PREPARATION OF A CYTOPLASMICALLY INCOMPATIBLE STRAIN OF CULEX PIPIENS FATIGANS FOR USE IN GENETIC CONTROL IN INDIAl. B. S. Krishnamurthy and H.

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1 WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE PREPARATION OF A CYTOPLASMICALLY INCOMPATIBLE STRAIN OF CULEX PIPIENS FATIGANS FOR USE IN GENETIC CONTROL IN INDIAl WHO/VBC/ ENGLISH ONLY INDEXED by 2 3 B. S. Krishnamurthy and H. Laven INTRODUCTION Toumanoff (1937) was one of the first to record genetic barriers in reciprocal crosses of ~ mosquitos. Ghelelovitch (1952) suspected cytoplasmic heredity of the factors responsible for the sterility observed in certain crosses involving different strains of C.p. molestus. Laven (1953) proved the factors responsible for uni/bidirectional sterility, resul ting from crosses of diffe,rent members of allopatric populations of the Culex pipiens complex, to be cytoplasmically inherited. He also showed that these strains,ha,d the ability to retain'this character unchanged indefinitely despite replacement of their genome. He has designated this character as crossing type and the phenomenon of total suppression of offspring due to blockage of the sperm before karyogamy with the egg nucleous, as cytoplasmic incompatibili ty. Laven (1967) has listed more' than 20 suph crossing types in the Culex pipiens complex. Furthermore, he has emphasized that it iis possible to construct one or more incompatible strains for a given population of Culexpipiens fatigans anywhere in the world. Desirable genetic traits can be introduced into an incompatible strain without changing the cytoplasmic incompatibility and thus it is possible to adapt strains from the temperate region to any ecological condition. :::Juen all ~tiapt~ strain having the cytoplasm of Paris and the genome of Fresno, California, UnitediStates,'of America, was prepared and success1ullytested in a field experiment for suppression, of ~ popul~tions in Burma (Laven, 1961a}. The ability to prepare such strains has eliminated the necessity of locating' such strains, in nature. Pal (1966) has cogently pointed out that release of such cytoplasmically incompatible males is an alternate to the current methods used in the induced sterile male technique. Incompatible strain Cytoplasmic incompatibility is one of the genetic methods to be evaluated at the Delhi WHO/ICMR Research Project on the Genetic Control of Mosquitos in New Delhi, India. A strain possessing bidirectional incompatibi,1ity. with the Delhi population of C. fatigans was needed. Such a strain was available and had been previously prepared at the WHO Reference Center, Mainz in It was maintained in the Parasitology baboratory of the University of Malaya, Kuala Lumpur, MalaYSia, and it was tested against C. fatigans from different places in India: Ahmedabad, Bangalore, Delhi and Madras. The reciprocal crosses with the Indian strains and D3 indicated bidirectional incompatibility with all the strains tested. Figure I details the results of the test crosses. Strain 03 was considered suitable for use in Delhi area. 1 The investigations at the Genetic Institute, Mainz were supported by a grant from VBC/WHO 'Geneva. 2 WHO/ICMR Research Unit on Genetic Control of Mosquitos, New Delhi, India. 3 Institute of Genetics, Johannes Gutenberg University, Mainz, Germany, The issue of this document does not constitute formal publication. It should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone ire responsible for views expressed in signed uticles. Ce document ne constitue pas une publication. II ne do it faire I'objet d'aucun compte rendu ou resume ni d'aucune citation sans I'autorisation de I'Organisation Mondiale de la Sante. les opinions exprimees dans, les articles signes n'engagent que leurs auteurs.

