The results obtained in studies on the vital staining of Anopheles larvae, conducted between December 1, 1937, and April 18, 1938, have been highly satisfactory. During these studies we have been able to stain larvae so that the staining could be easily recognized in the adults that emerge from the larvae. Staining may be recognized in the living mosquito by placing the specimen in a vial or capsule and examining under the dissecting microscope. It then is apparent in the tissues underlying the ventral sclerite of the neck. Or the staining may be recognized by dissecting the specimen and examining it under the dissecting microscope. It is then apparent in the walls of the abdomen and in the tissues of the thorax.
Fourth stage larvae were used in thirty-two lots, and third stage larvae in six lots. It is possible to stain third stage larvae sufficiently to be able to recognize the staining in the adults that emerge.
The stains giving the best results in these experiments were Giemsa's, Wright's, Methylene Blue, and Congo Red, in the order named. Giemsa's in dilution of 1:250 of Grubler's stock solution gave excellent results when the larvae were allowed to remain in the stain either seventy-two hours, or until the larvae had pupated. Staining was easily recognized in adults resulting from larvae so stained up to thirty-nine days after emergence, and in one case nine days after a blood meal. Every adult emerging from larvae stained with Giemsa's was unmistakably stained.
Wright's stain, in dilutions of 1:15000, 1:30000, and 1:75000, appears not to be to any appreciable extent toxic to larvae exposed to it for three to five days. Excellent staining was secured in adults developing from larvae stained with Wright's. Staining was easily recognized in adults so stained up to fifty-eight days after emergence, and in one specimen forty days after a blood meal.
Methylene Blue, in dilution of 1:1000 of a concentrated alcoholic solution, gave quite satisfactory staining in the adults emerging from larvae exposed to this stain. Staining was recognized in adults developing from larvae stained with Methylene Blue up to twenty-six days after emergence.
Congo Red, as used in these experiments, while not entirely satisfactory, might, with further study, be found to be a stain which could be used in a practical way in marking mosquito larvae.
Other stains used, but with less satisfactory staining, were, Brilliant Green, Eosin “Y”, Acid Fuchsin, Brom Thymol Blue, Safranine, Crystal Violet, Gentian Violet, Erythrosin, Basic Fuchsin, Uranin, Neutral Red, and Bismarck Brown.
No adults were obtained from larvae stained with Brilliant Green. With all other stains, however, staining was demonstrated to some extent in adults obtained from larvae stained. It is therefore possible to employ a number of stains in marking larvae. However, it appears significant that the stains with which the best results were obtained, Giemsa's, Wright's, and Methylene Blue, are dilutions of alcoholic solutions, whereas others were watery solutions.
There appears to be no difference in the way the different sexes take the stain. Males and females that emerge appear to be equally well stained.
Staining of larvae sufficiently to enable the stain to be recognized in the adults that emerge therefrom can be done without inflicting any excessive mortality on the larvae and without appreciably impairing the longevity of the adults that emerge.
Staining can be recognized in the adults after the assimilation and digestion of a blood meal.
Staining is apparently internal, as none could be discerned in the exoskeleton of the mosquitoes. Staining is permanent. It has been recognized in adults up to fifty-nine days after emergence. Attempts to dissolve it in acetone and in alcohol, and in both combined were unsuccessful. Only by digesting the tissues of the mosquito in a caustic solution were we able to liberate the stain. It is suggested that the staining is intracellular but further study is now in progress to ascertain the validity of this suggestion.
It is observed that many larvae used in these experiments remained in the larval stage or in the pupal stage for what appears to be an undue length of time. We are not prepared to offer an explanation for this phenomenon. However, since we were unable to control the temperature in the laboratory in which these experiments were conducted, the effects of winter temperatures can not be excluded as a factor.
Further study should be undertaken on this and certain other phases of this report. Our experiments are still in progress, including a field application of the information gained through these studies.