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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

2013 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 (C.) 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:
Research on development of artificial diets for the pink spotted lady beetle, Coleomegila (C.) maculata, yielded three new formulations using new ingredients recently coming into the market. These new diet formulations have shown promise by reducing development time and increasing adult size and evaluation of the new formulations is under way. Efforts to identify factitious food for the pink spotted lady beetle have yielded promising results using a mixture of eggs of the brine shrimp (Artemia franciscana) and of a powdered freshwater algae (Chlorella vulgaris). While the use of factitious food is more complicated and expensive than an artificial diet, it provides significant economic advantages over the state of the art rearing based on eggs from the Mediterranean flour moth. New laboratory strains of the pink spotted lady beetle have been bred. These strains include visible marker strains with variable coloration and spot patterning. These strains will be useful for de novo sequencing, for dissecting biochemical pathways and for testing gene expression in stress conditions. Modifications of existing artificial diet formulations for C. maculata have been successfully tested on other predatory species including the insidious flower bug, Orius insidiosus, and the predatory mite, Amblyoseiulus swirskii. Both of these species are produced commercially in Europe and the U.S. and there is a current cooperative agreement on this research. On the production of the predatory mite, Phytoseiulus persimilis, a new diet encapsulation method and improved diet formulation have been developed as part of a Cooperative Research and Development Agreement. On the study of semiochemicals associated with lady beetles, work is in progress on the isolation and identification of compounds present in cedar trees (e.g., Juniperus virginiana L.) that might stimulate oviposition in the pink spotted lady beetle. A toxic alkaloids (myrrhine) previously reported as a defensive chemical present in the egg shells of several species of lady beetles, have been found present in the egg shells of the pink spotted lady beetle. On the in vivo production of insect killing (entomopathogenic) nematodes, a new diet has been developed to improve the production of the nematode host, the yellow mealworm (Tenebrio molitor). This diet was developed using insect self-selection by multiple-choice of predetermined diet formulations. This novel method has promise for refining existing predator diets without the need of long and costly trial and error tests. On the classic biological control efforts against invasive stink bugs, continuous cultures of 4 imported and 7 native wasp egg parasitoid species have been established in quarantine. These egg parasitoids are currently being tested for their effectiveness against the brown marmorated stink bug (Halyomorpha halys), the kudzu bug (Megacopta cribraria) and the bagrada bug (Bagrada hilaris). Production of these invasive stink bug species has been accomplished by the continuous availability of eggs from the spined soldier bug (Podisus maculiventris), which has been maintained successfully in culture using yellow mealworm pupae.


4.Accomplishments
1. Improved artificial diets for the pink spotted lady beetle, Coleomegilla (C.) maculata, and the insidious flower bug, Orius (O.) insidiosus. Current rearing techniques of these 2 important predators consist of using mixtures of pollen and eggs from the Mediterranean flour moth (Angasta (=Ephestia) kuehniella). This method is expensive and labor intensive. Diets developed the previous year were improved and with the addition of globulins. The new formulation improved fecundity in C. maculata and reduced cannibalism in O. insidiosus. The new formulations constitute a significant improvement over diets developed during 2012. Biological control producers and organic growers will benefit from this discovery.

2. A new artificial diet for the predatory mite Amblyoseiulus (A.) swirskii. Current commercial production of this predatory mite depends on the production of other mite species used as prey. Production of additional species increases the costs of production of predatory mites and their final price for the consumer. An artificial diet will eliminate the need to produce additional mite species and will simplify the rearing methods substantially cutting cost of predator production. ARS scientists of the Biological Control of Pests Research Unit in Stoneville, MS, developed a semi-solid colloidal artificial diet capable to maintain growth and reproduction of A. swirskii for several generations without any apparent ill effects. The semi-solid nature of the diet makes it easy to present to the mites and facilitates its use during shipping and release of the predators increasing their life span. Producers of greenhouse grown crops, such as tomato, strawberry, squash, etc. will be potentially benefited by this discovery.

3. Using the self-selection technique to develop diet supplements for Tenebrio (T.) molitor. The yellow mealworm (T. molitor) is a commercially produced insect sold in the U.S. for a variety of purposes including mass producing insect killing nematodes. Improving artificial diets is a complicated process because altering one of the more than 100 components impacts the diet quality and the trial and error process can take many years to produce an optimal formulation. Using a new method based on self selection techniques ARS scientists of the Biological Control of Pests Research Unit at Stoneville, MS, were able to obtain an optimal combination of 4 diet ingredients to improve mealworm growth and reproduction. This is the first time that self selection has been used to develop diets in insects. This method shows great promise for development of artificial diets, especially on omnivore predators such as the pink spotted lady beetle, the insidious flower bug, and the spinned soldier bug, Podisus maculiventris. Nematode producing industry will benefit by improving the mealworm diet. Reducing costs of mealworm production is one of the fundamental objectives for commercial production of insect killing nematodes.

