A method for the inoculation of mosquito larvae

42.5

NOTES

A Method

for

the

Inoculation

In attempting to maintain the agents of viral and bacterial diseases of mosquito larvae by artificial inoculation we have encountered technical difficulties. Their small size, their body turgor, which even a puncture from a No. 37 hypodermic needle will release, and their susceptibility to potential secondary invaders, always numerous in an aquatic environment, are characteristics of mosquito larvae which must be overcome for our purposes. After experimenting with a variety of devices we now use a pair of simply modified jeweler’s or Swiss-style stainless steel, microdissecting forceps which have proven to be highly successful (Figs. 1 and 2). Since some interest in this technique has been shown by workers from other laboratories it seemed worthwbile to describe our method. This technique is of no value in obtaining quantitative data on dosages, but serves simply as

FIGS. 1 & 2. Jeweler’s opposing one off. Approx.

forceps 10 X.

which

have

been

of

Mosquito

Larvae

a means of maintaining, with a minimum of time and effort, laboratory strains of viruses and bacteria which are not readily cultured on artificial media. In addition to mosquito larvae of many species we have inoculated the larvae of Fannia canicularis, Musca domestica, and Galleria mellonella. Of these, mosquito larvae were by far the most susceptible to physical damage, but with our technique we have inoculated second-stage mosquito larvae and have had as high as 75% surviving the inoculation with 80% of the survivors developing patent infections. In inoculating Gulleria mellonella with the Tipula iridescent virus and with the Sericesthis iridescent virus we have had 100% surviving the inoculation with 100% developing patent infections. The techniques used in handling all these species have been essentially the same. Larvae to be inoculated were first bathed

modified

by

bending

one point

over

and

grinding

the

426

NOTES

FIG.

3. Fourth-stage

mosquito larva with patent bacterial infection

in an 800 ppm solution of a quaternary ammonium germicide (Roccal, Winthrop Laboratories) for 3 minutes, rinsed in three changes of sterile distilled water and placed on moist filter paper on the stage of a dissecting microscope. A suspensionof the organism to be injected was placed in a concavity of a sterile agglutination slide. The sterile tips of the forceps were first dipped into the suspensionof virus or bacteria and then used to pick up a larva by pinching the cuticle of its abdomen and transferring the larva to a paper cup of tap water. After an hour in the cup survivors were placed in

The

Relation of

showing site of inoculation.

regular rearing pans to incubate. Figure 3 shows a melanized spot on the third abdominal segment of a mosquito larva which indicates the site of inoculation. I am indebted to Dr. W. R. Kellen for his assistance with the photography and for his encouragement in writing this note.

TRUMAN B. CLARK University of California-State Department of Public Health Mosquito Project Fresno, California Accepted June 20, 1966

of pH to the Activity of Inclusion a Heliothis Nuclear Polyhedrosisl

In order to prevent dissolution of nuclear polyhedrosis inclusion bodies, the “pH of the suspendingmedia must not drop below 5 or go above 8.5” [G. H. Bergold, “Hand1 In cooperation with the Texas Agricultural Experiment Station, Texas A & M University, College Station, Texas.

Bodies

buch der Virusforschung” (K. F. Meyer and C. Hallauer, eds.), Vol. 4, pp. 60-142, 19581. This generalization has been subsequently confirmed for other nuclear polyhedroses [G. H. Bergold, in “Insect Pathology” (E. A. Steinhaus, ed.), Vol. 1, 413-456, 19631 as well as for cytoplasmic polyhedroses (K. M.