Glochidial Release Research Articles

Physiological effects of interacting native and invasive bivalves under thermal stress

Across many ecosystems in North America and Europe, native freshwater bivalves (Order Unionida) are threatened by fouling and competition for food by the invasive zebra mussel Dreissena polymorpha. In light of climate change, knowledge on the influence of water temperature on these competitive effects is important, yet poorly understood. This study examines the physiological impact of the interaction between D. polymorpha and the native European unionid Anodonta cygnea over a 28 day—period in response to water temperatures of 12, 19, and 25 °C by comparing their glycogen, glucose, lipid and protein concentrations. The laboratory experiment comprised three treatments: (1) fouling of A. cygnea by D. polymorpha, (2) both species present but not fouling; and (3) a control in which A. cygnea and D. polymorpha were placed separately. Increased water temperatures caused physiological stress in D. polymorpha as evident from reduced glycogen, glucose, lipid and protein concentrations. Dreissena polymorpha benefited from fouling of unionids, as individuals that fouled A. cygnea tended to have increased glycogen, glucose, lipid and protein concentrations. Competitive effects of D. polymorpha over the unionid bivalve species, however, were not intensified by elevated temperatures. Glochidia release, lower infestation intensity, and physiological stress of Dreissena at higher temperatures were likely confounding factors. The results of this study suggest that understanding the physiological consequences of species interactions at changing temperatures can be an important tool to assess future climate change impacts on freshwater bivalves and aquatic community structures.

Comparative diversity in glochidia of Australasian freshwater mussels

Most freshwater mussels have larvae (glochidia in Unionidae, Margaritiferidae and Hyriidae) that are parasitic on fishes. This study describes and compares the diversity of glochidia among 17 species of Australasian Hyriidae. Here, scanning electron microscopy was used to illustrate shell morphology, while patterns of diversity in size, shape and morphological characteristics were analysed quantitatively and qualitatively with Principal Component, Linear Discriminant, and Multiple Correspondence Analyses to separate species with varying degrees of discrimination. Results showed shell lengths ranged from 50 to 390 μm. Shape varied, from sub-oval to sub-triangular, bilaterally symmetrical to scalene, and hook morphology varied from unicuspid, bicuspid, tricuspid or complex with varying length and structure. Unique observations of this study include the consistent variation in glochidial release mechanisms among Australian subfamilies and New Zealand genera. Hyridellini species and Echyridella aucklandica (Gray and Dieffenbach, 1843) are released either freely or as “mesoconglutinates” (presumed brood lures), whereas Velesunioninae and Echyridella menziesii (Gray and Dieffenbach, 1843) release glochidia in “amorphous mucous conglutinates”. Hyridellini predominantly occur within perennially flowing rivers of coastal south-eastern Australia with generally low turbidity, whereas the Velesunioninae occur more typically in slower flowing, intermittent waters, many prone to extended periods of high turbidity. Thus, where Hyridellini occur, mesoconglutinates as visual brood lures may be a more efficient mechanism for infesting host fishes than the passive infestation strategy typical of velesunionine species frequenting more turbid waters. Finally, this study presents systematic descriptions of glochidia and a provisional key for identification of Australasian hyriid glochidia, making an important contribution to the current understanding of taxonomy and life history traits, both critical for hyriid conservation.

Reproductive phenology and the influence of temperature in two sympatric New Zealand freshwater mussel species

Context Phenology plays a key role in shaping population dynamics, community structure and evolutionary adaptations. For freshwater mussels that rely on a parasitic larval (glochidia) phase on fish, shifts in reproductive phenology driven by environmental conditions may result in mismatches between glochidia release and host fish availability. Aims We investigated intra- and interspecific reproductive timing variations in sympatric Echyridella aucklandica and E. menziesii, and identified thermal cues (accumulated degree days, ADD) associated with brooding and glochidia maturation. Methods Brooding activity and glochidia maturation were assessed fortnightly–monthly over 1 year within four New Zealand streams. Results The previously unknown phenology of E. aucklandica showed earlier brooding (May–July) and longer gravidity (9–11 months) than for E. menziesii (August; 6–7 months). Both species exhibited peak brooding in late austral spring–summer (November–December). ADD played a key role in regulating the timing of brooding onset in both species, as evidenced by the narrow ADD range observed across sites, and the relationship between ADD and brooding onset in both species. Conclusion The demonstrated link between ADD and reproductive phenology has broad implications in the context of climate change. Specifically, it raises concerns about potential timing mismatches in glochidia release and host-fish availability, which could affect the survival and reproductive success of freshwater mussels.

