Lambrecht, Frank L.

English

Cadernos Espinosanos (E-Journal)

Rev. Inst. Med. trop. São Paulo 9(1):11-17, janeiro-fevereiro, 1961 UDC 599.32 THE INFLUENCE OF CERT AIN ENVIRONMENT AL F ACTORS ON THE DEVELOPMENT OF EGGS AND SURVIV AL OF MIRACIDIA OF ECHINOSTOMA BARBOSA! LIE AND BASCH, 1966 Frank L. LAMBRECHT ( l) SUMMARY The environmental factors of temperature, pH concentration, buffer solutions and pressure were studied to determine their influence on the development and hatching of the eggs of Echinostoma barbosai. Combinations of three tempera-tures (23°C, 28°C and 31 °C) and five pH concentrations (8.5, 7.5, 6.5, 5.5 and 4. 5) showed that maximum development and yield of miracidia occur at 28°C and pH levels of around 7. 5 to 6. 5. However, under certain circumstances in natural conditions, the slower hatching and release of miracidia might have some advantages over a quick emergence in a single burst of all the miracidia at the sarne time. Eggs developed in buffered distilled water were susceptible to the chemical composition of the buffer system used. The survival of miracidia at various pH concentrations from eggs developed in different pH solutions seemed to depend on the pH level where the miracidia were placed after hatching rather than on the level where the embryo developed. Survival in all experiments was best at pH levels of 8. 5 to 7. 5. Pressure, tested up to the equivalent of 34. 6 ft of water, did not seem to affect embryonic development and hatching was normal. · INTRODUCTION The life cycle of Echinostoma barbosai was described by LIE & BASCH 4 from the snail Biomphalaria glabrata SA Y obtained from Recife, Brazil. The cercariae encyst in various snails; the adult worms develop in chicks, ducklings and pigeons. When E. barbosai and Schistosoma mansoni SAM· BON occur together in the sarne snail, rediae of the former interfere with the· development of sporocysts of the latter (LIE 3 ). Howe-ver, interaction between these two parasites is less effective than between rediae of E. audyi and sporocysts of certain trematodes in the snail Lymnaea rubiginosa MrCHELIN (LIE et al. 2 ). The studies mentioned revealed that cer-tain environmental factors, such as tempe-rature, pH level, composition of pH buffers, and pressure, influenced the development of the eggs of E. barbosai and the survival of the miracidia. These influences were the-refore investigated and are here described. This work was supported in part by the University of California International Center for Medical Research and Training (Hooper Foundation, San Francisco School of Medicine) -with Research Grant TW 00144 from the Office of International Research, National Institutes of Health, United States Public Health Service. (1) The G. W. Hooper Foundation, University of California Medical Center, San Francisco, Cali-fornia 94122, U.S.A. 11 LAMBRECHT, F. L. - The influence of certain environmental factors on the development of eggs and survival of miracidia of Echinostoma barbosai LIE and BASCH, 1966. Rev. Inst. Med. trop. São Pai.lo 9:11-17, 1967. MATERIALS AND METHODS Adult E. barbosai worms were collected from experimentally infected 2-week-old chicks that had been fed metacercariae 19 to 24 days previously. Chicks were killed with chloroform, the rectum was dissected out between the anus and the caecum, and its contents were removed under saline solution in a large Petri dish. From there the worms were transferred in clean saline or huffer solution to 2-inch Petri dishes. The quantity of infected material that was fed to the chicks was not standardized, so that infections with adult worms varied con-siderably from one host to another, ranging from O to 60. The number of eggs per worm also varied, usually between 50 and 150. Eggs were teased out of the worm tissues by means of fine needles under the final buffer solution in 2-inch