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New storage methods and improved trapping techniques for the parasitic nematode Neoaplectana carpocapsae
JOURSAL
OF INVERTEBRATE
PATHOLOGY
33, 155-
158 (1979)
New Storage Methods and Improved Trapping Techniques Parasitic Nematode Neoaplectana carpocapsae
for the
J F. HOWELL Yakima
Agricultural
Research Laboratop. Federal Department of Agriculture.
Research, Yakima,
Science and Education Washington 98902
Administration.
U. S
Received June 5. 1978 The parasitic nematode Neoaplectana carpocapsae had consistently high survival when it was stored at 3°C on moist filter paper ca. 1 year. Oxygen-enriched storage atmospheres were found unnecessary. During the second year of storage, mortality was a little higher, an average of ca. 37%. Storage for 19 months did not significantly reduce nematode infectivity. Survival during trapping was improved by controlling the number of cadavers per trap. KEY WORDS: Neoaplectana carpocapsae; storage method: DD-136: storage method for nematode; Nematode; Neoaplectana curpocapsae.
INTRODUCTION
and storing Neoaplectana carpocapsae (DD-136), a neoaplectanid nematode that is a parasite of several insects, was necessary to obtain sufficient material for laboratory and field testing. Dutky et al. (1964) noted that survival of the nematode was variable when it was stored in liquid; they also noted that the concentration of nematodes affected storage life and recommended 50,00O/ml of water held at 7°C. Chu and Ching (1975) tested survival of densities of from 350 to lOOO/ml water and demonstrated that as density increased so did survival. They obtained 80% survival with a density lOOO/ml, a concentration only l/50 that recommended by Dutky et al. (1964). However, both groups of investigators suggested that the survival rate and storage technique could be improved. Dutky et al. (1964) obtained larval nematodes migrating from the host cadaver by trapping them in a water moat and then separating them from the water by decanting or filtration. However, in my rearing of this nematode at the Yakima Agricultural Research Laboratory, survival has been generally poor in the nematode trap prior to storage and also in storage. Though, if sur-
vival was good in the trap, successful storage was more likely. Failure to obtain good results in storing nematodes in fluid media prompted me to develop an alternative storage method, which has proven useful for me both for storing and shipping.
Accumulating
MATERIALS
AND METHODS
Some ad hoc rearings seemed to indicate that the size of the nematode trap influenced survival. Therefore, two sizes of traps were tested: (1) a large glass Petri dish (200 x 55 mm); and (2) a standard Petri dish (90 x 12 mm). In the large containers Petri dish covers (90 x 12 mm) wrapped with VWR’ grade 613 qualitative filter paper were used initially (culture A) as islands for holding the inoculated dead host larvae (Dutky et al., 1964); and small plastic lids (45 x 3 mm) covered with filter paper were used for islands in the 90 x 12-mm Petri dishes. Subsequently (culture B), the small plastic lids were used as islands in both containers. For culture A, the large containers (200 x 55 mm) were covered with Para-film 1This paper reports the results of research only. Mention of a commercial product in this paper does not constitute a recommendation for use by the U.S. Department of Agriculture. 155 0022-201 l/79/020155-04$01.00/O Copyright c 1979 by Academic Press. Inc. All rights of reproduction m any form reserved.
156
J F. HOWELL
lids and the small Petri dishes (90 x 12 mm) with glass Petri dish covers; for culture B, Para-film lids were used exclusively with both sizes of container. A 1: 1000 solution of formaldehyde-water was used as the trap fluid, 50 ml in the large dish and 6 ml in the small. For culture A, the dead host larvae were laid out on the islands side-by-side so they covered all the surface available (8- 10 on the small and 32-50 on the large islands). For culture B, the cadavers were spaced so there was free space equal to a cadaver between each one (four to five per island). The host larvae were western Spodoptera yellowstriped armyworm, praejka (from J. E. Halfhill) for culture A and Heliothis zea (supplied by Ray Patana, Cotton Insects Biological Control Lab., Tucson, Ariz.) for culture B. The rearing area was maintained at 21” ? 3°C and 55 ? 5% RH with a 16-hr photophase. Collection of nematode larvae began 7 days postinoculation of the host larvae.
FIG. 1. Storage system for Neoaplecrana with stacking ridges, and desiccator jar.
