Temperature and (or) crowding (larval density) functioned as stressors in the induction of symptoms associated with the nucleopolyhedrosis virus of the European pine sawfly, Neodiprion sertifer, Subsamptes of larvae maintained at 30 and 35°C, with three levels of larval density each (20, 60, and 100/shoot) which had died under these conditions, revealed the presence of polyhedral inclusion bodies under microscopic examination. In contrast, larvae maintained at 25°C with the same three larval density levels experienced no symptoms of virus infection or mortality, The latter was consistent with field observations when temperatures during larval development ranged from 14°C to 27°C and larval densities were in the same general range

Coppel, H. C

Mohamed, M. A

Podgwaite, J. D

English

ValpoScholar

The Great Lakes Entomologist 
Volume 18 
Number 3 - Fall 1985 Number 3 - Fall 1985 Article 4 
October 1985 
Temperature and Crowding Effects on Virus Manifestation in 
Neodiprion Sertifer (Hymenoptera: Diprionidae) Larvae 
M. A. Mohamed 
University of Wisconsin 
H. C. Coppel 
University of Wisconsin 
J. D. Podgwaite 
USDA Forest Service 
Follow this and additional works at: https://scholar.valpo.edu/tgle 
 Part of the Entomology Commons 
Recommended Citation 
Mohamed, M. A.; Coppel, H. C.; and Podgwaite, J. D. 1985. "Temperature and Crowding Effects on Virus 
Manifestation in Neodiprion Sertifer (Hymenoptera: Diprionidae) Larvae," The Great Lakes Entomologist, 
vol 18 (3) 
Available at: https://scholar.valpo.edu/tgle/vol18/iss3/4 
This Peer-Review Article is brought to you for free and open access by the Department of Biology at ValpoScholar. 
It has been accepted for inclusion in The Great Lakes Entomologist by an authorized administrator of ValpoScholar. 
For more information, please contact a ValpoScholar staff member at scholar@valpo.edu. 
1985 THE GREAT LAKES ENTOMOLOGIST 115 
TEMPERATURE AND CROWDING EFFECTS ON VIRUS 
MANIFESTATION IN NEODIPRION SERTIFER 
(HYMENOPTERA: DIPRIONIDAE) LARVAE 
M, A. Mohamed,l H, C. Coppel,l and J, D, Podgwaite2 
ABSTRACT 
Temperature and (or) crowding (larval density) functioned as stressors in the induction 
of 
symptoms associated with the nucleopolyhedrosis virus 
of the European pine sawfly, 
Neodiprion sertifer, Subsamptes of larvae maintained at 30 and 35°C, with three levels of 
larval density each (20, 60, and 100/shoot) which had died under these conditions, 
revealed the presence of polyhedral inclusion bodies under microscopic examination. In 
contrast, larvae maintained at 25°C with the same three larval density levels experienced 
no symptoms of virus infection or mortality, The latter was consistent with field 
observations when temperatures during larval development ranged from 14°C to 27°C and 
larval densities were in the same general range. 
The vertical transmission of sawfly nucIeopolyhedrosis virus (NPV) between genera­
tions generally falls into two categories; environmental persistence and persistence in the 
host development sequence (Cunningham and Entwistle 1981). Environmental persistence 
has been well documented from foliage and soil samples; however, different explanations 
exist as to how the virus is maintained and spread by the adult sawflies. According to 
Cunningham and Entwistle (1981) there is no evidence that sawfly virus is transmitted 
from adults to progeny either within the egg or on it, even though Bird (1961) suggested 
ovarial transmission for both the European spruce sawfly, Gilpinia hercyniae (Hartig), and 
the European pine sawfly. Neodipriol1 sertifer (Geoffroy). It is established that infected 
adults are capable of contaminating the foliage with virus which may later be consumed 
by developing larvae. Bird (1961) did not favor the latent virus infection theory as a 
transmission possibility. Latent virus infection has been demonstrated in severallepidop­
teran species, manifesting itself in spontaneous outbreaks under various stressors includ­
ing temperature and crowding (Steinhaus 1958). With coniferous sawflies, the only 
documented case of a spontaneous epizootic in North America was with the European 
spruce sawfly (Bird and Elgee 1957). The causative factors of this outbreak are unknown, 
though it is generally accepted that the virus was introduced from Europe through the 
release of contaminated exotic parasitoids. This paper provides some evidence for the 
possibility of latent viral infection in N. sertifer and the roles of temperature and crowding 
in 
disease expression. 
MATERIALS AND METHODS 
Second-instar larvae 
of N. sertifer were selected from a "virus-free" population in the 
Kettle Moraine State Forest in southeastern Wisconsin. Red pine shoots, with foliage from 
the previous year, were cut at the base and inserted in waxed, I-pt containers filled with 
IDepartment of Entomology, University of Wisconsin, Madison, WI 53706, 

'USDA 
Forest Service, Northeastern Forest Experiment Station, Hamden, 
CT 06514. 

