A field trial on control of Culex pipiens fatigans wied. by release of males of strain integrating cytoplasmic incompatibility and a translocation

• WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE WHO/VBC/76. 635 ,. 'I I ,. ENGLISH ONLY (f(..t lt: .Y • ~lr 1 • ./ tl,U ,,._ A FIELD TRIAL ON CONTROL OF CULEX PIPIENS FATIGANS WIED. BY RELEASE OF MALES OF A STRAIN INTEGRATING CYTOPLASMIC INCOMPATIBILITY AND A TRANSLOCATION by 1 . 2 2 2 G. D. Brooks, C. F. Curt~s, K. K. Grover, B. S. Krishnamurthv, P. L. Rajagopalan, 2 L. S. Sharma, 2 •3 V. P. Sharma,2 D. Singh,~ K. R. P. SinghS2 M. Yasuno,2,4 M. A. Ansari, 2 T. Adak,5 H. V. Agarwa1, 5 C. P. Batra, R. K. Chandrahas,5 P. R. Malhotra,5 P. K. B. Menon,5 R. Menon,6 S. Das,5 R. K. Razdan5 and V. Vaidyanathan5 ABSTRACT INDEXED An integrated strain of C. p. fatigans Wied. (IS-31B) was used for the first time in attempts to control or suppress a natural population. Males were released at a rate of 5000 to 40 000 and 5000 to 20 000 per day in two villages. Some isolation was achieved around the first village by creation of a 3 km wide zone kept free of mosquito production by use of conventional larvicides. All released males were marked and the ratio of released (marked) to wild (unmarked) ranged from 17 to 225:1 in the first village and from 7 to 200:1 in the second. With these ratios and the known level of competitiveness of IS-31B males most matings in the villages were expected to be by IS-31B males. Sterility as measured in the egg rafts reached a high of 67.9% and 53.2% in weeks 5 and 4 after releases began in the first and seco~d villages respectively. The average level of sterility plateaued, and subsequently declined before the termination of releases. After consideration of various possible explanations of this phenomenon it was concluded that a (a) a decline in numbers of emer~ing females and (b) immigra- tion of already inseminated females in the later stages of the experiment were the most likely causes. Comparison of the rates of change in adult densities in the comparison villages indicated that a degree of population suppression was achieved in the release villages for a five-week period in late August and early September; thereafter all populations responded as expected for the seasonal trend. Further evidence for partial population suppression due to the releases was obtained from observations on breeding in the villages. 1 Project Leader, WHO/ICMR Research Unit on Genetic Control of Mosquitos, New Delhi, India. Present address: Department of Health, Education, and Welfare, Cent~r for Disease Control, Atlanta, Georgia, United States of America. 2 ~cientist Staff, WHO/ICMR Research Unit. 3 Present address: College of Agriculture, Udaipur University, Udaipur, Rajasthan, India. 4 Present address: Department of Parasitology, 5 Research Assistant, WHO/ICMR Research Unit. 6 Health Educator, WHO/ICMR Research Unit. The issue of this document does not constitute formal publication. lt should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone are responsible for views expressed in signed articles. University of Tokyo, Japan. Ce document ne constitue pas une publication. 11 ne doit faire l'objet d'aucun compte rendu ou resume ni d'aucune citation sans l'autorisation de !'Organisation Mondiale de la Sante. Les opinions exprimees dans les articles signes n'engagent que leurs auteurs. WHO/VBC/76.635 page 2 Introduction Following demonstration of the use of cytoplasmically incompatible males of Culex p~p~ens for suppression of a natural population of this species in Burma (Laven, 1967), Laven & Aslamkhan (1970) proposed use of cytoplasmic incompatibility combined with a male-linked translocation complex to form an integrated strain as a genetic control agent. The construction of such a strain was started in 1971 as part of a research programme testing the feasibility of genetic control of mosquitos by the WHO/ICMR Research Unit on Genetic Control of Mosquitos in Delhi, India. Prerequisites of the strain were: first, that the. males had to be cytoplasmically incompatible with Indian populations and, second, the strains would require a male-linked translocation causing partial sterility in matings within the strain to prevent build-up of a large population of the released strain after elimination of the indigenous population. Krishnamurthy & Laven (197~ were successful in developing a strain with European cytoplasm and chromosomes of Indian origin, which overcame the problem of adaptation of a foreign strain for use in the Indian environment, and retained its incompati- bility to the indigenous strain. The strain designated IS-31B was ready for field testing in 1973. After the termination of a release experiment using chemosterilized males (Yasuno et al., 1975), in July 1973, field testing of the strain was commenced in the months of August through November. The basic question to be answered was whether such a strain could inject sufficient ster~lity into a wild population to suppress the population. The study involved release of IS-31B males in three rural villages, one surrounded by a 3 km breeding free zone and the second located within the zone of the primary release village. The results were compared with two comparison villages, one with a similar 3 km breeding free zone and the second devoid of any artificially applied control measures. The study plan included the same elements of quality control of release material as were applied in a large- scale trial with chemosterilized males reported by Yasuno et al. (1975). Materials and methods Release sites The sites selected were the villages of Gommanhera, Jhiljhil, Pindwala Khurd and Paprawat in the Delhi Union Territory (Fig. 1), approximately 60 km south-west of New Delhi. The villages are located on the tops of small hillocks but even so are only a few metres above the surrounding terrain. The village Gommanhera, designated the primary release site, consists of 228 occupied residential houses and has a human population of 2012. There are 32 wells within the target area, with a total surface area of 2850 m2 • Jhiljhil, the second release site, is 2 km to the west and has 144 houses and a population of 677. Though there are only 26 wells their total surface area is 3089 m2 . In each case the release site was the specified village plus a 250 m wide belt surrounding it, i.e. a circle with a 500 m radius in all. Pindwala Khurd, the primary comparison village, is somewhat smaller than the release sites and contains only 88 houses with a human population of 770. This village is located approximately 4 km to the north-east of Gommanhera. Paprawat, the village used for observations of an undisturbed mosquito fauna, contains 249 residences and has a population of approximately 2000. This village is approximately 3-1/2 km to the north of Pindwala Khurd. The concept of breeding free zones was developed after failure to achieve high levels of sterility during releases by the Unit in 1972 and represents an attempt to create an artificial isolation of the study areas (Sharma, 1974). For this study a 3 km wide belt (Fig. 1) was used around both Gommanhera and Pindwala Khurd as in an experiment with chemosterilized males (Yasuno et al., 1975). The zones differed slightly from those of the earlier study in that they overlapped (Fig. 1) between the two villages. The terrain WHO/VBC/76.635 page 3 2 covered by the zones consists primarily of cultivated fields but within the 70 km area there are 15 villages and over 1600 open irrigation wells. Jhiljhil, the second release village, is situated within the zone surrounding Gommanhera. Selection of the villages was based on the fact that: (1) research had been carried out by the Unit in the general area for the past three years and much ecological and demographic data was available, and (2) the village of Gommanhera possessed the desirable feature of isolation in that there were villages on only three sides and it was relatively further from the urban area of Delhi than villages used in earlier studies by the Unit. The experiment was started at the beginning of August in the monsoon season and evaluation was completed by mid-December 1973. Mosquito populations in this part of rural India are generally steady during the monsoon season but show a gradual decrease to low levels by late November (Yasuno et al., 1973; Rajagopalan et al., 1976). Genetic stocks The integrated genetic strain (IS-31B) had cytoplasm of Paris origin and chromosomes of Delhi origin including a translocation complex linked to the~ (male determining) locus to yield partial sterility in matings within the strain. The details of preparation of this strain have been described by Krishnamurthy & Laven (1976). Public relations, mapping and insecticidal control Cooperation of the public in the villages was sought by personal contact between health education staff and village leaders as well as by daily contacts between field staff and individuals of the community. Discussions and demonstrations of various aspects of the release procedures