Identification of larval fish in mangrove areas of Peninsular Malaysia using morphology and DNA barcoding methods

The identification of larval fish has been an important morphological issue in marine biology due to the dramatic transformations that most species undergo from early larval stages to adulthood. Insufficient morphological diagnostic characters in larval fishes made it easy to misidentify them and a difficult process to key to genus and species level. The experiment aims to find out, by applying DNA barcoding, how consistent the morphological identifications can be among larval fish. Larval fish were mainly collected using plankton nets around mangrove areas in Pendas (Johor), Setiu (Terengganu), Pekan (Pahang) and Matang (Perak) Malaysia between April 2015 and October 2015. A total of 354 samples were morphologically identified, mostly to the family level and a few to the genus level. Larval fish ranged from 1.5 mm to 31 mm of total length, with the most abundant individuals being <3 mm. Among them, a total of 177 individuals were selected for DNA barcoding analyses. Molecular works involved polymerase chain reaction (PCR) and sequencing of mitochondrial Cytochrome c Oxidase I (COI) gene fragment (655 base pairs) methods. DNA barcoding enabled all samples to be identified down to species level. The overall genetic identities ranged from 91% to 100%. Morphological identification classified the specimens into 19 families and 11 genera while DNA barcoding identified them into 19 families 33 genera and 40 species. A comparison between the two methods showed a mismatched identification of 42.6% where the accuracy percentage for morphological identification was moderate for the family level (67.8%) but was low for genus level identification (30%). The DNA barcoding method also managed to successfully identify 86.4% of the samples up to their species level where morphological method has failed to do so. The most misidentified families in the study were Blenniidae, Sparidae, Apogonidae Ambassidae and Monachantidae while almost all samples from the family Gobiidae and Engraulidae were correctly identified to family level because of their distinct morphology. In conclusion, taxonomic studies of larval fish should continue using combination of both morphology and DNA barcoding methods. Morphological identification should be more conservative i.e., when in doubt, it is better to key only to family and not to the genus and species level. DNA barcoding is a better method for deeper taxonomic levels identification with the existence of robust sequence reference libraries and should be able to validate the accuracy of traditional larval fish identification.     

Inferring larval taxonomy and morphology in Maladera species (Coleoptera: Scarabaeidae: Sericini) using DNA taxonomy tools

Based on a comparative molecular study of scarab chafers we matched adult and larval instars to identify and describe unknown larvae of Sericini. Here, we use for the first time a two‐fold DNA taxonomy approach based on: (i) mitochondrial and nuclear DNA markers of a local sample (from Nepal) of adults and larvae, in combination with character and tree‐based species delimitation methods; and (ii) a global search of cytochrome c oxidase subunit I (cox1) sequences with GenBank data. In the latter analysis we used a sequence of a specimen that resulted in the first analysis conspecific with the larvae of Maladera affinis (Blanchard) as the query sequence in GenBank, and checked in a minimum evolution tree whether larva–adult matches from the local approach were altered through interference with other taxa of the worldwide database. Both approaches unambiguously identified the unknown larvae as belonging to M. affinis and Maladera cardoni (Brenske). Based on this robust framework of taxonomic identification we could associate names to the larval morphology of the third larval instar of these two Nepalese Maladera species, which are both known for their economical importance in agriculture. They are described here in detail and are compared with known related taxa, especially with Maladera castanea (Arrow).     

A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae)

Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the development and application of a single-step multiplex PCR for the identification of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are pests on many commercial crops, including grapevine, in which they transmit viral diseases. Morphological identification of mealybug species is usually time-consuming, requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized field-collected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.     