Nematodes were prepared for storage by filtering them from the trap fluid with a 13.5-cm-diameter porcelain vacuum filter equipped with 15-cm filter disks and then washing them twice in fresh formaldehyde-water solution (1: 1000). Samples were taken at this point to measure prestorage mortality. The filter disk containing the nematodes was either placed in 15-cmdiameter plastic dishes with lids (culture A) or on 15-cm-diameter watch glasses that were stored in a desiccator jar (culture B) (Fig. 1). (Each desiccator contained a saturated solution of ammonium phosphate [NH,H,PO,l for humidification; 93% RH at 20°C.) Each filter disk was wetted to just in excess of saturation with the formaldehyde- water solution. All storage was at 3” + 2°C. Dutky et al.‘s (1964) storage technique required purging the storage vessels with oxygen. To test the necessity of oxygenation 16 vessels were oxygenated and 12
carpocupsue consisting of filter disk, large watch glass
STORAGE TABLE RATE
OF SURVIVAL
Neoaplectana
1
OF THE PARASITIC
carpocapsae
TECHNIQUE
Two
IN
NEMATODE
SIZES OF TRAPS”
Percentage survivalb Days between
Trap size
collections
Small
Large
3 7 9 16
76.5 75.7 80.5 63.3
Culture A a 65.1 ab a 52.6 b a 58.3 ab ab 42.2 b
7 7 7 7 9
79.2 75.7 79.3 82.6 69.9
Culture B a 79.0 a a 75.5 a a 84.9 a a 88.6 a a 81.7 a
ferred to filter paper disks that were placed in plastic dishes containing six western yellowstriped armyworm larvae. After 4 days, insect mortality was scored, and the cadavers were dissected to determine the presence of reproducing female nematodes, a verification of cause of mortality. There were live replicates from each age group (check, 11 and 19 months storage), each replicate represented a separate collection of nematodes. The nematodes used in this study were originally obtained from S. Dutky, Insect Physiology Laboratory, Beltsville, Maryland. RESULTS
Average 79.6% a There were three and five replicates for each collection of cultures A and B respectively. b Means with common letters not significantly different at the 0.05 level of significance by a Duncan’s multiple range test.
were unoxygenated during storage; all nematodes were from culture A and stored for approximately 1 year. Nematodes taken from storage were tested to determine whether storage had altered infectivity. For this test, from 24,000 to 235,000 nematode larvae were trans-
Survival of culture A was somewhat higher in the smaller trap (Table l), but culture B was not affected significantly by trap size. Average percentage survival pre- and poststorage was similar for the first 11 months of storage (Table 2). However, survival after 18-19 months of storage was sometimes reduced. Infectivity was essentially 100% following storage (Table 3) as was the case for prestorage nematodes. DISCUSSION
Cultures A and B differed in the size of the island, number of insect cadavers per island, and the insect host. Nevertheless,
TABLE SURVIVAL
OF THE PARASITIC
NEMATODE
Test No.
No. of replicates
Average percentage survival prestorage
1 2 3 4 5 6
8 5 3 3 8 1
76.5 75.7 80.5 63.3 76.5 -
1 2 3
7 8 8
85.6 75.6 82.1
” Unoxygenated. b Oxygenated.
157
FOR N. CARPOCAPSAE
2
Neoaplectana
Months in storage (Period 1)
carpocapsae
ON MOIST
FILTER
PAPER
Average percentage survival
Months in storage (Period 2)
Average percentage survival
Culture A 11 11 10 10 11 9
69.7” 82.1” 86.7” 47.lb 51.7” 83.3”
19 19 18 18
50.8 15.0 74.4 52.1
Culture B” 6 5 4
90.7 73.2 73.0
158
J F. HOWELL TABLE COMPARISON
OF THE INFECTIVITY FOLLOWING
STORAGE
3
AND VIRULENCE PERIOD
OF
OF Neoaplertana
carpocapsae
11 AND 19 MONTHS
No. of insect larvae: Treatment Check 19 months storage 11 months storage
In test
Dead”