were used employing the techniques and experience gained in previous experiments in nearby villages (Singh et al., 1972; Singh & Brooks, 1974). Great care was taken to give a realistic picture without expressing over-optimism as to expected results. All mosquito breeding sites were located and mapped using the plane table technique. Each site was numbered to facilitate complete coverage by the survey, evaluation and control teams during the study. All wells and other breeding sites found positive for Culicine immatures in the 3 km zones surrounding the villages were treated with Abate. Weekly surveys and simultaneous treatments were maintained thereafter to ensure complete detection and elimination of mosquito production throughout the zones. Larval breeding found in vessels within villages in the breeding free ~one was controlled by removal of the water from the vessels. Specimens were collected routinely from positive receptacles and returned to the laboratory for identification. The only areas in the two zones excluded from treatment were the 500 m radius circles at Gommanhera, Jhiljhil and Pindwala Khurd indicated in Fig. 1. To reduce the population of inseminated females within the release villages and hence increase the chance of a rapid build-up of egg sterility, Gommanhera and Jhiljhil were fogged on two consecutive days before releases commenced with 8% malathion in diesel oil using a swing fog machine. Pindwala Khurd was similarly treated to maintain comparability. Ecological observations The mosquito population density was asse$sed weekly by handcatch index in the five villages as described by Yasuno et al. (1973). The absolute population was assessed weekly in Gommanhera and Pindwala Khurd, and fortnightly in Jhiljhil, by the mark-release-recapture method. As all material being released in Gommanhera and Jhiljhil was marked with standard colours (red in Gommanhera and yellow in Jhiljhil), releases of males for these estimates were marked with a different colour. For the estimates in Gommanhera and Jhiljhil, males of IS-31B were released, whereas males released in Pindwala Khurd were normal fertile males froma laboratory colony. Multipoint releases in houses within the village were used, as this procedure had been shown by Yasuno & Rajagopalan (1973), to give uniform dispersal within the built-up areas of villages. WHOjVBC/76.635 page 4 Hand collections of resting mosquitos provided the ratio of marked (released) to unmarked (wild) males. These indices, as reported in this study, are for unmarked (wild) mosquitos only. The ratio of marked (released) to unmarked (wild) males was also monitored in mating swarms by making collections with sweep-nets. All classifications as marked or unmarked, as well as species identifications were made after microscopic examination of the specimens. Estimates of pupal emergence in receptacles were made by counting the actual number of pupae. Estimates for wells, however, were made using a standardized sampling net technique (Rajagopalan et al., 1976). At intervals samples of the pupae were taken to the laboratory to emerge to check that all were C. p. fatigans and not other Culex species which are difficult to distinguish at the pupal stage. Release material The IS-31B strain was mass reared by the techniques described by Singh et al. (1974, 1975). Although heavy larval mortality was encountered in the initial stages of production, the strain adapted quickly to the rearing procedures and behaved similarly to Delhi normal strain with the exception of pupal yield, which did not exceed 80% of the input of 1st instars, compared to 90% in the normal strain. The release material was sexed using a grid system (Sharma et al., 1972) and held under ·standard conditions of temperature and humidity in the laboratory pending transport and release in the field. Caging, marking and transportation of mosquitos were carried out using techniques and equipment described by Singh et al. (1975) and following the procedures used in the large field operation described by Yasuno et al. (1975). Mass production of the IS-31B strain reached 150 000 male pupae per day. Production levels were maintained well above that required to meet the following daily release schedules: NUMBERS OF MALES RELEASED DAILY IN GOMMANHERA AND JHILJRIL VILLAGES Starting date Inside village Outside v~llage Gommanhera 4 August 5000 - 20 August 10000 - 27 August 20 000 - 14 September 20 000 20 000 Jhiljhil 2 September 5 000 - 11 September 10 000 - 23 September 10 000 10 000 Quality control As quality control is essential to the operational use of any sterile male release programme, stringent techniques were applied in this study as the study constituted the first application of an integrated genetic strain. Quality control checks were as follows: (a) Selection of the grid for sexing based on daily examination of samples of pupae drawn from each day's production and employment of a sequential analysis chart (Yasuno et al., 1975). WHO/VBC/76.635 page 5 (b) MOrtality of pupae and adults was measured by routine counting of dead pupae and after emergence, marking, transportation to the villages and release and also in samples drawn from the release cages and observed for seven days. Persistence of the marking was also evaluated in these samples. (c) Sterility due to the translocation was monitored by sampling 10 egg rafts daily from each cage of adults in the breeding colonies. (d) As a check for contamination and incompatibility of the strain, test crosses of IS~31B were made to a Delhi strain. To avoid errors in sexing and any complications due to polymorphism of cytoplasmic types in the Delhi strain, reciprocal crosses of young males and females of the IS-31B strain were in the later stages made to a Delhi cytoplasm strain derived from a single raft isolate and carrying a genetic marker. (e) To permit early detection of contamination in the release material by the Delhi strain or by non-translocated males, a system of routine checks for partial sterility in samples of lOO egg rafts from each of the adult IS-31B cages from both the mass reared and stock colonies was instituted. In the stock colonies any normal hatch rafts were eliminated from the parentage of the next generation. (f) Competitiveness of IS-31B males versus Delhi males for virgin Delhi females was tested in outdoor cages at a 1:1:1 ratio. Both unmarked and marked and 12-36 hours and 36-60 hours old IS-31B males were evaluated. (g) Further tests of the competitiveness of the IS-31B strain males were carried out through two field experiments during this study and are reported by Grover et al. (1976). Evaluation of sterility Evaluation of sterility induced in the wild population in each of the villages was carried out from egg rafts collected from natural breeding sites. Initially, breeding was found primarily in receptacles, but, from mid-September, eggs were found also in wells; therefore egg rafts were collected from both habitats from 15 September onwards. Egg raft collections from receptacles were made systematically from nearly all of the receptacles in which there was oviposition. It was found that C. tritaeniorhynchus shared the well breeding habitat with C. p. fatigans (Menon & Rajagopalan, 1974) but rarely was oviposition by this species observed in receptacles. The egg rafts and larvae of C. p. fatigans and C.tritaeniorhynchus were distinguisned by the following criteria taken together (i) shape of egg raft - C. p. fatisans rafts are tapered at each end while tritaeniorhynchus are flattened, (ii) appearance after hatching: C. p. fatigans egg rafts usually stay intact after hatching, maintaining the shape of the raft while in C. tritaeniorhynchus the eggs scatter after hatch, (iii) identification by first instar larvae: hatched first instar larvae were identified by the following comparative characteristics: C. p. fatigans (a) Siphon short with apex chitinized giving the appearance of a "black cap". (b) Pigmentation absent from head and dorsal surface of abdominal segments. C. tritaeniorhynchus (a) Siphon slightly longer without chitinized "black cap" appearance. (b) Pigment usually present on head and dorsal surface of abdominal segments. A few larvae from 10 hatched egg rafts (five classified as C. p. fatigans and five as C. tritaeniorhynchus) were collected daily and reared for third instar identification using the keys of Barraud (1934), Bram (1967) and Reuben (1969). These checks provided confirmation that the raft and first instar identification was being carried out accurately. Egg rafts WHO/VBC/76.635 page 6 were also collected from ground water pools around the release villages in the second and third week of September, hatched and identified. All were found to be C. tritaeniorhynchus. Egg rafts collected for sterility assessment were held for examination at 48 hours in laboratories set up in houses in villages and the number of fully hatched (>90%) and partially hatched (2-90%) rafts were recorded. Larvae from normal and partially hatched egg rafts were returned, as far