Proc. Nati. Acad. Sci. USAVol. 83, pp. 7335-7339, October 1986Ecology

Dispersal of the parasitic ciliate Lambornella clarki: Implicationsfor ciliates in the biological control of mosquitoes

(Aedes sierrensis/parasitic castration/oviposition behavior/longevity/treehole)

DAVID E. EGERTER, JOHN R. ANDERSON, AND JAN 0. WASHBURNDivision of Entomology and Parasitology, 201 Wellman Hall, University of California, Berkeley, CA 94720

Communicated by Ray F. Smith, June 20, 1986

ABSTRACT Lambornella clarki (Ciliophora: Tetrahymen-idae), an endoparasite ofAedes sierrensis (Diptera: Culicidae),is dispersed by infected adult mosquitoes. Invasion of theovaries induces parasitically castrated females to exhibitoviposition behavior and thereby actively disperse ciliatesthrough deposition into water. Oviposition behavior of infectedfemales is prolonged and mimics that of normal gravid femalesin their first gonotropic cycle. Adults of both sexes alsopassively disperse ciliates by dying on water surfaces, andinfected adults are more likely to die on water than uninfectedadults. Ciliates dispersed by infected adults can infect larvaeand form desiccation-resistant cysts. Parasite-induced disper-sal by hosts, desiccation-resistant cysts, an active host-seekinginfective stage, and high infection and mortality rates ailindicate significant biological control potential for these andrelated ciliates against container-breeding mosquitoes.

Ciliatosis in treehole-breeding mosquitoes appears to be awidespread phenomenon, having been reported from South-east Asia (1); the USSR (2); Europe (3); Africa (4); and inCalifornia (5-9), Louisiana (10), and Oregon (11) in theUnited States. However, except for taxonomic descriptionsand brief notes on occurrence, little is known aboutciliate-mosquito associations. We are currently investigatingone such association, that of the parasite Lambornella clarkiCorliss and Coats (Ciliophora: Tetrahymenidae) and itsnatural host, the western treehole mosquito, Aedes sierrensis(Ludlow) (Diptera: Culicidae). In previous investigations (8,9), we found L. clarki-infected mosquito larvae in approxi-mately one-half of the treeholes sampled at our principal fieldsite in Mendocino County in northern California. Such awidespread natural distribution ofthis parasite among cryptictreehole habitats suggested an effective mechanism of dis-persal. Our additional observations (8) of naturally occurringadult infections, and parasitic castration of female A. sier-rensis by L. clarki, led us to postulate that infected adult A.sierrensis are the agents of L. clarki dispersal and thatinfected female hosts may actively "oviposit" ciliates. Theexperiments reported here were undertaken to test thesehypotheses.

MATERIALS AND METHODSMosquitoes and Parasites. Unless otherwise indicated, all

A. sierrensis used in these experiments were from a labora-tory colony that originated from field collections in the SierraNevada foothills. This strain has been continuously main-tained in our laboratory at Berkeley since 1974, usingmethods described previously (12). We obtained infectedadult A. sierrensis by exposing larvae to infective ciliates in250-ml polyethylene cups filled with a dilute solution of

autoclaved treehole water and deionized water. Fifty recent-ly hatched first instar A. sierrensis larvae were added to thesecups along with 0.2 g of sterile larval food (finely groundPurina rat chow). Cultures were held in an incubator at 110Cwith a 14:10 light/dark photoperiod. Successfully eclosingadult mosquitoes were removed daily and placed blindly intocages used in the different experiments. Because infectedadults are morphologically indistinguishable from normalindividuals, and only -10% of all adults successfully eclosingfrom the laboratory cultures were infected with L. clarki,infected and normal individuals were identified in two ways:by the presence of L. clarki in water vials in the cages, andby dissection at the conclusion of each experiment.