Alive
30 30 30
5a 24 b 29 b
5 2 0
0 Values with common letters are not significantly multiple range test.
the data (and observation) imply that crowding of cadavers together on a large island (50 vs. 20) results in increased mortality among the nematodes. The trap fluid often became cloudy in such high density traps but remained clear in the low density situations. However, the nematode larvae that emerged were the same size in both cultures, i.e., ca. 500-550 pm. Dutky et al. (1964) reported a relationship between nematode survival in storage and nematode density. In the present tests, nematode density averaged ca. 20,00O/ml in the small traps per trap period compared and 3000-4000/ml in the large traps, but these differences in density in the traps did not influence survival. Dutky ef al. (1964) suggested that a large surface-to-volume ratio was needed so gas exchange would prevent anoxia during storage. They found that anoxia was delayed or reduced by providing oxygenenriched storage atmospheres. However, with the filter paper storage method described here, the surface-to-volume ratio was maximized and oxygenation did not improve storage life. Nematodes stored for either 11 or 19 months were highly infective (Table 3). Infected insect larvae cease to feed soon after they become infested with nematodes. However, the western yellowstriped armyworm is very cannibalistic in the absence of food. Consequently, if infestation by the nematode is delayed, cannibalism occurs. Table 3 shows that there was more cannibalism among insect larvae inoculated
Cannibalized 20 4 1
different at the 0.05 level of significance by a Duncan’s
with nematodes that had been stored 19 months than among larvae exposed to nematodes stored 11 months. Since the time necessary for insect larvae to become infected was greater with the older nematodes, prolonged storage appeared to affect infectivity. This change in infectivity was insignificant for cultural purposes since 100% of the larvae were usually infected; it simply took longer. Thus if the nematode is to be used as a pathogen for insect control, prolonged storage would have a significant and detrimental effect. No optimum or maximum storage capacity per unit (filter disk) was established. However, some units contained 500,000 nematodes, and I felt this was far below capacity. In conclusion, the described storage technique (saturated filter papers) gave consistent results; was space conservative, simple, and cheap and facilitated handling and transportation. However, in storage, moisture constantly coalesces on the sides of’the storage vessels and is slowly but continually lost from the filters. Therefore, periodic checks and the addition of moisture is necessary. It is important to keep the filters moist. Survival is not possible on dry filters. REFERENCES CHU. Y. I., AND CHING, H. A. 1975. The storage method of parasitic nematode DD-136. PIarzt Prof. Bull. Tahvan. 17, 115-120. DUTKY, S. R., THOMPSON, J. V., AKD CANTWELL, G. E. 1964. A technique for mass propagation of the DD-136 nematode. J. Insecr Pathol.. 6, 417-422.
OF INVERTEBRATE
PATHOLOGY
33, 155-
158 (1979)
New Storage Methods and Improved Trapping Techniques Parasitic Nematode Neoaplectana carpocapsae
for the
J F. HOWELL Yakima
Agricultural
Research Laboratop. Federal Department of Agriculture.
Research, Yakima,
Science and Education Washington 98902
Administration.
U. S
Received June 5. 1978 The parasitic nematode Neoaplectana carpocapsae had consistently high survival when it was stored at 3°C on moist filter paper ca. 1 year. Oxygen-enriched storage atmospheres were found unnecessary. During the second year of storage, mortality was a little higher, an average of ca. 37%. Storage for 19 months did not significantly reduce nematode infectivity. Survival during trapping was improved by controlling the number of cadavers per trap. KEY WORDS: Neoaplectana carpocapsae; storage method: DD-136: storage method for nematode; Nematode; Neoaplectana curpocapsae.
INTRODUCTION
and storing Neoaplectana carpocapsae (DD-136), a neoaplectanid nematode that is a parasite of several insects, was necessary to obtain sufficient material for laboratory and field testing. Dutky et al. (1964) noted that survival of the nematode was variable when it was stored in liquid; they also noted that the concentration of nematodes affected storage life and recommended 50,00O/ml of water held at 7°C. Chu and Ching (1975) tested survival of densities of from 350 to lOOO/ml water and demonstrated that as density increased so did survival. They obtained 80% survival with a density lOOO/ml, a concentration only l/50 that recommended by Dutky et al. (1964). However, both groups of investigators suggested that the survival rate and storage technique could be improved. Dutky et al. (1964) obtained larval nematodes migrating from the host cadaver by trapping them in a water moat and then separating them from the water by decanting or filtration. However, in my rearing of this nematode at the Yakima Agricultural Research Laboratory, survival has been generally poor in the nematode trap prior to storage and also in storage. Though, if sur-
vival was good in the trap, successful storage was more likely. Failure to obtain good results in storing nematodes in fluid media prompted me to develop an alternative storage method, which has proven useful for me both for storing and shipping.
