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Infection of the cereal leaf beetle, Oulema melanopa (Linnaeus) by Beauveria bassiana (Balsamo) vuillemin
JOURNAL
OF INVERTEBRATE
PATHOLOGY
7, 101-111
(1965)
NOTES Infection of the Cereal Leaf (Linnaeus) by Beauveria
Beetle,
Oulema
melanopu
(Balsamo)
bass&ma
Vuillemin’ The cereal leaf beetle, Oulema melanopa (Linnaeus) (= Lema melanopa Linnaeus) , is an Old World species with extensive geographic distribution, but which only recently has been introduced into the United States. The beetle was first discovered in southwestern Michigan (Anonymous, Coop. Econ. Znsect Rept., 12, 881, 1962) and later in adjacent counties in Indiana. During a fall survey in September 1963, Mr. Richard Shade, Department of Entomology, Purdue University, collected a single, apparently diseased beetle from beneath the bark of a tree. The specimen was submitted to our laboratory for diagnosis. The following is an account of the diagnosis and subsequent infection trials. Preliminary examination of the specimen indicated that it had succumbed as a result of a mycosis and standard isolation and identification techniques for fungi were employed during diagnostic studies. The growth characteristics of the unidentified fungus, on slants 1 This investigation was supported in United States Department of Agriculture No. 12-14-100-7175 (33).
as well as in slide culture, indicated that the fungus was a species of the hyphomycetous Fungi Imperfecti and in the genus Beauveria. The growth of the fungus was periodically examined and compared with previously identified cultures of Beauveria in our culture collection. In most respects, our isolate (Purdue Entomology Accession No. F-003 1) also compared with a modified description of Beauveria basstiana (Balsamo) Vuillemin [D. M. MacLeod, Can. J. Botany, 32, 818-890 (1954)]. On this basis, it was concluded that B. bassiana had naturally infected the cereal leaf beetle, apparently a new insect host for this species of fungus. The parasitic relationship between the fungus and the cereal leaf beetle was investigated by conducting infectivity tests utilizing field-collected first generation adults and subcultures of the original pure culture isolate. The insects were inoculated by placing them in a culture tube containing the sporulating fungus after which the tube was gently tapped. They were then removed to a sterile 100 X 15 mm petri dish containing a sterile filter paper moistened with sterile water and supplied with
part by Contract
TABLE CUMULATIVE
MORTALITY
OF THE CEREAL
LEAF
BEETLE,
Beauveria
1 O&ma melanopa bassiuna
Hours Treatment Fungus Control
24
48
72
0/2P o/25
o/25 0/2s
o/25
a Numerator indicates number dead; denominator b Died of other causes as determined by autopsy.
after
101
WITH
SPORES
OF
inoculation
a/25
indicates
AFTER INOCULATION
number
96
120
Total
IS/25 o/25
25/2.5 3/W
25/25 3/25h
treated;
summary
of five replications.
102
NOTES
food in the form of a small bouquet of green. house grown oat seedlings. The control groups were treated identically with the exception ot the administration of the inoculum. Beetles which died during the test were surface sterilized and reisolation of the fungus from these specimens was employed as a criterion of infectivity. The results of the laboratory infection trial are presented in Table 1. The virulence of this isolate appears quite dramatic and observations during the course of the test indicated a substantial reduction in feeding and activity by beetles treated with conidia; this was particularly noticeable after 48 hours incubation at 26’C. In a similar test of infectivity of the fungus on the larval stage of 0. melanopa, only one insect died of the mycosis.
Sericesthis
Iridescent Adult
Sericesthis Iridescent Virus (SIV) occurs naturally in the pasture scarab, Sericesthis. It also multiplies readily in cells of larvae of the greater wax moth, Galleria mellonella (Linnaeus), which have been artificially infected (E. A. Steinhausand R. Leutenegger,J. Insect Pathol., 5, 266-270, 1963; M. F. Day and E. H. Mercer, Asst. J. Biol. Sci., 17, 892-902, 1964). This note reports the infection of adult Galleria. The virus and viral deoxyribonucleic acid (DNA) was revealed by the Feulgen technique in the cytoplasm of many cells as characteristically red-staining material, whereas Feulgen-positive material was never present in the cytoplasm of the same cells in noninfected Galleria. SIV particles are uniform in size and shape and were easily recognized in sections examined in the electron microscope. SIV can thus be readily identified in infected cells of its host, either by light or electron microscopy. When last-instar or younger Galleria larvae
Since the described isolation and infectivity tests were conducted, we have repeatedly isolated B. bassiana from fall-collected adults and also from field-collected adults which had been transferred to insectary conditions and later succumbed to the mycosis. There appears to be, based on preliminary examination of these cultures, definitive strain differences between various isolates. Single spore isolations of these strains may provide us with a particularly virulent strain for use in control of 0. melanopa adults. J. D. PASCHKE Department of Entomology Purdue University Lafayette, Indiana Accepted November 18, 1964
Virus Infection Galleria
of
the
were inoculated with SIV and maintained at 20°C they pupated, produced up to 3.5-mg virus in 28 days, and died as pupae. However, when 6-day-old or older pupae were infected, adult moths were produced, and from these, iridescent pellets of virus could readily be obtained by differential centrifugation. This virus was highly infectious upon inoculation into Galleria larvae at dosagesof no more than 200 particles/larva. Complete serial sections (10 p) of infected adult female Galleria were stained by the Feulgen technique, followed by Light Green. The fat body and epidermal cells presented a remarkable appearance. The cytoplasm of every cell of these tissueswas intensely Feulgen-positive, demonstrating the presence of large amounts of viral DNA (Fig. 1). Virus was also present in the sarcoplasm (but not in the fibrillar region) of many musclecells, in hemocytes, in tracheal epithelium, in the heart, and in the nerve cell bodies of many,
OF INVERTEBRATE
PATHOLOGY
7, 101-111
(1965)
NOTES Infection of the Cereal Leaf (Linnaeus) by Beauveria
Beetle,
Oulema
melanopu
(Balsamo)
bass&ma
Vuillemin’ The cereal leaf beetle, Oulema melanopa (Linnaeus) (= Lema melanopa Linnaeus) , is an Old World species with extensive geographic distribution, but which only recently has been introduced into the United States. The beetle was first discovered in southwestern Michigan (Anonymous, Coop. Econ. Znsect Rept., 12, 881, 1962) and later in adjacent counties in Indiana. During a fall survey in September 1963, Mr. Richard Shade, Department of Entomology, Purdue University, collected a single, apparently diseased beetle from beneath the bark of a tree. The specimen was submitted to our laboratory for diagnosis. The following is an account of the diagnosis and subsequent infection trials. Preliminary examination of the specimen indicated that it had succumbed as a result of a mycosis and standard isolation and identification techniques for fungi were employed during diagnostic studies. The growth characteristics of the unidentified fungus, on slants 1 This investigation was supported in United States Department of Agriculture No. 12-14-100-7175 (33).
