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Reproduction of an inbred strain of Culex pipiens prevented by loss of Wolbachia pipientis
JOURNAL
OF INVERTEBRATE
Reproduction
of an inbred Strain of Culex pipiens Loss of Wolbachia pipientis
DOMINIC S.T. Depurtment
41, 184- 190 (1983)
PATHOLOGY
of Entomology,
Prevented
by
AWAHMUKALAH AND MARION A. BROOKS
Fisheries,
and Wildlife, Unitlersify St. Paul, Minnesota 55108
of Minnesota,
1980 Folw~ell
Avenue,
Received December 7, 1981; accepted August 16, 1982 The development time for an autogenous strain of Culex pipiens was prolonged after the mosquitoes were “cured” of their symbiotic Wolbachia pipientis with tetracycline. Eggs produced by aposymbiotic females caged with either aposymbiotic or normal males failed to hatch. Neither growth rate nor reproduction was improved by feeding the larvae a commensal flagellate, bovine blood cells, or adenylic acid. KEY WORDS: Culex pipiens; Wolbachiu pipientis; aposymbiosis; growth rate; sterility; chlortetracycline; mosquito control; egg hatch.
INTRODUCTION
Wolbachia pipientis, a symbiotic rickettsia discovered in the gonads of both sexes of Clrlex pipiens (Hertig and Wolbach, 1924), occurs in numerous strains of the C. pipiens complex in every stage of development (Yen and Barr, 1971, 1974; IrvingBell, 1974). The phenomenon of partial or complete sterility in crosses between laboratory strains of the C. pipiens complex, incompatibility” termed “cytoplasmic (Laven, 1957), was found to be regulated by W. pipientis (Yen and Barr, 1973). By rearing mosquito larvae in tetracycline, which eliminated the rickettsia from the gonads, the ensuing aposymbiotic adults of different strains became reproductively compatible and fertile crosses resulted (Yen and Barr, 1973: Potaro and Barr, 1975). Cytoplasmic incompatibility also occurs in wild populations of C. pipiens (Barr, 1980) and in a number of strains of the Aedes scutelfaris group as well: they too possess Wolbachia (Smith-White and Woodhill, 1954; Wade and MacDonald, 1976; Beckett et al., 1978; Wright and Wang, 1980; Wright and Barr, 1981). Yen (1975) pointed out that transovarially transmitted rickettsiae in mosquitoes are known only in the two complexes that show cytoplasmic incompatibility. Further, a causal relationship
between Wolbachia and cytoplasmic incompatibility has been demonstrated in a moth, Ephestia cautefla (Kellen et al., 1981). The purpose of the work reported here was to study the role of W. pipientis in the reproduction of C. pipiens. We worked with a single inbred strain that was “cured” with tetracycline. The treated larvae developed slowly and the aposymbiotic adults were incapable of producing viable offspring, a discovery that was unexpected in light of the findings mentioned above for crosses. Efforts were then made to find a nutritional factor that might improve growth and permit reproduction of the mosquitoes deprived of their symbiotic rickettsia. Although this strain of C. pipiens is autogenous, its reproduction tends to decline gradually unless the females are allowed to take a blood meal occasionally. The blood meal suffices to maintain autogenous oviposition for about ten more generations. Evidently certain nutrients can be passed to the larvae and carried forward for several generations, which is common in insects (Chapman, 1976). The question arose as to the ability of the aposymbiotic larvae to take up and conserve growth factors from the blood that would ordinarily be acquired from a female progenitor in anautogenous
184 0022-201 l/83/020184-07$01.50/O Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
APOSYMBIOTIC
strains. To test this possibility, we added defibrinated bovine blood to the rearing infusion for aposymbiotic larvae. Reproductive potential, nutritionally dependent, is often augmented by the metabolism of associated microorganisms. Insects utilizing diets deficient in essential factors in all life stages possess mutualistic microorganisms (Buchner, 1965). Mosquitoes are said to have no need for mutualists because larval nutrition provides essentials that are stored and carried forward to supplement the blood diet of the adult. In nature, larvae of various species of Culex and other mosquitoes are commonly (up to 90%) infected with Crithidia fasciculnta (Clark et al., 1964) acquired from the water. This flagellate is a member of one of the four genera of lower trypanosomatids that live almost exclusively in the guts of insects without alternate vertebrate hosts (Wallace, 1943). Any effect of the flagellates on the mosquito hosts is unknown, but there is reason to expect that the mosquitoes could derive some nutrients, either by absorption of the excretory products of the flagellates or by digestion of the entire protozoans. The possibility that commensal organisms or any components of their cell bodies might be nutritionally advantageous to the aposymbiotic insects was investigated by adding cultured flagellates to the rearing infusion. The third substance tested as a developmental factor for aposymbiotic mosquitoes was 5’-adenylic acid, found to be required for growth and adult development of normal C. pipierrs (Dadd, 1979). MATERIALS
AND
METHODS
Mosquito colotr~. A San Jose strain of Culex pipiens pipiem was kindly provided by Dr. A. Ralph Barr, School of Public Health, University of California, Los Angeles. It had been laboratory-reared for more than 2 years prior to our receiving it in October 1979. The female is autogenous, capable of ovipositing a single egg raft without a blood meal. Larvae were reared in tap water con-
