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This paper has integrated new and past data to elucidate how lipid, protein and glycogen metabolism contribute to generating the ATP required by the southern hemisphere lamprey Geotria australis during its ~ 13-15 months, non-trophic upstream spawning migration. Energy is required for maintenance, swimming, the development of gonads and secondary sexual characters and spawning and post-spawning activities. Plasma and muscle metabolites were measured in animals subjected to an exercise-recovery regime at the commencement and completion of the spawning run. The present study demonstrated the following. At all stages of the migration, plasma glucose and glycerol concentrations increased during exercise and then declined, whereas plasma FFAs exhibited the reverse trend. During exercise and recovery, alanine declined and ammonia increased in the plasma of early migrants, while the opposite occurred in mature males. Following exercise, muscle alanine rose and then declined in early migrants, but declined and then rose in mature males. The composite data emphasise that, while the same catabolic processes are employed by both sexes early in the migration, when animals are immature, they differ markedly between the sexes as they mature and then spawn, reflecting their different demands. Energy is supplied predominantly via anaerobic metabolism in early migrants, but by anaerobic and aerobic metabolism in prespawning females and by aerobic metabolism in mature males and spent females. Although proteolysis is limited early in the migration, it is employed extensively during maturation and particularly by females, which undergo a substantial reduction in length in the lead-up to spawning.

The pouched lamprey, Geotria australis Gray, 1851, has long been considered monotypic in the Geotriidae family with a wide southern temperate distribution across Australasia and South America. Recent studies have provided molecular and morphological evidence for a second Geotria species in South America; Geotria macrostoma (Burmeister, 1868). The aim of this study was to determine morphometric and physical characteristics of adult G. macrostoma that further differentiate this re-instated species of Geotriidae from G. australis. The diagnostic features discriminating immature adult G. macrostoma from G. australis when entering fresh water, are distinct differences in dentition, oral papillae and fimbriae counts and differences in coloration. In addition, G. macrostoma display greater growth of the prebranchial region and oral disc and has a deeper body depth and higher condition factor. All current ecological knowledge of the genus Geotria is based on Australasian populations, which may not be applicable to G. macrostoma. To ensure the conservation and protection of the Patagonian lamprey as a re-identified species, further investigations are needed to understand its life history, biology and ecology throughout its range.

Lampreys are extant members of the agnathan (jawless) vertebrates that diverged ~500 million years ago, during a critical stage of vertebrate evolution when image-forming eyes first emerged. Among lamprey species assessed thus far, the retina of the southern hemisphere pouched lamprey, Geotria australis, is unique, in that it possesses morphologically distinct photoreceptors and expresses five visual photopigments. This study focused on determining the number of different photoreceptors present in the retina of G. australis and whether each cell type expresses a single opsin class. Five photoreceptor subtypes were identified based on ultrastructure and differential expression of one of each of the five different visual opsin classes (lws, sws1, sws2, rh1, and rh2) known to be expressed in the retina. This suggests, therefore, that the retina of G. australis possesses five spectrally and morphologically distinct photoreceptors, with the potential for complex color vision. Each photoreceptor subtype was shown to have a specific spatial distribution in the retina, which is potentially associated with changes in spectral radiance across different lines of sight. These results suggest that there have been strong selection pressures for G. australis to maintain broad spectral sensitivity for the brightly lit surface waters that this species inhabits during its marine phase. These findings provide important insights into the functional anatomy of the early vertebrate retina and the selection pressures that may have led to the evolution of complex color vision.

The Ukrainian brook lamprey Eudontomyzon mariae is the most widespread lamprey species in eastern Europe. Although E. mariae is generally considered a derivative of Eudontomyzon danfordi, an exclusively freshwater parasitic species, it has alternatively been suggested that it was recently derived from a now extinct anadromous Black Sea ancestor. Several non-parasitic lampreys and the landlocked sea lamprey, which have recently evolved from anadromous ancestors, still develop a seawater-type mitochondria-rich cell (SW-MRC) in their gills. In contrast, this cell type is not present in the gills of either Lampetra aepyptera, a non-parasitic lamprey of ancient origin, or the parasitic Ichthyomyzon unicuspis and I. castaneus that likewise have long evolutionary histories in fresh water. Eudontomyzon mariae from the Vistula River in the Baltic River basin does not possess SW-MRC, which is inconsistent with a recent origin from an anadromous ancestor. Mitochondrial DNA sequence data were thus used to infer the relationship between different populations of E. mariae and E. danfordi, and to reconstruct the transition from anadromy to freshwater residency. The results suggest that E. mariae evolved independently in the Baltic, Black, and Caspian Sea basins, and not recently from an anadromous ancestor. Although E. mariae in the Danube River may have arisen relatively recently from E. danfordi (differing by 0.7–1.1% in cytochrome b gene sequence), other E. mariae populations (including in the Vistula River) are genetically closer (0.6%) to the hypothetical ancestor of both E. mariae and E. danfordi. That ancestor was probably a freshwater resident, since SW-MRCs are not rapidly lost following confinement in fresh water.

Despite their highly conserved body plan and larval stage, adult life history type in lampreys diverges on two main axes related to migration and feeding. Of the 41–45 recognized lamprey species, 18 species feed parasitically after metamorphosis and their juvenile (sexually immature) feeding phase lasts from 3–4 months to 2–4 years. Nine of these species are exclusively freshwater resident; five are exclusively or almost exclusively anadromous, and four (sea lamprey, European river lamprey, Arctic lamprey, and, to a lesser extent, Pacific lamprey) are largely anadromous but with established freshwater populations. The other 23–27 described species are non-parasitic “brook” lampreys which remain within their natal streams. They initiate sexual maturation during metamorphosis, and, because the non-trophic periods of metamorphosis and sexual maturation are superimposed, the parasitic feeding phase is eliminated; this makes them the only vertebrates known to have non-trophic adults. Body size at maturity varies dramatically among life history types, ranging from ~110 to 150 mm total length (TL) in non-parasitic species to 800–900 mm TL in the anadromous sea lamprey. Freshwater forms are typically intermediate in size, although those that inhabit small systems may be no larger than non-parasitic lampreys and others (particularly the Great Lakes sea lamprey) are quite large. Some anadromous species (most notably European river lamprey, Pacific lamprey, and Arctic lamprey) show considerable intraspecific variation, consisting of typical large-bodied forms and dwarf or “praecox” forms that appear to feed at sea for a reduced period of time. Establishment in fresh water is more common in species that are consistently small-bodied or those with praecox forms. The only exceptions are the very small-bodied western river lamprey (mean TL at maturity ~200 mm), which does not produce freshwater parasitic forms (although it has given rise to innumerable non-parasitic freshwater populations), and the sea lamprey which, despite its very large size, has successfully colonized the Great Lakes. Abundant prey of a suitable size range is critical for establishment of freshwater parasitic populations. However, even with abundant prey, abandonment of anadromy is expected only under circumstances where decreases in mortality and the costs associated with migration make the reduction in size at maturity, and the accompanying reduction in fecundity, worthwhile. Pacific lamprey generally fail to establish when isolated above recently constructed barriers, likely because the reservoirs in which they have been isolated are relatively small and because they appear to osmoregulate poorly in fresh water. However, because colonization of fresh water appears to select for individuals “pre-adapted” to feed and grow to maturity in fresh water (i.e., relying on existing genetic variation within the source population), probability of establishment would likely increase with the number of founders. The existence of three closely related freshwater parasitic species suggests that Pacific lamprey successfully colonized fresh water in the past. Whether sea lamprey colonized Lake Ontario and Lake Champlain post-glacially or in historic times is debated. At present, the “invasion-by-canal” hypothesis appears to be the most convincing, but definitive resolution should be possible with genome-level analyses. Given the decimation of the Great Lakes ecosystem by sea lamprey, it is critical to be able to predict the potential for anadromous lampreys to become invasive in other freshwater systems. Migratory type is rarely considered a species-specific character unless it is accompanied by identifiable morphological differences. In contrast, variability in feeding type has long been considered a species-specific character because size-assortative mating was thought to result in reproductive isolation between parasitic and non-parasitic forms. However, not all parasitic and non-parasitic forms appear to be reproductively isolated, and different species show different degrees of divergence from their presumed parasitic ancestor. “Paired” non-parasitic species are defined as those that are morphologically similar to a particular parasitic species in all aspects other than body size, and “relict” brook lampreys are those that cannot be obviously paired with extant parasitic forms. However, molecular analyses have: (1) identified the closest extant parasitic relative to these relict species (although a few “orphan” species still remain, where identification of the closest living relative still sheds little light on the identity of the parasitic ancestor); (2) shown that the distinction between paired and relict species is sometimes unclear; and (3) demonstrated that there is also considerable variation among paired species in the degree to which they are morphologically and genetically differentiated from their parasitic ancestors. We review this “speciation continuum,” particularly in European river and brook lamprey populations where recent genetic and genomic studies show significant gene flow between these species where they co-occur (i.e., refuting assumptions of complete reproductive isolation resulting from size-assortative mating) while also showing that there are genome-level differences between the feeding types (i.e., refuting the hypothesis of phenotypic plasticity). In sympatry, European river and brook lampreys appear to be partially reproductively isolated ecotypes that nevertheless maintain distinct phenotypes, because regions of the genome involved in reproductive isolation and local adaptation resist the homogenizing effect of introgression. Interestingly, the results of analyses used to reconstruct the demographic history of divergence in this species pair are inconsistent with recent and rapid divergence in sympatry following the recent glacial retreat; rather, they support divergence in allopatry ~200,000–250,000 years ago and re-establishment of secondary contact ~90,000 years ago. Some loci have been identified that differ between the forms (e.g., the vasotocin and gonadotrophin-releasing hormone 2 precursor genes), but an understanding of the genetic basis of life history evolution in lampreys remains elusive. There appear to be strong parallels between factors that promote or constrain loss of anadromy in parasitic species (and reduction in duration of the feeding phase) and those that lead to or limit the evolution of non-parasitism (i.e., total elimination of the feeding phase). Smaller-bodied parasitic species have been far more prolific in producing non-parasitic derivatives than others; western river lamprey are already so small at maturity that “skipping” right to a non-parasitic form represents a more profitable trade-off between mortality and fecundity than freshwater parasitism. In contrast, there is a conspicuous absence of brook lamprey derivatives from the large-bodied sea, pouched, and Caspian lampreys. Our comparisons suggest that 300 mm TL (with a 10–12× reduction in fecundity) is the cut-off above which shifts to non-parasitism would not be beneficial. Therefore, we predict that Great Lakes sea lamprey is not an “intermediate” freshwater parasitic form that will give rise to a non-parasitic derivative, because complete elimination of the parasitic feeding phase would represent too large (~40×) a reduction in fecundity.

