Sensitivity of bipartite network analyses to incomplete sampling and taxonomic uncertainty.

Bipartite network analysis is a powerful tool to study the processes structuring interactions in ecological communities. In applying the method, it is assumed that the sampled interactions provide an accurate representation of the actual community. However, acquiring a representative sample may be difficult as not all species are equally abundant or easily identifiable. Two potential sampling issues can compromise the conclusions of bipartite network analyses: failure to capture the full range of interactions (sampling completeness) and use of a taxonomic level higher than species to evaluate the network (taxonomic resolution). We asked how commonly used descriptors of bipartite antagonistic communities (modularity, nestedness, connectance, and specialization [H2 ']) are affected by reduced host sampling completeness, parasite taxonomic resolution, and their crossed effect, as they are likely to co-occur. We used a quantitative niche model to generate weighted bipartite networks that resembled natural host-parasite communities. The descriptors were more sensitive to uncertainty in parasite taxonomic resolution than to host sampling completeness. When only 10% of parasite taxonomic resolution was retained, modularity and specialization decreased by ~76% and ~12%, respectively, and nestedness and connectance increased by ~114% and ~345% respectively. The loss of taxonomic resolution led to a wide range of possible communities, which made it difficult to predict its effects on a given network. With regards to host sampling completeness, standardized nestedness, connectance, and specialization were robust, whereas modularity was sensitive (~30% decrease). The combination of both sampling issues had an additive effect on modularity. In communities with low effort for both sampling issues (50%-10% of sampling completeness and taxonomic resolution), estimators of modularity, and nestedness could not be distinguished from those of random assemblages. Thus, the categorical description of communities with low sampling effort (e.g., if a community is modular or not) should be done with caution. We recommend evaluating both sampling completeness and taxonomic certainty when conducting bipartite network analyses. Care should also be exercised when using nonrobust descriptors (the four descriptors for parasite taxonomic resolution; modularity for host sampling completeness) when sampling issues are likely to affect a dataset.

Mining various genomic resources to resolve old alpha-taxonomy questions: A test of the species hypothesis of the Proteocephalus longicollis species complex (Cestoda: Platyhelminthes) from salmonid fishes.

High-throughput sequencing strategies became commonly employed to study non-model parasites, but the corresponding genomes and transcriptomes were seldom mined following the original publication. Similar to the data generated with genome skimming techniques based on shallow-depth shotgun genomes, various genomic and transcriptomic resources can be screened for useful molecular phylogenetic markers traditionally characterised with Sanger sequencing. Here, we provide an example of a strategy using reduced-representation genomic as well as transcriptomic data to obtain broad insights into the molecular diversity of the cestode Proteocephalus longicollis, a common parasite of salmonids distributed throughout the Holarctic region. We extract popular mitochondrial and nuclear ribosomal markers from various genomic resources for hundreds of parasite specimens from multiple European whitefish populations and compare those with Proteocephalus representatives from other species of salmonids and various geographical regions. In contrast with the previous morphology-based assessments, molecular phylogeny reveals a high degree of genetic divergence between Proteocephalus isolates from different salmonids, contrastingly low genetic differentiation within the parasite's populations hosted by the European whitefish (Coregonus lavaretus species complex), and a sister species relationship of Proteocephalus from European whitefish and Proteocephalus percae, a parasite of European perch (Perca fluviatilis). Proteocephalus spp. from North American lake whitefish, brown trout and Arctic charr each formed clearly distinct lineages. These results advance our understanding of the interrelationships of the Proteocephalus-aggregate, a well-recognized clade of Holarctic freshwater fish proteocephalids, and support resurrection of some of the nominal species of Proteocephalus, including Proteocephalus exiguus La Rue, 1911 from North American coregonids and Proteocephalus fallax La Rue, 1911 from European C. lavaretus, reserving Proteocephalus longicollis (Zeder, 1800) exclusively for parasites of Salmo trutta.

Temporal stability of polymorphic Arctic charr parasite communities reflects sustained divergent trophic niches.