as possible, to the respective sources from which collected. Unhatched and partially hatched egg rafts and rafts thought to be from species other than C. p. fatigans were returned to the Unit laboratory for further examination and/or identifi- cation. Rafts with >2% egg hatch were classified as unhatched and these were further classified as incompatible (if there was at least one embryonated egg) or unembryonated (which was attributed to failure of insemination). Results Health education Evidence of success in achieving full cooperation among the villagers was evident by late August. Some problems were experienced, however, which included: (1) misunderstandings concerning hiring and discharge of locally recruited personnel (2) the death of two buffaloes in the village which the villagers claimed to have been caused by the use of insecticides in the area (3) objections by village women to release of mosquitos in or near their houses which caused annoyance for them while performing domestic duties (4) refusal of entry to certain unit personnel by some of the villagers on religious grounds and (5) difficulty in explaining why, in spite of the programme, the mosquito nuisance continued. The latter was particularly troublesome since the Anopheles population was at a seasonal peak during the experiment. Larvicidal operations Expansion of existing was started in late July. weekly surveys for culicine collections and receptacles detailed maps to include Pindwala Khurd and its breeding free zone Mapping was not entirely complete until mld-October. Results of breeding in wells, cement tanks, pools, pits, ditches, rain water in the breeding free zones are shown in Table la. The number of sites with breeding potential in the Gommanhera zone ranged from 720, in early August, to over 4800 in mid-September, after which it declined. In the Pindwala Khurd zone on the other hand, there were over 6000 potential breeding sites in the first week in August, but slowly declined to half that number by the last week in October. These variations are attributable to the appearance of ground pools, water in receptacles etc. only during the monsoon. Culicine egg rafts and larvae were found in numerous habitats throughout the season but production of pupae was limited by the larvicidal operations and occurred only in the months of August and September. The sites with pupal production were mainly habitats in agricultural fields and, as shown in Table lb, most of the Culex breeding in these sites was not C. p. fatigans. Thus the aim of eliminating adult emergence of C. p. fatigans in the breeding free zone was largely achieved. Adulticide operation Adulticide treatments to reduce the C. p. fatigans population inside the villages of Gommanhera and Pindwala Khurd, were carried out for two days prior to the first release. The municipal authorities were also conducting antimalaria spraying in Gommanhera at this time. As a result of these operations, the C. p. fatigans density as measured by hand catch index was reduced to 0.33 in Gommanhera, and 0.3 in Pindwala Khurd (Tables 2 and 3). In Jhiljhil village, located within the Gommanhera breeding free zone, where releases were started on 27 August, the village was also fogged on 25 August. As a result, the adult density was reduced from 1.97 on WHO/VBC/76.635 page 7 24 August to 0.2 on 28 August. Concurrently the mosquito density in the uncontrolled comparison village, Paprawat, was high with a hand catch index of 5.7. Population evaluation The weekly fluctuations of the population in Gommanhera as measured by relative and absolute density and the male to female ratio of wild (unmarked) mosquitos along with the estimated number emerging daily within the target area and the observed marked to unmarked male ratio are shown in Table 2a and Fig. 2. After fogging of the village the population remained at a low dens~ty for most of August but then increased nearly ten-fold in the week of 25 August. This increase was registered in both the hand catch index and the absolute population estimate and affected both sexes. Subsequently, the wild population gradually declined to a low density of less than 0.2 in November. . At the beginning of the study, the estimated number of adults emerging daily in the target area was 525, which increased to over 2000 per day by the third week of August. This number decreased to a stabilized level of slightly less than 1000 per day until mid-September. Thereafter the number dropped to less than lOO per day. With the exception of 20 October when a daily emergence of 160 was observed (primarily from one well), relatively little emergence occurred during late October and November. Breeding in the Gommanhera area was sustained primarily in receptacles from August through September. With the gradual drying up of receptacles in October, very few pupae were produced from these habitats. Although well breeding had begun by the last week of September, production in these sources remained at a low level. The data on population density in Jhiljhil are shown in Table 2b and Fig. 2. The wild mosquito density was 0.2 at first assessment and increased to mid-September. Thereafter the population slowly decreased and remained low. Daily emergence ranged between 160 to 250 during the first three weeks of September but an eight-fold decrease occurred the following week followed by very low production thereafter. The relative mosquito density in the comparison village of Pindwala Khurd steadily increased after fogging until the last week of September (Table 3). Thereafter, a gradual decline was observed. The estimated daily emergence remained relatively constant at a few hundred per day in August, September and October. Initially, breeding was primarily in receptacles and cement tanks but this was replaced by breeding in wells after subsidence of the rainy period. At Paprawat, the relative density fluctuated at a high level, when compared to the release villages (Tables 2 and 3). The density gradually increased until mid-September but showed a decreasing trend thereafter. In October and November hand catches were carried out in four other untreated villages to the south west of the Gommanhera breeding free zone. The results are shown in Table 4. Release material Female contamination of the release material remained below 0.2% throughout the study. Combined mortality of release material from pupal packaging, marking and transportation to the field averaged 2.2% with a range of ~ 2%. The level of sterility due to the translocation, i.e. in matings within the IS-31B strain, was initially about 72% with a range of + 10%. A slight decline in the sterility was noticed to 65% in October. During November in the mass rearing colonies there was a sharp decline to 55%, but the stock colonies showed no such decline at that time. WHO/VBC/76.635 page 8 Results of the test crosses are shown in Table Sa, No normal hatch rafts were present in May. However, in tests made in August a number of normal or partial hatch rafts were observed, indicating possible contamination. Data regarding the egg rafts samples derived from the stock colonies and mass reared ~olonies are presented in Table 5b, Seven of the rafts with normal hatch were cultured individually and the resulting adults were test crossed to the IS-31B and Delhi stocks. The tests showed that five were of Delhi crossing type, thus indicating contamination by the indigenous strain. The remaining two rafts were of the Paris crossing type without the trans- location. Data on competitiveness of the IS-31B males aged 12-36 hours against Delhi males for Delhi females are presented in Table 6a and the results of tests of IS-31B males aged 36-60 hours.are presented in Table 6b. Laboratory cage tests with integrated strain males aged between 36-60 hours against Delhi males of the same age are presented in Table 6c. Egg raft evaluation Data on the results of egg raft evaluation from Gommanhera are shown in Table 7a and Fig. 3. The proportion of incompatible rafts increased steadily until mid-September even though the marked released male:wild male ratio in the preceding weeks fluctuated greatly (Fig. 3). From mid-September there was a rise in the percentage of partially sterile egg rafts. The source of such rafts was probably mainly matings between IS-31B males and females, the latter being "contaminants" in the release material and/or IS-31B individuals born in the village as a result of the earlier generations of release. In September and October the percentage incompatibility among rafts collected in receptacles remained almost constant and in the initial collections from wells similar percentages incompatible were found. However, in late October and November, when all the rafts were laid in wells the percentage of incompatible rafts declined markedly, Checks continued to be appli€d throughout this period to ensure that rafts of species other than C. p, fatigans were not included in the samples for which data were quoted, Releases of IS-31B males were terminated on 9 November but about 30% of