The anadromous Geotria australis, one of only three lamprey species representing the early agnathan (jawless) stage of vertebrate evolution in Australia and New Zealand, is declining in abundance. Its adults were caught soon after they had entered rivers on their non-trophic upstream migration and maintained in laboratory tanks for 13–15 months through to spawning. As adult G. australis are susceptible to haemorrhagic septicaemia, they were treated prophylactically and maintained in 3-m3 aquaria supplied with a flow-through charcoal filtration system and UV steriliser. Air temperature and the light:dark regime were constantly adjusted to parallel those in the environment. Males developed the very large suctorial disc and gular pouch characteristic of maturity and both sexes matured at the same time as in the wild. While males frequently showed aggressive behaviour towards each other, the same male and female mated on several occasions. The male coiled around the female and, with his urogenital papilla close to the female’s cloaca, twisted and vibrated, leading to egg release. These eggs formed coagulated clusters as in the wild, with many progressing through to the eight-cell stage. Remarkably, numerous G. australis were still alive 95–392 days after the end of the short spawning period, and one male after a further 119 days. Postspawning survival would be facilitated inter alia by extensive proteolysis, reflected in a shortening of the body. The data in this paper emphasise that G. australis is a highly atypical lamprey and provides invaluable information for conserving this declining species.

This study demonstrates that the silver technique of Grimelius (Acta Soc. Med. Ups. 73:243–270, 68) is ideally suited for the study of cardiovascular chromaffin cells in lampreys. This method showed that in the Southern Hemisphere lamprey, Geotria australis, the distribution of chromaffin cells differs from that described for holarctic species. In G. australis, the chromaffin cells are found mainly in the sinus venosus, atrium, and nearby regions of the cardinal and jugular veins, and they are absent from the ventricle and conus arteriosus. The location and discreteness of the large accumulation of chromaffin cells in the lateral wall of the right posterior cardinal vein of adults resemble those of the precardiac axillary bodies of elasmobranchs. Chromaffin cells become more abundant during metamorphosis. The possible phylogenetic and functional significance of lamprey chromaffin cells is briefly discussed.

The morphology, cell types, and innervation of the several small papillae (x̄ = 17) and two larger papillae, which together form a ring just outside the fimbriae surrounding the suctorial disc of adult Geotria australis, have been studied using various histological stains, including silver impregnation, and scanning and transmission electron microscopy. The epithelium of all papillae consists almost entirely of mucigenic cells. The multivillous and oligovillous cells, which are found elsewhere in the lamprey epidermis, were not observed, and Merkel and polyvillous cells are rare. Free nerve endings are common, however, in the basal layers of the epidermis. Unlike the small papillae, the two large papillae contain a core of skeletal muscle and a prominent layer of dermal collagen. In the submucosa of these large papillae, the nerves form a dense, compact layer that contains many large and probably sensory axons. It is suggested that the oral disc papillae of adult G. australis are encapsulated mechanosensory structures that play a role in enabling the animal to locate and attach to a suitable point on host fishes or other surfaces.

Lampreys are one of two extant representatives of the earliest group of vertebrates, the agnathans or jawless fishes. The single species of the southern hemisphere lamprey family Geotriidae, Geotria australis, possesses the potential for pentachromatic color discrimination opposed to the mono- or dichromacy found in other lampreys. However, little is known of the retinal ganglion cell types that contribute to visual processing in G. australis. A quantitative morphological approach was used to distinguish and describe retinal ganglion cell types in G. australis. The morphology of retinal ganglion cells was revealed by retrograde biocytin labeling from the optic disc. Cells were digitally reconstructed, and somatic area and position and dendritic field size, density, tortuosity, and stratification were subjected to quantitative morphometric analyses. Cluster analysis, in conjunction with similarity profile analysis (SIMPROF), statistically identified five discrete monostratified retinal ganglion cell types, one of which may comprise two subtypes. Two bistratified types were identified separately, including a biplexiform and a bistratified subtype. The use of cluster analysis with SIMPROF provided a robust statistical technique for objectively identifying cell types whose characteristics were similar and significantly different from those of other types and thus provides an objective resolution of the problems posed by "lumpers vs. splitters" when designating cell types. The diversity of retinal ganglion cells suggests that visual information in the lamprey G. australis is processed in parallel streams, as in gnathostomes. These findings, together with the results of previous studies, indicate that the visual system of the lamprey G. australis represents the upper limit of visual complexity in extant agnathans.

The intestinal contents of adults of 12 of the 18 species of parasitic lampreys were examined microscopically and tested for blood to determine whether a species fed mainly on blood, flesh or blood and flesh. The diets of each species are shown to be related to characteristics of their dentition, buccal glands and velar tentacles. The trends exhibited by those relationships were used to hypothesize as to the diets of those six species for which there were anatomical but not dietary data. The dentition aids the suctorial disc in attaching the lamprey to its host and removing host material, while velar tentacles prevent material entering the branchial cavity and buccal glands produce lamphredin that has anticoagulant and lytic properties. In blood feeders, such as Petromyzon marinus and Mordacia species, the w-shaped transverse lingual lamina and hook-shaped longitudinal laminae bear numerous, fine cusps, which are ideal for rasping a hole in the host. In contrast, in flesh feeders, such as Lampetra fluviatilis and Geotria australis, the transverse lingual lamina is u shaped and the longitudinal laminae are straighter and possess at least one stout cusp, thereby facilitating the removal of host flesh through gouging. The buccal glands are generally larger in blood feeders than flesh feeders, presumably reflecting a need to produce anticoagulant continuously. Each velar tentacle contains a central cartilaginous rod, surrounded by a space which, during feeding, becomes engorged with blood and thus more rigid. They are small (≤1 mm long) and few (2-6) in blood feeders and large (typically ≥2 mm long) and numerous (3-42) in flesh feeders, which, in particular, require a mechanism for preventing solid material entering the branchial pouches and thus potentially clogging the gills. On the basis of recent cladistic analysis, blood feeding is ancestral to flesh feeding in Northern Hemisphere lampreys (Petromyzontidae).

Very few studies have described brain scaling in vertebrates throughout ontogeny and none in lampreys, one of the two surviving groups of the early agnathan (jawless) stage in vertebrate evolution. The life cycle of anadromous parasitic lampreys comprises two divergent trophic phases, firstly filter-feeding as larvae in freshwater and secondly parasitism as adults in the sea, with the transition marked by a radical metamorphosis. We characterized the growth of the brain during the life cycle of the pouched lamprey Geotria australis, an anadromous parasitic lamprey, focusing on the scaling between brain and body during ontogeny and testing the hypothesis that the vast transitions in behavior and environment are reflected in differences in the scaling and relative size of the major brain subdivisions throughout life. The body and brain mass and the volume of six brain structures of G. australis, representing six points of the life cycle, were recorded, ranging from the early larval stage to the final stage of spawning and death. Brain mass does not increase linearly with body mass during the ontogeny of G. australis. During metamorphosis, brain mass increases markedly, even though the body mass does not increase, reflecting an overall growth of the brain, with particularly large increases in the volume of the optic tectum and other visual areas of the brain and, to a lesser extent, the olfactory bulbs. These results are consistent with the conclusions that ammocoetes rely predominantly on non-visual and chemosensory signals, while adults rely on both visual and olfactory cues.

