Geography, phylogeny and host switch drive the coevolution of parasitic Gyrodactylus flatworms and their hosts.

BACKGROUND: Gyrodactylus is a lineage of monogenean flatworm ectoparasites exhibiting many features that make them a suitable model to study the host-parasite coevolutionary dynamics. Previous coevolutionary studies of this lineage mainly relied on low-power datasets (a small number of samples and a single molecular marker) and (now) outdated algorithms. METHODS: To investigate the coevolutionary relationship of gyrodactylids and their fish hosts in high resolution, we used complete mitogenomes (including two newly sequenced Gyrodactylus species), a large number of species in the single-gene dataset, and four different coevolutionary algorithms. RESULTS: The overall coevolutionary fit between the parasites and hosts was consistently significant. Multiple indicators confirmed that gyrodactylids are generally highly host-specific parasites, but several species could parasitize either multiple (more than 5) or phylogenetically distant fish hosts. The molecular dating results indicated that gyrodactylids tend to evolve towards high host specificity. Speciation by host switch was identified as a more important speciation mode than co-speciation. Assuming that the ancestral host belonged to Cypriniformes, we inferred four major host switch events to non-Cypriniformes hosts (mostly Salmoniformes), all of which occurred deep in the evolutionary history. Despite their relative rarity, these events had strong macroevolutionary consequences for gyrodactylid diversity. For example, in our dataset, 57.28% of all studied gyrodactylids parasitized only non-Cypriniformes hosts, which implies that the evolutionary history of more than half of all included lineages could be traced back to these major host switch events. The geographical co-occurrence of fishes and gyrodactylids determined the host use by these gyrodactylids, and geography accounted for most of the phylogenetic signal in host use. CONCLUSIONS: Our findings suggest that the coevolution of Gyrodactylus flatworms and their hosts is largely driven by geography, phylogeny, and host switches.

Strong mitonuclear discordance in the phylogeny of Neodermata and evolutionary rates of Polyopisthocotylea.

The genomic evolution of Polyopisthocotylea remains poorly understood in comparison to the remaining three classes of Neodermata: Monopisthocotylea, Cestoda, and Trematoda. Moreover, the evolutionary sequence of major events in the phylogeny of Neodermata remains unresolved. Herein we sequenced the mitogenome and transcriptome of the polyopisthocotylean Diplorchis sp., and conducted comparative evolutionary analyses using nuclear (nDNA) and mitochondrial (mtDNA) genomic datasets of Neodermata. We found strong mitonuclear discordance in the phylogeny of Neodermata. Polyopisthocotylea exhibited striking mitonuclear discordance in relative evolutionary rates: the fastest-evolving mtDNA in Neodermata and a comparatively slowly-evolving nDNA genome. This was largely attributable to its very long stem branch in mtDNA topologies, not exhibited by the nDNA data. We found indications that the fast evolution of mitochondrial genomes of Polyopisthocotylea may be driven both by relaxed purifying selection pressures and elevated levels of directional selection. We identified mitochondria-associated genes encoded in the nuclear genome: they exhibited unique evolutionary rates, but not correlated with the evolutionary rate of mtDNA, and there is no evidence for compensatory evolution (they evolved slower than the rest of the genome). Finally, there appears to exist an exceptionally large (≈6.3 kb) nuclear mitochondrial DNA segment (numt) in the nuclear genome of newly sequenced Diplorchis sp. A 3'-end segment of the 16S rRNA gene encoded by the numt was expressed, suggesting that this gene acquired novel, regulatory functions after the transposition to the nuclear genome. In conclusion, Polyopisthocotylea appears to be the lineage with the fastest-evolving mtDNA sequences among all of Bilateria, but most of the substitutions were accumulated deep in the evolutionary history of this lineage. As the nuclear genome does not exhibit a similar pattern, the circumstances underpinning this evolutionary phenomenon remain a mystery.

Mitogenomic evolutionary rates in bilateria are influenced by parasitic lifestyle and locomotory capacity.

