Relative susceptibilities of cyclops species from Rajasthan State to guinea worm (Dracunculus medinensis) larvae*

Bulletin ofthe World Health Organization, 63(5): 881 - 886 (1985) i World Health Organization 1985 Relative susceptibilities of cyclops species from Rajasthan State to guinea worm (Dracunculus medinensis) larvae* SAROJ BAPNA' The role of eight different species of cyclops as intermediate hosts of Dracunculus medinensis has been studied by investigating their relative preferencesfor the ingestion of free-living larvae, the time requiredfor larval penetration into the haemocoel, the number of larvae lethal to the copepods, and the threshold of tolerancefor successful development ofthe larvae within the cyclops. On the basis oflarval development up to the infective stage, four species of cyclops-Paracyclops fi'mbriatus, Thermocyclops oithonoides, Mesocyclops leuckarti sensu lato and Microcyclops varicans -were ranked as suitable hosts. Eucyclops serrulatus was partially refractory, and Tropocyclops prasinus and Ectocyclops rubescens were completely refractory. Guinea worm larvae werepathogenic to Macrocyclops distinctus. In dracunculiasis, the role of cyclops as an inter- mediate host is well established, but various aspects of the interactions between Dracunculus larvae and cyclops are still not clearly understood. In the present investigation some basic aspects of the relationship between the intermediate host and the parasite were examined. Observations on the relative preferences of various cyclops for larval ingestion, the time for successful penetration of larvae through the gut wall into the haemocoel, the number of larvae proving lethal, and the threshold of tolerance (the maximum number of larvae supported by a cyclops before it died) are reported in this article. MATERIALS AND METHODS The collection of cyclops was made from step wells, ponds, and lakes in certain areas (Banswara, Durgapur, and Udaipur) of Rajasthan State where dracunculiasis is endemic. The following eight species of cyclops were identified: Paracyclops fimbriatus, Thermocyclops oithonoides, Mesocyclops leuckarti sensu lato, Microcyclops varicans, Eucyclops serrulatus, Macrocyclops distinctus, Tropocyclops prasinus and Ectocyclops rubescens. Each species was * This article is based on part of a thesis for the Ph.D. degree awarded to the author by the University of Udaipur in 1982. Requests for reprints should be addressed to Dr R.C. Srivastava, Department of Agricultural Zoology and Entomology, Sukhadia University, A.R.S. Durgapura, Jaipur-3 02015, Rajasthan, India. ' Research Associate, Department of Zoonosis, Haffkine Institute, Acharya Donde Marg, Parel, Bombay 400012, India. maintained in separate glass aquaria in filtered pond water at 22 ± 2 °C. Hay infusions were provided as food twice a week (3). Only active and parasite-free adult females of the eight species of cyclops were used in the experiments. A regular supply of adult female guinea worms, Dracunculus medinensis was obtained from a local practitioner, within 2-6 hours of the worm's emergence from a human body. The free-living larvae were obtained by immersing the uterus of the worm in pond water, and a counted number of them were used for making larval suspensions of different densities. To obtain developing larvae from the haemocoel of infested cyclops, the latter were first placed in modified Moorthy's fixative (mercuric perchloride 0.25 g, sodium chloride 1.48 g, and distilled water 100 ml) (4) where they died within 2-5 minutes. The copepods were then carefully dissected to facilitate recovery of the larvae without injuring them. The living larvae were then examined to identify their developmental stage. Ingestion ofD. medinensis larvae by different species of cyclops One adult of each species of cyclops was exposed to 500 D. medinensis larvae in 50 ml of pond water for two hours and then transferred to a Petri dish containing filtered pond water. The total number of larvae recovered from each species of cyclops was counted after 24 hours. Each experiment was repeated 10 times with one species of cyclops. For the purpose of analysis of variance, the data were 4593 -881- S. BAPNA Table 1. Numbers of free-living larvae of D. medinensis ingested by eight species of cyclops collected in Rajasthan State, India Mean number Mean of Species of of larvae transformed cyclops ingested (x) value (a) Paracyclops fimbriatus (13.04) 3.68 Thermocyclops oithonoides (6.52) 2.65 Mesocyclops leuckarti sensu lato (6.26) 2.60 NSb Microcyclops varicans (3.99) 2.12 NS Eucyclops serrulatus (1.93) 1.56_ NS Macrocyclops distinctus (1.09) 1.26 Tropocyclops prasinus (0.87) 1.17 NS Ectocyclops rubescens (0.16) 0.81 Standard error 0.2057 Critical difference at 5% = 0.58 ° Values of y