Although confined to fresh water, non-parasitic species of lampreys and the landlocked parasitic sea lamprey, all of which were derived relatively recently from anadromous ancestors, still develop chloride cells, whose function in their ancestors was for osmoregulation in marine waters during the adult parasitic phase. In contrast, such cells are not developed by the non-parasitic least brook lamprey Lampetra aepyptera, which has been separated from its ancestor for >2 million years, nor by the freshwater parasitic species of the genus Ichthyomyzon. The length of time that a non-parasitic species or landlocked parasitic form or species has spent in fresh water is thus considered the overriding factor determining whether chloride cells are developed by those lampreys.

The lampreys (Petromyzontiformes), one of the two surviving groups of agnathan (jawless) vertebrates, currently consist of 41 recognized species. This group has an antitropical distribution, with the 37 species of Northern Hemisphere lampreys assigned to the Petromyzontidae, whereas the four species of Southern Hemisphere lampreys are separated into either the Geotriidae (one species) or Mordaciidae (three species). All lamprey species have a blind and microphagous, burrowing larva (ammocoete), which spends a number of years in the soft sediment of creeks and rivers, after which it undergoes a radical metamorphosis. Eighteen lamprey species then embark on an adult parasitic phase (nine at sea and nine in fresh water) during which they increase markedly in size, whereas the other 23 species do not feed as adults and remain in fresh water. On the basis of morphology, 17 of the 23 non-parasitic species each evolved from a particular parasitic species whose descendants are still represented in the contemporary fauna. The remaining six non-parasitic species, the so-called “southern relict” species, have no obvious potential ancestral parasitic species, implying they have diverged markedly from their parasitic ancestor or that the parasitic ancestor is now extinct. Many of the main taxonomic characteristics reside in features that are associated with parasitic feeding, for example, the type and arrangement of the teeth on the suctorial disc and tongue-like piston. The phylogenetic relationships, derived by maximum parsimony analyses of morphological and anatomical data for the 18 parasitic species, were similar in most respects to those obtained by subjecting molecular data (cytochrome b mitochondrial DNA sequence data) for those species to Bayesian analyses. However, in contrast to the results of morphological analyses, the genera Eudontomyzon and Lampetra were not monophyletic when using molecular analyses. When non-parasitic species were included in the molecular analyses, some of the six relict non-parasitic species formed clades with parasitic species which, from their morphology, had been allocated by taxonomists to different genera. More genes, and particularly nuclear genes, should be used to help resolve the basis for these differences between the morphological and molecular phylogenies.

This study explored the distribution of parathyroid hormone-related protein (PTHrP) and its mRNA in tissues of the lamprey Geotria australis, a representative of one of the two surviving groups of an early and jawless stage in vertebrate evolution. For this purpose, antibodies to N-terminal and mid-molecule human PTHrP were used to determine the locations of the antigen. Sites of mRNA production were demonstrated by in situ hybridisation with a digoxigenin-labelled riboprobe to exon VI of the human PTHrP gene. The results revealed that antigen and its mRNA were widely distributed among similar sites of tissue localisation to those described for mammalian and avian species. However, some novel sites of localisation, such as in the gill and notochord, were also found. Some differences in PTHrP localisation were noted among individuals at different intervals of the life cycle, indicating that the distributions of PTHrP, and possibly its roles, change with the stage of development in this species. The widespread tissue distribution in G. australis implies diverse physiological roles for this protein. The presence of PTHrP in the lamprey, a representative of a group of vertebrates, which apparently evolved over 540 million years ago, strongly suggests that it is a protein of ancient origin. In addition, the successful use of antibodies and probes based on the human sequence in the lamprey also provides evidence that the PTHrP molecule may have been conserved from lampreys through to humans.

Lampreys, together with hagfishes, are the sole survivors of the jawless (agnathan) stage in vertebrate evolution. The 34 species of northern hemisphere lampreys are placed in the Petromyzontidae, whereas each of the four species of southern hemisphere lampreys are separated into either the monospecific Geotriidae or monogeneric Mordaciidae. Eighteen species feed parasitically as adults in either marine or fresh waters, whereas 20 species are nonparasitic and have apparently evolved from parasitic species. This account of adaptive radiation is based on the hypothesis that the basal stock of contemporary lampreys possessed many characters that were similar to those of contemporary Ichthyomyzon and Petromyzon species, and that the Mordacia species and Geotria australis have been separated for a long period. Most of the morphological differences among the adults of northern hemisphere lampreys (i.e., variations in dentitional arrangement and size of buccal glands and velar tentacles) are related to whether the species feeds mainly on blood, as with Ichthyomyzon and Petromyzon species, or predominantly on flesh, as with the more derived Lampetra species. Mordacia species have apparently remained blood feeders, while G. australis has become a flesh feeder. The eyes of the adults of southern hemisphere lampreys are unique amongst lampreys, with those of G. australis possessing two types of cone photoreceptor (as well as a rod photoreceptor) and an irideal flap, while those of Mordacia mordax possess only a rod photoreceptor but contain a tapetum. These differences represent adaptations to life in bright surface waters by G. australis and to diurnal burrowing and nocturnal activity by Mordacia species. The restriction of larval intestinal diverticula to Mordacia species and G. australis may represent adaptations for increasing the ability to digest the particularly tough plant material that is found in southern hemisphere rivers. The bile duct of the larvae of southern hemisphere lampreys enters the apex of the above intestinal diverticula and thus, unlike the situation in northern hemisphere lampreys, does not play a role in forming the endocrine pancreas of adult lampreys during metamorphosis. This accounts for the absence of intermediate and caudal pancreatic cords in the adults of southern hemisphere species. The amino acid sequences of intestinal molecules in different lamprey taxa support the view that the lampreys are monophyletic and that polyploidy occurred early in or just prior to their evolution.

The phylogenetic relationships between the two southern hemisphere lamprey families (Geotriidae and Mordaciidae) and their northern hemisphere counterparts (Petromyzontidae) are unresolved. Insulin was isolated from an extract of islet-containing intestinal tissue from ammocoetes of the Australian lamprey, Mordacia mordax. Its primary structure was established as A-chain: GIVEQCCHRK10CSIYDMENYC20N and B-chain: SALMGTGGTH10LCGSHLVEAL20YVVCGQRGFF30 YTP[SKG]. Although the residues in parentheses are only tentatively assigned, mass spectrometry supports the proposed sequence and demonstrates that Mordacia proinsulin, unlike proinsulin from Geotria australis, is fully processed to mature insulin. Insulins from M. mordax and G. australis and from the northern hemisphere lampreys Petromyzon marinus and Lampetra fluviatilis share a pentapeptide extension to N-terminus of the B-chain (Ser-Ala-Leu-Xaa-Gly) that has never been found in the insulins of any other vertebrate class. This observation provides support for the claim that the Petromyzontiformes constitute a monophyletic group. M. mordax insulin differs from that of G. australis by 18 amino acid residues but by only four residues from the common sequence of P. marinus and L. fluviatilis insulin. These data are consistent with the view that Geotriidae and Mordaciidae have been separated for a long period and suggest that G. australis insulin has undergone an accelerated rate of molecular evolution.

The concentration of total non-haem iron and its ferritin iron component, and the activity of the enzyme superoxide dismutase (SOD), were measured in the livers of ammocoetes, metamorphosing animals (stages 1-7) and recently metamorphosed downstream migrants of the lamprey Geotria australis. Total non-haem iron in the liver rose significantly from 0.15-0.55 μg.mg wet weight(-1) in ammocoetes and metamorphosing stages 1-3 to 2.2-2.9 μg.mg(-1) in stages 5-7 and to 8.8 μg.mg(-1) in downstream migrants. The comparable values for ferritin iron were 0.06-0.26, 1.4-2.0, and 5.3 μg.mg(-1). Superoxide dismutase activity fell sharply from 0.39 μg.mg(-1) in large ammocoetes to between 0.07 μg.mg(-1) in stage 1 and 0.15 μg.mg(-1) in stage 6, before rising significantly to 0.26 μg.mg(-1) in stage 7 and 0.35 μg.mg(-1) in downstream migrants. The sharp fall in SOD activity at the beginning of metamorphosis is assumed to be related to the marked decline in plasma iron which occurs at the onset of this non-trophic phase in the life cycle. It is proposed that the subsequent increase in SOD activity in the liver of G. australis with increasing iron represents a mechanism aimed at reducing the potentially toxic effects of iron accumulation. This view is consistent with the significant and positive correlation found between both total non-haem and ferritin iron and SOD activity in the liver of non-trophic animals.

