Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish

Background

How and why animals lose eyesight during adaptation to the dark and food-limited cave environment has puzzled biologists since the time of Darwin. More recently, several different adaptive hypotheses have been proposed to explain eye degeneration based on studies in the teleost Astyanax mexicanus, which consists of blind cave-dwelling (cavefish) and sighted surface-dwelling (surface fish) forms. One of these hypotheses is that eye regression is the result of indirect selection for constructive characters that are negatively linked to eye development through the pleiotropic effects of Sonic Hedgehog (SHH) signaling. However, subsequent genetic analyses suggested that other mechanisms also contribute to eye regression in Astyanax cavefish. Here, we introduce a new approach to this problem by investigating the phenotypic and genetic relationships between a suite of non-visual constructive traits and eye regression.

Results

Using quantitative genetic analysis of crosses between surface fish, the Pachón cavefish population and their hybrid progeny, we show that the adaptive vibration attraction behavior (VAB) and its sensory receptors, superficial neuromasts (SN) specifically found within the cavefish eye orbit (EO), are genetically correlated with reduced eye size. The quantitative trait loci (QTL) for these three traits form two clusters of congruent or overlapping QTL on Astyanax linkage groups (LG) 2 and 17, but not at the shh locus on LG 13. Ablation of EO SN in cavefish demonstrated a major role for these sensory receptors in VAB expression. Furthermore, experimental induction of eye regression in surface fish via shh overexpression showed that the absence of eyes was insufficient to promote the appearance of VAB or EO SN.

Conclusions

We conclude that natural selection for the enhancement of VAB and EO SN indirectly promotes eye regression in the Pachón cavefish population through an antagonistic relationship involving genetic linkage or pleiotropy among the genetic factors underlying these traits. This study demonstrates a trade-off between the evolution of a non-visual sensory system and eye regression during the adaptive evolution of Astyanax to the cave environment.

     

Vibration attraction response is a plastic trait in blind Mexican tetra (Astyanax mexicanus), variable within subpopulations inhabiting the same cave

Animals evolve their sensory systems and foraging behaviours to adapt and colonize new and challenging habitats such as the dark cave environment. Vibration attraction behaviour (VAB) gives fish the ability to locate the source of a water disturbance in the darkness. VAB evolved in the blind Mexican cave tetra, Astyanax mexicanus. VAB is triggered in cavefish by vibration stimuli peaking at 35 Hz, which is within the main spectrum of water fluctuations produced by many prey crustaceans and insects. VAB has a genetic component and is correlated to an increased number of head mechanosensory neuromasts in the eye orbital region when compared to surface fish. Previous competitive prey capture assays have supported the advantage of VAB for foraging in the dark. Despite its putative adaptive function, VAB has been described as absent in some Astyanax cave populations (Tinaja and Molino) but present in others (Pachón, Piedras, Toro and Sabinos). Here we have tested the occurrence of VAB in the field and in multiple cave populations using a vibrating device in natural pools. Our results confirmed the presence of VAB in caves such as Pachón, Toro and Sabinos but showed that VAB is also present in the Tinaja and Molino cave populations, previously reported as VAB-negative in laboratory experiments. Thus, VAB is available throughout the range of hypogean A. mexicanus. However, and most notably, within a given cave the levels of VAB were highly variable among different pools. Fish at one pool may express no VAB, while fish at another nearby pool of the same cave may actively show VAB. While a variety of environmental conditions may foster this diversity, we found that individuals inhabiting pools with a high abundance of organic matter have reduced expression of VAB. In contrast, in pools with little organic debris where fish probably depend more on hunting than on scavenging, VAB is enhanced. Our results suggest that expression of VAB is a plastic trait whose variability can depend on local conditions. Such plasticity may be required within and among caves where high environmental variability between pools results in a diverse availability of food.     