Petri dishes using a stereoscopic microscope with a zoom lens. The buffer solution was replaced severa! ti-mes to remove debris and the Petri dish closed with its top half. Eggs were incuba-ted in the dark in ali experiments. The development of the embryo was exa-mined daily under the compound microscope with low power objective directly from the Petri dish, from which the top half had been removed. Sixteen stages of develop-ment were recognized based on those illus-trated for E. revolutum by JoHNSON 1 • Procedures varied for the study of the influence of different environmental factors and are described separately: l) Temperature and pH Experiments on temperature and pH were run simultaneously. The pH buffer of Mcllvane was used, consisting of appropria-te mixtures of 0.2 N Na2HPO, with 0.1 N citric acid. Five pH mixtures ( 4.5, 5.5, 6.5, 7.5 and 8.5) and three temperatures ( room temperature about 23ºC; incubation temperatures of 28ºC and 31°C) were tested. Stages of development of the eggs, simi-lar to those of J OHNSON 1, were determined daily and plotted against time. The result-ing curves are shown in Figs. 1 to 5. The final percentages of hatching after 28 days 12 are represented diagrammatically based on temperature ( Fig. 6) and on pH concen-trations (Fig. 7). 2) Buf fer solutions The experiments in 1 were ali carried out with the citric acid-dibasic phosphate system. Two other buffer systems were tried, one hased on the mixtures of mono-and dibasic phosphates, the other on Na acetate-acetic acid comhinations. 3) Survival o/ the núracidia. The survival of miracidia from eggs hal-ched in three different pH leveis was tested by their motility; the hatched miracidia were observed after being placed in homo-logous and various heterologous pH leveis. To do this, hatching of large batches of eggs in a given pH concentration was fol-lowed under the stereoscopic microscope. Under favorable circumstances, miracidia hatched in bursts of severa! dozen at a time. The freshly emerged miracidia were picked up by a Pasteur pipette fitted with a rubber bulb and were transferred into a small dish filled with the pH solution to be tested. A dish about 20 mm in diame-ter and 18 mm deep, for use with the Beckman pH meter,· was found most con-venient and was small enough to allow observation of ali the miracidia at one glance through the stereoscopic microscope. Between 10 and 20 miracidia were placed in each dish for observation. Tests were repeated to reduce errors and individual va-nat10n. The end of survival time was esti-mated to have been reached when more than 90% of the miracidia became 1mmo-bile and usually sank to the bottom. Survival was tested at three pH leveis: 8.5, 7.5 and 6.5. Miracidia liberated in each pH concentration were respectively pla-ced in five pH solutions: 8.5, 7.5, 6.5, 5.5 and 4.5. The buffer system used was the citric acid-dibasic phosphate combination. 4) Pressure To evaluate development and survival of eggs under conditions of deep water, expe-riments were set up to observe the effect of pressure on hatching time. Eggs col-lected in the way described here were di-LAMBRECHT, F. L. - The influence of certain environmental factors on the eggs and survival of miracidia of Echinostoma barbosai LIE and BASCH, 1966. trop. São Paulo 9:11-17, 1967. development of Rev. Inst. Med. :, iii e ',; ~ < :,: " < 1 100 90 80 70 60 50 40 30 20 10 pH 8.5 --- oi 23 ºC O 4 12 16 20 24 Fig. 1 DAYS 100 90 80 70 60 50 'º 30 20 10 16 12 28 100 90 80 70 60 ~~ / . .,,,, ---. ·-·-·---,L'✓ ·-------·- - - -I I 50 40 3P 20 I 1 I Í I i / j I 10 j / pH 7.5 --- oi 23 ºC - ot28 ºC -·- ot :;1 ºC H~:~CHING--j -----,t------------------------12 / .,,..,,/ B / li"' ✓-~-----✓ 4 t r 1 1 1 O 12 16 20 24 28 pH 6.5 ---ot 23 ºC - ot 28 ºC -·- ai 31 ºC Fig. 2 ' o~~--'-----'-~--'---1J_2 _ _j__J16'----'-----'20_..J...