Serum thyroid hormone concentrations were measured during the seven stages of metamorphosis (1–7) of the southern hemisphere lamprey, Geotria australis. The respective mean concentrations ± SEM of serum thyroxine (T4) and triiodothyronine (T3) fell from 31.73 ± 4.09 and 5.06 ± 0.70 nM in large ammocoetes sampled in February, at the time when metamorphosis was initiated, to 4.54 ± 0.36 and 1.03 ± 0.12 nM at stage 5. Although there was a small, but significant, recovery of serum T4 concentrations during stages 6 and 7, no such corresponding statistically significant rise occurred in serum T3 concentrations. Serum thyroid hormone concentrations in ammocoetes sampled during the period when metamorphosis was taking place, exhibited a marked seasonal increase between February and May–June (late autumn/early winter); serum T3 and T4 concentrations peaked in May–June and were, respectively, > 2 fold and > 8 fold higher than those recorded for samples in late February (mid summer). By mid-July the serum T4 and T3 levels had declined from the peak values. Ammocoetes taken from streams at 16°C in June and acclimated to aquaria water at 25°C or 6°C had significantly lower serum T3 and T4 concentrations at the higher temperature, and also a lower serum T4, but not T3 concentration, at the lower temperature. Treatment of separate groups of ammocoetes with either propylthiouracil or T3 for 70 days significantly depressed and raised respectively, the serum thyroid hormone and hepatic T3 concentrations and caused significant changes in the body weight, but did not induce the onset of metamorphosis.

The present study has characterized gonadotropic releasing hormone (GnRH)-like molecules in the brains of representatives of the two southern hemisphere families of lampreys, Geotriidae and Mordaciidae. Chromatographic and immunocytochemical evidence showed that the brains of Geotria australis and Mordacia mordax contain two forms of GnRH-like molecules. These two forms correspond to lamprey GnRH-I and -III, which were first sequenced from the brain of the anadromous sea lamprey Petromyzon marinus, a representative of the family Petromyzontidae that is found only in the northern hemisphere. In chromatographic studies (HPLC) using lamprey GnRH-I and -III antiserum, two early eluting GnRH forms coeluted with synthetic lamprey GnRH-I and -III standards. Our studies thus indicate that, despite their apparently long period of separation, the three families of extant lampreys have each retained both of the lamprey GnRH (-I and -III forms) molecules. Moreover, immunocytochemical localization of lamprey GnRH indicated that the pattern of its distribution in the adult brain of at least one of these southern hemisphere lampreys (G. australis) is similar to that previously described for P. marinus. Distribution of GnRH in the brain of larval G. australis was not as extensive as that in larval P. marinus, which may account for the later gonadal development in the former species. The fact that lamprey GnRH-I and -III are the dominant GnRH forms in all three families of lampreys implies that these neurohormones have an ancient origin.

Peptide tyrosine–tyrosine (PYY) has been isolated from the intestines of two species of reptile, the desert tortoise Gopherus agassizii (Testudines) and the Burmese python Python molurus (Squamata), from the primitive Actinopterygian fish, the bichir Polypterus senegalis (Polypteriformes) and from two agnathans, the Southern-hemisphere lamprey Geotria australis (Geotriidae) and the holarctic lamprey Lampetra fluviatilis (Petromyzontidae). The primary structure of bichir PYY is identical to the proposed ancestral sequence of gnathostome PYY (YPPKPENPGE10 | DAPPEELAKY20 | YSALR HYINL30 | ITRQRY). Tortoise and python PYY differ by six and seven residues, respectively, from the ancestral sequence consistent with the traditional view that the Testudines represent an earlier divergence from the primitive reptilian stock than the Squamates. The current views of agnathan phylogeny favor the hypothesis that the Southern-hemisphere lampreys and the holarctic lampreys arose from a common ancestral stock but their divergence is of a relatively ancient (pre-Tertiary) origin. The Geotria PYY-related peptide shows only two amino acid substitutions (Pro10→Gln and Leu22→Ser) compared with PYY from the holarctic lamprey Petromyzon marinus. This result was unexpected as Petromyzon PYY differs from Lampetra PYY deduced from the nucleotide sequence of a cDNA (Soderberg et al. J. Neurosci. Res. 1994;37:633–640) by 10 residues. However, a re-examination of an extract of Lampetra intestine revealed the presence of a PYY that differed in primary structure from Petromyzon PYY by only one amino acid residue (Pro10→Ser). This result suggests that the structure of PYY has been strongly conserved during the evolution of Agnatha and that at least two genes encoding PYY-related peptides are expressed in Lampetra tissues.

This paper describes the ocular morphology of young adults of the southern hemisphere lamprey Geotria australis, the sole representative of the Geotriidae, and makes comparisons with those of holarctic lampreys (Petromyzontidae). As previously reported for the holarctic lamprey Ichthyomyzon unicuspis [Collin and Fritzsch, 1993], the lens of G. australis is non-spherical and possesses a cone-shaped posterior that may be capable of mediating variable focus. The avascular retina of G. australis is well differentiated, containing three retinal ganglion cell populations, three layers of horizontal cells and three photoreceptor types. In contrast to petromyzontids that contain only two photoreceptor types (short and long), G. australis possesses one rod-like (R1) and two cone-like (C1 and C2) photoreceptors. Although the rod-like receptor in G. australis may be homologous with the short receptors of holarctic lampreys, the two cone-like receptors have morphological characteristics that differ markedly from those of the long receptors of their holarctic counterparts. The features which distinguish the two cone-like receptors from those of the long receptor type in holarctic lampreys are the characteristics of the mitochondria and the presence of large amounts of two different types of stored secretory material in the endoplasmic reticulum of the myoid (refractile bodies). The endoplasmic reticulum of each receptor type has a different shape and staining profile and is polymorphic, each showing a continuum of distension. It is proposed that the presence of two cone-like photoreceptors with different characteristics would increase the spectral range of G. australis and thus be of value during the parasitic phase, when this lamprey lives in the surface marine waters. The irideal flap, present in G. australis but not petromyzontids, would assist in reducing intraocular flare during life in surface waters. The results of this study, which are discussed in the context of the proposed evolution of lampreys, emphasise that it is important to take into account the characteristics of the eyes of southern hemisphere lampreys when making generalizations about the eyes of lampreys as a whole.

This study was aimed at determining whether the marked changes that occur in the diet during the life cycle of the lamprey Geotria australis, and thus also in the fatty acid composition of the triacylglycerol stores, are accompanied by alterations in the V(max) and apparent K0.5 of skeletal muscle carnitine palmitoyltransferase I (CPT I) towards the CoA derivatives of the fatty acids 16:0, 16:1n-7, 18:1n-9, 18:2n-6, and 22:6n-3. The activity of CPT I towards the last four of these acyl CoAs increased progressively from the microphagous larvae to young adults, prior to the onset of their marine trophic phase, to fully grown adults, which had ceased feeding or fish and embarked on their spawning run. For example, the V(max) of CPT I towards 18:1n-9 rose from 4.4, to 7.6 to 11.5 nmol · min-1 · mg mitochondrial protein-1 over the course of the three life-cycle stages. The values for the V(max) of lamprey muscle CPT I towards the various CoA esters were not directly correlated with the percentage contributions of the corresponding fatty acids to the triacylglycerol stores. Despite very pronounced differences in the biochemistry, physiology, and physical activity of each life-cycle stage, as well as in the fatty acid composition of the triacylglycerols, before and after the adult trophic phase, the V(max) for CPT I was always greatest for 16:1n-7 and 18:1n-9. Because the reactivity of CPT I towards the latter two monoenoic fatty acids is also high in teleost fishes and in rat, such a characteristic may have been conserved throughout vertebrate evolution.

