Molecular evidence for further overlooked species within the Gammarus fossarum complex (Crustacea: Amphipoda)

The freshwater amphipod Gammarus fossarum Koch, in Panzer, 1836 is a locally abundant keystone species mainly occuring in European headwaters but also in larger rivers. Genetic studies in the past 25 years have revealed three cryptic species within nominal G. fossarum (types A, B and C). Assignments of specimens to these types were based on allozyme and 16S markers. Today, a fragment of the cytochrome c oxidase subunit 1 (CO1) is primarily used as a genetic marker for species assignments (‘DNA Barcoding’), yet not a single CO1 sequence of G. fossarum is available in the Barcode of Life Database. We analysed new CO1 and 16S data for German, Hungarian and Croatian G. fossarum specimens and compared them with 16S and CO1 sequences of G. fossarum from GenBank. Thereby, we close the gap between traditional allozyme- and 16S-based species assignments and modern CO1 barcoding. Studying genetic variation in 55 specimens from 29 populations, we identified between 11 and 23 novel and genetically distinct clades using distance- and tree-based methods. Our results suggest that G. fossarum comprises several additional, yet unrecognised, species in particular from the Balkan region. Therefore, a taxonomic revision and biogeographic reconsideration of the G. fossarum complex is urgently needed.     

Environmental calcium and mitochondrial DNA polymorphism among local populations of Gammarus fossarum (Crustacea, Amphipoda)

Mitochondrial DNA sequence analysis was used to investigate genetic diversity and phylogeographic population structure among geographically isolated alpine populations of the amphipod Gammarus fossarum. This study was performed across a region with an important glacial history and substantial geographical variations in calcium levels, in order to evaluate the relative impact on the genetic diversity of the population history and this environmental factor known to be associated with local adaptation and population growth. Analysis of the nucleotide sequences for a 376-bp segment of the cytochrome c oxidase I gene in 84 specimens from seven different populations revealed 20 different haplotypes distributed into five major lineages that are not geographically structured. Analysis of molecular variance indicates that the populations from the crystalline massif are less diverse than those living on limestone, suggesting a global correlation between genetic diversity and calcium concentration of the water. However, the role of this environmental factor appears to be indirect, with smaller population size leading to lower genetic diversity.     

Isolation and characterization of the first eight microsatellite loci in Gammarus fossarum (Crustacea, Amphipoda) and cross-amplification in Gammarus pulex and Gammarus orinos

We characterized the first microsatellite markers for Gammarus fossarum. Eight loci gave satisfactory amplification patterns in two stream populations (Southern France) with number of alleles ranging from 2 to 10 and expected heterozygosity from 0.076 to 0.857. We performed cross-amplification in two closely related gammarid species, Gammarus pulex and Gammarus orinos. Among the eight tested microsatellite loci, four correctly amplified in G. pulex and three in G. orinos.     

The cannibalistic behaviour of two Gammarus species (Crustacea: Amphipoda)

This study examines the cannibalistic behaviour of the freshwater amphipods Gammarus duebeni celticus Stock & Pinkster, 1970 and G. pulex (L., 1758). In the first experiment, interactions were staged among all combinations of single adult males, single adult females, adults in the precopulatory mate-guarding phase and juveniles. Cannibalism by inter-moult individuals on newly moulted conspecifics occurred in all interaction categories in both species. Gammarus d. celticus , however, were significantly more cannibalistic than G. pulex. Cannibalism between and within sex and size categories (males > females > juveniles) was facilitated by the vulnerability of individuals at moult. Individuals of smaller size categories, however, did not cannibalize newly moulted conspecifics of larger size categories. Males were less cannibalistic on newly moulted females than on newly moulted males and juveniles and, when in the precopulatory condition, appeared to defend females from cannibalistic attacks. In a second experiment, stream conditions were simulated in the laboratory and replicated populations monitored for nine weeks. High levels of cannibalism, and the species and sex differences in cannibalism identified in the first experiment, were confirmed under these heterogeneous conditions. Cannibalism by males on their newly moulted female mating partners, termed 'reversed' sexual cannibalism, was further investigated. When males were deprived of foraging opportunities, cannibalism of precopulatory partners was significantly more frequent. The occurrence of 'reversed' sexual cannibalism is thus interpreted as a conflict between motivation to feed and motivation to mate.     