1
Mohamed et al.: Temperature and Crowding Effects on Virus Manifestation in Neodip
Published by ValpoScholar, 1985
116 THE GREAT LAKES ENTOMOLOGIST Vol. 18, No.3 
Table I. Percent mortality (x SE) of N. sertifer larvae from nucleopolyhedrosis virus at 
three levels and three temperature regimes. a 
Density (larvae/shoot) 
(DC) 
20 100 25 
30 35 
0% 77± 1% 
100% 0% 
84±7% 
100% 
0% 95±5% 
100% 
aAt each temperature/density combination there were two replicates for a total of 18 units. 
Table 2. Analysis of variance of virus-induced mortality in N. sertifer larvae. 
Effect F ratio p 
Density F(2,9) = 4.036 0.01 < P < 0.05 
Temperature F(2,9) = 1271 P < 0.005 
Temperature and density F(4,9) = 4.036 0.01 < P < 0.05 
water (400 ml). Larvae were transferred to the shoots at rates f 20, 60, and I Do/shoot. 
These densities were comparable to those observed in the field on similar units of foliage. 
Six replicates were provided at each density. Each unit was sealed with a glass lantern 
globe and covered with cheesecloth for ventilation. The units were placed in controlled 
chambers at 25 ± I DC, 30 1DC, and 35 ± 1°C, RH at 50-60%, and a 16: 8 photoperiod. 
The latter provided two replicates for each density and temperature combination or a total 
of 
six units in each chamber. 
Each unit was monitored for 
21 days with fresh foliage added when necessary. Dead 
larvae were removed at three-day intervals and stored singly in disposable test tubes at 
O°C. 
From these larvae, three were picked at random from each replicate 
at each 
temperature-density combination. Each larva was macerated in a sterile glass tissue 
homogenizer and examined for polyhedral inclusion bodies (PIB) with brightfield mi­
croscopy at 600X. The number of PIB/larva was counted using a Levy chamber. Larvae 
in 
the field were monitored concurrently for symptoms 
of NPV infections. 
Data were analysed using a multifactor analysis f variance. This allowed testing of the 
response (percent mortality), and PIB accumulation, as a function of temperature, density 
(crowding), or a combination of the two. 
RESULTS AND DISCUSSION 
Mortality due to NPV at the three levels of temperature and density (Table I) shows that 
there was no mortality due to NPV at 25°C. This was consistent with the absence of any 
symptoms of NPV infection of larvae in the field under daily average temperatures which 
ranged from 13.9 to 27.0DC. during the same time period. At 30DC, the virus manifested 
itself in a progressive differential mortality directly re ated to the increase in larval 
densities. And lastly, there was a small temperature-density interaction that appeared to 
affect the response. To quantify the latter, a multifactor analysis of variance (Table 2) 
showed that the density and temperature interaction on the response was significant at p 
< 0.05. However, if the significance level was set at p 0.01, it would appear that 
temperature alone was the significant factor in the induction of mortality by a latent virus. 
Neodiprion sertifer larvae are colonial feeders and one would expect that crowding (larval 
density) would have less effect than some of the other stressors. 
2
The Great Lakes Entomologist, Vol. 18, No. 3 [1985], Art. 4
https://scholar.valpo.edu/tgle/vol18/iss3/4
1985 THE GREAT LAKES ENTOMOLOGIST 117 
Table 3. Average number of polyhedral inclusion bodies (PIE) (lOglO units) observed in 
N. sertifer larvae at various densities or temperature conditions. 
Larval PIElIarva" PIE/larvab 
it ± SD °C it ± SD 
