654 JouRr.uLoF THEAtr{snIclr.l Mosqurro CoNrRor,Assocrluox Vol. 7, No. 4 GIEMSA STAIN AS A MARKER IN THE PITCHER-PLANT MOSQUITO, WYEOMYIA SMITHII CHERYL A. KLECKNER AND WILLIAM E. BRADSHAW Depdrtment of Biology, Uniuersity of Oregon,Eugene, OR 97403 ABSTRACT. Third and fourth instars of Wyeomyia srnithii were reared in Giemsa stain at 4 concentrationsbetween 4 x 10-?and 10*5g/liter. The mosquitoesretained the blue mark as adults and remainedmarked throughout their laboratory life. Concentration of Giemsasignificantly affectedeclosion successbut had no significant effect on mean days to pupation or days as a pupa, male or female adult longevity, per-capita female fecundity or fertility. Larval exposureto low concentrations (4 x 10-7 or 10-" g/liter) of Giemsa stain provides an effective lifetime tag for otherwise indistinguishable laboratory populations. We undertook this study of Wyeomyinsmithii eclosionwas recordedby removing and sexing (Coq.)to determinewhether Giemsa stain would the pupal exuviae. Adults had constant access be an effective,easily detectablemarker for this to raisins and a cut leaf of their host plant, mosquito in laboratory populations. A reliable Sarraceniapurpured Linn., in a 70-ml glassjar marker for Wy. smithii would allow us to deter- half-filled with distilled water. The bottom of mine relative longevity of adults from distinct the cage was lined with filter paper kept moist populations when placed in mixed population with distilled water. cages receiving different treatments, Experi- We conducted 2 experiments. In the first, ments were designed to reveal the effect of within one day ofeclosion, adults were squashed Giemsa concentration,experienced during de- betweenslide and coverslip,sexed and examined velopment from larval diapauseto pupation, on against a white background under a dissecting adult eclosion success,longevity, fecundity, fer- microscope (15-30x magnification) for the tility and persistenceof the marker in the adults. Giemsastain. In this experiment 2 trials of third Overwintering larvae of Wyeomyia smithii instars were run at each of the 4 concentrations; (June were collectedin Maine 1988)and main- trials of fourth instars consisted of 2 replicates tained in the Iaboratory for 3 generations.Third each at concentrations of 10-6 and 4 x 10-u gl instar diapausewas induced by a short-day pho- liter and one trial each at 4 X 10-? and 10-5 (L:D : + toperiod 8:16) at 21 0.5"C and main- Giemsaconcentration. In the secondexperiment tained for 6 months prior to experiments.Larvae the adults remained in the cagesand were main- which molted to the fourth instar and the third tained until all adults had died. All dead adults instars were compared in 2 experiments. were removed daily, squashed,sexed and exam- The Giemsastain was preparedby adding the ined for the blue marker using a dissecting mi- appropriate amount of Giemsa powder (Fisher croscope.Eggs were removed from leaf and jar, ScientificCo., Pittsburgh,PA) to 1 liter of dis- pupal paper liner, tilled water and heating to ensure dissolution. dish and rinsed into distilled petri We tested 4 concentrationsof Giemsa stain: water in a 150 x 25 mm dish and counted. larvae were removed from the diapause regime Eggswere kept for 10 daysand all hatchedlarvae and placedin Giemsa(4 x 10-?,10-6, 4 x 10-6, were counted. Trials consistedof 2 replicates of and 10-5 g Giemsa/liter) solution. Experiments third instars in each of the 4 Giemsaconcentra- were conductedwith a daily sine-wavethermo- tions and in distilled water (controls); fourth period(12-28'C, mean :2t"C),80% RH anda instar trials included 2 replicatesat 4 x 10-6and long-dayphotoperiod (L:D = 18:6).Replicates at 10-6g/Iiter, one trial at 4 x l0-1 and at 10-5 of 30 larvae in 125 ml of Giemsasolution per g/liter Giemsaconcentration, and 2 control rep- petri dish were compared with controls reared licatesin distilled water. in distilled water. Larvae were fed (3 times/wk) Frequencieswere evaluated using the G-test an emulsion of guinea pig chow and freeze-dried for independence(Sokal and Rohlf 1969).Data brine shrimp (3:1 by volume). When cleaning were analyzedwith the nonparametric Kruskal- was necessaryfresh Giemsa solution was added Wallis test (SAS Institute 1985). to maintain a constantconcentration. The Giemsa marker was easily observed by Pupaewere collectedevery Monday, Wednes- using the technique describedabove. In the first day and Friday, transferred to plastic dessert experiment where the adults were examined on disheshalf-filled with unstained, distilled water the day of emergence,the blue Giemsastain was and placed in cylindrical screenedcages (200 x readily visible in L00Voof the individuals that 150 mm) in the same room as the larvae. Adult had been exposedto the stain as larvae, regard- DECEMBER1991 OpnRarroNar,ero ScroNrruc NorES Iessof