Fishing for hosts: Larval spurting by the endangered thick-shelled river mussel, Unio crassus.

Fishing for hosts: Larval spurting by the endangered thick-shelled river mussel, Unio crassus.

Host Selection of Potomida semirugata (Unionidae: Bivalvia) in Reproduction Strategy

Potomida semirugata, which belongs to the Unionidae family, require a host fish to complete their life cycle. In this study, the host fish preference of this species was investigated. Besides, mussel species and hence the attachment state of larvae on the host fish were also investigated. Five different fish species namely Cyprinus carpio, Oreochromis niloticus, Clarias gariepinus, Anguilla anguilla, and Carasobarbus luteus were used as host candidates. In this study, five glass aquaria each containing 140 liters of water without any substrate were used and 3 mussels and 10 fish were placed in each aquarium. The aquaria were monitored for 120 days and the glochidia release on the fish was determined by examining gill tissue, dorsal and caudal fins by using a stereomicroscope. Results indicated that C. carpio and O. niloticus were more preferable in terms of mussel-host fish relationship. The best host fish was determined as C. carpio in terms of gill-fullness and juvenile mussel transformation at the ground of the aquarium. These observations provide significant insight and information regarding the effective culture of freshwater mussels.

Reproductive Phenology of Elliptio complanata in an Upper Susquehanna River Tributary of New York

Elliptio complanata (Eastern Elliptio) is typically an abundant species in lotic systems throughout the northeastern US; however, as with many other freshwater mussel species, some populations of Eastern Elliptio are in decline. Freshwater mussels have complex life cycles, which are important to understand for their conservation and management. The goal of this study was to determine the timing of Eastern Elliptio spawning, brooding, and glochidia release. Throughout the spawning season, we used gonad and gill extracts and drift nets to track the timing of reproduction in Otego Creek, NY, a tributary to the Susquehanna River. Females began brooding fertilized eggs by mid-May, and by early June, all females collected were brooding fertilized eggs or d-shaped glochidia. The temperature was 18 °C when all females contained glochidia. Peak glochidia-drift occurred ∼1 week after we recorded the highest levels of brooding and continued at low levels for several weeks. Phenology modeling helped us to determine that accumulated thermal units was the best predictor of reproductive activity. Our work highlights the environmental cues responsible for spawning, brooding, and glochidia release in a population of Eastern Elliptio. This empirical approach to predicting reproductive activity has great potential as a tool for timing the collection of brood stock for propagation or other important conservation measures.

The Effects of Seasonal Temperature and Photoperiod Manipulation on Reproduction in the Eastern Elliptio Elliptio complanata

The eastern elliptio Elliptio complanata is a species of freshwater mussel common to streams and rivers of the Atlantic Coast. Egg fertilization, larval brooding, and glochidial release are reported to occur within a period of several weeks during early to midsummer. In this study, mussels were exposed to manipulated photoperiod and water temperatures to prolong the availability of glochidia for use in artificial propagation and research. Brooding mussels were collected from Pine Creek, Tioga County, PA, in late December and were housed in groups subjected to one of four environmental treatments: natural temperature and photoperiod, 6-wk delay in natural conditions, 12-wk delay in natural conditions, and natural temperature and photoperiod with a winter low of 10°C. Reproductive activity was monitored for 1 y. Mussels subjected to natural conditions released mature glochidia between 16°C and 19°C with a photoperiod of 15 h of light. Temperature and photoperiod delays of 6 and 12 wk delayed reproduction proportional to the treatment, and constant 10°C winter low temperatures slightly shifted the timing of glochidial release. Survival during the study was high (96%–100%). Data indicate that the seasonal availability of E. complanata glochidia can be extended 3-fold using photoperiod and temperature manipulation.

