| It's Gut Check Time! |
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In this article we will introduce you to the methods that we have developed for studying predation. Specifically, we will discuss:
1. Why insect predators are important.
Insect predators are partially responsible for keeping insect pest populations in check. Predators, along with parasites and pathogens are Mother Nature's best pest control agents. Prior to the insecticide revolution in the 1940's it was estimated that 7.0% of the world's crops were destroyed by insect pests. Today some authorities estimate our losses at 13.0%. This increase in crop destruction over the last half century is due, in part, to the destruction of insect pests' natural enemies by pesticides. These problems, coupled with increasing environmental awareness have forced agriculturists to seek more environmentally safe and cost-effective pest control strategies.
2. Why insect predators are so difficult to study.
Observing insect predators directly in the field is much more difficult than observing large vertebrate predators such as lions, tigers, or bears -- Oh My!! Most insect predators are small, elusive, and/or nocturnal making field observations difficult. Furthermore, direct field observations are disruptive to the natural predator behaviors, time consuming, uncomfortable (it can get to 117?F in the summer), and unreliable. We have circumvented these difficulties by developing an indirect method for evaluating predator/prey interactions.
3. What is an "indirect" study of predation.
An indirect study of predation is studying a predator's feeding preference without actually seeing the predator feed on its prey. The immunoassay techniques we employ were pioneered by researchers in medical fields. These immunoassays are virtually identical to tests used to screen human patients for early signs of disease and pregnancy. However, instead of screening patients for the presences of disease-specific proteins or pregnancy- specific hormones we screen predator gut contents for the presence of prey- specific proteins.
4. Advantages of indirect predator studies.
Gut content immunoassays avoid pitfalls inherent in direct observation examinations. Immunoassays provide researchers with a method to determine which predators have the greatest impact on suppressing populations of key insect pests. The immunoassays used are simple, quick (1,000 predators/day), sensitive (less then one prey can be detected), and cost effective (ca. $0.05/predator).
5. Disadvantages of indirect predator studies.
The disadvantage of using immunological methods to study predation is that these methods are not quantifiable (i.e., unable to determine how many pests the predator ate). Uncontrollable variables make precise quantitative assessments impossible. Biotic factors such as predator/prey phenology, number of prey eaten, and prior metabolic status can all affect the quantitative outcome on an immunoassay. Furthermore, prey digestive rates of each predator species must be determined before a reliable qualitative estimate of predation can be made.
6. Target Pests
Pink bollworm (PBW) 
and sweetpotato whitefly (SPW) 
are the two pests that we have focused most of our research efforts toward. We selected these pests because they are the two most economically significant pests of cotton in the western U.S. Annual economic losses incurred by PBW averaged 26% of the cotton crop value from 1966 to 1980 in California's Imperial Valley. Since 1980, cotton production there has decreased from over 100,000 acres to less than 15,000 acres principally due to PBW.
Cotton boll damage by PBW
SPW has been a pest in the west since the early 1980's, and with the recent introduction of a new species (silverleaf whitefly), SPW has become the major pest of cotton and many other crops. It has been estimated that SPW was responsible for over $100 million of total crop loss in California and Arizona in 1991.
Both of these pests are showing an ever-increasing resistance to pesticides, unfortunately their natural enemies (predators and parasites) are not. Sound scientific data on biological control of these pests with predators may help alleviate the overuse of insecticides on the cotton insect complex.
7. A step-by-step guide to our gut content ELISA.
A. Field Collection
The first step toward evaluating predators is to collect them. To do this we used a super duper bug vacuum that was developed by Dr. Joe Ellington. We collected predators by simply vacuuming the cotton field. After vacuuming we immediately placed the predators on dry ice.
When we return to the lab we sort all of the predators to species and run an enzyme-linked immunosorbent assay (ELISA) on each individual. The steps of the ELISA are as follows:
- Predators are ground in phosphate buffered saline (PBS) and a 100 ?l aliquot is placed in the well of a 96-well ELISA plate for incubation overnight.
- Wells are emptied and blocked with 350 ?l of 1% non- fat dry milk. After 30 min. the milk is discarded.
- A 50 ?l aliquot of prey-specific MAb is added to each well of the assay plate for 1 hour.
- The MAb is discarded and the plates are rinsed with PBS. Fifty ?l of goat anti-mouse secondary antibody conjugated to alkaline phosphatase diluted to 1:500 in 1% non-fat milk is added to the wells and incubated for 1 hour.
- Plates are rinsed and 50 ?l of 1.0 mg/ml p- nitrophenyl phosphate substrate is added to each well.
- After 2 hours the absorbance of each well is read with an ELISA microplate reader. Positive reactions for the presence of prey antigens in a predators gut leaves a yellow colored product in the wells whereas negative reactions remain clear.
An ELISA plate with several positive reactions.
8. Surprises from our research.
Much of our research verified what we had suspected all along. For instance, we hypothesized that populations of ladybird beetles 
, big-eyed bugs 
, and minute pirate bugs 
would have a high frequency of individuals scoring positive for the presence of PBW and SPW prey in their guts. We thought this because there was a high number of these individuals in our fields (they must be eating something) and laboratory studies suggested that they feed on these two pests.
Because our gut content immunoassays are rapid and inexpensive, we had the opportunity to screen potential predator species that we might have overlooked by conventional methods. For example, we examined the major insect pest Lygus hesperus.
Although the lygus bug is considered a major insect pest it has been reported to exhibit some predatory activity. Our immunoanalysis of this "pest" showed that it is a major predator on both SPW and PBW. In fact, is was clearly the best predator of PBW. Our results suggest that we need to assess carefully the beneficial impact of lygus in the cotton agroecosystem.
Another finding that would not have occurred by conventional methods was the identification of the collops beetle as a major predator of SPW. Collops are typically not encountered very frequently in cotton fields in Arizona and subsequently they have been overlooked as a potential beneficial. However, our immunoanalysis suggest that 60% of the individuals we examined contained SPW egg antigen in their guts (almost twice the frequency of the next best predator species). Again, if we were studying predation by conventional methods, this species might have been overlooked. We now should focus our attention on thoroughly studying the basic biology of collops. Then perhaps we can conserve and enhance these natural enemy populations in the field or maybe even augment their populations.