Polymorphic Arctic charr Salvelinus alpinus populations frequently display distinct differences in habitat use, diet, and parasite communities. Changes to the relative species densities and composition of the wider fish community have the potential to alter the habitat niche of sympatric Arctic charr populations. This study evaluated the temporal stability of the parasite community, diet, and stable isotopes (δ13C, δ15N) of three sympatric Arctic charr morphs (piscivore, benthivore, and planktivore) from Loch Rannoch, Scotland, in relation to changes to the fish community. All Arctic charr morphs displayed distinct differences in parasite communities, diet, and stable isotope signatures over time, despite the establishment of four new trophically transmitted parasite taxa, and increased fish and zooplankton consumption by the piscivorous and planktivore morphs, respectively. Native parasite prevalence also increased in all Arctic charr morphs. Overall, Loch Rannoch polymorphic Arctic charr morph populations have maintained their distinct trophic niches and parasite communities through time despite changes in the fish community. This result indicates that re-stocking a native fish species has the potential to induce shifts in the parasite community and diet of Arctic charr morphs.

Phylogeography and cryptic species structure of a locally adapted parasite in New Zealand.

The phylogeographic patterns of many taxa on New Zealand's South Island are characterized by disjunct distributions that have been attributed to Pleistocene climatic cycles and the formation of the Southern Alps. Pleistocene glaciation has been implicated in shaping the contemporary genetic differentiation between populations of the aquatic snail Potamopyrgus antipodarum. We investigated whether similar phylogeographic patterns exist for the snail's locally adapted trematode parasite, Atriophallophorus winterbourni. We found evidence for a barrier to gene-flow in sympatry between cryptic, but ecologically divergent species. When focusing on the most common of these species, disjunct geographic distributions are found for mitochondrial lineages that diverged during the Pleistocene. The boundary between these distributions is found in the central part of the South Island and is reinforced by low cross-alpine migration. Further support for a vicariant origin of the phylogeographic pattern was found when assessing nuclear multilocus SNP data. Nuclear and mitochondrial population differentiation was concordant in pattern, except for populations in a potential secondary contact zone. Additionally, we found larger than expected differentiation between nuclear- and mitochondrial-based empirical Bayes FST estimates (global FST : 0.02 vs. 0.39 for nuclear and mitochondrial data, respectively). Population subdivision is theoretically expected to be stronger for mitochondrial genomes due to a smaller effective population size, but the strong difference here, together with mitonuclear discordance in a putative contact zone, is potentially indicative of divergent gene flow of nuclear and mitochondrial genomes.

Next-generation cophylogeny: unravelling eco-evolutionary processes.

A fundamental question in evolutionary biology is how microevolutionary processes translate into species diversification. Cophylogeny provides an appropriate framework to address this for symbiotic associations, but historically has been primarily limited to unveiling patterns. We argue that it is essential to integrate advances from ecology and evolutionary biology into cophylogeny, to gain greater mechanistic insights and transform cophylogeny into a platform to advance understanding of interspecific interactions and diversification more widely. We discuss key directions, such as incorporating trait reconstruction and considering multiple scales of network organization, and highlight recent developments for implementation. A new quantitative framework is proposed to allow integration of relevant information, such as quantitative traits and assessment of the contribution of individual mechanisms to cophylogenetic patterns.

Conspecific migration and environmental setting determine parasite infracommunities of non-migratory individual fish.

Parasite infracommunities tend to be stochastic in nature, although environmental characteristics such as the type of water source in streams and host traits can have an effect on the biotic assemblages and by extension the parasite fauna. We examined the effect of water source and the rate of adult fish migration on the metazoan parasite infracommunities of conspecific juvenile brown trout, Salmo trutta L. among streams flowing into Lake Lucerne (Switzerland). Juvenile (1 to 2-year old) fish harboured higher parasite species richness in groundwater-fed than in surface water-fed streams, whereas the rate of fish migration did not affect infracommunity richness. Heteroxenous species were more common in groundwater-fed streams with high and medium rates of trout migration, whereas infracommunities in surface water-fed streams and streams with low rates of fish migration were dominated by one monoxenous parasite or lacked infections. Similarity in the parasite infracommunity composition of juvenile trout across streams was explained by the interaction between type of water source and adult migration rates. Our conclusions support that similarity in the parasite composition of resident freshwater conspecifics can be predicted by the local environmental settings and host migratory behaviour, whereas parasite richness is mainly influenced by the environmental characteristics.

