640 MICROBIAL DISEASES OP INSECTS 



AsTiPiciAi. — ^The virulence of B. acridiorum decreases very rapidly in culture 

 and in order to obtain the desired destruction at locusts it is absolutely necessary 

 to employ cultures of the highest possible virulence as an attenuated virus immunizes 

 the locust and renders it refractory to a culture of the highest virulence when applied 

 later. 



The virulence is increased by successive passages through locusts or nymphs; 

 twelve series of passages are made using twelve locusts in each series. The cul- 

 ture to be rejuvenated is mixed with a few cubic centimeters of sterile water or broth. 

 Injections are made with a syringe having a very fine sharp-pointed needle. The 

 insect is seized with the left hand, the ventral portion toward the operator, and the 

 needle of the syringe inserted between the second and third anterior abdominal 

 segments at the point of intersection with one of the longitudinal ridges, horizon- 

 tally in the direction of the head to a depth of about 3 mm. for an adult insect, a 

 little less for a nymph. The point of the needle should enter the abdominal cavity, 

 not merely pierce the tegument as in the latter case the effect would be nil. If 

 the needle is inserted too deep the internal organs wiU be injured. A very fine- 

 pointed bent pipette could be employed equally* well. One or two drops of the emul- 

 sion of the old culture are injected. 



As- soon as the locusts in the first series become sick or preferably are nearly 

 dead, press the abdomen between the fingers and collect in a watch glass the blackish 

 liquid which issues from the anus. Inject a drop of this liquid into the abdominal 

 cavity of the second series of locusts, following the same technic and observing the 

 same precautions as for those of the first series. These insects will die in a shorter 

 period cif time. Obtain as previously, in a watch glass the intestinal liquid of three 

 or four of the first dead locusts of the second series, dilute half with water and sterile 

 broth and inoculate the third series. To inoculate the fourth series, use the intes- 

 tinal Uquid of the first dead of the third series diluted to a third; a fifth series with 

 the hquid diluted to a fourth and continue with the series in this way. It is excep- 

 tional that it will be necessary to proceed further than the twelve series. The viru- 

 lence of B. acridiorum is increased sufficiently if death occurs eight hours after injec- 

 tion. One-hundredth of a cubic centimeter of virus at its maximum virulence in- 

 jected into a locust will cause the characteristic diarrhoea in two hours and death 

 an hour later. This method of increasing the virulence takes five to six days and 

 this period of time has to be taken into consideration when it is necessary to employ 

 the culture on a practical scale. 



When the acridian to be infected belongs to a different species than that for which 

 the virulence of B. acridiorum has been previously augmented, a large number of 

 passages may be necessary as a culture virulent for one species may not be able to 

 infect another species. In one case fifty-two passages were necessary in order to 

 kill Staurdnautus maroccanas (Algeria) in eight hours while for the same insect at 

 Cyprus only twelve passages were necessary. It is also desirable that the first 

 few series consist of a large number of insects, as there will be apt to be some 

 which will be more sensitive to the virus. Their natural resistance can be weakened 

 by fasting for several days before inoculation. The intestinal contents should not 

 be diluted until the virus will kill within fifteen hours. 



