24 AN INTRODUCTION TO THE STUDY OF VIRUSES 
assumed that all the insects received approximately the same dose of 
virus. After feeding on the aster they were transferred to rye seedlings 
which are immune to the aster yellows virus. Thereafter the insects 
were removed in batches at regular intervals and macerated with 
water to form a paste. This was inoculated at various dilutions into 
the bodies of known virus-free leaf-hoppers, and the leaf-hoppers 
were colonized on healthy aster seedlings. Such a procedure is neces- 
sary because the aster yellows virus cannot be transmitted mechanically 
by sap-inoculation and the technique is based on the earlier work of 
Storey (1932) with the streak virus of maize. Black found that the 
longer the leaf-hoppers remained on the rye seedlings before being 
ground up and diluted, the more they could be diluted whilst still 
being infective when injected into the transmitting leaf-hopper. 
Insects macerated before the fourth day gave no infection, whereas 
those macerated on the twelfth day gave infection when diluted 
1: 1000. Black interprets this as evidence that the virus multiplied 
in the insects when colonized on the rye plant; but Bawden (1943) 
has criticized this interpretation on the ground that the number of 
successful inoculations is usually greater if the extracts of macerated 
insects are diluted 1: tooo than if diluted 1: 100 or 1:10. He 
suggests that this is due to the presence of an inhibitor (Black, 1939) 
which becomes less effective with dilution. This criticism would be 
valid enough if the macerated insects were being inoculated to the test 
plant, for only under these conditions does the inhibitor come into 
action, but since they are inoculated into a virus-free insect which then 
infects the plant, the presence of the inhibitor is immaterial and the - 
quantity of virus present is the vital factor. 
Kunkel (1937) has carried out experiments on the same leaf-hopper 
and virus and has also obtained results which he considers support 
the theory of virus multiplication in the insect. He exposed infective 
insects to high temperatures for varying periods and noted the effect 
of this on the insect’s power to transmit the virus. After exposure for 
one day to a temperature of 32°C, the leaf-hoppers lost the power to 
infect healthy plants but quickly regained infectivity, without recourse 
to a fresh source of virus, when the temperature was lowered. After 
exposure for a week to high temperatures, the power to infect was 
regained but much more slowly, whilst insects exposed for twelve 
days failed to regain it at all. Kunkel interprets these results as indi- 
cating a reduction by heat of the virus content of the insect’s body 
below the minimum dose necessary for infection. He considers that 