Assembly rules of helminth parasite communities in grey mullets: combining components of diversity.

Organisms aggregate in ecological communities. It has been widely debated whether these associations are explained by deterministic or, in contrast, random processes. The answer may vary, depending on the level of an organisational scale (α, ß and γ) and the facet of diversity considered: taxonomic, functional and phylogenetic. Diversity at the level of a sampling unit (i.e. host individual) is the α diversity; ß diversity represents the extent of dissimilarity in diversity among sampling units (within a level of an organisational scale, ß1; between levels of an organisational scale, ß2); and the total diversity of a system is γ diversity. Thus, the combination of facets and levels of a scale may be useful to disentangle the mechanisms driving the composition of a parasite community. Using helminth parasite taxonomic, functional, and a proxy for phylogenetic diversity of three species of grey mullets (Teleostei: Mugilidae) from the Mediterranean Sea, we show that random and deterministic processes of different nature explain the assemblage of parasite communities. The parasite community at a host individual (α) was invariably a random subset of the total diversity in the community for the three facets of diversity. At the ß1 level, taxonomic diversity was lower than expected by chance, whereas functional diversity and the proxy for phylogenetic diversity were random. At the ß2 level, diversity patterns suggested environmental filtering of the parasite assemblage: species, trait, and phylogenetic compositions of parasite communities seemed to depend primarily on the species of host, but also on the locality and season. Our study shows that parasite communities are not totally understood if any of the components (i.e. facets and levels) of diversity is neglected.

Random Tanglegram Partitions (Random TaPas): An Alexandrian Approach to the Cophylogenetic Gordian Knot.

Symbiosis is a key driver of evolutionary novelty and ecological diversity, but our understanding of how macroevolutionary processes originate extant symbiotic associations is still very incomplete. Cophylogenetic tools are used to assess the congruence between the phylogenies of two groups of organisms related by extant associations. If phylogenetic congruence is higher than expected by chance, we conclude that there is cophylogenetic signal in the system under study. However, how to quantify cophylogenetic signal is still an open issue. We present a novel approach, Random Tanglegram Partitions (Random TaPas) that applies a given global-fit method to random partial tanglegrams of a fixed size to identify the associations, terminals, and nodes that maximize phylogenetic congruence. By means of simulations, we show that the output value produced is inversely proportional to the number and proportion of cospeciation events employed to build simulated tanglegrams. In addition, with time-calibrated trees, Random TaPas can also distinguish cospeciation from pseudocospeciation. Random TaPas can handle large tanglegrams in affordable computational time and incorporates phylogenetic uncertainty in the analyses. We demonstrate its application with two real examples: passerine birds and their feather mites, and orchids and bee pollinators. In both systems, Random TaPas revealed low cophylogenetic signal, but mapping its variation onto the tanglegram pointed to two different coevolutionary processes. We suggest that the recursive partitioning of the tanglegram buffers the effect of phylogenetic nonindependence occurring in current global-fit methods and therefore Random TaPas is more reliable than regular global-fit methods to identify host-symbiont associations that contribute most to cophylogenetic signal. Random TaPas can be implemented in the public-domain statistical software R with scripts provided herein. A User's Guide is also available at GitHub.[Codiversification; coevolution; cophylogenetic signal; Symbiosis.].

Towards a Unified Functional Trait Framework for Parasites.

Trait-based research holds high potential to unveil ecological and evolutionary processes. Functional traits are fitness-related characteristics of individuals, which are measured at individual level and defined without using information external to the individual. Despite the usefulness of the functional approach to understand the performance of individuals in ecosystems, and parasitism being the most common life-history strategy on Earth, studies based on functional traits of parasites are still scarce. Since the choice of functional traits is a critical step for any study, we propose a core list of seven functional traits of metazoan parasites, related to three universal challenges faced by organisms (dispersal, establishment, and persistence), and give guidelines to define appropriate functional traits in future parasite community studies.