Accumulating
MATERIALS
AND METHODS
Some ad hoc rearings seemed to indicate that the size of the nematode trap influenced survival. Therefore, two sizes of traps were tested: (1) a large glass Petri dish (200 x 55 mm); and (2) a standard Petri dish (90 x 12 mm). In the large containers Petri dish covers (90 x 12 mm) wrapped with VWR’ grade 613 qualitative filter paper were used initially (culture A) as islands for holding the inoculated dead host larvae (Dutky et al., 1964); and small plastic lids (45 x 3 mm) covered with filter paper were used for islands in the 90 x 12-mm Petri dishes. Subsequently (culture B), the small plastic lids were used as islands in both containers. For culture A, the large containers (200 x 55 mm) were covered with Para-film 1This paper reports the results of research only. Mention of a commercial product in this paper does not constitute a recommendation for use by the U.S. Department of Agriculture. 155 0022-201 l/79/020155-04$01.00/O Copyright c 1979 by Academic Press. Inc. All rights of reproduction m any form reserved.
156
J F. HOWELL
lids and the small Petri dishes (90 x 12 mm) with glass Petri dish covers; for culture B, Para-film lids were used exclusively with both sizes of container. A 1: 1000 solution of formaldehyde-water was used as the trap fluid, 50 ml in the large dish and 6 ml in the small. For culture A, the dead host larvae were laid out on the islands side-by-side so they covered all the surface available (8- 10 on the small and 32-50 on the large islands). For culture B, the cadavers were spaced so there was free space equal to a cadaver between each one (four to five per island). The host larvae were western Spodoptera yellowstriped armyworm, praejka (from J. E. Halfhill) for culture A and Heliothis zea (supplied by Ray Patana, Cotton Insects Biological Control Lab., Tucson, Ariz.) for culture B. The rearing area was maintained at 21” ? 3°C and 55 ? 5% RH with a 16-hr photophase. Collection of nematode larvae began 7 days postinoculation of the host larvae.
FIG. 1. Storage system for Neoaplecrana with stacking ridges, and desiccator jar.
Nematodes were prepared for storage by filtering them from the trap fluid with a 13.5-cm-diameter porcelain vacuum filter equipped with 15-cm filter disks and then washing them twice in fresh formaldehyde-water solution (1: 1000). Samples were taken at this point to measure prestorage mortality. The filter disk containing the nematodes was either placed in 15-cmdiameter plastic dishes with lids (culture A) or on 15-cm-diameter watch glasses that were stored in a desiccator jar (culture B) (Fig. 1). (Each desiccator contained a saturated solution of ammonium phosphate [NH,H,PO,l for humidification; 93% RH at 20°C.) Each filter disk was wetted to just in excess of saturation with the formaldehyde- water solution. All storage was at 3” + 2°C. Dutky et al.‘s (1964) storage technique required purging the storage vessels with oxygen. To test the necessity of oxygenation 16 vessels were oxygenated and 12
carpocupsue consisting of filter disk, large watch glass
STORAGE TABLE RATE
OF SURVIVAL
Neoaplectana
1
OF THE PARASITIC
carpocapsae
TECHNIQUE
Two
IN
NEMATODE
SIZES OF TRAPS”
Percentage survivalb Days between
Trap size
collections
Small
Large
3 7 9 16
76.5 75.7 80.5 63.3
Culture A a 65.1 ab a 52.6 b a 58.3 ab ab 42.2 b
7 7 7 7 9
79.2 75.7 79.3 82.6 69.9
Culture B a 79.0 a a 75.5 a a 84.9 a a 88.6 a a 81.7 a
ferred to filter paper disks that were placed in plastic dishes containing six western yellowstriped armyworm larvae. After 4 days, insect mortality was scored, and the cadavers were dissected to determine the presence of reproducing female nematodes, a verification of cause of mortality. There were live replicates from each age group (check, 11 and 19 months storage), each replicate represented a separate collection of nematodes. The nematodes used in this study were originally obtained from S. Dutky, Insect Physiology Laboratory, Beltsville, Maryland. RESULTS
Average 79.6% a There were three and five replicates for each collection of cultures A and B respectively. b Means with common letters not significantly different at the 0.05 level of significance by a Duncan’s multiple range test.
were unoxygenated during storage; all nematodes were from culture A and stored for approximately 1 year. Nematodes taken from storage were tested to determine whether storage had altered infectivity. For this test, from 24,000 to 235,000 nematode larvae were trans-
Survival of culture A was somewhat higher in the smaller trap (Table l), but culture B was not affected significantly by trap size. Average percentage survival pre- and poststorage was similar for the first 11 months of storage (Table 2). However, survival after 18-19 months of storage was sometimes reduced. Infectivity was essentially 100% following storage (Table 3) as was the case for prestorage nematodes. DISCUSSION
Cultures A and B differed in the size of the island, number of insect cadavers per island, and the insect host. Nevertheless,
TABLE SURVIVAL
OF THE PARASITIC
NEMATODE
Test No.