were obtained using the formula y = Tx + T. b The results that are statistically not significantly (NS) different at the 5% probability level are linked by brackets. transformed using Bartlett's formula: y = A =X+ (where y is the transformed value and x denotes the number of larvae ingested by the cyclops ) (14). Time requiredfor the larvae to penetrate the gut wall and reach the haemocoel in different species of cyclops One cyclops of each species was exposed to two D. medinensis larvae in 5 ml of water in separate receptacles; each treatment was repeated 20 times so that 20 observations could be made. As soon as the larvae were ingested, each cyclops was transferred to a Petri dish containing 10 ml of filtered pond water. Each infested cyclops was examined at intervals of 15 minutes to determine the time taken by the larvae to penetrate the gut wall and reach the haemocoel. In the haemocoel, the wriggling movements of the larvae made them easily identifiable by microscopic examination. The time between ingestion of a larva and its presence in the haemocoel of different species of cyclops was noted. Lethal larval dose and the maximum number of larvae supported by different species of cyclops Ten adult females from each species of cyclops were first exposed to 100 D. medinensis larvae in 25 ml of water for 2 hours and then transferred to 100-ml beakers containing 50 ml of filtered pond water. In the control exeperiments the cyclops were not exposed to the larvae in each case. After 15 days, each surviving cyclops was dissected and the total number of larvae and the developmental stage of each larva were noted. Thus, the maximum number of larvae that could successfully develop in a cyclops was determined. The super-infested cyclops, which died during the course of the study, were dissected (1-6 hours after their death) and the number of larvae counted, thus giving an index of the lethal dose of larvae for each species of cyclops. RESULTS Ingestion ofD. medinensis larvae by different species of cyclops The free-living larvae of D. medinensis were swallowed by the cyclops, most species of which became infested within a few minutes. Ingestion of larvae was highest in P.fimbriatus and lowest in E. rubescens. The statistical significance of the numbers of larvae ingested by the different species of cyclops studied is indicated in Table 1. Time taken by the larvae topenetrate the gut wall and reach the haemocoel in different species ofcyclops The time required for penetration of the gut wall varied according to the species of cyclops (Table 2). All the larvae (20 observations in each case) successfully penetrated the gut wall of P.fimbriatus, T. oithonoides, M. leuckarti, and M. varicans and reached the haemocoel. In E. serrulatus and M. distinctus, however, penetration was successful in only 16 and 13 out of 20 observations, respectively. In T.prasinus, 19 observations of the ingested larvae showed failure to penetrate the gut wall within 24 hours; only one larva was seen in the haemocoel after 2 hours. In the 20 observations of E. rubescens, no larva was seen in the haemocoel, indicating complete failure of larval penetration of the gut wall. Lethal larval dose and the maximum number of larvae supported by different species of cyclops The super-infested cyclops were sluggish, showed a tendency to remain at the bottom of the containers, and usually died within a week. On the other hand, cyclops that were not heavily infested were active and swam frequently towards the water surface. Data on the lethal larval dose and the maximum number that could survive and/or develop in 882 SUSCEPTIBILITY OF CYCLOPS TO GUINEA WORM LARVAE Table 2. Times required by D. medinensis larvae to penetrate the gut wall apd reach the haemocoel in different species of cyclops No. of Minimum Maximum Average positive time time time Species of cyclops observations (minutes) (minutes) (minutes) Paracyclops fimbriatus 20 45 90 58.5 Thermocyclops oithonoides 20 60 240 75.0 Mesocyclops leuckarti sensu lato 20 60 120 76.5 Microcyclops varicans 20 75 180 99.0 Eucyclops serrulatus 16 75 300 108.0 Macrocyclops distinctus 13 90 300 132.0 Tropocyclops prasinus 1 120 - - Ectocyclops rubescens - - different species of cyclops are presented in Table 3. The following observations were made: -Two adults of P.fimbriatus died 2 days after infestation, and 3 more died after 4 days, the larval number exceeding 18 in each case. Among the surviving adults, the number of larvae in the haemocoel ranged from 4 to 16. In one case, the cyclops had 16 larvae, 9 of which had completed the second moult and reached the third larval stage, while the rest were in the second stage. -Four of the 10 T. oithonoides (407o) contained 10 to 15 larvae and died within a week; the surviving