Culex
pipirns
185
taining a pinch of a mixture of four parts ground rabbit pellets and one part brewer’s yeast. Air was bubbled through the infusion as necessary to prevent excessive turbidity. The autumn- winter temperature in the insectary was 21-23°C with a relative humidity of 40-50%; the spring-summer temperature was 25-28°C with a relative humidity of 50-55%. Clean dishes of water containing 100 pupae were placed in meshed cages, 35 cm on each side, for adult emergence, which was about 90% successful. The cages were enclosed in wet terry cloth and plastic film to raise the humidity for the adults to 60-75%. Sugar cubes were furnished and the lighting regime was set for 14 L:lO D. The females were allowed to oviposit in the emergence water. Egg rafts were collected morning and evening and placed in fresh dishes of infusion to hatch. Under these conditions, autogenous egg production began to decline after about ten generations, at which time it became necessary to provide young chicks or house sparrows. The blood meal resulted in the production of a second, larger egg raft by each female, followed by another sequence of renewed vigor in autogeny. Aposymbiotic mosquitoes. A stock solution of chlortetracycline hydrochloride (Sigma, Lot llOF-0526), 0.5 mg/ml, was diluted l/20 or l/l0 in tap water to produce larval rearing waters containing 0.025 and 0.05 mg/ml, respectively. Egg rafts or lday-old larvae were put into the antibiotic waters and food was added. The water was not changed thereafter but fresh water was added as necessary to maintain the level. In both antibiotic treatments, the length of time to develop from egg to adult, the number of egg rafts laid, and the number of eggs hatching were recorded and compared to the same parameters for normal mosquitoes. To confirm the loss of symbiotes following antibiotic treatment, adults were examined microscopically. A random selection of five of each sex were dissected in
186
AWAHMUKALAH
Belar’s saline solution (Breland, 1961) and ovaries or testes were smeared on microscope slides. The smears were stained with Machiavello’s (1937) stain. Viewed under high dry or oil immersion objectives, the pleomorphic rickettsial bodies, if present, appear bright red against a contrasting blue background. Flagellate culture. Crithidia fasciculata (ATCC 12857) was obtained from the American Type Culture Collection, Rockville, Maryland. The flagellates were maintained by fortnightly subculture in blood agar slants. These consisted of 5 ml of brain-heart infusion agar containing 0.5 ml of defibrinated bovine blood and overlaid with 2 ml of brain-heart infusion broth. The cultures were incubated at 26”C, with appropriate examination at each subculture to confirm their purity. Nutritional supplements for aposymbiotic mosquitoes. For the reasons discussed above, three candidates for growth factors were fed to larvae under treatment with tetracycline. Detibrinated bovine blood was added to the rearing infusions at levels of 0.5 ml/200 ml and 1.0 ml/200 ml, giving final concentrations of 0.25 and 0.50%. On blood agar slants, C. fascicufata reached populations of 107/ml and were added to the mosquito infusions at the rate of 2 ml/200 ml. The flagellate is not found indigenously in this mosquito colony. Adenylic acid (adenosine 5’-monophosphate; Pfaltz and Bauer) was made as a stock solution of 10m3 M in distilled water. Dilutions of lo-’ and 10-j M were made in 200 ml of rearing infusion. In all cases, either egg rafts or l-day-old larvae from stock cultures were put into dishes of test materials containing food, antibiotic, and supplement. The water was not changed but additional water was added as needed until the larvae pupated. Records were made of the growth of the larvae in antibiotic with and without the three supplements. A dish of 50-100 pupae from each treatment was set in each adult emer-
AND
BROOKS
gence cage and examined twice daily for the appearance of adults to ascertain the time required to complete development. After the egg rafts were produced, they were observed for hatching. Mating combinations. Reproductive ability of adult mosquitoes from treated and untreated larvae was tested by confining ten males and ten females in each cage in the following combinations: Female
Male
Normal Aposymbiotic Normal Aposymbiotic
Normal Aposymbiotic Aposymbiotic Normal
In every combination, replicated five times, a record was made of the number of egg rafts laid (the maximum possible was one per female) and the number of eggs that hatched. RESULTS
Tetracycline in the larval rearing infusion eliminated W. pipientis from the gonads, prolonged development time, decreased survival of larvae, and prevented hatching of eggs produced by the adult stage. Data obtained over a period of many generations at summer and winter temperatures indicated the mean time for the normal mosquitoes to develop from egg to adult was 10 days in summer and 17 days in winter (Fig. l), with approximately 90% survival to the adult stage. The time required for development in the lower concentration of antibiotic was significantly prolonged, being 13 days in summer and 20 days in winter. The time for development in the higher concentration of antibiotic was prolonged still further, being 15 days in summer and 23 days in winter. The symbiotic rickettsiae could not be demonstrated in stained smears of gonads of adults that developed from either concentration of antibiotic. Larval mortality in the antibiotic, both summer and winter, was slightly higher
187
Cu1e.r pipirns
APOSYMBIOTIC
.w.
S.S.
ABC Fig.
