Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

Benthic nematodes biodiversity patterns along the gradient of the Tagus estuary (Western Coast of Portugal): Link with abundances distribution pattern of the nonindigenous bivalve Ruditapes philippinarum

The non-indigenous bivalve Ruditapes philippinarum is native to the western Pacific Ocean and it has been one of the most widely introduced species for aquaculture purposes in Europe. In Portugal its presence is known in several estuaries for more than two decades and its populations have increased greatly along the last years1. Currently it’s one of the dominant benthic species in some areas of the Tagus estuary (264,1±470,8 ind./30s tow). Studies on the impacts of invasive bivalves on meiofaunal communities are scarce and restricted to the harvesting effect. Meiofauna of the Tagus estuary is poorly known and possible impacts of the introduction of R. philippinarum were never assessed.

Fixing, storage time and DNA extraction applied to dT-RFLP for ecological and molecular studies of marine nematodes assemblages

The application of molecular methods offers an alternative faster than traditional methods based on morphology It is nearly impossible to process all the samples in short period using traditional methods, and the deterioration of marine sediments rapidly occurs The dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows a rapid assessment of biodiversity changes of nematodes assemblages The use of a not suitable fixing, storage time and DNA extraction could be a limitation in molecular analysis like dT-RFLP and real time PCR.Objetives: the best fixative •the level of DNA degradation over the time •the best DNA extraction method for marine nematodes and suitable for dT-RFLP analysis

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Histopathology from liver of tuvira (Gymnotus spp.) parasitized by larvae of nematodes.

Abstract: The aim of this study was to evaluate the histological changes in the liver of thirty-five Gymnotus spp. parasitized by endohelminths collected between April 2012 to October 2013 in commercial bait fish farming of Pantanal basin. Histological cuts of 7?m were stained with hematoxylin-eosin for parasites research and liver changes and have also been submitted to the Perls histochemical method for evaluation of hemosiderosis (Fe+++) based on the incidence degree and severity of change (Grade I, II and III) and tests for the presence of central melanomacrophages. Parasites identified were: Brevimulticaecum sp. with a prevalence of 22,9%, Eustrongylides sp 17,1%, Contracaecum type I 68,7%, Contracaecum type II 5,7%, Contracaecum type III 5,7% and larvae of Anisakidae 11,4%. Histological analysis showed intense disorganization of hepatic parenchyma with degenerate hepatocytes due to high parasitic infection, changes that can be deleterious and compromise the organism functioning, being harmful to the health of evaluated animals. Also evidencing normal tissue interleaved with different stages of Fe+++ deposit in grades II and III, injuring or destroing the cell. Histopathological changes in the tuvira?s liver suggested a chronic response and the development of a balance relation between tuvira and parasitism by endohelminth identified in this study. There are also a testimony to the health condition of commercial bait fish farming on current ecosystem conditions.

Protostrongylids nematodes as a tool to study the biogeography of wild mammals

The European brown hare (Lepus europaeus Pallas, 1778) is an important game species, distributed across Europe and introduced in other regions. Recently, a geographically isolated population, closely related to an ancestral lineage of Lepus europaeus meridiei, was found on Pianosa Island, off the coast of Tuscany, Italy (Mengoni et al., 2018). Thus, the unique opportunity to explore the evolution and genetic structure of its helminth parasites was added to its exceptional isolation condition. Various lungworm species within the genus Protostrongylus (Nematoda: Protostrongylidae) are described in European brown hares. Our aim was to analyze the parasite population through morphological and molecular approaches in order to study the biogeography of the European brown hares (L. e. meridiei) population from Pianosa Island. Moreover, we investigated the morphology of a monospecific genus, i.e. Orthostrongylus, considering its quite intrigant descriptive history and its still unclear and debated classification. Nuclear and mitochondrial markers were used based on their resolution power and expected polymorphism; the whole Internal Transcribed Spacer 1 and 2 (ITS), including the 5.8S rRNA sequence and the Large Subunit (28S) were used, as nuclear genes, for confirmation of the species identification. Conversely, the cytochrome oxidase c subunit I (COI) was used, as mithocondrial genes, to assess interspecific genetic relationships. Molecular analysis corroborated the morphological identification since all the generated ITS and LSU sequences were 100% consistent with the species Protostrongylus oryctolagi and Orthostrongylus macrotis. The paucity of molecular data existent about this genus of parasites underlines the need for more insight’s studies. An in-depth analysis of broncho-pulmonary parasites and the host-parasites relationships along with the improvement of the use of mitochondrial genes, as well as the assessment of new polymorphic markers could contribute to an extensive understanding of parasites fauna and taxonomy, as well as their relationship with wild mammals’ hosts.