cyclops contained 3 to 9 larvae. One cyclops, which had ingested 9 larvae, showed the bi- and trifid tail condition in 5 and 2 larvae, respectively, while the remaining 2 had initiated the first moult. - The mortality rate among super-infested M. leuckarti was similar to that in T. oithonoides (407o); the number of larvae present in the dead cyclops ranged from 9 to 15. Among the surviving cyclops, 2 to 7 larvae were recovered from the haemocoel; all these larvae were in the third larval stage in all the cyclops examined. -M. varicans was sensitive to super-infestation because 7 out of the 10 infested cyclops (70%) died during the course of the study; 1-5 larvae were recovered from each dead cyclops. However, in the surviving cyclops a minimum of 1 and a maximum of 4 larvae were found in the haemocoel; all these larvae had completed the second moult. -Six of the 10 super-infested E. serrulatus (6001o) died within 4 days; 5-8 larvae were recovered from the Table 3. Lethal dose and maximum number of D. medinensis larvae supported by different species of cyclops' Maximum number of No. of cyclops No. of larvae larvae supported Species of cyclops surviving lethal to cyclops by a cyclops Paracyclops fimbriatus 5 18 16 Thermocyclops oithonoides 6 10 9 Mesocyclops leuckarti sensu lato 6 9 7 Microcyclops varicans 3 7 4 Eucyclops serrulatus 4 5 4 Macrocyclops distinctus 0 1 Tropocyclops prasinus 8 1 1 b Ectocyclops rubescens 10 ° Ten cyclops were exposed to the larvae in each treatment. b Alive but no sign of moulting could be observed in this larva. 883 S. BAPNA haemocoel of these dead cyclops. In those that survived, the number of larvae ranged from 1 to 4; most of these larvae had reached the second instar but in a few cases both second and third larval stages were observed. -There was 100%o mortality among infested M. distinctus within a period of 5 days; the number of larvae in the dead specimens ranged from 1 to 4. -Two out of the 10 exposed T.prasinus died within 2 days and were found to have 1 and 4 larvae in them. Among the surviving cyclops, only one was found to have a living larva which showed no sign of moulting. -No larvae were present in any of the 10 adults of E. rubescens that were examined for 20 days after exposure. DISCUSSION Although Fryer in 1957 suggested that the larger forms of cyclops tended to be carnivorous and that smaller species like Microcyclops and Eucyclops were herbivorous (2), the present study has shown that both M. varicans and E. serrulatus could easily ingest guinea worm larvae under laboratory conditions. Further, in the case of M. varicans the ingested larvae underwent two successive moults in the body cavity, .whereas in E. serrulatus the development of ingested larvae was not synchronous. These findings in M. varicans and E. serrulatus may reflect a change in the behaviour of different strains of these species in different ecological conditions. The time required for larvae to reach the haemocoel varied in the different species of cyclops. In the case of E. rubescens, the larvae failed to penetrate the gut wall; in T. prasinus, with one exception, most of the ingested larvae failed to penetrate the gut wall. Although the factors responsible for larval penetration are not clear, possible explanations include larval injury due to the feeding habits of the cyclops or the physiological condition of the gut of these species of cyclops (6); in addition, the anatomical structure of the gut wall may offer physical resistance to the larvae. Although in nature, super-infestation of cyclops by guinea worm larvae is probably rare, this phenomenon has frequently been observed under laboratory conditions. Reports of the relative susceptibility of different species of cyclops to guinea worm larval infestation are conflicting. A maximum of 15 larvae in Cyclops vernalis (10) and 20 larvae in C. leuckarti (S) were observed in the haemocoel. In both cases, the cyclops were active. However, Thermocyclops nigerians was very sensitive to super- infestation and did not survive if it harboured more than 4 larvae (8). Similarly, it has been reported that C. vernalis americanus could not withstand 5 larvae (7). From our results, however, P.fimbriatus, T. oithonoides, M. leuckarti and M. varicans may be regarded as fully susceptible to D. medinensis since the numbers of larvae within the threshold of tolerance could successfully undergo development to the third (infective) stage. In E. serrulatus the development of most larvae reached the second instar, with very few larvae developing to the third instar. In T. prasinus and E. rubescens, the ingested larvae did not develop at all, indicating that these species are unsuitable as intermediate hosts. With regard to M. distinctus, the 100070 mortality rate