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Microsporidian bioassay technique for third-instar Pseudaletia unipuncta larvae
JOURNAL
OF INVERTEBRATE
PATtIOLOGY
27,397-398
(1976)
Microsporidian Bioassay Technique for Third-lnstar ~se~~~/etj~ ~~j~~n~t~ Larvae In general, early-instar lepidopterous larvae can be infected by a smaller dose of microsporidian spores than late-instar larvae when held under similar conditions. However, the techniques often used to inoculate late-instar larvae are inappropriate for the first, second, or third instars. Bioassays designed to determine various dosage-response phenomena such as lethal doses, effective doses, infective doses, and lethal times or to establish host ranges of microsporidian species require a sensitive, reproducible method that provides the greatest probability of success. Microsporidian infections have been induced expe~mentally in insects by various means including surface application of spores to artificial media (W. M. Brooks, J. Znvertebr. Puthol., 16, 390-399, 1970), contamination of the insect’s mouthparts (W. R. Kellen and J. E. Lindegren, J. invertebr. Palhol. 21, 293-300, 1973), mixing of spores within baits (R. E. McLaughlin, R. J. Daum, and M. R. Bell, J. Invertebr. Pathol. 12, 168-174, 1968; J. E. Henry, K. Tiahrt, and E. A. Oma, J. Znvertebr. Pathol. 21, 263-272, 1973) loopfeeding of aqueous spore suspensions (J. V. Maddox, unpubl.), force-feeding of spore suspensions with a microapplicator (G. L. Nordin, and J. V. Maddox, J. Invertebr. Pathol. 24, l-1 3, 1974), and injection of spores into the host’s hemocoel (A. H, Undeen, and J. V. Maddox, J. Invertebr. Pathol. 22, 258-265, 1973). ‘Each of these methods has inherent limitations. This paper describes a surface application technique for administering Nosema necatrix spores to third-instar Pseadalet~a un~~~nctu larvae using inexpensive reusable bioassay containers. Each bioassay unit consisted of a I-oz disposable plastic medicine cup’ partially filled ‘Supplied Paso, Texas
by Thunderbird 799 12.
Container
Corporation,
El
with a congealed semidefined diet, one glass tube (7 x 25 mm) made from a Pasteur pipet by scoring and breaking the glass at the neck const~ction, and a small cotton stopper for inserting into the broken end of the bioassay tube (Fig. 1). Because early-instar larvae consume little of the total diet surface in the cup, both the spore dose and the larvae must be confined to a smaller area. The confining area was equivalent to the inside diameter of a Pasteur pipet (5.0 mm) and was delineated by inserting the top of the pipet approximately 10 mm into the diet surface near the center of the cup and then carefully withdrawing the pipet. Care was taken to insure that the inoculation area was completely isolated from the remainder of the medium so that the inoculum was not dispersed beyond the limits set. Next, a 2-~1 aliquot from a serial decimaldilution series of N. necatrix spores (see Table 1) was dispensed on to the center of the inoculation area with a Hamilton syringe and allowed to air dry (5-10 min). Larvae were then placed individually in the bioassay tubes fitted with stoppers. The tubes were then inserted into the diet over the inoculated surface until the top of each tube was slightly below the rim of the medicine cup. Cups were left uncapped until the entire inoculated area was consumed. If the cups were tightly sealed prior to this time, excess moisture accumulated within the tubes. Furthermore, the inverted position allowed the frass to fall away from the diet surface permitting easy inspection of the feeding progress. Plastic food crispers (7pa x lOpa x 3% in.) were convenient for holding the bioassay units during the course of experimentation. Larvae were reared at 24°C on a 16-hr-light, 8-hrdark photophase. When all the inoculated area had been consumed, the tubes were removed so that the larvae were free to feed upon the re-
397 Copyright All rights
D 1976 by Academic Press, of reproduction in any form
Inc. reserved.
398
NOTES
Infective
Spore dose per Larva 106 10s 104 103
102 0
Dose-Response
Number treated
of Third-Instar Surface Application
Number consuming diet within 48 hr.
30 30 30 30 30 30
‘Percentage of infection Abbott’s formula.