The ultrastructure of the sinus gland of Gammarus oceanicus (Crustacea: Amphipoda)

Summary The sinus gland of Gammarus oceanicus, like that of other crustaceans, is composed of three elements: neurosecretory axons, glial cells and stromal sheath. Five neurosecretory axon types are identified on the basis of granule diameter, shape, and electron density, and axon matrix density. Exocytosis appears to be the major release mechanism of neurosecretory material. The preterminal regions of neurosecretory axons contain axoplasmic reticulum and neurotubules. Their arrangement in the axon and relationship with one another suggest a transport function. Multilamellar bodies are found in the terminal regions of neurosecretory axons. They arise from mitochondria and may be involved in granulolysis.The technical assistance of G.A. Bance, statistical assistance of D. MacCharles and D.W. Hagen, and financial support provided by the University of New Brunswick Research Fund to K.H. are gratefully acknowledged     

Zu den Ursachen der unterschiedlichen pH-Empfindlichkeit von Gammarus pulex L., G. fossarum KOCH (Amphipoda) und Aselus aquaticus L. (Isopoda)

By means of ion chromatography the extensive and significantly higher loss of ions of G. pulex and G. fossarum in acid water (pH 3.0) as compared to neutral water is proved. While cations are being accumulated in the water, some ions (Na⁺, Cl⁻) leave the hemolymph in important percentage, others increase highly after being set free from the cells (K⁺ and Ca²⁺). The loss of cations with Asellus aquaticus in acid water (pH 3) is clearly lower than with Gammarus, and it is not significantly higher than when the animals are put into neutral water.     

Differential parasite (Trematoda) encapsulation in Gammarus aequicauda (Amphipoda)

Because resistance to parasites usually has a cost for host species, it is theoretically expected that, in case of multi-infection, host immune responses should vary according to the levels of parasite pathogenicity. The crustacean gammarid Gammarus aequicauda is the second intermediate host of 4 trematode species. Three of these parasites always encyst in the abdomen of gammarids and have no particular effect on the host. However, 1 of these species is sometimes able to encyst in the cerebroid ganglia of the gammarid and strongly alter its behavior in a way that increases its predation risk by aquatic birds, the definitive hosts. In accordance with the hypothesis that the level of parasite pathogenicity influences the likelihood and the degree of host reaction, cases of melanization in our gammarid collection almost exclusively concern the cerebral metacercariae. Our results also indicate that this melanization is able to cancel the behavioral alterations induced by the parasite, suggesting that the cause of the manipulation is not the physical presence of metacercariae in the brain.     

Assortative pairing in Gammarus insensibilis (Amphipoda) infected by a trematode parasite

We have investigated the influence of Microphallus papillorobustus (Trematoda) on the reproductive biology and mating patterns of its intermediate host Gammarus insensibilis (Amphipoda). Infected Gammarus species show altered behaviour which renders them more susceptible to predation by Charadriiform birds, the parasite's definitive hosts. In a natural population of G. insensibilis, mean parasite intensity was higher for unpaired individuals than for paired individuals. Fecundity was reduced in infected amphipods. Size-assortative pairing was significant, although infected males were found with smaller females compared to uninfected males of the same size. There was also a positive assortative pairing by parasitic prevalence. Vertical segregation between infected and uninfected individuals, male-male competition for access to uninfected females, and female choice may explain assortative mating for prevalence. This study provides the first empirical evidence that parasites can have a direct effect on patterns of mating in gammarids.     

The dynamics of predation on Gammarus spp. (Crustacea: Amphipoda)