Morphological and molecular characterisation of a new species of Gyrodactylus von Nordmann, 1832 (Monogenoidea: Gyrodactylidae) of cichlid fishes (Perciformes) from Mexico.

Gyrodactylus mojarrae n. sp. is described from the gills of the Neotropical cichlids Thorichthys maculipinnis, Rocio octofasciata, Vieja zonata and V. fenestrata from several localities across southern Mexico. The new species is erected on the basis of the morphology of the haptoral elements (anchors, bars and hooks), and its phylogenetic position within the Gyrodactylidae is evaluated based on the analysis of partial sequences of the ITS1, 5.8 rRNA gene and ITS2. Gyrodactylus mojarrae n. sp. differs from other congeneric species by having hooks with a straight shaft and recurved point, pointed toe and convex heel, presence of reduced superficial anchor roots, by the number and disposition of spinelets of the male copulatory organ, and the absence of shield and lateral processes of the superficial bar. Molecular circumscription of isolates of G. mojarrae n. sp. from different host and hydrological basins showed that these isolates are conspecific and represent a distinct lineage from other congeners, including newly sequenced isolates of Gyodactylus sp. A and Gyrodactylus sp. B from Astyanax mexicanus (Characidae) and Gobiomorus dormitor (Eleotridae), respectively. Genetic affinities of Gyodactylus sp. A and B indicate that they might represent undescribed species infecting freshwater fish from the Americas.

Neither Diplectanum nor specific: a dramatic twist to the taxonomic framework of Diplectanum (Monogenea: Diplectanidae).

The taxonomy of the genus Diplectanum has been exclusively based on morphological features, with 28 nominal species parasitic on perciform fishes recognised. We used molecular data, to our knowledge for the first time, to evaluate the taxonomic framework of Diplectanum, infer the relationships amongst species attributed to this genus, re-assess the degree of host specificity and explore the population genetic structure of Diplectanum spp. parasitising Mediterranean sciaenids, which are potential target fish species for aquaculture diversification in the region. A minimum of 10 specimens of Diplectanum spp. were sequenced per host species (Argyrosomus regius, Sciaena umbra, Umbrina canariensis and Umbrina cirrosa) and locality (Burriana, Sant Carles de la Ràpita and Santa Pola (Spain)) together with five individuals of the type species Diplectanum aequans. Sequences of partial 28S rDNA and internal transcribed spacer region of Diplectanum spp. were analysed together with those from other Diplectanidae spp. in GenBank using Bayesian inference and maximum likelihood phylogenetic methods. Population genetic analyses were performed using cytochrome c oxidase subunit I gene sequences for a diplectanid species with a wide distribution across host species and localities. Results showed that Diplectanum was not monophyletic, nor were the specimens infecting sciaenids. Instead they formed two separate clades, 26.1-28.6% divergent for the internal transcribed spacer and 13.2-16.9% for the 28S region from D. aequans. Altogether, our results suggest that these specimens represent two distinct new genera from Diplectanum and five putative species with low host specificity. It is likely that morphological variability has led to the description of more species than were detected by molecular methods. In contrast to other monogeneans, Diplectaninae gen. spp. are chiefly generalists. Nonetheless, intraspecific genetic divergence in the internal transcribed spacer region of Diplectaninae gen. spp., and population genetic analyses of one presumed generalist species, Diplectaninae gen. sp. 1.2, showed significant variation between subpopulations living on different hosts. The intraspecific genetic structure by host also suggests different cross-infection potential amongst sciaenid species.

Author Correction: Evaluation of three methods for biomass estimation in small invertebrates, using three large disparate parasite species as model organisms.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Evaluation of three methods for biomass estimation in small invertebrates, using three large disparate parasite species as model organisms.