incompatible rafts were observed during the next two weeks and this declined to 5.6% by the second week of December. Data on the pre- and post-release sterility in the population of Jhiljhil (secondary release village) are presented in Table 7b and Fig, 4. Some incompatible rafts were observed in the population before releases which can be attributed to IS-31B males migrating from Gommanhera or to females inseminated by IS-31B males in Gommanhera which migrated, or both. During the period when 5000 males were released, the proportion of incompatible rafts rose to about 42%. After two additional increases in release number, i.e. to 10 000 and 20 000 per day, incompatibility rose to 53.2% but in most subsequent weeks it declined despite continuation of releases. On 12 November 10 000 virgin females were released into the village in order to carry out a test of mating competitiveness of IS-31B (Grover et al. 1976). The greater availability of virgin females to mate with IS-31B in the village, as a result of this release, was associated with a temporary increase in egg raft incompatibility but the number of rafts laid following this large release was surprisingly small. Partially sterile rafts appeared in Jhiljhil only on four occasions and in no case exceeded 1.6% of the rafts examined. Egg raft collection from receptacles showed a considerable drop in early October in this village and after 25 October almost all data were from wells. In Pindwala Khurd (comparison village), the egg rafts collected followed the expected seasonal trends but were relatively more numerous than in the release village. A few incompatible egg rafts were obtained each week (Table 7c). These egg rafts are presumably WHO/VBC/76.635 page 9 the product of emigrants from the release village. Some sterile but not incompatible egg rafts were also observed, In these egg rafts all the eggs were fully embryonated and the reason for this type of sterility is uncertain. Such egg rafts were excluded from calculations of incompatibility, Test of the genetic nature of the rafts with normal hatch laid in the release village Samples of a few larvae were collected from each of 40 hatching rafts from Gommanhera on 30 August and from 30 hatching rafts in Jhiljhil on 21 September. They were reared as single raft cultures and each culture was identified as morphologically identical to C. p, fatigans. Test matings of the females from each culture to IS-31B males showed incompatibility in each case, Test matings of the males to females of the laboratory colony of Delhi origin gave full fertility and fully fertile Fl male progeny. Males and females from the collection of rafts in Jhiljhil were also mixed with IS-31B males in laboratory cages at a 1:1:1 ratio. Close to 50% incompatible rafts were obtained. The larvae from the compatible rafts from this competition test were reared and the males and females once more mixed with IS-31B males at a 1:1:1 ratio and again approximately 50% incompatible rafts were obtained, Predation by Culex (Lutzia) fuscanus The presence of egg rafts and larvae of the larval predator Culex (Lutzia) fuscanus was recorded during surveys of receptacles for pupae and for egg rafts. It was found that when C. (Lutzia) larvae were present they prevented C. p. fatigans pupal production from the rafts laid in that receptacle, Data on the presence of C. (Lutzia) are shown in Table 8. Though the percentage of receptacles infested was low it was found from observations in Gommanhera in September and early October that a large proportion of the C. p, fatigans rafts were laid in receptacles infested with C. (Lutzia). Emigration from Gommanhera Collections were made in several villages and considerable numbers were found of marked individuals which had been released in Gommanhera and had emigrated to other villages (Table9). Discussion The feasibility of mass production, sexing, marking and transportation of an integrated strain for operational release was demonstrated, However, the maximum output from the space available was somewhat less than with a normal strain because of the reduced larval survival and the requirement for keeping more adult colonies to yield a given output of viable eggs, From previous studies by the unit it has become evident that quality control of the release material is an important part of any release programme. Results of such observations during this experiment showed that the sterility level of the translocation in the !S-31B strain declined slightly over the study period. Further studies of this phenomenon are reported elsewhere (Krishnamurthy et al. 1975), In this experiment the sterility level of the translocation was .not of much practical importance because the partially sterile egg rafts collected from the field always constituted only a minority of the total rafts collected (Fig, 3). The IS-31B strain was slightly contaminated by the Delhi strain in August as shown in Table 4. Precautions taken to prevent further contamination were successful and replacement of stocks with uncontaminated material caused the contamination to disappear. The percentage of contamination was never large enough to have an appreciable effect on results in the field. WHO/VBC/76.635 page 10 As mentioned some of the normal hatch rafts found in the IS-31B colony yielded progeny with Delhi cytoplasmic type. These are recognizable as Delhi strain contaminants. However, two other normal hatch rafts had Paris cytoplasm, and it seems probable that these were a result of contamination by the Delhi strain and matings of the De 19 cytoplasmic type (uni- directionally compatible with Paris) which exists in the Delhi strain (Subbarao et al. 1974). An alternative explanation of crossovers between the translocation and the ~ locus seems unlikely because later studies with a purified IS-31B colony gave no more instances of normal hatch rafts. The adult population densities in Gommanhera and Pindwala Khurd were routinely estimated by two methods - hand catch index and mark-release-recapture. There was a highly statistically significant correlation between the two parameters (Tables 2 and 3). Because of likely differences in carrying capacity of the breeding sites in the study areas it might be misleading to compare gross or relative densities in the different villages especially in view of the fact that four untreated villages to the south west of Gommanhera had very much lower hand catch indices than those of Pindwala Khurd and Paprawat when they were sampled in October (Tables 3 and 4). It is safer therefore to base comparisons on trends in population levels, on the assumption that the relative densities in the villages would follow similar trends in seasonal change, as was found in earlier studies conducted in two ecologically differing villages in this area (Yasuno et al. 1973). The wild mosquito population in Pindwala Khurd estimated by mark-release-recapture increased fifteen-fold during August and September and that in the other comparison village, Paprawat, fluctuated but increased 2.1 fold during the month of September (Table 3). At Gommanhera, the measures of density of wild mosquitos increased nearly ten-fold between 18 August and 25 August, but after 25 August the population gradually declined at an average weekly rate of 19%. The population trends for Gommanhera and Jhiljhil appeared similar in this gradual decline (Fig. 2). There was a considerable degree of sterility in egg rafts during this period, and it seems very probable that the downward trends in the populations of Gommanhera and Jhiljhil during September were the result of the release of the IS-31B males. From late September onward, however, there was a decrease in population in all four villages and little indication of a faster decline in the release village than observed in the comparison villages. Additional evidence indicating the effect of the introduced sterility in causing partial population suppression was obtained from the data on immature stages. Table 2 indicates very sharp declines in pupal emergence in Gommanhera and Jhiljhil from peaks in August to near zero or zero in late October. However, in the control village, Pindwala Khurd, there was no such downward trend and the peak value of pupal emergence was observed in October (Table 3). The number of egg rafts laid in receptacles in the two release villages declined during September and October to zero in November (Table 7). This can largely be explained by the previously reported shift of C. fatigans breeding in this area from receptacles to wells at the end of the monsoon season (Yasuno et al. 1973) and this trend was also seen in the control village Pindwala Khurd (Table 7). However, the decline and disappearance of receptacle oviposition was steeper and more complete in the release villages. It seems probable that the presence of C. (Lutzia) fuscanus in receptacles was one of the important seasonal influences causing a decline in successful breeding in receptacles. It was usually found that if C. (Lutzia) fuscanus was present in a receptacle, all