Possible Hybridization of Brazilian Planorbid Snails and its Importance in Population Dynamics

This study focuses on the possibility of experimental hybridization among host snail species for Schistosoma mansoni in Brazil, with morphological characterization of the hybrids found. By using albinism as a genetic marker, intraspecific crossbreedings were performed between two strains of each species involved, in addition to interspecific crossbreedings; the only viable crossbreeding was between pigmented Biomphalaria glabrata (Paulista, PE) and albino B. tenagophila (Joinville, SC), with the formation of F1 and F2 generations. All offspring in F1 displayed black eyes and a renal ridge on the mantle, while F2 displayed dissociated morphological traits. With regard to reproduction, F1 was more efficient than F2. The experiment’s results suggest post-zygotic reproductive isolation.

Interspecific crossbreedings between geographically isolated species were described by Barbosa: B. alexandrina (Cairo, Egypt) and B. glabrata (Recife, Pernambuco, Brazil) in 1956;B. straminea (Pernambuco, Brazil) and B. peregrina (Ecuador); B. tenagophila (São Paulo, Brazil) and B. peregrina (Ecuador) (Barbosa et al. 1958). Formation of fertile hybrid offspring was observed in these crossbreedings. The same author in 1964 commented on the natural hybridization of B. glabrata and B. tenagophila, reporting intermediate forms, explained by the proximity of the respective populations. Later on, Barbosa (1973) observed this process of natural hybridization in the Northeast of Brazil, between B. glabrata and B. straminea. Breeding sites previously colonized exclusively by B. glabrata were replaced by B. straminea over the course of three years. During the process of competitive exclusion, mixed forms were found, possibly resulting from interspecific crossbreeding. Such intermediate forms were no longer found during later field studies, showing their inability to persist in nature.
This study's objectives were to observe the possibility of experimental hybridization between host species for Schistosoma mansoni in Brazil and to study the reproductive capacity and subsequent morphological characterization of the hybrid forms. schistosomiasis mansoni in Brazil (Fig. 1). In crossbreeding experiments, according to Newton (1954), albino individuals produce pigmented offspring when submitted to crossbreeding with pigmented populations. The first phase of the experiment involved rearing ten specimens from each strain in isolation for approximately one year. This procedure allowed the strains to maintain homozygosis. Approximately 15 days after eclosion, the offspring of these snails were used in the crossbreeding experiments. They were reared by pairs, according to Fig. 1, in cylindrical recipients measuring 7.5 by 8 cm with a capacity of 250 ml of water, for a period of 30 days. At the end of this period, the albino specimens were separated from the pigmented ones and their offspring were observed for 24 weeks. The following parameters were observed: reproductive parameters-fecundity (number of egg-masses; number of eggs/egg masses; number of eggs/snail) and fertility parameters (production of fertile and sterile eggs; eclosion rate) and phenotypic parameters-pigmentation of eyes, pigmentation of mantle, and internal morphology, observing differential traits.

Intra
The number of egg-masses in albino specimens was observed daily, as were other aspects pertaining to fecundity. Egg-masses were separated weekly and observed as to the production of fertile and sterile eggs. Records were also kept during this same period on the number of snails with black and white eyes in each egg-masses. The number of eclosed snails was recorded every six days.
Part of F1, coming from interspecific crossbreedings, was reared in isolation (10 pairs) and grouped (30 specimens) at approximately 15 days of age under the same conditions as in the previous experiment, observing the same parameters in this generation. Another part was submitted to anatomical dissection in order to establish the presence or absence of the renal ridge, using histological cross sections.

