Gut content metabarcoding of specialized feeders is not a replacement for environmental DNA assays of seawater in reef environments.

In tropical marine ecosystems, the coral-based diet of benthic-feeding reef fishes provides a window into the composition and health of coral reefs. In this study, for the first time, we compare multi-assay metabarcoding sequences of environmental DNA (eDNA) isolated from seawater and partially digested gut items from an obligate corallivore butterflyfish (Chaetodon lunulatus) resident to coral reef sites in the South China Sea. We specifically tested the proportional and statistical overlap of the different approaches (seawater vs gut content metabarcoding) in characterizing eukaryotic community composition on coral reefs. Based on 18S and ITS2 sequence data, which differed in their taxonomic sensitivity, we found that gut content detections were only partially representative of the eukaryotic communities detected in the seawater based on low levels of taxonomic overlap (3 to 21%) and significant differences between the sampling approaches. Overall, our results indicate that dietary metabarcoding of specialized feeders can be complimentary to, but is no replacement for, more comprehensive environmental DNA assays of reef environments that might include the processing of different substrates (seawater, sediment, plankton) or traditional observational surveys. These molecular assays, in tandem, might be best suited to highly productive but cryptic oceanic environments (kelp forests, seagrass meadows) that contain an abundance of organisms that are often small, epiphytic, symbiotic, or cryptic.

Analysis of the complete mitochondrial genome sequence of Japanese butterflyfish, Chaetodon nippon (Chaetodontiformes, Chaetodontidae).

Japanese butterflyfish (Chaetodon nippon) belong to the family Chaetodontidae and order Chaetodontiformes. It has circular mitochondrial genome of 16,507 bp in length with 55.4% of A + T content and has 37 genes, including 22 tRNA, 2 rRNA, and 13 protein-coding genes, in addition to a control region. The results of phylogenetic analysis indicated that the C. nippon, C. wiebeli, C. auripes, C. auriga, C. octofasciatus, C. speculum, and C. modestus are closely related to each other. The findings of this study will provide useful genetic information for further phylogenetic and taxonomic classifications of Chaetodontidae.

Rapid resource depletion on coral reefs disrupts competitor recognition processes among butterflyfish species.

Avoiding costly fights can help conserve energy needed to survive rapid environmental change. Competitor recognition processes help resolve contests without escalating to attack, yet we have limited understanding of how they are affected by resource depletion and potential effects on species coexistence. Using a mass coral mortality event as a natural experiment and 3770 field observations of butterflyfish encounters, we test how rapid resource depletion could disrupt recognition processes in butterflyfishes. Following resource loss, heterospecifics approached each other more closely before initiating aggression, fewer contests were resolved by signalling, and the energy invested in attacks was greater. By contrast, behaviour towards conspecifics did not change. As predicted by theory, conspecifics approached one another more closely and were more consistent in attack intensity yet, contrary to expectations, resolution of contests via signalling was more common among heterospecifics. Phylogenetic relatedness or body size did not predict these outcomes. Our results suggest that competitor recognition processes for heterospecifics became less accurate after mass coral mortality, which we hypothesize is due to altered resource overlaps following dietary shifts. Our work implies that competitor recognition is common among heterospecifics, and disruption of this system could lead to suboptimal decision-making, exacerbating sublethal impacts of food scarcity.

Complete mitochondrial genome of brown-banded butterflyfish Chaetodon modestus (Chaetodontiformes, Chaetodontidae) and phylogenetic analysis.

The complete mitochondrial genome of the Chaetodon modestus (Temminck and Schlegel, 1844) was first determined in this study, which is 16,490 bp in length, containing 13 protein-coding genes, two rRNA genes, and 22 tRNA. Out of 37 mitochondrial genes, except for ND6 and eight tRNA (Pro, Glu, Ser, Tyr, Cys, Asn, Ala, Gln) genes were encoded on the L-strand, the others were encoded on the H-strand. The overall base composition includes A (28.0%), T (28.7%), G (16.7%), ad C (26.6%). The phylogenetic tree was built using the maximum-likelihood approach to provide a relationship within Chaetodontidae, which might be valuable for species management.

The complete mitochondrial genome of threadfin butterflyfish, Chaetodon auriga (Chaetodontiformes: Chaetodontidae) and phylogenetic analysis.

Chaetodon auriga (Forsskal, 1775) belongs to the family Chaetodontidae and the order Chaetodontiformes. Here, we report the complete mitochondrial genome of C. auriga assembled using the Illumina MiSeq platform. The circular mitochondrial genome of C. auriga is 16,527 bp long, has an A + T content of 54.53%, and contains 37 genes (13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes), and a non-coding region. The overall nucleotide composition was A: 28.19%, T: 26.34%, G: 16.27%, and C: 29.20%. The mitochondrial genome of C. auriga contributes to revealing the phylogenetic relationships among species of the Chaetodontidae family.

The complete mitochondrial genome of Chaetodon speculum (Chaetodontiformes, chaetodontidae).