TABLE 1 Pseudaletia unipuncta of Spores to Artificial
Number infected
Infected’ (%)
21 18 16 8 4 0
87.5 69.2 59.2 32.0 14.3
24 26 27 25 28 27 calculated
from
Larvae to Noserna Media
number
consuming
FIG. I. Schematic diagram of bioassay unit in cross section, inverted position. (A), Medium within I-oz medicine cup; (B), bioassay tube made from a Pasteur pipet; (C), cotton stopper; (D), diet surface area receiving inoculum.
mainder of the diet within the capped medicine cups for 2 weeks. Then, all alive and dead larvae were examined microscopically for evidence of infection. Bioassay tubes 70-
bO-
Calculated probit 6.16 5.62 5.07 4.53 3.99
rlecatvix
by
95’5 Fiducial limits (probits) Lower
Upper
5.71 5.30 4.83 4.24 3.56
6.62 5.93 5.31 4.82 4.41
0 entire
diet
surface
within
48 hr:
corrected
by
and stoppers were autoclaved, cleaned, and reused in later experiments. Table 1 presents the results of the bioassay with the lower and upper 95% fiducial limits for each dose. The infective dose-response regression line (y = 2.8436 + 0.5440x) with a standard error of 0.3773 is shown in Figure 2. The ID,, value was 7,313 spores/larva with 95% lower and upper fiducial limits of 99 1 and 50,406 spores/larva, respectively. Although this technique has been used successfully on microsporidian bioassays, other types of pathogens such as occluded viruses may be bioassayed by the same technique provided that the test larvae readily accept an artificial medium and are surface feeders during the early instars. This technique is particularly useful when only a small amount of inoculum is available for testing. This paper (No. 75-7-34) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. The author wishes to thank Dr. J. V. Maddox of the Illinois Natural History Survey, Urbana, Illinois, for probit analysis computer program time.
G. L. FIG. 2. Infective dose-response third instar Pseudaletia unipuncta cat& with 95% fiducial limits.
regression line for larvae to Nosema ne-
Department of Entomology University of Kentucky Lexington, Kentucky 40506 Received March 5, 1975
NORDIN
OF INVERTEBRATE
PATtIOLOGY
27,397-398
(1976)
Microsporidian Bioassay Technique for Third-lnstar ~se~~~/etj~ ~~j~~n~t~ Larvae In general, early-instar lepidopterous larvae can be infected by a smaller dose of microsporidian spores than late-instar larvae when held under similar conditions. However, the techniques often used to inoculate late-instar larvae are inappropriate for the first, second, or third instars. Bioassays designed to determine various dosage-response phenomena such as lethal doses, effective doses, infective doses, and lethal times or to establish host ranges of microsporidian species require a sensitive, reproducible method that provides the greatest probability of success. Microsporidian infections have been induced expe~mentally in insects by various means including surface application of spores to artificial media (W. M. Brooks, J. Znvertebr. Puthol., 16, 390-399, 1970), contamination of the insect’s mouthparts (W. R. Kellen and J. E. Lindegren, J. invertebr. Palhol. 21, 293-300, 1973), mixing of spores within baits (R. E. McLaughlin, R. J. Daum, and M. R. Bell, J. Invertebr. Pathol. 12, 168-174, 1968; J. E. Henry, K. Tiahrt, and E. A. Oma, J. Znvertebr. Pathol. 21, 263-272, 1973) loopfeeding of aqueous spore suspensions (J. V. Maddox, unpubl.), force-feeding of spore suspensions with a microapplicator (G. L. Nordin, and J. V. Maddox, J. Invertebr. Pathol. 24, l-1 3, 1974), and injection of spores into the host’s hemocoel (A. H, Undeen, and J. V. Maddox, J. Invertebr. Pathol. 22, 258-265, 1973). ‘Each of these methods has inherent limitations. This paper describes a surface application technique for administering Nosema necatrix spores to third-instar Pseadalet~a un~~~nctu larvae using inexpensive reusable bioassay containers. Each bioassay unit consisted of a I-oz disposable plastic medicine cup’ partially filled ‘Supplied Paso, Texas
by Thunderbird 799 12.
