WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE
WHO/VBC/75.596 ENGLISH ONLY
SEGREGATION OF CYTOPLASMIC INCOMPATIBILITY/COMPATIBILITY PROPERTIES IN CULEX PIPIENS FATIGANS by Sarala K. Subbarao,l B. S. Krishnamurthy, C. F. Curtis T. Adak and R. K. Chandrahas WHO/ICMR Research Unit on Genetic Control of Mosquitos New Delhi, India
ABSTRACT Maternally inherited variants, which arose within a laboratory colony of Culex pipiens fatigans have been studied by rearing single raft cultures. Segregation of the cytoplasmic incompatibility/compatibility properties of the male progeny of individual females was demonstrated. Also, from the daughters of individual females sub-lines could be derived within which all the males showed the same incompatibility or compatibility properties. Tetracycline treatment produced lines which superficially simulated the segregation phenomenon, but the tetracycline lines all ultimately reverted to the cytoplasmic compatibility type of the strain which was submitted to the treatment. The types of variations in cytoplasmic incompatibility properties which have been reported are discussed. INTRODUCTION Cytoplasmic incompatibility is a naturally occurring phenomenon in the Culex p1p1ens complex. It has been shown by a backcrossing programme that this is a maternally inherited character without any involvement of chromosomal genes (Laven, 1957). Pal (1966) suggested that cytoplasmic incompatibility could be used in the control of mosquito populations. Paris cytoplasm was reported to be bi-directionally incompatible with Delhi cytoplasm (Krishnamurthy & Laven, 1972). However, Subbarao et al. (1974, 1975) found polymorphism in cytoplasmic types in Culex pipiens fatigans populations from various parts of the Indian sub-continent. Males of the minority type (De 19) were found to be compatible with all the cytoplasmic types tested. The majority type, whose males are incompatible with Paris females, could be separated from the De 19 type by rearing single raft cultures. All the males from anyone of the cultures showed the same compatibility properties. In addition to the polymorphism observed in natural populations, Subbarao et al. (1975) reported the cytoplasmic variants ISB 20 and ISB 49, which arose in a laboratory colony of the IS3lB strain which has faris cytoplasm (Krishnamurthy & Laven, 1974). This paper reports studies on ISB 20 and ISB 49 which revealed the occurrence of segregation among the progeny of individual females. A similar phenomenon has been briefly reported by French (1970).
1
Present address: !
Vector Control Research Centre, 22 Sham Nath Marg, Delhi 110054,
India.
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WHO/VBC/75.596 page 2 Yen & Barr (1973) treated larvae with tetracycline and derived a line whose males showed universal compatibility and whose females showed universal incompatibility with untreated mosquitos. Electron microscopy revealed that these mosquitos were free from rickettsiae, while the untreated mosquitos contained large numbers of rickettsiae (Yen & Barr, 1973, 1974) and they referred to the progeny of treated female mosquitos as aposymbiotic lines. The authors proposed that these rickettsial organisms are responsible for the cytoplasmic incompatibility phenomenon. The present paper also reports studies on tetracycline treatment of both larval and adult mosquitos. Stocks The following stocks have been used in the experiments: Delhi (De): De 2 De 19 (Pa)De/De IS3lB (Ha)De/De (Ba)De/De (Pr)De/De a laboratory colonized strain of Delhi origin (Singh et al., 1975). Single raft isolate of the Delhi majority type isolated from Delhi (De). Single raft isolate of the Delhi minority type isolated from Delhi (De). Paris cytoplasm with Delhi genome (Krishnamurthy & Laven, 1974). Paris cytoplasm with Delhi genome incl~ding a male-linked translocation (Krishnamurthy & Laven, 1974). Hamburg cytoplasm with Delhi genome (Krishnamurthy & Laven, 1974). Bangkok cytoplasm type A with Delhi genome (Krishnamurthy & Laven, 1974). Prague cytoplasm with Delhi genome (Subbarao et al., 1975).