C. p. fatigans larvae were consumed. Thus the percentage of C. p. fatigans predated depended on the proportion of C. p. fatigans rafts laid in C. (Lutzia) fuscanus infested receptacles. The percentage of receptacles infested in Gommanhera and Pindwala Khurd were approximately the same and from the available data there is no reason to suppose that these were differential rates of predation between the two villages. Thus the conclusion seems valid that the faster decline in C. p. fatigans breeding in Gommanhera compared with the control village was due to the introduced sterility, WHO/VBC/76.635 page 11 Comparison of the data for Gommanhera on adult densities (Fig. 2) pupal emergence (Table 2) and numbers of egg rafts colle~ted (Table 7) shows qualitative resemblance in the trends during the experiment in that all parameters peaked towards the end of August and then declined. More detailed examination, however, reveals the anomaly that the very sharp peak in the two measures of adults in the week ending 25 August followed a much less dramatic peak in pupal emergence. The peak of adults cannot be explained by an influx of mated females, since the proportion of egg raft incompatibility rose steadily during this period. The observe'd large adult peak was associated with less than a two-fold increase in the number of rafts collected in receptacles. No obvious explanation of this anomaly is available. The following hypotheses were considered to explain why the percentage of incompatible rafts did not rise above 70% in either of the release villages. (a) Insufficient numbers and/or competitiveness of the released males An insufficient number of released males seems unlikely in view of the lack of correlation of the percentage incompatibility achieved and the numbers released (Figs. 3 and 4). The observed proportion of marked (i.e. IS-31B) males in the Gommanhera population was above 90% in every week except one, from 8 August until releases terminated (Fig. 3) and similar very high ratios existed in Jhiljhil from early September onward. Two tests of the mating competitiveness of IS-31B males were carried out using the principle devised by Dame et al. (1964) of releasing and recapturing marked females. Results of these studies are reported in detail elsewhere (Graver et al. 1976); in summary, they indicated that IS-31B males were well able to induce high levels of incompatibility in females known to have mated in the village in which IS-31B males were present. The two experiments gave a mean estimate of competitiveness! of released, marked IS-31B males of 38.5%. This estimate has been applied to the observed marked to unmarked ratios in Gommanhera and Jhiljhil to obtain estimates of the effective proportion of IS-31B, i.e. the ratio of matings in the village expected to be by IS-31B. For example for the week ending 8 September the effective ratio in Gommanhera = (30.7 x 0.385):"! = 11.8:1, i.e. the effective proportion of IS-31B - was 11.8/12.8 = 92.5%. The results are plotted in Figs. -3 and 4. -Wii:h the exception of the week ending 25 August, the effective proportion in Gommanhera was always over 86% and from mid- September it was over 93% until after releases were terminated. Similar high effective ratios were seen in Jhiljhil. Thus it is concluded that there were ample males with adequate competitiveness to have induced extremely high levels of sterility in an isolated breeding population. (b) Existence of a genetic variant not susceptible to control by IS-31B If there was a variant in the wild population causing females to be either compatible with IS-31B (Paris cytoplasm) males or unwilling to mate with males of this type, the expected effect of releases of IS-31B would be a rise of percentage incompatibility to a maximum followed by a decline as the type which was susceptible to mating and sterilization was selectively eliminated from the population. A decline in sterility was indeed observed in the later stages of the releases in both Gommanhera and Jhiljhil. However, the mating tests on the female products of fertile rafts sampled from the Gommanhera and Jhiljhil populations ruled out the possibility that there were appreciable numbers of a cytoplasmic type in the wild population compatible with IS-31B (Paris cytoplasm) males. It should be pointed out that a polymorphism of cytoplasmic type has subsequently been discovered in wild populations of C. p. fatigans in India (Subbarao et al. 1974, 1975), but this only affects the reciprocal cross of Indian males to Paris females. This mating was of negligible importance in this experiment, as indicated by the small number of partial hatch rafts which were recorded (Fig. 3 and 4) • 1 Mean probability of each IS-31B male mating compared with mean probability of each wild male mating. WHO/VBC/76.635 page 12 The tests with females from the hatching rafts in Gommanhera and Jhiljhil gave no evidence for the existence of sibling species comparable to those in the Anopheles gambiae complex (Davidson et al. 1967), and the competition tests gave no indication for a variety in the population which tends to mate assortatively and to discriminate against the IS-31B strain. The possibility has not yet been completely excluded that such a situation exists, but only affects mating under wild conditions. However, the high percentage of incompatible rafts achieved in the field mating competitiveness tests (Grover et al. 1976) indicate that if such a variety is present, it is rare in the wild populations from which the female material used for the competitiveness tests was derived. (c) Decline of adult emergence rate Since mosquito females are generally monogamous and store sperms for later use, there are always lags between changes in the composition of mating populations of males and corresponding changes in the composition of the eggs laid. The length of the lag period depends on the rate at which new females emerge, mate and "dilute" the population of previously inseminated females and the rate at which the latter die off. In this experiment the rate of emergence declined sharply in the later stages and it might be suggested that consequently the egg laying population was dominated by old females surviving from the time when the proportion of fully fertile rafts was reduced to 30% in Gommanhera and that thereafter no appreciable decrease in fertile rafts could be expected. The data for Gommanhera shown in Table 2 provide evidence with which a test of this hypothesis can be made and the calculations are shown in Table 10. For simplicity a time unit of one week is used and females are assumed to mature one week after emergence. A loss rate of 15% per day, or 68.2% per week, is assumed (Yasuno & Rajagopalan, 1973). The table starts in the week when maximum incompatibility was reached and the survivors from those alive in that week are assumed to have continued laying rafts 68% of which were incompatible, while females emerging and mating after that week are assumed to have laid 95% incompatible rafts, as would be expected in an isolated population with a 52:1 ratio of IS-31B wild type and with 38.5% competitiveness of IS-31B males. As indicated in Table 10 there was sufficient emergence in Gommanhera in late September and October to have led to a decline in the proportion of fertile rafts to 6% if the population had been isolated. A further calculation based on the extreme assumption of no adult deaths, used only the estimated adult female population in the week of 12 September of 3808 (Table 10) and the total of about 5180 female emergents from that week until the end of October (Table 10). This calculation indicated that a decline to 16% fertile rafts would have been expected by the end of October. A factor, omitted from Table 10, which would have been expected to further "dilute" the fertile females surviving from before mid-September were the "contaminant" females released with the males. With releases of 40 000 per day and a contami- nation rate of about 0.2%, about 80 females would have been released per day and the total number of these released between mid-September and the end of October would have approximately equalled the total adult female population in mid-September. The IS-31B females could only give partially sterile or incompatible rafts and if their survival and fecundity in the wild was normal they would have introduced a further reduction by about one-third in the proportion of fertile rafts that can be explained by the survival of old females. Thus though the declining rate of emergence of young females in Gommanhera must have contributed to the decline in the rate of increase in egg raft sterility it was insufficient to explain the fact that the observed proportion of fertile rafts could not be reduced below 30% and it certainly cannot explain the rise in proportion of fertile rafts in the later part of the release period and after the releases were terminated. By similar calculations it can be shown that there was sufficient emergence in Jhiljhil during September to lead one to expect a rise in incompatibility above the plateau at 40-50% that was observed. (d) Immigration of inseminated females Yasuno et al. (1975) have concluded that immigration of inseminated females was an important factor in a village experiment with chemosterilized male C. p. fatigans. The WHO/VBC/76.635 page 13 inadequacy of the above three hypotheses, as well as the following evidence, led to the conclusion that immigration was important in the later months of this experiment with IS-31B also: 1. c. p. fatigans egg rafts and larvae continued to appear in wells and receptacles in the 3 km wide zones around Gommanhera despite the fact that emergence of C. p. fatigans pupae was virtually prevented there (Table 1). The egg rafts can only have been laid by immigrant females from beyond the breeding free zone and it seems reasonable to conclude some would have reached Gommanhera also. 2. Marked males which had been released in Gommanhera were recaptured in many other villages (Table 9). The greatest distance of recapture was 11 km from the release point, which demonstrates the ability of C. p. fatigans to move even longer distances than previously recorded (Afridi and Majid, 1938; Yasuno et al. 1975). 