Larval diet influence on oviposition behaviour inSpodoptera littoralis

Females ofSpodoptera littoralis Boisd. (Lepidoptera: Noctuidae) with different feeding experiences during their larval development were tested for their ovipositional response to methanol extracts of larval frass and semisynthetic diets. The effect of the following frass, diet and diet component extracts was tested: (a) frass fromS. littoralis orAgrotis segetum larvae fed on a potato-based diet; (b) frass fromS. littoralis larvae fed on a wheat germ-based diet; (c) potato and wheat germ-based diets; and (d) potatoes and wheat germ. Ovipositing females without prior experience of the potato diet were deterred by extracts of: (1) larval frass from either species fed on potato diet; (2) the potato-based diet; (3) potato. Also females with experience of the potato diet during only a part of their larval development were deterred from oviposition by frass of larvae reared on the potato diet and by the diet itself. However, for females reared on the potato diet for their entire larval development, oviposition was no longer deterred by either of the three extracts listed above. Extracts of: (1) frass from larvae of either species reared on wheat germ diet: (2) the wheat germ diet; or (3) wheat germ did not significantly affect oviposition. Females with ablated antennae were still deterred by frass extracts from larvae fed on potato diet, when they had been reared on the wheat germ diet. In feeding experiments, larvae of larval stage one and of larval stage three-four reared on either of the two diets preferred to feed on the wheat germ diet. However, the preference was significantly stronger for larvae with no prior contact with the potato diet. The effect of larval experience on the loss of oviposition-deterring activity by extracts of larval frass, diets and diet components is discussed in view of induction and selection.     

Using DNA barcoding and phylogenetics to identify Antarctic invertebrate larvae: Lessons from a large scale study

Ecological studies of the diversity and distribution of marine planktonic larvae are increasingly depending on molecular methods for accurate taxonomic identification. The greater coverage of reference marine species on genetic databases such as GenBank and BoLD (Barcoding of Life Data Systems; www.boldystems.org); together with the decreasing costs for DNA sequencing have made large scale larval identification studies using molecular methods more feasible. Here, we present the development and implementation of a practical molecular approach to identify over 2000 individual marine invertebrate larvae that were collected in the Ross Sea, Antarctica, during the austral summer over five years (2002-2007) as part of the LGP (Latitudinal Gradient Project). Larvae for molecular ID were morphologically identified to belong to the Phyla Mollusca, Echinodermata, Nemertea and Annelida (Class Polychaeta), but also included unidentified early developmental stages which could not be assigned a specific taxon (e.g., eggs, blastulae). The use of a 100μm mesh plankton net makes this one of the first larval identification studies to simultaneously consider both embryos and larvae. Molecular identification methods included amplification of up to three molecular loci for each specimen, a pre-identification step using BLAST with GenBank, phylogenetic reconstructions and cross-validation of assigned Molecular Operational Taxonomic Units (MOTUs). This combined approach of morphological and molecular methods assigned about 700 individuals to 53 MOTUs, which were identified to the lowest possible taxonomic level. During the course of this long-term study we identified several procedural difficulties, including issues with the collection of larvae, locus amplification, contamination, assignment and validation of MOTUs. The practical guidelines that we describe here should greatly assist other researchers to conduct reliable molecular identification studies of larvae in the future.     

‘Direct PCR’ optimization yields a rapid,cost‐effective,nondestructive and efficient method for obtaining DNA barcodes without DNA extraction

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction (‘direct PCR’) and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.     

Investigation on Natural Diets of Larval Marine Animals Using Peptide Nucleic Acid-Directed Polymerase Chain Reaction Clamping

The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected.     

Application of real-time PCR for simultaneous identification and quantification of larval abalone

A paucity of direct studies of marine invertebrate larval dispersal motivated the development of a high-throughput method for identification and quantification of pinto abalone (Haliotis kamtschatkana) larvae in seawater. DNA extracted from sample retentate provided template to screen for species-specific cytochrome oxidase I (COI) mitochondrial DNA sequence via quantitative PCR (QPCR) technology. Primers and a dual-labeled probe were designed and used to identify and quantify DNA from the target species in blind tests of unknown samples alongside a standard template quantity series. Quantity estimates derived from QPCR standard curves were verified via direct enumeration of larvae using light microscopy. Multiplex reactions containing an internal positive control minimized underestimation of quantity and false negatives via partial or full PCR inhibition, respectively. Planned controlled field release and collection experiments to examine larval dispersion patterns via sampling over short and long postrelease times anticipate similar QPCR assays for other marine invertebrate species to aid investigations of larval dispersal in the marine environment.     