__2J_,--'--_j,s DAYS Fig. 3 100 ,y 90 80 70 60 50 pH 5.5 --- 01 23 ºC 'º - ot 28 "C 30 -·- oi 31 ''C 20 10 'o~--'-~-'----'-- .L -1J_2 _ _j__lL6 _ _j__2J_o _ _j__2L,-'-__J28 OAYS Fig. 4 100 90 :, 80 iii 70 e 5 60 :, 50 :,: pH 4.5 --- ot 23 ºC -ai 28 ºC -·- oi 31 ºC Fig. 5 Figs. 1-5 - Curves showing development of embryo in egg (lower section) and percentage of hatched eggs (shown above "hatching" line). Embl'.'.yonic stages based on JOHNSON, 1920. 13 C) ~ :i:: u 1-<( :i:: ~ LAMBRECHT, F. L. - The influence of certain environmental factors on the development of eggs and survival of miracidia of Echinostoma barbosai LIE and BASCH, 1966. Rev. Jnst. Med. trop. São Pai<lo 9:11-17, 1967. vided into two batches in separate Petri dishes in pH 7.5 buffered water. Experi-ments were carried out in the incubator at 28ºC with one batch used as a control. The second Petri dish was placed inside a pressure jar, known as an anaerobic bacte-riological incubation jar, closed by means of a metal, screw-top lid and provided with an air outlet and a pressure gauge. Once closed, the pressure inside the jar was raised by connecting the outlet with an air-pressu-re system to obtain the desired pressure; then the valve was closed and the whole jar placed in an incubator. 100 90 80 70 60 50 40 30 20 10 o 23 ºC [J pH 8.5 ■ pH 7.5 lilllJ pH 6.5 0 pH 5.5 ~ pH 4.5 Fig. 6 - Percentage of eggs hatching 28 days after their remova! from the worms for each pH levei, at temperatures of 23°, 28° and 31 °c. 100 90 80 Temperatura EJ23 ºC ■ 28 ºC ~ 31 ºC pH Fig. 7 - Percentage of days after their remova! at pH leveis of 8.5, 7.5, for each of the three 5.5 4.5 eggs hatching 28 from the worms, 6.5, 5.5 and 4.5 tempera tures. 15 14 13 12 "' 11 ► ~ 10 9 8 7 6 5 8.5 Temperature -at 23 ºC --- at 28 ºC -·- at 31 ºC· 7.5 6.5 pH 5.5 Fig. 8 - Number of days for eggs to start hatching at the three temperatures and the ~ > > "' :::,"' õ "' "' :::, o ::e 4.30 4.00 3.30 3.00 2.30 2.00 1.30 1.00 0.30 five pH leveis tested. 0 pH 8.5 ■ pH 7.5 l!llJ pH 6.5 4.5 0.00 8.5 7.5 6.5 5.5 4.5 SURVIVAL at pH Fig. 9 - Time of survival in hours of freshly hatched miracidia at room temperature at combinations of pH leveis of the incubated egg and of the buffer tested. RESULTS 1) T emperature and pH The experiments, which were repeated with very similar results as part of other ex-periments, showed that: a) The highest percentage of hatching eggs occurred at 28°C at pH levels of 7.5, 6.5 and 5.5; the highest yields were 95% at pH 6.5. b) Mo-re constant results for all pH levels occur-red at 23ºC, although the peak at this 14 LAMBRECHT, F. L. - The influence of certain environmental factors on the development of eggs and survival of miracidia of Echinostoma barbosai LIE and BASCH, 1966. Rev. Inst. Med. trop. São Paulo 9:11-17, 1967. temperature was only 751% ( at pH 8.5 and 5.5). c) Results at 31 ºC were definitely Iess satisfactory than at 23 and 28°C. Ave-rages of hatching for all pH levels were calculated for each temperature, with the following results: at 28ºC - 71 % ; at 23ºC - 65'%; at 31 ºC - 40%. d) Optimum hatching under the conditions of these ex-periments occurred at 28ºC at the 6.5 to 7.5 pH levels, when 90 to 95% of eggs hatched. This is clearly shown in Fig. 7. Averages for all temperatures at each pH level were: at pH 8.5 - 611%; at pH 7.5 - 71'%; at pH 6.5 - 73'%; at pH 5.5 -56%; at pH 4.5 - 31'%. The speed of embryonic development, that is, the time lapse between teasing out the eggs and the start of hatching, is shown in Table I. TABLE I pH Jevel Tempera ture Day ha tching in Centigrade starts 8.5 23 11 28 6 31 5 7.5 23 10 28 6 31 5 6.5 23 10 28 6 31 5 5.5 23 11 28 7 31 9 4.5 23 15 28 9 31 9 The more alkaline solutions, i.e. those between pH 6.5 and 8.5, seemed the most favorable, and the temperatures of 28 and · 31 ºC shortened hatching time. As previous-ly mentioned, poorer