F. PIMENTEL-SOUZA, N.D.C. BARBOSA* and D-F- RESENDE**

Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas,

Universidade Federal de Minas Gerais, 30I60 Belo Horizonte, MG, Brasil

*Estação de Pesqlúsa e Desenvolvimento Ambiental,

CEMIG, 38120 Conceição das Alagoas, .MG, Brasil

**Centro de Pesquisas René Rachou, FIOCUZ, Ministério

da Saúde, 30I90 Belo Horizonte, MG, Brasil

I. The reproduction of lhe snail Biomphalaria glabrata, one of the intermediate hosts of Schistosoma mansoni, was evaluated under Iaboratory conditions by measuring egg production, number ofovipositions, number of eggs per oviposition, and hatching rate using a Latin square design with five different temperature treatments. This permitted the elimination of occasional variation in oviposition due to manipulation or a priori group.

2. Egg production and spawning rates were higher at temperatures between 20.0 and 27.5°C than at 17.5°C. Number of eggs per spawning and hatching rate did not vary with temperature.

3. During a 3-day adaptation phase, a short-term thermal effect appeared in which some indices of reproductive rates differed significantly from those of the experimental phase.

Key words: Schistosoma mansoni vector, Biomphalaria glabrata, oviposition, temperature, adaptation.

lntroduction
Schistosomiasis is an endemic disease that affects tens of millions of people throughout the world. One way to fight the disease is to avoid contamination through intermediate hosts, primarily certain planorbid snails. One of the preventive strategies is to centrol the population of these transmitting mollusks. However, it has been known for some time that the population is expanding rather than contracting (Olivier, 1955). In Brazil it has recently been shown that the endemic zone is advancing southwards, preceded by invasion of these regions by infested snail vectors and infected people (Paraense and Correa, 1987; Teles and Vaz, 1987). A siimilar phenomenon has been occurring in Africa (Appleton, I977a). Mathematical models of population control have been developed in an attempt to discover mechanisms that will prevent the expansion of these snail populations (Coutinho, 1985).

The colonizing potential of these snails depends, among other factors, on their fertility rate. Based on the hypothesis advanced by Brumpt (I94I), evidence has been presented that B. glabrata attains optimum fertility at temperatures between 20 and 25°C (Barbosa et aI., 1987). The same is true for B. alexandrina and B. truncatus (EI-Hassan, 1974). According to Michelson (1961), B. glabrata is most fertile at 25°C, although the World Health Orgattization (OMS, 1957) places the optimum fertility of lhe snail betwecn 18 and 28°C. A great decrease in fertility has been reported to occur in B. glabrata below 20°C (Brumpt, 1941; Michelson, 1961; Paulini and Camey, 1964) and sterility has been reported to occur at 30°C, a temperature where increased growth is also observed (Brumpt, 1941; Michelson, 1961). Appleton (1977b) reported greater reproduction, growth and survival for B. pfeifferi at 25°C, with a marked fall in all of these parameters at higher temperatures. In contrast, Shiff and Garnett (1967) obtained greater reproductive rates at 27°C. Gordon et al. (1934) reported that B. pfefferi became sterile at temperatures higher than 35°C. Gaud and Dupuy (1955) reported maximum oviposition by B. truncatus at 22.7°C, although these investigators did not specify what statistical analysis was used and did not include a control for growth.

The concept of wide variability ú1 oviposition among these snails has been encountered in the literature since the report by Standen (1951) and has been cited by others together with the phenomenon of reproduction distributed throughout the year (Rey, 1956; Paulini and Camey, 1964; Kawazoe, 1975; Barbosa, 1984; Barbosa et al., 1987). It is also known that the length of time required to deplete a snail's egg-laying potential is inversely dependent on rearing density and directly dependent on their growth rate during the experimental period (Ritchie et al., 1966). Because of the wide variability in sexual behavior, Schall (1980) was unable to perform statistical analysis of annual differences in frequency of cross-copulation behavior. In order to be able to study the variation in fertility over an annual cycle and to determine the influence of illumination, Barbosa et al. (1987) applied the Latin square design, thus avoiding the effect of intrinsic variability on snail reproduction.

The objective of the present study was to determine the influence of temperature (17.5-27.5°C) on the fertility of. B. glabrata. The Latin square design was used so as to avoid any influence of variability in egg-laying that might arise from handling and from a priori groups.

Material and Methods
The study was conducted on 50 selected, uninfected, melanic adult (approximately 90 days post-hatching) snails from the same oviposition, with an initial mean diameter of 12 mm. The animals were from a stock that has been reared under laboratory conditions in the Schistosoma Research Unit (GIDE) at the Institute of Biological Sciences (ICB), UFMG for more than 25 years. Snails were fed lettuce which was renewed at the end of each experimental phase, at the time of egg collection.

