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I: Biological Control (12 Articles) |
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I.3 Laboratory Bioassays of Nosema locustaeMichael B. Hildreth, Chris W. Brey, Billy W. Fuller, and R. Nelson Foster Introduction IntroductionThe use of living insect pathogens as biocontrol agents for insects requires that the virulence (killing power) of these agents must be monitored occasionally, especially just prior to their distribution into the environment. Evaluation of an agent's virulence can be accomplished through the use of laboratory bioassays involving the target insects (raised in the laboratory) and the biocontrol agents that are to be tested. The first biocontrol agent registered by the U.S. Environmental Protection Agency for grasshopper pests was the protozoan Nosema locustae. Grasshoppers acquire N. locustae infections by eating its spore stage. N. locustae infects the fat bodies of grasshoppers and is only mildly pathogenic to its host. For several years, our lab at South Dakota State University (SDSU) has been bioassaying the viability and virulence of N. locustae spores supplied commercially to the U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine (USDA, APHIS, PPQ) Grasshopper Integrated Pest Management (GHIPM) Project. Laboratory-reared third-instar Melanoplus sanguinipes grasshoppers were used as the target insect for these bioassays, and the grasshoppers were fed the Nosema spores on small disks cut from romaine lettuce. The grasshoppers were then kept in the lab for 20 days, and LD50 (the calculated dose of pathogen or toxin that kills half of the bioassayed grasshoppers) values were calculated based upon the percentage of grasshoppers that had died by the end of the time period. When Nosema is used to control grasshoppers, spores are typically applied on rangelands with a wheat-bran bait. Lettuce bioassays can be used only to measure the viability of spores prior to the spores' addition to wheat bran. The purpose of this chapter is to describe the protocol used in our laboratory to measure the virulence of N. locustae spores stored in water and applied to lettuce disks, and also to describe a bioassay protocol that we've used for measuring the virulence of these spores after their addition to wheat bran. Representative results from these bioassays are reported in this chapter. Lettuce BioassayMethods. - Nosema locustae spores used for these studies were provided by various commercial sources. All spores were stored in distilled water at -4°F (-20°C) until use. Melanoplus sanguinipes grasshoppers used in the studies were a lab-reared Canadian strain that had been maintained at SDSU for several years. These grasshoppers were reared according to the recommendations provided by Henry (1985). The lettuce bioassay was based upon recommendations supplied by John Henry (personal communication). Spores were counted in a hemocytometer (a special slide used in hospitals to count blood cells) and applied to freshly cut lettuce disks approximately one-third inch (7 mm) in diameter disks using a 10 mL (microliter) pipettor. Six dosages of N. locustae spores in 10 mL distilled water (e.g., 0, 1 x 104, 104.5, 105, 105.5, 106 spores; 1 x 104.5 is equal to 3.162 x 104 or 31,620) were added to the disks (120 disks per dosage), and allowed to dry for 1 to 4 hours. Each disk was fed individually to a third-instar grasshopper that had been previously starved for 1 day in glass vials at approximately 86 °F (30 °C). To distribute the grasshoppers into vials, the insects needed to be cooled briefly from ambient 86 °F (30 °C) to approximately 39 °F (4 °C). Before adding the appropriate lettuce disks to the vials, the vials were randomly sorted and divided into the appropriate six dosage groups. Once 80 grasshoppers from each group had eaten an entire disk, they were placed in groups of 5 into 16 bioassay tubes (8 inches or 20 cm long, 2.75 inches or 7 cm in diameter) constructed of 0.08-inch (0.02-mm) sheet acetate with screened ends. Generally, grasshoppers ate an entire disk within 2 hours or did not eat it even after 12 hours. The 16 bioassay tubes were divided into 4 replicates of 4 tubes each. In the bioassay tubes, grasshoppers were fed laboratory-reared rye grass daily along with triple sulfa-coated rolled oats (Henry and Oma 1975) and maintained under continuous fluorescent illumination at approximately 86 °F (30 °C). Each day, we counted the number of dead grasshopper carcasses in the bioassay tubes. Grasshoppers frequently cannibalized other grasshoppers in the tubes, and portions of carcasses often were found. Therefore, we verified the number of living grasshoppers remaining in each tube to not overlook cannibalized individuals. We calculated LD50 values by using the software package POLO-PC (LeOra Software, Inc., Cary, NC). Results.