Trypsin-like enzymes from two morphotypes of the 'living fossil' Triops (Crustacea: Branchiopoda: Notostraca)

Trypsin-like enzymes from two morphotypes (here called short and long) of the 'living fossil' Triops of Baja California Sur, Mexico were studied. Adults of both morphotypes were obtained from outdoor static cultures using dry soil from the natural habitats as a source of cysts and culture substrate. Individual and pooled extracts were made from dissected digestive tubes. The effect of pH and temperature on the trypsin activity was studied using N-alpha-benzoyl-DL-Arg-p-nitroanilide (BAPNA) as substrate. The highest proteolytic activity was found at the same pH with extracts of both morphotypes. At this pH, there was greater proteolytic activity at a lower temperature with the short morphotype extract than with the long morphotype extract. Substrate-SDS-PAGE zymograms showed bands of activity. Short morphotype extracts produced six bands; five of them were serine proteases of which three were trypsin-like enzymes. Long morphotype extracts revealed eight bands; six of them were serine proteases of which three were trypsin-like enzymes.     

pH-related Hatching Success of Triops cancriformis (Crustacea: Branchiopoda: Notostraca)

Six different pH ranges were tested for the hatching rate of Triops cancriformis cysts. Cumulative hatching numbers approach to approximately 50% of cysts. The highest hatching rates, co-occurring with the shortest hatching times, were recorded in the neutral range. No hatching took place at pH values 9 and above. This corresponds with the most frequent habitats of this species, snowmelt pools of river flood plains as well as aestival pools caused by heavy rainfalls. Occurrences in sodic alkaline pans can be explained by relatively low pH values at the time of inundation.     

Heart ultrastructure in Lepidurus arcticus pallas (Crustacea,Branchiopoda, Notostraca)

Summary The heart of Lepidurus arcticus consists of an epicardium and a single layer of strongly polarized myocardial cells, 10–50 m thick, with the myofibrillar part facing the epicardium. The Z-bands are diffuse and some Z-material forms attachment plaques. Relaxed sarcomeres show a hexagonal arrangement of thick filaments and 6 thin filaments in orbit, but filaments often diverge in their orientation.The sarcolemma invaginates from both the epicardial and the endocardial side of the cell, forming clefts and T-tubules. The sarcoplasmic reticulum is loosely reticular, cisternae associate with sarcolemma to form large and typical peripheral and interior couplings. The latter are of the button-to-button type and they tend to be located at the A-I level.This work was supported by grants from the Norwegian Research Council for Science and Humanities     

Orientation responses of Triops granarius (Lucas) (Branchiopoda: Notostraca) and Streptocephalus ssp. (Branchiopoda: Anostraca)

Summary Triops granarius and Streptocephalus spp. show an orthokinetic response and swim faster in light than in darkness. Triops is negatively, Streptocephalus positively phototactic. The lethal temperature for 24 hours' exposure is 34°C for Triops, 35°C for Streptocephalus. Temperatures around 29°–30°C are preferred by both to 20°C or 33°C. Phototaxis overrides these temperature reactions in Streptocephalus but not in Triops. Both swim against water currents: Triops does not become habituated to mechanical stimuli upon responses to which its survival may depend.

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Résumé Le Triops granarius et le Streptocephalus spp. ont une réaction orthocinétique et se déplacent plus vite dans la lumière que dans le noir. Le Triops est négativement phototactique, le Streptocephalus l'est positivement. La température mortelle pour une exposition de 24 h. est 34°C pour le Triops et 35°C pour le Streptocephalus. Tous les deux préfèrent des températures de l'ordre de 29°–30°C plutôt que de 20°C ou 33°C. Le phototactisme domine ces réactions aux température chez le Streptocephalus mais pas chez le Triops. Tous les deux se déplacent contre les courants: le Triops ne peut pas s'accoutumer aux excitents méchaniques, même si sa vie dépend de sa réaction à ces excitents.

