Tom Moens

Observations on the feeding ecology of estuarine nematodes

Observations on living estuarine nematodes show that previous feeding type classifications do not accurately represent the trophic structure of an intertidal mudflat in the Westerschelde Estuary (Netherlands). A new scheme with six major nematode feeding guilds is p roposed: (1) microvores; (2) ciliate feeders; and (3) deposit feeders sensu stricto are all nematodes without a distinct buccal armature. In the first two groups bacteria and protozoa, respectively are the major particulate food sources, while other items are included in the diet of the third. The three other categories are recognized among the nematodes with a buccal armature: (4) epigrowth feeders; (5) facultative predators; and (6) predators. Diatoms and other microalgae are an important particulate food for many epigrowth feeders. The importance of bacteria as a food source for these nematodes remains poorly documented. A strictly or mainly predatory behaviour has been described for only few species from the study area. Several nematodes, however, are facultative predators. The predatory strategy of Calyptronema maxweberi, as described in this paper, suggests the use of a paralysing or lethal secretion in prey capture, which, to our knowledge, is the first report for aquatic nematodes. Furthermore, the importance of sources other than particulate food in free-living aquatic nematodes is stressed. Our observatior., show that many aquatic nematodes are in fact opportunistic feeders, which may change feeding strategies in response to available food.

Dispersal and gene flow in free-living marine nematodes

Dispersal and gene flow determine connectivity among populations, and can be studied through population genetics and phylogeography. We here review the results of such a framework for free-living marine nematodes. Although field experiments have illustrated substantial dispersal in nematodes at ecological time scales, analysis of the genetic diversity illustrated the importance of priority effects, founder effects and genetic bottlenecks for population structuring between patches <1 km apart. In contrast, only little genetic structuring was observed within an estuary (<50 km), indicating that these small scale fluctuations in genetic differentiation are stabilized over deeper time scales through extensive gene flow. Interestingly, nematode species with contrasting life histories (extreme colonizers vs persisters) or with different habitat preferences (algae vs sediment) show similar, low genetic structuring. Finally, historical events have shaped the genetic pattern of marine nematodes and show that gene flow is restricted at large geographical scales. We also discuss the presence of substantial cryptic diversity in marine nematodes, and end with highlighting future important steps to further unravel nematode evolution and diversity.

Carbon flows through a benthic food web: Integrating biomass, isotope and tracer data.

The herbivorous, detrital and microbial pathways are major components of marine food webs. Although it is commonly recognized that these pathways can be linked in several ways, elucidating carbon transfers between or within these pathways remains a challenge. Intertidal flat communities are driven by a wide spectrum of organic matter sources that support these different pathways within the food web. Here we reconstruct carbon pathways using inverse analysis based on mass balancing, stable isotope signatures and tracer data. Data were available on biomass, total carbon production and processing, integrated diet information from stable isotope signatures and the transfer of recently produced carbon through the food web from an isotope tracer study. The integration of these data improved the quality of the inverse food web reconstruction considerably, as demonstrated explicitly by an uncertainty analysis. Deposition of detritus (detrital pathway) from the water column and subsequent assimilation and respiration by bacteria and to a lesser extent by microbenthos (microbial pathway) dominated the food web. Secondary production was dominated by bacteria (600 mg C m −2 d −1 ), but transfer to higher trophic levels was limited to 9% and most bacterial carbon was recycled back to dissolved organic carbon (DOC) and detritus. Microbenthos secondary production (77 mg C m −2 d −1 ) was supported by DOC (73%) and detritus (26%) and was entirely transferred up the food web. The higher trophic levels consisting of nematodes, meiobenthos (copepods, ostracods and foraminifera) and macrobenthos fed highly selectively and relied primarily on microphytobenthos and pelagic primary production (herbivorous pathway). Deposit feeding is a common feeding mode among these sediment dwelling fauna, but detritivory was negligible due to this selective feeding. This strong resource selectivity implies that the herbivorous and detrital-microbial pathways function more or less autonomously, with limited interaction.

Phylogeography of the **Rhabditis (Pellioditis) marina** species complex: evidence for long-distance dispersal, and for range expansions and restricted gene flow in the northeast Atlantic

Pinpointing processes that structure the geographical distribution of genetic diversity of marine species and lead to speciation is challenging because of the lack of obvious dispersal barriers and the likelihood of substantial (passive) dispersal in oceans. In addition, cryptic radiations with sympatric distributions abound in marine species, challenging the allopatric speciation mechanism. Here, we present a phylogeographical study of the marine nematode species complex Rhabditis (Pellioditis) marina to investigate processes shaping genetic structure and speciation. Rhabditis (P.) marina lives on decaying macroalgae in the intertidal, and may therefore disperse over considerable distances. Rhabditis (P.) marina consists of several cryptic species sympatrically distributed at a local scale. Genetic variation in the COI gene was screened in 1362 specimens from 45 locations around the world. Two nuclear DNA genes (ITS and D2D3) were sequenced to infer phylogenetic species. We found evidence for ten sympatrically distributed cryptic species, seven of which show a strong genetic structuring. A historical signature showed evidence for restricted gene flow with occasional long‐distance dispersal and range expansions pre‐dating the last glacial maximum. Our data also point to a genetic break around the British Isles and a contact zone in the Southern Bight of the North Sea. We provide evidence for the transoceanic distribution of at least one cryptic species (PmIII) and discuss the dispersal capacity of marine nematodes. The allopatric distribution of some intraspecific phylogroups and of closely related cryptic species points to the potential for allopatric speciation in R. (P.) marina.