1
D Development
E
FG of
HI
J
CUl.SX
K
LMN pipienr
OP larvae
FIG. 1. The days required for development of the mosquitoes from egg to adult stage are shown for larvae reared under the following conditions: Spring-Summer temperatures (S.S.): (A) normal (symbiotic) in routine rearing procedure; (B) in low tetracycline (0.025 mgiml); (C) in high tetracycline (0.05 mgiml); (D) in low tetracycline + 2 x IO’ Crithidiu fascicduta; (E) in high tetracycline + 2 x 10’ C. fusciculara: (F) in low tetracycline + 0.25% deftbrinated bovine blood; (G) in high tetracycline + 0.25% detibrinated bovine blood; (H) in low tetracycline + lo-” M adenosine 5’-monophosphate: (I) in low tetracycline + lo-” M adenosine 5’-monophosphate; (J) in high tetracycline + 10-j M adenosine S’-monophosphate; (K) in high tetracycline + 10m4M adenosine 5’-monophosphate. Fall-Winter temperatures (F.W.): (L) normal; (M) in low tetracycline; (N) in high tetracycline; (0) in low tetracycline + 2 x 10’ C. fasciculata; (P) in high tetracycline + 2 x lo7 C. fbsciculatrr. The vertical lines represent the standard deviations of the means. The difference in growth rate between normal (A and L) and antibiotic-treated larvae was significant at the 5% level. The difference between antibiotic-treated larvae with or without nutritional supplements was not significant.
than it was normally, particularly during the first 2 days. Adult emergence was significantly lower than that of normal mosquitoes, being only 70-80% successful. In every case, the adults oviposited egg rafts, but none of the eggs hatched.
Efforts to Improve Development of Aposymbiotic Mosquitoes Effect offeeding bovine blood to lurvae.
This experiment was performed in summer when the temperature was 25-28°C and
188
AWAHMUKALAH
normal mosquitoes completed their development in less than 10 days. Larvae treated with antibiotic required 12 and 13 days. Upon adding detibrinated blood to the infusions, there was no improvement in growth rate (Fig. 1). The results were the same for 0.25 and 0.50% blood. The blood cells lysed in the water and so were not available as particulate matter, yet some cell components were probably ingested along with other particulate matter in the infusion. The blood did not benefit reproduction as none of the eggs hatched. Effect of adding cultured C. fasciculata to the larval rearing infusions. Suspensions of 2 x lo7 flagellates were fed to larvae in both concentrations of antibiotic, in summer and in winter, and caused no improvement in development time (Fig. 1). Control larvae that were given 2 ml of sterile broth also showed no change. There was no sign of pathology in the larvae as a result of the flagellate infections. Examinations of ten larval guts showed flagellates were present in seven of them. After the 7th day in the larval infusion with antibiotic, flagellates were still present in appreciable numbers. Eggs produced by these mosquitoes after they became adults failed to hatch. Effect of feeding adenylic acid to larvae. This was done in summer temperatures when larvae in antibiotic were developing in 12 or 13 days. Larvae in low tetracycline fed either 10m4 or 10m5 M adenylic acid developed in 12 days, while in high tetracycline with adenylic acid, they developed in 12 or 13 days (Fig. 1). Thus, these growth rates were no better than the rates for treated larvae not fed adenylic acid. There was complete failure of the eggs produced by these mosquitoes to hatch. Mating Combinations These results, shown in Table 1, were obtained from five replications combining ten males and ten females in each cage for every bidirectional pairing. Ail of the egg rafts were produced autogenously by females not given any of the nutritional
AND
BROOKS
supplements described above for larvae. The greatest number of egg rafts was produced by the normal insects: 46 egg rafts from 50 females mated to normal males, with more than 89% of them hatching. The 50 normal females mated to aposymbiotic males laid 33 egg rafts, of which 91% hatched. The numbers of egg rafts produced by the aposymbiotic females (41 and 34, respectively) were within the range of the numbers produced by normal females but the hatchability was zero. As pointed out above, eggs produced by aposymbiotic females that had been fed dietary supplements in the larval stage also failed to hatch. Throughout these experiments performed over a period of 2 years, only two egg rafts produced by aposymbiotic females showed any hatch, namely, one larva from each, and both of these individuals developed into male adults. DISCUSSION
The concept of cytoplasmic incompatibility in C. pipiens is not applicable in our work since eggs and sperm are always compatible in inbreeding of this strain. Nonviability of eggs from aposymbiotic females mated to either aposymbiotic or normal males suggests that the major significance of the rickettsial agent is in the promotion of egg, i.e., embryonic, development. Wright and Barr (1981), in describing the appearance of incompatible eggs of Aedes polynesiensis, stated that they resemble eggs of aposymbiotic female C. pipiens crossed with infected males of another strain in that very little embryonic development occurred. The authors suggested that the basis of incompatibility may be that an egg swiftly neutralizes the sperm from an incompatible strain. In our work, the viability of eggs from normal females mated to aposymbiotic males indicates that the symbiote is not necessary for male fertility and that the sperm are not neutralized in an egg containing rickettsiae. On the other hand, there may be an adverse effect of symbiotes that enter an egg with-
APOSYMBIOTIC
C&x
189
pipivns
TABLE 1 MATING EXPERIMENTS Matings 10 normal 10 normal 10 normal 10 normal 10 normal
F x 10 normal F x 10 normal F x 10 normal F x 10 normal F x 10 normal Total
10 normal 10 normal IO normal IO normal 10 normal
F x 10 apo. F x 10 apo. F x 10 apo. F x 10 apo. F x 10 apo. Total
IO aposymbiotic IO aposymbiotic 10 aposymbiotic 10 aposymbiotic 10 aposymbiotic Total
Number of egg rafts laid
Number of egg rafts hatched
10 9 8 10 9 46
9 8 7 9 8 41
90 89 88 90 89 89
6 6 6 8 7 33
4 6 6 7 7 30
67 100 100 88 100 91
8 9 8 9 7 41
0 0 0 0 0 0
0 0 0 0 0 0
7 7 6 8 6 34
0 0 0 0 0 0
0 0 0 0 0 0
M M M M M
M M M M M
F F F F F
x x x x x
10 normal 10 normal 10 normal 10 normal 10 normal
IO aposymbiotic F 10 aposymbiotic F 10 aposymbiotic F 10 aposymbiotic F 10 aposymbiotic F Total
x x x x x
10 apo. 10 apo. 10 apo. 10 apo. 10 apo.
