Rivulus marmoratus is the only known hermaphroditic fish species naturally self-fertilizing. Tissue grafts between wild-caught fish and their uniparental laboratory descendants give the autograft reaction, indicating propagation by selfing in the wild also. Only hermaphrodites have been found in the wild locally, although selfing through more than 10 uniparental laboratory generations yielded a few primary male gonochorists, under 5% in contrast to over 95% that were hermaphrodites. Females seem to be non-existent. 2. Two series of experiments were undertaken to identify a possible environmental factor able to cause a deviation to the male phenotype during sex differentiation, on the working hypothesis that low male incidence in clones composed otherwise of hermaphrodites indicated a lability in the sex-determining mechanism through which the genotype normally produces the hermaphrodite phenotype. 3. Individuals of two clones, each in its own jar throughout life, were exposed to the eight combinations of bright or dim light, sea water or fresh water, high or low temperature (Experimental Series One). Exposure was from not later than the ¾ blastoderm stage until sexual maturity at high temperature or five months post-hatching at low. 4. Over seven times the number of males previously encountered were obtained all but one from low-temperature treatments. Male production was correlated with low-temperature rearing despite alternative light intensities and salinities and structural-functional abnormalities (prolapsed oviduct, pharyngeal hyperplasia, kyphosis) peculiar to different dim-light, salinity-temperature combinations, and partly attributable to hormonal derangements. Mortalities were high enough to present the formal dilemma of a differential male induction versus hermaphrodite mortality at low temperature and vice versa at high, but this dilemma was resolved by Experimental Series Two. 5. The Experimental Series One fish were monitored daily up to 1,376 days post-hatching, by which time almost 60% of the hermaphrodites had changed to functional secondary male gonochorists, the rest dying or killed as hermaphrodites, some each year. Primary males remain unchanged except for senile degeneration. Secondary males arise mostly late in laboratory-prolonged life, by involution of the ovarian component of the ovotestes with further evolution of the testicular component, the caudal ocellus fading or vanishing as they become orange like the primary males. 6. In Experimental Series Two, mortalities were low and the structural-functional abnormalities were absent. All individuals were kept at the same intermediate salinity and light intensity: Group A, at moderate temperature throughout to maturity; Group B, at the same temperature through hatching, at low temperature the first five months post-hatching, thereafter at the moderate temperature; Group C, at the moderate temperature up to stages from optic vesicle formation to outset of blood circulation, then at low temperature through eclosion and for five months post-eclosion. Group-C embryos being cut from their chorions to minimize deaths from hatching failure. 7. The Group-A eggs yielded 100% hermaphrodites, the Group-B eggs, 92% hermaphrodites and 8% deaths, the Group-C eggs, 72% males, 18% hermaphrodites, and 10% deaths. Exposure to low temperature from as late as outset of blood circulation produced males. 8. The uniqueness of the present experiments and results, exclusion of alternative explanations, significance of the temperature effect per se, and the implications of these findings for the interpretation of intersexuality in fishes are discussed at length.