The evidence that parasitic animals exhibit elevated mitogenomic evolutionary rates is inconsistent and limited to Arthropoda. Similarly, the evidence that mitogenomic evolution is faster in species with low locomotory capacity is limited to a handful of animal lineages. We hypothesised that these two variables are associated and that locomotory capacity is a major underlying factor driving the elevated rates in parasites. Here, we study the evolutionary rates of mitogenomes of 10,906 bilaterian species classified according to their locomotory capacity and parasitic/free-living life history. In Bilateria, evolutionary rates were by far the highest in endoparasites, much lower in ectoparasites with reduced locomotory capacity and free-living lineages with low locomotory capacity, followed by parasitoids, ectoparasites with high locomotory capacity, and finally micropredatory and free-living lineages. The life history categorisation (parasitism) explained ≈45%, locomotory capacity categorisation explained ≈39%, and together they explained ≈56% of the total variability in evolutionary rates of mitochondrial protein-coding genes in Bilateria. Our findings suggest that these two variables play major roles in calibrating the mitogenomic molecular clock in bilaterian animals.

The mitochondrial genome of Heterosentis pseudobagri (Wang & Zhang, 1987) Pichelin & Cribb, 1999 reveals novel aspects of tRNA genes evolution in Acanthocephala.

BACKGROUND: Acanthocephala is a clade of obligate endoparasites whose mitochondrial genomes (mitogenomes) and evolution remain relatively poorly understood. Previous studies reported that atp8 is lacking from acanthocephalan mitogenomes, and that tRNA genes often have nonstandard structures. Heterosentis pseudobagri (Arhythmacanthidae) is an acanthocephalan fish endoparasite for which no molecular data are currently available, and biological information is unavailable in the English language. Furthermore, there are currently no mitogenomes available for Arhythmacanthidae. METHODS: We sequenced its mitogenome and transcriptome, and conducted comparative mitogenomic analyses with almost all available acanthocephalan mitogenomes. RESULTS: The mitogenome had all genes encoded on the same strand and unique gene order in the dataset. Among the 12 protein-coding genes, several genes were highly divergent and annotated with difficulty. Moreover, several tRNA genes could not be identified automatically, so we had to identify them manually via a detailed comparison with orthologues. As common in acanthocephalans, some tRNAs lacked either the TWC arm or the DHU arm, but in several cases, we annotated tRNA genes only on the basis of the conserved narrow central segment comprising the anticodon, while the flanking 5' and 3' ends did not exhibit any resemblance to orthologues and they could not be folded into a tRNA secondary structure. We corroborated that these are not sequencing artefacts by assembling the mitogenome from transcriptomic data. Although this phenomenon was not observed in previous studies, our comparative analyses revealed the existence of highly divergent tRNAs in multiple acanthocephalan lineages. CONCLUSIONS: These findings indicate either that multiple tRNA genes are non-functional or that (some) tRNA genes in (some) acanthocephalans might undergo extensive posttranscriptional tRNA processing which restores them to more conventional structures. It is necessary to sequence mitogenomes from yet unrepresented lineages and further explore the unusual patterns of tRNA evolution in Acanthocephala.

Using PhyloSuite for molecular phylogeny and tree-based analyses.

Phylogenetic analysis has entered the genomics (multilocus) era. For less experienced researchers, conquering the large number of software programs required for a multilocus-based phylogenetic reconstruction can be somewhat daunting and time-consuming. PhyloSuite, a software with a user-friendly GUI, was designed to make this process more accessible by integrating multiple software programs needed for multilocus and single-gene phylogenies and further streamlining the whole process. In this protocol, we aim to explain how to conduct each step of the phylogenetic pipeline and tree-based analyses in PhyloSuite. We also present a new version of PhyloSuite (v1.2.3), wherein we fixed some bugs, made some optimizations, and introduced some new functions, including a number of tree-based analyses, such as signal-to-noise calculation, saturation analysis, spurious species identification, and etc. The step-by-step protocol includes background information (i.e., what the step does), reasons (i.e., why do the step), and operations (i.e., how to do it). This protocol will help researchers quick-start their way through the multilocus phylogenetic analysis, especially those interested in conducting organelle-based analyses.

Positive selection-driven fixation of a hominin-specific amino acid mutation related to dephosphorylation in IRF9.