Gammarus spp. (Crustacea: Amphipoda) are widespread throughout a diverse range of marine, freshwater and estuarine/brackish habitats, often dominating benthic macroinvertebrate communities in terms of both numbers and/or biomass. Gammarus spp. are the dominant macroinvertebrate prey items of many fish, whether as a seasonal food source or a year-round staple. Selective predation by fish on Gammarus spp. is often linked to parasitism and the body size of the prey. Gammarus spp. populations are under increasing threat from both pollution and replacement/displacement by introduced species. Loss of populations and species invasions/replacements could have significant impacts on native predator species if the predator(s) cannot successfully adapt their feeding patterns to cope with non-indigenous Gammarus prey species. Despite this, many fish predation studies do not identify Gammarus prey to species level. This lack of precision could be important, as Gammarus spp. exhibit wide variations in physiochemical tolerances, habitat requirements, abilities to invade and susceptibility to replacement. Although rarely acknowledged, the impacts of nonpiscean predators (particularly macroinvertebrates) on Gammarus prey species may frequently be stronger than those exerted by fish. A major aim of this review is to ascertain the current importance of Gammarus as a prey species, such that the implications of changes in Gammarus spp. populations can be more accurately assessed by interested groups such as ecologists and fisheries managers. We also review the dynamics of Gammarus spp. as prey to a diverse array of mammals, birds, amphibians, insects, flatworms, other crustaceans such as crabs and crayfish and, perhaps most importantly, other Gammarus spp.     

Parasite altered micro-distribution of Gammarus pulex (Crustacea: Amphipoda)

In a river survey, Gammarus pulex amphipods both unparasitised and parasitised with the acanthocephalan Echinorhynchus truttae were distributed similarly with respect to flow regimen, tending to be more abundant in faster, shallower, riffle patches. However, there was a higher prevalence of parasitism in faster, shallower areas than in slower, deeper areas and abundance correlated with macrophyte coverage for unparasitised but not parasitised amphipods, indicating subtle differences in habitat usage. A laboratory 'patch' simulation indicated that parasitism influenced micro-distribution. There were higher proportions of unparasitised amphipods in/under stone substrates and within weed. In contrast, there were higher proportions of parasitised amphipods in the water column and at the water surface. As the experiment progressed, unparasitised but not parasitised amphipod habitat usage shifted from those micro-habitats above the substrate and in the water column to those in/under the substrates. Experiments also demonstrated that parasitised amphipods were more active and had a greater preference for illumination. Previous studies of the effects of acanthocephalan parasitism of amphipod hosts have focussed on how drift behaviour is altered, now we show that subtle differences in micro-habitat usage could translate to greatly increased vulnerability to fish predation. We discuss how aggregation of parasitised individuals within specific habitats could promote parasite transmission.     

Calcium balance in premoult and post-moult Gammarus pulex (Amphipoda)

SUMMARY. Adult Gammarus pulex lose about 42% body calcium into solution over a 2–3-day period preceding the moult. A further 54% body calcium is lost with the exuviae, leaving c . 4% in the newly moulted animal.
Nearly all of the body calcium in a stage A (post moult) animal is contained in the hepatopancreas and a transient increase in the calcium content of this tissue is seen in some individuals. The haemolymph calcium compartment is heavily depleted at this stage. In stage B animals the hepatopancreas calcium level has returned to, or even fallen below, the intermoult level while the haemolymph calcium concentration remains lower than in intermoult animals.
Animals switch from a strongly negative calcium balance to a state of rapid calcium uptake immediately following moult. Uptake at 10°C proceeds at a rate of between 3 and 10 μmol g⁻¹ h⁻¹ depending on the calcium concentration of the external medium. The lower rate is found in starved animals in 0.1 mM calcium and this concentration is probably close to a minimum for the satisfactory restoration of body calcium. Recalcification is completed in 10–14 days in 0.1 mM calcium and is apparently enhanced by the presence of food in the form of a portion of oak leaf together with the cast exuviae. In 1.0 mM calcium the recalcification period is shortened to 3–4 days. This is considerably longer than the time recorded for French populations of this species (Vincent, 1969), although reasons for this are offered.     

Comparative study of mathematical models for the relationship between water temperature and brood development time of Gammarus fossarum and G. roeseli (Crustacea: Amphipoda)

SUMMARY. 1. The relationship between water temperature ( T°C ) and brood development time (d days for embryonic development time plus 'post-hatch time') was investigated experimentally using animals from four populations of Gammarus fossarum (populations 1–4) and two populations of G. roeseli (populations 5 and 6) in Austrian streams.
2. Eleven mathematical models were examined as suitable functions to describe the relationship between d and T . The models were compared with respect to their goodness-of-fit and suitability for further quantitative, statistical analyses of intra- and interspecific differences between natural populations of G. fossarum and G. roeseli .
3. Statistical analyses were performed with logarithmically transformed values of d , in order to provide homogeneous variances between the different constant temperatures used. The following three-parameter model was found to be the most suitable for the data:
This equation was fitted in its linear form:      