M M M M M
M M M M M
out symbiotes, but we favor the view that the symbiotes are essential for the completion of embryonic development. It is clear that only one half of the normal complement of symbiotes, that from the female, is required for embryonic development. Since we did not observe the mosquitoes closely for copulatory behavior, it may be that the aposymbiotic females were not inseminated due to lack of a sex attractant. The production of factors by microbial symbiotes that act as sex pheromones is known in other cases. For example, a bacterial symbiote in a Scarabeid beetle produces a sex attractant (Hoyt et al., 1971), and ingestion of streptomycin by a bark beetle inhibits pheromone processing by symbiotic bacteria (Byers and Wood, 1981). Possibly the female gonadal symbiote in C. pipiens has an influence on the production of a substance that acts as a sex attractant.
Percentage hatch
Our efforts to find a nutritional factor that would improve growth rate and egg viability of the aposymbiotic mosquitoes were all unproductive. We tried to determine if blood consumed by larvae might be carried forward as an emergency reserve to aid in vitellogenesis during autogeny, which in itself is of survival value in an emergency. However, there seemed to be nothing in blood proteins that was useful for either growth or egg viability. We obtained no support for the possibility that the flagellates could act as mutualists for the aposymbiotic mosquitoes. Adenylic acid in amounts that are essential in a defined medium for normal C. pipiens did not improve growth nor benefit reproduction. However, in all these experiments there remains a complication that needs to be addressed, viz., what effect does the antibiotic have on other microbial flora that may normally contribute growth factors?
190
AWAHMUKALAH
Apart from using cytoplasmic incompatibility as a control for the C. pipiens complex, as has been suggested (Barr, 1966; Laven, 1967), manipulation of aposymbiosis in small bodies of water with tetracycline might be promoted as a strategy for population suppression. According to Mitscher (1978), oxytetracycline, 0.2 mg/ml at 37°C andpH 7.0, has a half life of 26 hr. Thus the antibiotic probably could persist long enough, especially at temperatures lower than 37”C, to produce sterile female mosquitoes. The precise stage in development at which the antibiotic is effective needs to be determined. However, more should be learned about the pathological effects of antibiotic on other aspects of the mosquito’s physiology and environment to understand its action. Elimination of the rickettsial symbiotes may not be solely responsible for the adverse effects we observed. ACKNOWLEDGMENTS This work was supported in part by Research AI 09914 from the National Institutes of Health, Public Health Service, to Marion A. Brooks. paper is No. 12,030, Scientific Journal Series, nesota Agricultural Experiment Station.
Grant U.S. The Min-
REFERENCES BARR, A. R. 1966. Cytoplasmic incompatibility as a means of eradication of C&r pipiens L. Proc. Calif. Mosq.
Cont.
Assoc.
34, 32-35.
A. R. 1980. Cytoplasmic incompatibility in natural populations of a mosquito, Cltlex pipiens L. Nature (London), 283, 71-72. BECKETT, E. B., BOOTHROYD, B., AND MACDONALD, W. W. 1978. A light and electron microscope study of rickettsia-like organisms in the ovaries of mosquitoes of the Aedes scutellaris group. Ann. Trap.
BARR,
Med.
Parasitol..
72, 277-283.
BRELAND, 0. P. 1961. Studies on the chromosomes of mosquitoes. Ann. Entomol. Sot. Amer. 54, 360-375. BUCHNER, P. 1965. “Endosymbiosis of Animals with Plant Microorganisms” (Translated from German). Wiley, New York. BYERS, 3. A., AND WOOD, D. L. 1981. Antibioticinduced inhibition of pheromone synthesis in a bark beetle. Science, 213, 763-764. CHAPMAN, R. F. 1976. “The Insects. Structure and Function.” Elsevier, New York. CLARK, T. B., KELLEN, W. R., LINDEGREN, J. E.. AND SMITH, T. A. 1964. The transmission of Crithidia~~.scicrrlata Leger 1902 in Culiseta incidem (Thomson). J. Pr~~toz~r>/.. 11. 400-402.
AND BROOKS DADD, R. H. 1979. Nucleotide, nucleoside and base nutritional requirements of the mosquito Clrle.r pipiens. J. Insect
Physiol..
25, 353-359.
HERTIG, M.. AND WOLBACH, S. B. 1924. Studies on rickettsia-like microorganisms in insects. J. Med. Res.
44, 329-374.