The arms race between humans and pathogens drives the evolution of the human genome. It is thus expected that genes from the interferon-regulatory factors family (IRFs), a critical family for anti-viral immune response, should be undergoing episodes of positive selection. Herein, we tested this hypothesis and found multiple lines of evidence for positive selection on the amino acid site Val129 (NP_006075.3:p.Ser129Val) of human IRF9. Interestingly, the ancestral reconstruction and population distribution analyses revealed that the ancestral state (Ser129) is conserved among mammals, while the derived positively selected state (Val129) was fixed before the "out-of-Africa" event ~ 500,000 years ago. The motif analysis revealed that this young amino acid (Val129) may serve as a dephosphorylation site of IRF9. Structural parallelism between homologous genes further suggested the functional effects underlying the dephosphorylation that may affect the immune activity of IRF9. This study provides a model in which a strong positive Darwinian selection drives a recent fixation of a hominin-specific amino acid leading to molecular adaptation involving dephosphorylation in an immune-responsive gene.

Inverted base composition skews and discontinuous mitochondrial genome architecture evolution in the Enoplea (Nematoda).

BACKGROUND: Within the class Enoplea, the earliest-branching lineages in the phylum Nematoda, the relatively highly conserved ancestral mitochondrial architecture of Trichinellida is in stark contrast to the rapidly evolving architecture of Dorylaimida and Mermithida. To better understand the evolution of mitogenomic architecture in this lineage, we sequenced the mitogenome of a fish parasite Pseudocapillaria tomentosa (Trichinellida: Capillariidae) and compared it to all available enoplean mitogenomes. RESULTS: P. tomentosa exhibited highly reduced noncoding regions (the largest was 98 bp), and a unique base composition among the Enoplea. We attributed the latter to the inverted GC skew (0.08) in comparison to the ancestral skew in Trichinellidae (-0.43 to -0.37). Capillariidae, Trichuridae and Longidoridae (Dorylaimida) generally exhibited low negative or low positive skews (-0.1 to 0.1), whereas Mermithidae exhibited fully inverted low skews (0 to 0.05). This is indicative of inversions in the strand replication order or otherwise disrupted replication mechanism in the lineages with reduced/inverted skews. Among the Trichinellida, Trichinellidae and Trichuridae have almost perfectly conserved architecture, whereas Capillariidae exhibit multiple rearrangements of tRNA genes. In contrast, Mermithidae (Mermithida) and Longidoridae (Dorylaimida) exhibit almost no similarity to the ancestral architecture. CONCLUSIONS: Longidoridae exhibited more rearranged mitogenomic architecture than the hypervariable Mermithidae. Similar to the Chromadorea, the evolution of mitochondrial architecture in enoplean nematodes exhibits a strong discontinuity: lineages possessing a mostly conserved architecture over tens of millions of years are interspersed with lineages exhibiting architectural hypervariability. As Longidoridae also have some of the smallest metazoan mitochondrial genomes, they contradict the prediction that compact mitogenomes should be structurally stable. Lineages exhibiting inverted skews appear to represent the intermediate phase between the Trichinellidae (ancestral) and fully derived skews in Chromadorean mitogenomes (GC skews = 0.18 to 0.64). Multiple lines of evidence (CAT-GTR analysis in our study, a majority of previous mitogenomic results, and skew disruption scenarios) support the Dorylaimia split into two sister-clades: Dorylaimida + Mermithida and Trichinellida. However, skew inversions produce strong base composition biases, which can hamper phylogenetic and other evolutionary studies, so enoplean mitogenomes have to be used with utmost care in evolutionary studies.

The Role of Intestinal Microbiota in Regulating the Metabolism of Bile Acids Is Conserved Across Vertebrates.

In mammals, bile acid (BA) concentrations are regulated largely by the gut microbiota, and a study has shown that some metabolic responses to the gut microbiota are conserved between zebrafish and mice. However, it remains unknown whether the influence of specific intestinal microbes on BA metabolism is conserved between higher and lower vertebrates (i.e., mammals and fish). In the present study, Citrobacter freundii GC01 isolated from the grass carp (Ctenopharyngodon idella) intestine was supplemented to the fish and mice feed. We found the changes in the bile acid profile, especially significant changes in secondary BAs in both grass carp and mice fed on C. freundii. Also, lipid metabolism was significantly affected by C. freundii. Analysis of liver transcriptome sequencing data and validation by RT-qPCR revealed that the CYP7A1 gene was significantly up-regulated in both grass carp and mice. In addition, the overexpression of HNF4B from grass carp resulted in a significant increase in the expression level of CYP7A1. Generally, our results suggest that the metabolism of BAs by intestinal microbiota is conserved across vertebrates. Furthermore, specific intestinal bacteria may regulate the bile salt synthesis through CYP7A1 and that HNF4B might be an important regulator of BA metabolism in fish.