Host status of some selected Brazilian upland rice cultivars to Meloidogyne incognita race 4 and Rotylenchulus reniformis

Nowadays, rice is among the most preferred crops for rotation with soybean and cotton in the large producing areas of Central Brazil. Nevertheless, the host status of the Brazilian upland rice cultivars for Meloidogyne incognita race 4 and Rotylenchulus reniformis has not been investigated and remains unknown. This study dealt with the assessment of the host response of some selected Brazilian upland rice cultivars to these nematodes under glasshouse conditions. The host status for each tested interaction was based on the nematode reproduction factor (RF) and number of nematodes (g root)(-1). Two experiments with M. incognita race 4, referred to as trial I (initial population (IP) = 4000) and trial 2 (IP = 800), included, respectively, 14 cultivars (cvs AN Cirad 141, BRS Monarca, BRS Primavera, AN Cambara, BRS Pepita, BRS Curinga, BRS Sertaneja, IAPAR 9, IAPAR 62, IAPAR 63, IAPAR 64, IAPAR 117, IAC 201, IAC 202) and 19 cultivars (the same ones in Experiment 1 plus cvs BRS Maravilha, BRS Talento, BRS Bonanca, Ricetec Ecco, BRS Soberana). Except for cv. BRS Pepita, rated as resistant, the cultivars were rated as susceptible or moderately susceptible (RF means ranged from 1.09 to 12.56). In a third experiment with R. reniformis (IP = 1800) that included the same cultivars as in Experiment I, all cultivars were rated as resistant (RF means ranged from 0.01 to 0.29).

Pathogenicity and reproductive fitness of Pratylenchus brachyurus on cowpea

The lesion nematode Pratylenchus brachyurus is widespread in cowpea plantations throughout the tropics and sub-tropics. However, the pathogenicity of P. brachyurus on cowpea has been scarcely studied. In this work, it was demonstrated in two glasshouse experiments that an isolate (Pb-20) of P brachyurus was pathogenic to cowpea cv. IPA-206, adversely affecting the plant growth and pod formation and filling. Initial population levels of 5000 and 15 000 nematodes per plant caused reduction of root growth and typical decay of root tissue. The third experiment demonstrated that all six cowpea cultivars selected for evaluation supported reproduction of three isolates of P. brachyurus (Pb-20, Pb-21 and Pb-23) in their roots, although the reproduction factor values obtained indicated that they were dissimilar in their reproductive fitness. Low resistance to R brachyurus was reported for at least one tested cultivar, but apparently of an insufficient degree to be effective for field management of the nematode.

Meiobenthic community underneath the carcass of a stingray: a snapshot after natural death

The impact of large food falls and carrion on meiobenthic communities remains little understood. The objective of the present study was to investigate whether the carcass of a stingray, encountered fortuitously in an Australian estuary, affects the underlying meiobenthic community, in particular nematode assemblages. The integrity of the skeleton and the low redox values observed under the carcass suggest that the cadaver had been slowly and chiefly decomposed by microbes. The abundance and number of meiofaunal taxa, as well as nematode abundance and nematode-species richness, were significantly lower under the carcass when compared to samples outside the carcass. Nonetheless, a few nematode species, typical of hypoxic/anoxic sediments, were more abundant under the carcass. Interestingly, all these species were absent or rare in samples near, but not under, the carcass, suggesting that they may take advantage of the reduced environment created by the carcass and the consequent lack of competition to prosper. As observed for other marine environments, carcasses in estuaries create a microhabitat that supports a characteristic meiobenthic fauna, distinct from those inhabiting the surrounding sediments, but similar to those of reduced habitats.