HOYT, C. P., OSBORNE, G. 0.. AND MULCOCK, A. P. 1971. Production of an insect sex attractant by symbiotic bacteria. Nature (London), 230, 472-473. IRVING-BELL, R. J. 1974. Cytoplasmic factors in the gonads of Culex pipirns complex mosquitoes. L[fij Sri., 14, 1149-1151. KELLEN, W. R., HOFFMAN, D. F.. AND KWOCK, R. A. 1981. Wolbachiu sp. (Rickettsiales: Rickettsiaceae) a symbiont of the almond moth, Ephestiu matella: Ultrastructure and influence on host fertility. J. Intjertebr. Pathol., 37, 273-283. LAVEN, H. 1957. Vererbrung durch Kerngene und das Problem der ausserkaryotischen Vererbung bei Culex pipiens, II. Ausserkaryotische Vererbung. Z. Vererbungsl.. 88, 478-516. LAVEN, H. 1967. A possible model for speciation by cytoplasmic isolation in the Cttlex pipiens complex. Bull. W.H.O., 37, 263-266. MACCHIAVELLO, A. 1937. Estudios sobre tifus exantematico. III. Un nuevo metodo para tenir rickettsia. Ret,. Chil. Hig. Med. Pm,.. 1, lOl- 106. MITSCHER. L. A. 1978. “The Chemistry of the Tetracycline Antibiotics.” Dekker. New York. POTARO, J. K., AND BARR. A. R. 1975. “Curing” Wolbarhia infections in Clr1e.upipiens. J. Med. Entornol.. 12, 265. SMITH-WHITE, S.. AND WOODHILL, A. R. 1954. The nature and significance of non-reciprocal fertility in Ardes scutellaris and other mosquitoes. Proc. Linn. Sot. N. S. W., 79, 163-176. WADE, J. O., AND MACDONALD, W. W. 1976. Compatible and incompatible crosses within the Aedes scatellaris group. Trans. R. Sot. Trap. Med. H!g., 71, 109. WALLACE, F. G. 1943. Flagellate parasites of mosquitoes with special reference to Crithidia fascicalata Leger, 1902. J. Parasitol.. 29, 196-205. WRIGHT, J. D., AND BARR, A. R. 1981. Wolbachia and the normal and incompatible eggs of Aedes polynesiensis (Diptera: Culicidae). J. Inl~ertebr. Pathol.. 38, 409-418. WRIGHT, J. D.. AND WANG, B. 1980. Observations on Wolbachiae in mosquitoes. J. Invertebr. Pathol.. 35, 200-208. YEN, J. H. 1975. Transovarial transmission of rickettsia-like microorganisms in mosquitoes. Ann. N.Y. Acad. Sci.. 266, 152- 161. YEN. J. H., AND BARR, A. R. 1971. New hypothesis of the cause of cytoplasmic incompatibility in Culex pipiens L. Nature (London), 232, 657-658. YEN, J. H., AND BARR, A. R. 1973. The etiological agent of cytoplasmic incompatibility in Culex pipiens.
YEN,
J. Invertebr.
Pathol..
22, 242-250.
A. R. 1974. Incompatibility in C’rrlex pipiens. 111 “The Use of Genetics in Insect Control” (R. Pal and M. J. Whitten, eds.). Elsevier, Amsterdam. J. H.,
AND
BARR,
OF INVERTEBRATE
Reproduction
of an inbred Strain of Culex pipiens Loss of Wolbachia pipientis
DOMINIC S.T. Depurtment
41, 184- 190 (1983)
PATHOLOGY
of Entomology,
Prevented
by
AWAHMUKALAH AND MARION A. BROOKS
Fisheries,
and Wildlife, Unitlersify St. Paul, Minnesota 55108
of Minnesota,
1980 Folw~ell
Avenue,
Received December 7, 1981; accepted August 16, 1982 The development time for an autogenous strain of Culex pipiens was prolonged after the mosquitoes were “cured” of their symbiotic Wolbachia pipientis with tetracycline. Eggs produced by aposymbiotic females caged with either aposymbiotic or normal males failed to hatch. Neither growth rate nor reproduction was improved by feeding the larvae a commensal flagellate, bovine blood cells, or adenylic acid. KEY WORDS: Culex pipiens; Wolbachiu pipientis; aposymbiosis; growth rate; sterility; chlortetracycline; mosquito control; egg hatch.
INTRODUCTION
Wolbachia pipientis, a symbiotic rickettsia discovered in the gonads of both sexes of Clrlex pipiens (Hertig and Wolbach, 1924), occurs in numerous strains of the C. pipiens complex in every stage of development (Yen and Barr, 1971, 1974; IrvingBell, 1974). The phenomenon of partial or complete sterility in crosses between laboratory strains of the C. pipiens complex, incompatibility” termed “cytoplasmic (Laven, 1957), was found to be regulated by W. pipientis (Yen and Barr, 1973). By rearing mosquito larvae in tetracycline, which eliminated the rickettsia from the gonads, the ensuing aposymbiotic adults of different strains became reproductively compatible and fertile crosses resulted (Yen and Barr, 1973: Potaro and Barr, 1975). Cytoplasmic incompatibility also occurs in wild populations of C. pipiens (Barr, 1980) and in a number of strains of the Aedes scutelfaris group as well: they too possess Wolbachia (Smith-White and Woodhill, 1954; Wade and MacDonald, 1976; Beckett et al., 1978; Wright and Wang, 1980; Wright and Barr, 1981). Yen (1975) pointed out that transovarially transmitted rickettsiae in mosquitoes are known only in the two complexes that show cytoplasmic incompatibility. Further, a causal relationship
between Wolbachia and cytoplasmic incompatibility has been demonstrated in a moth, Ephestia cautefla (Kellen et al., 1981). The purpose of the work reported here was to study the role of W. pipientis in the reproduction of C. pipiens. We worked with a single inbred strain that was “cured” with tetracycline. The treated larvae developed slowly and the aposymbiotic adults were incapable of producing viable offspring, a discovery that was unexpected in light of the findings mentioned above for crosses. Efforts were then made to find a nutritional factor that might improve growth and permit reproduction of the mosquitoes deprived of their symbiotic rickettsia. Although this strain of C. pipiens is autogenous, its reproduction tends to decline gradually unless the females are allowed to take a blood meal occasionally. The blood meal suffices to maintain autogenous oviposition for about ten more generations. Evidently certain nutrients can be passed to the larvae and carried forward for several generations, which is common in insects (Chapman, 1976). The question arose as to the ability of the aposymbiotic larvae to take up and conserve growth factors from the blood that would ordinarily be acquired from a female progenitor in anautogenous