Twenty-five 200-ml beakers, each containing a pair of snails, were divided into 5 groups. Water filtered through fine sand and charcoal in order to eliminate chlorine and impurities was added up to a level of 10 cm. A mixture of sterilized earth and lime (8:1) was placed on the bottom of each beaker. The beakers were covered with transparent plastic and placed in groups of 5 in five 14-liter aquaria filled with water at thermostat-controlled temperatures designated A to E (17.5, 20.0, 22.5, 25.0 and 27.5°C, respectively). In order to control water temperature in the aquaria to within 0.5°C only by heating, the first aquarium was placed in a cold room at 15°C and the second in an air-conditioned room at 19°C. The remaining aquaria were left in the laboratory at room temperature (about 20°C). In all cases, the variations in room temperature were always below the reference value. Data collection was started 17 days before midwinter in 1985 and was continued for 18 days afterwards.

Treatment sequences were picked at random for each snail group according to the principle of the Latin square design (Barbosa et al., 1987), as follows:

The snails were allowed to adapt at each temperature for 3 days. Eggs were collected at 9:00 A-M- on the third day. Each experimental phase extended from the third to the seventh day, when eggs were again collected at about 9:00 A-M- During this period, temperature was measured 6 times a day at about 2-h intervals, starting at 8:00 A.M.. During the adaptation phase, temperature was measured 6 times on the first day and once on the morning of the third.

Environmental lighting was adjusted so as to provide approximately 13 lux to the beakers, as measured with a photometer of our own making (Schall, 1980; Barbosa, 1984; Barbosa et al., 1987). Eggs were collected at the end of each stage in the experimental sequence, placed in water in plastic cups under constant illumination with approximately 22 lux and at room temperature, and observed daily for approximately 30 days (or until hatching) with the aid of a magnifying glass (Zeiss, Jena). Snails appear to develop only from viable eggs (Barbosa et al., 1987).

Statistical analysis
Data were analyzed statistically according to the procedures recommended for the Latin square design, with five replicates (Snedecor and Cochran, 1980), not balanced and incomplete, which is a kind of analysis of variance, using the Statistical Analysis System (SAS, 1985). Data were processed with a modal 4341 IBM computer, at the Scientific Computation Laboratory, UFMG. The Duncan test was used for multiple comparisons of the means and identification of the differences. The level of significance was set at 5%. In the design, temperature represents the treatment and the groups and sequences of treatment application represent the different sources of variation controlled in the experiment. The measurements made for each beaker were considered to be replicates (Armitage, 1977; Montgomery, 1984).

When a snail died, its companion was also removed. Thus, 3 pairs were removed from group 3 and 3 from group 4. A preliminary analysis of the data indicated group 3 to be the outlier since, starting from the second experimental period, the mean number of viable eggs laid each day by each snail was less than 1.0 (and sometimes zero) for the 4 surviving snails. For this reason, this group was excluded from analysis of the results. The egg-laying values concerning the removal of snails from group 4 during the experiment were also considered missing.

Except for total number of eggs and number of viable eggs per spawning, the data were submitted to square root transformation before analysis in order to normalize the distribution of the variances. The square root was selected because count data usually follow a Poisson distribution (Kendell et al., 1983).

Results
Experimental phase
The number of viable eggs per snail per day was affected by temperature and period but not by the difference among individuals (groups or beakers). A significantly lower number of viable eggs per snail per day was observed only at 17.5°C (P<0.05, Duncan test; Table 1). Between 20 and 27.5°C, there was a steady but statistically nonsignificant increase (Table 1). ln terms of sequence of exposure to different temperatures, the number of viable eggs per snail per day reached a maximum (21.4) during the second period (data not shown).

Table 1 - Variations in parameters of reproduction as a function of temperature in B. glabrata.Groups of 10 snaiIs (5 pairs in 200 ml each) were exposed to a given temperature for 3 days (adaptation phase) at 13 lux. Eggs were collected and then oviposition parameters were measured again alter 4 days at the same temperature (experimental phase). Adaptation and experimental phases at each temperature were repeated in a random sequence, until each group had been exposed to all 5 temperatures. Hatching rates are based on a 30-day observation of each cluster of eggs maintained at 22 lux and at room temperature. Viability is based on the development of a young snail inside the egg. Data am reported as means per snail per day. Means of each group not sharing a common superscript an different (P < 0.05, Duncan test).

