Use of Radiography in Behavioral Studies of Turfgrass-Infesting Scarab Grub Species (Coleoptera: Scarabaeidae)

The behavior of turfgrass-infesting scarab grubs in response to soil physical properties may affect the stress that each species exerts on turfgrass and the efficacy of control tactics. To gain a more realistic picture of the events that occur within the soil matrix, we have developed a nondestructive X-ray technique to study soil insect movement and behavior in simulated and natural soil blocks in the laboratory Laboratory studies using this technique were done to determine the effect of some soil physical factors on scarab grub movement patterns. Species-specific differences were demonstrated in the responses of four scarab grub species (Japanese beetle, Popillia japonica Newman; European chafer, Rhizotrogus mqalis (Razoumowsky); oriental beetle, Anomala orientalis Waterhouse; and northern masked chafer Cyclocephala borealis (Arrow) ) to changing temperature and moisture conditions. Studies also were done to determine the effect of soil moisture on the movement and persistence of an insecticide (isofenphos) applied to turfgrass and its effect on European chafer grub movement and mortality This study showed that isofenphos was relatively nonmobile under our experimental conditions, and that insecticide efficacy depended on factors, such as soil moisture, that influence the position of grubs in the soil profile. We believe that a better understanding of the interactions among grub behavior, insecticide persistence, and movement, as illustrated by this research, will improve our ability to manage scarab grubs in turfgrass and will be applicable to additional soil systems. I N THE NORTHEASTERN United States, turfgrass is subject to intense feeding pressure from a complex of scarab beetles that cause extensive damage as immatures (Tashiro 1987). Although the adult behavior of many members is of the scarab pest complex and has been well studied, the species-specific behavior of the immatu re stages has been generally ignored. However, species-specific differences of scarab grubs in response to the physical properties of soil may affect the stress that each species exerts on turfgrass and the efficacy of control tactics. Information about the behavior of scarab grubs is anecdotal in most instances because of the difficulty in studying the behavior of soil insects under natural conditions. There is a general perception that such research, if not impossible, is difficult, tedious, and not cost-effective when compared with similar studies of aboveground insects. This attitude has resulted in the soil ecosystem being treated as a black box in which the consequences rather than the processes of insect behavior are measured. The control of soil insects that damage turfgrass and horticultural commodities has been inconsistent since the loss of the cyclodiene insecticides (Harris 1972, 1982; Baker 1986). One reason for this inconsistency is the lack of understanding of the interaction of the control tactic (chemical, biological, or genetic), the cropping system, the target insect, and physical (soil moisture, temperature, texture, and compaction) and biotic (presence of microbial flora and other organisms) environment of the soil. Although each of these factors has been independently studied by basic and applied researchers, their interdependence has been ignored in most studies. Understanding the behavior of soil insects found in turfgrass is important to pest management because of the difficulty in moving soil insecticides down into the soil of established turfgrass. If thatch is in the upper soil profile, binding and degradation may increase with several soil insecticides (Niemczyk & Krueger 1982, Niemczyk & Chapman 1987). Physical factors that make target insects move as little as 1 cm into the soil profile may put these insects out of the effective active zone of chemical and biological control agents. The failure in predicting soil insect damage and the costs of satisfactory control dictate a more systematic and comprehensive approach to soil insect research. Paramount to such an approach is the ability to monitor the response of soil insects to static and dynamic environmental factors in the soil and to manipulate these factors to determine changes in insect response. Understanding the fundamental differences in behavioral response among species of soil insects to a variety of typical soil factors will enhance our ability to predict the stress each species will inflict on a particular crop. An improved understanding of the interaction of the control agent and the target species with the soil environment should lead to changes in management practices that increase the level of overlap between control agent and target pest. In this paper, we describe a technique that uses radiography which permits us to study soil insect behavior using nondestructive sampling and to make repeated observations over short intervals. We used this technique to study the movement behavior of several scarab grub species, and we present data that demonstrate the influence of soil moisture and soil temperature on vertical g rub movement. We also used radiography and analytical chemical analysis to study the interaction of a turf insecticide, a