rojas, M.G., Morales Ramos, J.A., Allen, M.L., Spencer, B. 2011. Stethorus punctillum (Coleoptera: coccinellidae). Biological Control: A Guide to Natural Enemies in North America.

Walker III, W.B., Allen, M.L. 2010. Expression and RNA interference of salivary polygalacturonase genes in Lygus lineolaris (Miridae: Hemiptera). Journal of Insect Science. 173(10):1-13.

Walker III, W.B., Allen, M.L. 2011. RNA interference mediated knockdown of IAP in Lygus lineolaris induces mortality in adult and pre-adult life stages. Entomologia Experimentalis et Applicata. 138:83-92.

Riddick, E.W., Wu, Z. 2010. Lima bean – lady beetle interactions: hooked trichomes affect survival of Stethorus punctillum larvae. Biocontrol. 56:55-63.

Riddick, E.W., Wu, Z. 2010. Potential long-term storage of the predatory mite Phytoseiulus persimilis. Biocontrol. 55(5):639-644.

Last Modified: 4/24/2014
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