This value was significantly different from those obtained during aIl other periods (P<0.05, Duncan test), and the minimum values were obtained during the first and last periods (10.5 and 10.6, respectively). These data show that this 35-day experiment was conducted during a period of high and relatively stable reproductive performance (Ritchie et aI., 1966). The lack of differences among individuals (groups and beakers) revealed uniform variability in egg-laying among snails submitted to the same conditions (excluding, of course, group 3).

Number of spawnings and total number of eggs per snail per day were similarly affected by sequence (data not shown) and by temperature, with the lowest values occurring at 17.5°C and the highest at 22.5°C (P<0.05, Duncan test; Table 1).

Total number of eggs per spawning and number of viable eggs per spawning per snail per day did not vary significantly with sequence or with temperature in the range studied (P<0.05, Duncan test). The data for viable eggs suggest a small, statistically nonsignificant decrease in this parameter with sequence of exposure to different temperatures, with a variation from 4.0 in the first period to 2.8 in the fourth (data not shown). The intrinsic variability was uniform and did not depend on temperature. These data suggest that these parameters may be controlled by morphological characteristics of the snail which are related to shell size (Gerken, 1977).

There was no significant difference in hatching note in relation to temperature, period or individual. However, anaIysis of the residues obtained in the analysis of variance to estimate the adjustment of the theoretical statistical model suggested the existence of different populations with 0, 1, 2 and 3 hatchings per spawning, corresponding to very small percentages of viable eggs in the total number of eggs.

Adaptation phase
During adaptation the number of viable eggs per snail per day differed from that observed in the previous phase by showing aIso group and beaker effects (P<o.05, Duncan test). Hatching, total number of eggs per snail per day and spawning per snail per day exhibited a group effect when compared with the experimental phase (data not shown). The total number of eggs per spawning per snail per day varied with sequence of exposure to different temperatures, and beaker effects in relation to the experimental phase (P<0.05, Duncan test; data not shown), and the number of viable eggs per spawning per snail per day showed temperature (Table 1) and group effects (data not shown) in relation to the experimental phase (P<0.05, Duncan test). The difference among groups indicated the extrinsic effect of laboratory manipulation, probably due to the different sequences of exposure to thermal treatment. This demonstrated that the adaptation phase was properly selected, since the effect of extrinsic variability due to the short-terra thermal effect was stabilized during the phase. Indeed, the adaptation phase also served to stabilize the effects of the other parameters. It can be seen that total number of eggs per spawning was affectedby a short-terra thermal effect, probably acting either on the ootestis or on the canals that transport the eggs.

Discussion
The present study demonstrated that the highest fertility of the snail B. glabrata occurred at temperatures between 20 and 27.5°C, a significant decrease occurring only at 17.5°C. Most of the investigators cited in the Introduction assumed that maximum fertility occurs around 22.5 or 25.0°C. These values seem to be those found as mean values when fertility is observed in the field and in the laboratory without temperature control, with temperature ranging from a maximum of 30°C to a minimum of 17.5°C, when acute decreases in fertility may occur.

No statistically significant differences in fertility were detected among experimental groups or among beakers, demonstrating the absence of intrinsic variability in fertility among these snails of the GIDE strain. It would be interesting to confirm this result using other natural or laboratory strains. However, the demonstration of a difference in fertility among groups and/or beakers during the adaptation phase indicates that, on a short-term basis, oviposition may be highly sensitive to small variations in handling, possibly revealing stress in adapting to different thermal treatments. Thus, the observation reported by Standen (1951) of wide variability in oviposition appears to be meaningful only in field or uncontrolled laboratory situations. The observation may reflect the lack of standardized maintenance techniques used for snails at the time.

The highest frequencies of sighting for B. glabrata in the field occurred between 27 and 32°C (Chernin, 1967), and were probably linked to the optimum temperature for snailgrowth, which is 30°C. If so, then capacity for reproduction may have been accelerated because of snail size (Ritchie et al., 1966) and not simply by the temperature effect, since castration was found to occur at 30°C. Today it is understood that the hypothesis of Hyman (1967) that 25°C may be the best temperature for permanent maintenance of B. glabrata probably represents a compromise between the best growth and good fertility. Similarly, the reproductive potential of the snail may be of a long-term type, with low egg-laying density, or of a short-term type, with high egg-laying density (Ritchie et al., 1966). The highest egg-laying densities for other schistosomiasis vectors were also observed at 25 to 33°C, i.e. for B. pfeifferi in the field (Gordon et al., 1934) and in the laboratory (Shiff and Garrett, 1967), and at 25°C for B. alexandrina and B. truncatus (El-Hassan, 1974).