Invertebrate biomass is considered one of the main factors driving processes in ecosystems. It can be measured directly, primarily by weighing individuals, but more often indirect estimators are used. We developed two indirect and non-destructive approaches to estimate biomass of small invertebrates in a simple manner. The first one was based on clay modelling and the second one was based on image analysis implemented with open-source software. Furthermore, we tested the accuracy of the widely used geometric approximation method (third method). We applied these three different methods to three morphologically disparate model species, an acanthocephalan worm, a crustacean and a flatworm. To validate our indirect estimations and to test their accuracy, we weighed specimens of the three species and calculated their tissue densities. Additionally, we propose an uncomplicated technique to estimate thickness of individuals under a microscope, a required measurement for two of the three indirect methods tested. The indirect methods proposed in this paper provided the best approximation to direct measurements. Despite its wide use, the geometric approximation method showed the lowest accuracy. The approaches developed herein are timely because the recently increasing number of studies requiring reliable biomass estimates for small invertebrates to explain crucial processes in ecosystems.

Microphallus ochotensis sp. nov. (Digenea, Microphallidae) and relative merits of two-host microphallid life cycles.

A new digenean species, Microphallus ochotensis sp. nov., was described from the intestine of Pacific eiders (Somateria mollissima v-nigrum) from the north of the Sea of Okhotsk. It differs from other microphallids in the structure of the metraterm, which consists of two distinct parts: a sac with spicule-like structures and a short muscular duct opening into the genital atrium. Mi. ochotensis forms a monophyletic clade together with other congeneric species in phylograms derived from the 28S and ITS2 rRNA gene. Its dixenous life cycle was elucidated with the use of the same molecular markers. Encysted metacercariae infective for birds develop inside sporocysts in the first intermediate host, an intertidal mollusc Falsicingula kurilensis. The morphology of metacercariae and adults was described with an emphasis on the structure of terminal genitalia. Considering that Falsicingula occurs at the Pacific coast of North America and that the Pacific eider is capable of trans-continental flights, the distribution of Mi. ochotensis might span the Pacific coast of Alaska and Canada. The range of its final hosts may presumably include other benthos-feeding marine ducks as well as shorebirds. We suggest that a broad occurrence of two-host life cycles in microphallids is associated with parasitism in birds migrating along sea coasts. The chances that migrating birds would stop at a site where both first and second intermediate hosts occur are relatively low. The presence of a single molluscan host in the life cycle increases the probability of transmission.

A new genus and two new species of dactylogyrid monogeneans from gills of Neotropical catfishes (Siluriformes: Doradidae and Loricariidae).

A new genus of dactylogyrid monogeneans (Ancyrocephalinae), Paracosmetocleithrum n. gen., is erected to accommodate P. trachydorasi n. sp. from Trachydoras paraguayensis (Siluriformes: Doradidae) in the Upper Paraná River basin, Brazil. The new genus differs from Neotropical dactylogyrids in the presence of a well-developed ornamentation in the middle portion of the ventral bar, and a sclerotized patch on the surface of the dorsal bar with an inconspicuous medial process that possesses two submedial projections arising from the tapered ends of this patch. In addition, Demidospermus rhinelepisi n. sp. is described from Rhinelepis aspera (Siluriformes: Loricariidae). The new species, which is the fifth species of the genus described from loricariids, can be differentiated from congeners by the possession of a sclerotized patch attached to the middle portion of the ventral bar, and by morphology of the accessory piece, which presents broad ends, tapering in the centre, rounded proximal end, distal end folding on both sides with folds extending to approximately ¾ of the accessory piece length. Molecular data on both new species are also provided and species composition of Demidospermus, recently revealed as polyphyletic by molecular studies including the present one, is discussed.

Life History, Systematics and Evolution of the Diplostomoidea Poirier, 1886: Progress, Promises and Challenges Emerging From Molecular Studies.