Whole-genome sequencing identifies rare missense variants of WNT16 and ERVW-1 causing the systemic lupus erythematosus.

Systemic lupus erythematosus (SLE, OMIM 152700) is a rare autoimmune disease with high heritability that affects ~0.1% of the population. Previous studies have revealed several common variants with small effects in European and East Asian SLE patients. However, there is still no rare variant study on Chinese SLE patients using the whole-genome sequencing technology (WGS). Here, we designed a family based WGS study to identify novel rare variants with large effects. Based on large-scale allele frequency data from the gnomAD database, we identified rare protein-coding gene variants with disruptive and sequence-altering impacts in SLE patients. We found that the burden of rare variants was significantly higher than that of common variants in patients, suggesting a larger effect of rare variants on the SLE pathogenesis. We identified the pathogenic risk of rare missense variants with significant odds ratios (p < 0.05) in two genes, including WNT16 (NC_000007.14:g.121329757G > C, NP_057171.2:p.(Ala86Pro) and 7 g.121329760G > C, NP_057171.2:p.(Ala87Pro)), which explains five out of seven patients covering all three families but are absent from all controls, and ERVW-1 (NC_000007.14:g.92469882A > G, NP_001124397.1:p.(Leu167Pro), rs74545114; NC_000007.14:g.92469907G > A, NP_001124397.1:p.(Arg159Cys), rs201142302; NC_000007.14:g.92469919G > A, NP_001124397.1:p.(His155Tyr), rs199552228), which explains the other two patients. None of these variants were identified in any of the controls. These associations are supported by known gene expression studies in SLE patients based on literature review. We further tested the wild and mutant types using the luciferase assays and qPCR in cells. We found that WNT16 can activate the canonical Wnt/ß-catenin pathway while the mutant cannot. Additionally, the wild ERVW-1 expression can be significantly up-regulated by cAMP while the mutant cannot. Our study provides the first direct genetic and in vitro evidence for the pathogenic risk of mutant WNT16 and ERVW-1, which may facilitate the design of precision therapy for SLE.

Slow crabs - fast genomes: Locomotory capacity predicts skew magnitude in crustacean mitogenomes.

Base composition skews (G-C/G+C) of mitochondrial genomes are believed to be primarily driven by mutational pressure, which is positively correlated with metabolic rate. In marine animals, metabolic rate is also positively correlated with locomotory capacity. Given the central role of mitochondria in energy metabolism, we hypothesised that selection for locomotory capacity should be positively correlated with the strength of purifying selection (dN/dS), and thus be negatively correlated with the skew magnitude. Therefore, these two models assume diametrically opposite associations between the metabolic rate and skew magnitude: positive correlation in the prevailing paradigm, and negative in our working hypothesis. We examined correlations between the skew magnitude, metabolic rate, locomotory capacity, and several other variables previously associated with mitochondrial evolution on 287 crustacean mitogenomes. Weakly locomotory taxa had higher skew magnitude and ω (dN/dS) values, but not the gene order rearrangement rate. Skew and ω magnitudes were correlated. Multilevel regression analyses indicated that three competing variables, body size, gene order rearrangement rate, and effective population size, had negligible impacts on the skew magnitude. In most crustacean lineages selection for locomotory capacity appears to be the primary factor determining the skew magnitude. Contrary to the prevailing paradigm, this implies that adaptive selection outweighs nonadaptive selection (mutation pressure) in crustaceans. However, we found indications that effective population size (nonadaptive factor) may outweigh the impact of locomotory capacity in sessile crustaceans (Thecostraca). In conclusion, skew magnitude is a product of the interplay between adaptive and nonadaptive factors, the balance of which varies among lineages.

Evolutionary history of inversions in directional mutational pressures in crustacean mitochondrial genomes: Implications for evolutionary studies.