The relative importance of genetic and nongenetic inheritance in relation to trait plasticity in Callosobruchus maculatus

A trait's response to natural selection will reflect the nature of the inheritance mechanisms that mediate the transmission of variation across generations. The relative importance of genetic and nongenetic mechanisms of inheritance is predicted to be related to the degree of trait plasticity, with nongenetic inheritance playing a greater role in the cross‐generational transmission of more plastic traits. However, this prediction has never been tested. We investigated the influence of genetic effects and nongenetic parental effects in two morphological traits differing in degree of plasticity by manipulating larval diet quality within a cross‐generational split‐brood experiment using the seed beetle Callososbuchus maculatus. In line with predictions, we found that the more plastic trait (elytron length) is strongly influenced by both maternal and paternal effects whereas genetic variance is undetectable. In contrast, the less plastic trait (first abdominal sternite length) is not influenced by parental effects but exhibits abundant genetic variance. Our findings support the hypothesis that environment‐dependent parental effects may play a particularly important role in highly plastic traits and thereby affect the evolutionary response of such traits.     

Evolution of Space Dependent Growth in the Teleost Astyanax mexicanus

The relationship between growth rate and environmental space is an unresolved issue in teleosts. While it is known from aquaculture studies that stocking density has a negative relationship to growth, the underlying mechanisms have not been elucidated, primarily because the growth rate of populations rather than individual fish were the subject of all previous studies. Here we investigate this problem in the teleost Astyanax mexicanus, which consists of a sighted surface-dwelling form (surface fish) and several blind cave-dwelling (cavefish) forms. Surface fish and cavefish are distinguished by living in spatially contrasting environments and therefore are excellent models to study the effects of environmental size on growth. Multiple controlled growth experiments with individual fish raised in confined or unconfined spaces showed that environmental size has a major impact on growth rate in surface fish, a trait we have termed space dependent growth (SDG). In contrast, SDG has regressed to different degrees in the Pachón and Tinaja populations of cavefish. Mating experiments between surface and Pachón cavefish show that SDG is inherited as a dominant trait and is controlled by multiple genetic factors. Despite its regression in blind cavefish, SDG is not affected when sighted surface fish are raised in darkness, indicating that vision is not required to perceive and react to environmental space. Analysis of plasma cortisol levels showed that an elevation above basal levels occurred soon after surface fish were exposed to confined space. This initial cortisol peak was absent in Pachón cavefish, suggesting that the effects of confined space on growth may be mediated partly through a stress response. We conclude that Astyanax reacts to confined spaces by exhibiting SDG, which has a genetic component and shows evolutionary regression during adaptation of cavefish to confined environments.     

Grandparental effects in marine sticklebacks: transgenerational plasticity across multiple generations

Nongenetic inheritance mechanisms such as transgenerational plasticity (TGP) can buffer populations against rapid environmental change such as ocean warming. Yet, little is known about how long these effects persist and whether they are cumulative over generations. Here, we tested for adaptive TGP in response to simulated ocean warming across parental and grandparental generations of marine sticklebacks. Grandparents were acclimated for two months during reproductive conditioning, whereas parents experienced developmental acclimation, allowing us to compare the fitness consequences of short‐term vs. prolonged exposure to elevated temperature across multiple generations. We found that reproductive output of F1 adults was primarily determined by maternal developmental temperature, but carry‐over effects from grandparental acclimation environments resulted in cumulative negative effects of elevated temperature on hatching success. In very early stages of growth, F2 offspring reached larger sizes in their respective paternal and grandparental environment down the paternal line, suggesting that other factors than just the paternal genome may be transferred between generations. In later growth stages, maternal and maternal granddam environments strongly influenced offspring body size, but in opposing directions, indicating that the mechanism(s) underlying the transfer of environmental information may have differed between acute and developmental acclimation experienced by the two generations. Taken together, our results suggest that the fitness consequences of parental and grandparental TGP are highly context dependent, but will play an important role in mediating some of the impacts of rapid climate change in this system.     

