Telomere length is highly heritable and independent of growth rate manipulated by temperature in field crickets.

Many organisms are capable of growing faster than they do. Restrained growth rate has functionally been explained by negative effects on lifespan of accelerated growth. However, the underlying mechanisms remain elusive. Telomere attrition has been proposed as a causal agent and has been mostly studied in endothermic vertebrates. We established that telomeres exist as chromosomal-ends in a model insect, the field cricket Gryllus campestris, using terminal restriction fragment and Bal 31 methods. Telomeres comprised TTAGGn repeats of 38 kb on average, more than four times longer than the telomeres of human infants. Bal 31 assays confirmed that telomeric repeats were located at the chromosome-ends. We tested whether rapid growth between day 1, day 65, day 85, and day 125 is achieved at the expense of telomere length by comparing nymphs reared at 23°C with their siblings reared at 28°C, which grew three times faster in the initial 65 days. Surprisingly, neither temperature treatment nor age affected average telomere length. Concomitantly, the broad sense heritability of telomere length was remarkably high at ~100%. Despite high heritability, the evolvability (a mean-standardized measure of genetic variance) was low relative to that of body mass. We discuss our findings in the context of telomere evolution. Some important features of vertebrate telomere biology are evident in an insect species dating back to the Triassic. The apparent lack of an effect of growth rate on telomere length is puzzling, suggesting strong telomere length maintenance during the growth phase. Whether such maintenance of telomere length is adaptive remains elusive and requires further study investigating the links with fitness in the wild.

Evidence for genetic isolation and local adaptation in the field cricket Gryllus campestris.

Understanding how species can thrive in a range of environments is a central challenge for evolutionary ecology. There is strong evidence for local adaptation along large-scale ecological clines in insects. However, potential adaptation among neighbouring populations differing in their environment has been studied much less. We used RAD sequencing to quantify genetic divergence and clustering of ten populations of the field cricket Gryllus campestris in the Cantabrian Mountains of northern Spain, and an outgroup on the inland plain. Our populations were chosen to represent replicate high and low altitude habitats. We identified genetic clusters that include both high and low altitude populations indicating that the two habitat types do not hold ancestrally distinct lineages. Using common-garden rearing experiments to remove environmental effects, we found evidence for differences between high and low altitude populations in physiological and life-history traits. As predicted by the local adaptation hypothesis, crickets with parents from cooler (high altitude) populations recovered from periods of extreme cooling more rapidly than those with parents from warmer (low altitude) populations. Growth rates also differed between offspring from high and low altitude populations. However, contrary to our prediction that crickets from high altitudes would grow faster, the most striking difference was that at high temperatures, growth was fastest in individuals from low altitudes. Our findings reveal that populations a few tens of kilometres apart have independently evolved adaptations to their environment. This suggests that local adaptation in a range of traits may be commonplace even in mobile invertebrates at scales of a small fraction of species' distributions.

Slower senescence in a wild insect population in years with a more female-biased sex ratio.

Life-history theories of senescence are based on the existence of a trade-off in resource allocation between body maintenance and reproduction. This putative trade-off means that environmental and demographic factors affecting the costs of reproduction should be associated with changes in patterns of senescence. In many species, competition among males is a major component of male reproductive investment, and hence variation in the sex ratio is expected to affect rates of senescence. We test this prediction using nine years of demographic and behavioural data from a wild population of the annual field cricket Gryllus campestris. Over these generations, the sex ratio at adulthood varied substantially, from years with an equal number of each sex to years with twice as many females as males. Consistent with the predictions of theory, we found that in years with a greater proportion of females, both sexes experienced a slower increase in mortality rate with age. Additionally, phenotypic senescence in males was slower in years when there were more females. Sex ratio did not affect the baseline mortality rate in males, but females suffered higher age-independent mortality rates when males were in short supply.

Wild cricket social networks show stability across generations.