     

Molecular phylogeny of the Branchiopoda (Crustacea)--multiple approaches suggest a 'diplostracan' ancestry of the Notostraca

The evolutionary history of Branchiopoda (Crustacea) traditionally has attracted considerable interest due to the diversity of the group. Recently molecular methods have been applied to the study of branchiopod systematics with some success, but central questions, such as the phylogenetic position of Laevicaudata and Notostraca, and the intrinsic cladoceran phylogeny, remain unanswered. We examined the phylogeny of Branchiopoda by using two genes, mitochondrial 16S rRNA and nuclear 28S rRNA, which previously have seen little use for inferring branchiopod phylogeny. The number of ingroup taxa included was 42, representing all eight extant branchiopod orders. The data were analyzed using parsimony, maximum likelihood, and Bayesian Inference of phylogeny. Some higher-level taxa were supported in all analyses of the combined data: Phyllopoda, Cladoceromorpha, Cladocera, and Gymnomera. Other higher-level taxa were not supported in any trees: Diplostraca and Conchostraca. A case is made for a possible diplostracan ingroup position of Notostraca based on our data and on previously published molecular and morphological evidence. The recent discovery of a Devonian branchiopod, which is morphologically an intermediate between a notostracan and a 'conchostracan', is congruent with a diplostracan ancestry of Notostraca.     

Reproductive isolation and genetic differentiation in North American species of Triops (Crustacea: Branchiopoda: Notostraca)

Electrophoretic analysis of 31 populations, ranging from California to Kansas, indicates that the North American tadpole shrimp Triops longicaudatus (LeConte) is actually a mixture of at least two reproductively isolated species. In the central United States, the predominant species is T. longicaudatus, which is found typically in ephemeral prairie pools. In the southwestern United States, the predominant species is Triops newberryi (Packard), which characteristically inhabits large playa pools. The two species coexist occasionally and in sympatric situations they are reproductively isolated from each other. The two species are genetically distinct at a level greater than is typical of conspecific populations. These genetic differences are correlated with subtle morphological differences and profound differences in reproductive biology. Both species represent complexes of bisexual and unisexual populations. Bisexual populations of T. longicaudatus are usually composed of males and females in approximately equal frequencies; bisexual populations of T. newberryi are composed of males in low frequencies and self-compatible hermaphrodites. Unisexual populations of both species apparently consist entirely of self-compatible hermaphrodites. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evidence of cross-fertilization in a gonochoric population of the tadpole shrimp Triops numidicus (Crustacea: Branchiopoda: Notostraca)

Cross-fertilization was evident in a gonochoric population of the notostracan Triops numidicus, in which a thick, hard eggshell had been suspected of preventing the sperm from reaching the egg. Most of the eggs from copulated females hatched into larvae, but the eggs from virgin females did not develop. In the larvae, paternal DNA fragments were detected by AFLP. In histological sections, several spermatozoa were found in the shell of newly oviposited eggs in the brood pouches of copulated females, suggesting that the shell was still soft enough to be penetrated by spermatozoa. These results showed that copulation and subsequent fertilization achieved by penetration of sperm through the newly deposited eggshell were indispensable for the normal development of the eggs.     

Experimental test of the ecosystem impacts of the keystone predator Triops cancriformis (Branchiopoda: Notostraca) in temporary ponds

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SEM studies on the early larval development of Triops cancriformis (Bosc)(Crustacea: Branchiopoda, Notostraca)

We investigated early larval development in the notostracan Triops cancriformis (Bosc, 1801–1802) raised from dried cysts under laboratory conditions. We document the five earliest stages using scanning electron microscopy. The stage I larva is a typical nauplius, lecithotropic and without trunk limbs. The stage II larva is feeding and has trunk limb precursors and a larger carapace. Stage III larvae have larger trunk limbs and a more adult shape. Stage IV larvae have well developed trunk limbs, and stage V larvae show atrophy of the antennae. We describe the ontogeny of selected features such as trunk limbs and carapace, discuss ontogeny and homologization of head appendages, follow the development of the feeding mechanism, and discuss trunk limb ontogeny.     

Draft genomes and genomic divergence of two Lepidurus tadpole shrimp species (Crustacea,Branchiopoda, Notostraca)

Crustaceans of the order Notostraca (Branchiopoda) are distributed worldwide and are known for the remarkable morphological stasis between their extant and Permian fossil species. Moreover, these crustaceans show relevant ecological traits and a wide range of reproductive strategies. However, genomic studies on notostracans are fairly limited. Here, we present the genome sequences of two notostracan taxa, Lepidurus arcticus and Lepidurus apus lubbocki. Taking advantage of the small genome sizes (~0.11 pg) of these taxa, genomes were sequenced for one individual per species with one run on the Illumina HiSeq X platform. We finally assembled 73.2 Mbp (L. arcticus) and 90.3 Mbp (L. apus lubbocki) long genomes. Assemblies cover up to 84% of the estimated genome size, with a gene completeness >97% for both genomes. In total, 13%–16% of the assembled genomes consist of repeats, and based on read mapping, L. apus lubbocki shows a significantly lower transposable element content than L. arcticus. The analysis of 2,376 orthologous genes indicates an ~7% divergence between the two Lepidurus taxa, with a nucleotide substitution rate significantly lower than that of Daphnia taxa. K_a/K_s analysis suggests purifying selection in both branchiopod lineages, raising the question of whether the low substitution rate of Lepidurus is correlated with morphological conservation or is linked to specific biological traits. Our analysis demonstrates that, in these organisms, it is possible to obtain high‐quality draft genomes from single individuals with a relatively low sequencing effort. This result makes Lepidurus and Notostraca interesting models for genomic studies at taxonomic, ecological and evolutionary levels.     