Evidence for multiple genetic forms with similar eyeless phenotypes in the blind cavefish, Astyanax mexicanus

A diverse group of animals has adapted to caves and lost their eyes and pigmentation, but little is known about how these animals and their striking phenotypes have evolved. The teleost Astyanax mexicanus consists of an eyed epigean form (surface fish) and at least 29 different populations of eyeless hypogean forms (cavefish). Current alternative hypotheses suggest that adaptation to cave environments may have occurred either once or multiple times during the evolutionary history of this species. If the latter is true, the unique phenotypes of different cave-dwelling populations may result from convergence of form, and different genetic changes and developmental processes may have similar morphological consequences. Here we report an analysis of variation in the mitochondrial NADH dehydrogenase 2 (ND2) gene among different surface fish and cavefish populations. The results identify a minimum of two genetically distinctive cavefish lineages with similar eyeless phenotypes. The distinction between these divergent forms is supported by differences in the number of rib-bearing thoracic vertebrae in their axial skeletons. The geographic distribution of ND2 haplotypes is consistent with roles for multiple founder events and introgressive hybridization in the evolution of cave-related phenotypes. The existence of multiple genetic lineages makes A. mexicanus an excellent model to study convergence and the genes and developmental pathways involved in the evolution of the eye and pigment degeneration.     

Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans

Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS–PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.     

PATERNAL CARE: DIRECT AND INDIRECT GENETIC EFFECTS OF FATHERS ON OFFSPRING PERFORMANCE

Knowledge of how genetic effects arising from parental care influence the evolution of offspring traits comes almost exclusively from studies of maternal care. However, males provide care in some taxa, and often this care differs from females in quality or quantity. If variation in paternal care is genetically based then, like maternal care and maternal effects, paternal effects may have important consequences for the evolution of offspring traits via indirect genetic effects (IGEs). IGEs and direct–indirect genetic covariances associated with parental care can contribute substantially to total heritability and influence predictions about how traits respond to selection. It is unknown, however, if the magnitude and sign of parental effects arising from fathers are the same as those arising from mothers. We used a reciprocal cross‐fostering experiment to quantify environmental and genetic effects of paternal care on offspring performance in the burying beetle, Nicrophorus vespilloides. We found that IGEs were substantial and direct–indirect genetic covariances were negative. Combined, these patterns led to low total heritabilities for offspring performance traits. Thus, under paternal care, offspring performance traits are unlikely to evolve in response to selection, and variation in these traits will be maintained in the population despite potentially strong selection on these traits. These patterns are similar to those generated by maternal care, indicating that the genetic effects of care on offspring performance are independent of the caregiver's sex.     

The role of maternal and paternal effects in the evolution of parental quality by sexual selection

Genetic models of maternal effects and models of mate choice have focused on the evolutionary effects of variation in parental quality. There have been, however, few attempts to combine these into a single model for the evolution of sexually selected traits. We present a quantitative genetic model that considers how male and female parental quality (together or separately) affect the expression of a sexually selected offspring trait. We allow female choice of males based on this parentally affected trait and examine the evolution of mate choice, parental quality and the indicator trait. Our model reveals a number of consequences of maternal and paternal effects. (1) The force of sexual selection owing to adaptive mate choice can displace parental quality from its natural selection optimum. (2) The force of sexual selection can displace female parental quality from its natural selection optimum even when nonadaptive mate choice occurs (e.g. runaway sexual selection), because females of higher parental quality produce more attractive sons and these sons counterbalance the loss in fitness owing to over-investment in each offspring. (3) Maternal and paternal effects can provide a source of genetic variation for offspring traits, allowing evolution by sexual selection even when those traits do not show direct genetic variation (i.e. are not heritable). (4) The correlation between paternal investment and the offspring trait influenced by the parental effects can result in adaptive mate choice and lead to the elaboration of both female preference and the male sexually selected trait. When parental effects exist, sexual selection can drive the evolution of parental quality when investment increases the attractiveness of offspring, leading to the elaboration of indicator traits and higher than expected levels of parental investment.     

Adaptive evolution of eye degeneration in the Mexican blind cavefish

The evolutionary mechanisms responsible for eye degeneration in cave-adapted animals have not been resolved. Opposing hypotheses invoking neural mutation or natural selection, each with certain genetic and developmental expectations, have been advanced to explain eye regression, although little or no experimental evidence has been presented to support or reject either theory. Here we review recent developmental and molecular studies in the teleost Astyanax mexicanus, a single species consisting of a sighted surface-dwelling form (surface fish) and many blind cave-dwelling forms (cavefish), which shed new light on this problem. The manner of eye development and degeneration, the ability to experimentally restore eyes, gene expression patterns, and comparisons between different cavefish populations all provide important clues for understanding the evolutionary forces responsible for eye degeneration. A key discovery is that Hedgehog midline signaling is expanded and inhibits eye formation by inducing lens apoptosis in cavefish embryos. Accordingly, eyes could have been lost by default as a consequence of natural selection for constructive traits, such as feeding structures, which are positively regulated by Hh signaling. We conclude from these studies that eye degeneration in cavefish may be caused by adaptive evolution and pleiotropy.     