Longevity. We conducted all experiments with adult A.sierrensis in an insectary at 220C, "30% relative humidity,and a 14:10 light/dark photoperiod. Potentially infectedadults of both sexes were maintained individually in 0.5-litercardboard cages provided with a sucrose cube and a vialholding -25 ml of autoclaved dilute treehole water. Theseadults were paired with known uninfected mates to determinewhether venereal transmission ofL. clarki was possible. Onegroup of potentially infected females was allowed to engorgeon a human host to ascertain the effects of a blood meal onsurvivorship and behavior. Each caged adult was checkeddaily and dissected upon death to determine whether itharbored L. clarki or was uninfected; spermathecae offemales were examined to determine insemination status. Wealso noted whether mosquitoes had died in the water vials orelsewhere in the cages, and water from all vials was examinedat x 10 to x40 for L. clarki.

Dispersal. In addition to recording ciliate dispersal intowater vials in the longevity study, experiments were con-ducted with 21 infected females to examine temporal andspatial characteristics of ciliate dispersal. Fourteen of thesemosquitoes were isolated as described above and their watervials were checked every 2 days. We replaced any vialscontaining ciliates, and when females were found dead, theywere dissected. The remaining 7 infected females were eachmaintained in screen-topped, 2.5-liter cylindrical cardboardcages fitted in the bottom with five identical water vials(center and four cardinal points). We examined the vials dailyfor 10 days and replaced any in which ciliates were present.At the end of the 10-day period, all 7 females were dissected.

Oviposition Behavior of Normal Gravid Females. To com-pare egg deposition of normal gravid females with L. clarkideposition by parasitized females, we maintained the formerfemales in the same 5-vial testing chambers described above.To control for possible laboratory-selected changes inoviposition behavior, we tested both laboratory colonyfemales and females reared from larvae collected fromnatural treeholes. All females were blood-fed on the samehuman host 5 days post-eclosion, after which each wasplaced in a 5-vial cage. We checked the vials every 24 hr andreplaced any that contained eggs. Oviposition behavior wasassessed by the following parameters: (i) days from blood-feeding to first oviposition; (it) number of days over which

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Table 1. Longevity of L. clarki-infected and normal A. sierrensis

Infected Normal

x, days x, daysn (±SD) n (±SD) P

Male 72 19.8 133 52.0 <0.001(±14.2) (±23.3)

FemaleNon-blood-fed 30 26.1 116 42.7 <0.001

(±16.7)* (±18.2)Blood-fed 14 31.7 23 33.5 0.36

(+18.0)* (±11.3)*

*No significant differences among these groups; t test, P > 0.05.

oviposition occurred; (iii) number of vials used as ovipositionsites; (iv) number of vials used each day oviposition oc-curred; and (v) percentage of total eggs oviposited into thevial containing most eggs.

Infectivity of Adult-Dispersed Ciliates. Two replicategroups of potentially L. clarki-infected adults (50 males and50 females in each) were used to determine the infectivity ofadult-dispersed ciliates to larval hosts. These mosquitoes hadfree access to sucrose cubes and a 250-ml polyethylene cupfilled with autoclaved dilute treehole water. They were givena blood meal once a week and the water cups were changedevery week following blood-feeding. Upon removal from thegroup cages, samples of water from the surface and bottomof the cups were examined at x 10 to x40 for L. clarkitrophozoites and desiccation-resistant cysts. Subsequently, abioassay was performed by adding 50 first-instar A. sierrensislarvae and 0.2 g of larval food to each cup. We allowed theselarvae to mature in an 110C incubator under a 14:10 light/darkphotoperiod for -5 weeks, whereupon they were sacrificedand examined microscopically for L. clarki infections. Theexperiment was ended after 6 weeks, when the majority ofmosquitoes had died.

RESULTSLongevity. Infected male and female A. sierrensis had

decreased longevity when compared to normal adults (Table1). Blood-fed normal females had decreased longevity com-pared to non-blood-fed normal females, but no significantdifferences in longevity were observed among the non-blood-fed infected, blood-fed infected, and blood-fed normal fe-males (Table 1). Thus, infection with L. clarki and blood-feeding were associated with similar decreases in longevity,and there appeared to be no synergistic effect between the

Table 2. Dispersal of L. clarki by adult A. sierrensis

Total DispersalSex infected Passive Active Total %

Males 72 4 (15) 4 5.6Females 44 9 (6) 21 30 68.2

Passive dispersal refers to adults dying on water surfaces andreleasing ciliates through decomposition; active dispersal refers tofemales that, subsequent to ciliate invasion of the reproductive tract,exhibit oviposition behavior, thereby depositing ciliates into water.Numbers in parentheses are number of adults dying in water vials butnot releasing ciliates prior to dissection. All adults were dissectedwithin 24 hr after death.

two. Although every female was inseminated (indicating thatmating had occurred), no evidence of venereal transmissionof L. clarki was observed.