-An example of the typical results obtained from the 22 bioassays conducted in our lab during the past 5 years is shown in figure I.3-1. A few of the uninfected control grasshoppers always died during the 20 days of each bioassay. For all of the 22 bioassays, generally less than 20 percent of the control grasshoppers died before the end of the bioassay. Inoculation of grasshoppers with increasing numbers of N. locustae spores consistently increased the mortality rate for grasshoppers infected with 1 x 106 , 1 x 105.5, and 1 x 105 spores. Grasshoppers inoculated with 1 x 106 Nosema spores typically started to die sooner than the control grasshoppers within the first 8 days postinoculation (p.i.); the largest number of deaths normally occurred between days 10 and 14 p.i. By 20 days p.i., 70 to 100 percent of the grasshoppers infected with 1 million (1 x 106) spores had died among the various bioassays performed. It typically took grasshoppers inoculated with 1 x 105.5 spores longer to die than it did for grasshoppers infected with 1 x 106, and fewer grasshopper had died by the 20-day bioassay period (generally 40 to 90 percent). The mortality rate for grasshoppers dosed with 1 x 105 spores tended to separate gradually from the control mortality, and usually became consistently apparent only after 16 days p.i. Mortality in grasshoppers infected with the two lower concentrations (1 x 104 and 104.5) did not consistently differ from those of the controls even at 20 days p.i. The calculated LD50 for the bioassay shown in figure I.3-1 was 1.19 x 105 at 20 days p.i.
Figure I.3-1-Percent mortality of a 1990 tube bioassay involving third-instar Melanoplus sanguinipes grasshoppers treated with varying dosages of Nosema locustae spores on lettuce disks (e.g., 0, 1 x 104 , 104.5 , 105 , 105.5, 106) and maintained for 20 days postinoculation at approximately 86 °F (30 °C). Solid triangle = 0 spores/grasshopper, open square = 1 x 104 spores/grasshopper, open triangle = 1 x 104.5 spores/grasshopper, solid square = 1 x 105 spores/grasshopper, open circle = 1 x 105.5 spores/ grasshopper, and solid circle = 1 x 106 spores/grasshopper.
Bran BioassayMethods. - To calculate the theoretical quantity of spores present on average-size flakes of commercially formulated bran, we filtered several grams of the bran through a series of wire sieves with diminishing pore sizes. Most of the flakes were collected on three sieves with pore sizes of 2.36 mm (mesh 8), 2.00 mm (mesh 10), and 1.70 mm (mesh 12). From each of these sieves, 100 flakes were weighed individually. The quantity of spores on each group of flakes was calculated based upon the assumption that each pound of bran contained 1 x 109 spores. As described in the results section, the theoretical concentration of spores on flakes of commercially formulated bran (an average 1-mg flake should contain 2.2 x 103 spores) was roughly 100 times lower than the concentration of spores easily detected in laboratory bioassays (1 x 105.5 or 3.16 x 105 spores could easily be detected based upon their effect on grasshopper deaths). Therefore, in order to bioassay spores on a single bran flake, it was necessary to formulate new bran with spores at a concentration 100 times that of commercially formulated bran (1011 spores/lb instead of 109 spores/lb). The spores were sprayed onto wheat bran while continually mixing the bran with a small cement mixer. These spores had been recently recovered from grasshoppers and bioassayed on lettuce in our lab (LD50 value was 3.29 x 105). In addition to the spores, the spray solution contained 0.2 percent weight to volume (w/v) hydroxymethyl cellulose in distilled water. Hydroxymethyl cellulose is thought to help the spores stick to the bran (Henry et al. 1973). An aerosol sprayer was used to spray the solution on the bran. The treated