Paternal but not maternal age influences early-life performance of offspring in a long-lived seabird

Variability in demographic traits between individuals within populations has profound implications for both evolutionary processes and population dynamics. Parental effects as a source of non-genetic inheritance are important processes to consider to understand the causes of individual variation. In iteroparous species, parental age is known to influence strongly reproductive success and offspring quality, but consequences on an offspring fitness component after independence are much less studied. Based on 37 years longitudinal monitoring of a long-lived seabird, the wandering albatross, we investigate delayed effects of parental age on offspring fitness components. We provide evidence that parental age influences offspring performance beyond the age of independence. By distinguishing maternal and paternal age effects, we demonstrate that paternal age, but not maternal age, impacts negatively post-fledging offspring performance.     

EFFECTS OF MATERNAL AND PATERNAL ENVIRONMENT AND GENOTYPE ON OFFSPRING PHENOTYPE IN SOLIDAGO ALTISSIMA L.

To predict the possible evolutionary response of a plant species to a new environment, it is necessary to separate genetic from environmental sources of phenotypic variation. In a case study of the invader Solidago altissima, the influences of several kinds of parental effects and of direct inheritance and environment on offspring phenotype were separated. Fifteen genotypes were crossed in three 5 × 5 diallels excluding selfs. Clonal replicates of the parental genotypes were grown in two environments such that each diallel could be made with maternal/paternal plants from sand/sand, sand/soil, soil/sand, and soil/soil. In a first experiment (1989) offspring were raised in the experimental garden and in a second experiment (1990) in the glasshouse. Parent plants growing in sand invested less biomass in inflorescences but produced larger seeds than parent plants growing in soil. In the garden experiment, phenotypic variation among offspring was greatly influenced by environmental heterogeneity. Direct genetic variation (within diallels) was found only for leaf characters and total leaf mass. Germination probability and early seedling mass were significantly affected by phenotypic differences among maternal plants because of genotype ( genetic maternal effects ) and soil environment ( general environmental maternal effects ). Seeds from maternal plants in sand germinated better and produced bigger seedlings than seeds from maternal plants in soil. They also grew taller with time, probably because competition accentuated the initial differences. Height growth and stem mass at harvest (an integrated account of individual growth history) of offspring varied significantly among crosses within parental combinations ( specific environmental maternal effects ). In the glasshouse experiment, the influence of environmental heterogeneity and competition could be kept low. Except for early characters, the influence of direct genetic variation was large but again leaf characters (= basic module morphology) seemed to be under stricter genetic control than did size characters. Genetic maternal effects, general environmental maternal effects, and specific environmental maternal effects dominated in early characters. The maternal effects were exerted both via seed mass and directly on characters of young offspring. Persistent effects of the general paternal environment ( general environmental paternal effects ) were found for leaf length and stem and leaf mass at harvest. They were opposite in direction to the general environmental maternal effects, that is the same genotypes produced “better mothers” in sand but “better fathers” in soil. The general environmental paternal effects must have been due to differences in pollen quality, resulting from pollen selection within the male parent or leading to pre- or postzygotic selection within the female parent. The ranking of crosses according to mean offspring phenotypes was different in the two experiments, suggesting strong interaction of the observed effects with the environment. The correlation structure among characters changed less between experiments than did the pattern of variation of single characters, but under the competitive conditions in the garden plant height seemed to be more directly related to fitness than in the glasshouse. Reduced competition could also explain why maternal effects were less persistent in the glasshouse than in the garden experiment. Evolution via selection of maternal effects would be possible in the study population because these effects are in part due to genetic differences among parents.     