2 WHO/VBC/ page 2 Reserve strain The field use of males of such an incompatible strain in large numbers, does not preclude the inadvertant release of a few females which poses the threat of establishment of the incompatible strain. A reserve strain which has bidirectional incompatibility with the released strain is required to eliminate the incompatible strain if such an eventuality occurs. Hence it is mandatory that a reserve strain or back up strain must have bidirectional incompatibility with the cytoplasmic incompatible strain (03) and preferably with the local Delhi strain, as well. As no such strain was readily available it was decided that one be prepared. Efforts were initiated at the Genetic Institute Mainz in the summer of The details are given below: The candidate strain should not only be incompatible with the 03 but should have such qualities as survival and viability in the ecological conditions of the area where the strain is intended to be used. For the selectjon of a suitable donor of plasma C. pipiens strains known to possess incompatibility were chosen from various geographical areas. The inter- and intra-specific crossing relationship between themselves and with the target strains, Delhi and D3, were determined by test crosses. The strains chosen for plasma were "London" from United Kingdom, "Paris" from France, "Hamburg" and "Oggelshausen" from North and South Germany "Scauri" from Italy and "Cairo" from Egypt. The donor of chromosomes also needs the characteristics of viability and survival under Indian conditions. Therefore tropical/sub-tropical strains from Africa, Dar-es-Salaam, India "Anupgarh" and "Suratgarh" and South-East Asia "Bangkok" were utilized. Their inter-relationship as well as their crossing type with 03 and Delhi populations were determined. The results of the test crosses are schematically presented in Fig. II. l~e data indicates that none of the known cytoplasmically incompatible strains have bidirectional incompatibility with both 03 strain and Delhi Culex fatigans population. However, two strains, "Hamburg" and "Scauri" do have bidirectional incompatibility with 03 strain and a unidirectional incompatibility with the Delhi strain. The crossing relationship of these strains is shown in Fig. III. The strain "Scauri" was observed to possess a low capacity to inseminate and a low viability and was considered unsuitable. The Hamburg strain on the other hand behaved normally and is also vigorous. To verify the above observations, incompatible strains were prepared using both the donors of plasma "Scauri" and "Hamburg" and four candidate strains of donors of genome. The strains prepared were: Female x Male Female x Male Hamburg x Jkmgkok Scauri x I3angkok x!jar-as-salaam x Dar-es-Salaam x,"'ova x Nova x Fresno x Fresno All crosses were compatible and produced viable progeny and eliminated the use of an intermediary strain necessary to transfer tile genome to the donor of plasma if the above crosses had been incompatible. Characteristics of the above eight strains were studied for three generations. In view of the crossing relationship of Hamburg and Scauri with 03 and Delhi strains being the same, the best amongst them originating from Hamburg was selected for replacement of the genome and was alternately back crossed with males of Bangkok and Dar-es-Salaam strains. The backcrossing was continued for six generations alternating the males of the two strains and an inextricable mix up of genome was achieved which ensured the continued benefits of heterosis thus making it viable even under exacting ecological conditions.

3 WHO/VBC/ page 3 CONCLUS IONS The studies have substantiated that incompatible strains to suit any specific requirement of ecological conditions can be achieved in a comparatively short period saving on time and effort required in detecting such strains in nature. In order to determine the pathways used in preparation of incompatible strains, determination of the crossing relationship of the strains involved with the target strains as well as the inter-relationship of the donors of plasma and genome was emphasized. ACKNOWLEDGEMENT The authors gratefully acknowledge the kind assistance rendered by the staff of WHO Reference Centre, Mainz, Germany in connexion with the preparation of the cytoplasmic incompatible strain D4. REFERENCES Ghelelovitch S. (1952) Laven H~ (1953) Laven H. (1967a) Laven H. (1967b) Pal R. (1966) Toumanoff C. (1937) Sur Ie determinisme g6n6tique de la ster ili,t6 dans les croisements entre diff6rente,s" ' souches de Culex autogenicus Roubaud. C.R. Acad. Sci. (Paris) 234: Reziprok unterschiedliche Kreuzbarkeit."von ' Stechmuchen (Culicides) and ihre Deutung ala, plasmatiche Vererbung, Z. indo Abst. V~rerb:' 85 : '. Eradication of Culex E,. fatigans through cytoplasmic incompatibility. Nature 21'6 No A Speciation and Evolution in Culex pipi~ns. Chapter 7 p Genetics of Ins~ct Vectors, edited by Wright, J.W. & Pal. -R. Elsevier Publishing Company, Amsterdam;~ Genetic control of vectors of disease with special reference to Culex pipiensfati~aul~~,~: WHO/VBC/ (Mimiographed document)~ Essais preliminaires d'inter-croisement de St. albopictus Skuse avec, St. argentea Poiret s. fasciata Theob. Bull., Soc. med. chir. Indoch. 51,

4 " FIGURE I '0 ::E ill ::z:: IlG 0 ct>... < "" n I:II ~ ~ w (Xl (!) RESUl TS OF TEST CROSSES BETWEEN. 03 AND INDIAN STRAINS OF C. FATIGANS ~~ AA De Ah Mo De Ah Mo '- D3 INCOMPATIBLE STRAIN I - Wff/~ Ah I-I -t-~-t- I 1- I-t-I-L- V//////.A + De DELHI AtfMEDABAD Mo MADRAS Bo BANGALORE INCOMPA TlBLE CROSS + COMPATIBLE CROSS

5 FIC.UBE 1I RESULTS OF TEST CROSSES OF CULEX PIPIENS/ FA TIG ANS FOR SELECTION OF A STRAIN AS DONOR OF CYTOPLASM AND OF THE GENOME ~ Lo Pea. Hea. 07 Sc: C4 Dc Os A 'It S... B4 DES LEC.END Lo ~ Lo LONDON Pea. + ~ Po. PARIS HQ. - - ~ H4. HAM8UBC. 0,/ ~ , OC.C.ELSHAUSEN Sc ~ Sc SCAURI C ~ - + C4. CAIRO De ~ De DELHI 03 + I± ~ D!t INCOMPA T1BLE A.,. + - ~ "... ANUPGARH S", ~ + + Su SURATC.ARH i&ea ~ + 8ea. 8ANC.KOK DES =+++~ DES OAR-E-SALAM + COMPATI8LE CROSS INCOMPATIBLE CROSS FIGURE I II D3 - Incompatible strain De = Delhi Ha Hamburg Sc = Scauri '0 ~ Il> :c ':i~ <: "'0; -S- '" w ()) '"