Dispersal of the parasitic ciliate Lambornella clarki: Implications

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 83, pp , October 1986 Ecology Dispersal of the parasitic ciliate Lambornella clarki: Implications for ciliates in the biological control of mosquitoes (Aedes sierrensis/parasitic castration/oviposition behavior/longevity/treehole) DAVID E. EGERTER, JOHN R. ANDERSON, AND JAN 0. WASHBURN Division of Entomology and Parasitology, 201 Wellman Hall, University of California, Berkeley, CA Communicated by Ray F. Smith, June 20, 1986 ABSTRACT Lambornella clarki (Ciliophora: Tetrahymenidae), an endoparasite ofaedes sierrensis (Diptera: Culicidae), is dispersed by infected adult mosquitoes. Invasion of the ovaries induces parasitically castrated females to exhibit oviposition behavior and thereby actively disperse ciliates through deposition into water. Oviposition behavior of infected females is prolonged and mimics that of normal gravid females in their first gonotropic cycle. Adults of both sexes also passively disperse ciliates by dying on water surfaces, and infected adults are more likely to die on water than uninfected adults. Ciliates dispersed by infected adults can infect larvae and form desiccation-resistant cysts. Parasite-induced dispersal by hosts, desiccation-resistant cysts, an active host-seeking infective stage, and high infection and mortality rates ail indicate significant biological control potential for these and related ciliates against container-breeding mosquitoes. Ciliatosis in treehole-breeding mosquitoes appears to be a widespread phenomenon, having been reported from Southeast Asia (1); the USSR (2); Europe (3); Africa (4); and in California (5-9), Louisiana (10), and Oregon (11) in the United States. However, except for taxonomic descriptions and brief notes on occurrence, little is known about ciliate-mosquito associations. We are currently investigating one such association, that of the parasite Lambornella clarki Corliss and Coats (Ciliophora: Tetrahymenidae) and its natural host, the western treehole mosquito, Aedes sierrensis (Ludlow) (Diptera: Culicidae). In previous investigations (8, 9), we found L. clarki-infected mosquito larvae in approximately one-half of the treeholes sampled at our principal field site in Mendocino County in northern California. Such a widespread natural distribution ofthis parasite among cryptic treehole habitats suggested an effective mechanism of dispersal. Our additional observations (8) of naturally occurring adult infections, and parasitic castration of female A. sierrensis by L. clarki, led us to postulate that infected adult A. sierrensis are the agents of L. clarki dispersal and that infected female hosts may actively "oviposit" ciliates. The experiments reported here were undertaken to test these hypotheses. MATERIALS AND METHODS Mosquitoes and Parasites. Unless otherwise indicated, all A. sierrensis used in these experiments were from a laboratory colony that originated from field collections in the Sierra Nevada foothills. This strain has been continuously maintained in our laboratory at Berkeley since 1974, using methods described previously (12). We obtained infected adult A. sierrensis by exposing larvae to infective ciliates in 250-ml polyethylene cups filled with a dilute solution of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact autoclaved treehole water and deionized water. Fifty recently hatched first instar A. sierrensis larvae were added to these cups along with 0.2 g of sterile larval food (finely ground Purina rat chow). Cultures were held in an incubator at 110C with a 14:10 light/dark photoperiod. Successfully eclosing adult mosquitoes were removed daily and placed blindly into cages used in the different experiments. Because infected adults are morphologically indistinguishable from normal individuals, and only -10% of all adults successfully eclosing from 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, and by 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 infected adults of both sexes were maintained individually in 0.5-liter cardboard cages provided with a sucrose cube and a vial holding -25 ml of autoclaved dilute treehole water. These adults were paired with known uninfected mates to determine whether venereal transmission of L. clarki was possible. One group of potentially infected females was allowed to engorge on a human host to ascertain the effects of a blood meal on survivorship and behavior. Each caged adult was checked daily and dissected upon death to determine whether it harbored L. clarki or was uninfected; spermathecae of females were examined to determine insemination status. We also noted whether mosquitoes had died in the