184 0022-201 l/83/020184-07$01.50/O Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
APOSYMBIOTIC
strains. To test this possibility, we added defibrinated bovine blood to the rearing infusion for aposymbiotic larvae. Reproductive potential, nutritionally dependent, is often augmented by the metabolism of associated microorganisms. Insects utilizing diets deficient in essential factors in all life stages possess mutualistic microorganisms (Buchner, 1965). Mosquitoes are said to have no need for mutualists because larval nutrition provides essentials that are stored and carried forward to supplement the blood diet of the adult. In nature, larvae of various species of Culex and other mosquitoes are commonly (up to 90%) infected with Crithidia fasciculnta (Clark et al., 1964) acquired from the water. This flagellate is a member of one of the four genera of lower trypanosomatids that live almost exclusively in the guts of insects without alternate vertebrate hosts (Wallace, 1943). Any effect of the flagellates on the mosquito hosts is unknown, but there is reason to expect that the mosquitoes could derive some nutrients, either by absorption of the excretory products of the flagellates or by digestion of the entire protozoans. The possibility that commensal organisms or any components of their cell bodies might be nutritionally advantageous to the aposymbiotic insects was investigated by adding cultured flagellates to the rearing infusion. The third substance tested as a developmental factor for aposymbiotic mosquitoes was 5’-adenylic acid, found to be required for growth and adult development of normal C. pipierrs (Dadd, 1979). MATERIALS
AND
METHODS
Mosquito colotr~. A San Jose strain of Culex pipiens pipiem was kindly provided by Dr. A. Ralph Barr, School of Public Health, University of California, Los Angeles. It had been laboratory-reared for more than 2 years prior to our receiving it in October 1979. The female is autogenous, capable of ovipositing a single egg raft without a blood meal. Larvae were reared in tap water con-
Culex
pipirns
185
taining a pinch of a mixture of four parts ground rabbit pellets and one part brewer’s yeast. Air was bubbled through the infusion as necessary to prevent excessive turbidity. The autumn- winter temperature in the insectary was 21-23°C with a relative humidity of 40-50%; the spring-summer temperature was 25-28°C with a relative humidity of 50-55%. Clean dishes of water containing 100 pupae were placed in meshed cages, 35 cm on each side, for adult emergence, which was about 90% successful. The cages were enclosed in wet terry cloth and plastic film to raise the humidity for the adults to 60-75%. Sugar cubes were furnished and the lighting regime was set for 14 L:lO D. The females were allowed to oviposit in the emergence water. Egg rafts were collected morning and evening and placed in fresh dishes of infusion to hatch. Under these conditions, autogenous egg production began to decline after about ten generations, at which time it became necessary to provide young chicks or house sparrows. The blood meal resulted in the production of a second, larger egg raft by each female, followed by another sequence of renewed vigor in autogeny. Aposymbiotic mosquitoes. A stock solution of chlortetracycline hydrochloride (Sigma, Lot llOF-0526), 0.5 mg/ml, was diluted l/20 or l/l0 in tap water to produce larval rearing waters containing 0.025 and 0.05 mg/ml, respectively. Egg rafts or lday-old larvae were put into the antibiotic waters and food was added. The water was not changed thereafter but fresh water was added as necessary to maintain the level. In both antibiotic treatments, the length of time to develop from egg to adult, the number of egg rafts laid, and the number of eggs hatching were recorded and compared to the same parameters for normal mosquitoes. To confirm the loss of symbiotes following antibiotic treatment, adults were examined microscopically. A random selection of five of each sex were dissected in
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Belar’s saline solution (Breland, 1961) and ovaries or testes were smeared on microscope slides. The smears were stained with Machiavello’s (1937) stain. Viewed under high dry or oil immersion objectives, the pleomorphic rickettsial bodies, if present, appear bright red against a contrasting blue background. Flagellate culture. Crithidia fasciculata (ATCC 12857) was obtained from the American Type Culture Collection, Rockville, Maryland. The flagellates were maintained by fortnightly subculture in blood agar slants. These consisted of 5 ml of brain-heart infusion agar containing 0.5 ml of defibrinated bovine blood and overlaid with 2 ml of brain-heart infusion broth. The cultures were incubated at 26”C, with appropriate examination at each subculture to confirm their purity. Nutritional supplements for aposymbiotic mosquitoes. For the reasons discussed above, three candidates for growth factors were fed to larvae under treatment with tetracycline. Detibrinated bovine blood was added to the rearing infusions at levels of 0.5 ml/200 ml and 1.0 ml/200 ml, giving final concentrations of 0.25 and 0.50%. On blood agar slants, C. fascicufata reached populations of 107/ml and were added to the mosquito infusions at the rate of 2 ml/200 ml. The flagellate is not found indigenously in this mosquito colony. Adenylic acid (adenosine 5’-monophosphate; Pfaltz and Bauer) was made as a stock solution of 10m3 M in distilled water. Dilutions of lo-’ and 10-j M were made in 200 ml of rearing infusion. In all cases, either egg rafts or l-day-old larvae from stock cultures were put into dishes of test materials containing food, antibiotic, and supplement. The water was not changed but additional water was added as needed until the larvae pupated. Records were made of the growth of the larvae in antibiotic with and without the three supplements. A dish of 50-100 pupae from each treatment was set in each adult emer-