BACKGROUND: A central part of an animal's environment is its interactions with conspecifics. There has been growing interest in the potential to capture these interactions in the form of a social network. Such networks can then be used to examine how relationships among individuals affect ecological and evolutionary processes. However, in the context of selection and evolution, the utility of this approach relies on social network structures persisting across generations. This is an assumption that has been difficult to test because networks spanning multiple generations have not been available. We constructed social networks for six annual generations over a period of eight years for a wild population of the cricket Gryllus campestris. RESULTS: Through the use of exponential random graph models (ERGMs), we found that the networks in any given year were able to predict the structure of networks in other years for some network characteristics. The capacity of a network model of any given year to predict the networks of other years did not depend on how far apart those other years were in time. Instead, the capacity of a network model to predict the structure of a network in another year depended on the similarity in population size between those years. CONCLUSIONS: Our results indicate that cricket social network structure resists the turnover of individuals and is stable across generations. This would allow evolutionary processes that rely on network structure to take place. The influence of network size may indicate that scaling up findings on social behaviour from small populations to larger ones will be difficult. Our study also illustrates the utility of ERGMs for comparing networks, a task for which an effective approach has been elusive.

Behaviour in captivity predicts some aspects of natural behaviour, but not others, in a wild cricket population.

Examining the relevance of 'animal personality' involves linking consistent among- and within-individual behavioural variation to fitness in the wild. Studies aiming to do this typically assay personality in captivity and rely on the assumption that measures of traits in the laboratory reflect their expression in nature. We examined this rarely tested assumption by comparing laboratory and field measurements of the behaviour of wild field crickets (Gryllus campestris) by continuously monitoring individual behaviour in nature, and repeatedly capturing the same individuals and measuring their behaviour in captivity. We focused on three traits that are frequently examined in personality studies: shyness, activity and exploration. All of them showed repeatability in the laboratory. Laboratory activity and exploration predicted the expression of their equivalent behaviours in the wild, but shyness did not. Traits in the wild were predictably influenced by environmental factors such as temperature and sunlight, but only activity showed appreciable within-individual repeatability. This suggests that some behaviours typically studied as personality traits can be accurately assayed in captivity, but the expression of others may be highly context-specific. Our results highlight the importance of validating the relevance of laboratory behavioural assays to analogous traits measured in the wild.

Neighbouring populations, opposite dynamics: influence of body size and environmental variation on the demography of stream-resident brown trout (Salmo trutta).

In organisms such as fish, where body size is considered an important state variable for the study of their population dynamics, size-specific growth and survival rates can be influenced by local variation in both biotic and abiotic factors, but few studies have evaluated the complex relationships between environmental variability and size-dependent processes. We analysed a 6-year capture-recapture dataset of brown trout (Salmo trutta) collected at 3 neighbouring but heterogeneous mountain streams in northern Spain with the aim of investigating the factors shaping the dynamics of local populations. The influence of body size and water temperature on survival and individual growth was assessed under a multi-state modelling framework, an extension of classical capture-recapture models that considers the state (i.e. body size) of the individual in each capture occasion and allows us to obtain state-specific demographic rates and link them to continuous environmental variables. Individual survival and growth patterns varied over space and time, and evidence of size-dependent survival was found in all but the smallest stream. At this stream, the probability of reaching larger sizes was lower compared to the other wider and deeper streams. Water temperature variables performed better in the modelling of the highest-altitude population, explaining over a 99 % of the variability in maturation transitions and survival of large fish. The relationships between body size, temperature and fitness components found in this study highlight the utility of multi-state approaches to investigate small-scale demographic processes in heterogeneous environments, and to provide reliable ecological knowledge for management purposes.

The relationship between the body and air temperature in a terrestrial ectotherm.

Ectotherms make up the majority of terrestrial biodiversity, so it is important to understand their potential responses to climate change. Often, models aiming to achieve this understanding correlate species distributions with ambient air temperature. However, this assumes a constant relationship between the air temperature and body temperature, which determines an ectotherm's thermal performance. To test this assumption, we develop and validate a method for retrospective estimation of ectotherm body temperature using heat exchange equations. We apply the model to predict the body temperature of wild field crickets (Gryllus campestris) in Northern Spain for 1985-2019 and compare these values to air temperature. We show that while air temperature impacts ectotherm body temperature, it captures only a fraction of its thermal experience. Solar radiation can increase the body temperature by more than 20°C above air temperature with implications for physiology and behaviour. The effect of solar radiation on body temperature is particularly important given that climate change will alter cloud cover. Our study shows that the impacts of climate change on species cannot be assumed to be proportional only to changing air temperature. More reliable models of future species distributions require mechanistic links between environmental conditions and thermal ecophysiologies of species.

Fertilisation and early developmental barriers to hybridisation in field crickets.