water vials or elsewhere in the cages, and water from all vials was examined at x 10 to x40 for L. clarki. Dispersal. In addition to recording ciliate dispersal into water vials in the longevity study, experiments were conducted with 21 infected females to examine temporal and spatial characteristics of ciliate dispersal. Fourteen of these mosquitoes were isolated as described above and their water vials were checked every 2 days. We replaced any vials containing ciliates, and when females were found dead, they were dissected. The remaining 7 infected females were each maintained in screen-topped, 2.5-liter cylindrical cardboard cages fitted in the bottom with five identical water vials (center and four cardinal points). We examined the vials daily for 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 compare egg deposition of normal gravid females with L. clarki deposition by parasitized females, we maintained the former females in the same 5-vial testing chambers described above. To control for possible laboratory-selected changes in oviposition behavior, we tested both laboratory colony females and females reared from larvae collected from natural treeholes. All females were blood-fed on the same human host 5 days post-eclosion, after which each was placed in a 5-vial cage. We checked the vials every 24 hr and replaced any that contained eggs. Oviposition behavior was assessed by the following parameters: (i) days from bloodfeeding to first oviposition; (it) number of days over which

2 7336 Ecology: Egerter et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Longevity of L. clarki-infected and normal A. sierrensis Infected Normal x, days x, days n (±SD) n (±SD) P Male <0.001 (±14.2) (±23.3) Female Non-blood-fed <0.001 (±16.7)* (±18.2) Blood-fed (+18.0)* (±11.3)* *No significant differences among these groups; t test, P > oviposition occurred; (iii) number of vials used as oviposition sites; (iv) number of vials used each day oviposition occurred; and (v) percentage of total eggs oviposited into the vial containing most eggs. Infectivity of Adult-Dispersed Ciliates. Two replicate groups of potentially L. clarki-infected adults (50 males and 50 females in each) were used to determine the infectivity of adult-dispersed ciliates to larval hosts. These mosquitoes had free access to sucrose cubes and a 250-ml polyethylene cup filled with autoclaved dilute treehole water. They were given a blood meal once a week and the water cups were changed every week following blood-feeding. Upon removal from the group cages, samples of water from the surface and bottom of the cups were examined at x 10 to x40 for L. clarki trophozoites and desiccation-resistant cysts. Subsequently, a bioassay was performed by adding 50 first-instar A. sierrensis larvae and 0.2 g of larval food to each cup. We allowed these larvae to mature in an 110C incubator under a 14:10 light/dark photoperiod for -5 weeks, whereupon they were sacrificed and examined microscopically for L. clarki infections. The experiment was ended after 6 weeks, when the majority of mosquitoes had died. RESULTS Longevity. Infected male and female A. sierrensis had decreased longevity when compared to normal adults (Table 1). Blood-fed normal females had decreased longevity compared to non-blood-fed normal females, but no significant differences in longevity were observed among the non-bloodfed infected, blood-fed infected, and blood-fed normal females (Table 1). Thus, infection with L. clarki and bloodfeeding 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 Dispersal Sex infected Passive Active Total % Males 72 4 (15) Females 44 9 (6) Passive dispersal refers to adults dying on water surfaces and releasing ciliates through decomposition; active dispersal refers to females 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 but not releasing ciliates prior to dissection. All adults were dissected within 24 hr after death. two. Although every female was inseminated (indicating that mating had occurred), no evidence of venereal transmission of L. clarki was observed. Dispersal. Ciliates occurred in water vials of infected males and females that died on the water surface (Table 2; passive dispersal; n = 13; 4 males, 9 females), and death in water vials for all mosquitoes was significantly correlated with infection (x2, P < 0.02). Ciliates were also present in water vials of infected females found dead on the floor of their cages (Table 2; active dispersal; n = 21; 12 non-blood-fed and 9 blood-fed); all females were parasitically castrated by ciliates occupying the ovaries. While a total of 30 of 44 infected females (68.2%) dispersed L. clarki in the longevity experiment, only 4 of 72 infected males (5.6%) did