bran was then allowed to dry and was stored at 39 °F (4 °C). Attempts were made to bioassay the 100x-treated bran using the same approach used for the lettuce bioassay. One week after formulation of the 100x bran, third-instar grasshoppers were cooled as described above and distributed individually into glass vials. The grasshoppers were starved for 24 hours, randomized, and divided into four groups. Treated bran flakes of different sizes (sieved through mesh 8, 10, or 12) were added to each appropriate vial. Untreated control flakes (sieved only through mesh size 10) were added to the tubes containing control grasshoppers. Once 80 grasshoppers from each group had consumed all bran flakes, they were placed in groups of 5 into 16 bioassay tubes and maintained as described for the lettuce bioassay. Results from the single-flake bran bioassay study suggested that each grasshopper needed to consume additional bran before any effect could be detected. Therefore, an attempt was made to enable each grasshopper to consume a maximum quantity of treated bran before inclusion in a second bioassay. For that bioassay, 100 grasshoppers were maintained in a large screened rearing cage (30 x 32 x 55 cm) for 48 hours. The only food source during this time was 2.0 g of control or treated bran contained in a standard petri dish. After 24 hours, the uneaten bran was replaced with fresh. Weights were determined from each container of bran and compared to the weights of similar bran maintained similarly just outside the cage. At the end of the bioassay period, the grasshoppers were maintained in bioassay tubes as described for the single-flake bioassay. Results. - The average weight for each size of Nolo Bait® bran flakes and the estimated number of spores per flake are shown in table I.3-1. The average values ranged from 1.42 mg for larger flakes sieved through mesh 8 to 0.625 mg for flakes sieved through mesh 12. If 1 x 109 spores are added to each pound of bran, then each milligram of flakes should contain 2.20 x 103 spores; therefore, the largest flake weighed in this study (2.2 mg) should contain 4.85 x 103 spores.
Table I.3-1-Average weight in milligrams for each size of bran flakes and estimated spores per flake
Figure I.3-2 illustrates the mortality rates of grasshoppers fed only one flake of 100x-treated bran from each of the various sieves. Because the average flake of bran weighed 1.05 mg, it should contain approximately 2.32 x 105 spores. After 30 days, the mortality rates from the experimental groups of grasshoppers were not significantly greater than that of the controls. In fact, fewer of the grasshoppers receiving the small flakes of experimental bran died than did the control. Unfortunately, however, the mortality rate for the control grasshoppers in this experiment was twice that of previous experiments, and may have obscured any small effects caused by Nosema.
Figure I.3-2-Initial tube bioassay involving N. locustae-treated wheat bran flakes given individually to third-instar M. sanguinipes maintained for 34 days postinoculation. Solid triangle = grasshoppers given an untreated flake of bran; open triangle = grasshoppers given a treated bran flake that passed through a mesh 7 sieve but not the mesh 8 sieve; open square = flake passed through mesh 8 but not mesh 10; solid square = flake passed through mesh 10 but not mesh 12. Spores had been added to the bran at a concentration of 1 x 1011 spores per pound of bran.
Grasshoppers given as much of the 100x-treated bran as they wanted for 2 days consumed an average of 2.56 mg on the first day and 1.20 mg on the second. Therefore, each experimental grasshopper consumed an average of 3.76 mg of treated bran (roughly 6 small flakes) or 8.27 x 105 spores by the end of the second day. At the end of 2 days, control grasshoppers consumed less than half of the bran consumed by the experimental grasshoppers (fig. I.3-3). Mortality at 30 days p.i. was 75 percent higher for experimental grasshoppers than for those receiving control bran (fig. I.3-4). Mortality rates increased significantly in the experimental grasshoppers after 14 days p.i.
Figure I.3-3-Consumption of control and experimental (Nosema-treated) bran by 400 grasshoppers in each group during the first and second day of the inoculation period. Values are expressed in grams consumed per grasshopper.