BACKGROUND: Post-mating interactions between the reproductive traits and gametes of mating individuals and among their genes within zygotes are invariably complex, providing multiple opportunities for reproduction to go awry. These interactions have the potential to act as barriers to gene flow between species, and may be important in the process of speciation. There are multiple post-mating barriers to interbreeding between the hybridising field crickets Gryllus bimaculatus and G. campestris. Female G. bimaculatus preferentially store sperm from conspecific males when mated to both conspecific and heterospecific partners. Additionally, conspecific males sire an even greater proportion of offspring than would be predicted from their sperm's representation in the spermatheca. The nature of these post-sperm-storage barriers to hybridisation are unknown. We use a fluorescent staining technique to determine whether barriers occur prior to, or during embryo development. RESULTS: We show that eggs laid by G. bimaculatus females mated to G. campestris males are less likely to begin embryogenesis than eggs from conspecific mating pairs. Of the eggs that are successfully fertilised and start to develop, those from heterospecific mating pairs are more likely to arrest early, prior to blastoderm formation. We find evidence for bimodal variation among egg clutches in the number of developing embryos that subsequently arrest, indicating that there is genetic variation for incompatibility between mating individuals. In contrast to the pattern of early embryonic mortality, those hybrids reaching advanced stages of embryogenesis have survival rates equal to that of embryos from conspecific mating pairs. CONCLUSIONS: Post-sperm-storage barriers to hybridisation show evidence of genetic polymorphism. They are sufficiently large, that if the species interbreed where they are sympatric, these barriers could play a role in the maintenance of reproductive isolation between them. The number of eggs that fail to develop represents a substantial cost of hybridization to G. bimaculatus females, and this cost could reinforce the evolution of barriers occurring earlier in the reproductive process.

Multiple post-mating barriers to hybridization in field crickets.

Mechanisms that prevent different species from interbreeding are fundamental to the maintenance of biodiversity. Barriers to interspecific matings, such as failure to recognize a potential mate, are often relatively easy to identify. Those occurring after mating, such as differences in the how successful sperm are in competition for fertilisations, are cryptic and have the potential to create selection on females to mate multiply as a defence against maladaptive hybridization. Cryptic advantages to conspecific sperm may be very widespread and have been identified based on the observations of higher paternity of conspecifics in several species. However, a relationship between the fate of sperm from two species within the female and paternity has never been demonstrated. We use competitive microsatellite PCR to show that in two hybridising cricket species, Gryllus bimaculatus and G. campestris, sequential cryptic reproductive barriers are present. In competition with heterospecifics, more sperm from conspecific males is stored by females. Additionally, sperm from conspecific males has a higher fertilisation probability. This reveals that conspecific sperm precedence can occur through processes fundamentally under the control of females, providing avenues for females to evolve multiple mating as a defence against hybridization, with the counterintuitive outcome that promiscuity reinforces isolation and may promote speciation.

Environment and mate attractiveness in a wild insect.

The role of female choice in sexual selection is well established, including the recognition that females choose their mates based on multiple cues. These cues may include intrinsic aspects of a male's phenotype as well as aspects of the environment associated with the male. The role of the spatial location of a potential mate has been well studied in territorial vertebrates. However, despite their role as laboratory models for studies of sexual selection, the potential for insects to choose their mates on the basis of location has scarcely been studied. We studied a natural population of individually tagged crickets (Gryllus campestris) in a meadow in Northern Spain. Adults typically move between burrows every few days, allowing us to examine how pairing success of males can be predicted by the burrow they occupy, independent of their own characteristics. We observed the entirety of ten independent breeding seasons to provide replication and to determine whether the relative importance of these factors is stable across years. We find that both male ID and the ID his burrow affect the likelihood that he is paired with a female, but the burrow has a consistently greater influence. Furthermore, the two factors interact: the relative attractiveness of an individual male depends on which burrow he occupies. Our finding demonstrates a close interaction between naturally and sexually selected traits. It also demonstrates that mate choice studies may benefit from considering not only obvious secondary sexual traits, but also more cryptic traits such as microhabitat choice.

Fine-scale population structure, inbreeding risk and avoidance in a wild insect population.