The distribution and ultrastructure of the mitochondria-rich (MR) cells in the gills of larval (ammocoetes) and adult lampreys (Petromyzon marinus and Geotria australis) have been studied. One type of MR cell, which is found only in ammocoetes, occurs in groups on and between gill lamellae. Freeze-fracture replicas show that the apical membrane of this ammocoete MR cell contains globular particles. The second type of MR cell, which is present in both ammocoetes and adults in freshwater, is located between lamellae and at the base of the filament. This cell usually occurs singly and is typically intercalated between ammocoete MR cells in larval lampreys and between pavement cells and pavement and chloride cells in adult lampreys. It contains rod-shaped particles in either the apical membrane (subtype A) or, far less frequently, the lateral membrane (subtype B) and in membranes of cytoplasmic vesicles and tubules. These features characterize this intercalated MR cell as a member of a group of MR cells that are also found in urinary epithelia of tetrapods and the amphibian epidermis, where they are involved in H+ and HCO3- secretion. Because this type of MR cell disappears when the young adult lamprey enters the sea and reappears immediately after the fully grown adult re-enters freshwater on its spawning run, it is presumably essential for osmoregulation in freshwater. On the basis of electrophysiological studies on frog skin, it is proposed that the subtype A of the branchial intercalated MR cell of lampreys provides the driving force for the Na+ uptake by active H+ secretion. By analogy with urinary epithelia, the subtype B cells may exchange Cl- for HCO3-.

Thin-section and freeze-fracture electron microscopy have been used to determine the types of junctions that are present in the notochord of adult lampreys and thereby propose ways in which the functional properties of the notochord are integrated. The notochord consists of a fibrous sheath, which surrounds a single layer of small 'epithelial' cells and a central core of large vacuolated cells. The epithelial and vacuolated notochordal cells are all linked by numerous desmosomes, which are the predominant type of intercellular junction in the notochord. Furthermore, the notochordal epithelium is linked to the inner surface of the notochordal sheath by many hemidesmosomes, which are associated with patches of basal lamina-like material. The above linkages, produced by desmosomes and hemidesmosomes, together with the tensile strength of the notochordal sheath, must be crucial for maintaining the structural integrity of the notochord during the sinuous swimming movements exhibited by lampreys. The communicating (gap) junctions, that are present between vacuolated cells, would facilitate metabolic coupling between these cells. Zonulae occludentes ('tight junctions') were not found between notochordal epithelial cells, indicating that there is no effective paracellular barrier to the diffusion of macro-molecules, ions and small polar solutes at the outermost layer of the notochordal cells.

The Vmax of rat muscle mitochondrial CPT I toward the coenzyme A derivatives of 16:0, 16:1n-7, 18:1n-9, and 22:6n-3 were far lower than those recorded previously for this enzyme in rat liver at the same temperature (37°C). However, the Vmax of 7.0 nmol · min−1 · mg mitochondrial protein−1 for linoleoyl CoA (18:2n-6), which was the greatest recorded for the five acyl CoAs examined in muscle, was similar to that in liver. These comparisons presumably reflect a difference in the essential fatty acid requirements of these two rat tissues. Although the Vmax values for CPT I in the musculature of a lower vertebrate (larval lamprey) at 20°C were similar to those exhibited toward the coenzyme A derivatives of 16:0, 16:1n-7, 18:1n-9, and 22:6n-3 by the CPT I of rat musculature at 37°C, the corresponding Vmax toward 18:2n-6 (3.2 nmol · min−1 · mg mitochondrial protein−1) was lower. The latter relatively low activity may spare from oxidation this essential fatty acid, which is in low abundance in the diet of larval lampreys. Although the Vmax values toward the four nonessential fatty acids in larval lamprey muscle were similar to those in rat muscle, the corresponding K0.5 values were lower, thus indicating that the musculature of larval lampreys has a high capacity for energy generation through β-oxidation.

O2 uptake and CO2 excretion by the branchial and postbranchial regions of adults of the lamprey Geotria australis have been measured in humidified air at 15 and 5°C. At 15°C, the mean standard V(O2) for whole animals was 31.9 μl·gm-1·hr-1, which is similar to the interpolated V(O2) in water at the same temperature and time of year. The corresponding mean standard V(CO2) was 25.2 μl·gm-1·hr-1, producing an RQ of 0.79. The branchial region was responsible for ~87% of O2 uptake and ~80% of CO2 excretion, which indicates that the gills of lampreys retain their integrity in air and thereby facilitate gas exchange. The continuous pumping of the branchial chamber would also facilitate gas exchange across the gills. The RQ is far higher in the postbranchial than branchial region, i.e., 1.26 vs. 0.72. The proportionately greater excretion of CO2 than uptake of O2 by the postbranchial than branchial region is presumably related to a combination of the greater capacitance of tissue for CO2 than O2 and the presence of a thicker tissue barrier in the skin of the trunk and tail than in the lamellae of the gills. At 5°C, the mean standard V(O2) and V(CO2) for whole animals declined markedly to 13.1 and 13.5 μl·gm-1·hr-1, respectively. The corresponding rise in RQ from 0.79 at 15°C to 1.03 at 5°C implies that aerobic metabolism now utilizes solely carbohydrate, rather than lipid and some carbohydrate. The transfer to solely carbohydrate metabolism at 5°C may be triggered by a need to supplement aerobic metabolism with anaerobic metabolism to compensate for an apparent shortfall in oxygen uptake produced by inadequate ventilation of the gills.

Males of the southern hemisphere lamprey Geotria australis develop a large gular pouch during sexual maturation. The wall of this pouch contains (I) an epidermis comprising typical lamprey epidermal cells, (II) a thick dermis consisting of layers of collagen fibres, with intervening oxytalan fibres, and a vascular network, and (III) a hypodermis. The hypodermis contains active fibroblasts embedded in an extracellular matrix, comprising collagen fibrils, some of which form dense bundles, together with oxytalan fibres and an amorphous material that contains glycosaminoglycans. The hypodermis also contains arteries, which are innervated and confluent with a large anastomosing system of sinuses. Since these sinuses open at intervals into the large central cavity of the pouch, it is proposed that, at maturity, they discharge blood into the central lumen of the pouch, thereby causing the pouch to become distended. The degree of such distension would be regulated by the limited elasticity of the dermal collagen fibres. The dermis is thickest in those regions where the pouch is most susceptible to abrasion. The Weibel-Palade bodies, which are abundant in the sinus endothelia, may facilitate tissue repair where necessary. The gular pouch is thus a specialized structure, which may play a role in courtship or the spawning act.

The localization of exocrine pancreatic (zymogen) cells in the epithelium of the anterior intestine of the larvae of holarctic lampreys (Petromyzontidae) is considered the most primitive arrangement of these cells in vertebrates (Barrington, '72). In contrast, the zymogen cells are located in prominent forward-projecting diverticular extensions of the anterior intestine in larvae of the two monogeneric southern hemisphere lamprey families (Geotriidae and Mordaciidae). During the present study, tryptic inhibitory activity was not found in the two diverticula of Geotria australis, whereas a trypsin inhibitor of the Kazal type was found in the diverticulum of Mordacia mordax, eluting at the same chromatographic position as the inhibitor of rat pancreas. It is suggested that a trypsin inhibitor is not required in G. australis, because its wide duct-like diverticula would facilitate a relatively steady dispersal of trypsin into the anterior intestine. In contrast, a trypsin inhibitor would be of value to M. mordax since the highly folded epithelial lining and narrow central canal of its diverticulum would presumably lead to some trypsin being retained in the diverticulum. Such a protective mechanism against unbridled proteolysis would be complemented by the relatively very low level of trypsin activity found in the diverticulum of M. mordax. Consideration of our data in the context of the proposed scheme for the phylogeny of lamprey taxa suggests that a trypsin inhibitor has been evolved in the larval exocrine pancreatic tissue of one of the lamprey families, i.e., Mordaciidae, independent of that developed in the exocrine pancreas of gnathostomes.

Mordacia mordax is one of the two anadromous parasitic lamprey species of the southern hemisphere family Mordaciidae. Its adults possess two lateral buccal glands and one central buccal gland. When the tongue-like piston is retracted, the buccal glands occupy much of the opening of the oral cavity at the rear of the buccal cavity. The glands contain numerous tube-like, ductless secretory units, which discharge directly into the buccal cavity. Their secretory epithelial cells contain numerous granules, some of which are zymogen-like, while others have a beaded, spiralled appearance. The similarity of the latter to mast cell granules suggests that they may likewise produce an anticoagulant, which would be valuable to a presumed blood feeder such as M. mordax. The mucus produced by these cells could act as a carrier for the secretions and as an adhesive for promoting retention of t he secretions on the host's surface. When the young adults is transferred to salt water, the buccal glands increase their production and discharge of secretions. Since the glands are not enclosed in musculature, their secretions are probably discharged by mechanical pressure applied by the forward movement of the head of the tooth-bearing piston into the buccal cavity. An account is given of the way in which the location, number, glandular organization, secretory granules, and type of secretion of the buccal glands of M. mordax, and thus presumably also their mode of function, differ markedly from those of members of the other lamprey family found in the southern hemisphere, and of all holarrctic lampreys. © 1995 Wiley-Liss, Inc.