as well as in slide culture, indicated that the fungus was a species of the hyphomycetous Fungi Imperfecti and in the genus Beauveria. The growth of the fungus was periodically examined and compared with previously identified cultures of Beauveria in our culture collection. In most respects, our isolate (Purdue Entomology Accession No. F-003 1) also compared with a modified description of Beauveria basstiana (Balsamo) Vuillemin [D. M. MacLeod, Can. J. Botany, 32, 818-890 (1954)]. On this basis, it was concluded that B. bassiana had naturally infected the cereal leaf beetle, apparently a new insect host for this species of fungus. The parasitic relationship between the fungus and the cereal leaf beetle was investigated by conducting infectivity tests utilizing field-collected first generation adults and subcultures of the original pure culture isolate. The insects were inoculated by placing them in a culture tube containing the sporulating fungus after which the tube was gently tapped. They were then removed to a sterile 100 X 15 mm petri dish containing a sterile filter paper moistened with sterile water and supplied with
part by Contract
TABLE CUMULATIVE
MORTALITY
OF THE CEREAL
LEAF
BEETLE,
Beauveria
1 O&ma melanopa bassiuna
Hours Treatment Fungus Control
24
48
72
0/2P o/25
o/25 0/2s
o/25
a Numerator indicates number dead; denominator b Died of other causes as determined by autopsy.
after
101
WITH
SPORES
OF
inoculation
a/25
indicates
AFTER INOCULATION
number
96
120
Total
IS/25 o/25
25/2.5 3/W
25/25 3/25h
treated;
summary
of five replications.
102
NOTES
food in the form of a small bouquet of green. house grown oat seedlings. The control groups were treated identically with the exception ot the administration of the inoculum. Beetles which died during the test were surface sterilized and reisolation of the fungus from these specimens was employed as a criterion of infectivity. The results of the laboratory infection trial are presented in Table 1. The virulence of this isolate appears quite dramatic and observations during the course of the test indicated a substantial reduction in feeding and activity by beetles treated with conidia; this was particularly noticeable after 48 hours incubation at 26’C. In a similar test of infectivity of the fungus on the larval stage of 0. melanopa, only one insect died of the mycosis.
Sericesthis
Iridescent Adult
Sericesthis Iridescent Virus (SIV) occurs naturally in the pasture scarab, Sericesthis. It also multiplies readily in cells of larvae of the greater wax moth, Galleria mellonella (Linnaeus), which have been artificially infected (E. A. Steinhausand R. Leutenegger,J. Insect Pathol., 5, 266-270, 1963; M. F. Day and E. H. Mercer, Asst. J. Biol. Sci., 17, 892-902, 1964). This note reports the infection of adult Galleria. The virus and viral deoxyribonucleic acid (DNA) was revealed by the Feulgen technique in the cytoplasm of many cells as characteristically red-staining material, whereas Feulgen-positive material was never present in the cytoplasm of the same cells in noninfected Galleria. SIV particles are uniform in size and shape and were easily recognized in sections examined in the electron microscope. SIV can thus be readily identified in infected cells of its host, either by light or electron microscopy. When last-instar or younger Galleria larvae
Since the described isolation and infectivity tests were conducted, we have repeatedly isolated B. bassiana from fall-collected adults and also from field-collected adults which had been transferred to insectary conditions and later succumbed to the mycosis. There appears to be, based on preliminary examination of these cultures, definitive strain differences between various isolates. Single spore isolations of these strains may provide us with a particularly virulent strain for use in control of 0. melanopa adults. J. D. PASCHKE Department of Entomology Purdue University Lafayette, Indiana Accepted November 18, 1964
Virus Infection Galleria
of
the
were inoculated with SIV and maintained at 20°C they pupated, produced up to 3.5-mg virus in 28 days, and died as pupae. However, when 6-day-old or older pupae were infected, adult moths were produced, and from these, iridescent pellets of virus could readily be obtained by differential centrifugation. This virus was highly infectious upon inoculation into Galleria larvae at dosagesof no more than 200 particles/larva. Complete serial sections (10 p) of infected adult female Galleria were stained by the Feulgen technique, followed by Light Green. The fat body and epidermal cells presented a remarkable appearance. The cytoplasm of every cell of these tissueswas intensely Feulgen-positive, demonstrating the presence of large amounts of viral DNA (Fig. 1). Virus was also present in the sarcoplasm (but not in the fibrillar region) of many musclecells, in hemocytes, in tracheal epithelium, in the heart, and in the nerve cell bodies of many,