3. The observed ratio of marked to unmarked males was about the same as the ratio of numbers released to the number emerging in August, but in September and October and in the pooled data for females (Table 11) there was a large discrepancy, suggesting that the mosquitos emerging in the village were not the only source of unmarked individuals in the village in these months. Alternative explanations in terms of reduced survival of released males or loss of detectable marking were not supported by laboratory quality control tests. Also field data reported by Yasuno et al. (1975) indicated very reliable persistence of detectable colour on mosquitos marked by the same method as used in this experiment. Conclusion In conclusion this experiment demonstrated that in the favourable conditions found in Gommanhera in August the IS-31B strain was able rapidly to introduce incompatibility into the rafts laid in the village and it appears that sufficient sterility was induced to cause a population decline in September, contrary to the seasonal trend in the comparison villages. However, because of changing conditions of population movement, continued releases in September and October did not lead to elimination of fertile rafts. Consequently the second part of the concept of using an integrated strain - the phase in which the translocation is required to control the residual population of the released strain - was not put to the test. This experiment was run with the same basic design as an experiment earlier in 1973 with chemosterilized males (Yasuno et al. 1975). It was hoped that the two experiments might provide data on which to judge the relative merits of the two systems of genetic control. However, in both experiments the extent of sterility induced was found to vary with season, apparently because of fluctuations in the extent of isolation of the village populations. Since the two experiments had, for logistical reasons, to be run in different seasons and in villages at different distances from the urban area of Delhi, they do not provide clear-cut comparative data on the efficiency of the two systems. It can only be stated that under favourable environmental conditions both systems are capable of indueing high levels of sterility into wild populations. ACKNOWLEDGEMENTS The authors are grateful to Prof. Dr H. Lavenfor his help and advice not only in developing the IS-31B strain but also in carrying out the field trials. WHO/VBC/76.635 page 14 REFERENCES Afridi, M. K. & Majid, A. (1938) Observations on the range of dispersal of Culex fatigans and its infiltration into Delhi urban area, J. Malar. Inst. India, !• 155-167 Barraud, F. J. (1934) The fauna of British India, including Ceylon and Burma. Diptera Vol. V. Family Culicidae. Tribes Magashinini and Culicini. Taylor and Francis, London Bram, R. (1967) Contributions to the mosquito fauna of southeast Asia-!!. The genus Culex in Thailand (Diptera: Culicidae). Contr. Am. ent. Inst., l• No. 1, 296 Dame, D. A. Woodard, D. B., Ford, H. R. & Weidhaas, D. E. (1964) Field behaviour of sexually sterile Anopheles quadrimaculatus, Mbsq. News, 24, 6-14 Davidson, G., Paterson, H. E., Coluzzi, M., Mason, G. F. & Micks, D. W. (1967) The Anopheles gambiae complex Ch. 6 in The Genetics of Insect Vectors of Disease, ed. J. W. Wright and R. Pal, Elsevier, Amsterdam Graver, K. K., Curtis, C. F., Sharma, V. P., Singh, K. R. P., Dietz, K., Agarwal, H. v., Razdan, R. K. & Vaidyanathan, V. (1976) Competitiveness of chemosterilized males and cytoplasmically incompatible translocated males of Culex pipiens fatigans ~ied. in the field. Bull. ent. Res., 66, 469-480 Krishnamurthy, B. S. & Laven, H. (1974) Development of cytoplasmically incompatible and integrated (translocated incompatible) strains of Culex pipiens fatigans for use in genetic control. J. Genet. 62, 117-129 Krishnamurthy, B. S., Curtis, C. F., Subbarao, S. K., Adak, T. & Chandrahas, R. K. (1975) Studies on the induction of high sterility male linked translocations in Culex p. fatigans, WHO/VBC/75.559 Laven, H. (1967) Eradication of Culex fatigans through cytoplasmic incompatibility, Nature, 216, 383-384 Laven, H. & Aslamkhan, M. (1970) Control of Culex p1p1ens pipiens and Culex p. fatigans with integrated genetical systems, Pak. J. Sci., 22, 303-312 Menon, P. K. B. & Rajagopalan, P. K. (1974) A note on Culex tritaeniorhynchus Giles in Delhi villages, WHo/vBc774.497 Rajagopalan, P. K., Yasuno, M. & Menon, P. K. B. U976) Density effect on survival of immature stages of Culex pipiens fatigans in breeding sites in Delhi villages. Ind. J. Med. Res., 64, 688-708 Reuben, R. (1969) A redescription of Culex vishnui Theo with notes on C. pseudovishnui Colless and C. tritaeniorhynchus Giles, from southern India, Bull. Ent. Res. 58(3), 643-652 Sharma, V. P., Patterson, R. S. & Ford, H. R. (1972) A device for the rapid separation of male and female mosquito pupae, Bull. Wld Hlth Org., 47, 429-432 Sharma, V. P. (1974) A review of the work done on the protection of the experimental areas from infiltration of mosquitos with particular reference to genetic control of Culex pipiens fatigans Wied. in Delhi Union Territory villages, Jour. Com. Dis., ~. 127-135 Singh, D., Patterson, R. S., Yasuno, M. & Jolly, R. (1972) Genetic control of mosquitos. The importance of an educational diagnosis, Intl. J. Hlth. Ed., XV, 269-274 WHO/VBC/76.635 page 15 Singh, D. & Brooks, G. D. (1974) The role of health education in the program of research on genetic control of mosquitos, Jour. Corn. Dis., ~. 142-144 Singh, K. R. P., Brooks, G. D. & Ansari, M. A. (1974) Mass rearing of mosquitos, J. Com. Dis., ~. 121-126 Singh, K. R. P., Patterson, R. S., LaBrecque, G. C. & Razdan, R. K. (1975) Mass rearing of Culex fatigans, Jour. Com. Dis., l(l), 31-53 Singh, K. R. P., Razdan, R. K., Vaidyanathan, V. & Malhotra, P. R. (1975) Caging, marking and transportation of Culex pipiens fatigans Wied. for large scale genetic control operations, Jour. Corn. Dis., l(4) 269-279 Subbarao, S. K., Curtis, C. F., Singh, K. R. P. & Krishnamurthy, B. S. (1974) Variation in cytoplasmic crossing type in populations of Culex fatigans from the Delhi area, Jour. Com. Dis., ~. 80-82 Subbarao, S. K., Krishnamurthy, B. S., Curtis, C. F., Singh, K. R. P., Adak, T. & Chandrahas, R. K. (1975) Further studies on variation of cytoplasmic incompatibility in the Culex pipiens complex WHO/VBC/75.543, Ind. J. Med. Res. (In press) Yasuno, M., Kazmi, S. J., LaBrecque, G. C. & Rajagopalan, P. K. (1973) Seasonal changes in larval habitats and population density of Culex fatigans in Delhi villages, WHO/VBC/73.429, Ind. J. Med. Res. (In press) Yasuno, M. & Rajagopalan, P. K. (1973) Population estimation of Culex fatigans in Delhi. WHO/VBC/73.431, J. Com. Dis. (In press) Yasuno, M., Rajagopalan, P. K., Russel, S. & LaBrecque, G. C. (1973) Dispersal of Culex fatigans in Delhi, villages, Bull. Wld Hlth Org., 48, 317-321 Yasuno, M., Macdonald, W. W., Curtis, C. F., Grover, K. Rajagopalan, P. K., Sharma, L. S., Sharma, V. P., Singh, D., Singh, K. R. P., Agarwal, H. V., Das, S., Kazmi, S. J., Menon, P. K. B., Menon, R., Razdan, R. K. & Vaidyanathan, V. (1975) A release experiment with chemosterilized male Culex i iens fati ans Wied. in a village surrounded by a breeding free zone. WHO/VBC 75.522, Ind. J. Med. Res. (In press) TA BL E 1 ta 1 . !O l.!J &l l. o re e a 1. n 1n c n e o re ea 1. n g -r r e e z o n e s a r o u n a liQ :om an ne ra 26 J un e 2- 8 9- 14 16 -2 1 23 -2 30 ~4 6- 11 13 -1 8 20 -2 5 27 -1 3- 8 10 -1 5 17 -2 2 23 -2 9 1- 7 8- 13 15 -2 1 22 -2 7 29 -3 5- 10 Zo ne 10 J ul y Ju ly Ju ly Ju ly Ju ly A ug . A ug . A ug . A ug . Se pt . Se pt . Se pt . Se pt . Se pt . O ct . O ct . O ct . O ct . N ov . N ov . G om m an he ra N o. o f br ee di ng pl ac es !