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gence cage and examined twice daily for the appearance of adults to ascertain the time required to complete development. After the egg rafts were produced, they were observed for hatching. Mating combinations. Reproductive ability of adult mosquitoes from treated and untreated larvae was tested by confining ten males and ten females in each cage in the following combinations: Female
Male
Normal Aposymbiotic Normal Aposymbiotic
Normal Aposymbiotic Aposymbiotic Normal
In every combination, replicated five times, a record was made of the number of egg rafts laid (the maximum possible was one per female) and the number of eggs that hatched. RESULTS
Tetracycline in the larval rearing infusion eliminated W. pipientis from the gonads, prolonged development time, decreased survival of larvae, and prevented hatching of eggs produced by the adult stage. Data obtained over a period of many generations at summer and winter temperatures indicated the mean time for the normal mosquitoes to develop from egg to adult was 10 days in summer and 17 days in winter (Fig. l), with approximately 90% survival to the adult stage. The time required for development in the lower concentration of antibiotic was significantly prolonged, being 13 days in summer and 20 days in winter. The time for development in the higher concentration of antibiotic was prolonged still further, being 15 days in summer and 23 days in winter. The symbiotic rickettsiae could not be demonstrated in stained smears of gonads of adults that developed from either concentration of antibiotic. Larval mortality in the antibiotic, both summer and winter, was slightly higher
187
Cu1e.r pipirns
APOSYMBIOTIC
.w.
S.S.
ABC Fig.
1
D Development
E
FG of
HI
J
CUl.SX
K
LMN pipienr
OP larvae
FIG. 1. The days required for development of the mosquitoes from egg to adult stage are shown for larvae reared under the following conditions: Spring-Summer temperatures (S.S.): (A) normal (symbiotic) in routine rearing procedure; (B) in low tetracycline (0.025 mgiml); (C) in high tetracycline (0.05 mgiml); (D) in low tetracycline + 2 x IO’ Crithidiu fascicduta; (E) in high tetracycline + 2 x 10’ C. fusciculara: (F) in low tetracycline + 0.25% deftbrinated bovine blood; (G) in high tetracycline + 0.25% detibrinated bovine blood; (H) in low tetracycline + lo-” M adenosine 5’-monophosphate: (I) in low tetracycline + lo-” M adenosine 5’-monophosphate; (J) in high tetracycline + 10-j M adenosine S’-monophosphate; (K) in high tetracycline + 10m4M adenosine 5’-monophosphate. Fall-Winter temperatures (F.W.): (L) normal; (M) in low tetracycline; (N) in high tetracycline; (0) in low tetracycline + 2 x 10’ C. fasciculata; (P) in high tetracycline + 2 x lo7 C. fbsciculatrr. The vertical lines represent the standard deviations of the means. The difference in growth rate between normal (A and L) and antibiotic-treated larvae was significant at the 5% level. The difference between antibiotic-treated larvae with or without nutritional supplements was not significant.
than it was normally, particularly during the first 2 days. Adult emergence was significantly lower than that of normal mosquitoes, being only 70-80% successful. In every case, the adults oviposited egg rafts, but none of the eggs hatched.
Efforts to Improve Development of Aposymbiotic Mosquitoes Effect offeeding bovine blood to lurvae.
This experiment was performed in summer when the temperature was 25-28°C and
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normal mosquitoes completed their development in less than 10 days. Larvae treated with antibiotic required 12 and 13 days. Upon adding detibrinated blood to the infusions, there was no improvement in growth rate (Fig. 1). The results were the same for 0.25 and 0.50% blood. The blood cells lysed in the water and so were not available as particulate matter, yet some cell components were probably ingested along with other particulate matter in the infusion. The blood did not benefit reproduction as none of the eggs hatched. Effect of adding cultured C. fasciculata to the larval rearing infusions. Suspensions of 2 x lo7 flagellates were fed to larvae in both concentrations of antibiotic, in summer and in winter, and caused no improvement in development time (Fig. 1). Control larvae that were given 2 ml of sterile broth also showed no change. There was no sign of pathology in the larvae as a result of the flagellate infections. Examinations of ten larval guts showed flagellates were present in seven of them. After the 7th day in the larval infusion with antibiotic, flagellates were still present in appreciable numbers. Eggs produced by these mosquitoes after they became adults failed to hatch. Effect of feeding adenylic acid to larvae. This was done in summer temperatures when larvae in antibiotic were developing in 12 or 13 days. Larvae in low tetracycline fed either 10m4 or 10m5 M adenylic acid developed in 12 days, while in high tetracycline with adenylic acid, they developed in 12 or 13 days (Fig. 1). Thus, these growth rates were no better than the rates for treated larvae not fed adenylic acid. There was complete failure of the eggs produced by these mosquitoes to hatch. Mating Combinations These results, shown in Table 1, were obtained from five replications combining ten males and ten females in each cage for every bidirectional pairing. Ail of the egg rafts were produced autogenously by females not given any of the nutritional
