Journal of the American Control Association, l5(l):72-73, 1999 Copyright @ 1999 by the American Mosquito Control Association, Inc.

COLONIZATION OF NIGRIPALPUS THEOBALD (DIPTERA: CULICIDAE) BY STIMULATION OF MATING USING MALES OF OTHER MOSQUITO SPECIES

J. W. KNIGHT nNo J. K. NAYAR

Florida Medical Entomology Laboratory, IFAS, llniversity of Florida, 200 9th Street, SE, Vero Beach, FL 3296:2

ABSTRACT' Culex nigripalpzs Theobald (Diptera: Culicidae) was recolonized successfully by cohabiting Aedes taeniorhynchus males or males of other mosquito species starting in a large outdoor cage under natural Iight-dark cycles and temperatures and ending in a 1-ft.3 cage under artificial light-dark cycles at 24"C without added stimulation.

KEY WORDS Culex nigripalpus, Culex salinarius, Culex restuans, male Aedes taeniorhynchus, coloniza- tion, mating stimulation

Culex nigripalpus Theobald is an important vec- larvae were identified a few hours after a hatch tor of diseases of humans and is a major pest of (Dodge 1966), and larvae were reared to the adult livestock in rural south-central Florida. It is incrim- stage in the laboratory by the method of Nayar inated as a vector of St. Louis encephalitis in hu- (1968). Simultaneously, Ae. taeniorhynchus adults mans, eastern equine encephalitis in horses, dog from a laboratory colony were reared in the labo- heartworm () in dogs, wild tur- ratory by the method of Nayar (1967). Only Ae. key malaria (Plasmodium hermani) avian pox virus taeniorhynchus males, separated from females, of wild turkeys in wild turkeys, and avian malaria were used to stimulate copulation of Cx. nigripal- (Plasmodium elongatum) in hawks and owls (Nayar pus adults. 1982, Nayar et al. 1998). Even though this species Newly emerged adults of Cx. nigripalprr,r arrd was colonized in the mid-1960s (Haeger 1963, Pro- newly emerged males of Ae. taeniorhynchus wete vost 1969) and was maintained at the Florida Med- released in cages for mating trials. Adult mosqui- ical Entomology Laboratory (FMEL) in Vero toes were provided a 107o sucrose solution in cot- Beach, FL, for about 2O years before the colony ton wicks. After they were allowed to mate for 8- was lost, few details have been published on its 10 days, the adult mosquitoes were fed blood over- colonization. In the earliest attempts to colonize C-x. night on restrained chickens. Ten days after blood- nigripalpus in the laboratory, Haeger (1963) ob- feeding, groups of 100 females were placed in served low sperm transfer during mating under ar- l-pint plastic cups with lids and provided with oak- tificial conditions in the laboratory. Subsequently, leaf infusion for egg laying overnight. Egg rafts he obtained significant insemination from Cx. ni- were collected the next day and put in individual gripalpus copulations when he provided the stim- tubes containing water and a small amount of liver ulus of swarming and mating Aedes taeniorhynchus powder and yeast (1:3) mixture as a source of food. in the same cage (Provost 1969). Later, Haeger and Egg rafts that hatched were counted and recorded. O'Meara (1970) were able to introduce wild genes In the first series of trials. the wild adults of Cx. into an established Cx. nigripalpr.r colony by mat- nigripalpus (2,000+) and males of Ae. taeniorhyn- ing colony females with wild males. Earlier, Niel- chus (2,M+) were released at emergence into a sen and Haeger (1960) showed that, in mosquitoes, large, screened, wood-framed, walk-in cage (6 ft. male swarming and mating frequently occur simul- long X 6 ft. wide x 8 ft. high) on a cement floor taneously, especially under laboratory conditions, under a Lexan-paneled roof enclosure under a can- even though swanning is not required for mating. opy of oak trees providing natural light-dark cycles These studies suggested that, in mosquitoes, male and temperature. Additional humidity was provided swarming may be an important factor in stimulating when the relative humidity of the air dropped below mating under artificial caged conditions. A new col- 707o. Sixteen percent of the egg rafts collected from ony of Cx. nigripalpas has recently been estab- these females hatched. In subsequent 4 trials, cage- lished in our laboratory by cohabiting males of Ae. m ted Cx. nigripalpus progeny were mixed with taeniorhynchzs or of other species of mosquitoes adults reared from wild egg rafts to populate the in the cage. This paper presents a procedure for cage adequately for mating in the above manner establishing and maintaining a laboratory colony of outdoors. About 6O7o of the egg rafts hatched dur- Cx. nigripalpus. ing these trials. In the final trial in the large cage Egg rafts of wild Cx. nigripalprs were collected outdoors, only cage-mated (colony) progeny of Cx. in black plastic pans (12 in. wide x 18 in. long X nigripalpus were used, and SOVo of the egg rafts 9 in. high) containing oak-leaf infusion at the hatched. After this trial outdoors, mating was at- FMEL. The wild egg rafts were hatched, first-instar tempted in 3-ft.3 cages indoors in a room with large