The ecological and evolutionary importance of fine-scale genetic structure within populations is increasingly appreciated. However, available data are largely restricted to wild vertebrates and eusocial insects. In addition, there is the expectation that most insects tend to have such large- and high-density populations and are so mobile that they are unlikely to face inbreeding risks through fine-scale population structuring. This has made the growing body of evidence for inbreeding avoidance in insects and its implication in mating systems evolution somewhat enigmatic. We present a 4-year study of a natural population of field crickets. Using detailed video monitoring combined with genotyping, we track the movement of all adults within the population and investigate genetic structure at a fine scale. We find some evidence for relatives being found in closer proximity, both across generations and within a single breeding season. Whilst incestuous matings are not avoided, population inbreeding is low, suggesting that mating is close to random and the limited fine-scale structure does not create significant inbreeding risk. Hence, there is little evidence for selective pressures associated with the evolution of inbreeding avoidance mechanisms in a closely related species.

Testing the effect of early-life reproductive effort on age-related decline in a wild insect.

The disposable soma theory of ageing predicts that when organisms invest in reproduction they do so by reducing their investment in body maintenance, inducing a trade-off between reproduction and survival. Experiments on invertebrates in the lab provide support for the theory by demonstrating the predicted responses to manipulation of reproductive effort or lifespan. However, experimental studies in birds and evidence from observational (nonmanipulative) studies in nature do not consistently reveal trade-offs. Most species studied previously in the wild are mammals and birds that reproduce over multiple discrete seasons. This contrasts with temperate invertebrates, which typically have annual generations and reproduce over a single season. We expand the taxonomic range of senescence study systems to include life histories typical of most temperate invertebrates. We monitored reproductive effort, ageing, and survival in a natural field cricket population over ten years to test the prediction that individuals investing more in early-reproduction senesce faster and die younger. We found no evidence of a trade-off between early-life reproductive effort and survival, and only weak evidence for a trade-off with phenotypic senescence. We discuss the possibility that organisms with multiple discrete breeding seasons may have greater opportunities to express trade-offs between reproduction and senescence.

Comparing individual and population measures of senescence across 10 years in a wild insect population.

Declines in survival and performance with advancing age (senescence) have been widely documented in natural populations, but whether patterns of senescence across traits reflect a common underlying process of biological ageing remains unclear. Senescence is typically characterized via assessments of the rate of change in mortality with age (actuarial senescence) or the rate of change in phenotypic performance with age (phenotypic senescence). Although both phenomena are considered indicative of underlying declines in somatic integrity, whether actuarial and phenotypic senescence rates are actually correlated has yet to be established. Here we present evidence of both actuarial and phenotypic senescence from a decade-long longitudinal field study of wild insects. By tagging every individual and using continuous video monitoring with a network of up to 140 video cameras, we were able to record survival and behavioral data on an entire adult population of field crickets. This reveals that both actuarial and phenotypic senescence vary substantially across 10 annual generations. This variation allows us to identify a strong correlation between actuarial and phenotypic measures of senescence. Our study demonstrates age-related phenotypic declines reflected in population level mortality rates and reveals that observations of senescence in a single year may not be representative of a general pattern.

Cell proliferation in the forebrain and midbrain of the sea lamprey.

Cell proliferation in the forebrain and midbrain of the sea lamprey (Petromyzon marinus L.) was investigated by proliferation cell nuclear antigen (PCNA) immunocytochemistry, with BrdU labeling as a complementary technique. Correspondence between proliferation regions and areas of early neuronal differentiation was also assessed using antibodies against HNK-1 early differentiation marker. The brain of late embryos shows a homogeneously thick ventricular zone (VZ) containing PCNA-immunoreactive (PCNA-ir) nuclei. In early prolarvae, several discontinuities formed by PCNA-negative cells, and differences among regions in VZ thickness, become apparent. In late prolarvae and early larvae, these differences in VZ thickness and appearance, as well as the presence of PCNA-negative discontinuities, allowed us to correlate proliferation domains and neuroanatomical regions. In larvae, the number of PCNA-ir cells in the VZs diminish gradually, although a few PCNA-ir cells are present in the ependyma of most regions. In late larvae, proliferation becomes confined to a few ventricular areas (medial pallium, caudal habenula, ventral preoptic recess near the optic nerve, and tuberal portion of the posterior hypothalamic recess). During metamorphosis there appears to be no proliferation, but in upstream adults a few PCNA-ir cells are observed in the most caudal habenula. The characteristics of the proliferative regions revealed in lamprey with PCNA immunocytochemistry show notable differences from those observed in other vertebrates, and these differences may be related to the peculiar life cycle of lampreys.

Comparing pre- and post-copulatory mate competition using social network analysis in wild crickets.