grub species, and the simulated turf environment. Our aim was to illustrate how a combination of laboratory research techniques in model systems can be useful in improving our understanding of field phenomena. Radiography in Soil Insect Research Although there have been field studies that look at the movement of soil insects over Long periods (Forbes 1907, Griddle 1918, McCollock & Hayes 1923, Mail 1930, Dowdy 1944, Hawley 1949, LaFrance 1968, Fisher et al. 1975), these studies considered the soil insects found i n the top 5 cm as being at the surface layer. Detailed k110w1edge of the movement of grubs within the top 5 cm over short periods can have a profound effect on pest management. Laboratory studies of the movement and feeding behavior of soil insects often were done outside the soil (Fulton 1928) or between thin sandwiches of glass plates and sterile, homogeneous sand (Doane et al. MICHAEL G. VILLANI is a n mistantprofessor of soil insect ecology in the Department of EntomologY, New York State Agricultural Experintent Station, Gene~ja N.Y 14456. ROBERT J. WRIGHT is a n entomologist formerly with Cornell hi~lersity at Long Island HorticulturaL Research Lab, Ritlerhead N.Y He is curvent<y an, assistant professor with Department of Entomology, University of Nebraska, Lincoln, Nebr 68583, serving as extension IPM specialist. 1975), which strongly modified insect movement patterns. Villani & Gould (1986) developed an X-ray technique that makes it possible to study soil insect movement and behavior within heterogeneous soil blocks. prior use of this technique to monitor soil insect behavior has been limited to small arenas (30 by 4 by 15 cm) in simulated soils. also required the use of expensive X-ray film and substantial darkroom time. We expanded this technique to study several species of scarab grubs in larger arenas (up to 35 by 12 by 43 cm with large grubs as subjects; smaller grubs require proportionally thinner arenas) and with field-collected soil blocks, and we used a paper rather than film base (this is approximately 90% cheaper, requires only 5 s to develop, and does not require a wet darkroom for processing). Radiographs of soil blocks are of sufficient quality for us to identify the location of grubs within the soil profile. Soil blocks retain their field characteristics (compaction, heterogeneity, and endemic floral and faunal communities) and therefore allow careful monitoring and manipulation of the system "or long periods. The radiograph5 were taken with the Hewlett-Packard Faxitron (model 43855B, Hewlett-Packard Company, Palo Alto, Calif.). This unit is a self-contained, radiationshielded cabinet X-ray system designed to give high-resolution radiographs of small to medium-sized objects. Millamperage is rated 3.00 and voltage is continuously variable with a range from 10 through 130 kV (kilovoltage); this output allows for visualization of 2.5-cm steel plate, 15-cm aluminum plate, or their equivalent. Exposures for these studies were at 70 kV for 20 s per exposure. An Industrex Instant Processor (model P-1, Kodak, Rochester, N.Y.) was used to produce fully processed radiographs on sheets of Kodak Industrex Instant 600 paper in 10 s. The radiographs can be viewed in direct light. Fig. 1-5 are examples of laboratory radiographs of scarab grubs. Fig. 1 shows a simulated soil block, and Fig. 2-5 are field-collected soil blocks (35 by 8 by 43 cm). Scrab Grub Movement Monitored by Radiography All studies were conducted in Plexiglas arenas (35 by 5 by 43 cm) filled with sieved loamy sand soil (organic matter content, 2.4%; soil pH 6.9). These dimensions allowed the scarab grubs to move in all directions until the constraints of the walls were met. We needed a method for presenting data gathered through our radiography studies that provided a direct view of the location of the grub population in the soil Profile. The use of boxplots (McGill et al. 1978) provided us with such a tool. These boxplots provide more useful information than does the use of means and variances (they allow identification of outliers, which may be important in studies of insect behavior), and they are more amenable to interpretation than is simple plotting of frequency distribution data. Effects of Moisture Fluctuation. Many earlier reports (reviewed by Tashiro [1987]) indicate that grub species respond to soil moisture gradients differently. Our study examined the species-specific responses of four scarab grub species to conditions that simulate irrigation (or rainfall) and drought. We seeded each arena with about 3 g of a mixture (of 49% red fescue, Festuca rubra L.; 19% Kentucky bluegrass, Poapratensis L.; 15% perennial ryegrass, Loliunz perenne L.; 14% Chewings fescue, P rubra commutata Gaud; and 3% other) and then held at 20°C with a 12:12 (L:D) photoperiod for 7-10 wk. Each arena was then infested with 10 third-instar grubs of one of four species (European chafer [EC], Rhizotrogzls majalis (Razoumowsky); Japanese beetle DB], Popillia japonica Newman; oriental beetle [OBI, Anomala orientalis Waterhouse; and northern masked chafer [MC], or Cyclocephala borealis (Arrow) ), with a to. tal of 30 grubs of each species (3 arenas for each scarab grub species). Grubs were placed on the turfgrass and allowed to dig down. Grubs that did not dig