Population dynamics of Triops cancriformis (Crustacea: Branchiopoda: Notostraca) of the Espolla temporary pond in the northeastern Iberian peninsula

The population dynamics of Triops cancriformis in Espolla temporary pond (NE Iberian peninsula) were studied during 1996 and 1997, which encompassed six flooded periods. Data were collected on each individual's size, sex, and, if female, on number of eggs in the oostegopodes. Male-biased sex ratios were found only in the drying-out phase and variations in fecundity were strongly related to hydroperiod duration. Sex ratio variation during the drying-out phase can be attributed to female mortality because the very low recruitment observed does not support the hypothesis of an increase of males. Two hypotheses are advanced to account for female mortality: (1) differential reproductive effort, and (2) size selective predation by herons. This population is characterised by low values of maximum densities compared with other notostracan populations, and by higher densities in the spring–summer hydroperiods than in the winter ones.     

Male occurrence in Austrian populations of Triops cancriformis (Branchiopoda, Notostraca) and ultrastructural observations of the male gonad

Abstract. Recent reports of occasional males in Austrian populations of Triops cancriformis have promoted interest in an analysis of the ultrastructure of the male gonad. It appears as a double thick and well‐defined tubular structure running along the midgut, inside the hemocoel. It is composed of two portions: the wall and the lumen. The former is made up of germ cells that are widespread and intermixed with somatic cells. The lumen is ～200 μm wide and acts as the collecting site of spermatids and sperm. The germ cells are recognizable by their rounded appearance and by the occurrence of exceptionally long synaptonemal complexes in their nuclei. Their maturation implies a volume elongation and an aggregation in a characteristic “cyst” arrangement. The cysts migrate towards the lumen, where they break open, releasing spermatids and sperm. At the end of this process the lumen is filled with maturing gametes. The sperm have very condensed chromatin nuclei and partially reduced cytoplasm where the most striking features are the axoneme and strewn microtubules together with evident pseudopodia. Our morphological data support the full functionality of males in T. cancriformis. However, further work on gametogenesis, distribution of sexes, and genetic analysis of breeding experiments are needed to reach a fuller understanding of the role of males in middle and northern European populations of this species.     

Discriminating between the Effects of Founding Events and Reproductive Mode on the Genetic Structure of Triops Populations (Branchiopoda: Notostraca)

Crustaceans that initially colonize a freshwater temporary pond can strongly bias the subsequent genetic composition of the population, causing nearby populations to be genetically distinct. In addition, these crustaceans have various reproductive modes that can influence genetic differentiation and diversity within and between populations. We report on two species of tadpole shrimp, Triops newberryi and Triops longicaudatus “short”, with different reproductive modes. Reproduction in the tadpole shrimp can occur clonally (parthenogenesis), with self fertilization (hermaphroditism), or through outcrossing of hermaphrodites with males (androdioecy). For all these reproductive modes, population genetic theory predicts decreased genetic diversity and increased population differentiation. Here we use mitochondrial control region (mtCR) sequences and nuclear microsatellite loci to determine if the difference in reproductive mode affects the high genetic structure typical of persistent founder effects. Previous authors indicated that T. newberryi is androdioecious because populations are composed of hermaphrodites and males, and T. longicaudatus “short” is hermaphroditic or parthenogenetic because males are absent. In our data, T. newberryi and T. longicaudatus “short” populations were highly structured genetically over short geographic distances for mtCR sequences and microsatellite loci (T. newberryi: Φ_ST = 0.644, F _ST = 0.252, respectively; T. l. “short”: invariant mtCR sequences, F _ST = 0.600). Differences between the two Triops species in a number of diversity measures were generally consistent with expectations from population genetic theory regarding reproductive mode; however, three of four comparisons were not statistically significant. We conclude the high genetic differentiation between populations is likely due to founder effects and results suggest both species are composed of selfing hermaphrodites with some level of outcrossing; the presence of males in T. newberryi does not appreciably reduce inbreeding. We cannot exclude the possibility that males in T. newberryi are non-reproductive individuals and the two species have the same mating system.     