20 4.74 ± 0.61 (n 12)A 30 4.87 ± 0.54 (n 18) 
60 4.80 ± 0.46 (n = 12)A 35 4.31 0.33 (n 18) 
100 4.23 ± .28 (n = 12)B 
aMeans followed by same letter not significantly different, and, those by different letters 
significantly different at P = 0.05 (df = 33) by Least Significant Difference Test. 
"Means significantly different P < 0.001, df = 28 (t-test with unequal variances). 
To analyse the accumulation of PIE in NPV -killed larvae, three larvae/replicate were 
subsampled at random from each temperature (30°C and 35°C) and density combination. 
Each of the 36 larvae was checked microscopically for the number of PIE present. Using 
10glO 
values to normalize the data, analysis 
of variance showed that temperature-density 
interaction on the accumulation of virus in the larvae was not statistically significant 
(F(2,30) = 2.87, p > 0.05). However, either density or temperature was significant in 
larval virus accumulation (F(2,30) = 9.21, p < 0.01; F(l,30) = 15.43, P < 0.01, 
respectively). Examination of the individual means (Table 3) showed that more virus was 
accumulated in the larvae at the lower densities (20 and 60 larvae/colony) and temperature 
(30°C) than at the higher density (100 larvae/Colony) and temperature (35CC). These 
results suggest that temperature or density may limit the net accumulation of virus in the 
larvae. 
Given the conditions of this experiment where the most stringent care was exercised in 
maintaining sterility in all objects and instruments used, and the choice of larvae from an 
area with no known history of NPV, it is possible to conclude that N. sertifer does harbor 
a latent virus. The conditions under which it manifests itself in the laboratory are harsh, 
particularly with regard to high temperatures. Thus, it is unlikely that epizootics caused 
by latent virus will be observed in the current range of this species where temperatures 
above 30aC are infrequent. It is possible, however, that latent virus could limit the 
southern range of N. sertifer where temperatures may average above 30°C. 
ACKNOWLEDGMENTS 
Research supported by the College of Agricultural and Life Sciences, University of 
Wisconsin-Madison, in part by the Wisconsin Department of Natural Resources through 
the School of Natural Resources, and in part through funds provided by the USDA Forest 
Service, Northeastern Forest Experiment Station, Cooperative study 4500-FS-NE­
2202.77. The authors acknowledge the technical assistance of Odette Mohamed and the 
editorial suggestions of J. C. Cunningham. 
LITERATURE CITED 
Bird, F. T. 1961. Transmission of some insect viruses with particular reference to ovarial 
transmission and its importance in the development of epizootics. J. Insect Pathol. 
3:352-380. 
Bird, F. T. and D. E. Elgee. 1957. A virus disease and introduced parasites as factors 
controlling the European spruce sawfly, Diprion hercyniae (Htg.) in central New 
Brunswick. Canadian Entomol. 89:371-378. 
3
Mohamed et al.: Temperature and Crowding Effects on Virus Manifestation in Neodip
Published by ValpoScholar, 1985
118 THE GREAT LAKES ENTOMOLOGIST Vol. 18, NO.3 
Cunningham, J. C. and P. F. Entwistle. 1981. Control of sawflies by baculovirus. 
p. 
380--407 
in H. D. Burges (ed.). Microbial control of pests and plant diseases 
1970-1980. Academic Press, London-New York. 
Steinhaus, E. A. 	 1958. Crowding as a possible stress factor in insect disease. Ecology 
39:503-514. 
4
The Great Lakes Entomologist, Vol. 18, No. 3 [1985], Art. 4
https://scholar.valpo.edu/tgle/vol18/iss3/4