Chaetodontidae species feeding observations showed that they mostly fed on different coral species. Among them, Chaetodon speculum (Cuvier, 1831) is one of most important genera of Chaetodontidae, C. baronessa and C. bennetti appeared to ingest annelid worms during the course of coral feeding, whereas gut contents of C. punctatofasciatus and C. speculum were dominated by crustaceans. However, the systemically classification and taxonomic studies have so far been limited. In this study, we report the complete mitochondrial genome sequence of C. speculum. The mitogenome has 16,537 base pairs (54.4% A + T content) and made up of total of 37 genes (13 protein-coding, 22 transfer RNAs and 2 ribosomal RNAs), and a putative control region. This study will provide useful genetic information for future phylogenetic and taxonomic classification of Chaetodontidae.

Insights into the Natural Defenses of a Coral Reef Fish Against Gill Ectoparasites: Integrated Metabolome and Microbiome Approach.

Understanding natural defense mechanisms against parasites can be a valuable tool for the development of innovative therapies. We have previously identified a butterflyfish species (Chaetodon lunulatus) that avoids gill monogenean parasites while living amongst closely related parasitized species. The metabolome and microbiome of several sympatric butterflyfish species from the island of Moorea (French Polynesia) were previously described. In this study, we used the previously generated datasets in an attempt to identify metabolites and bacteria potentially involved in parasite defense mechanisms. We investigated the interplay between the gill mucus metabolome and microbiome of the non-susceptible C. lunulatus versus sympatric butterflyfish species that were always found parasitized in the Central and Eastern Indo-Pacific. After observing significant differences between the metabolome and bacteria of susceptible versus non-susceptible fish, we obtained the discriminant metabolites and operational taxonomic units (OTUs) using a supervised analysis. Some of the most important discriminant metabolites were identified as peptides, and three new peptides derived from ß-subunit hemoglobin from C. lunulatus (CLHbß-1, CLHbß-2, and CLHbß-3) were purified, characterized and synthesized to confirm their structures. We also identified specific bacterial families and OTUs typical from low-oxygen habitats in C. lunulatus gill mucus. By using a correlation network between the two datasets, we found a Fusobacteriaceae strain exclusively present in C. lunulatus and highly correlated to the peptides. Finally, we discuss the possible involvement of these peptides and Fusobacteriaceae in monogenean avoidance by this fish species.

Neobenedenia melleni (Monogenea: Capsalidae) in ornamental reef fish imported to Brazil / Neobenedenia melleni (Monogenea: Capsalidae) em peixes ornamentais de recife importados para o Brasil

Abstract The capsalid monogenean Neobenedenia melleni is known as a lethal pathogen for captured marine teleost ornamental fish, if left untreated. This study reports the occurrence of N. melleni parasitizing four species of ornamental reef fish imported into Brazil and maintained in quarantine: Arabian angelfish (Pomacanthus asfur ), yellowbar angelfish (Pomacanthus maculosus), regal angelfish (Pygoplites diacanthus), and bluecheek butterflyfish (Chaetodon semilarvatus). Ten days after the beginning of quarantine, some fish showed behavioral alterations, such as irritability, and corneal opacity, which were rapidly diagnosed to be caused by monogenean parasites by body surface scraping. The fish from the same batch were treated with two applications of 2 mg L-1 praziquantel each at an interval of four days. Seven days after the first treatment, the mucus surface of the fish was re-examined, which did not reveal the parasites presence being delivered for commercialization.

Resumo Neobenedenia melleni é conhecido como um parasito letal para peixes teleósteos ornamentais marinhos capturados se não tratado. Este estudo relata a ocorrência de N. melleni parasitando quatro espécies de peixes ornamentais importados para o Brazil: Arabian angelfish (Pomacanthus asfur), yellowbar angelfish (Pomacanthus maculosus), regal angelfish (Pygoplites diacanthus) e bluecheek butterflyfish ( Chaetodon semilarvatus). Dez dias depois do início da quarentena, foram observados irritabilidade e opacidade na córnea dos peixes que, foram diagnosticadas com N. melleni na superfície corporal. Todos os peixes foram tratados com duas aplicações de praziquantel 2 mg L-1 em intervalo de quatro dias. Sete dias após o início do tratamento, um novo exame parasitológico foi realizado o qual não revelou a presença dos parasitos, sendo liberados para comercialização.

The complete mitochondrial genome of Chaetodon wiebeli (Chaetodontiformes, Chaetodontidae).

Chaetodon wiebeli is one of the most important genera of Chaetodontidae. However, the systemic classification and taxonomic studies have so far been limited. In this study, we report the complete mitochondrial genome sequence of C. wiebeli. The mitogenome has 16,523 bp (54.3% A + T content) and is made up of a total of 37 genes (13 protein-coding, 22 transfer RNAs, and 2 ribosomal RNAs), and a putative control region. This study will provide useful genetic information for future phylogenetic and taxonomic classification of Chaetodontidae.

Ice ages and butterflyfishes: Phylogenomics elucidates the ecological and evolutionary history of reef fishes in an endemism hotspot.