Members of the Diplostomoidea mature in amniotes and employ vertebrates, annelids and molluscs as second intermediate hosts. Diplostomoid life cycles generally follow a three-host pattern typical of digeneans, but novelties have arisen in some species, including obligate four-host life cycles, vertical transmission, and intracellular parasitism. In this review, we summarize the basic biology of diplostomoids with reference to molecular studies, and present challenges, gaps and areas where molecular data could address long-standing questions. Our analysis of published studies revealed that most molecular surveys find more diplostomoid species than expected, but this tendency is influenced by how much effort goes into examining specimens morphologically and the number of sequenced worms. To date, molecular work has concentrated disproportionately on intraspecific or species-level diversity of larval stages in the Diplostomidae in temperate northern regions. Although the higher taxonomy of the superfamily is recognized to be in need of revision, little molecular work has been conducted at this level. Our phylogenetic analysis indicates several families and subfamilies require reconsideration, and that larval morphotypes are more reflective of evolutionary relationships than definitive hosts. The host associations of adult diplostomoids result from host-switching processes, whereas molecular surveys indicate that larval diplostomoid metacercariae have narrow ranges of second intermediate hosts, consistent with coevolution. Molecular data are often used to link diplostomoid developmental stages, and we provide data from adult Neodiplostomum and Mesoophorodiplostomum that correct earlier misidentifications of their larval stages and propose alternatives to collecting definitive hosts.

Molecular characterisation of four echinostomes (Digenea: Echinostomatidae) from birds in New Zealand, with descriptions of Echinostoma novaezealandense n. sp. and Echinoparyphium poulini n. sp.

Morphological and molecular characterisation of echinostome specimens (Digenea: Echinostomatidae) recovered in one Anas platyrhynchos L. and one Cygnus atratus (Latham) (Anseriformes: Anatidae) from New Zealand revealed the presence of two known species, Echinostoma miyagawai Ishii, 1932 and Echinoparyphium ellisi (Johnston & Simpson, 1944) and two species new to science. Comparative morphological and phylogenetic analyses supported the distinct species status of Echinostoma novaezealandense n. sp. ex Branta canadensis (L.), A. platyrhynchos and C. atratus, and Echinoparyphium poulini n. sp. ex C. atratus. Echinostoma novaezealandense n. sp., a species of the "revolutum" species complex characterised by the possession of a head collar armed with 37 spines, keyed down to E. revolutum but was distinguished from the latter in having a much narrower body with almost parallel margins, longer oesophagus, wider cirrus-sac, larger seminal vesicle, much smaller ventral sucker, ovary, Mehlis' gland and testes, more anteriorly located ovary and testes, and distinctly smaller eggs (81-87 × 42-53 vs 106-136 × 55-70 µm). This new species appears similar to Echinostoma acuticauda Nicoll, 1914 described in Australia but differs in having a longer forebody, more posteriorly located ovary and testes, and much smaller eggs (81-87 × 42-53 vs 112-126 × 63-75 µm). Echinoparyphium poulini n. sp. is differentiated from the four species of Echinoparyphium possessing 37 collar spines considered valid as follows: from E. chinensis Ku, Li & Chu, 1964 in having a much smaller body, four (vs five) angle spines and simple seminal vesicle (vs bipartite); from E. schulzi Matevosyan, 1951 in having a less robust body at a comparable body length, much smaller ventral sucker, ovary and testes, and longer but narrower eggs (87-109 × 50-59 vs 70-85 × 60-84 µm); and from the two smaller forms, E. serratum Howell, 1968 and E. aconiatum Dietz, 1909, in a number of additional metrical features correlated with body size and especially in the possession of much larger collar spines. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for representative isolates of the four species and analysed together with sequences for Echinostoma spp. and Echinoparyphium spp. available on GenBank. Phylogenetic analyses based on the mitochondrial nad1 gene revealed congruence between the molecular data and species identification/delineation based on morphology; this was corroborated by the 28S rDNA sequence data.

Parasciadicleithrum octofasciatum n. gen., n. sp. (Monogenoidea: Dactylogyridae), parasite of Rocio octofasciata (Regan) (Cichlidae: Perciformes) from Mexico characterised by morphological and molecular evidence.