Fecal DNA analysis for identifying species and sex of sympatric carnivores: a noninvasive method for conservation on the Tsushima Islands, Japan

Fecal analysis is a useful tool for the investigation of food habits and species identity in mammals. However, it is generally difficult to identify the species based on the morphological features and contents of feces deposited by mammals of similar body size. Therefore we developed noninvasive DNA analysis methods using fecal samples for identification of the species and sex of four small sympatric carnivores living on the Tsushima Islands of Japan: the leopard cat (Felis bengalensis), Japanese marten (Martes melampus), Siberian weasel (Mustela sibirica), and feral cat (Felis catus). Based on DNA sequence data from previous phylogenetic studies, we designed species-specific primers for polymerase chain reaction (PCR) amplification of the partial mitochondrial cytochrome b gene (112-347 bp) to identify the species and primers for the partial SRY gene (135 bp) to determine the sex. Due to the adjustment of PCR conditions, those specific DNA fragments were successfully amplified and then applied for species and sex identification. Nucleotide sequences obtained from the PCR products corresponded with cytochrome b sequences of the carnivore species expected. The protocol developed could be a valuable tool in the management and conservation of the four carnivore species occurring on the Tsushima Islands.     

High-throughput molecular identification of fish eggs using multiplex suspension bead arrays

The location and abundance of fish eggs provide information concerning the timing and location of spawning activities and can provide fishery-independent estimates of spawning biomass. However, the full value of egg and larval surveys is severely restricted because many species' eggs and larvae are morphologically similar, making species-level identification difficult. Recent efforts have shown that nearly all species of fish may be identified by mitochondrial DNA (mtDNA) sequences (e.g. via 'DNA barcoding'). By taking advantage of a DNA barcode database, we have developed oligonucleotide probes for 23 marine fish species that produce pelagic eggs commonly found in California waters. Probes were coupled to fluorescent microspheres to create a suspension bead array. Biotin-labelled primers were used to amplify the mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal rRNA genes from individual fish eggs. The amplicons were then hybridized to the bead array, and after the addition of a reporter fluorophore, samples were analysed by flow cytometry with Luminex 100 instrumentation. Probes specifically targeted eggs that are abundant and/or from morphologically indistinguishable species pairs. Results showed that the 33 different probes designed for this study accurately identified all samples when PCR was successful. Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput and lower cost than DNA sequencing. The increasing availability of DNA barcode data for numerous fish faunas worldwide suggests that bead arrays could be developed and widely used for fish egg, larval and tissue identifications.     

Genetic identification of asteroid larvae from Tasmania, Australia, by PCR–RFLP

Studies of seastar larvae in the Derwent River (Tasmania) were hampered by identification uncertainties. A genetic test was developed, based on PCR amplification of a 1300 bp mitochondrial DNA region followed by digestion with three restriction enzymes. The restriction profiles of adults of 14 seastar species were determined. The test was validated for larvae as laboratory-raised larvae of two species had the appropriate composite haplotypes. Approximately 80% of planktotrophic seastar larvae from Derwent River plankton samples were identified as the recently introduced northern Pacific seastar, Asterias amurensis . The two other larval seastars identified were Coscinasterias muricata and Patiriella regularis .     

PCR-based species identification of Agriotes larvae

Click beetle larvae within the genus Agriotes (Coleoptera: Elateridae), commonly known as wireworms, are abundant ground-dwelling herbivores which can inflict considerable damage to field crops. In Central Europe up to 20 species, which differ in their distribution, ecology and pest status, occur in arable land. However, the identification of these larvae based on morphological characters is difficult or impossible. This hampers progress towards controlling these pests. Here, we present a polymerase chain reaction (PCR)-based approach to identify, for the first time, 17 Agriotes species typically found in Central Europe. Diagnostic sequence information was generated and submitted to GenBank, allowing the identification of these species via DNA barcoding. Moreover, multiplex PCR assays were developed to identify the nine most abundant species rapidly within a single-step reaction: Agriotes brevis, A. litigiosus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus, A. lineatus and A. proximus. The latter two species remain molecularly indistinguishable, questioning their species status. The multiplex PCR assays proved to be highly specific against non-agrioted elaterid beetles and other non-target soil invertebrates. By testing the molecular identification system with over 900 field-collected larvae, our protocol proved to be a reliable, cheap and quick method to routinely identify Central European Agriotes species.     