Inversions of the origin of replication (ORI) in mitochondrial genomes produce asymmetrical mutational pressures that can cause strong base composition skews. Due to skews often being overlooked, the total number of crustacean lineages that underwent ORI events remains unknown. We analysed skews, cumulative skew plots, conserved sequence motifs, and mitochondrial architecture of all 965 available crustacean mitogenomes (699 unique species). We found indications of an ORI in 159 (22.7%) species, and mapped these to 23 ORI events: 16 identified with confidence and 7 putative (13 newly proposed, and for 5 we improved the resolution). Two ORIs occurred at or above the order level: Isopoda and Copepoda. Shifts in skew plots are not a precise tool for identifying the replication mechanism. We discuss how ORIs can produce mutational bursts in mitogenomes and show how these can interfere with various types of evolutionary studies. Phylogenetic analyses were plagued by artefactual clustering, and ORI lineages exhibited longer branches, a higher number of synonymous substitutions, higher mutational saturation, and higher compositional heterogeneity. ORI events also affected codon usage and protein properties. We discuss how this may have caused erroneous interpretation of data in previous studies that did not account for skew patterns.

A chromosome-level genome assembly of Cairina moschata and comparative genomic analyses.

BACKGROUND: The Muscovy duck (Cairina moschata) is an economically important duck species, with favourable growth and carcass composition parameters in comparison to other ducks. However, limited genomic resources for Muscovy duck hinder our understanding of its evolution and genetic diversity. RESULTS: We combined linked-reads sequencing technology and reference-guided methods for de novo genome assembly. The final draft assembly was 1.12 Gbp with 29 autosomes, one sex chromosome and 4,583 unlocalized scaffolds with an N50 size of 77.35 Mb. Based on universal single-copy orthologues (BUSCO), the draft genome assembly completeness was estimated to be 93.30 %. Genome annotation identified 15,580 genes, with 15,537 (99.72 %) genes annotated in public databases. We conducted comparative genomic analyses and found that species-specific and rapidly expanding gene families (compared to other birds) in Muscovy duck are mainly involved in Calcium signaling, Adrenergic signaling in cardiomyocytes, and GnRH signaling pathways. In comparison to the common domestic duck (Anas platyrhynchos), we identified 104 genes exhibiting strong signals of adaptive evolution (Ka/Ks > 1). Most of these genes were associated with immune defence pathways (e.g. IFNAR1 and TLR5). This is indicative of the existence of differences in the immune responses between the two species. Additionally, we combined divergence and polymorphism data to demonstrate the "faster-Z effect" of chromosome evolution. CONCLUSIONS: The chromosome-level genome assembly of Muscovy duck and comparative genomic analyses provide valuable resources for future molecular ecology studies, as well as the evolutionary arms race between the host and influenza viruses.

Disrupted architecture and fast evolution of the mitochondrial genome of Argeia pugettensis (Isopoda): implications for speciation and fitness.

BACKGROUND: Argeia pugettensis is an isopod species that parasitizes other crustaceans. Its huge native geographic range spans the Pacific from China to California, but molecular data are available only for a handful of specimens from North-American populations. We sequenced and characterised the complete mitogenome of a specimen collected in the Yellow Sea. RESULTS: It exhibited a barcode (cox1) similarity level of only 87-89% with North-American populations, which is unusually low for conspecifics. Its mitogenome is among the largest in isopods (≈16.5 Kbp), mostly due to a large duplicated palindromic genomic segment (2 Kbp) comprising three genes. However, it lost a segment comprising three genes, nad4L-trnP-nad6, and many genes exhibited highly divergent sequences in comparison to isopod orthologues, including numerous mutations, deletions and insertions. Phylogenetic and selection analyses corroborated that this is one of the handful of most rapidly evolving available isopod mitogenomes, and that it evolves under highly relaxed selection constraints (as opposed to positive selection). However, its nuclear 18S gene is highly conserved, which suggests that rapid evolution is limited to its mitochondrial genome. The cox1 sequence analysis indicates that elevated mitogenomic evolutionary rates are not shared by North-American conspecifics, which suggests a breakdown of cox1 barcoding in this species. CONCLUSIONS: A highly architecturally disrupted mitogenome and decoupling of mitochondrial and nuclear rates would normally be expected to have strong negative impacts on the fitness of the organism, so the existence of this lineage is a puzzling evolutionary question. Additional studies are needed to assess the phylogenetic breadth of this disrupted mitochondrial architecture and its impact on fitness.

Architectural instability, inverted skews and mitochondrial phylogenomics of Isopoda: outgroup choice affects the long-branch attraction artefacts.