results percentage-wise were obtained at 31°C. Figure 8 shows de-velopment curves ohtained when time was plotted against pH leveis. The rate of hatching in some experiments was gradual, any time within the 28 days of observation; in others maximum hatching took place shortly after the first eggs started to emerge, usually when hatching conditions were favorable. Examples of "drawn-out" curves were seen at pH 4.5, 5.5 and 8.5 at 23°C and at pH 7.5 at 31 ºC (Figs. 1, 2, 4 and 5) . ln ali these experiments the number of eggs that were in process of hatching was still increasing, although slowly, at the end of the 28 days of obser-vation. This phenomenon has certain sur-vival value for the species: if, under highly favorable conditions, the sudden liberation of large numbers of hatching miracidia has a relatively high potential of infective value through sheer numbers at a given time, this also means that within the life span of this stage ( not more than about 4 hours, as sub-sequent experiments showed) ali suitable snail hosts must be found and invaded to msure survival of any particular batch of eggs. On the other hand, with slow emer-gence, the lower number of available mira-cidia is compensated for by their presence over a longer period of time, i.e., abundance is replaced by Ionger time of exposure. 2) Buf j er solutwns The two buffer systems tried out gave far less satisfactory results than the citric acid-dibasic phosphate system used in pre-vious experiments. ln fact, no develop-ment resulted when the Na acetate - acetic acid- combination was used, and results were very poor for the mono- and dibasic phos-phate mixtures at pH 4.5, 5.5 and 6.5. The mono- and dibasic phosphate buffer gave somewhat better results at pH 7.5 and 8.5, but still yielded fewer miracidia than the citric acid-phosphate combination. Compa-nson between these two buffer systems at 28°C showed: Citric acid - phosphate: 80% hatching after 8 days. Phosphate - phosphate: 63% hatching after 8 days. Citric acid - phosphate : 921% hatching after 11 days. Phosphate - phosphate: 74% hatching after 11 days. Embryos developed normally in tapwater at a pH of 7 .2; 821% hatched after 11 days 15 LAMBRECHT, F. L. - The influence of certain environmental factors on the development of eggs and survival of miracidia of Echinostoma barbosai LIE and BASCH, 1966. Rev. Inst. Med. trop. São Paulo 9:11-17, 1967. at 28°C. Development in distilled water at pH 5. 7 was also normal, 75% of the eggs hatching after 11 days. These results were very similar to those obtained with buffered solutiohs at the sarne pH levels (Figs. 2 and 4). Both ion-concentration and chemi-cal composition seemed important to the de-velopment of the embryos. 3) Survival of miracidia Hatchings from pH 5.5 and 4.5 were also attempted, but they yielded so · few miraci-dia at a given time that the regular tests described were practically impossible. Ho-wever, in one experiment the hatched mira-cidia from a pH 5.5 solution could be follo-wed at pH 8.5, 7.5 and 6.5 levels, and all survived about 30 minutes, which is conside-rably less time than in more alkaline so-lutions. Results, diagrammatically presented m Fig. 9, show greater survival at the higher alkaline levels of 8.5 and 7.5, decreasing rapidly at lower pH levels. It is noteworthy that survival time depended on the pH of the environment in which the miracidia were placed after hatching rather than at time the eggs ( embryo) developed. ln only two cases did the homologous pH systems ( of the eggs and of the miracidia) seem to pro-vide optimum survival conditions, namely those of pH 7.5 and 6.5. ln the lower ran-ges, pH 7.5-hatched miracidia did some-what better than those hatched from the two other pH levels. 