Experimental procedure To avoid the effects of aging in causing partial compatibility (Singh et al., 1976) all matings were carried out with two to three day old males. Chicks were provided as a blood source on the fourth day after mating and egg rafts (which are clumps of about 150 eggs laid by individual females) were collected on the ninth day. The hatchability of the egg rafts was scored after two days and incompatible rafts were identified by their characteristic mixture of unhatched embryonated and unembryonated eggs. Single raft cultures In order to study samples of progeny from individual females, egg rafts were reared in individual bowls. Some of the males and females from each culture were inbred and the remainder were test-crossed with various cytoplasmic types. Tetracycline treatment (a) Larval treatment: larvae of the (Pa)De/De strain were reared in concentrations of 0.025 and 0.05 mg/ml tetracycline, the solution being changed on alternate days. The larval developmental time was considerably extended, especially at the higher concentration. Treated males and females were inbred and males were test-crossed with Delhi females. Progeny of treated inbred lines were test-crossed with all the available cytoplasmic types. (b) Adult treatment: in the normal rearing procedure cotton pads with 1% glucose solution are provided for feeding adults and for experimental purposes 0.1 or 1 mg/ml tetracycline was added to the solution for the first four days of adult life.
WHO/VBC/75.596 page 3 RESULTS Variation in IS31B colony As reported by Subbarao et al. (1975) the males from 50 single raft cultures from the IS31B colony were test-crossed with Delhi females to determine their crossing type. Males from two of the rafts, designated ISB 20 and ISB 49 showed compatibility, while males from the other 48 cultures showed full incompatibility with Delhi females (Fig. 1). Males of ISB 49 showed full compatibility with females of all cytoplasmic types available in this unit, but females of ISB 49 retained the Paris compatibility and incompatibility properties (Fig. 1). ISB 20 females, like ISB 49 females, retained Paris compatibility/incompatibility properties, but among males of ISB 20, unlike ISB 49, there was a segregation of individuals showing compatible or incompatible properties with respect to De 2, De 19, Ba and Ha females (Fig. 2). Fifteen single raft cultures were reared from ISB 20 (designated ISB 20 (1) - (15» to investigate the genetic constitution of the ISB 20 line. Females and males were inbred and males were test-crossed with De 2 females (Fig. 2). Six lines (with a total of 111 rafts) gave males with full compatibility, two lines (with a total of 30 rafts) gave males with full incompatibility, while the remaining seven lines showed a segregation of males, like the original ISB 20 line. Two of these segregating sub-lines ISB 20(7) and ISB 20(9) were tested further by rearing single raft cultures, ISB 20(7)a-m and ISB 20(9)a-o and test-crossing the males (Fig. 3). In lines ISB 20(7)g and ISB 20(9)f it was found that male progeny were, respectively, all compatible and all incompatible with De 2 females but, in the other lines, segregation continued. The latter lines were pooled and maintained as a laboratory stock culture without further testing for four to five months. On re-testing at the end of that period, all males were found to be compatible with De 2 females. Meanwhile, to test whether the properties of ISB 20 and ISB 49 were cytoplasmically or chromosomally inherited, males and females of each type were outcrossed to the normal (Pa)De/De stock. When the males of the ISB 20 and ISB 49 stocks were outcrossed, the male progeny showed the properties of the (Pa)De/De stock, i.e. bi-directional incompatibility with De 2. Conversely, when females of ISB 49 and of several of the sub-lines of ISB 20 were outcrossed the male progeny retained the properties of the line to which their maternal parent belonged. This remained true when four successive backcrosses of females to (Pa)De/De males were carried out. Studies with tetracycline (i) Larval treatment of (Pa)De/De strain:
There was heavy larval and adult mortality from the 0.05 mg/ml treatment and the few rafts obtained from the s4rviving adults failed to hatch. In the matings between treated males and females fromO.025mg/ml treatment, abnormally small rafts with very low hatchability were obtained. Treated males were also test-crossed with De females and the rafts showed a mean hatch of.20% (range 0.7-67%), i.e. the incompatibility barrier had been partially broken. The Fl male progeny from treated females x treated males were compatible with both De and Pa females (Fig. 4). The Fl female progeny were incompatible with De 2 males but a majority of females were compatible with Pa males. Thus, the majority of individuals appeared to belong to the cytoplasmic type which has been designated ISB 49, while the minority resembled Yen and Bar's aposymbiotic lines in that the females were incompatible with the Pa type, i.e. the strain from which they were derived.