RESULTS
Viable hybrid offspring were only obtained with the interspecific crossbreeding of pigmented B. glabrata (Paulista, PE) and albino B. tenagophila (Joinville, SC), with the formation of F1 and F2. Crossbreeding of albino B. straminea (R3) and pigmented B. tenagophila (Vila Velha, ES) was the only one to maintain reproductive isolation. The other crossbreedings produced hybrid offspring, but the specimens died before reaching sexual maturity.
All offspring of the crossbreeding between pigmented B. glabrata (Paulista, PE) and albino B. tenagophila (Joinville, SC) had black eyes, pigmented mantles of the spotted "Wildtype" described by Richards (1967), and a renal ridge on the mantle (Figs 2, 3), with the latter confirmed by histological cross section (Fig. 4).
Hybrid offspring from generation F2 reared separately had 67.7% of snails with black eyes and 32.2 % with red eyes, or a ratio of 2:1. Snails reared together showed 78.9% with black eyes and 21% with red eyes, or a ratio of some 3:1. Comparing the number of snails with red as opposed to black eyes reared separately or grouped showed a significant difference (X 2 = 11.31; p > 0.05), with a predominance of black-eyed snails reared in groups. Table I shows fecundity results and Table II fertility results for ten specimens of albino B. tenagophila (Joinville, SC) submitted to intraspecific crossbreeding with pigmented B. tenagophila (Vila Velha, ES) and interspecific crossbreeding with pigmented B. glabrata (Paulista, PE). Comparing reproductive parameters for albino B. tenagophila submitted to intra-and interspecific crossbreeding, no significant difference was observed with regard to fecundity: number of eggmasses (t = 0.11, p = 0.91, g.l.= 13); number of eggs/egg-masses (t = -1.72, p = 0.13, g.l. = 7) ; number of eggs/snail (t = -1.51, p = 0.17, g.l. = 8) and fertility: number of fertile eggs (t = -1.69, p = 0.13, g.l.=7); number of sterile eggs (t = 0.57, p = 0.59, g.l. = 5), and number of eclosed snails (t = -1.66, p = 0.13, g.l. = 8). Tables III and IV show the fecundity and fertility results for generation F2 reared in isolation and grouped. A proportional equivalence was observed for values obtained for the number of eggs/ egg-masses at week 7 for the two situations (Table  III). In terms of fertility, both in isolation and grouped, production of fertile eggs and eclosion of snails were achieved.
Another trait observed in the offspring of the albino specimen was the pigmented mantle. All offspring of both intraspecific and interspecific crossbreedings were individuals characterized as the spotted "Wildtype" (Richards 1967). This phenotype was similar to that found by Richards (1969), where crossbreedings of "albino" individuals with those of the diffuse "Wildtype" led to the formation of "Wildtype" individuals with the dominant spotted pattern.
As for the histology of the mantle, hybrid offspring from interspecific crossbreeding between albino B. tenagophila (Joinville, SC) and pigmented B. glabrata (Paulista, PE) had the renal ridge, a trait of B. glabrata. This trait had been observed previously in nature by Barbosa (1957), who found specimens of B. tenagophila (São Gonçalo, RJ) with the renal ridge and/or a pigmented line on the mantle. The author suggested that the breeding site was a transition zone with the presence of intermediate forms resulting from a possible crossbreeding with B. glabrata, although this species was not found nearby. In addition, previous research suggests that certain species are natural hybrids that became efficient. Wium-Andersen (1973) suggests that B. alexandrina is a hybrid of B. pfeiferi and B. sudanica, while Simões et al. (1985) suggests that B. intermedia is a hybrid of B. straminea and B. peregrina.
In the current study, both the phenotypic and genotypic traits of hybrids may be diluted through gene exchanges. The fecundity and fertility results in the fertile hybrid offspring show that generation F2 was not as efficient as generation F1. Such results can be explained by the allopatric speciation theory, involving the formation of a normal, vigorous, and fertile hybrid F1 generation, but a weak F2 generation with the presence of sterile individuals (Krebs 1985).
This study showed that the interspecific crossbreeding of albino B. tenagophila (Joinville, SC) and pigmented B. glabrata (Paulista, PE) was feasible, with the formation of fertile hybrid offspring. The phenotypes displayed by generation F1 and F2 indicated the existence of simple Mendelian inheritance for these traits in the planorbid populations. With regard to reproductive aspects, the fecundity and fertility of the generations decreased, tending to form sterile individuals, thus suggesting post-zygotic reproductive isolation.