Sexual selection results from variation in success at multiple stages in the mating process, including competition before and after mating. The relationship between these forms of competition, such as whether they trade-off or reinforce one another, influences the role of sexual selection in evolution. However, the relationship between these 2 forms of competition is rarely quantified in the wild. We used video cameras to observe competition among male field crickets and their matings in the wild. We characterized pre- and post-copulatory competition as 2 networks of competing individuals. Social network analysis then allowed us to determine 1) the effectiveness of precopulatory competition for avoiding postcopulatory competition, 2) the potential for divergent mating strategies, and 3) whether increased postcopulatory competition reduces the apparent reproductive benefits of male promiscuity. We found 1) limited effectiveness of precopulatory competition for avoiding postcopulatory competition; 2) males do not specifically engage in only 1 type of competition; and 3) promiscuous individuals tend to mate with each other, which will tend to reduce variance in reproductive success in the population and highlights the trade-off inherent in mate guarding. Our results provide novel insights into the works of sexual competition in the wild. Furthermore, our study demonstrates the utility of using network analyses to study competitive interactions, even in species lacking obvious social structure.

Dynamics of among-individual behavioral variation over adult lifespan in a wild insect.

Investigating patterns of among and within-individual trait variation in populations is essential to understanding how selection shapes phenotypes. Behavior is often the most flexible aspect of the phenotype, and to understand how it is affected by selection, we need to examine how consistent individuals are. However, it is not well understood whether among-individual differences tend to remain consistent over lifetimes, or whether the behavior of individuals relative to one another varies over time. We examined the dynamics of 4 behavioral traits (tendency to leave a refuge, shyness, activity, and exploration) in a wild population of field crickets (Gryllus campestris). We tagged individuals and then temporarily removed them from their natural environment and tested them under laboratory conditions. All 4 traits showed among-individual variance in mean levels of expression across the adult lifespan, but no significant differences in how rapidly expression changed with age. For all traits, among-individual variance increased as individuals got older. Our findings reveal seldom examined changes in variance components over the adult lifetime of wild individuals. Such changes will have important implications for the relationship between behavioral traits, life-histories, and fitness and the consequences of selection on wild individuals.

Ontogeny of gamma-aminobutyric acid-immunoreactive neurons in the rhombencephalon and spinal cord of the sea lamprey.

The development of neurons expressing gamma-aminobutyric acid (GABA) in the rhombencephalon and spinal cord of the sea lamprey (Petromyzon marinus) was studied for the first time with an anti-GABA antibody. The earliest GABA-immunoreactive (GABAir) neurons appear in late embryos in the basal plate of the isthmus, caudal rhombencephalon, and rostral spinal cord. In prolarvae, the GABAir neurons of the rhombencephalon appear to be distributed in spatially restricted cellular domains that, at the end of the prolarval period, form four longitudinal GABAir bands (alar dorsal, alar ventral, dorsal basal, and ventral basal). In the spinal cord, we observed only three GABAir longitudinal bands (dorsal, intermediate, and ventral). The larval pattern of GABAir neuronal populations was established by the 30-mm stage, and the same populations were observed in premetamorphic and adult lampreys. The ontogeny of GABAergic populations in the lamprey rhombencephalon and spinal cord is, in general, similar to that previously described in mouse and Xenopus.

Early development of the retina and pineal complex in the sea lamprey: comparative immunocytochemical study.

Lampreys have a complex life cycle, with largely differentiated larval and adult periods. Despite the considerable interest of lampreys for understanding vertebrate evolution, knowledge of the early development of their eye and pineal complex is very scarce. Here, the early immunocytochemical organization of the pineal complex and retina of the sea lamprey was studied by use of antibodies against proliferating cell nuclear antigen (PCNA), opsin, serotonin, and gamma-aminobutyric acid (GABA). Cell differentiation in the retina, pineal organ, and habenula begins in prolarvae, as shown by the appearance of PCNA-negative cells, whereas differentiation of the parapineal vesicle was delayed until the larval period. In medium-sized to large larvae, PCNA-immunoreactive (-ir) cells were numerous in regions of the lateral retina near the differentiated part of the larval retina (central retina). A late-proliferating region was observed in the right habenula. Opsin immunoreactivity appears in the pineal vesicle of early prolarvae and 3 or 4 days later in the retina. In the parapineal organ, opsin immunoreactivity was observed only in large larvae. In the pineal organ, serotonin immunoreactivity was first observed in late prolarvae in photoreceptive (photoneuroendocrine) cells, whereas only a few of these cells appeared in the parapineal organ of large larvae. No serotonin immunoreactivity was observed in the larval retina. GABA immunoreactivity appeared earlier in the retina than in the pineal complex. No GABA-ir perikaryon was observed in the retina of larval lampreys, although a few GABA-ir centrifugal fibers innervate the inner retina in late prolarvae. First GABA-ir ganglion cells occur in the pineal organ of 15-17 mm larvae, and their number increases during the larval period. The only GABA-ir structures observed in the parapineal ganglion of larvae were afferent fibers, which appeared rather late in development. The time sequence of development in these photoreceptive structures is rather different from that observed in teleosts and other vertebrates. This suggests that the unusual development of the three photoreceptive organs in lampreys reflects specialization for their different functions during the larval and adult periods.