A dermal capillary network was found in three species of hagfishes (Myxine glutinosa, Paramyxine atami, Eptatretus stoutii) and in the adults of three species of lampreys (Geotria australis, Lampetra japonica, Petromyzon marinus). However, it was found neither in the ammocoetes of any of the species examined, nor in the adults of the lamprey Lampetra fluviatilis. The complex arrangement of dermal capillaries described for adult L. fluviatilis by Czopek & Sawa (1971) was apparently an artefact. The dermal capillaries of hagfishes and lampreys are lined by a continuous endothelium, comprising flattened cells that possess most of the usual complement of organelles found in the endothelia of higher vertebrates. The pericapillary space contains proteoglycans, glycoproteins and tubular microfibrils (diam. 11–15nm) and, in hagfishes, also irregularly arranged collagen fibrils (diam. 40–120 nm). The thickness of the endothelium varied amongst species, ranging from c. 100nm in G. australis and P. marinus to over 500 nm in L. japonica and all three species of hagfishes. In comparison with the endothelial cells of lampreys, those of hagfishes have a better developed basement membrane, possess small desmosomes in the flange-like intercellular contact regions, and contain numerous plasmalemmal smooth tubules, rather than the smooth vesicles that are typically present in endothelial cells. In addition, the hagfish dermal capillaries have pericytes. Amongst hagfishes, the dermal capillary network is far better developed in Myxine glutinosa and Paramyxine atami, which burrow and may therefore have to rely to some degree on cutaneous respiration, than in Eptatretus stoutii which does not burrow. However, a dermal capillary network is probably important for all species of hagfishes since it would enable the precursors required for synthesizing mucus to be transported to the epidermis, where mucous cells are very abundant. The particularly well-developed dermal capillary network in Geotria australis may facilitate the transport of oxygen through the unusually thick dermis of their migrating adults as they move around barriers on their upstream migration, and must thus presumably have to employ cutaneous respiration.

This paper investigates whether elastic fibre-like structures are present in the ventral aorta of hagfishes and lampreys. Fibres, which are morphologically similar to the elastic fibres of gnathostomatous (jawed) vertebrates, are shown to be present in the ventral aorta of the hagfish Paramyxine atami, and also, but to a lesser extent, the dorsal aorta of this species and the ventral aorta of another hagfish, Eptatretus stouti.The ‘elastic’ tissue formed irregular sheet-like aggregates, comprising well-defined amorphous material surrounded by tubular microfibrils, whose diameters ranged from 10 to 15 nm. While this tissue was most abundant in the same region of the aortae as that occupied by the elastica interna in the blood vessels of gnathostomes, it was also found deeper in the wall of these blood vessels. Although tubular microfibrils were found in the ventral aorta of the hagfish Myxine glutinosaand the lamprey Geotria australis, these were never associated with well–defined, amorphous material. This parallels the results of previous studies on Myxine glutinosaand another species of lamprey, Petromyzon marinus.Thus, the elastic fibre–like tissues found in the ventral aortae of P. atamiand E. stoutiprovide the first examples of such structures in this region in agnathan vertebrates.

Thin sections and freeze-fracture replicas of the water-blood barrier in the gill lamellae of adult lampreys (Geotria australis, Lampetra fluviatilis) demonstrate that the occluding junctions between epithelial pavement cells differ markedly from those between “endothelial” pillar cells in the structure and arrangement of their strands. The zonulae occludentes between pavement cells typically consist of complex networks of 4–6 strands, the mean number of which undergoes a small but significant decline when the animal is acclimated to seawater. In comparison, the occluding junctions between pillar cells are less elaborate and may represent maculae or fasciae, rather than zonulae occludentes. They do not apparently undergo a change when the animal enters saltwater. The results indicate that the main part of the paracellular diffusion barrier to proteins and ions is located in the epithelium rather than the endothelium. Communicating (gap) junctions are present between adjacent pavement cells, between pavement and basal cells and between pillar cells. These findings suggest that the epithelial cells and the pillar cells in the water-blood barrier of lampreys both form ‘functional syncytia’. The results are discussed in the context of ion-transporting epithelia in other aquatic vertebrates.

1. 1. The proportion of polyunsaturated fatty acids in the triacylglycerols and phospholipids of muscle and ovary were far lower in adult Lampetra planeri than adult Lampetra fluviatilis. 2. 2. The most abundant fatty acids in the triacylglycerols were 16:1 in the muscle and ovary of adult L. planeri and 18:1 in the muscle and 22:6 in the ovary of L. fluviatilis. 3. 3. The above interspecific differences reflect marked differences in the fatty acid compositions of the lipids ingested and stored during the respective preceding trophic phases, i.e. during microphagous larval life in L. planeri and during feeding on fish flesh in L. fluviatilis.

Adults of the Southern hemisphere lamprey Geotria australis were subjected to an exercise/recovery regime at the commencement and end of their 12–15 month non-trophic, upstream spawning migration. In early (immature) migrants and pre-spawning females, muscle glycogen was markedly depleted during exercise, but became rapidly replenished. As muscle lactate rose during exercise and peaked 1–1.5 h into the recovery period, and therefore after muscle glycogen had become replenished, it cannot be the direct source for that replenishment. However, both plasma lactate and glycerol (but not muscle glycerol and glucose) rose sharply during exercise and then declined markedly during the first 0.5 h of recovery and thus exhibited the opposite trend to that of muscle glycogen, implying that these limited pools of glycogenic precursors contribute to glycogen replenishment. Although plasma glucose rose following exercise, and consequently could also be a precursor for muscle glycogen replenishment, it remained elevated even after muscle glycogen had become replenished. While resting pre-spawning females and mature males retained high muscle glycogen concentrations, this energy store became permanently depleted in females during spawning. In mature males, muscle glycogen remained high and lactate low during the exercise/recovery regime, whereas muscle glycerol declined precipitously during exercise and then rose rapidly. In summary, vigorous activity by G. australis is fuelled extensively by anaerobic metabolism of glycogen early in the spawning run and by pre-spawning females, but by aerobic metabolism of its energy reserves in mature males.

Thin sections and freeze-fracture replicas have been used to study the structure of the zonulae occludentes of the branchial chloride cells in young adults of the anadromous lamprey Geotria australis, caught during their downstream migration to the sea and after acclimation to full-strength seawater (35‰). The chloride cells in the epithelium of the gill filaments of both freshwater- and seawater-acclimated animals form extensive multicellular complexes. In freshwater animals, the majority of chloride cells (64%) are covered by pavement cells and are thus not exposed to the external environment. Most of the other chloride cells are separated from each other by pavement cells or their processes. The zonulae occludentes between chloride cells and pavement cells and between adjacent chloride cells are extensive and characterised by a network of 4 (range 3–7) superimposed strands. In seawater-acclimated animals, the pavement cells cover only 30% of the chloride cells and their processes no longer occur between chloride cells. Whereas the zonulae occludentes between chloride cells and pavement cells are still extensive, those between chloride cells are shallow and comprise only a single strand or two parallel strands. The zonulae occludentes between the chloride cells of lampreys acclimated to seawater are similar to those in the gills of teleosts in seawater, and are thus considered to be leaky and to provide a low-resistance paracellular pathway for the passive transepithelial movement of Na+.

Spermatogenesis during post-metamorphic life in the anadromous parasitic lamprey Geotria australis was examined in histological sections of testes of animals collected in the field or maintained in the laboratory. The fully metamorphosed downstream migrants of G. australis possessed a very small testis in which the germ cells consisted of irregularly arranged prospermatogonia. By the end of the subsequent protracted marine trophic phase, the testis had increased in cross-sectional area by about 20 times and become organised into well-defined cysts. Since approximately 40% of these cysts possessed spermatocytes and 12% contained spermatids or early sperm, meiosis is initiated prior to the non-feeding upstream migration, which in G. australis lasts for the unusually long period of 15-16 months. The incidence of cysts with meiotic and post-meiotic stages decreased during the first 3 months of the spawning run before starting to rise during subsequent months. Spermiogenesis was never completed in any animals caught in the field during the first 9 months of the upstream migration or in those subsequently held in the laboratory up to and beyond November, the month when spawning naturally takes place. The phasing and asynchrony of spermatogenesis during post-metamorphic life in G. australis differs markedly from the situation in Northern Hemisphere lampreys.

Summary The distribution of proteoglycans in the sheath and epithelial cells of the lamprey notochord had been studied using electron microscopy on material stained with the dye cupromeronic blue (CMB). CMB-precipitates, which indicate the presence of sulphated proteoglycans, were found in the inner collagenous but not the outer “elastica externa” regions of the notochord sheath. Precipitates were also found in the zone of the basement membrane of the notochord epithelial cells, in intercellular spaces within the notochord epithelium, and in intracellular spherical bodies of the epithelial cells. These results indicate that the proteoglycans in the notochord sheath are produced by the notochord cells. The combined presence of sulphated proteoglycans and collagen type II in the notochord sheath parallels the situation found in the cartilage of higher vertebrates.