How stabilizing selection and nongenetic inheritance combine to shape the evolution of phenotypic plasticity

Relatively little is known about whether and how nongenetic inheritance interacts with selection to impact the evolution of phenotypic plasticity. Here, we empirically evaluated how stabilizing selection and a common form of nongenetic inheritance—maternal environmental effects—jointly influence the evolution of phenotypic plasticity in natural populations of spadefoot toads. We compared populations that previous fieldwork has shown to have evolved conspicuous plasticity in resource‐use phenotypes (“resource polyphenism”) with those that, owing to stabilizing selection favouring a narrower range of such phenotypes, appear to have lost this plasticity. We show that: (a) this apparent loss of plasticity in nature reflects a condition‐dependent maternal effect and not a genetic loss of plasticity, that is “genetic assimilation,” and (b) this plasticity is not costly. By shielding noncostly plasticity from selection, nongenetic inheritance generally, and maternal effects specifically, can preclude genetic assimilation from occurring and consequently impede adaptive (genetic) evolution.     

Blind cavefish and heat shock protein chaperones: a novel role for hsp90alpha in lens apoptosis

Lens apoptosis plays a central role in cavefish eye degeneration. Heat shock proteins (hsps) can regulate apoptosis; therefore, we examined the relationship between constitutive hsp70 and hsp90 expression and lens apoptosis. The model system is Astyanax mexicanus, a teleost species consisting of an eyed surface-dwelling (surface fish) form and numerous blind cave-dwelling (cavefish) forms. Optic primordia are formed in the cavefish embryo but they subsequently undergo lens apoptosis, arrest in development and degenerate. Astyanax hsp90 and hsp70 DNAs were isolated to use as probes to compare gene expression during surface fish and cavefish development. Hsp90beta, which encodes one of two hsp90 isoforms, was not expressed in the surface fish or cavefish lens, whereas hsp70 was expressed in the lens of both forms, suggesting that neither is directly involved in lens apoptosis. In contrast, hsp90alpha, the other hsp90 isoform, was expressed in the cavefish but not the surface fish lens. Hsp90alpha expression peaked shortly before the beginning of lens apoptosis in three convergent cavefish populations, suggesting a close relationship with lens apoptosis. The absence of hsp90beta in the lens allowed us to use geldanamycin and radicicol, specific inhibitors of hsp90 chaperone function, to determine whether lens cell death requires hsp90alpha expression. Both inhibitors blocked TUNEL labeling in the cavefish lens, suggesting that hsp90alpha is required for apoptosis. In contrast to their effects on the lens, these inhibitors induced TUNEL labeling in the surface epidermis, presumably due to effects on hsp90beta function, implying that the two-hsp90 isoforms may have contrasting roles in cell survival. We conclude that hsp90alpha plays a novel role in lens apoptosis and cavefish eye degeneration.     

Complete elimination of maternal mitochondrial DNA during meiosis resulting in the paternal inheritance of the mitochondrial genome in Chlamydomonas species

Summary. The non-Mendelian inheritance of organellar DNA is common in most plants and animals. In the isogamous green alga Chlamydomonas species, progeny inherit chloroplast genes from the maternal parent, as paternal chloroplast genes are selectively eliminated in young zygotes. Mitochondrial genes are inherited from the paternal parent. Analogically, maternal mitochondrial DNA (mtDNA) is thought to be selectively eliminated. Nevertheless, it is unclear when this selective elimination occurs. Here, we examined the behaviors of maternal and paternal mtDNAs by various methods during the period between the beginning of zygote formation and zoospore formation. First, we observed the behavior of the organelle nucleoids of living cells by specifically staining DNA with the fluorochrome SYBR Green I and staining mitochondria with 3,3′-dihexyloxacarbocyanine iodide. We also examined the fate of mtDNA of male and female parental origin by real-time PCR, nested PCR with single zygotes, and fluorescence in situ hybridization analysis. The mtDNA of maternal origin was completely eliminated before the first cell nuclear division, probably just before mtDNA synthesis, during meiosis. Therefore, the progeny inherit the remaining paternal mtDNA. We suggest that the complete elimination of maternal mtDNA during meiosis is the primary cause of paternal mitochondrial inheritance. Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 901-0213, Japan.     