WHO/VBC/7S.S96 page 4 In the F2 generation males varied in their compatibility with De and females again showed either compatibility or incompatibility with Paris males (Fig. 4). To study this variation further, 15 single raft cultures, TTl-IS, from the F generation were reared and the males and females from each were test-crossed with De femafes and Pa males respectively (Fig. 4). Twelve cultures were found to regain Paris cytoplasmic characters, i.e. the males were incompatible with De and the females were compatible with Pa. In two lines, TTS and TT7, males were compatible with De females but females were incompatible with Pa males. At the next generation males and females from these two lines were test mated with females and males of all cytoplasmic types available in this unit (Fig. 4). Males from both lines were found to be compatible with all cytoplasmic types, but females were incompatible with the Pa type from which they were derived and with several other types. However, these were partially compatible with De 19 males: the mean hatchability was 8.4% with a range from 0-25.3% hatch. Eggs from unhatched and partially hatched egg rafts from this cross were predominantly unembryonated. The fertility in matings within the TTS and TT7 lines was almost normal (mean egg hatchability of 88.8%). As shown in Fig. 4, the single raft culture TT9 showed a mixture of female types and further single raft isolations from it produced lines which had reverted to Paris properties and lines which showed segregation among males similar to that of the ISB 20 line described above. However, subsequent results (not shown in Fig. 4) indicated reversion of the TT9 line to the Pa type. (ii) Adult treatment of (Pa)De/De
Following 1 mg/ml tetracycline treatment only one of the rafts laid by the treated females hatched, but following 0.1 mg/ml tetracycline treatment, all the rafts hatched. Progeny from both the treatments were reared and the males and females were then testcrossed with De females and Pa males respectively. Males derived from parents treated with 1 mg/m1 showed compatibility with De females and the females were compatible with Pa males. These properties resemble the ISB 49 isolate described above. Some of the males from the 0.1 mg/ml treatment showed compatibility with De females and others showed incompatibility but all females were compatible with Pa males, i.e. the results resembled those with ISB 20. However, after two generations of breeding without any further treatment of both 1.0 mg/ml and 0.1 mg/m1 treated lines, it was found that the compatibility of the males with De females had reverted to the Pa cytoplasmic type, i.e. to the properties of the stock from which they were derived. DISCUSSION Subbarao et al. (1975) showed that the origin of the ISB 20 and ISB 49 types was a mutation-like process from a previously invariable colony. The present paper demonstrates that the properties of the two new variants ISB 20 and ISB 49 were maternally inherited. ISB 49 provides an interesting analogy with the naturally occurring De 19 variant of Indian populations (Subbarao et al., 1974, 1975). The males of both of these types are apparently universally compatible, and the females show typical compatibility/incompatibility properties. In the case of ISB 49 the properties of the females resemble those of Pa and in the case of De 19 they resemble the majority Delhi type, De 2. In the ISB 20 line there was variation between the compatibility properties of the sons of an individual mother. In addition the line gave rise to pure sub-lines within which all males showed the same properties with respect to females of several other cytoplasmic types. The segregations both between the sons of an individual female and between the lines derived from the daughters of one female suggests the co-existence of different cytoplasmic determinants in an individual and a process of unequal assortment. Very large numbers of rickettsiae are indicated in electron micrographs of C. pipiens oocytes (Yen & Barr, 1974). It may be that the process of unequal assortment acts not at the level of