72 Mencu 1999 ScreNrtnrcNots 73 windows for natural light-dark cycles and main- ACKNOWLEDGMENTS tained at 24"C. In 2 trials in 3-ft.3 cages, Cx. nigri- palpus adults were stimulated to mate by the ad- This is Florida Agricultural Experimental Sta- dition of Ae. taeniorhynchus males. In these trials, tion, Journal Series R-06286. We thank Jamie A. lSVo and Zl%o of the egg rafts hatched. At 24"C, Jennings for her technical assistance in conducting many of the Cx. nigripalpus survived up to 2 this study. months. Colonized Cx. nigripalprs were adapted to a 1- REF'ERENCES CITED ft.3 cage during the next 4 trials. In all these trials, Ae. taeniorhynchus males were added to cages to Dodge, H. R. 1966. Studies on mosquito larvae. II. The first-stage larvae of North stimulate mating. Egg rafts collected from these fe- American Culicidae and of world Anophelinae. Can. Entomol. 98 :33'7-393. males showed increases in egg hatching from 87o Haeger, J. S. 1963. Progress in colonization of Culex ni- in the flrst 2 trials to 40Vo in the 3rd trial and to gripalpus. Rep. 34th Annu. Meeting Fla. Anti-Mosquito 76Vo in the 4th trial. At present, we are maintaining Assoc. p. 82-85. our laboratory colony of Cx. nigripalpus in l-ft.3 Haeger, J. S. and G. F O'Meara. 1970. Rapid incorpo- cages in a bio-room maintained at 24"C with light- ration of wild genotype of Culex nigripalpus (Diptera: dark cycles of 12 h, and,Ae. taeniorhynchzs males Culicidae) into laboratory-adapted strain. Ann. Ento- are no longer added to stimulate mating. mol. Soc. Am. 63:1390-1391. Nayar, J. K. 1967. Pupation rhythm in Aedes taeniorhyn- We point out here that, during the process of lab- cy'las (Wiedemann). II. Ontogenetic timing, rate of de- oratory colonization of this species, some loss in velopment, and endogenous diurnal rhythm of pupation. genetic variability may have occurred, even though Ann. Entomol. Soc. Am. 6O:946-971. -fheo- it has not yet been demonstrated. However, genetic Nayar, J. K. 1968. Biology of Culex nigripalpus variability of this species can be substantially in- bald (Diptera: Culicidae). Part l: effect of rearing con- creased by incorporation of wild genes by mating ditions on growth and the diurnal rhythm of pupation colonized females with wild males, as was previ- and emergence. J. Med. Entomol. 5:39-46. ously demonstrated by Haeger and O'Meara Nayar, J. K. 1982. Bionomics and physiology of Culex (l970). nigripalpus (Diptera: Culicidae) of Florida: an impor- tant vector of diseases. Florida Agricultural Experimen- Other species of newly emerged males, such as tal Stations, IFAS, University of Florida, Bull. 827:1- Aedes albopictus allLdAnopheles quadrimaculatus, 73. Gainesville, FL. added to cages with newly emerged Cx. nigripalpus Nayar, J. K., J. W. Knight and S. R. Telford. 1998. Vector also stimulated mating and a higher percentage of ability of mosquitoes for isolates of Plasmodium elon- egg raft hatching was found than without their ad- gatum from raptors in Florida. J. Parasitol. 84:542-546. dition. These studies show that the addition of Ae. Nielsen, E. T and J. S. Haeger. 1960. Swarming and mat- taeniorhynchas males or males of other species of ing in mosquitoes. Entomol. Soc. Am. Misc. Publ. l: 7 r-95. mosquitoes apparently stimulate swarming of Cx. Provost, M. W. 1969. The natural history of Culex ni- nigripalpus to the extent that they mate with caged gripalpus. 1n.' St. Louis encephalitis in Florida-ten females in the laboratory. We have successfully years of research, surveillance and control programs, colonized Cx. nigripalpus, Culex salinarius, and 46-62. Floid,a State Board of Health Monogr. 12. Iack- Culex restuans by this technique. sonville, FL.