Meiobenthic communities of seagrass beds (Zostera capricorni) and unvegetated sediments along the coast of New South Wales, Australia

Seagrass beds have higher biomass, abundance, diversity and productivity of benthic organisms than unvegetated sediments. However, to date most studies have analysed only the macrofaunal component and ignored the abundant meiofauna present in seagrass meadows. This study was designed to test if meiobenthic communities, especially the free-living nematodes, differed between seagrass beds and unvegetated sediments. Sediment samples from beds of the eelgrass Zostera capricorni and nearby unvegetated sediments were collected in three estuaries along the coast of New South Wales, Australia. Results showed that sediments below the seagrass were finer, with a higher content of organic material and were less oxygenated than sediments without seagrass. Univariate measures of the fauna (i.e. abundance, diversity and taxa richness of total meiofauna and nematode assemblages) did not differ between vegetated and unvegetated sediments. However multivariate analysis of meiofaunal higher taxa showed significant differences between the two habitats, largely due to the presence and absence of certain taxa. Amphipods, tanaidacea, ostracods, hydrozoans and isopods occurred mainly in unvegetated sediments, while kinorhyncs, polychaetes, gastrotrichs and turbellarians were more abundant in vegetated sediments. Regarding the nematode assemblages, 32.4% of the species were restricted to Z. capricorni and 25% only occurred in unvegetated sediments, this suggests that each habitat is characterized by a particular suite of species. Epistrate feeding nematodes were more abundant in seagrass beds, and it is suggested that they graze on the microphytobenthos which accumulates underneath the seagrass. Most of the genera that characterized these estuarine unvegetated sediments are also commonly found on exposed sandy beaches. This may be explained by the fact that Australian estuaries have very little input of freshwater and experience marine conditions for most of the year. This study demonstrates that the seagrass and unvegetated sediments have discrete meiofaunal communities, with little overlap in species composition. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of drill cuttings discharge on meiofauna communities of a shelf break site in the southwest Atlantic

The present study investigates the effects of drill cutting discharges on the structure of meiofauna communities in an area of the shelf break at Campos Basin, Southeast Brazil. Drilling activities were operated, in a first phase, with water-based fluid and, in a second phase, with synthetic fluid paraffin-based (NAF-III). A total of 135 samples taken at a pre-drilling situation (MS1) and two post-drilling moments (MS2 and MS3-3 and 22 months post-drilling, respectively) were analyzed. Effects on meiofauna were dependent on two main factors: 1-the impact received during drilling operation, if water-based or synthetic/water-based drilling fluid and 2-the background state, if it already presented signs of previous drilling activities or not. Based on univariate and multivariate analysis, there were evidences that the most affected area after drilling was those under the influence of synthetic-based fluid and that already had signs of previous drillings activities. The region impacted only by water-based fluid was less affected and the only one that completely recovered after 22 months. Nematodes and copepods had different responses to the impact. While copepods flourish in the impacted area and recovered 22 months after drilling, nematodes were adversely affected shortly after drilling and the community structure only recovered where hydrocarbons had been depleted.