- 11 4 21 2 45 1 61 5 56 5 72 0 1 10 8 2 18 1 2 67 8 4 65 9 4 87 3 4 56 6 4 38 2 2 71 2 1 67 3 2 03 3 1 83 4 1 31 4 1 19 6 1 26 7 N o. + f or e gg - r a ft s 3 5 8 9 4 20 32 91 11 2 14 1 20 6 49 20 5 45 56 60 52 38 30 27 + f or l ar va e 1 11 2 3 15 6 0 91 18 8 38 8 64 2 56 6 74 3 33 0 15 3 12 1 77 33 13 22 + f or pu pae ~ 0 0 0 0 0 1 0 15 25 40 0 5 7 2 0 0 0 0 0 0 Pi nd w al a K hu rd N o. o f br ee di ng ~ ex am . 1 38 6 2 53 6 2 83 2 3 61 2 2 93 7 6 00 4 5 78 4 5 60 8 3 85 3 5 93 4 5 10 1 4 96 2 4 47 6 3 71 1 3 67 7 3 41 2 3 40 3 3 39 1 3 26 0 3 15 0 + f or e gg -r af ts 40 70 98 10 5 13 5 12 7 13 4 24 1 13 5 18 5 11 9 16 6 17 7 11 3 12 9 15 7 14 8 95 94 70 + f or l ar va e 53 52 59 77 15 71 19 3 16 6 10 5 19 7 79 24 5 73 35 22 31 31 14 10 10 + f or pu pae ~ 0 0 0 0 0 1 25 33 16 24 8 2 2 0 0 0 0 0 0 0 (b ) Id en ti fi ca ti on o f C ul ic in e la rv ae i n Se pt em be r a n d O ct ob er 1 97 3 in t he b re ed in g fr ee z o n e s a ro u n d G om m an he ra a n d Pi nd w al a K hu rd T ot al T ot al n u m be r o f sa m pl es e x a m in ed H ab it at s ha bi ta ts C ul ex f at ig an s C ul ex f at ig an s w it h C ul ex ( Lu tz ia ) C ul ex f at ig an s w it h o th er sa m pl ed (o nl y) fa sc an us C ul ex s pp . W el ls 28 7 0 0 Ce m en t ta n ks 28 3 2 2 Po nd s, d it ch es a n d fi el ds 35 2 9 2 8 D ra in s 37 5 2 2 R ec ep ta cl es 12 2 53 31 11 T ot al 56 7 77 37 23 - - - a N o. o f pu pa e re c o rd ed i s pr im ar il y fr om r e c e pt ac le s s u rv e ys . B re ed in g w as de st ro ye d by e m pt yi ng t he c o n ta in er s. Id en ti fi ca ti on o f c u li ci ne l ar va e in t he b re ed in g fr ee z o n e s, Se pt em be r th ro ug h N ov em be r. b O th er C ul ex s pp . o n ly 21 21 33 3 28 27 43 0 12 -1 7 19 -2 4 26 -2 9 N ov . N ov . N ov . 1 26 1 1 12 9 1 14 5 27 17 14 20 1 4 0 0 0 3 28 1 3 19 6 3 25 1 44 51 49 4 2 0 0 0 0 Pe rc en ta ge h ab it at s c o n ta in in g C. fa ti ga ns br ee di ng 25 .0 25 .0 5. 4 24 .3 77 .8 24 .1 6 - P os it iv e s it es r e c o rd ed f or p up ae w e re m a in ly h ab it at s in a gr ic ul tu ra l fi el ds . Sp ec ie s id en ti fi ca ti on i n su c h ha bi ta ts s ho w ed C . p. fa ti ga ns t o c o n s ti tu te o n ly 5 .4 % o f th e to ta l br ee di ng i n ra in -w at er c o ll ec ti on s. W he re C . p. fa ti ga ns b re ed in g ha bi ta ts w e re lo ca te d th ey w e re de st ro ye d. Th e pr od uc ti on o f pu pa e re fe rs t o C ul ex s pp . th us , e m e rg en ce o f C. p. fa ti ga ns w as c o n s id er ed i ns ig ni fi ca nt . ,, I 'g ~ ()Q 0 ID ~ . . . . . , O '> C" l - :::, . 0'> 0' > w " ' TABLE 2. (a) Gommanhera I Unmarked mosquitos Week ending H. C. M:F index ratio ECOLOGICAL DATA ON THE "TARGET" POPULATIONS (wild) Estimated!? Marked:unmarked male ratio Abs. daily in a emergence village Pop.- WHO/VBC/76.635 page 17 Estimated daily loss rate of released males (%) ~uly 26 1.46 1:2.7 - - - - Aug. 4£ 0.33 1:7.3 220 525 2.8:1 - 11 0.32 1:18 145 550 35:1 - 18 0.43 1:3.3 985 2 180 17-:1 25.12 25 4.1 1:1.9 29 000 1 060 2.2:1 - Sept. 1 0.9 1:2.2 4 570 940 18.7:1 - 8 1.7 1:3.8 10 680 820 30.7:1 16.6 15 0.9 1:5 4 570 830 67.3:1 22 0.8 1:1.2 3 810 280 41.4:1 23.3 29 0.5 1:0.93 1 520 85 60.6:1 67.0 Oct. 6 0.5 1:0.83 1 520 50 47.1:1 25.5 13 0.4 1:0.5 756 70 66.3:1 35.0 20 0.5 1:0.94 1 820 16<>= 63.7:1 - 27 0.23 1:0.75 xx§ 5 161:1 - Nov. 3 0.21 1:0.9 XX 10 172:1 - 10 0.2 1:2 XX 10 225:1 - 17 0.13 1:1 XX 15 93:1 - 24 0.25 1:3 XX 35 17:1 - (b) Jhiljhil ~ug. 11 1.03 1:1.4 - 440 - -25~ 1.97 1:2.3 - 80 - - ~ept. 1 0.2 1:5 3 000 200 7:1 - 8 0.8 1:2 - 225 .10.3:1 - 15 1.03 1:1.2 2 700 250 13:1 - 22 0.9 1:1 - 160 19:1 - 29 0.63 1:1.3 2 450 20 32:1 - pet. 6 0.66 1:1 60 27:1 - 13 0.6 1:1.1 2 400 40 35:1 - 20 0.7 1:8 35 200:1 - 27 0.8 1:3.8 0 112:1 - ~ov. 3 0.93 1:1.8 0 17:1 - 10 0.6 1:4.7 0 58:1 - 17 0.3 1:2 0 40:1 - 24 0.25 1:7 10 64:1 - a The regression equation of H.C. index on absolute population in Gommanherawas y=7.64 x -2.3 (F0 = 62.2, P<o.ool). b Breeding occurred primarily in receptacles until end of week 13 October and in wells thereafter. c Insecticidal treatment in villages. d Popula.tion too low for absolute population estimation. e Most of the pupae produced in one. well. WHO/VBC/7 6. 635 page 18 TABLE 3. Week ending July 25 August ~ 11 18 25 September 1 8 15 22 29 October 6 13 20 27 November 3 10 17 24 ECOLOGICAL DATA FROM THE COMPARISON VILLAGES Pindwala Khurd Paprawat H. C. M:F Absolute Daily H. C. M:F index ratio populatio~ emergence index ratio 1.7 0.3 1:9 1 010 75 5.7 1:3 0.5 1:6.5 1 250 125 - - 1.9 1:6 2 860 500 1.1 1:3 1.5 1:4.6 2 395 150 - - 1.9 1:1.7 2 860 245 3.1 1:4.5 2. 1 1:2 3 085 250 2.8 1:2.4 3.2 1:1.2 4 350 270 4. 7 . 1: 1. 9 5.1 1:2.5 6 505 290 5.2 1:0.8 12.9 1:1.3 15 500 300 6.5 1:1.1 10.0 1:1. 1 12 170 290 2.7 1:0.7 8.5 1:2.3 10 400 760 4.8 1: 1. 1 7.5 1:1.1 9 300 660 4.8 1:0.8 5.8 1:1.3 7 340 414 2.2 1:0.74 5.8 1:2 - - 2.5 1:1.3 4.9 1:1.4 - - 3.4 1: 1. 1 2.6 1:1.3 - - 2.5 1:2 1.1 1:2.4 - - 2.5 1:1.4 ~The regression equation of H.C. index on absolute population in Pindwala Khurd was y = 1.5 x + 0.67 (F0 = 158, P< .0001). £ Insecticidal treatment on 2-3 August in Pindwala Khurd.· TABLE 4. DENSITY (HAND CATCH INDEX) IN FOUR VILLAGES LOCATED ON THE OUTSIDE FRINGE OF THE BREEDING FREE ZONE October 13 October 20 October 27 November 3 Deorala 0.2 1.1 0.6 0.5 Mankroula 0.8 0.8 0 0.5 Sarangpur 1.6 2.5 0.8 0.6 Galibpur 2.3 2.9 0.5 1.2 WHO/VBC/76.63S page 19 TABLE S. RESULTS OF QUALITY CONTROL TESTS ON THE IS-31B STRAIN OF C, P. FATIGANS DURING THE RELEASE PROGRAMME (a) Test crosses of IS-31B and Delhi strain Type of crosses No. of embryonated egg rafts from all tests Month Females Males Total Normal hatch Incompatible Partial hatch May De IS-31B 67 0 67 0 May IS-31B De 103 0 103 0 August De IS-31B S03 20 474 9 August IS-31B De 460 11 436 13 September De IS-31B 168 0 168 0 September IS-31B De 173 0 173 0 September IS-31B (De)~ Rl 109 0 109 0 (De)~ Rl IS-31B 39 0 39 0 Note: De = Laboratory Delhi strain. (De)~ Rl = Strain derived from a single raft isolate of a strain with Delhi cyto- plasm and homozygous for the ruby eye gene. (b) Examination of egg rafts from the IS-31B colonies Month Total No. normal No. incompatible No. unembryonated No. partial hatch rafts hatch (%) rafts (%) (%) rafts ( %) Stock -- ·- ·-. August 17 34S 0(0) 182(l.OS) 146(0.84) 17 017(98.3) September 27 271 6(0.022) 214(0.78) S94(2.18) 26 4S7(97.1) October 7 109 2(0.028) 10(0.14) 64(0.90) 7 033(98.8) Total 51 72S 8(0.01S) 406(0.79) 804( 1. ss) 50 S07(97.8) Mass rearins colonies August 1 S99 8(0.S) 37(2.32) 9(0.S6) 1 S4S(96. 7) September 6 352 6(0.094) 93(1. 46) 87(1.37) 6 166(97.0) October 2 373 14(0.S9) 9(0.38) 12(0.Sl) 2 338(98.4) Total 10 324 28(0.27) 139(1.35) 108 ( 1.04) 10 049(97.2) WHO/VBC/76.635 page 20 TABLE 6. RESULTS OF CAGE TESTS TO DETERMINE THE COMPETITIVENESS OF IS-31B MALES VERSUS DELHI MALES Field cage tests (a) Unmarked IS-31B males (12-36 hours old) versus unmarked Delhi laboratory males (12-36 hours old) at a ratio of 1:1 No. of embryonated Date rafts collected Total Normal Incom- Partial 1973 hatch patible hatch* 8.8 91 39 52 24.8 289 168 114 5.9 166 109 57 17.# 147 97 50 17.9.£ 255 165 90 2. 11.!!: 705 340 365 ~ IS-31B males from mass reared colony. b - IS-31B males from genetic stock colony. 1 7 0 0 0 0 x2 (expectation of lOO% competitive) 1. 6(not sig.) 10. 3(P< 0.001) 15.7 (P < 0.001) 14. 5 ( p < 0. 001) 21. 4 ( p < 0 • 00 1 ) 0.9(not sig.) (b) Unmarked IS-31B males (36-60 hours old) versus unmarked Delhi males (12-36 hours old) at a ratio of 1:1 22.7 46 24 22 0 0.02(not sig.) 29.7 166 68 98 0 5.05(P< 0.05) 5.9 118 57 61 0 0.08(not sig.) 2.11 781 402 379 0 0. 68 (not sig.) 2.11 725 289 436 0 2 • 98 ( p < 0 . 00 1 ) 2.11 831 427 404 0 0.64(not sig.) Laboratory cage tests (c) IS-31B males (36-60 hours old) versus laboratory Delhi males (36-60 hours old) at a ratio of approximately 1:1 Untreated 291 149 142 0 0.6l(not sig.) 336 174 162 0 0.43(not sig.) Marked 321 201 120 0 19. 9(P( 0.001) 297 191 106 0 '34. 3 (P< 0.001) Transported 241 125 116 0 0.16(not sig.) to and from 270 141 129 0 0.25(not sig.) release site Partial hatch rafts were either due to released misclassified females of IS-31B or double matings. (a) Gommanhera Week (dates) No. exam. Pre-release 30 July - 3 August 561 Release Aug, 4-10 294 11-17 754 18-24 1 161 25-31 793 Sept. 1-7 791 8-14 811 15-21 519 22-28 384 29- 191 Oct. 5 6-12 109 13-19 46 20-26 8 27- 4 Nov. 2 3-9 0 10-16 0 17-23 0 24-30 0 Dec. 1-7 0 8-14 0 (b) Jhiljhil Pre-release Aug. 4-10 64 11-17 76 18-24 85 Release Aug. 27- 176 Sept. 2 3-9 291 10-16 351 17-23 241 24-30 169 Oct. 1-7 118 8-14 74 15-21 13 22-28 5 29- G I Nov. 4 5-11 3 12-18 0 19-25 1 26- 2 De e. 2 3-9 0 10-14 0 (c) Pindwala Khurd July 30- 120 Aug. 3 4-10 236 11-17 573 18-24 468 25-31 686 Sept. 1-7 794 8-14 1 149 15-21 848 22-28 797 29- 884 Oct. 5 6-1;2 646 13-19 273 20-26 121 27- 34 Nov. 2 3-9 29 10-16 29 17-23 38 24-30 28 Dec. 1-7 13 8-14 2 TABLE 7. EGG RAFT STERILITY IN THE C, FATIGANS POPULATIONS Origin of egg rafts Receptacles Wells No. No, No, % No, No, No, No, 7. Incomp. PS UE Incomp. exam. Incomp. PS UE Incomp, 0 - - - - - - - - 11 1 2 3.7 - - - - - 99 5 17 13.1 - - - - - 437 10 8 37.6 - - - - - 370 10 6 46.6 - - - - - 446 9 1 56.4 - - - - - 551 11 4 67.9 - - - - - 306 4 0 58.9 237 153 3 0 64,55 241 11 3 62.8 276 172 8 0 62,31 120 6 0 62.8 190 105 8 1 55.26 67 4 0 61.5 94 47 9 0 50,00 29 4 0 63.0 126 54 14 1 42,85 6 1 0 75,0 96 44 8 0 ; 45,83 0 0 0 o.o 125 34 4 0 i 27.20 I 0 0 0 - 101 26 15 0 ! 