Temperature and Crowding Effects on Virus Manifestation in Neodiprion Sertifer (Hymenoptera: Diprionidae) Larvae

Valparaiso University

The Great Lakes Entomologist Volume 18 Number 3 - Fall 1985 Number 3 - Fall 1985 Article 4 October 1985 Temperature and Crowding Effects on Virus Manifestation in Neodiprion Sertifer (Hymenoptera: Diprionidae) Larvae M. A. Mohamed University of Wisconsin H. C. Coppel University of Wisconsin J. D. Podgwaite USDA Forest Service Follow this and additional works at: https://scholar.valpo.edu/tgle  Part of the Entomology Commons Recommended Citation Mohamed, M. A.; Coppel, H. C.; and Podgwaite, J. D. 1985. "Temperature and Crowding Effects on Virus Manifestation in Neodiprion Sertifer (Hymenoptera: Diprionidae) Larvae," The Great Lakes Entomologist, vol 18 (3) Available at: https://scholar.valpo.edu/tgle/vol18/iss3/4 This Peer-Review Article is brought to you for free and open access by the Department of Biology at ValpoScholar. It has been accepted for inclusion in The Great Lakes Entomologist by an authorized administrator of ValpoScholar. For more information, please contact a ValpoScholar staff member at scholar@valpo.edu. 1985 THE GREAT LAKES ENTOMOLOGIST 115 TEMPERATURE AND CROWDING EFFECTS ON VIRUS MANIFESTATION IN NEODIPRION SERTIFER (HYMENOPTERA: DIPRIONIDAE) LARVAE M, A. Mohamed,l H, C. Coppel,l and J, D, Podgwaite2 ABSTRACT Temperature and (or) crowding (larval density) functioned as stressors in the induction of symptoms associated with the nucleopolyhedrosis virus of the European pine sawfly, Neodiprion sertifer, Subsamptes of larvae maintained at 30 and 35°C, with three levels of larval density each (20, 60, and 100/shoot) which had died under these conditions, revealed the presence of polyhedral inclusion bodies under microscopic examination. In contrast, larvae maintained at 25°C with the same three larval density levels experienced no symptoms of virus infection or mortality, The latter was consistent with field observations when temperatures during larval development ranged from 14°C to 27°C and larval densities were in the same general range. The vertical transmission of sawfly nucIeopolyhedrosis virus (NPV) between genera­tions generally falls into two categories; environmental persistence and persistence in the host development sequence (Cunningham and Entwistle 1981). Environmental persistence has been well documented from foliage and soil samples; however, different explanations exist as to how the virus is maintained and spread by the adult sawflies. According to Cunningham and Entwistle (1981) there is no evidence that sawfly virus is transmitted from adults to progeny either within the egg or on it, even though Bird (1961) suggested ovarial transmission for both the European spruce sawfly, Gilpinia hercyniae (Hartig), and the European pine sawfly. Neodipriol1 sertifer (Geoffroy). It is established that infected adults are capable of contaminating the foliage with virus which may later be consumed by developing larvae. Bird (1961) did not favor the latent virus infection theory as a transmission possibility. Latent virus infection has been demonstrated in severallepidop­teran species, manifesting itself in spontaneous outbreaks under various stressors includ­ing temperature and crowding (Steinhaus 1958). With coniferous sawflies, the only documented case of a spontaneous epizootic in North America was with the European spruce sawfly (Bird and Elgee 1957). The causative factors of this outbreak are unknown, though it is generally accepted that the virus was introduced from Europe through the release of contaminated exotic parasitoids. This paper provides some evidence for the possibility of latent viral infection in N. sertifer and the roles of temperature and crowding in disease expression. MATERIALS AND METHODS Second-instar larvae of N. sertifer were selected from a "virus-free" population in the Kettle Moraine State Forest in southeastern Wisconsin. Red pine shoots, with foliage from the previous year, were cut at the base and inserted in waxed, I-pt containers filled with IDepartment of Entomology, University of Wisconsin, Madison, WI 53706, 'USDA Forest Service, Northeastern Forest Experiment Station, Hamden, CT 06514. 1Mohamed et al.: Temperature and Crowding Effects on Virus Manifestation in NeodipPublished by ValpoScholar, 1985116 THE GREAT LAKES ENTOMOLOGIST Vol. 18, No.3 Table I. Percent mortality (x SE) of N. sertifer larvae from nucleopolyhedrosis virus at three levels and three temperature regimes. a Density (larvae/shoot) (DC) 20 100 25 30 35 0% 77± 1% 100% 0% 84±7% 100% 0% 95±5% 100% aAt each temperature/density combination there were two replicates for a total of 18 units. Table 2. Analysis of variance of virus-induced mortality in N. sertifer larvae. Effect F ratio p Density F(2,9) = 4.036 0.01 < P < 0.05 Temperature F(2,9) = 1271 P < 0.005 Temperature and density F(4,9) = 4.036 0.01 < P < 0.05 water (400 ml). Larvae were transferred to the shoots at rates f 20, 60, and I Do/shoot. These densities were comparable to those observed in the field on similar units of foliage. Six replicates were provided at each density. Each unit was sealed with a glass lantern globe and covered with cheesecloth for ventilation. The units were placed in controlled chambers at 25 ± I DC, 30 1DC, and 35 ± 1°C, RH at 50-60%, and a 16: 8 photoperiod. The latter provided two replicates for each density and temperature combination or a total of six units in each chamber. Each unit was monitored for 21 days with fresh foliage added when necessary. Dead larvae were removed at three-day intervals and stored singly in disposable test tubes at