Container
Corporation,
El
with a congealed semidefined diet, one glass tube (7 x 25 mm) made from a Pasteur pipet by scoring and breaking the glass at the neck const~ction, and a small cotton stopper for inserting into the broken end of the bioassay tube (Fig. 1). Because early-instar larvae consume little of the total diet surface in the cup, both the spore dose and the larvae must be confined to a smaller area. The confining area was equivalent to the inside diameter of a Pasteur pipet (5.0 mm) and was delineated by inserting the top of the pipet approximately 10 mm into the diet surface near the center of the cup and then carefully withdrawing the pipet. Care was taken to insure that the inoculation area was completely isolated from the remainder of the medium so that the inoculum was not dispersed beyond the limits set. Next, a 2-~1 aliquot from a serial decimaldilution series of N. necatrix spores (see Table 1) was dispensed on to the center of the inoculation area with a Hamilton syringe and allowed to air dry (5-10 min). Larvae were then placed individually in the bioassay tubes fitted with stoppers. The tubes were then inserted into the diet over the inoculated surface until the top of each tube was slightly below the rim of the medicine cup. Cups were left uncapped until the entire inoculated area was consumed. If the cups were tightly sealed prior to this time, excess moisture accumulated within the tubes. Furthermore, the inverted position allowed the frass to fall away from the diet surface permitting easy inspection of the feeding progress. Plastic food crispers (7pa x lOpa x 3% in.) were convenient for holding the bioassay units during the course of experimentation. Larvae were reared at 24°C on a 16-hr-light, 8-hrdark photophase. When all the inoculated area had been consumed, the tubes were removed so that the larvae were free to feed upon the re-
397 Copyright All rights
D 1976 by Academic Press, of reproduction in any form
Inc. reserved.
398
NOTES
Infective
Spore dose per Larva 106 10s 104 103
102 0
Dose-Response
Number treated
of Third-Instar Surface Application
Number consuming diet within 48 hr.
30 30 30 30 30 30
‘Percentage of infection Abbott’s formula.
TABLE 1 Pseudaletia unipuncta of Spores to Artificial
Number infected
Infected’ (%)
21 18 16 8 4 0
87.5 69.2 59.2 32.0 14.3
24 26 27 25 28 27 calculated
from
Larvae to Noserna Media
number
consuming
FIG. I. Schematic diagram of bioassay unit in cross section, inverted position. (A), Medium within I-oz medicine cup; (B), bioassay tube made from a Pasteur pipet; (C), cotton stopper; (D), diet surface area receiving inoculum.
mainder of the diet within the capped medicine cups for 2 weeks. Then, all alive and dead larvae were examined microscopically for evidence of infection. Bioassay tubes 70-
bO-
Calculated probit 6.16 5.62 5.07 4.53 3.99
rlecatvix
by
95’5 Fiducial limits (probits) Lower
Upper
5.71 5.30 4.83 4.24 3.56
6.62 5.93 5.31 4.82 4.41
0 entire
diet
surface
within
48 hr:
corrected
by
and stoppers were autoclaved, cleaned, and reused in later experiments. Table 1 presents the results of the bioassay with the lower and upper 95% fiducial limits for each dose. The infective dose-response regression line (y = 2.8436 + 0.5440x) with a standard error of 0.3773 is shown in Figure 2. The ID,, value was 7,313 spores/larva with 95% lower and upper fiducial limits of 99 1 and 50,406 spores/larva, respectively. Although this technique has been used successfully on microsporidian bioassays, other types of pathogens such as occluded viruses may be bioassayed by the same technique provided that the test larvae readily accept an artificial medium and are surface feeders during the early instars. This technique is particularly useful when only a small amount of inoculum is available for testing. This paper (No. 75-7-34) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. The author wishes to thank Dr. J. V. Maddox of the Illinois Natural History Survey, Urbana, Illinois, for probit analysis computer program time.
G. L. FIG. 2. Infective dose-response third instar Pseudaletia unipuncta cat& with 95% fiducial limits.
regression line for larvae to Nosema ne-
Department of Entomology University of Kentucky Lexington, Kentucky 40506 Received March 5, 1975
NORDIN