Brooding and glochidia release in Margaritifera auricularia (Spengler, 1793) (Unionoida: Margaritiferidae)

We describe the gill anatomy of Margaritifera auricularia, including a histological analysis of the marsupium, its brooding capacity and the length of the maturation process, providing information on the hitherto unknown reproductive strategy of French populations. Ova and developing embryos from the Charente and Creuse river populations were found in March, with glochidial release occurring in April. Development from first cleavage to glochidial maturity took 25–37 days. Compared with the Spanish populations, the reproductive period begins some weeks later. The estimated number of larvae per gravid mussel was around 2,000,000. We expect the findings presented here will improve captive breeding projects that aim to conserve the few remaining populations of this endangered species in Europe.

Classes de comprimento e descrição histológica das gônadas de Diplodon ellipticus (Wagner, 1827) (Mollusca, Bivalvia, Hyriidae) em um lago artificial, Morretes, Paraná, Brasil

This study aimed to analyze the frequency by length classes and the histological organization of gonads of Diplodon ellipticus at an artificial lake located in the town of Morretes, Parana, Brazil. Six bimonthly sampling campaigns were conducted within the period from July 2009 to May 2010, with capture of 150 specimens. The total length of shells was measured for determining the frequencies of length classes and an analysis of the presence of marsupials in branchiae was performed. Sex determination was performed through histological analysis of cross sections of the visceral mass. We registered 8 length classes, with an interval of 9 mm, and the modal class varied between 50 and 59 mm. The population is predominantly dioecious, with occurrence of hermaphrodite specimens at low frequency. Marsupials were registered among females (86%) in all sampling campaigns and in 2 hermaphrodite specimens. Histological analyses showed that spermatogenesis and oogenesis occur over the year, indicating that D. ellipticus has a strategy that provides continuous reproduction, with peaks of glochidia release.

Vertical Migration and Reproductive Patterns of a Long-Term Brooding Freshwater Mussel, Villosa constricta (Bivalvia: Unionidae) in a Small Piedmont Stream

We delineated a permanent 15 m by 9 m reach of a mussel bed in a small piedmont stream in the Cape Fear River Basin of North Carolina, USA. A total of 14 surveys were conducted at the study site from May 2005 to September 2006 at time intervals ranging from 2 weeks to 3 months. The study area was divided into fifteen 1-m-wide transects, and each transect was thoroughly searched twice during each survey event for any mussels on the substrate surface. We recorded species identification, length, gravidity (for known females) and replaced the mussel in the exact spot it was found. A pilot study was conducted to determine detection success with one, two, and three passes per transect and detection success was monitored on all transects throughout the study. We estimate that two passes over these transects yielded approximately 90% of the mussels on the sediment surface. Vertical migration patterns of Villosa constricta, and in particular females, were highly seasonal. Additional within-season variation could not be explained by seasonal patterns alone. Larger individuals were recaptured more frequently. Female mussels became gravid from August through March indicating that spawning and glochidial release took place over an extended period. In 2005, glochidial release was 1-2 months later than in 2006 and lasted through June. In 2006, glochidial release began before 7 February in 2006 and lasted through April. Smaller V. constricta (23-28 mm) were more likely to be gravid, and about half of the individual females were observed to spawn in consecutive years.

Effects of temperature and photoperiod on lure display and glochidial release in a freshwater mussel

Freshwater mussels use an array of strategies to transfer their parasitic larvae (glochidia) to fish hosts. We examined the effects of temperature, photoperiod, and female gravidity on mantle lure display and conglutinate release by Ligumia subrostrata (Say, 1831) in 2 laboratory experiments. In the 1 st experiment, we examined the use of these strategies in 4 temperature treatments (5, 15, 25, 35uC) and 3 photoperiods (10:14, 12:12, and 14:10 h light: dark). In the 2 nd experiment, we observed infection strategies under ambient conditions with flow-through pond water. Water temperature appeared to be the primary cue governing use of these strategies. Lure display occurred over a protracted period, but the highest display frequency occurred between ,11 and 20uC. Lure display declined rapidly above this range and ceased altogether .28uC. Release of conglutinates increased coincident with the decrease in lure display but, at ambient temperatures, occurred over a protracted period similar in duration to lure display. Females that were not gravid at the beginning of the experiment did not display lures, and gravid females whose gills were flushed of glochidia displayed for only a short period during which frequency of display was much lower than gravid individuals. Ligumia subrostrata exhibits a temperature-mediated switch between alternate host strategies. We present evidence that lure display is a primary strategy for host infection and conglutinate release is a secondary, bet-hedging strategy to reduce wastage of glochidia that ultimately must be cleared from the gills at the end of the reproductive season.