Direct PCR of indigenous and invasive mosquito species: a time‐ and cost‐effective technique of mosquito barcoding

Millions of people die each year as a result of pathogens transmitted by mosquitoes. However, the morphological identification of mosquito species can be difficult even for experts. The identification of morphologically indistinguishable species, such as members of the Anopheles maculipennis complex (Diptera: Culicidae), and possible hybrids, such as Culex pipiens pipiens/Culex pipiens molestus (Diptera: Culicidae), presents a major problem. In addition, the detection and discrimination of newly introduced species can be challenging, particularly to researchers without previous experience. Because of their medical importance, the clear identification of all relevant mosquito species is essential. Using the direct polymerase chain reaction (PCR) method described here, DNA amplification without prior DNA extraction is possible and thus species identification after sequencing can be achieved. Different amounts of tissue (leg, head; larvae or adult) as well as different storage conditions (dry, ethanol, ?20 and ?80 °C) and storage times were successfully applied and showed positive results after amplification and gel electrophoresis. Overall, 28 different indigenous and non‐indigenous mosquito species were analysed using a gene fragment of the COX1 gene for species differentiation and identification by sequencing this 658‐bp fragment. Compared with standard PCR, this method is time‐ and cost‐effective and could thus improve existing surveillance and control programmes.     

From feces to data: A metabarcoding method for analyzing consumed and available prey in a bird‐insect food web

Diets play a key role in understanding trophic interactions. Knowing the actual structure of food webs contributes greatly to our understanding of biodiversity and ecosystem functioning. The research of prey preferences of different predators requires knowledge not only of the prey consumed, but also of what is available. In this study, we applied DNA metabarcoding to analyze the diet of 4 bird species (willow tits Poecile montanus, Siberian tits Poecile cinctus, great tits Parus major and blue tits Cyanistes caeruleus) by using the feces of nestlings. The availability of their assumed prey (Lepidoptera) was determined from feces of larvae (frass) collected from the main foraging habitat, birch (Betula spp.) canopy. We identified 53 prey species from the nestling feces, of which 11 (21%) were also detected from the frass samples (eight lepidopterans). Approximately 80% of identified prey species in the nestling feces represented lepidopterans, which is in line with the earlier studies on the parids' diet. A subsequent laboratory experiment showed a threshold for fecal sample size and the barcoding success, suggesting that the smallest frass samples do not contain enough larval DNA to be detected by high‐throughput sequencing. To summarize, we apply metabarcoding for the first time in a combined approach to identify available prey (through frass) and consumed prey (via nestling feces), expanding the scope and precision for future dietary studies on insectivorous birds.     

DNA barcoding and morphological identification of neotropical ichthyoplankton from the Upper Paraná and São Francisco

The identification of fish larvae from two neotropical hydrographic basins using traditional morphological taxonomy and DNA barcoding revealed no conflicting results between the morphological and barcode identification of larvae. A lower rate (25%) of correct morphological identification of eggs as belonging to migratory or non‐migratory species was achieved. Accurate identification of ichthyoplankton by DNA barcoding is an important tool for fish reproductive behaviour studies, correct estimation of biodiversity by detecting eggs from rare species, as well as defining environmental and management strategies for fish conservation in the neotropics.     

A new strategy for species identification of planktonic larvae: PCR-RFLP analysis of the internal transcribed spacer region of ribosomal DNA detected by agarose gel electrophoresis or DHPLC

Planktonic survey is important for understanding the dynamicsand structure of populations or communities. However, the smallsize of early planktonic larvae, usually <500 µm, makesit difficult or impossible to discriminate closely related taxabased on morphological characters. We developed a new strategyfor species identification of planktonic larvae, namely, polymerasechain reaction–restriction fragment length polymorphism(PCR–RFLP) analysis of the internal transcribed spacer(ITS) region detected by agarose gel electrophoresis or denaturinghigh-performance liquid chromatography (DHPLC). A total of fourrestriction enzymes were selected for PCR–RFLP analysis.Based on any one of four restriction maps, 12 commercial shellfishspecies could be differentiated from each other in agarose gelanalysis. But more accurate results were obtained in DHPLC analysis.As an example of larval identification, hybrid larvae were successfullyidentified by their showing diagnostic peaks of both parentsin DHPLC analysis. These results suggest that this strategycan meet the demands for plankton survey and studies of hybridization.     