Ontogeny of gamma-aminobutyric acid-immunoreactive neuronal populations in the forebrain and midbrain of the sea lamprey.

Although brain organization in lampreys is of great interest for understanding evolution in vertebrates, knowledge of early development is very scarce. Here, the development of the forebrain and midbrain gamma-aminobutyric acid (GABA)-ergic systems was studied in embryos, prolarvae, and small larvae of the sea lamprey using an anti-GABA antibody. Ancillary immunochemical markers, such as proliferating cell nuclear antigen (PCNA), calretinin, and serotonin, as well as general staining methods and semithin sections were used to characterize the territories containing GABA-immunoreactive (GABAir) neurons. Differentiation of GABAir neurons in the diencephalon begins in late embryos, whereas differentiation in the telencephalon and midbrain was delayed to posthatching stages. In lamprey prolarvae, the GABAir populations appear either as compact GABAir cell groups or as neurons interspersed among GABA-negative cells. In the telencephalon of prolarvae, a band of cerebrospinal fluid-contacting (CSF-c) GABAir neurons (septum) was separated from the major GABAir telencephalic band, the striatum (ganglionic eminence) primordium. The striatal primordium appears to give rise to most GABAir neurons observed in the olfactory bulb and striatum of early larval stages. GABAir populations in the dorsal telencephalon appear later, in 15-30-mm-long larvae. In the diencephalon, GABAir neurons appear in embryos, and the larval pattern of GABAir populations is recognizable in prolarvae. A small GABAir cluster consisting mainly of CSF-c neurons was observed in the caudal preoptic area, and a wide band of scattered CSF-c GABAir neurons extended from the preoptic region to the caudal infundibular recess. A mammillary GABAir population was also distinguished. Two compact GABAir clusters, one consisting of CSF-c neurons, were observed in the rostral (ventral) thalamus. In the caudal (dorsal) thalamus, a long band extended throughout the ventral tier. The nucleus of the medial longitudinal fascicle contained an early-appearing GABAir population. The paracommissural pretectum of prolarvae and larvae contained a large group of non-CSF-c GABAir neurons, although it was less compact than those of the thalamus, and a further group was found in the dorsal pretectum. In the midbrain of larvae, several groups of GABAir neurons were observed in the dorsal and ventral tegmentum and in the torus semicircularis. The development of GABAergic populations in the lamprey forebrain was similar to that observed in teleosts and in mouse, suggesting that GABA is a very useful marker for understanding evolution of forebrain regions. The possible relation between early GABAergic cell groups and the regions of the prosomeric map of the lamprey forebrain (Pombal and Puelles [ 1999] J. Comp. Neurol. 414:391-422) is discussed in view of these results and information obtained with ancillary markers.

Proliferating cell nuclear antigen (PCNA) immunoreactivity and development of the pineal complex and habenula of the sea lamprey.

The development of the pineal complex and the habenula of the sea lamprey was studied with proliferating cell nuclear antigen (PCNA) immunocytochemistry. The pineal organ and the habenula primordia appeared in late embryos, and neuron differentiation began in prolarvae, as indicated by the presence of PCNA-negative cells. The parapineal primordium could not be distinguished until early prolarval stages, and cell differentiation was delayed to the larval period. Although the number of cycling (PCNA-immunoreactive) cells gradually decreased during the larval period in the three organs studied, their patterns of differentiation were different. We conclude that the unusual developmental pattern observed is related with the complex life cycle of lampreys.