Survival of Glochidial Larvae of the Freshwater Pearl Mussel, Margaritifera laevis (Bivalvia: Unionoida), at Different Temperatures: A Comparison Between Two Populations With and Without Recruitment

The viability of free-living glochidia of the freshwater pearl mussel (Margaritifera laevis) was studied in the laboratory at water temperatures of 10 degrees C, 15 degrees C and 20 degrees C. To obtain glochidia, gravid female mussels were collected from the Chitose River, inhabited by adult and juvenile mussels, and from the Abira River, where only adult mussels were found. Daily survival rates of glochidia from each population at various water temperatures were significantly different, and survival time was longest at the lowest temperature in each population. Maintenance of some field mussel populations might become difficult at higher water temperatures due to the short survival time of glochidia and expected low density of host fish. Daily survival rates of glochidia were compared between the Abira population at 15 degrees C and the Chitose population at 20 degrees C, since these temperatures were close to the mean water temperature during the period of glochidial release in the respective rivers. Daily mean survival rates were significantly different between the Abira population at 15 degrees C and the Chitose population at 20 degrees C. Mean glochidial survival rate for the Chitose population changed from 85.3% to 66.2% from 9 to 13 h, whereas that for the Abira population dropped suddenly from 80.4% to 34.2% from 10 to 14 h after the initiation of experiment. Absence of juveniles in the Abira River might have been caused by the low glochidial viability. Survival times of free-living glochidia in Margaritiferidae tend to be shorter than in other families in Unionoida. A trade-off is suggested between high fertility and low glochidial survival rate in Margaritiferidae.

Reproduction based on local patch size of Alasmidonta heterodon and dispersal by its darter host in the Mill River, Massachusetts, USA

Abstract We studied the reproduction and shell size structure of the endangered dwarf wedgemussel (Alasmidonta heterodon) in the Mill River, Massachusetts, at sites of different mussel abundance, and assessed the dispersal capacity of its host fish, the tessellated darter (Etheostoma olmstedi). We quantified 4 phases of reproduction at 5 study sites of contrasting adult mussel abundance (3 low-abundance sites, 1 intermediate, 1 high), including gravidity, glochidial release, host infection rate, and juvenile recruitment. The ratio of gravid to nongravid individuals was higher at the high-abundance site, and comparatively lower at the intermediate- and low-abundance sites. Glochidial release, infection rates on host darters, and juvenile recruitment were directly proportional to mussel abundance. The highest glochidia density (0.12/m3) occurred at the high-abundance site. Mean infection rates during early May to late June ranged from 31% at the high-abundance site to 8% at the intermediate- and 0 to 2% at ...

The identity and biology of unio mancus laMarck, 1819 (=U. Elongatulus) (bivalvia: unionidae) in the Iberian Peninsula

Genetic variability, shell morphology, anatomy, reproductive season, glochidium morphology and host fish of several Iberian populations of Unio mancus Lamarck, 1819 (=U. elongatulus C. Pfeiffer, 1825) were examined. The genetics of three allopatric populations were studied, including two ‘races’ previously described for this species on the Iberian Peninsula. All shared the same haplotype of the COI mitochondrial gene. It is proposed that all known Iberian Mediterranean populations of the genus Unio correspond to Unio mancus. All mature specimens studied were dioecious. Glochidial release occurred between March and August. The barbel Barbus graellsii was the natural host of the U. mancus glochidia in the studied area. In artificial infestations performed in aquaria, seven fish species, all native, carried the glochidia throughout their complete metamorphosis. This process occurred in 141 degree/days. Newly released juveniles were spherical and had a thin marginal rim of new shell.

Timing of spawning and glochidial release in Scottish freshwater pearl mussel (Margaritifera margaritifera) populations

Summary1. The timing of reproduction was investigated in six Scottish freshwater pearl mussel populations from 1993 to 2002. Gravid females were examined and the release of mussel larvae (glochidia) was monitored.2. Annual spawning (oviposition) and spat (glochidial release) events occurred during June to July and June to September, respectively.3. Between‐river differences in timing seem to be related to water temperature. Mussels in the warmest rivers tend to spawn and spat first, and vice‐versa.4. Thermal variations also seem to influence the timing of reproduction within rivers, which can be delayed by several weeks during cold years. At least 3000°‐days occur between annual episodes of glochidial release.5. The timing of spawning is determined gradually, probably by a thermal summation effect.6. The release stage occurs as a sudden, synchronised event, with most of the glochidia spat over 1–2 days, indicating that it is triggered by an environmental cue. Sudden changes in water temperature and/or river level often result in spats, and the underlying mechanism may be respiratory.

