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Mosquito Variability: Did the Mosquito do it?

Studies by the Yellow Fever Board, Cuba 1900.
This summer marks 100 years since the discovery of Aedes aegypti transmission of yellow fever (YF) virus. The events in this discovery began on May 24, 1900 when the Adjutant General appointed a Board, consisting of Drs. Walter Reed, Aristides Agramonte, James Carroll and Jesse Lazear. Their mission was to study infectious diseases in U. S. troops in Cuba at the close of the Spanish-American War. The Board focused on YF. On July 21, 1900, Reed and Agramonte evaluated the case history of a U. S. soldier who had been a prisoner confined in the guardhouse since June 6. He showed YF symptoms on June 12 and died of the disease on June 18. None of eight other prisoners, all sharing the same cell became sick, and one had slept in the same bunk that had previously been used by the dead prisoner. Three men who handled the linen of all YF patients remained in perfect health. Thus the Board focused on mosquito transmission for YF proposed nearly 20 years previously by Dr. Carlos Findlay. Despite numerous attempts, Findlay had been unable to show mosquito transmission of YF. The work by Ross and Manson with malaria and mosquitoes were known to the Board adding credibility for mosquito transmission of YF.

The first experiments used Ae. aegypti that had fed on YF patients three days previously and then fed on human recipient volunteers. No disease occurred. We now know that this incubation period was too short for these mosquitoes to have virus in their salivary glands at three days post-infection. The Board however was discouraged. Dr. Lazear applied an Ae. aegypti female to himself on August 16. It had fed on an infected patient on August 6. Ten days incubation might be sufficient virus to infect the salivary glands. Yet Lazear remained in fine health. The Board was becoming less convinced of mosquito transmission.

Now for an important Ae. aegypti female. It was hatched in the laboratory and was blood-fed on a YF case in the second day of a severe illness on Aug 15, 1900. It had also fed on patients on Aug. 21, 23, and 25. On August 27, Lazear commented to Carroll that this mosquito was refusing to take blood, seemed weak, and was likely to die. Dr. Carroll decided to try feeding it himself to avoid losing it. Lazear held the tube containing it for a period against Carroll's forearm. Then Carroll held the tube against his own forearm and the mosquito began to blood feed. On August 30, 1900 Carroll felt ill, and by September 2 had severe YF. Luckily he survived. Walter Reed was in Washington D.C. during this period writing a report on Typhoid. His letter (Figure 1) was written after he learned of Carroll's recovery. Note the closing line - "Did the mosquito do it?" At the time, even Carroll believed he might have contracted YF during his work in the autopsy room or in the hospital, and not from this mosquito.

Four days after Carroll's experiment, Jesse Lazear met Pvt. William E. Dean, Troop B, 7th Calvary. Deandid knew about the experiments and Carroll asked if he would allow himself to be bitten. Dean had not been in the tropics before and had not been off the YF free base for nearly two months - a perfect non-immune. Dean did not believe in mosquito transmission, and Lazear and Agramonte were skeptical as well. Several Ae. aegypti females, all previously fed on YF patients, were used on Dean. By September 8 Dean had YF and he too survived. Lazear and Agramonte cabled Reed that mosquito transmission had been confirmed. However, bad news was to follow. On Sept. 25, 1900 Dr. Jesse Lazear died of YF. He had become ill on Sept. 18, and had related that he had observed a wild Ae. aegypti feeding on his hand as he was blood feeding a captive mosquito on a YF patient in hospital on Sept 14. Rather then interrupting his experiment, he watched as the wild mosquito engorged itself. He had thought he was immune in view of the failed experimental transmission attempt on himself on Aug. 16. Reed returned to Cuba in November and Reed, Agramonte and Carroll initiated experiments to substantiate the mosquito transmission theory. Experiments using human subjects, some succumbing to YF, that conclusively proved the mosquito - YF link.

What lessons can we learn today from the early experiments? First, experimental conditions are critical. Many YF transmission attempts failed because the need for viral replication in the mosquito was unknown. We now know that, depending on temperature, 10-14 days is needed for YF virus to infect mosquito salivary glands. Second, the choice of the individual mosquito was critical. We now know that Ae. aegypti, like all mosquito species, show ndividual variation in a variety of traits, including susceptibility to infection. Some Ae. aegypti females are incapable of being infected and transmitting YF due to their genetic makeup. Therefore, the choice of an individual mosquito, like the one infecting James Carroll, played a critical role in demonstrating mosquito transmission. It is likely that Jesse Lazear's first transmission attempt failed because he used a resistant Ae. aegypti.

Lessons for Modern Day Mosquito Control

What does this mean to us today? We know that there are differences between mosquito species, and accurate vector identification is important (see Dr. Lounibos' accompanying essay). There are differences in many important characteristics, i.e., host preferences, activity periods, habits, vector ability, insecticide resistance, etc. What we sometimes ignore is that there are population and geographic differences in these same traits within a species. Culex nigripalpus is widely distributed throughout Florida, yet we have no understanding of population or geographic variation. The same is true for Aedes taeniorhynchus and other Florida pest mosquitoes. Do west coast Cx. nigripalpus respond differently to pesticides, control strategies, vector status etc. compared to east coast mosquitoes, to those in the south? The similarity among pest mosquitoes is virtually unknown. We urgently need this information.One method to estimate geographic variation is to analyze mosquito genetic markers. There now exists an extensive literature on the importance of such studies (see W. J. Tabachnick and W. C. Black.1995. Making a case for molecular population genetic studies of arthropod disease vectors using molecular techniques. Parasitology Today 11:27-30). Polymerase chain reaction methods to detect DNA markers in individual mosquitoes are very useful, and their evaluation in Florida mosquito pests will be a first step in being able to apply the nascent field of genomics to mosquito control. As we develop new vector control methods, it will be essential to have an understanding of the mosquito material used in experiments and how this material applies to different regions of Florida. Knowing the genetic relatedness of west coast and east coast Cx. nigripalpus for example, will allow experimental findings to be applied to the field. When mosquitoes are evaluated for pesticide resistance, we need to know the genetic nature of the tested mosquitoes. How do they compare to the field? What do they represent? This information is critical to determine the applicability of the results to different Florida regions. Understanding mosquito genetic variation is essential to providing efficient, effective, and proper mosquito control and vector borne disease intervention in Florida.

Walter J. Tabachnick, Director
Florida Medical Entomology Laboratory

Figure above is a copy of correspondence from Dr. Walter Reed to Dr. James Carroll asking "Did the mosquito do it?"