so (Table 2). The 21 females used to examine spatial and temporal patterns of parasite dispersal all contained ciliates in their reproductive tracts when dissected. In the first group of 14 females, 4 died without dispersing ciliates. However, the remaining 10 females dispersed ciliates into their water vials on one or more occasions (x = times; range, 1-15 times; n = 10) over a period ranging from 2 to 40 days (x = 21.8 ± 12.6 days; n = 10). Ciliates first appeared in water vials days post-eclosion (x = days; n = 10). The remaining 7 females (individually isolated in large cages with five identical water vials) deposited ciliates into a mean of 12.7 ± 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 vials used for ciliate deposition on the days when deposition occurred was 1.8 ± 0.9 vials (range, 1-4 vials; n = 50 days) (Table 3). Infected females deposited ciliates into as many as 4 of 5 vials each day, and even on the 10th day 3 females dispersed ciliates into 3 of 5 vials. Oviposition Behavior of Normal Gravid Females. No significant differences (t test, P > 0.05) were observed in any of the five parameters used to assess the oviposition behavior of Table 3. Oviposition behavior of gravid laboratory colony and wild A. sierrensis females, and parasite-dispersal behavior of L. clarki-infected females Colony Wild Infected Parameters x SD n x SD n x SD n Time, days No. of days No. of vials No. of vials per day % total eggs No 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 t test. Time = days from blood-feeding to first oviposition; No. of days = total number from first to last oviposition; No. of vials = total number used to complete oviposition; No. of vials per day = number used as oviposition sites each day oviposition observed; % total eggs = percentage of total eggs laid in vial containing most eggs; all blood-feeding on day 5 post-eclosion. Infected females parasitically castrated 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 were present during 10-day test period. n = number of females tested except for No. of vials per day, where n = number of days oviposition or ciliate deposition occurred.

3 Ecology: Egerter et al. Proc. Natl. Acad. Sci. USA 83 (1986) ~~~~~~~~~~~~~~~~.. 4 As r....* OWN t.i _, * 4 AS A. le. 0 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 wild females (Table 3). Oviposition was first observed on average 7.7 ± 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 of 2.8 ± 1.9 vials (n = 20, laboratory colony) as oviposition sites and a mean of 1.9 ± 1.0 vials (n = 30 days, laboratory colony) each day oviposition was observed (Table 3). Even though the majority offemales (12 of 20, laboratory colony) laid their eggs in more than one vial, an average of 88.1% ± 14.6% (n = 20, laboratory colony) of the total eggs laid by each female was laid in a single vial (Table 3). Infectivity of Adult-Dispersed Ciliates. Infected adults dispersed L. clarki ciliates, which produced infection rates

4 7338 Ecology: Egerter et al. ranging from 2.0% to 89.4% in larvae exposed in the bioassay (x = 49.5% %; n = 7), in cups that were removed during the first 4 weeks of the experiment. We did not observe free-swimming trophozoites of L. clarki in cups that were removed after 4 weeks, and no infected larvae were obtained when reared in these cups. Dead mosquitoes were present on the water surface of all cups removed from cages throughout the 6 weeks of the experiment. In addition to free-swimming ciliates, a high density of desiccation-resistant cysts was observed at the bottom of one cup taken from the cages at the end of the 2nd week. DISCUSSION While the present work clearly shows diminished longevity for both sexes of adult A. sierrensis infected with L. clarki, survivorship was sufficient for effective dispersal of L. clarki by two mechanisms: (i) active dispersal by parasitically castrated females; and (it) passive dispersal by infected mosquitoes of both sexes that die on the water and, with decomposition, release ciliates. Invasion of the female reproductive tract resulted in ovarial distention (as in egg development), and females exhibited oviposition behavior whereby ciliates were actively dispersed through deposition into water. Dissection of infected mosquitoes has revealed that L. clarki enters the ovaries as early as 72 hr post-eclosion (unpublished data), and that the ovaries 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) do parasitized females exhibit oviposition behavior and actively disperse ciliates into water. Since infected mosquitoes tended to die in water vials, and since it is normal behavior for wild A. sierrensis adults to rest in treeholes, such infected adults also may serve as a natural means of