concentration. In the secondexperiment, mean adult longevity ranged from 5.5 to 17.0 days. The blue Giemsa marker persisted throughout the adult life span in 100% of the individuals that had been exposedto the stain as larvae, regardlessof concentration, but was z not visible in any ofthe unexposedcontrols. a The concentration of Giemsasignificantly af- 9ro fected eclosion success(G with a df: 8L.46;P O < 0.001) (Fig. 1). The Kruskal-Wallis test (x' t! with 4 df) showedno significant effect of Giemsa concentration on mean days to pupation (12 : 5.35;P > 0.05) or days as a pupa (X2: 2.31;P > 0.05)(Table 1). Similarly, the Kruskal-Wallis G:81.a6 test showedno significant effect of Giemsacon- P <0.001 centration on longevity of adult males (12 : 4.13;P > 0.05)or females(x' : 7.73;P > 0.05), o 4 10 40 100 per-capitafemale fecundity (x' : 6.13;P > 0.05) or fertility (x' :6.55; P > 0.05). GIEMSACONCENTRATION X 1O-7GILITER In our study, long exposureof 2-4 wk during Fig. 1. Eclosionsuccess of Wyeomyiasmithii pupae larval development effectively marked Wyeo- exposedto Giemsastain as third (O) and fourth (O) rnyia smithii larvae. At low concentrations (4 X instars,including controls (0 g/liter).Concentration 10-7and 10-6g/liter) the dye affects emergence of Giemsasignificantly affected eclosion (P < 0.001). minimally and yet produces 100% marking that persists throughout adult lifetime. The stain also has a minimal effect on life history char- indicated that some growth retardation was ev- acters of Wy. stnithii. Previous studies (Weath- ident in the treated larvae. Our studies show no ersbeeand Hasell 1938,Haddow 1942,Joslyn et significant effects of Giemsa concentration on al. 1985,Joslyn and Fish 1986,Kay and Mot- developmenttime or on longevity, fecundity or tram L986) with Giemsa did not investigate the fertility of adult Wy. smithii. However, as shown effects of the procedure on longevity, fecundity in Table 1, there were parallel (albeit nonsig- or fertility of the treated adults. Weathersbee nificant) rises in larval development times and and Hasell (1938)and Kay and Mottram (1986) declines in male and female longevity, female Table 1. Life-history parametersof Wyeomyiasmithii exposedto different concentrations of Giemsa stain, including controls reared in distilled water (0 g/liter). Values are parameter means. t0 40 100 x'(K-w)* Days to pupation D.JA 3rd instar 22 26 26 30 29 4th instar 19 20 18 20 24 Days as pupa 2.31 3rd instar 87887 4th instar 798qE Male longevity (days) 4.r3 3rd instar 10 10 t2 66 4th instar t2 16 12 10# Femalelongevity (days) 3rd instar 13 15 13 10 11 4th instar 14 L7 12 10# Fecundity (eggsfemale) 6.13 3rd instar 46 34 17 17 4th instar 48 34 60 0# Fertility (%hatnh) 6.55 3rd instar 93 92 79 00 4th instar 99 75 100 0# f No adults eclosedfrom these treatments. * Kruskal-Wallis test for equality of Giemsa effects;with 4 df minimum significant (P < 0.0S)x2 = g.4g. 656 JounNer,oF THE AunRrcex Mosqurro CoNrRoLAssocrarroN Vor. ?,No. 4 fecundity and egg hatchability among mosqui- purea,pp.161-189. In: J. H. Frank and L. P. Loun- toes which had been exposed as larvae to a ibos (eds.),Phytotelmata: terrestrial plants as hosts Giemsaconcentration greater than 10-6 g/liter. for aquatic insect communities. Plexus Publ., Med- We would therefore caution against long expo- ford, NJ. Bradshaw, sures to higher than 10-6 g/liter Giemsa. Used W. E. and C. M. Holzapfel.1983. Predator- mediated, non-equilibrium coexistenceof tree-hole judiciously, prolonged exposure to low concen- mosquitoesin southeasternNorth America. Oecol- trations of Giemsa can provide a good tag for ogia 57:239-256. laboratory populations of. Wyeornyia srnithii Copeland,R. S. and G. B. Craig, Jr. 1990.Habitat which are otherwise indistinguishable as adults. segregationamong treehole mosquitoes(Diptera: Although our experiments were not designed Culicidae) in the Great Lakes region of the United to test field use, the Giemsa self-marking tech- States.Ann. Entomol. Soc.Am. 83:1063-1073. nique has potential field applicability. An effec- Haddow,A. J.1942. The mosquitofauna and climate tive field marker would aid in dispersal studies. of native huts at Kisumu, Kenya. Bull. Entomol. Res.33:91-142. Giemsa stain could be used to determine which Joslyn, D. J., L. B. Conrad and P. T. Slavin. 1985. individual adult mosquitoesemerge from differ- Development and preliminary field testing of the ent speciesof pitcher plants (Bradshaw 1983)or Giemsa self-marker for the salt marsh mosquito different sub-habitats among tree hole mosqui- Aedessollicitans (Walker) (Diptera:Culicidae). Ann. toes (e.g.,Bradshaw and Holzapfel 1983,Cope- Entomol. Soc. Am. 78.,20-23. land and Craig 1990). Joslyn, D. J. and D. Fish. 1986.Adult dispersalof We thank Christina Holzapfel, Jeffrey J. Aedescomrnums using Giemsa self-marking. J. Am. Hard, Sam Donovan and John M.