The majority strand of mitochondrial genomes of crustaceans usually exhibits negative GC skews. Most isopods exhibit an inversed strand asymmetry, believed to be a consequence of an inversion of the replication origin (ROI). Recently, we proposed that an additional ROI event in the common ancestor of Cymothoidae and Corallanidae families resulted in a double-inverted skew (negative GC), and that taxa with homoplastic skews cluster together in phylogenetic analyses (long-branch attraction, LBA). Herein, we further explore these hypotheses, for which we sequenced the mitogenome of Asotana magnifica (Cymothoidae), and tested whether our conclusions were biased by poor taxon sampling and inclusion of outgroups. (1) The new mitogenome also exhibits a double-inverted skew, which supports the hypothesis of an additional ROI event in the common ancestor of Cymothoidae and Corallanidae families. (2) It exhibits a unique gene order, which corroborates that isopods possess exceptionally destabilized mitogenomic architecture. (3) Improved taxonomic sampling failed to resolve skew-driven phylogenetic artefacts. (4) The use of a single outgroup exacerbated the LBA, whereas both the use of a large number of outgroups and complete exclusion of outgroups ameliorated it.

Disentangling the interplay of positive and negative selection forces that shaped mitochondrial genomes of Gammarus pisinnus and Gammarus lacustris.

We hypothesized that the mitogenome of Gammarus lacustris (GL), native to the Qinghai-Tibet Plateau, might exhibit genetic adaptations to the extreme environmental conditions associated with high altitudes (greater than 3000 m). To test this, we also sequenced the mitogenome of Gammarus pisinnus (GP), whose native range is close to the Tibetan plateau, but at a much lower altitude (200-1500 m). The two mitogenomes exhibited conserved mitochondrial architecture, but low identity between genes (55% atp8 to 76.1% cox1). Standard (homogeneous) phylogenetic models resolved Gammaridae as paraphyletic, but 'heterogeneous' CAT-GTR model as monophyletic. In indirect support of our working hypothesis, GL, GP and Gammarus fossarum exhibit evidence of episodic diversifying selection within the studied Gammaroidea dataset. The mitogenome of GL generally evolves under a strong purifying selection, whereas GP evolves under directional (especially pronounced in atp8) and/or relaxed selection. This is surprising, as GP does not inhabit a unique ecological niche compared to other gammarids. We propose that this rapid evolution of the GP mitogenome may be a reflection of its relatively recent speciation and heightened non-adaptive (putatively metabolic rate-driven) mutational pressures. To test these hypotheses, we urge sequencing mitogenomes of remaining Gammarus species populating the same geographical range as GP.

PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies.

Multigene and genomic data sets have become commonplace in the field of phylogenetics, but many existing tools are not designed for such data sets, which often makes the analysis time-consuming and tedious. Here, we present PhyloSuite, a (cross-platform, open-source, stand-alone Python graphical user interface) user-friendly workflow desktop platform dedicated to streamlining molecular sequence data management and evolutionary phylogenetics studies. It uses a plugin-based system that integrates several phylogenetic and bioinformatic tools, thereby streamlining the entire procedure, from data acquisition to phylogenetic tree annotation (in combination with iTOL). It has the following features: (a) point-and-click and drag-and-drop graphical user interface; (b) a workplace to manage and organize molecular sequence data and results of analyses; (c) GenBank entry extraction and comparative statistics; and (d) a phylogenetic workflow with batch processing capability, comprising sequence alignment (mafft and macse), alignment optimization (trimAl, HmmCleaner and Gblocks), data set concatenation, best partitioning scheme and best evolutionary model selection (PartitionFinder and modelfinder), and phylogenetic inference (MrBayes and iq-tree). PhyloSuite is designed for both beginners and experienced researchers, allowing the former to quick-start their way into phylogenetic analysis, and the latter to conduct, store and manage their work in a streamlined way, and spend more time investigating scientific questions instead of wasting it on transferring files from one software program to another.

Identification of neuropeptides from eyestalk transcriptome profiling analysis of female oriental river prawn (Macrobrachium nipponense) under hypoxia and reoxygenation conditions.