No. of replicates
Average percentage survival prestorage
1 2 3 4 5 6
8 5 3 3 8 1
76.5 75.7 80.5 63.3 76.5 -
1 2 3
7 8 8
85.6 75.6 82.1
” Unoxygenated. b Oxygenated.
157
FOR N. CARPOCAPSAE
2
Neoaplectana
Months in storage (Period 1)
carpocapsae
ON MOIST
FILTER
PAPER
Average percentage survival
Months in storage (Period 2)
Average percentage survival
Culture A 11 11 10 10 11 9
69.7” 82.1” 86.7” 47.lb 51.7” 83.3”
19 19 18 18
50.8 15.0 74.4 52.1
Culture B” 6 5 4
90.7 73.2 73.0
158
J F. HOWELL TABLE COMPARISON
OF THE INFECTIVITY FOLLOWING
STORAGE
3
AND VIRULENCE PERIOD
OF
OF Neoaplertana
carpocapsae
11 AND 19 MONTHS
No. of insect larvae: Treatment Check 19 months storage 11 months storage
In test
Dead”
Alive
30 30 30
5a 24 b 29 b
5 2 0
0 Values with common letters are not significantly multiple range test.
the data (and observation) imply that crowding of cadavers together on a large island (50 vs. 20) results in increased mortality among the nematodes. The trap fluid often became cloudy in such high density traps but remained clear in the low density situations. However, the nematode larvae that emerged were the same size in both cultures, i.e., ca. 500-550 pm. Dutky et al. (1964) reported a relationship between nematode survival in storage and nematode density. In the present tests, nematode density averaged ca. 20,00O/ml in the small traps per trap period compared and 3000-4000/ml in the large traps, but these differences in density in the traps did not influence survival. Dutky ef al. (1964) suggested that a large surface-to-volume ratio was needed so gas exchange would prevent anoxia during storage. They found that anoxia was delayed or reduced by providing oxygenenriched storage atmospheres. However, with the filter paper storage method described here, the surface-to-volume ratio was maximized and oxygenation did not improve storage life. Nematodes stored for either 11 or 19 months were highly infective (Table 3). Infected insect larvae cease to feed soon after they become infested with nematodes. However, the western yellowstriped armyworm is very cannibalistic in the absence of food. Consequently, if infestation by the nematode is delayed, cannibalism occurs. Table 3 shows that there was more cannibalism among insect larvae inoculated
Cannibalized 20 4 1
different at the 0.05 level of significance by a Duncan’s
with nematodes that had been stored 19 months than among larvae exposed to nematodes stored 11 months. Since the time necessary for insect larvae to become infected was greater with the older nematodes, prolonged storage appeared to affect infectivity. This change in infectivity was insignificant for cultural purposes since 100% of the larvae were usually infected; it simply took longer. Thus if the nematode is to be used as a pathogen for insect control, prolonged storage would have a significant and detrimental effect. No optimum or maximum storage capacity per unit (filter disk) was established. However, some units contained 500,000 nematodes, and I felt this was far below capacity. In conclusion, the described storage technique (saturated filter papers) gave consistent results; was space conservative, simple, and cheap and facilitated handling and transportation. However, in storage, moisture constantly coalesces on the sides of’the storage vessels and is slowly but continually lost from the filters. Therefore, periodic checks and the addition of moisture is necessary. It is important to keep the filters moist. Survival is not possible on dry filters. REFERENCES CHU. Y. I., AND CHING, H. A. 1975. The storage method of parasitic nematode DD-136. PIarzt Prof. Bull. Tahvan. 17, 115-120. DUTKY, S. R., THOMPSON, J. V., AKD CANTWELL, G. E. 1964. A technique for mass propagation of the DD-136 nematode. J. Insecr Pathol.. 6, 417-422.