The studies of the effect of temperature cited in the previous paragraph can be criticized for lack of a growth central, an omission which leads to an erroneous conclusion for temperatures close to 30°C. At this temperature, faster growth may be expected, with a consequent acceleration in the capacity for reproduction within a short period of time. In the present study this limitation was avoided by using the Latin square design to eliminate a priori group and experimental sequence effects at aIl temperatures. The effect we attribute to temperature is based on a random distribution of groups and sequences. In this experiment, all snails starred at the same size but final snail size was not measured. However, in previous studies in which we applied the Latin square design (Barbosa, 1984; Barbosa et al., 1987), shell diameters increased from 12.1 to 14.0 mm over 35 days, with no difference among studies acne at the beginning and at the end of different seasons of the year.

Table 2 summarizes data reported in the literature for B. glabrata oviposition under laboratory conditions comparable to ours. The experiments of Magalhães and Carvalho (1969) and Kawazoe (1975) were conducted on pairs of snails that were only slightly smaller than ours. If we allow for the small differences in size, the data obtained by these investigators tend to be similar to ours. In the next two studies, groups of several snails were

Table 2 - Temperature effects on average reproductive output of B. glabrata reported in laboratory studies performed under similar conditions.
*Estimated from reported data.

exposed simultaneously to different temperatures (one temperature per group. Slightly higher fertility was obtained by Paulini and Camey (1964), probably because they used slightly larger snails at a lower density than ours, and much lower fertility was obtained by Luttermoser (1943), who worked with temperatures close to that which produces sterility. The fertility rates obtained by Ritchie et al. (1966) with isolated snails were much higher, but consistent with their use of larger snails. However, the values obtained by Gerken (1977) with groups of 200 snails, all of the same size, were IA and 2.5 times ours. This difference may be explained by their use of larger snails and more light, which was estimated to be more than 100 lux (vs 13 lux in the present study). Indeed, in a previous study we found that a variation from 0.02 to 100 lux caused a three- fold increase in egg production (Barbosa et al.,1987).

B. glabrata is known to lay more eggs at night. Egg hatching per snail per day seems to be synchronized with solar rhythm, since hatching rate was highest under a12-h light:12-h dark lighting schedule, though spawning was even more favored by 100 lux lighting, with respective values of 0.75 and 0.85 (notsignificant difference; Barbosa et al., 1987). Inversion of the natural light cycle did not change the hatching rate, but when lighting was changed from 4-h light:4-h dark to 0.02 lux darkness, hatching rate per snail per day fell significantly from 0.53 to 0.22 (data from Barbosa et al., 1987; see also Cole, 1925; Paulini and Camey, 1964). This observation may explain the very low values obtained in this experiment, with a mean hatching value of 0.14 per snail per day, since the eggs were stored without solar rhythm and under constant low lighting of 22 lux after collection. Under constant lighting there was also a decrease in cross-copulation compared with the frequency observed for the natural lightittg cycle (Pimentel-Souza et al., 1988). If this leads to greater self-fecundation, it could have long-term negative repercussions on the genetic patrimory. The influence of lighting may be of interest in terms of public health by offering new, non-polluting alternatives in the fight against snail reproduction, such as shadowing brook banks and lake shores with trees or plastic sheets, or covering water streams in metropolitan regions.

Acknowledgments
The authors are grateful to I.B.M. Sampaio for useful comments, to GIDE, UFMG, for supplying the snails, to E.D. Bontempo for technicaI assistance, and to A.J-F. Ribeiro, a CNPq fellow, for participating in data analysis.