Figure I.3-4-Tube bioassay involving N. locustae-treated wheat bran given ad lib (from a petri dish) to third-instar M. sanguinipes maintained for 30 days postinoculation. Solid triangle = grasshoppers given untreated flakes of bran; open triangle = grasshoppers given 100 x strength treated bran.
ConclusionsThe LD50 values determined through the use of lettuce bioassays described in this chapter are generally similar to values reported in other studies. For example, Mussgnug and Henry (1979) calculated the LD50 for N. locustae in their study of M. sanguinipes to be 1.5 x 105 spores based upon a bioassay conducted for 24 days. In lettuce bioassays conducted at SDSU, spore quantities below 1 x 105 did not exhibit mortality rates that were consistently higher than those of the controls. Because the average bran flake from commercially prepared Nosema-treated bran theoretically contains only 2.32 x 103 spores, each grasshopper would need to ingest 43 flakes of treated bran to become inoculated with 1.0 x 105 spores. Melanoplus sanguinipes grasshoppers that were given only bran flakes during a 2-day period consumed an average of approximately six flakes of bran. In field studies, it is unlikely that many wild grasshoppers ingested more than 40 flakes of Nosema-treated bran; therefore, other factors must have influenced the reported effectiveness of N. locustae in the field (Henry 1971). By formulating bran with N. locustae spores at a concentration 100 times that which is generally sold commercially (1011 spores/lb versus 109 spores/lb), it was possible to measure mortality rates caused by the resultant Nosema infections. The results generally are consistent with those reported by Reuter et al. 1990 (unpubl.) when the 100x rate-compared to the standard rate and untreated populations only-resulted in significant mortality to one of two field-treated species tested in cages. Previous Article
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I Contents References CitedHenry, J. E. 1971. Experimental application of Nosema locustae for control of grasshoppers. Journal of Invertebrate Pathology 18: 389-194. Henry, J. E. 1985. Melanoplus spp. In: Singh, P.; Moore, R. F., eds. Handbook of insect rearing. Amsterdam: Elsevier Science Publishing B.V.: 451-464. Henry, J. E.; Oma, E. A. 1974. Effects of prolonged storage of spores on field applications of Nosema locustae (Microsporida: Nosematidae) against grasshoppers. Journal of Invertebrate Pathology 23: 371-377. Henry, J. E.; Oma, E. A. 1975. Sulfunamide antibiotic control of Malameba locustae (King and Taylor) and its effect on grasshoppers. Acrida 4: 217-226. Henry, J. E.; Tiahrt, K.; Oma, E. A. 1973. Importance of timing, spore concentrations, and spore carrier levels in application of Nosema locustae (Microsporida: Nosematidae) for control of grasshoppers. Journal of Invertebrate Pathology 21: 263-272. Mussgnug, G. L.; Henry, J. E. 1979. Compatibility of malathion and Nosema locustae Canning in Melanoplus sanguinipes. Acrida 8: 77-81. References Cited-UnpublishedReuter, K C.; Foster, R. N.; Hildreth, M.; Colletto, D.; Cushing, W. J.; Pucelik, M. J.; Kahler, D.; Houston, R.; Scott, A. 1990. Preliminary investigations of the effect of a greatly increased rate of Nosema locustae on rangeland grasshopper populations. In: Cooperative Grasshopper Integrated Pest Management Project, 1990 annual report. Boise, ID: U.S. Department of Agriculture, Animal and Plant Health Inspection Service: 169-174. Previous Article • Next Article • Section I Contents
Bioassays of Nosema locustae: An Outline of ProceduresI. Purpose of the outline is to describe two protocols to measure the virulence of Nosema locustae spores. A. First Protocol: used for spores stored in water B. Second Protocol: used for spores already adhered to bran II. Lettuce Bioassay A. Protocol
B. Results
III. Bran Bioassay A. Protocol
B. Results
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