Lampreys are one of the two surviving groups of jawless vertebrates, whose ancestors arose more than 540 million years ago. Some species, such as Geotria australis, are anadromous, commencing life as ammocoetes in rivers, migrating downstream to the sea, and migrating back into rivers to spawn. Five photoreceptor types and five retinal cone opsin genes ( LWS, SWS1, SWS2, RhA, and RhB) have previously been identified in G. australis. This implies that the ancestral vertebrates possessed photopic or cone- based vision with the potential for pentachromacy. Changes in the morphology of photoreceptors and their spectral sensitivity are encountered during differing aquatic phases of the lamprey lifecycle. To understand the molecular basis for these changes, we characterized the visual pigments and measured the relative levels of opsin expression over two lifecycle phases that are accompanied by contrasting ambient light environments. By expressing recombinant opsins in vitro, we show that SWS1, SWS2, RhA, and RhB visual pigments possess lambda max values of 359, 439, 497, and 492 nm respectively. For the LWS visual pigment, we predict a lambda max value of 560 nm based on key spectral tuning sites in other vertebrate LWS opsins. Quantitative reverse transcriptasepolymerase chain reaction reveals that the retinal opsin genes of G. australis are differentially regulated such that the visual system switches from a broad sensitivity across a wide spectral range to a much narrower sensitivity centered around 490 - 500 nm on transition from marine to riverine conditions. These quantitative changes in visual pigment expression throughout the lifecycle may directly result from changes in the lighting conditions of the surrounding milieu.

The morphology and spectral absorption characteristics of the retinal photoreceptors in the southern hemisphere lamprey Geotria australis (Agnatha) were studied using light and electron microscopy and microspectrophotometry. The retinae of both downstream and upstream migrants of Geotria contained two types of cone photoreceptor and one type of rod photoreceptor. Visual pigments contained in the outer segments of these three photoreceptor types had absorbance spectra typical of porphyropsins and with wavelengths of maximum absorbance (downstream/upstream) at 610/616 nm (long-wavelength-sensitive cone, LWS), 515/515 nm (medium-wavelength-sensitive cone, MWS), and 506/500 nm (medium-wavelength-sensitive rod). A "yellow" photostable pigment was present in the myoid region of all three types of photoreceptor in the downstream migrant. The same short-wavelength-absorbing pigment, which prevents photostimulation of the beta band of the visual pigment in the outer segment, was present in the rods and LWS cones of the upstream migrant, but was replaced by a large transparent ellipsosome in the MWS cones. Using microspectrophotometric and anatomical data, the quantal spectral sensitivity of each photoreceptor type was calculated. Our results provide the first evidence of a jawless vertebrate, represented today solely by the lampreys and hagfishes, with two morphologically and physiologically distinct types of cone photoreceptors, in addition to a rod-like photoreceptor containing a colored filter (a cone-like characteristic). In contrast, all other lampreys studied thus far have either (1) one type of cone and one type of rod, or (2) a single type of rod-like photoreceptor. The evolution or retention of a second type of cone in adult Geotria is presumably an adaptation to life in the brightly lit surface waters of the Southern Ocean, where this species lives during the marine phase of its life cycle. The functional significance of the unique visual system of Geotria is discussed in relation to its life cycle and the potential for color vision.

In addition to its novel, colour-regulating hormonal role in teleosts, the melanin-concentrating hormone (MCH) serves as a neuromodulatory peptide in all vertebrate brains. In gnathostome vertebrates, it is produced in several neuronal cell groups in the hypothalamus. The present work examines the organisation of the MCH system in the brain of lampreys, which separated from gnathostome vertebrates at an early stage in evolution. In all three lamprey genera examined-Petromyzon, Lampetra, and Geotria spp.-MCH perikarya were found in one major anatomical site, the periventricular dorsal hypothalamic nucleus of the posterior hypothalamus. Axons from these cell bodies projected medially into the ventricular cavity, and laterally to the neuropile of the lateral hypothalamus. From here, they extended anteriorly and posteriorly to the fore- and hindbrain. Other fibres extended dorsomedially to the habenular nucleus. In Lampetra, but not in Petromyzon, MCH fibres were seen in the pituitary neurohypophysis, most prominantly above the proximal pars distalis. The hypothalamic region in which the MCH perikarya are found forms part of the paraventricular organ (PVO), which is rich in monoamines and other neuropeptides. The association of MCH neurones with the PVO, which occurs also in many other nonmammalian vertebrates, may reflect the primary location of the MCH system. These MCH neurones were present in ammocoetes, postmetamorphic juveniles, and adults. They were more heavily granulated in adults than in young lampreys but showed no marked change in secretory appearance associated with metamorphosis or experimental osmotic challenge to indicate a role in feeding or osmoregulation. In sexually maturing Lampetra fluviatilis, however, a second group of small MCH neurones became detectable in the telencephalon, suggesting a potential role in reproduction and/or behaviour.

The standard rates of O2 consumption of larval Mordacia mordax (weight range 1.3-2.3 g), after these ammocetes had been in humidified air for 18 hr, were 26.8, 46.3 and 71.2 microL x g(-1) x hr(-1) at 10, 15 and 20 degrees C, respectively. The corresponding rates of CO2 excretion were 20.7, 35.6 and 54.1 microL x g(-1) x hr(-1). The RQs at the three temperatures were essentially identical (0.76 or 0.77) and similar to that of adults of the lamprey Geotria australis in air at 15 degrees C. The above RQs for ammocoetes, which are probably similar to those that would be recorded in water, are consistent with the view that the aerobic respiration of these animals relies predominantly on lipid as an energy source, but that some energy is derived from carbohydrate and/or protein. The RQs for larval and adult lampreys in air lie well within the range recorded for amphibious fishes in air.

Current views on Agnathan phylogeny favor the hypothesis that the genera of holarctic lampreys belong to a single family (Petromyzontidae) and form an interrelated progression in which Petromyzon is near to Ichthomyzon at the base of the phylogenetic tree and Lainpetra is the most derived. A stock similar to that of contemporary Ichthomyzon is considered to have given rise to the southern hemisphere lamprey Geotria australis, the sole member of the Geotriidae. In the present study, two molecular forms of glucagon were isolated from an extract of G. australis intestine that differed in structure by six amino acid residues. One form shows two amino acid substitutions (Leu14 → Met and Ala29 → Ser) compared with the single molecular form of glucagon isolated from the sea lamprey Petromyzon marinus and the second form shows three substitutions (Asp15 → Glu, Ser16 → Ala, Ile24 → Thr) compared with the single glucagon isolated from the river lamprey Lampetra fluviatilis. As Petromyzon and Lampetra glucagons differ by six amino acid residues, the data suggest that a duplication of the glucagon gene occurred prior to or early in lamprey evolution. Although both genes are strongly expressed in G. australis, the expression of one gene predominates in P. marinus while that of the other gene predominates in L. fluviatilis. Previous work has shown that, in the islet organ of G. australis, preprosomatostatin is processed almost exclusively to somatostatin=33. However, the present study demonstrates that somatostatin-14 is the major molecular form in G. australis intestine with somatostatin-33 present only as a minor component. This result demonstrates a tissue-dependent pathway of posttranslational processing of p reprosomatostatin in the Geotria enteropancreatic system.

Iodinated atrial natriuretic peptide (ANP) binding sites were examined in the gills and ventral aorta of the adult upstream-migrating lamprey Geotria australis using tissue section autoradiography, in vitro competition analysis and affinity cross-linking, while guanylate cyclase assays were performed on gill membranes of both adult and juvenile lampreys. A partial natriuretic peptide (NP) receptor sequence was amplified using reverse transcription/ polymerase chain reaction (RT-PCR). The results indicated that there was specific NP binding to the aortic endothelium and to pillar cell regions in the axial plate and secondary lamellae. In competition studies, 50 % of NP binding was abolished by 4 nmol l−1 rat ANP, 35 nmol l−1 porcine C-type NP (CNP) and 45 nmol l−1 C-ANF (a truncated ANP). Affinity cross-linking followed by SDS–PAGE demonstrated two binding sites at 205 and 65 kDa under non-reducing conditions and at 85 and 65 kDa under reducing conditions. Guanylate cyclase assays demonstrated that, while no NP-stimulated GC activity occurred in adult lampreys, NP-stimulated enhancement of cyclic GMP accumulation was found in juveniles in fresh water and more particularly in salt water. RT-PCR amplified a 471 base pair fragment with 68 % amino acid sequence homology to the eel natriuretic peptide receptor D (NPR-D). This study suggests that NP binding sites in the adult gill and aorta are of an NPR-C/D type, whereas an additional GC-coupled site exists in juveniles.