Anthelmintic effects of condensed tannins on Trichostrongylus colubriformis in experimentally infected sheep

Recent surveys have identified anthelmintic effects from many bioactive substances particularly from condensed tannin (CT) sources. The aims of the present study were to investigate the potential anthelmintic effects of condensed tannins (CT) on Trichostrongylus colubriformis in experimentally infected sheep and the nutritional consequences on animals. Twenty helminth-free lambs were divided into five groups of four animals. Groups I to IV were artificially infected with 6,000 third stage larvae (L3) of T. colubriformis. Group I was the infected control and group V was the uninfected control. Twenty-eight days post-infection (p.i.) lambs from GII were supplemented with tanniniferous sorghum (350 g/animal/day, during seven days); GIII were drenched with Acacia mearnsii extract (15% CT) for just one day and GIV during two days (1.6 g extract/kg BW). At day 36 p.i., animals from infected group (GI to GIV) were slaughtered. Faecal egg counts (FEC) values present a reduction on GII when compared with GI at day 29 p.i. (P < 0.05) and between GIII and GI at day 35 and 36 p.i. (P < 0.05). The values of egg hatchability and number of L3 recovered from the faeces were not statistical analyzed (there was no duplicate data), however there was a considerable reduction between the values from treated and control group. The use of CT on diet did not cause significant difference on blood parameters, body-weight and carcass-weight (P > 0.05). No difference was related on total worm burden between treatments; however, GIV presented lower number of females than GI (P < 0.05). The use of CT could be a promising alternative source to reduce the pasture contamination and to control T. colubriformis infection in sheep.

Predation potential and biology of Protogamasellopsis posnaniensis Wisniewski & Hirschmann (Acari: Rhodacaridae)

Rhodacaridae are cosmopolitan mites mentioned as predators, although nothing is known about their potential as biological control agents. One of the objectives of the work reported in this paper was to evaluate the potential of Protogamasellopsis posnaniensis (Acari: Rhodacaridae) as predator of representative species of insects of the families Sciaridae (Bradysia matogrossensis (Lane)) and Thripidae (Frankliniella occidentalis (Pergande)), of mites of the family Acaridae (Tyrophagus putrescentiae (Schrank) and Rhizoglyphus echinopus (Fumouze & Robin) and of nematodes of the family Rhabditidae (Protorhabditis sp.). Another objective was to determine the biological cycle of P. posnaniensis when fed the prey on which it performed best in the preceding predation test. The study was conducted in a laboratory where the experimental units were maintained at 25 +/- 1 degrees C, 97 +/- 3% RH and in the dark. Although the predator was able to kill all prey species considered in this study, the most favorable prey were T. putrescentiae, F. occidentalis and Protorhabditis sp. Survivorship of the predator in predation tests was always 98% or higher. Life table biological parameters when the predator was fed T. putrescentiae were: R(o) = 109.29; T = 19.06 days; lambda = 1.28 e r(m) = 0.32 female/female/day. Despite preying upon larvae of B. matogrossensis, eggs of the former can also be killed by the latter. The results indicated that A posnaniensis is a promising biological control agent, deserving additional studies on its possible use for the control of soil pests. (C) 2008 Elsevier Inc. All rights reserved.

Crop rotation in center-pivot for phytonematode control: density variation, pathogenicity and crop loss estimation

Crop rotation in center-pivot for phytonematode control: density variation, pathogenicity and crop loss estimation A field study conducted over three consecutive years, on a farm using crop rotation system under center-pivot and infested with the nematodes Pratylenchus brachyurus, P. zeae, Meloidogyne incognita, Paratrichodorus minor, Helicotylenchus dihystera, Mesocriconema ornata and M. onoense, demonstrated that intensive crop systems provide conditions for the maintenance of high densities of polyphagous phytonematodes. Of the crops established on the farm (cotton, maize, soybean and cowpea), cotton and soybean suffered the most severe crop losses, caused respectively by M. incognita and P. brachyurus. Since maize is a good host for both nematodes, but tolerant of M. incognita, its exclusion from cropping system would be favorable to the performance of cotton, soybean and cowpea. Results from experiments carried out in controlled conditions confirmed the pathogenicity of P. brachyurus on cotton. Additional management with genetic resistance was useful in fields infested with M. incognita, although the soybean performance was affected by low resistance of the cultivars used for P. brachyurus. In conclusion, crop rotation must be carefully planned in areas infested with polyphagous nematodes, specifically in the case of occurrence of two or more major pathogenic nematodes.