inoculating treeholes with 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 dispersal agents of L. clarki. Since all adults were dissected within 24 hr of death, we may have removed the infected males (n = 15) and females (n = 6) that died in the water vials without releasing ciliates before L. clarki could escape the cadavers (Table 2). Thus, our estimates of the passive (and predominantly male) contribution to total dispersal may be artificially low. However, because parasitized females actively disperse ciliates on multiple occasions, infected female A. sierrensis do indeed appear to be the dominant means by which L. clarki is dispersed. Examination of the oviposition behavior of normal gravid females in their first gonotropic cycle under our laboratory conditions revealed a tendency for eggs to be partitioned among several containers. That is, when offered more than one oviposition site, females tended to lay only a portion of their total eggs in one site (Table 3). In addition, females typically laid only a portion of their eggs on a single day (Table 3). This type of oviposition behavior has survival value in that instead of placing progeny in one site that may not be suitable, eggs are placed in several potential breeding sites, thus increasing the odds of using a site favorable to progeny (13). Such a behavioral strategy may be especially critical in A. sierrensis and other mosquito species that breed in containers-ephemeral resources subject to much variation. Comparison of normal oviposition behavior with the dispersal of L. clarki by parasitically castrated females under identical laboratory conditions demonstrated that deposited ciliates were also partitioned among several containers. Infected females visited the vials much more frequently than normal ovipositing females (Table 3), but the pattern of Proc. Natl. Acad. Sci. USA 83 (1986) behavior, as measured by the number of vials visited daily on the days that oviposition or ciliate deposition occurred, was not significantly different (t test, P > 0.05; Table 3). Therefore, although the behavior is prolonged, ciliate dispersal by infected females essentially mimics the oviposition behavior of normal gravid females. The results of our laboratory experiments revealed that L. clarki ciliates dispersed by adult A. sierrensis are both infective to larval hosts and capable of forming desiccationresistant cysts. L. clarki survives the summer drying of treeholes in these desiccation-resistant cysts, as does its mosquito host in desiccation-resistant eggs. Persistence within treeholes is excellent, and 90% of treeholes remain positive for L. clarki from year to year (9). Ciliate excystment and mosquito egg hatch occur synchronously when treeholes are flooded by winter rains (unpublished data). Free-swimming trophozoites are the infective stage, and these actively seek out and infect larval hosts via cuticular encystment (7). We have found that the successful eclosion of L. clarkiinfected A. sierrensis adults is a natural and common occurrence, and that infected adults are indeed the agents of parasite dispersal. Control of A. sierrensis and other container-breeding mosquitoes is difficult because breeding sites are cryptic, widely dispersed, and often inaccessible. Some authors have concluded that ciliate pathogens have little potential as biological control agents of mosquitoes (14, 15), while others have cited a need for more information before an accurate assessment can be made (1, 16-18). Our research findings indicate much promise for L. clarki and its relatives as manipulated biological control agents of container-breeding mosquitoes. Natural dispersal via infected mosquitoes is one aspect of the biology of L. clarki that greatly enhances its biological control potential. Three other observations suggest that L. clarki and related parasitic ciliates have much potential as biological control agents of mosquitoes: (i) a desiccation-resistant cyst stage enables ciliates to survive summer drying and to persist in mosquito breeding sites; (ii) in contrast to many other potential biological control agents of mosquitoes, 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 African sister species of L. clarki, Lambornella stegomyiae, indicate a host range that includes at least two genera of mosquitoes (19). We therefore believe these enigmatic ciliates are worthy of closer inspection as biological control agents of mosquitoes. We thank Drs. W. Sousa and Y. Tanada for comments on earlier drafts of this report, and Dr. R. Smith for communicating the manuscript on our behalf. The research was supported by a National Institutes of Health grant to J.R.A. 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