Life history and reproductive capacity of Gammarus fossarum and G. roeseli (Crustacea: Amphipoda) under naturally fluctuating water temperatures: a simulation study

SUMMARY 1. Mathematical functions developed in long‐term laboratory experiments at different constant temperatures were combined with daily water temperatures for 1991–93 in eight Austrian streams and rivers to simulate the complex life histories and reproductive capacities of two freshwater amphipods: Gammarus fossarum and G. roeseli. The functions describe brood development times, hatching success, times taken to reach sexual maturity, growth, and fecundity. The sex ratio was assumed to be 0.5 and an autumn–winter reproductive resting period was based on observations of six river populations. Simulations included summer‐cold mountain streams, summer‐warm lowland rivers, watercourses fed by groundwater or influenced by heated effluents, and varying amplitudes of change within each year. 2. A fortran 77 computer program calculated growth from birth to sexual maturity of first‐generation females born on the first day of each calendar month in 1991, and the numbers of offspring successfully released from the maternal broodpouch in successive broods. At the 1991–93 regimes of temperature, individual G. fossarum released 127–208 offspring and G. roeseli released 120–169 in seven or eight successive broods during life spans of less than 2 years in six rivers. Life spans extended into a third year in the relatively cool River Salzach (mean temperature 7.5 °C). They were not completed in the very cold River Steyr (mean 5.6, range 2.5–7.9 °C), where G. fossarum produced five broods (totalling 120 offspring) and G. roeseli only two broods (totalling 28 offspring) in the 3‐year period. Except in the Steyr, some offspring grew rapidly to maturity and produced several second‐generation broods during the simulation period; in the warmest rivers some third‐generation broods were also produced. Birth dates, early or late in the year, influenced the subsequent production of broods and young, depending on temperature regimes in particular rivers. Total numbers of offspring produced by the second and third generations represent the theoretical reproductive capacities of G. fossarum and G. roeseli. Minimum and maximum estimates mostly ranged from 100 to 17 300, were larger for G. fossarum except in the warmest river (March), where temperatures rose above 20 °C for 56–78 days in summer, and largest (maximum 37 600) in the River Voeckla heated by discharge from a power‐station (mean 11.5 °C). Results from the simulations agree with preliminary assessments of relative abundances for G. fossarum and G. roeseli in several of the study rivers, but in some one or both species appear to be absent. On a wider scale, the present study confirms that G. fossarum is potentially more successful than G. roeseli in cool rivers but indicates that neither species is likely to maintain viable populations in cold rivers strongly influenced by snow and ice‐melt. 3. The potential impacts of future river warming by increases of 1, 2 and 3 °C, due to climate change, vary according to river site, date of fertilisation, the extent of temperature increase, and the species of Gammarus. For Austrian rivers with mean temperatures in the range c. 7–10 °C, future warming would result in modest changes in the life histories and reproductive capacities of both G. fossarum and G. roeseli; the former would find improved temperature conditions in watercourses that are currently very cold throughout the year, and both would find warm rivers less tolerable. 4. The high potential reproductive capacity of gammarids, with rapid production of numerous successive broods when sexual maturity is finally achieved, indicates adaptation to high mortality during the relatively long period of growth to sexual maturity, and provides scope for an opportunistic strategy of emigration from centres of population abundance to colonise new territory when conditions are favourable. Rapid expansion of populations is desirable to combat the effects of environmental catastrophes, both frequent and short‐term floods and droughts, and more long‐term climatic changes that have occurred several times in glacial–interglacial periods during the current Ice Age.     