Dispersal. Ciliates occurred in water vials of infected malesand females that died on the water surface (Table 2; passivedispersal; n = 13; 4 males, 9 females), and death in water vialsfor all mosquitoes was significantly correlated with infection(x2, P < 0.02). Ciliates were also present in water vials ofinfected females found dead on the floor of their cages (Table2; active dispersal; n = 21; 12 non-blood-fed and 9 blood-fed);all females were parasitically castrated by ciliates occupyingthe ovaries. While a total of 30 of 44 infected females (68.2%)dispersed L. clarki in the longevity experiment, only 4 of 72infected males (5.6%) did so (Table 2).The 21 females used to examine spatial and temporal

patterns of parasite dispersal all contained ciliates in theirreproductive tracts when dissected. In the first group of 14females, 4 died without dispersing ciliates. However, theremaining 10 females dispersed ciliates into their water vialson one or more occasions (x = 7.9 + 4.6 times; range, 1-15times; n = 10) over a period ranging from 2 to 40 days (x =21.8 ± 12.6 days; n = 10). Ciliates first appeared in water vials11-23 days post-eclosion (x = 14.7 + 4.9 days; n = 10). Theremaining 7 females (individually isolated in large cages withfive identical water vials) deposited ciliates into a mean of12.7 ± 2.7 vials (n = 7) over a mean of 7.1 ± 1.0 days (n =7) during the 10 test days (Table 3). The mean number of vialsused for ciliate deposition on the days when depositionoccurred was 1.8 ± 0.9 vials (range, 1-4 vials; n = 50 days)(Table 3). Infected females deposited ciliates into as many as4 of 5 vials each day, and even on the 10th day 3 femalesdispersed ciliates into 3 of 5 vials.

Oviposition Behavior of Normal Gravid Females. No sig-nificant differences (t test, P > 0.05) were observed in any ofthe five parameters used to assess the oviposition behavior of

Table 3. Oviposition behavior of gravid laboratory colony and wild A. sierrensis females, andparasite-dispersal behavior of L. clarki-infected females

Colony Wild InfectedParameters x SD n x SD n x SD n

Time, days 7.7 1.9 20 7.8 1.6 14No. of days 1.6 1.0 20 2.0 1.3 14 7.1 1.0 7No. of vials 2.8 1.9 20 2.9 2.1 14 12.7 2.7 7No. of vials per day 1.9 1.0 30 1.8 0.9 23 1.8 0.9 50% total eggs 88.1 14.6 20 86.7 16.1 14No significant differences between any parameters measured for laboratory colony females and

field-collected laboratory-reared females; t test, P > 0.05; percentages arcsine transformed prior to ttest. Time = days from blood-feeding to first oviposition; No. of days = total number from first to lastoviposition; No. of vials = total number used to complete oviposition; No. of vials per day = numberused as oviposition sites each day oviposition observed; % total eggs = percentage of total eggs laidin vial containing most eggs; all blood-feeding on day 5 post-eclosion. Infected females parasiticallycastrated by L. clarki; parameters measured represent total days when ciliates were present in vials,total number of vials containing ciliates, and number of vials containing ciliates each day ciliates werepresent during 10-day test period. n = number of females tested except for No. of vials per day, wheren = number of days oviposition or ciliate deposition occurred.

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FIG. 1. Ovaries and oviducts freshly dissected out of two 16-day post-eclosion A. sierrensis females; (Left) from uninfected female; (Right)from L. clarki-infected female parasitically castrated by hundreds of ciliates occupying the reproductive tract. (Bar = 0.25 mm.)

laboratory colonized females versus laboratory-reared wildfemales (Table 3). Oviposition was first observed on average7.7 ± 1.9 days post-blood-feeding (n = 20, laboratory colony)and was completed a mean of 1.6 ± 1.0 days (n = 20,laboratory colony) later (Table 3). Females used a mean of2.8± 1.9 vials (n = 20, laboratory colony) as oviposition sitesand a mean of 1.9 ± 1.0 vials (n = 30 days, laboratory colony)

each day oviposition was observed (Table 3). Even thoughthe majority offemales (12 of 20, laboratory colony) laid theireggs in more than one vial, an average of 88.1% ± 14.6% (n= 20, laboratory colony) of the total eggs laid by each femalewas laid in a single vial (Table 3).