二化螟绒茧蜂对二化螟及其寄主植物挥发物的趋性反应

利用Y-型嗅觉仪研究了二化螟绒茧蜂Cotesia chilonis对寄主植物（水稻或茭白）、二化螟Chilo suppressalis幼虫、虫粪及虫害苗挥发物的行为反应。健康植株、二化螟幼虫和虫粪的挥发物对二化螟绒茧蜂具有显著引诱作用。在虫害苗与健康苗挥发物之间,二化螟绒茧蜂显著地偏好虫害苗,但当去除虫害苗中的幼虫和虫粪后,寄生蜂对去虫苗与机械损伤苗的选择无显著差异;在虫害苗与有虫健康苗之间,寄生蜂显著趋向虫害苗,表明虫害苗本身释放的挥发物对二化螟绒茧蜂引诱作用与机械损伤苗无显著差异,但与二化螟幼虫或虫粪挥发物之间可能具有协同增效作用。水稻苗经机械损伤或损伤后以二化螟幼虫唾液处理,其挥发物对二化螟绒茧蜂的引诱作用无显著改变。二化螟绒茧蜂对不同为害程度水稻挥发物的选择无显著差异。二化螟绒茧蜂对两种寄主植物的健康苗、虫害苗、取食两种植物的幼虫及虫粪的挥发物的选择无显著差异。结果表明,二化螟绒茧蜂栖境定位和寄主选择过程中所利用的挥发物主要来自寄主植物、二化螟幼虫和虫粪以及虫害苗与幼虫和虫粪的协同作用。     

Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocols

Worldwide, American foulbrood (AFB) is the most devastating bacterial disease of the honey bee (Apis mellifera). Because the distinction between AFB and powdery scale disease is no longer considered valid, the pathogenic agent has recently been reclassified as one species Paenibacillus larvae, eliminating the subspecies designations Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens. The creamy or dark brown, glue-like larval remains of infected larvae continue to provide the most obvious clinical symptom of AFB, although it is not conclusive. Several sensitive and selective culture media are available for isolation of this spore-forming bacterium, with the type of samples that may be utilized for detection of the organism being further expanded. PCR methods for identification and genotyping of the pathogen have now been extensively developed. Nevertheless, biochemical profiling, bacteriophage sensitivity, immunotechniques and microscopy of suspect bacterial strains are entirely adequate for routine identification purposes.     

Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species

Biodiversity studies require species level analyses for the accurate assessment of community structures. However, while specialized taxonomic knowledge is only rarely available for routine identifications, DNA taxonomy and DNA barcoding could provide the taxonomic basis for ecological inferences. In this study, we assessed the community structure of sediment dwelling, morphologically cryptic Chironomus larvae in the Rhine-valley plain/Germany, comparing larval type classification, cytotaxonomy, DNA taxonomy and barcoding. While larval type classification performed poorly, cytotaxonomy and DNA-based methods yielded comparable results: detrended correspondence analysis and permutation analyses indicated that the assemblages are not randomly but competitively structured. However, DNA taxonomy identified an additional species that could not be resolved by the traditional method. We argue that DNA-based identification methods such as DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of the taxonomic assessment in biodiversity and community ecology studies.     

Identifying freshwater mussels (Unionoida) and parasitic glochidia larvae from host fish gills: a molecular key to the North and Central European species

Freshwater mussels (order Unionoida) represent one of the most severely endangered groups of animals due to habitat destruction, introduction of nonnative species, and loss of host fishes, which their larvae (glochidia) are obligate parasites on. Conservation efforts such as habitat restoration or restocking of host populations are currently hampered by difficulties in unionoid species identification by morphological means. Here we present the first complete molecular identification key for all seven indigenous North and Central European unionoid species and the nonnative Sinanodonta woodiana, facilitating quick, low-cost, and reliable identification of adult and larval specimens. Application of this restriction fragment length polymorphisms (RFLP) key resulted in 100% accurate assignment of 90 adult specimens from across the region by digestion of partial ITS-1 (where ITS is internal transcribed spacer) polymerase chain reaction (PCR) products in two to four single digestions with five restriction endonucleases. In addition, we provide protocols for quick and reliable extraction and amplification of larval mussel DNA from complete host fish gill arches. Our results indicate that this new method can be applied on infection rates as low as three glochidia per gill arch and enables, for the first time, comprehensive, large-scale assessments of the relative importance of different host species for given unionoid populations.