The oriental river prawn, Macrobrachium nipponense, is a commercial freshwater prawn species in China. It is highly sensitive to hypoxia, and this has posed a challenge to its intensive culturing. To date, the effects of hypoxia on reproduction in female prawns are not entirely clear, as are the underlying mechanisms of the effects of hypoxia. In this work, comparative transcriptome and gene expression analyses of the eyestalk were performed in M. nipponense females under hypoxia and reoxygenation conditions. Sequencing and de novo assembly of the combined reads yielded 43,583 unigenes with an average length of 1726 bp. A total of 711 genes were found to be differentially expressed in the eyestalk under the hypoxia and reoxygenation conditions. With the help of functional and pathway enrichment analysis of the differentially expressed genes, a novel set of transcripts that were associated with several important functions, such as hormone biosynthesis and progesterone-mediated oocyte maturation, were identified. Additionally, ten neuropeptides were identified based on the differentially expressed transcripts, and they were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription PCR (RT-PCR) analyses. Three neuropeptide genes were expressed in the neural tissue and ovary of the prawns; this indicates that they were involved in reproductive processes. In particular, RNA interference (RNAi) short neuropeptide F dramatically promoted ovary maturation, as indicated by the gonad somatic index. While the present findings do indicate that hypoxia affects reproductive function in M. nipponense females, in-depth functional analyses of the candidate neuropeptides should be conducted in the future to understand their role in hypoxia adaptation and the associated mechanisms that affect the reproductive capacity of this species.

Mitochondrial genomes and 28S rDNA contradict the proposed obsoletion of the order Tetraonchidea (Platyhelminthes: Monogenea).

Due to the incongruence of morphology-based hypotheses and scarcity of molecular data, validity of the order Tetraonchidea remains contentious. The only complete mitogenome currently available for the entire order is that of Paratetraonchoides inermis (Tetraonchoididae). To study the phylogeny of Tetraonchidea from mitogenomic perspective, we sequenced the first mitogenome for the family Tetraonchidae: Tetraonchus monenteron (Tetraonchidea). To get a nuclear-data perspective, we also sequenced nuclear 28S rDNA gene of both species. The mitogenome of T. monenteron does not have high A + T content, nor tRNA pseudo-genes, both of which were unique features reported in P. inermis. However, T. monenteron exhibits a unique gene order, with a large number of tRNA rearrangements in comparison to P. inermis and other monogeneans. Phylogenetic analyses conducted using Bayesian inference and maximum likelihood methods, complemented with partitioning, consistently support the sister-group relationship of T. monenteron (Tetraonchidae) and P. inermis (Tetraonchoididae). This is also partially supported by the 28S rDNA data and two morphologic apomorphies. This close relationship of Tetraonchidae and Tetraonchoididae challenges the latest major morphology-based classification, which proposed obsoletion of the Tetraonchidea order, and grouped Tetraonchoididae into the Gyrodactylidea clade. The validity of this order shall have to be further confirmed with more data.

Morphology is not a reliable taxonomic tool for the genus Lernaea: molecular data and experimental infection reveal that L. cyprinacea and L. cruciata are conspecific.

BACKGROUND: Species belonging to the genus Lernaea are cosmopolitan parasites that can infect many different freshwater fish hosts. Due to a high degree of morphological intraspecific variability and high levels of interspecific similarities, their classification is extremely difficult and controversial. Although the suitability of the shape of cephalic horns has been questioned decades ago by some experimental infection studies, this character still plays the central role in the identification of Lernaea spp. METHODS: We used the nominal species Lernaea cyprinacea and Lernaea cruciata to test the hypothesis that the shape of the anchor can exhibit host-induced morphological variability, and that the two taxa may be synonymous. RESULTS: We examined 517 wild or farmed specimens of five host fish species (four cyprinids and a mosquitofish), and found that all 16 parasite specimens collected from mosquitofish could be morphologically identified as L. cruciata, whereas the remaining 25 parasite specimens were all identified as L. cyprinacea. We experimentally infected goldfish and mosquitofish specimens with offspring (copepodids) of a single L. cyprinacea specimen: the adult parasites from goldfish were morphologically identified as L. cyprinacea, and those from mosquitofish as L. cruciata. We then used molecular data to corroborate that all these specimens are conspecific. CONCLUSIONS: Our results suggest that L. cyprinacea and L. cruciata may be synonyms, misidentified as different species as a result of host-induced morphological variation. Given the current shortage of molecular data for the genus Lernaea, in order to resolve the taxonomy of this genus (determine the exact number of species), future studies should aim to sequence as much molecular data as possible, and conduct further experimental infections.