Infectivity of Adult-Dispersed Ciliates. Infected adults dis-persed L. clarki ciliates, which produced infection rates

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ranging from 2.0% to 89.4% in larvae exposed in the bioassay(x = 49.5% + 32.7%; n = 7), in cups that were removed duringthe first 4 weeks of the experiment. We did not observefree-swimming trophozoites of L. clarki in cups that wereremoved after 4 weeks, and no infected larvae were obtainedwhen reared in these cups. Dead mosquitoes were present onthe water surface of all cups removed from cages throughoutthe 6 weeks of the experiment. In addition to free-swimmingciliates, a high density of desiccation-resistant cysts wasobserved at the bottom ofone cup taken from the cages at theend of the 2nd week.

DISCUSSIONWhile the present work clearly shows diminished longevityfor both sexes of adult A. sierrensis infected with L. clarki,survivorship was sufficient for effective dispersal ofL. clarkiby two mechanisms: (i) active dispersal by parasiticallycastrated females; and (it) passive dispersal by infectedmosquitoes of both sexes that die on the water and, withdecomposition, release ciliates.

Invasion of the female reproductive tract resulted in ovarialdistention (as in egg development), and females exhibitedoviposition behavior whereby ciliates were actively dis-persed through deposition into water. Dissection of infectedmosquitoes has revealed that L. clarki enters the ovaries asearly as 72 hr post-eclosion (unpublished data), and that theovaries become fully distended with ciliates =10 days later(Fig. 1). Not until the ovaries are distended with ciliates(comparable to the size of ovaries containing mature eggs) doparasitized females exhibit oviposition behavior and activelydisperse ciliates into water. Since infected mosquitoes tendedto die in water vials, and since it is normal behavior for wildA. sierrensis adults to rest in treeholes, such infected adultsalso may serve as a natural means of inoculating treeholeswith ciliates.Although the sex ratio of infected males to infected females

was not significantly different from 1:1 (x2, P > 0.05),infected females were much more important as dispersalagents of L. clarki. Since all adults were dissected within 24hr of death, we may have removed the infected males (n = 15)and females (n = 6) that died in the water vials withoutreleasing ciliates before L. clarki could escape the cadavers(Table 2). Thus, our estimates of the passive (and predomi-nantly male) contribution to total dispersal may be artificiallylow. However, because parasitized females actively disperseciliates on multiple occasions, infected female A. sierrensisdo indeed appear to be the dominant means by which L. clarkiis dispersed.Examination of the oviposition behavior of normal gravid

females in their first gonotropic cycle under our laboratoryconditions revealed a tendency for eggs to be partitionedamong several containers. That is, when offered more thanone oviposition site, females tended to lay only a portion oftheir total eggs in one site (Table 3). In addition, femalestypically laid only a portion of their eggs on a single day(Table 3). This type of oviposition behavior has survivalvalue in that instead of placing progeny in one site that maynot be suitable, eggs are placed in several potential breedingsites, thus increasing the odds of using a site favorable toprogeny (13). Such a behavioral strategy may be especiallycritical in A. sierrensis and other mosquito species that breedin containers-ephemeral resources subject to much varia-tion.Comparison of normal oviposition behavior with the dis-

persal of L. clarki by parasitically castrated females underidentical laboratory conditions demonstrated that depositedciliates were also partitioned among several containers.Infected females visited the vials much more frequently thannormal ovipositing females (Table 3), but the pattern of

behavior, as measured by the number of vials visited daily onthe days that oviposition or ciliate deposition occurred, wasnot significantly different (t test, P > 0.05; Table 3). There-fore, although the behavior is prolonged, ciliate dispersal byinfected females essentially mimics the oviposition behaviorof normal gravid females.The results of our laboratory experiments revealed that L.