4. Optimal density for growth of the yellow mealworm, Tenebrio (T.) molitor. To optimize the production of mealworms in a minimal room space, mealworm larvae are commonly maintained in trays at high densities. This reduces requirements of valuable temperature and humidity controlled space. However, high densities may be detrimental to production. ARS scientists of the Biological Control of Pests Research Unit at Stoneville, MS, determined that the increase of larvae density induces a decrease in mealworm food conversion efficiency. Reduction in food conversion efficiency may translate in a reduction of production or an increase in production time countering the economic benefit of optimizing rearing space. Based on the research findings it is possible to establish an optimal density to save space while maintaining an acceptable efficiency of food conversion. Producers of mealworms will be beneficiated by these findings.

5. Develop a cost effective and time-efficient system for mass-producing the pink-spotted lady beetle. Current production of the pink spotted lady beetle relays on the use of eggs from the Mediterranean flour moth. Production of moth eggs is expensive and new alternatives are needed to produce lady beetles at an economic price. ARS scientists of the Biological Control of Pests Research Unit at Stoneville, MS, discovered that decapsulated eggs of the brine shrimp, Artemia (A.) franciscana, support satisfactory development of Coleomegilla (C.) maculata immature to the adult stage. Although C. maculata fecundity is less when fed solely on shrimp eggs, addition of a powdered freshwater algae (Chlorella vulgaris)/synthetic pollen appears to boost the reproduction of C. maculata. The utilization of A. franciscana eggs and powdered algae/synthetic pollen as food for C. maculata eliminates the need to grow live plants in rearing systems. In addition, rearing of C. maculata is more cost-effective when using A. franciscana eggs rather than Mediterranean flour moth eggs. Biological control producers and organic growers will benefit from this discovery.

6. Genetically useful strains of the pink spotted lady beetle, Coleomegilla (C.) maculata. While basic biological methods to raise the pink spotted lady beetle have been improved over many years, methods to raise specific strains for genetic research have not previously been addressed. To address this need, ARS scientists of the Biological Control of Pests Research Unit at Stoneville, MS, have developed several new laboratory strains of C. maculata. These strains include visible marker strains with variable coloration and spot patterning. Additionally an isofemale selected strain has been established by sequential bottlenecking (7X isofemale). This strain maintains the Wild (pink) phenotype. These strains will be useful for de novo sequencing, for dissecting biochemical pathways (particularly connected with pigmentation of cuticle and eyes) and for testing gene expression in stress conditions. Biotechnology scientists, biological control producers, ecologists, and industry will benefit from these strains and the genetic data they will yield.

7. Defensive compounds on the egg surface of the pink spotted lady beetle, Coleomegilla (C.) maculata. Defensive compounds have been identified from many lady beetles, but primarily from adult or larval specimens. Augmentation and conservation biological control programs are hampered by intraguild predation, particularly of eggs. RS scientists of the Biological Control of Pests Research Unit at Stoneville, MS, identified the defensive hydrocarbon “precoccinelline”, a stereoisomer of myrrhine. Literature indicates that defensive alkaloid myrrhine dominates the defensive hydrocarbon mixture of some lady beetle species. Thus eggs could be the most efficient source of this compound for research and potential industrial development. Fire ants destroyed but did not consume C. maculata eggs when presented; however, ants voraciously consumed plant bug (Lygus spp.) eggs. The defensive compound myrrhine seems to deter predators from feeding. Scientists may now study how the compound contributes to conspecific cannibalism and intraguild predation, and assess the compound as a pest deterrent. These discoveries will benefit ecologists, biological control producers, and pest control science.

8. Continuous culture of 4 imported and 7 native wasp egg parasitoids. The brown marmorated stink bug (Halyomorpha halys), the kudzu bug (Megacopta cribraria), and the bagrada bug (Bagrada hilaris) are exotic invasive species with no known natural enemies in the United States. These invasive species produce extensive damage in commercially important crops including various fruit crops, soy, cabbage, cauliflower, mustard, broccoli, and radish. Additionally they are known as household pests because of their tendency to overwinter inside houses. ARS scientists of the Biological Control of Pests Research Unit at Stoneville, MS, have been able to continuously rear in quarantine 4 imported and 7 native wasp species, parasitoids of true bug eggs, for possible application against these 3 exotic invasive insects. Effective biological control of these pests could reduce their economic impact. Potential beneficiaries include growers and the general public.


Review Publications
Riddick, E.W., Wu, Z., Rojas, M.G. 2013. Is Tetranychus urticae suitable prey for development and reproduction of the naive Coleomegilla maculata?. Insect Science. doi:10.1111/1744-7917.12033.

Morales Ramos, J. A., Rojas, M. G., Shapiro Ilan, D. I., Tedders, W. L. 2013. Use of nutrient self selection as a diet refining tool in Tenebrio molitor (Coleoptera: Tenebrionidae). Bulletin of Entomological Research. 48(3):206-221.

Last Modified: 9/29/2014
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