Host Fishes and Infection Strategies of Freshwater Mussels in Large Mobile Basin Streams, USA

We investigated host fishes, timing and modes of glochidial release, and host-attraction strategies for 7 species of freshwater mussels from the Buttahatchee and Sipsey rivers (Mobile Basin), Alabama and Mississippi, USA. We determined hosts as fish species that produced juvenile mussels from laboratory-induced glochidial infections. We established the following primary mussel/host relationships: Elliptio arca with Etheostoma artesiae and Percina nigrofasciata; Fusconaia cerina with 6 species of minnows (Cyprinidae); Lampsilis ornata with Micropterus salmoides; Medionidus acutissimus with 8 species of darters (Percidae); Obovaria unicolor with Ammocrypta beani, A. meridiana ,a ndEth- eostoma artesiae; Pleurobema decisum with Cyprinella venusta ;a ndQuadrula asperata with Ictalurus punc- tatus. For most mussel species, host use was similar to that of closely related species, indicating that, in general, this trait is highly conserved at the generic level. Four mussel species used host-attraction strategies that targeted their specific host fish. Fusconaia cerina and P. decisum released glochidia in conglutinates that elicited feeding responses from fishes in the field and in the laboratory. Gravid female Lampsilis ornata and M. acutissimus displayed mantle lures. Host-attraction strategies were less apparent for E. arca and Q. asperata, but these species released glochidia in association with copious mucous secretions, which may serve to entangle fishes, facilitating host infection. No host-attraction strategy was apparent for O. unicolor.

GILL EVACUATION AND RELEASE OF GLOCHIDIA BY UNIO PICTORUM AND UNIO TUMIDUS (BIVALVIA: UNIONIDAE) UNDER THERMAL AND HYPOXIC STRESS

This study aimed to elucidate the mechanism of glochidial release in Unio pictorum (L., 1758) and U. tumidus Philipsson, 1788, and thus interpret the confused reports of unionine reproduction in the literature. Both species release mature, hooked glochidia on mucous threads, resembling a pearl necklace. Individual threads can reach 15 cm in length and carry c. 500 glochidia. Under hypoxic conditions both species released conglutinate-like structures comprising c. 500 eggs and immature, unhooked glochidia. The conglutinate-like structures released by U. pictorum and U. tumidus are coloured yellow and white, respectively. Increased temperature did not induce the release of conglutinates. The conglutinate-like structures are unlikely to be involved in increasing reproductive success by attracting host fishes. Rather, the premature evacuation of the gill contents may relieve the respiratory burden on adult mussels under hypoxic stress. Ambient hypoxia during the reproductive season may help explain the irregular recruitment success of relatively long-lived mussels, such as U. pictorum and U. tumidus.

<i>Margaritifera auricularia</i> (Unionoidea, Margaritiferidae), the giant freshwater pearl mussel rediscovered in Spain