into the soil within 1 h were replaced. Within 48 h after infestation, the arenas were placed in continuous darkness at 20°C for the duration of the experiments. The scarab grubs were collected in the fall or spring from turfgrass at different sites (one site per species in each study) in New York from areas that had not been treated with insecticides that year. Grubs collected in the fall were stored at 10°C in soil covered with sod for up to 4 mo until they were used in these studies. Grubs collected in the spring were used within 2 wk of their collection. Test 1. Response to Irrigation. Soil moisture was measured gravimetrically at intervals throughout the soil profile at the beginning and end of the study (0 and 315 h after infestation [HAI]). Infested arenas were Xrayed at 4, 8, 12, 24, 32, 48, 56, 72, 80, 104, 128, 152, 176, 200, and 224 HAI. Water (100 ml/arena) was added at 32 and 200 HAI to simulate irrigation of a soil with 1.0 cm of water. This is the amount recommended in New York after application of soil insecticides for control of scarab grubs in turfgrass (Smith & Wilson 1987). At the end of the study, each box was X-rayed and then taken apart, and the position of each grub was recorded. Any dead grubs identified at this time were traced back in time by examining X-rays that had been made up to the point when the grub stopped changing positions. All data following the last change in position were removed from the analysis. Fig. 6a shows the changes in moisture throughout the soil profile from the start of the experiment to the final sampling. Water was added to the arenas at 32 h , and it moved slowly through the soil profile. Parallel studies that used probes to measure soil moisture tension at 2.5,5, 10, and 20 cm indicated that the moisture front reached the 10-cm point in approximately 48 h and did not move beyond that point for the duration of the study Soil moisture remained fairly constant in the upper profiles of the arenas until approximately hour 200, when additional water was added. The rate of movement of each species and the changes in population distribution can be displayed easily with boxplots. Fig. 7 illustrates the information provided by the boxplot display of the grub position data. Although not shown, boxplots can be used to determine statistical differences within species over time and among species for a single period of observation (McGill et al. 1978). The X-ray procedure allowed us to monitor the response of the same population to changing moisture conditions over time (Fig. 8). In terms of the responses of the medians over time, all species moved upward after water was added. However, wide differences in response can be seen in the four grub species in terms of the response of population medians and in the degree of variation around the median. Of particular note in these boxplots is the initial movement of all grub species downward from regions of low soil moisture to regions of higher soil moisture. In all cases, the shapes of the curves are similar, but the magnitude of the movement and the rate of population response are species specific. The most dramatic movement patterns are in the response of EC to dry soil conditions and the protracted movement of the population back toward the soil surface. We believe that EC grubs that contact the moisture front move rapidly to the surface. Relatively slow population response (as indicated by the extended lower tail of the boxplots) can be attributed to lack of contact between grubs and moisture in the lower soil profile. The movement of all species downward in the profile toward the end of the study may be a response to slightly lower soil moisture in the upper 2.5 cm of soil (Fig. 8) and also may involve a behavioral response to the prepupal state. Test 2. Response to Drought Simulation. Procedures for the second test were similar to those in Test 1, except that the soil was allowed to dry during the study period. This study was conducted to determine whether Fig. 1. Radiograph of simulated uniform-texture soil (loamy sand) containing two species of scarab grub. Two moisture levels can be seen (E); differences in radiograph tone density are attributable to differences in soil matrix density. The lighter upper surface indicates greater levels of soil moisture. The moisture fills most microand macro pores. Tbe lower stratum contains less moisture and more air in the soilpores, which increases the penetration of X-rays to the film. Two large Phyllophaga sp. grubs (A) are seen in their arenus; three smaller JB grubs (B) are closer to the surface of the soil (D). Although the soil conditions are not ideal to show tunneling, one Phyllophaga sp. burrow (C) can be seen. Note the apparent stratijication by species and the location of the Phyllophaga sp. at the soil moisture interface. 134 BULLETIN OF THE ESA Fig. 2. Radiograph of a soil block takm from moist, stratged soil of a weedy orchard. Tho grub species (A, Phyllophaga sp.; B, EC) were inwoduced to the top of the soil (F) 3 d before the radiograph was made. A, third-instar Phyllophaga sp.; B, third-instar EC; C, rock; D, interface of topsoil and sandy horizon (cracking is due to collection); 4 soil surface; G, burrows of soil organisms captured in soil block, most likely small earthworms.