Exploring the Use of Cytochrome Oxidase c Subunit 1 (COI) for DNA Barcoding of Free-Living Marine Nematodes

Background The identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition. Methodology Both partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance. Conclusion The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

Dual stable isotope abundances unravel trophic position of estuarine nematodes

The role and quantitative importance of free-living nematodes in marine and estuarine soft sedimentsremain enigmatic for lack of empirical evidence on the feeding habits and trophic position of most nematodespecies. Here we use natural abundances of carbon and nitrogen stable isotopes of some abundantnematode species/genera from estuarine intertidal sediments to assess their trophic level and major foodsources. In all stations, d15N of diierent dominant nematode species/genera spanned a range of 3.6 to6.3 ppt, indicating that at least two trophic levels were represented. The large nematodes Enoplus brevis,Enoploides longispiculosus and Adoncholaimus fuscus consistently had high d15N, in line with mouth-morphologybased predictions and empirical evidence on their predacious feeding modes. Daptonema sp., Metachromadoraremanei, Praeacanthonchus punctatus and Chromadoridae (dominated by Ptycholaimellus ponticus) hadcomparatively lower d15N, and d13C suggesting that microphytobenthos (MPB) is their major carbonsource, although freshly sedimented particulate organic matter may also contribute to their nutrition insilty sediments. The trophic position of Sphaerolaimus sp., a genus with documented predacious feedingmode, was ambiguous. Ascolaimus elongatus had d15N signatures indicating a predacious ecology, which isat variance with expectations from existing feeding type classi¢cations. Our study shows thatdespitelimitations imposed by the biomass requirements for EA-IRMS (elemental analyserisotope ratio massspectrometry)natural isotope abundances of carbon and nitrogen are powerful tools to unravel trophicstructure within nematode communities. At the same time, the prominence of diierent trophic levelsresults in a large span of d15N, largely invalidating the use of nitrogen isotope abundances to assess foodsources and trophic level of whole nematode communities.

Temperature and salinity constraints on the life cycle of two brackish-water nematode species

The present study investigates the influence of salinity and temperature on the life history of two estuarine bacterivorous nematode species, Pellioditis marina and Diplolaimelloides meyli, isolated from the mesohaline zone of the Westerschelde Estuary, SW Netherlands. Gravid females and adult males were inoculated in petri dishes containing agar layers of nine (for P. marina) or five (for D. meyli) different salinities, from almost freshwater to higher than marine, and incubated at a temperature of 20°C, to study the impact of salinity; agar layers with a salinity of 20‰, incubated under each of six different temperatures from 5 to 30°C, served to study the effect of temperature. Daily and total fecundity, development time and sex ratio were quantified, and preadult mortality was estimated. The results are compared to those of a partner study on the influence of salinity and temperature on respiration, assimilation and scope for production in the same nematode species. Salinity had relatively minor effects on fecundity, development times and sex ratio in both species, but strongly impacted juvenile viability at the extremes of the salinity range: at salinities close to 0 and 40‰, preadult mortality was more than 80% in P. marina; it was 100% at 5‰ in D. meyli. Both species had an (near) optimal fitness at salinities of 10 to 30‰. Temperature had a pronounced influence on both nematodes over the entire range studied. Diplolaimelloides meyli still reproduced and matured at temperatures exceeding 30°C, while P. marina had an upper temperature limit for reproduction of 25°C. Development times of D. meyli were more temperature-dependent than those of P. marina: the mean development time from adult to adult for the latter nematode ranged from 2 days at 25°C to 7 days at 9°C. The development time of D. meyli increased from 7 days at 25–30°C to 63 days at 10°C, temperature below which no reproduction occurred. Female-biased sex ratios were found in D. meyli at low temperatures and in P. marina under optimal salinity conditions. The life history results largely agree with the predicted scope for production, but discrepancies were found near the extremes of the abiotic range of both species. It is emphasized that the ranges observed are characteristic of populations, not of species; they may to an extent have been influenced by culture conditions. A comparison of the present results with literature data on other P. marina populations demonstrates that some populations of this species may still reproduce successfully under conditions which are lethal to other populations, raising the question as to whether cryptic species rather than populations of a single species are involved.