References

• Appleton CC (I977a). The influence of temperature on the Iife-cycle and distribution of B. pfeifferi in South-Eastern Africa. International Journal of Parasitology, 7: 335- 345.
• Appleton CC (1977b). The influence of above-optimal constant temperature on South African B. pfeifferi. Transactions of the Royal Society of Tropical Medicine and Hygiene, 71: 140-143.
• Armitage P (1977). Statistical Methods in Medical Research. Blackell`s, Oxford, 1-504,
• Barbosa NDC (1984). Ritmos circadiano e anual da atividade locomotora e da postura de ovos do caramujo B. glabrata, sob diferentes condições de iluminação. Master's thesis. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte.
• Barbosa NDC, Pimentel-Souza F & Sampaio IBM (1987). The effect of seasonal temperature and experimental illumination on reproductive rate in the snail B. glabrata. Brazilian Journal of Medical and Biological Research, 201 685- 696.
• Brumpt E (1941). Observations biologiques diverses concernant Planorbis glabratus, hôte intermédiaire de S. mansoni. Annales de Parasitologie, 18: 9-45.
• Chernin E (1967). Behavior of B. glabrata and of other snails in a the thermal gradient. Journal of Parasitology, 53: 1233-1240.
• Cole WH (1925). Egg-laying in two species of PLanorbis. American Naturalist, 59: 284-286.
• Coutinho FAR (1985). Sobre a regulação de populações de parasitas. Ciência e Cultura, 37: 57-60.
• EI-Hassan AA (I947). Laboratory studies on the direct effect of temperature on B. truncatus and B. alexandrina, the snail intermediate hosts of Schistosomes in Egypt. Folia Parasitologica, 21: 181-187.
• Gaud JR & Dupuy R (1955). Rythmes de dévelopment de B. truncatus en élevage au laboratoire. Annales de Parasitologie, 30: 62-68.
• Gerken SE (1977). Efeitos da alimentação e da densidade populacional sobre o crescimento, a sobrevivência e a fecundidade de B. glabrata, Master’s thesis. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte.
• Gordon RM, Davey TH & Peaston H (1934). The transmission of humor bilharziases in Sierra Leone, with an account of the Iife-cycle of the Schistosomes concerned, S. mansoni and S. haematobium. Annals of Tropical Medicine and Parasitology, 28: 323-418.
• Hyman LH (1967), The Invertebrates. Vol. VI, Mollusca I. McGraw-Hill, New York.
• Kawazoe U (1975). Alguns aspectos de biologia de B. glabrata e B. tenagophila. Master’s thesis. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte.
• Kendell MS, Stuart A & Ord JR (1983). The Advanced Theory of Statistics. Charles Griffin, London.
• Luttermoser GW (1943), A note on the Iife cycle of A. glabratus, a snail intermediate host of. S. mansoni. Journal of Parasitology, 29:231-232.
• Magalhães LA & Carvalho JF (1969). Estudo da postura e de duas populações de planorbídeos. Revista da Sociedade Brasileira de Medicina Tropical, 3:245-247.
• Michelson EM (1961). The effects of temperature on growth and reproduction of B. glabrata in the Iaboratory. American Journal of Hygiene, 73:66-74.
• Montgomery DC (1984). Design and Analysis of Experiments. John Wiley, New York.
• Olivier L (1955). The naturaI history and control of the snails that transmit the Schistosomes of man. American Journal of Tropical Medicine and Hygiene, 4:415-423.
• Organización Mundial de la Salud (OMS) (1957). Grupo de estudio sobre ecología de los moluscos huéspedes intermediários de la bilharzíasis. Serie de Informes Técnicos, Geneva.
• Paraense WL & Correa LR (1987). Probable extension of Schistosomiasis mansoni to southernmost Brazil. Memórias do Instituto Oswaldo Cruz, 82: 577.
• Paulini E & Camey T (1964). Observações sobre a biologia da A. glabrata. III. Influência da temperatura do ambiente sobre a frequência da postura. Revista Brasileira de Malariologia e Doenças Tropicais, 10: 499-504.
• Pimentel-Souza F, Schall VT, Barbosa NDC, Schettino M & Lautner Jr R (1988). Influence of experimental illumination and seasonal variation on crossbreeding mating in the snail B. glabrata. Memórias do Instituto Oswaldo Cruz, 839:79-85.
• Rey L (1956). Contribuição para o comportamento da morfologia, biologia e ecologia dos planorbídeos brasileiros transmissores da esquistossomose. Serviço Nacional de Educação Sanitária. Rio de Janeiro.
• Ritchie L, Hernandes A & Rosa-Amador R (1966). Biologicals potentials of A. glabrata. Life span and reproduction. American Journal of Tropical Medicine and Hygiene, 15: 614-617.
• Schall VT (1980). Comportamento do caramujo B. glabrata em um gradiente luminoso. Master’s thesis. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte.
• Shiff CJ & Garnett B (1967). The influence of temperature on the intrinsic rate of natural increase of the freshwater snail B. pfeifferi Archiv. fur Hydrobiologie, 62:429-438.
• Snedecor GW & Cochran WG (1980). Statistical Methods. Iowa State University Press, Ames.
• Standen OD (1951). Some observations upon the maintenance of A. glabrata in the laboratory. Annals of Tropical Medicine and Parasitology, 45: 80-83.
• Statistical Analysis System (SAS) (1985). Stat Guide for Personal computers. 6th edn. S.A.S. Institute, Cary, NC.
• Teles HMS & Vaz VF (1987). Notas sobre a distribuição de B. glabrata no Estado de São Paulo, Brasil. Revista de Saúde Pública, São Paulo, 21:508-512.