WHO/VBC/75.596 page 5 the rickettsiae themselves, but rather may involve cytoplasmic factors which control the rickettsiae. It is suggested that these controlling particles are present in small numbers so that their assortment would be appreciably affected by "sampling error". Sagar & Ramanis (1968, 1970) have analysed mathematically their data on cytoplasmic variants in Chlamydomonas based on the model of Schensted (1958). Though there is some quantitative information in our Culex p. fatigans data (Figs. 1, 2 and 3) we are not able to frame any detailed hypothesis on the segregation process. The occurrence of several sub-lines of ISB 20 whose males were fully compatible with De 2 females suggests that the ISB 49 variant is probably a secondary derivative of the original ISB 20 "mutation". As already indicated the segregating ISB 20 stock is no longer available since the laboratory stock culture has come to entirely resemble ISB 49. As shown in Fig. 4, from treatment of larvae with 0.025 mg/ml tetracycline, two lines (TT5 and TT7) were derived whose properties resembled those of the aposymbiotic lines of Yen & Barr (1973), i.e. the males were universally compatible and the females were compatible with males of their own lines but not with males of the Pa strain from which they were derived. It was found that these females were partially compatible with the naturally occurring De 19 type and there therefore, appears to be a certain degree of resemblance between the De 19 and the aposymbiotic type, but it should be recalled that De 19 females show conventional compatibility/incompatibility properties, unlike aposymbiotic females. The partial resemblance of De 19 males to the aposymbiotic type strongly suggests that it will not be possible to find a cytoplasmic type whose females are incompatible with it (Subbarao et al., 1975). This would have important practical consequences for the application of cytoplasmic incompatibility for genetic control because it would allow "recombination" or a released cytoplasm with indigenous genome (Curtis, 1976). As noted in Fig. 4, ISB 49 females. The results reported (i) De 2 and Pa; (ii) ISB 20; Electron microscopic studies on differed from De 19 in being incompatible with aposymbiotic suggest a gradation of properties in males as follows: (iii) ISB 49; (iv) De 19; and (v) aposymbiotic. these five types are in progress.
The effects of tetracycline treatment of larvae were not uniform; in addition to the TT5 and TT7 (aposymbiotic) derivatives of the treatment, other types were found (Fig. 4) some of which superficially resembled ISB 20. Unlike the latter stock all these tetracycline derivatives ultimately reverted to the parental Pa type. However, from ISB 20, in addition to the Pa type revertants (ISB 20(13), ISB 20(14) and ISB 20(9)f in Figs. 2 and 3), there were ISB 49 type sub-lines (ISB 20(1), (2), (5). (6), (8), and (15) in Fig. 2 and ISB 20(7)g in Fig. 3) and lines where segregation of male types continued for further generations (ISB 20(7)a-f and h-m, and ISB 20 (9)a-e and g-o in Fig. 3). Adult treatments with tetracycline at different concentrations gave lines which initially simulated ISB 49 and ISB 20. However, once again all the tetracycline lines reverted to Fa type after a few generations, unlike ISB 49 and ISB 20. The effect of tetracycline on adults could be of importance in a genetic control programme because the chickens kept for blood feeding of mosquitos have to be dosed with antibiotics to protect them from intestinal infections. Studies are in progress of the effects on incompatibility of feeding mosquitos on chickens dosed with various concentrations of tetracycline. This paper and other recent publications have shown that the earlier "all-or-none" picture of cytoplasmic incompatibility is complicated by the following phenomenon: (i) Polymorphism of cytoplasmic types in wild populations, e.g. De 19 and De 2 in Indian populations (Subbarao et al., 1974 and 1975) and a similar phenomenon in Bangkok (Krishnamurthy & Laven, 1974).
WHO/VBC/7').596 page 6 (ii) Segregation between the sons of a single female in their compatibility properties,
e.g. ISB 20 (Fig. 1).