clarki ciliates dispersed by adult A. sierrensis are bothinfective to larval hosts and capable of forming desiccation-resistant cysts. L. clarki survives the summer drying oftreeholes in these desiccation-resistant cysts, as does itsmosquito host in desiccation-resistant eggs. Persistence with-in treeholes is excellent, and 90% oftreeholes remain positivefor L. clarki from year to year (9). Ciliate excystment andmosquito egg hatch occur synchronously when treeholes areflooded by winter rains (unpublished data). Free-swimmingtrophozoites are the infective stage, and these actively seekout and infect larval hosts via cuticular encystment (7).We have found that the successful eclosion of L. clarki-

infected A. sierrensis adults is a natural and common occur-rence, and that infected adults are indeed the agents ofparasite dispersal. Control of A. sierrensis and other con-tainer-breeding mosquitoes is difficult because breeding sitesare cryptic, widely dispersed, and often inaccessible. Someauthors have concluded that ciliate pathogens have littlepotential as biological control agents of mosquitoes (14, 15),while others have cited a need for more information before anaccurate assessment can be made (1, 16-18). Our researchfindings indicate much promise for L. clarki and its relativesas manipulated biological control agents of container-breed-ing mosquitoes. Natural dispersal via infected mosquitoes isone aspect of the biology of L. clarki that greatly enhances itsbiological control potential. Three other observations suggestthat L. clarki and related parasitic ciliates have much poten-tial as biological control agents of mosquitoes: (i) a desicca-tion-resistant cyst stage enables ciliates to survive summerdrying and to persist in mosquito breeding sites; (ii) incontrast to many other potential biological control agents ofmosquitoes, ciliates actively seek out larval hosts; and (iii)field infection and mortality rates are high (refs. 8 and 9;unpublished data). Furthermore, collections of the Africansister species of L. clarki, Lambornella stegomyiae, indicatea host range that includes at least two genera of mosquitoes(19). We therefore believe these enigmatic ciliates are worthyof closer inspection as biological control agents of mosqui-toes.

We thank Drs. W. Sousa and Y. Tanada for comments on earlierdrafts of this report, and Dr. R. Smith for communicating themanuscript on our behalf. The research was supported by a NationalInstitutes of Health grant to J.R.A. (A120245).

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2. Dzerzhinsky, V. A., Nam, E. A. & Dubitskii, A. M. (1976)Parazitologia 10, 381-382.

3. Grasse, P.-P. & Boissezon, P. De (1929) Bull. Soc. Zool. Fr.54, 187-191.

4. Muspratt, J. (1947) Parasitology 38, 107-110.5. Kellen, W. R., Wills, W. & Lindegren, J. E. (1961) J. Insect

Pathol. 3, 335-338.6. Sanders, R. D. (1972) Proc. Calif. Mosq. Control Assoc. 40,

66-68.7. Clark, T. B. & Brandl, D. G. (1976) J. Invert. Pathol. 23,

341-349.8. Egerter, D. E. & Anderson, J. R. (1985) J. Invert. Pathol. 46,

296-304.9. Washburn, J. 0. & Anderson, J. R. (1986) J. Invert. Pathol.,

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12. Schwan, T. G. & Anderson, J. R. (1980) Mosquito News 40,263-269.

13. Rozeboom, L. E., Rosen, L. & Ikeda, J. (1973) J. Med.Entomol. 10, 397-399.

14. McLaughlin, R. E. (1971) in Microbial Control ofInsects andMites, eds. Burges, H. D. & Hussey, N. W. (Academic, NewYork), pp. 151-172.

15. Henry, J. E. (1981) Annu. Rev. Entomol. 26, 49-73.

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16. Corliss, J. 0. & Coats, D. W. (1976) Trans. Am. Microsc. Soc.95, 725-739.

17. Canning, E. U. (1982) Parasitology 84, 119-149.18. Clark, T. B. (1985) in Biological Control of Mosquitoes,

eds. Chapman, H. C., Barr, A. R., Laird, M. & Weidhaas,D. E. (Am. Mosquito Control Assoc., Fresno, CA), Bull. 6,pp. 56-58.

19. Muspratt, J. (1945) J. Entomol. Soc. S. Afr. 8, 13-20.

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