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supplements described above for larvae. The greatest number of egg rafts was produced by the normal insects: 46 egg rafts from 50 females mated to normal males, with more than 89% of them hatching. The 50 normal females mated to aposymbiotic males laid 33 egg rafts, of which 91% hatched. The numbers of egg rafts produced by the aposymbiotic females (41 and 34, respectively) were within the range of the numbers produced by normal females but the hatchability was zero. As pointed out above, eggs produced by aposymbiotic females that had been fed dietary supplements in the larval stage also failed to hatch. Throughout these experiments performed over a period of 2 years, only two egg rafts produced by aposymbiotic females showed any hatch, namely, one larva from each, and both of these individuals developed into male adults. DISCUSSION
The concept of cytoplasmic incompatibility in C. pipiens is not applicable in our work since eggs and sperm are always compatible in inbreeding of this strain. Nonviability of eggs from aposymbiotic females mated to either aposymbiotic or normal males suggests that the major significance of the rickettsial agent is in the promotion of egg, i.e., embryonic, development. Wright and Barr (1981), in describing the appearance of incompatible eggs of Aedes polynesiensis, stated that they resemble eggs of aposymbiotic female C. pipiens crossed with infected males of another strain in that very little embryonic development occurred. The authors suggested that the basis of incompatibility may be that an egg swiftly neutralizes the sperm from an incompatible strain. In our work, the viability of eggs from normal females mated to aposymbiotic males indicates that the symbiote is not necessary for male fertility and that the sperm are not neutralized in an egg containing rickettsiae. On the other hand, there may be an adverse effect of symbiotes that enter an egg with-
APOSYMBIOTIC
C&x
189
pipivns
TABLE 1 MATING EXPERIMENTS Matings 10 normal 10 normal 10 normal 10 normal 10 normal
F x 10 normal F x 10 normal F x 10 normal F x 10 normal F x 10 normal Total
10 normal 10 normal IO normal IO normal 10 normal
F x 10 apo. F x 10 apo. F x 10 apo. F x 10 apo. F x 10 apo. Total
IO aposymbiotic IO aposymbiotic 10 aposymbiotic 10 aposymbiotic 10 aposymbiotic Total
Number of egg rafts laid
Number of egg rafts hatched
10 9 8 10 9 46
9 8 7 9 8 41
90 89 88 90 89 89
6 6 6 8 7 33
4 6 6 7 7 30
67 100 100 88 100 91
8 9 8 9 7 41
0 0 0 0 0 0
0 0 0 0 0 0
7 7 6 8 6 34
0 0 0 0 0 0
0 0 0 0 0 0
M M M M M
M M M M M
F F F F F
x x x x x
10 normal 10 normal 10 normal 10 normal 10 normal
IO aposymbiotic F 10 aposymbiotic F 10 aposymbiotic F 10 aposymbiotic F 10 aposymbiotic F Total
x x x x x
10 apo. 10 apo. 10 apo. 10 apo. 10 apo.
M M M M M
M M M M M
out symbiotes, but we favor the view that the symbiotes are essential for the completion of embryonic development. It is clear that only one half of the normal complement of symbiotes, that from the female, is required for embryonic development. Since we did not observe the mosquitoes closely for copulatory behavior, it may be that the aposymbiotic females were not inseminated due to lack of a sex attractant. The production of factors by microbial symbiotes that act as sex pheromones is known in other cases. For example, a bacterial symbiote in a Scarabeid beetle produces a sex attractant (Hoyt et al., 1971), and ingestion of streptomycin by a bark beetle inhibits pheromone processing by symbiotic bacteria (Byers and Wood, 1981). Possibly the female gonadal symbiote in C. pipiens has an influence on the production of a substance that acts as a sex attractant.
Percentage hatch
Our efforts to find a nutritional factor that would improve growth rate and egg viability of the aposymbiotic mosquitoes were all unproductive. We tried to determine if blood consumed by larvae might be carried forward as an emergency reserve to aid in vitellogenesis during autogeny, which in itself is of survival value in an emergency. However, there seemed to be nothing in blood proteins that was useful for either growth or egg viability. We obtained no support for the possibility that the flagellates could act as mutualists for the aposymbiotic mosquitoes. Adenylic acid in amounts that are essential in a defined medium for normal C. pipiens did not improve growth nor benefit reproduction. However, in all these experiments there remains a complication that needs to be addressed, viz., what effect does the antibiotic have on other microbial flora that may normally contribute growth factors?
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Apart from using cytoplasmic incompatibility as a control for the C. pipiens complex, as has been suggested (Barr, 1966; Laven, 1967), manipulation of aposymbiosis in small bodies of water with tetracycline might be promoted as a strategy for population suppression. According to Mitscher (1978), oxytetracycline, 0.2 mg/ml at 37°C andpH 7.0, has a half life of 26 hr. Thus the antibiotic probably could persist long enough, especially at temperatures lower than 37”C, to produce sterile female mosquitoes. The precise stage in development at which the antibiotic is effective needs to be determined. However, more should be learned about the pathological effects of antibiotic on other aspects of the mosquito’s physiology and environment to understand its action. Elimination of the rickettsial symbiotes may not be solely responsible for the adverse effects we observed. ACKNOWLEDGMENTS This work was supported in part by Research AI 09914 from the National Institutes of Health, Public Health Service, to Marion A. Brooks. paper is No. 12,030, Scientific Journal Series, nesota Agricultural Experiment Station.
Grant U.S. The Min-
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