4) Pressure The first experiment on pressure was car-ried out at 5 lb/sq in, the next at 15 lb/sq in ( equal to about 11.5 ft and 34.6 ft of water respectively). ln both experiments the time of development and hatching of the eggs were the sarne as for the control and the percentage of hatched eggs after a given time was about the sarne. CONCLUSIONS This series of simple tests indicates that the development of the eggs of Echinostoma barbosai and the· survival of the :µiiracidia 16 are best at pH 6.5 to pH 7.5, at tempera-tures around 28ºC. Moreover, development was not impaired when pressure was applied to simulate a depth of about 35 ft of water. It is assumed that the pH levels and tempe-ratures found favorable in these tests are those representing conditions in the current natural environment of this parasite. While repeated experiments largely confirmed the results noted here, some batches of eggs de-viated in hatching and survival time. The reasons for these discrepancies have not been investigated. It would be interesting to obtain measurements of pH, temperature and che-mical composition from various natural ha-bitats of this parasite in the field and com-pare them with laboratory results. RESUMO Influência de certos fatôres ambientais sô-bre o desenvolvimento de ovos e a sobrevi-vência de miracídios de Echinostoma bar-bosai Lrn ,& BASCH, 1966. Fatôres ambientais como a temperatura, concentração hidrngeniônica e pressão, fo-ram estudados quanto à sua influência sôbre o desenvolvimento e a eclosão de ovos do Echinostoma barbosai. Combinações de três temperaturas (23ºC, 28ºC e 3lºC) e cinco concentrações de pH (8,5 - 7,5 - 6,5 - 5,5 e 4,5) demonstraram que o maior desen-volvimento e a produção máxima de mira-cídios ocorrem a 28ºC e níveis de pH em tôrno de 7,5 e 6,5. No entanto, em certas circunstâncias, a eclosão lenta e uma pro-dução reduzida de miracídios podem em condições naturais, apresentar vantagens em relação a uma emergência múltipla, numa só eclosão sincrônica, de todos os miracídios; Ovos desenvolvidos em água destilada tam-ponada mostraram-se sensíveis à composrçao química do tampão utilizado. A sobrevi-vência de. miracídios em diferentes soluções de pH parecia depender mais do nível de pH em que se colocavam os miracídiós após a eclosão do ôvo, do que da concentração hidrogeniônica em que se desenvolveu o embrião. Em todos os experimentos, a so-brevivência foi maior em níveis de pH de 8,5 a 7,5. Pressão, testada até o equivalen-te de 34,6, não pareceu afetar o desenvolvi-mento embrionário e a eclosão foi normal. LAMBRECHT, F. L. - The influence of certain environmental factors on the development of eggs and survival of miracidia of Echinostoma barbosai LIE and BASCH, 1966. Rev. Inst. Med. trop. Seio Paulo 9:11-17, 1967. ACKNOWLEDGMENT I wish to thank Professor K. J. Lrn for giving me the opportunity to carry out these experiments as a member of the research team working under his direction. REFERENCES 1. JOHNSON, J. C. - The life cycle of Echi-nostoma revoliitiim (Froelich). Univ. Calif. Piibl. Zool. 19:335-388, 1920. 2. LIE, K. J.; BASCH, P. F. & UMATHEVY, T. - Antagonism between two species of tre-matodes in the sarne sn.ail. Natiire (London) 206: 422-423, 1965. 3. LIE, K. J. - Antagonistic interaction bet-ween Schistosoma mansoni sporocysts and echinostome rediae in the snail A iistralorbis glabratus. Nature (London) (In press). 4. LIE, K. J. & BASCH, P. F. - The life his-tory of Echinostoma barbosai sp. n. (Echi-nostomatidae: Trema toda). J. Parasit. (In press). Recebido para publicação em 28/7/1966. 17 

Influência de certos fatores ambientais sobre o desenvolvimento de ovos e a sobrevivência de miracídios de Echinostoma barbosai Lie & Basch, 1966.

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The influence of certain environmental factors on the development of eggs and survival of miracidia of Echinostoma barbosai Lie and Basch, 1966

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