The spermatozoon of a 'living fossil': Tubiluchus troglodytes (Priapulida)

The spermatozoon of Tubiluchus troglodytes, the first priapulid formally described from the Mediterranean Sea has a head composed of an acrosome and a nucleus. The acrosome is divided in two branches coiled around the nucleus. The nucleus is basally columnar, but apically generates two rods helically coiled one around the other. The midpiece is formed by an axoneme with 27 accessory microtubules, surrounded by three mitochondria. An annulus separates the midpiece from the tail that contains a 9 + 2 axoneme surrounded by nine accessory microtubules. The spermatozoon of T. troglodytes is similar to that of the other two species known from the genus, and completely different from the 'primitive' one of the other priapulids. Since Tubiluchus is considered the most basal of the extant priapulids, and the only genus with an internal fertilization, it may be that in priapulids the external fertilization is a derived character.     

Mitochondrial and nuclear DNA variability in the living fossil Triops cancriformis (Bosc, 1801) (Crustacea, Branchiopoda, Notostraca)

The living fossil Triops cancriformis comprises bisexual (either gonochoric or hermaphroditic) and unisexual populations. Genetic surveys have recently revealed a general trend of low differentiation of 12S and 16S mitochondrial genes. We, therefore, surveyed further mitochondrial (COI gene and control region) and nuclear markers (dinucleotide microsatellites) to assess the genetic variability and to establish any relationship with the different reproductive modes found in European populations. The mitochondrial analyses confirmed the pattern of low variability. Hence, the low mitochondrial genetic variability appears as a common feature of the genus Triops. The microsatellite analysis found that Italian populations are monomorphic or exhibit little polymorphism, while other European samples display a higher degree of polymorphism and private alleles. Spanish, Austrian and Italian populations show patterns of Hardy-Weinberg disequilibrium that could be explained by the mode of reproduction, or by a higher frequency of null alleles in these populations. The low diversity and differentiation among Italian populations lead us to question the Monopolization Hypothesis. One microsatellite locus appears to be sex-linked, with heterozygotes detected only in males and hermaphrodites.     

Predation in a temporary pond with special attention to the trophic role of Triops cancriformis (Crustacea: Branchiopoda: Notostraca)

Temporary ponds are described as environments with a low predation pressure. Notostracans inhabit these types of ponds, and some populations acquire a high proportion of larger individuals, whose feeding behaviour is mainly predatory. The predation nature of Triops cancriformis is not widely accepted, because its diet is still partly controversial. We analysed the diet of one population of T. cancriformis in a Mediterranean temporary pond (Espolla pond, NE Iberian peninsula) to evaluate its predation behaviour. The gut content of the individuals bigger than 10 mm is mainly composed of detritus, plant fibres and microcrustaceans (cladocerans, ostracods and copepods). The prey number increases with the body size of individual T. cancriformis as previously described. Sex-biased predation was observed for one copepod prey, but not for the other. Predation pressure in that community was monitored along six hydroperiods as the percentage of predator biomass (not only T. cancriformis) in relation to the non-predator biomass. The proportion of predator biomass is high, and this contrasts with the low predation pressure expected for a temporary system with short hydroperiods.     

Structure and habits of the 'living fossil' pelecypod Neotrigonia

Neotrigonia (Molluscs, Pelecypoda). the only surviving genus of the once diverse and widespread Trigoniacea, was collected live from depths of 6–80 m on sandy mud bottoms off southeastern Australia. The two species collected. Neotrigonia gemma Iredale, 1924 and Neotrigonia margaritacea (Lamarck), 1804, are structurally similar and differ mainly in size and coloration.
Neotrigonia has such primitive features as an unfused mantle and filibranchiate ctenidia. Its foot is large, active and muscular and the ctenidial ciliary pattern resembles that of the Unionacea. The shell is non-streamlined and has pronounced radial omamentation.
Neotrigonia lives infaunally near the sediment surface, filter feeds. and is capable of fairly rapid burrowing. The burrowing facility, conferred by the foot, is unexpected in light of shell morphology. This suggests that shell morphology alone is not necessarily a reliable guide to the life habits of fossil trigoniaceans.