O°C. From these larvae, three were picked at random from each replicate at each temperature-density combination. Each larva was macerated in a sterile glass tissue homogenizer and examined for polyhedral inclusion bodies (PIB) with brightfield mi­croscopy at 600X. The number of PIB/larva was counted using a Levy chamber. Larvae in the field were monitored concurrently for symptoms of NPV infections. Data were analysed using a multifactor analysis f variance. This allowed testing of the response (percent mortality), and PIB accumulation, as a function of temperature, density (crowding), or a combination of the two. RESULTS AND DISCUSSION Mortality due to NPV at the three levels of temperature and density (Table I) shows that there was no mortality due to NPV at 25°C. This was consistent with the absence of any symptoms of NPV infection of larvae in the field under daily average temperatures which ranged from 13.9 to 27.0DC. during the same time period. At 30DC, the virus manifested itself in a progressive differential mortality directly re ated to the increase in larval densities. And lastly, there was a small temperature-density interaction that appeared to affect the response. To quantify the latter, a multifactor analysis of variance (Table 2) showed that the density and temperature interaction on the response was significant at p < 0.05. However, if the significance level was set at p 0.01, it would appear that temperature alone was the significant factor in the induction of mortality by a latent virus. Neodiprion sertifer larvae are colonial feeders and one would expect that crowding (larval density) would have less effect than some of the other stressors. 2The Great Lakes Entomologist, Vol. 18, No. 3 [1985], Art. 4https://scholar.valpo.edu/tgle/vol18/iss3/41985 THE GREAT LAKES ENTOMOLOGIST 117 Table 3. Average number of polyhedral inclusion bodies (PIE) (lOglO units) observed in N. sertifer larvae at various densities or temperature conditions. Larval PIElIarva" PIE/larvab it ± SD °C it ± SD 20 4.74 ± 0.61 (n 12)A 30 4.87 ± 0.54 (n 18) 60 4.80 ± 0.46 (n = 12)A 35 4.31 0.33 (n 18) 100 4.23 ± .28 (n = 12)B aMeans followed by same letter not significantly different, and, those by different letters significantly different at P = 0.05 (df = 33) by Least Significant Difference Test. "Means significantly different P < 0.001, df = 28 (t-test with unequal variances). To analyse the accumulation of PIE in NPV -killed larvae, three larvae/replicate were subsampled at random from each temperature (30°C and 35°C) and density combination. Each of the 36 larvae was checked microscopically for the number of PIE present. Using 10glO values to normalize the data, analysis of variance showed that temperature-density interaction on the accumulation of virus in the larvae was not statistically significant (F(2,30) = 2.87, p > 0.05). However, either density or temperature was significant in larval virus accumulation (F(2,30) = 9.21, p < 0.01; F(l,30) = 15.43, P < 0.01, respectively). Examination of the individual means (Table 3) showed that more virus was accumulated in the larvae at the lower densities (20 and 60 larvae/colony) and temperature (30°C) than at the higher density (100 larvae/Colony) and temperature (35CC). These results suggest that temperature or density may limit the net accumulation of virus in the larvae. Given the conditions of this experiment where the most stringent care was exercised in maintaining sterility in all objects and instruments used, and the choice of larvae from an area with no known history of NPV, it is possible to conclude that N. sertifer does harbor a latent virus. The conditions under which it manifests itself in the laboratory are harsh, particularly with regard to high temperatures. Thus, it is unlikely that epizootics caused by latent virus will be observed in the current range of this species where temperatures above 30aC are infrequent. It is possible, however, that latent virus could limit the southern range of N. sertifer where temperatures may average above 30°C. ACKNOWLEDGMENTS Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, in part by the Wisconsin Department of Natural Resources through the School of Natural Resources, and in part through funds provided by the USDA Forest Service, Northeastern Forest Experiment Station, Cooperative study 4500-FS-NE­2202.77. The authors acknowledge the technical assistance of Odette Mohamed and the editorial suggestions of J. C. Cunningham. LITERATURE CITED Bird, F. T. 1961. Transmission of some insect viruses with particular reference to ovarial transmission and its importance in the development of epizootics. J. Insect Pathol. 3:352-380. Bird, F. T. and D. E. Elgee. 1957. A virus disease and introduced parasites as factors controlling the European spruce sawfly, Diprion hercyniae (Htg.) in central New Brunswick. Canadian Entomol. 89:371-378. 3Mohamed et al.: Temperature and Crowding Effects on Virus Manifestation in NeodipPublished by ValpoScholar, 1985118 THE GREAT LAKES ENTOMOLOGIST Vol. 18, NO.3 Cunningham, J. C. and P. F. Entwistle. 1981. Control of sawflies by baculovirus. p. 380--407 in H. D. Burges (ed.). Microbial control of pests and plant diseases 1970-1980. Academic Press, London-New York. Steinhaus, E. A. 	 1958. Crowding as a possible stress factor in insect disease. Ecology 39:503-514. 4The Great Lakes Entomologist, Vol. 18, No. 3 [1985], Art. 4https://scholar.valpo.edu/tgle/vol18/iss3/4

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Temperature and Crowding Effects on Virus Manifestation in Neodiprion Sertifer (Hymenoptera: Diprionidae) Larvae

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