Based on an integrative taxonomic approach, combining morphological characters and partial sequences of the 28S rRNA gene, a new genus and species, Parasciadicleithrum octofasciatum, is proposed to accommodate dactylogyrids infecting the gills of Rocio octofasciata (Cichlidae) from a tributary of the Lacantún River basin, Chiapas State, southern Mexico. Morphologically, the new genus resembles members of Sciadicleithrum (Dactylogyridae) in the presence of membranes on the anterior margins of both ventral and dorsal haptoral bars. However, Parasciadicleithrum octofasciatum n. sp. shows morphological differences with respect to a subset of species of Sciadicleithrum occurring in the same host group and geographic area, such as the body size and gonad dimensions (length and width of germarium and testis, respectively, smaller in the new genus). Phylogenetic analyses performed herein based on Maximum Likelihood and Bayesian Inference criteria, showed that Parasciadicleithrum octofasciatum n. sp. represents an undescribed taxon morphologically very similar to species of Sciadicleithrum from which it can be consistently distinguished on the basis of molecular data. P. octofasciatum n. sp. appeared genetically distant from Sciadicleithrum spp. and nested with dactylogyrid parasites of African cichlids. Parasciadicleithrum n. gen. showed divergence levels in the 28S rDNA sequences of 25-26% with respect to Sciadicleithrum spp. Therefore, on the basis of molecular evidence mainly, and morphological differences highlighted above, the erection of the new genus is proposed. The evolutionary and ecological factors that may have influenced the associations between Neotropical cichlids and their dactylogyrid parasites are briefly discussed.

Improving detection of avian malaria from host blood: a step towards a standardised protocol for diagnostics.

Avian malaria, caused by Plasmodium spp., has been linked to the mortality and population-level declines in native birds in some regions. While molecular diagnostic methods have greatly improved our ability to detect infections of both human and bird malaria, failing to identify false negatives remains an important handicap, particularly for avian malaria due to host DNA presence in the bird blood cells. In an attempt to improve the accuracy of diagnostics by PCR, we evaluated the performance of a commercial silica-membrane-based DNA extraction kit by modifying the protocol with four unpooled elution volume alternatives. Our results suggest that the best template is the DNA extract obtained from the second eluate of a first 50 µL elution step. In one case, the only band visible was from this second eluate and, thus, may not have been identified as positive for Plasmodium spp. if a different elution protocol had been followed. Our results are likely explained by the concept of size exclusion chromatography by which particles of different sizes will elute at different rates. Overall, first elution templates may consist of a lower ratio of parasite to host DNA, while second eluates may contain a higher parasite to host DNA ratio. A low ratio of parasite to host DNA is a concern in detecting chronic infections, in which birds typically carry low levels of parasitemia, making accurate diagnostics imperative when identifying reservoirs of disease that could lead to spillback events.

Molecular approaches to trematode systematics: 'best practice' and implications for future study.

To date, morphological analysis has been the cornerstone to trematode systematics. However, since the late-1980s we have seen an increased integration of genetic data to overcome problems encountered when morphological data are considered in isolation. Here, we provide advice regarding the 'best molecular practice' for trematode taxonomy and systematic studies, in an attempt to help unify the field and provide a solid foundation to underpin future work. Emphasis is placed on defining the study goals and recommendations are made regarding sample preservation, extraction methods, and the submission of molecular vouchers. We advocate generating sequence data from all parasite species/host species/geographic location combinations and stress the importance of selecting two independently evolving loci (one ribosomal and one mitochondrial marker). We recommend that loci should be chosen to provide genetic variation suitable to address the question at hand and for which sufficient 'useful' comparative sequence data already exist. Quality control of the molecular data via using proof-reading Taq polymerase, sequencing PCR amplicons using both forward and reverse primers, ensuring that a minimum of 85% overlap exists when constructing consensus sequences, and checking electropherograms by eye is stressed. We advise that all genetic results are best interpreted using a holistic biological approach, which considers morphology, host identity, collection locality, and ecology. Finally, we consider what advances next-generation sequencing holds for trematode taxonomy and systematics.