X- and Y-chromosome linked paternal effects on a life-history trait

Males and females usually invest asymmetrically in offspring. In species lacking parental care, females influence offspring in many ways, while males only contribute genetic material via their sperm. For this reason, maternal effects have long been considered an important source of phenotypic variation, while paternal effects have been presumed to be absent or negligible. The recent surge of studies showing trans-generational epigenetic effects questions this assumption, and indicates that paternal effects may be far more important than previously appreciated. Here, we test for sex-linked paternal effects in Drosophila melanogaster on a life-history trait, and find substantial support for both X- and Y-linked effects.     

Experimental parasite infection reveals costs and benefits of paternal effects

Forces shaping an individual's phenotype are complex and include transgenerational effects. Despite low investment into reproduction, a father's environment and phenotype can shape its offspring's phenotype. Whether and when such paternal effects are adaptive, however, remains elusive. Using three‐spined sticklebacks in controlled infection experiments, we show that sperm deficiencies in exposed males compared to their unexposed brothers functionally translated into reduced reproductive success in sperm competition trials. In non‐competitive fertilisations, offspring of exposed males suffered significant costs of reduced hatching success and survival but they reached a higher body condition than their counterparts from unexposed fathers after experimental infection. Interestingly, those benefits of paternal infection did not result from increased resistance but from increased tolerance to the parasite. Altogether, these results demonstrate that parasite resistance and tolerance are shaped by processes involving both genetic and non‐genetic inheritance and suggest a context‐dependent adaptive value of paternal effects.     

THE COADAPTATION OF PARENTAL AND OFFSPRING CHARACTERS

Parents often have important influences on their offspring's traits and/or fitness (i.e., maternal or paternal effects). When offspring fitness is determined by the joint influences of offspring and parental traits, selection may favor particular combinations that generate high offspring fitness. We show that this epistasis for fitness between the parental and offspring genotypes can result in the evolution of their joint distribution, generating genetic correlations between the parental and offspring characters. This phenomenon can be viewed as a coadaptive process in which offspring genotypes evolve to function with the parentally provided environment and, in turn, the genes for this environment become associated with specific offspring genes adapted to it. To illustrate this point, we present two scenarios in which selection on offspring alone alters the correlation between a maternal and an offspring character. We use a quantitative genetic maternal effect model combined with a simple quadratic model of fitness to examine changes in the linkage disequilibrium between the maternal and offspring genotypes. In the first scenario, stabilizing selection on a maternally affected offspring character results in a genetic correlation that is opposite in sign to the maternal effect. In the second scenario, directional selection on an offspring trait that shows a nonadditive maternal effect can result in selection for positive covariances between the traits. This form of selection also results in increased genetic variation in maternal and offspring characters, and may, in the extreme case, promote host-race formation or speciation. This model provides a possible evolutionary explanation for the ubiquity of large genetic correlations between maternal and offspring traits, and suggests that this pattern of coinheritance may reflect functional relationships between these characters (i.e., functional integration).     

Biparental mitochondrial DNA inheritance in the parasitic trematode Schistosoma mansoni

The maternal inheritance of mitochondrial DNA (mtDNA) in eukaryotic organisms occurs because of the selective destruction of paternal mtDNA molecules that may be present in the zygote. The elimination of sperm mtDNA is less efficient in interspecific crosses, and biparental inheritance of mtDNA has been observed in a variety of species. Because interspecific crosses are likely to be extremely rare in nature, parental inheritance of mtDNA has been deemed of little relevance to population genetics. The mtDNA of the parasitic trematode Schistosoma mansoni was examined for its utility in addressing epidemiological questions related to the transmission and spread of schistosomiasis. Prior to embarking on such experiments, we sought to confirm the mode of inheritance of this molecule using the highly polymorphic mtDNA minisatellite as a marker. In 3 separate crosses, mtDNA apparently identical to paternal DNA was observed in some individuals of the F2 and F3 generations. These observations thus suggest the intraspecific paternal inheritance of mtDNA across multiple generations in Schistosoma mansoni.