1962] 
Bush — Genus Anastrepha 
89 
racks over moist sand in well ventilated wooden boxes. A sample 
of each collection was reared to the adult stage to confirm preliminary 
identification. Some species, such as A. ludens , A. mornbinpraeoptans , 
A. f rater cuius , and A. serpentina , were also reared on a laboratory 
diet of ground carrots and yeast (Finney, 1956). Eggs of these 
species were collected from females which were induced to oviposit 
in wax impregnated cheese cloth shells, formed and pigmented to 
represent fruit (McPhail and Guiza, 1956). For most cytological 
investigations only larvae in the prepupal stage were used. Other 
larval stages had suitable but fewer metaphase plates. 
The supraoesophageal and suboesophageal ganglion were used for 
the evaluation of all karyotypes with the exception of those of A. 
spatulata whose host and larva are not known, though the adult is 
collected in large numbers at certain times of the year. Adult 
spermatogonial metaphase plates were therefore used to establish the 
karyotype of this species. Attempts were made to obtain suitable 
oogonial metaphase plates, but these were unsuccessful. Larval and 
adult tissues were dissected out in normal saline (0.75 NaCl) and 
transferred immediately to a saturated solution of coumarin in 
distilled water for six to ten minutes following the technique of 
Sharma and Bal (1953) and Manna (1956). The majority of the 
species, including those treated statistically, were pretreated in cou- 
marin for seven minutes. Care had to be taken not to exceed ten 
minutes as chromosomes tended to become condensed and unsuitable 
for study (Fig. 8). However, the shortening effect of coumarin, if 
used judiciously, makes it possible to obtain well flattened metaphase 
plates that show the structural features of the chromosomes distinctly. 
Without the use of coumarin, chromosomes remained bunched and 
no structural detail could be observed. 
Tissue that had been pretreated in coumarin was then transferred 
either directly into aceto-orcein (2% orcein in 45% glacial acetic 
acid) for 30 minutes to one hour, or hydrolyzed in iN HC 1 for 30 
seconds to one minute at room temperature prior to staining. Hydroly- 
sis improved the over-all qualities of the preparations. Squashes were 
then made in a drop of aceto-orcein on albuminized slides using 
coverslips treated with a silicon anti-wetting agent, such as Desicote 3 , 
and made permanent following the simple and rapid quick-freeze 
method of Schultz et al. ( 1949) , as modified by Conger and Fairchild 
(i953). 
3 Beckman Desicote 18772, Beckman Scientific Instruments Division, Fuller- 
ton, California. 