Lampreys are one of the two surviving groups of the agnathan (jawless) stages in vertebrate evolution and are thus ideal candidates for elucidating the evolution of visual systems. This study investigated the retinal amino acid neurochemistry of the southern hemisphere lamprey Geotria australis during the downstream migration of the young, recently-metamorphosed juveniles to the sea and during the upstream migration of the fully-grown and sexually-maturing adults to their spawning areas. Glutamate and taurine were distributed throughout the retina, whilst GABA and glycine were confined to neurons of the inner retina matching patterns seen in most other vertebrates. Glutamine and aspartate immunoreactivity was closely matched to Müller cell morphology. Between the migratory phases, few differences were observed in the distribution of major neurotransmitters i.e. glutamate, GABA and glycine, but changes in amino acids associated with retinal metabolism i.e. glutamine and aspartate, were evident. Taurine immunoreactivity was mostly conserved between migrant stages, consistent with its role in primary cell functions such as osmoregulation. Further investigation of glutamate signalling using the probe agmatine (AGB) to map cation channel permeability revealed entry of AGB into photoreceptors and horizontal cells followed by accumulation in inner retinal neurons. Similarities in AGB profiles between upstream and downstream migrant of G. australis confirmed the conservation of glutamate neurotransmission. Finally, calcium binding proteins, calbindin and calretinin were localized to the inner retina whilst recoverin was localized to photoreceptors. Overall, conservation of major amino acid neurotransmitters and calcium-associated proteins in the lamprey retina confirms these elements as essential features of the vertebrate visual system. On the other hand, metabolic elements of the retina such as neurotransmitter precursor amino acids and Müller cells are more sensitive to environmental changes associated with migration.

When covered by moistened lint-free gauze, the larvae (ammocoetes) of the lamprey Geotria australis survived, without apparent discomfort, for 4 days in water-saturated air at 10, 15 and 20 degrees C. In air, the mean standard rates of O2 consumption of medium to large ammocoetes of G. australis (mean = 0.52 g) at 10, 15 and 20 degrees C were 14.5, 35.7 and 52.1 microliters.g-1.h-1, respectively. At 15 degrees C, the slope of the relationship between log O2 consumption (microliter O2.h-1) and log body weight for ammocoetes over a wide range in body weight was 0.987. The Q10s for rate of O2 consumption between 10 and 15 degrees C, 15 and 20 degrees C and 10 and 20 degrees C were 4.9, 2.9 and 3.6, respectively. Our results and observations of the ammocoetes suggest that, when out of water, larval G. australis derives most of its O2 requirements from cutaneous respiration, particularly at lower temperatures. This would be facilitated by the small size and elongate shape (and thus a relatively high surface-to-volume ratio), low metabolic rate, thin dermis, extensive subdermal capillary network and high haemoglobin concentration of larval G. australis.

An extract of the islet organ of the southern-hemisphere lamprey Geotria australis contained a high concentration of somatostatin-like immunoreactivity (34 nmol/g) but only trace amounts of insulin-like immunoreactivity. The primary structure of Geotria somatostatin-33 (AVQEAGGAAM10PPPGQRDRKA20GCKNFFWKTF30SSC) shows almost no similarity to somatostatins from the holarctic lampreys Petromyzon marinus and Lampetra fluviatilis in the N-terminal region but the functionally important C-terminal region, including the substitution Thr31 --Ser, is the same. Insulin was not identified in the extract but proinsulin and an incompletely processed form with an intact A-chain/C-peptide junction were purified and partially characterized. The primary structure of the insulin region of Geotria proinsulin was established as A-chain; GIVEKCCHNR10CSIYQ MESYC20N; B-chain: SALTGSGGNY10LCGSYLVDAL20YLACGPRGFF30YTSTPV. This sequence contains 17 amino acid substitutions compared with the identical insulins from P. marinus and L. fluviatilis but the unusual extension to the N-terminus of the B-chain (SALTG) is present. Compared with mammalian insulins, Geotria insulin contains several substitutions of strongly conserved residues such as Gln(A5) --Lys in the putative receptor-binding region, Glu(B26) --Pro important in dimerization, and Leu(A13) --Ile, His(B10) --Tyr, and His(B21) --Tyr important in hexamerization. Geotria proinsulin contains an Arg-Arg processing site at the B-chain/C-peptide junction but we speculate either that the Lys-Arg processing site at the C-peptide/A-chain junction is absent or that the Geotria pancreas is unable to synthesize a SPC2-type prohormone convertase. Our results are consistent with the view that G. australis and holarctic lampreys arose from a common stock but have been separated for a considerable period.

Molecular investigation of the origin of colour vision has discovered five visual pigment (opsin) genes, all of which are expressed in an agnathan (jawless) fish, the lamprey Geotria australis. Lampreys are extant representatives of an ancient group of vertebrates whose origins are thought to date back to at least the early Cambrian, approximately 540 million years ago [1.]. Phylogenetic analysis has identified the visual pigment opsin genes of G. australis as orthologues of the major classes of vertebrate opsin genes. Therefore, multiple opsin genes must have originated very early in vertebrate evolution, prior to the separation of the jawed and jawless vertebrate lineages, and thereby provided the genetic basis for colour vision in all vertebrate species. The southern hemisphere lamprey Geotria australis (Figure 1A,B) possesses a predominantly cone-based visual system designed for photopic (bright light) vision [2. S.P. Collin, I.C. Potter and C.R. Braekevelt, The ocular morphology of the southern hemisphere lamprey Geotria australis Gray, with special reference to optical specializations and the characterisation and phylogeny of photoreceptor types. Brain Behav. Evol. 54 (1999), pp. 96–111.2. and 3.]. Previous work identified multiple cone types suggesting that the potential for colour vision may have been present in the earliest members of this group. In order to trace the molecular evolution and origins of vertebrate colour vision, we have examined the genetic complement of visual pigment opsins in G. australis.

The cell types within the endocrine pancreatic tissue and anterior intestine of larvae and juveniles of representatives of the two southern-hemisphere families (Mordaciidae and Geotriidae) were compared, using immunohistochemistry and antisera against insulin (lamprey, bovine), two somatostatins (SST-14, -34), two PP-family peptides (aPY, NPY), and salmon glucagon and glucagon-like peptide (GLP). Cells of the islets and some anterior intestinal cells in larval Mordacia mordax showed intense immunoreactivity (IR) to the two insulin antisera. In contrast, immunoreactivity to these antisera in the islets of larval Geotria australis was restricted to antibovine insulin and even then the staining was weak. The islet cells did not IR with other antisera, but IR to aPY and NPY antisera was noted in a few intestinal cells of both species and cells in the intestine of G. australis were positively stained with antiSST-14 and/or -34. The single islet organ of adults of both species consisted only of antiinsulin-IR, B cells, and D cells, which were IR with only antiSST-14. Although IR was not seen in islet tissue to antisera against aPY, NPY, glucagon, and GLP, four cell types were identified in the intestinal epithelium in both species based on their IR to these antisera and the two antiSSTs. A fifth cell type IR to the two insulin antisera was recognized in adult M. mordax. The types and IR of endocrine cells in the enteropancreatic system of two southern-hemisphere lamprey families are compared with those of the Petromyzontidae, the single family of holarctic lampreys. Differences are discussed in relation to variations in hormone processing and whether they are a consequence of varied ontogenic and phylogenetic history among the extant Petromyzontiformes.

Freeze-fracture replicas demonstrate that the apical membrane of the chloride cells of young adult lampreys (Geotria australis) undergoes a structural change when the animal is acclimated from freshwater to seawater. Although the particles on both the P face and the E face of this membrane are randomly distributed in freshwater animals, they are usually arranged in clusters on both fracture faces in seawater-acclimated animals. Light optical diffraction analysis demonstrates that the particles forming the clusters are periodically arranged. Although comparable clusters of particles have not been recorded in other epithelial membranes that are engaged in transporting ions into a hypertonic environment, they may be involved in the final step of Cl- secretion by the chloride cells of lampreys in seawater.

Lampreys differ from the other extant agnathan group, the hagfishes, in possessing a fat column above the nerve cord. It is suggested that this region of fat is just one of many sites of lipid storage that have been developed to provide energy reserves for the lengthy non-trophic periods found during the life cycle of lampreys. The incorporation of this fat within an extension of the nerve cord sheath suggests that it could act as a protective dorsal cushion for the nerve cord, which would otherwise be more exposed due to the absence of complete vertebral arches. The development of the fat column as the principal blood cell forming structure in the adult can apparently be related to certain changes that take place during metamorphosis, particularly in the circulatory system.