recorded after larval ingestion indicates that guinea worm larvae are highly pathogenic to this species. Among the susceptible species of cyclops, there were differences in both the number of larvae lethal to them and the threshold of tolerance, depending on the species. The reasons for these differences are not known, but the availability of food to the developing larvae in the intermediate host and the complex immune processes governing host/parasite inter- relationships may be involved. In the present investigations, D. medinensis larvae were found to be pathogenic to M. distinctus. One explanation for this is that waste products from the larvae, during their development or moulting, pass into the haemocoel of the intermediate host, M. distinctus, where they cause physiological changes leading to death of the host. This explanation, if correct, may be related to the observations of Moorthy (5) and Muller (7), who found that immature cyclops infected with the larvae of D. medinensis failed to develop to the adult stage. The observation of only a single live larva, without any sign of moulting, in the body cavity of T.prasinus suggests that the factor necessary for the initiation of moulting may be secreted by the host (cyclops) or the latter may provide a stimulus to the developing larvae to secrete a specific moulting factor. This view is supported (by analogy) by the findings of Rogers (9) and Davey (1) on animal parasitic nematodes, but more work is needed to elucidate the mechanism -of these interactions. In India, some 12.2 million people living in over 10 000 villages in 80 districts of seven states have been reported to be vulnerable to dracunculiasis (12), and treatment of infected areas of water with temephos has been recommended for controlling the cyclops. Considering the deleterious effects of both acute and cumulative toxicity of insecticides (13) and legislation on the very selective use of insecticides to avoid pollution of potable water sources (11), we believe that alternative vector control strategies should be developed to reduce the use of toxic chemicals. The present study in Rajasthan has shown that, among the cyclops faunal complex in the Banswara, 884 SUSCEPTIBILITY OF CYCLOPS TO GUINEA WORM LARVAE 885 Dungarpur, and Udaipur districts, only P.fimbriatus, I. oithonoides, M. leuckarti sensu lato and M. varicans can act as true intermediate hosts for guinea worm larvae. It is therefore suggested that selective chemical control operations should be carried out in water bodies with only these vectors of guinea-worm disease, thereby minimizing the chances of pollution. ACKNOWLEDGEMENTS I am grateful to the Indian Council of Medical Research, Government of India, for the award of a Fellowship and am much indebted to Dr R. C. Srivastava, Department of Agricultural Zoology and Entomology, Sukhadia University, A.R.S. Jaipur, for guidance and valuable criticism on the manuscript. I extend my sincere thanks to Dr Boxshell, British Museum, London, for the identification of different species of cyclops. RESUMt SENSIBILITE RELATIVE DES ESPECES DE CYCLOPS DE L'ETAT DU RAJASTHAN AUX LARVES DE LA FILAIRE DE MEDINE (DRACUNCULUS MEDINENSIS) La qualite d'hote intermediaire de Dracunculus medinensis a ete etudi&e chez huit especes de Cyclops en se fondant sur les parametres suivants: preferences relatives pour l'ingestion de larves libres, dur6e necessaire A la penetration des larves dans l'hemocele, nombre de larves letales pour les copepodes et seuil de tolerance pour le developpement complet des larves dans l'organisme du Cyclops. En prenant comme crit&re le developpement larvaire jusqu'au stade infestant, on a pu 6tablir que quatre espoces constituent des h6tes convenables, A savoir Paracyclops fimbriatus, Thermocyclops oithonoides, Mesocyclops leuckarti sensu lato et Microcyclops varicans. Eucyclops serrulatus s'est montre partiellement refractaire tandis que Tropocyclops prasinus et Ectocyclops rubescens l'etaient totalement. Les larves de la filaire etaient patho- genes vis-A-vis de Macrocyclops distinctus. En Inde, environ 12,2 millions de personnes habitant plus de 10 000 villages repartis entre 80 districts de sept Etats seraient vulnerables A la dracunculose, et l'on y a recommande pour detruire les Cyclops le traitement par le temephos des etendues d'eau contaminees. Vu que les insec- ticides ont des effets nocifs, par leur toxicite tant aigue que cumulative, et qu'ils tombent sous le coup d'une legislation tres rigoureuse visant a eviter la pollution des sources d'eau potable, les auteurs considerent qu'il convient de mettre sur pied d'autres strategies de lutte antivectorielle afin de reduire l'utilisation des produits chimiques toxiques. 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