Margaritifera auricularia was described by Spengler in 1793. Since then specimens have been found in several localities in Western Europe and North Africa, often as fossils. However no living specimens have been scientifically recorded since 1917 (Haas, 1917). This author was the only one to study live specimens, collected in the Ebro River in Spain, publishing a short paper about its anatomy (Haas, 1924). As no more specimens were ever found, no data exist about its reproductive habits, spawning season nor is there a description of its larval stages. In a recent survey of Spanish Rivers to inventory the Habitats Directive (Directive 92/43/CEE) non-insect invertebrate species (Ramos, 1998), we found living populations of this giant pearl mussel in a very ancient channel (Imperial Canal) of the Ebro River (Araujo & Ramos, 1996) and in the Ebro (Alvarez Halcon, 1998), both in Aragon. More recently, there has been news about other M. auricularia populations living in the lower course of the Ebro river and in one of its irrigation channels (Altaba, 1997), both in Catalonia. An interesting theory exists about the specificity between M. auricularia and the Western European sturgeon Accipenser sturio (Linnaeus, 1758), a relict fish in European rivers and extinct in Spain. Both species occur together in Pleistocene deposits (Preece, 1988) and both have been declining since the first half of this century. With this theory in mind, we introduced several sturgeons into an aquarium with mussels, realizing that all mussels began to spawn. In the aquarium the mussels released all their larval stages, from unfertilized eggs to mature glochidia, passing through all intermediate stages. The glochidium of M. auricularia is very small (about 140 microns) and presents very small teeth. It is of the hookless type, as in other known species of the genus (Araujo & Ramos, 1998). This kind of glochidium specifically attaches itself to the gill filaments of the fish, whereas the hooked ones, typical of other genera, also attach themselves to the fins. Inmediately after the glochidia were released, we observed how they attached to the fish gills. We excised the gill filaments of the sturgeons 1, 2, 3, 4, 5 hours and 2, 3, 5, 13, 34 and 60 days after the begining of glochidia release. Now we are studying histological and scanning samples of these gill filaments in order to investigate the encystment process and to discover the period the glochidia need to complet their metamorphosis. Timing and length of the glochidial release period in the wild has been determined by collecting drift samples. With these data, fish were collected by electrofishing in order to investigate their susceptibility to the glochidia. The large size of all the specimens from the Imperial Canal population and the longevity achieved by species of this genus (around 100 years) suggest that this population may not have reproduced for many years. This leads us to ask wether the species has lost its reproductive capability, if it is unable to complete larval maturation or do the juveniles simply have a different habitat that we have not been able to find up to now? In situ observations suggest that the species should be more common, as occurs with other related genera (i. e. Unio, Psilunio, Anodonta) in the study area, if any kind of fish were compatible with

Host Fishes and Reproductive Biology of 6 Freshwater Mussel Species from the Mobile Basin, USA

Host fishes were identified for 6 species of freshwater mussels (Unionidae) from the Black Warrior River drainage, Mobile Basin, USA: Strophitus subvexus, Pleurobema furvum, Ptychobranchus greeni, Lampsilis perovalis, Medionidus acutissimus, and Villosa nebulosa. Hosts were determined as those that produced juvenile mussels from glochidial infestations in the laboratory. The following mussel-fish-host relationships were established: Strophitus subvexus with 10 species including Cyprinidae, Catostomidae, Fundulidae, Centrarchidae, and Percidae; Pleurobema furvum with Campostoma oligolepis, Cyprinella callistia, C. venusta, Semotilus atromaculatus, and Fundulus olivaceus; Ptychobranchus greeni with Etheostoma bellator, E. douglasi, Percina nigrofasciata, and Percina sp. cf. caprodes; Lampsilis perovalis with Micropterus coosae, M. punctulatus, and M. salmoides; Medionidus acutissimus with Fundulus olivaceus, Etheostoma douglasi, E. whipplei, Percina nigrofasciata, and Percina sp. cf. caprodes; and Villosa nebulosa with Lepomis megalotis, Micropterus coosae, M. punctulatus, and M. salmoides. Fundulus olivaceus served as host for 3 species and carried glochidia for long periods for 2 other species, suggesting that topminnows may serve as hosts for a wide variety of otherwise host-specialist mussel species. Host relationships for the species tested are similar to congeners. Methods of glochidial release, putative methods of host-fish attraction, and gravid periods are described for the 6 species.

Reproductive Biology of Four Species of Freshwater Mussels (Molluscs: Unionidae) in the New River, Virginia and West Virginia

ABSTRACT The gametogenic cycle, spawning and glochidial release periods, and age at sexual maturity were determined for four unionid species from the New River in Virginia and West Virginia: mucket, Actinonaias lieamentina; spike, Elliptio dilatata; purple wartyback Cyclonaias tuberculata; and pistolgrip, Tritogonia verrucosa. The mucket is a long-term brooder, spawning in mid-summer, brooding glochidia throughout fall and winter, and releasing them in spring. The spike, purple wartyback, and pistolgrip are short-term brooders. Spawning began in mid-March and continued into May for T. verrucosa, into June for C. tuberculata, and into July for E. dilatata. Glochidia were released upon maturation, beginning in mid-April and continuing through June for T. verrucosa, into August for E. dilatata, and extending from March through June for C. tuberculata. All four species are sexually mature at 4 to 6 years of age.