ON THE CULTIVATION OF FREE-LIVING MARINE AND ESTUARINE NEMATODES

Although a large body of literature exists on the systematics and ecology of free-living marine and brackish-water nematodes, key questions on the nature and magnitude of interactions between nematodes and other organisms in the benthos remain unanswered. Relatively few authors have investigated live nematodes in food web studies or in experiments dealing with the nematodes’ response to a varying environment. It is mainly for the latter purpose that attempts have been made to maintain, rear and cultivate selected species. This paper describes the methodology used for the maintenance, rearing, and eventual permanent agnotobiotic cultivation of a variety of estuarine nematodes. Spot plates, where small samples of sediment or macrophyte material are inoculated on a sloppy agar layer, have been used for the purpose of maintenance and initial cultivation. Those species that reproduce on spot plates are then selected for monospecific cultivation on agar layers with different nutrient enrichments and with micro-organisms cotransferred from the spot plates as food. Mixtures of bacto and nutrient agar prepared in artificial seawater were specifically suitable for the xenic cultivation of nine bacterivorous and, when supplied with Erdschreiber nutrients, two algivorous/bacterivorous nematode species. Up to three generations of five other nematode species have been reared under laboratory conditions, and several more were kept alive and active for variable periods of time on agar. Generation times observed on spot plates forAdoncholaimus fuscus andOncholaimus oxyuris were substantially shorter than previously published estimates and suggest a correspondingly higher predatory and scavenging potency for these and related enoplids. A procedure for the long-term storage of nematodes at −80°C with glycerol as a cryoprotectant was successfully used forDiplolaimella dievengatensis, Panagrolaimus sp. 1, andPellioditis marina, but not forDiplolaimelloides meyli. The authors have also summarized the existing literature on the cultivation of marine and brackish-water nematodes. Continuous cultivation appears to have been successful mainly for Aufwuchs and epiphytic nematodes; only few sediment-dwellers have been established in permanent culture. Of only just over 30 species that have ever been cultivated, more than half belong to one family (Monhysteridae) and three are Rhabditida, an order poorly represented in the marine environment. Four species have been grown in monoxenic and one in axenic culture, the latter though with limited success. It is concluded that our understanding of the basic nutritional requirements of marine nematodes is as yet insufficient, and that the culture techniques which have so far mainly deployed agar or liquid substrates, while being suitable for the cultivation of Aufwuchs and epiphytic nematodes, do not accurately enough mimic gradients specific of the natural habitat of many sediment-dwellers.

Temperature, salinity and food thresholds in two brackish-water bacterivorous nematode species: assessing niches from food absorption and respiration experiments

Abstract Respiration and food assimilation of two estuarine bacterivorous nematodes, the rhabditid Pellioditis marina and the monhysterid Diplolaimelloides meyli , were measured at a range of temperatures, salinities, and food densities. The aim of this study was to identify the fundamental niche of both species in their natural habitat, and to investigate the relative importance of food and abiotic factors in determining presence and success of nematode species in the highly dynamic estuarine tidal environments of macrophyte detrital habitats. Of the three factors studied, salinity least impacted P. marina and D. meyli . Respiration and assimilation in both species showed only minor variation in the salinity range of 10 to 30‰. Respiration decreased at marine salinities in P. marina , and increased in both species at oligohaline salinities down to a stress-induced maximum around a salinity of 5‰, then steeply declined towards freshwater conditions. Temperature heavily affected both species, but Q 10 -values in D. meyli were considerably higher than in P. marina , suggesting the former species to be particularly well adapted to fine-tuning its energy expenditure as a function of temperature. The highest respiration and assimilation rates in both species were at 25°C. At still higher temperatures, metabolic rates were depressed, but while P. marina was entirely inactivated above 30°C, D. meyli continued to respire and assimilate food up to 35°C. The scope for production, calculated as the net difference between assimilation and respiration rates (both expressed in units of C), was 0 at 5°C and increased to a maximum at 25°C in both nematodes; it declined at higher temperatures, but remained positive up to 35°C in D. meyli . Significant food assimilation in both nematodes occurred only at bacterial densities above 10 8 cells ml −1 . Assimilation rate reached a maximum at 5×10 8 cells ml −1 in D. meyli , and remained constant at higher densities. P. marina , by contrast, had a well defined peak assimilation at a food density of 2.5×10 9 cells ml −1 , with lower rates at both lower and higher food densities. This contrasts with observations on ingestion rate, and suggests a food density-dependent assimilation efficiency. The present results suggest that tolerances of exposure to salinity and temperature extremes on a daily rather than seasonal basis, may be of higher significance in niche differentiation between both species. Their fundamental niche further appears to be determined by the range of (near) optimal food conditions, which is narrow in P. marina but comparatively broader in D. meyli .

Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae)

Derycke, S., De Ley, P., De Ley, I.T., Holovachov, O., Rigaux, A. & Moens, T. (2010). Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae).—Zoologica Scripta, 39, 276–289.