(iii) Segregation of sub-lines from the daughters of one female, the males of anyone sub-line showing invariable properties, e.g. French (1970) and ISH 20(7)g and ISH 20(9)f in Fig. 3. (iv) Partial compatibility of young wild type males leading to partial egg hatch of individual rafts, e.g. Hanford females x Scauri males (Harr, 1970). (v) (vi) (vii) Partial compatibility of aged males (Singh et aI., 1976). Partial compaU bil i ty of aposyrnbiotic females x De 19 males (Fig. 4). Partial compatibility of newly tetracycline treated males (Fig. 4) . ACKNOWLEDGEMENTS We are grateful to R. C. Juyal, D. C. Joshi and Satish Kumar for their technical assistance. We thank Dr G. D. Brooks and Dr K. R. P. Singh for their encouragement throughout this work and Dr K. R. P. Singh for his comments on the manuscript. REFERENCES Barr, A. R. (1970) Partial compatibility and its effects on eradication by the incompatible male method, Proc. 37th Ann. Conf. Calif. Mosq. Control Assoc. Inc. 1976, pp. 19-24 Curtis, C. F. (1976) Population replacement in Culex fatigans by means of cytoplasmic incompatibility. 2. Field cage experiments with overlapping generations, Bull. WId Hlth Org. (In press) French, W. L. (1970) Evidence for the segregation of cytoplasmic genes in Culex pipiens an advanced form of animal life, Genetics, 64, 22 (Abstract) Krishnamurthy, B. S. & Laven, H. (1972) Preparation of a cytoplasmically incompatible strain of Culex pipiens fatigans for use in genetic control in India, WHO/VBC/72.389 (unpublished WHO document) Krishnamurthy, B. S. & Laven, H. (974) Development of cytoplasmically incompatible and integrated (translocated incompatible) strains of Culex pipiens fatigans for use in genetic control, WHO/VBC/74.496 (unpUblished WHO document) Laven,H. (1957) Vererbung durch Kerngene und das Problem del' ausserkaryotischen Vererbung bei Culex pipiens II. Ausserkaryotische Vererbung Z., Vererbungsl, 88, 478-516 Pal, R. (1966) Genetic control of vectors of disease with special reference to Culex pipiens fatigans, WHO/Vector Control/66.198 (unpublished WHO document) Sager, R. & Ramanis, Z. (1968) The pattern of segregation of cytoplasmic genes in Chlamydomonas, Proc. Nat. Acad. Sci. U.S., .§,l, 324-331 Sager, R. & Ramanis, Z. (1970) Methods of genetic analysis of chloroplast DNA in Chlamydomonas. In: Boardman, N. K., Linnane, A. E. & Smille, R. M., eds, Autonomy and bio enesis of mitochondia and chioro lac;ts, Amsterdam, North Holland Schensted, I. v. (19.58) Nudel of subnucleilr scgrcgatiun in the macronucleus of Ciliates, Amer. Nilt., 92, 161-170
WHO/VBC/75.596 page 7 Singh, K. R. P., Curtis, C. F. & Krishnamurthy, B. S. (1976) Partial loss of cytoplasmic incompatibility with age in males of Culex fatigans Wied., Ann. troPe Med. Parasit., (In press) Singh, K. R. P. et ale (1975) Mass rearing of Culex pipiens fatigans Wied., J. Com. Dis., l(l), 31-56 Subbarao, S. K. et ale (1974) Variation in cytoplasmic crossing type in population of C. p. fatigans Wied from Delhi area, J. Com. Dis., ~, 80-82 Subbarao, S. K. et al. (1975) Further studies on variation of cytoplasmic incompatibility in the Culex pipiens complex, WHO/VBC/75.543 (unpublished WHO document) Yen, J. H. & Barr, A. R. (1974) The etiological agent of cytoplasmic incompatibility in Culex pipiens L., J. Invert. Pathol., 22, 242-250 Yen, J. H. & Barr, A. R. (1974) Incompatibility in Culex p1p1ens. In: Pal, R. & Whitten, M. J., eds, The use of genetics in insect control, Elsevier/North Holland, Amsterdam, Chap. 6, pp. 97-118
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