25,74 0 0 0 - 57 17 4 0 29,82 0 0 0 - 15 5 0 0 i 33,33 0 0 0 - 48 9 0 0 18.75 0 0 0 - 135 24 2 0 I 17.77 0 0 0 - 213 12 7 4 I 5,63 l 1 0 0 1.6 - - - - - 2 0 0 2.6 - - - - - 3 0 1 3.5 - - - - - 11 0 0 6,2 - - - - - 123 2 0 42.2 - - - - - 131 0 0 37.3 - - - - - 115 2 4 47.7 - - - - - 90 0 0 53.2 - - - - - 52 0 0 44.1 - - - - - 36 2 0 48,6 - - - - - 8 0 0 61.5 19 7 0 0 36,8 3 0 0 60.0 20 5 0 0 25,0 0 0 0 - 70 11 1 0 15.7 0 0 0 o.o 8 2 0 0 25.0 0 0 0 - 5 1 0 0 20.0 1 0 0 100.0 5 4 0 0 80.0 0 0 0 o.o 17 2 0 0 11.8 0 0 0 - 37 2 0 1 5.4 0 0 0 - 29 1 0 0 3.4 0 0 0 o.o - - - - - 0 0 0 o.o - - - - - 0 0 0 o.o - - - - - 2 0 1 0.4 - - - - - 0 0 5 o.o - - - - - 5 3 5 0.6 - - - - - J 3 4 0.6 - - - - - 2 0 0 0.2 - - - - - 4 0 0 0.5 - - - - - 2 0 1 0.2 - - - - - 3 0 0 0.5 90 0 0 0 0.1 2 0 0 o. 7 361 3 0 0 0.8 0 0 3 o.o 461 8 0 0 1.7 0 0 1 0,0 310 10 0 2 3.2 2 0 0 6,9 273 15 0 1 5.5 0 0 0 0.0 307 7 3 1 2,3 1 0 0 2,6 225 10 0 1 4.4 1 0 0 3.6 145 9 0 0 6,2 0 0 0 o.o 137 2 0 0 1.4 0 0 0 o.o 190 3 1 3 1.6 No, i exam. i I 561 294 754 1 161 i 793 I 791 i 811 i 7 56 660 381 203 I I 172 104 129 101 57 15 I 48 135 213 64 76 85 176 291 351 241 169 118 74 32 25 70 11 5 6 19 37 29 120 236 573 468 686 794 1 149 848 797 884 736 634 582 344 283 336 263 173 150 192 wHojvsc/76.635 page 21 Total ! No. i or, I Incomp, i Inc;mp. I I 0 o.o I 11 3. 7 99 ' 13,1 437 I 37,6 I 370 46,6 446 56.4 551 67.9 459 60.7 413 62.6 225 I 59,0 114 56. 1 83 48.2 50 48.1 34 ! 26,4 26 25. 7 17 i 29.8 5 33.3 I 9 18,7 24 17.8 12 5,6 1 1.6 2 2.6 3 3.5 11 6.2 123 42,2 131 37,3 115 47.7 90 53,2 52 44,1 36 48,6 15 46.9 8 32.0 11 15,7 2 18,2 1 20.0 5 83.3 2 10.5 2 5.4 1 3.4 0 o.o 0 0,0 0 o.o 2 0.4 0 o.o 5 0.6 7 o. 6 2 0.2 4 0. 5 2 0.2 3 0.4 5 0.8 8 1.4 10 2.9 17 5,6 7 2,1 11 4.2 10 5, 8 2 1.3 3 1.6 IVHO/VBC/76. 635 page 22 TABLE 8. BREEDING STATUS OF RECEPTACLES IN GOMMANHERA AND PINDWALA KHURD FOR C. FATIGANS AND CULEX (LUTZIA) SP. GOMMANHERA of I Pc)Sitive for Week No. c. f. receptacles ending with water No. % Aug. 4 463 23 5.0 11 1 233 135 10.94 18 1 110 212 19.09 25 1 132 149 13.16 Sept. 1 1 369 ! 202 14.76 8 698 164 23.49 15 1 449 229 15.8 22 942 179 19.0 29 603 77 12.76 Oct. 6 714 50 7.0 13 476 27 5.67 C. f. Culex fatigans C.L. Culex (Iutzia) sp. PINDWALA KHURD Positive for of Positive for c. L. No. c. f. receptacles No. % with water No. % 1 o.2 11 0.89 204 16 7.84 8 0.72 192 33 17.18 8 o. 71 193 6 0.31 7 0.51 286 27 9.44 6 0.86 122 26 21.31 2 0.14 237 34 14.34 7 0.74 112 27 2.41 5 0.83 114 21 18.42 28 3.92 89 14 15.73 5 1.05 147 11 7.48 I Positive for C.L. No. % 2 0.98 2 1.04 2 1. 03 2 0.69 0 0 2 0.84 1 0.89 0 0 1 1.12 0 0 TABLE 9. Village Daryapur Deoraula Mankraula Galibpur Pindwala Kalan Karkhari Round Karkhari Jatmal Hasanpur Shikaripur Aslatpur Paprawat Prindwala Khurd Jhiljhil Dhulsiras Galibpur Sarangpur WHO/VBC/76.635 page 23 RECAPTURE OF MARKED MALES RELEASED IN GOMMANHERA Distance from No. of release village Date marked males (km) 2.5 28/9 1 3.8 9/10 4 17/10 2 3.8 9/10 3 17/10 1 4.4 9/10 1 17/10 4 5.4 19/10 1 3.5 19/10 2 5.7 19/10 3 2.9 19/10 9 2.9 19/10 1 2.9 19/10 12 8.2 8/10 1 4.9 16/10 11 4/7 6 2.2 24/8 1 17/9 12 25/9 6 3/10 3 11/10 4 11.2 31/10 1 Recaeture of Jhiljhil released males 2.5 9/10 1 17/10 1 1.6 9/10 3 17/10 3 TA BL E 10 . CA LC UL AT IO N OF T HE E XP EC TE D DE CL IN E IN P RO PO RT IO N OF F ER TI LE R AF TS A SS UM IN G AN I SO LA TE D PO PU LA TI ON A ND U SI NG T HE D AT A FR OM TA BL E 2 ON T HE A BS OL UT E PO PU LA TI ON O F FE M AL ES IN T HE W EE K OF 1 2 SE PT EM BE R, AN D TH E ES TI M AT ED D AI LY E M ER GE NC E IN S UC CE ED IN G W EE KS . A D AI LY L OS S RA TE O F 15 % I S AS SU M ED . No .~ D ai ly W ee kl y No .~ N o. o f m a tu re T ot al N o. ~ P ro po rt io n o f m a tu re fe m al es W ee k s u r v iv in g fr om s u r v iv in g fr om ~ e m e rg ed (fr om b ef or e (m ed ian d at e) th os e a li ve o n e m e r ge nc e e m e r ge nc e p re vi ou s w e e k' s s in ce 1 2 Se pt em be r a n d a ft er Fr om a ft er Fr om b ef or e o f fe m al es o f fe m al es 12 Se pt em be r e m e r ge nc e a n d s ti ll s u r v iv in g 12 S ep te m be r) 12 Se pt em be r 12 S ep te m be r 12 S ep te m be r 3 . 8 41 5 2 90 5 0 0 + 3 8 08 = 3 80 8 O% lO O% 98 0 - - - - - - - . . 93 1 - - t > 93 1 + 0~ 31 - - - {> 93 1 + 1 2 21 = 2 15 2 19 S ep te m be r 1 22 1 14 0 43 % 57 % 1 1 - - - - ¥ 20 S ep te m be r 43 30 1 - - - - = :: 3 14 - - t > 3 14 + 2 98 = 61 0 - - 1 > 6 10 + 3 91 = 1 00 1 61 % 39 % 2 O ct ob er t 5 25 17 5 96 - - - i> 9 6 + 195 ~91 --1 > 29 1 + 1 25 = 41 3 70 % 30 % 10 O ct ob er £ 35 24 5 - - - - - - - . . 56 - - - - - - [> 56 + 9 3 ~49 -- 1 > 14 9 + 4 0 = 18 9 79 % 21 % 17 O ct ob er 80 56 0 - - - - - - - . . 78 - - - - - - [> 78 + 48~ 26 - - 1 > 12 6 + 1 3 = 13 9 90 % 10 % 24 O ct ob er . 4 2 14 - - - - - . . 17 9 - - - - - - [> 17 9 + 40~ 19 - - 1 > 21 9 + 4 = 22 3 98 % 2% - - - - - - - - - · - - - - - - N ot e: -- -- -- -- -- -- -- -- ~~ in di ca te s a pp li ca ti on o f th e a ss u m e d 15 % d ai ly l os s r a te - - - - - - - - - - - - - - - - - {: >. in di ca te s tr a n sf er o f a fi gu re u n c ha ng ed . E xp ec te d % fu ll y fe rt il e e gg r a ft s 32 20 15 13 11 8 6 - - - - - - - - ~ ~ ~~ " '" ' - 1' -C 'l ':: l- a- a- " ' en WHO/VBC/76.635 page 25 TABLE ll. MONTHLY AVERAGES OF OBSERVED RATIOS OF MARKED TO UNMARKED MALES AND FEMALES IN GOMMANHERA AND RATIOS EXPECTED ON THE ASSUMPTION OF AN ISOLATED POPULATION FROM THE NUMBERS RELEASED AND NUMBER EMERGING Sex Month Observed ratio Expected ratio Male August 93:1 104:1 Male September 46:1 118:1 Male October 72:1 1 078:1 Female August-October 0.012:1 0.13:1 WHo/v~>c/76.635 page 26 r- I I I I I \ I I I I ' I z .. a: ::> a.. a: <t 0 '~ ~ c:t :r: ~ 0 <t 0 w a: 0 CJ) 0 FIG. 1 w I a: ::> a..z a:<t ~..J ct<t ..,~ .0 BREEDING FREE ZONES SURROUNDING THE TARGET VILLAGES OF GOMMANHERA AND JHILJHIL AND THE COMPARISON VILLAGE OF PINDWALA KHURD. ~ I a:o / =>a: I / %=> <t ( ., ~I ..J I . ,._:;~ ~ l . "· OD I 1 I I a: I u w I I z <t (!) 1., z 0::: \...... ::::> :.c i <tO.. ~ ..JZ 3:<1: I -I &> / a:~ / ,, "... a: ::::> o..o ~a: a:=> <XI ( 0~ <I E ~ <D If) If) <D 0 11 E u ·'· w ..J <t u CJ) " \ t I / r·....... _) i '· ........... / I ~ /.-·- FIG. 2 POPULATION TRENDS OF CULEX PIPIENS FATIGANS IN THE VILLAGES OF GOMMANHERA, JHILJHIL, PINDWALA KHURD AND PAPRAWAT, AUGUST THROUGH NOVEMBER 1973. RELEASES STARTED ON 4th AUGUST IN GOMMANHERA AND 2nd SEPTEMBER IN JHILJHIL. 25th AUGUST. Q 0 0 tb ADULTICIDE WAS APPLIED IN JHILJHIL ON Q / ,/ / \ \ \ \ \ \ ) / ..J z '"':) ..J / z / ...,..,... / '---- - -- -_-> 10 6 X30N I HOl VO ONVH <t a: w z z <t :lE 0 (!) 0 ~ C\1 t::;:.: 0 oz 0. C\1~ (.) 1()0 (J) C\1 C\1 C\1 ...,: 100.. -w (/) 10 C\1 CD • -(!) ::::> =<t WHO/VBC/76.635 page 27 WHO/VBC/76.635 page 28 FIG. 3 PROPORTIONS OF INCOMPATIBLE AND PARTIALLY STERILE EGG RAFTS AND OBSERVED AND EFFECTIVE PROPORTIONS OF RELEASED IS-31B MALES IN THE CULEX PIPIENS FATIGANS POPULATION OF GOMMANHERA, AUGUST THROUGH NOVEMBER 1973. 95% CONFIDENCE LIMITS ARE ATTACHED TO THE RAFT PROPORTIONS BASED ON THE SAMPLE SIZES AND THE BINOMIAL DISTRIBUTION. THE HISTOGRAM INDICATES THE RELEASE RATE OF IS-31B. z 0 ~ Q.CI) OUJ OC..J 4.<t ..,::E >m ~;f; w!!.! IL ILIL WO ., z 0 i= 0: o., n. Id O..J 0:-t 4.;:! ora . . I . . . . .. : .. ········«> . . . . I I I I Cl) . I 1- . I IL . "' I 1----a: I ~ •• 'j~ '. {/1 _J . / f~ :~ . I \ 0~ --~~ oac . ' ;!;IL I ' ~~ ~ I .> -· I ~ ~ I I I I I ( I --t . I . I . \ . t I ~ •,' I \ I -H- f.! + LL "' GC + w 'I ..J ii: t w t; I ~ / -? "' i\i GC w ID 0 0 q " ~ ILl > 0 0 v L _ ___i__ L. ... ;;:; ..,. C\1 GC w m !:: 0 1- 0 0 Q "' CO N ~ '-···· .. .J ~-o:~'~ w!!! Cl) mLL oo "' 0 2 0 110 ·~·-·-t-· .............. . 0 ., ······· 0 .... .... 0 CO 0 "' :il!lY.lN:ii:>Y:ild 0 .. 0 "' 0 N 0 Ill N N N !!! m ..... ., :::> (!) :::> "' 0 oo 0 0 oo 0 o.o 0 -N -"' __ J __ i.__J.......J 0 0 FIG. 4 PROPORTION OF INCOMPATIBLE RAFTS AND OBSERVED AND EFFECTIVE PROPORTIONS OF RELEAsED IS-31B MALES IN THE CULEX PIPIENS FATIGANS POPULATION OF JHILJHIL, AUGUST THROUGH NOVEMBER 1973. THE HISTOGRAM INDICATES THE RELEASE RATE OF IS-31B. fll .,.a: .J .J ~1&.1 Ill 1&.1 I ::E I&.I!S 1&.1 .J::E I (.) !!o I 1&.1 l-a: 1. ~ 0 :I&. N ::Ein / / Ol- .,...-"' (.)I&. z"' .... ..... -a: .,... ..... j..- a: 41, GDI&.I -ao ' ' ::E ' 1&.1 ' > ' 0 ' z .... ' ;• .,. I I I I I ' 1\i~ Ill ' 0 ' 1- (.) , =o \ oq, z 0 ~ ~ ~en Q.l&.l O.J a: Cl ~ ILZ ~~~~ ~ j:ii; c.> en ~- <I ........ 1&.10 ----~J fll 1&.1 .J Cl z 0 Ill I&J->.., a: en 1&.1 en Ill .... 0 0 0 0 0 Cll ., .... \ en 1- en / .... 1&.1 Cl .J / a: (.) ..-f-1 Cl 1&.11- ' iif:!; ~ -u 1- 1&.1 ' : a: ' ::E 2 ~ 0 (.) 0 ~ a: ~-1 .... 0 ., 0 .. 3f)VlN301:13d * * -- 0 N * .,. .... N oa: Nl&.l Ill • ..,1&.1 -I- Q. 1&.1 en CD 0 .., N 1- Ill .,.; -:::~ Cl 0 0 0 0 0 0 0 c5 c5 N I 0 0 0 c) WHO/VBC/76.635 page 29