2013 Annual Report
1a.Objectives (from AD-416):
The broad objective is to improve agricultural sustainability. A specific objective is to increase use of biological control (BC) which can reduce dependence on synthetic insecticides and minimize their influence on environmental quality. The goal of this Specific Cooperative Agreement is to reduce production costs and optimize field performance of a BC agent, the predatory pentatomid, Podisus maculiventris. This is a native species, selected to ameliorate problems associated with exotic BC agents. For effective mass-rearing, research is designed to improve the nutritional quality of the artificial culture medium to optimize BC field performance and to identify phenotypic characteristics that optimize production costs and field performance. We have reported results of research showing that nutritional inputs influence important traits, including fecundity (egg production), developmental rates and tolerance to cold-storage. This is a particularly important trait in lowering production costs for predatory BC agents. To leverage our successful research on nutritional improvements, we are combining nutritional research with classical genetics to select for economically valuable traits in the predatory pentatomid. Progress in this area depends on assessing genetic variation within and between predator populations. P. maculiventris occurs throughout the U.S. We are collecting local populations in Missouri and working with cooperators to collect populations along east-west and north-south transects of the U.S. We have already established a productive collaboration with University of Nebraska, to apply a standard protocol to determine genetic variability of a domesticated colony maintained in BCIRL for over 130 generations. The protocol uses amplified fragment length polymorphism technology (AFLP), a highly sensitive and reproducible tool widely used to determine genetic variation. Our first study documented a low level of genetic variability in the domesticated BCIRL population. We are now working on genetic variation in natural P. maculiventris collected from Missouri sites. We will expand this work to include populations along the transects just mentioned.
Aside from documenting the necessary genetic diversity required to selectively improve this BC agent, AFLPs associated with high fecundity and other important production traits will be developed into recognizable molecular markers to direct future selection programs, called marker assisted selection. This is crucial for effective selection programs. Without molecular markers the presence/absence of a desired phenotypic trait (such as high fecundity) and response to changes in nutritional quality cannot be recorded until insects have reached sexual maturity; marker assisted selection speeds the process because the presence/absence of the target trait can be recorded very early in the insect life cycle.
This Specific Cooperative Agreement will leverage established nutritional improvements, generate important new information and yield a novel approach to the improvement and use of BC programs at the national and global levels.
1b.Approach (from AD-416):
We will use amplified fragment length polymorphism (AFLP), a powerful, PCR-based molecular marker tool, to determine genetic variability in P. maculiventris populations. AFLPs have demonstrated utility in a wide variety of different studies on an array of different organisms.
There are several advantages to using AFLPs over other molecular genetics techniques. First, no previous genetic information about an organism is required for the use of AFLPs as the primers used are universal. This makes AFLPs more convenient than a technique such as DNA sequencing, where a priori genetic knowledge is required to create specific primers. Unlike RAPDs, AFLPs are reliably repeatable, which means that results produced by different laboratories will be uniform. AFLPs are also advantageous because the process is easy to learn and, after the initial cost of equipment, the reagents are relatively inexpensive. AFLPs are a fast and cost effective way to process a large number of samples. Techniques such as DNA sequencing may be prohibitively expensive with a large number of samples. Additionally, AFLPs utilize the entire genome as opposed to one or two specific genes or sites as is the case with RFLPs or microsatellites. Several different primer sets may be used to generate a large number of markers, increasing the statistical power of data analysis. Finally, the large number of amplified loci make AFLPs ideal for intra-specific studies and organisms in which genetic variability is low.
AFLPs are ideal for this project as the facilities for this molecular technique are already in place. A 4200 IR Sequencer (LI-COR Biosciences, Lincoln, NE) is available for use in the insect genetics laboratory at the University of Nebraska. The requisite reagents and other equipment are also in place. In addition, the insect genetics laboratory has a great deal of experience with AFLPs and has used this technique successfully for many years.
Genetic variability among field collected spined soldier bugs from Michigan, Mississippi, Missouri, Florida and laboratory reared colonies was examined. The majority of the genetic variation was found within populations as compared to between populations. Rearing populations of differing origins for multiple generations in a laboratory environment resulted in a convergence in genetic diversity. However, laboratory-reared populations retained sufficient genetic variation to support the use of laboratory-reared insects in wide-spread field release efforts. The findings of this work relate to the parent project objective 1 “Select and develop beneficial lines of a predator, the spined soldier bug, Podisus maculiventris, for improved bionomic traits such as increased female fecundity, sex ratio, development time, and shelf life.”