Development of Ascarophis sp. (Nematoda: Cystidicolidae) to maturity in Gammarus deubeni (Amphipoda)

Experimentally transmitted Ascarophis sp. (Spirurida) developed to adult worms in the invertebrate host, Gammarus deubeni (Amphipoda), collected in the intertidal zone in Passamaquoddy Bay, New Brunswick, Canada. The morphological development and growth of larval stages is very similar to other cystidicolids, which are found as adults in fish. Unlike virtually all other Spirurida, which require a vertebrate definitive host, infective larvae of Ascarophis sp. migrate from the invertebrate host musculature into the hemocoel where they molt twice to become adults. Gravid females appear at 80 days and 69 days post-infection at 10-12 C and 18-20 C, respectively. While there is little evident host reaction to the parasite within the muscle tissue, within the hemocoel there is hemocytic reaction to shed nematode cuticles, released eggs, and sometimes the worm itself, including some melanization. The worms are morphologically similar to Ascarophis sp. from G. oceanicus in the Baltic and White seas and among Ascarophis species from fish is most similar to A. arctica. It is suggested that Ascarophis sp. no longer requires a vertebrate host and is transmitted between amphipods either through death and disintegration of infected amphipods and dispersal of the nematode eggs, or more likely through cannibalism or necrophagy.     

Anatomy of the extrinsic gut musculature of Gammarus minus (Crustacea: Amphipoda)

Inserting on the buccal and esophageal foregut of Gammarus minus are numerous pairs of serially arranged dorsal dilator muscles, a single pair of lateral muscles, and two pairs of posterior muscles. Muscles of the cardiac stomach include three dorsal sets, a single pair associated with the pterocardiac ossicles, and two pairs inserting on the ventral aspect. A single pair of muscles inserts on the lateral aspect of the pyloric stomach. The extrinsic muscles of the foregut originate from exoskeletal apodemes of the cephalothoracic cuticle, sockets of the mandible, and a maxillary bridge that lies just ventral to the cardiac stomach. The extrinsic musculature of the hindgut is restricted to the rectal region and consists of paired dorsal and ventral series in an X-configuration. A single unpaired muscle inserts on the ventral midline. Extrinsic muscles of the hindgut originate from the integument of the last pleonic segment. The general arrangement of extrinsic gut muscles in G. minus is similar but not identical to that of other amphipods studied. However, the pattern is quite different from that of other malacostracans.     

Factors affecting the microdistribution of Gammarus pulex (Amphipoda): an experimental study

SUMMARY. 1. The tnierodistribution of Gammarus species is size assortative: larger animals are associated with larger substrate particles. Using an artificial stream with scrubbed substrate we investigate the hypothesis that the assortative mechanism is stimulated by current avoidance rather than food searching.
2. In uniformly mixed substrate, G. pulex tend to move upstream, but in patchy substrate they are found in a predictable substrate grade. There is no discrimination between patches by different size classes of G. pulex in static water. The size assortative field pattern can only be reproduced under flow conditions in the laboratory. It occurs in the absence of food and irrespective of the sequence or location of substrate patches.
3. As density increases, G. pulex are displaced from the optimal patches but move into the open stream rather than into the cover of alternative patch types.
4. We discuss the significance of these results and suggest that some earlier models explaining mechanisms for microdistribution patterns tnay have been unnecessarily complex. Contrasts between natural and simulated stream situations and their experimental advantages are noted.     

Spatial distribution of cryptic species diversity in european freshwater amphipods (Gammarus fossarum) as revealed by pyrosequencing

In order to understand and protect ecosystems, local gene pools need to be evaluated with respect to their uniqueness. Cryptic species present a challenge in this context because their presence, if unrecognized, may lead to serious misjudgement of the distribution of evolutionarily distinct genetic entities. In this study, we describe the current geographical distribution of cryptic species of the ecologically important stream amphipod Gammarus fossarum (types A, B and C). We use a novel pyrosequencing assay for molecular species identification and survey 62 populations in Switzerland, plus several populations in Germany and eastern France. In addition, we compile data from previous publications (mainly Germany). A clear transition is observed from type A in the east (Danube and Po drainages) to types B and, more rarely, C in the west (Meuse, Rhone, and four smaller French river systems). Within the Rhine drainage, the cryptic species meet in a contact zone which spans the entire G. fossarum distribution range from north to south. This large-scale geographical sorting indicates that types A and B persisted in separate refugia during Pleistocene glaciations. Within the contact zone, the species rarely co-occur at the same site, suggesting that ecological processes may preclude long-term coexistence. The clear phylogeographical signal observed in this study implies that, in many parts of Europe, only one of the cryptic species is present.