For tropical marine species, hotspots of endemism occur in peripheral areas furthest from the center of diversity, but the evolutionary processes that lead to their origin remain elusive. We test several hypotheses related to the evolution of peripheral endemics by sequencing ultraconserved element (UCE) loci to produce a genome-scale phylogeny of 47 butterflyfish species (family Chaetodontidae) that includes all shallow water butterflyfish from the coastal waters of the Arabian Peninsula (i.e., Red Sea to Arabian Gulf) and their close relatives. Bayesian tree building methods produced a well-resolved phylogeny that elucidated the origins of butterflyfishes in this hotspots of endemism. We show that UCEs, often used to resolve deep evolutionary relationships, represent an important tool to assess the mechanisms underlying recently diverged taxa. Our analyses indicate that unique environmental conditions in the coastal waters of the Arabian Peninsula probably contributed to the formation of endemic butterflyfishes. Older endemic species are also associated with narrow versus broad depth ranges, suggesting that adaptation to deeper coral reefs in this region occurred only recently (<1.75 Ma). Even though deep reef environments were drastically reduced during the extreme low sea level stands of glacial ages, shallow reefs persisted, and as such there was no evidence supporting mass extirpation of fauna in this region.

The complete mitochondrial genome of Chaetodon octofasciatus (Perciformes: Chaetodontidae) and phylogenetic studies of Percoidea.

The complete mitochondrial genome of this species was first determined in this study, which is 16,485 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, a putative control region, and 1 origin of replication on the light-strand. The overall base composition includes C(28.2%), A(28.3%), T(27.4%), and G(16.1%). Moreover, the 13 PCGs encode 3796 amino acids in total, 12 of which use the initiation codon ATG except COI that uses GTG. Most of them have TAA as the stop codon, whereas ND3 ends with TAG, and three protein-coding genes (COII, ND4, and Cytb) ended with the incomplete stop codon represented as a single T. The phylogenetic tree based on the Neighbor Joining method was constructed to provide relationship within Percoidea, which could be a useful basis for management of this species.

Characterisation of the gill mucosal bacterial communities of four butterflyfish species: a reservoir of bacterial diversity in coral reef ecosystems.

While recent studies have suggested that fish mucus microbiota play an important role in homeostasis and prevention of infections, very few studies have investigated the bacterial communities of gill mucus. We characterised the gill mucus bacterial communities of four butterflyfish species and although the bacterial diversity of gill mucus varied significantly between species, Shannon diversities were high (H = 3.7-5.7) in all species. Microbiota composition differed between butterflyfishes, with Chaetodon lunulatus and C. ornatissimus having the most similar bacterial communities, which differed significantly from C. vagabundus and C. reticulatus. The core bacterial community of all species consisted of mainly Proteobacteria followed by Actinobacteria and Firmicutes. Chaetodonlunulatus and C. ornatissimus bacterial communities were mostly dominated by Gammaproteobacteria with Vibrio as the most abundant genus. Chaetodonvagabundus and C. reticulatus presented similar abundances of Gammaproteobacteria and Alphaproteobacteria, which were well represented by Acinetobacter and Paracoccus, respectively. In conclusion, our results indicate that different fish species present specific bacterial assemblages. Finally, as mucus layers are nutrient hotspots for heterotrophic bacteria living in oligotrophic environments, such as coral reef waters, the high bacterial diversity found in butterflyfish gill mucus might indicate external fish mucus surfaces act as a reservoir of coral reef bacterial diversity.

Neobenedenia melleni (Monogenea: Capsalidae) in ornamental reef fish imported to Brazil

Abstract The capsalid monogenean Neobenedenia melleni is known as a lethal pathogen for captured marine teleost ornamental fish, if left untreated. This study reports the occurrence of N. melleni parasitizing four species of ornamental reef fish imported into Brazil and maintained in quarantine: Arabian angelfish (Pomacanthus asfur ), yellowbar angelfish (Pomacanthus maculosus), regal angelfish (Pygoplites diacanthus), and bluecheek butterflyfish (Chaetodon semilarvatus). Ten days after the beginning of quarantine, some fish showed behavioral alterations, such as irritability, and corneal opacity, which were rapidly diagnosed to be caused by monogenean parasites by body surface scraping. The fish from the same batch were treated with two applications of 2 mg L-1 praziquantel each at an interval of four days. Seven days after the first treatment, the mucus surface of the fish was re-examined, which did not reveal the parasites presence being delivered for commercialization.

Resumo Neobenedenia melleni é conhecido como um parasito letal para peixes teleósteos ornamentais marinhos capturados se não tratado. Este estudo relata a ocorrência de N. melleni parasitando quatro espécies de peixes ornamentais importados para o Brazil: Arabian angelfish (Pomacanthus asfur), yellowbar angelfish (Pomacanthus maculosus), regal angelfish (Pygoplites diacanthus) e bluecheek butterflyfish ( Chaetodon semilarvatus). Dez dias depois do início da quarentena, foram observados irritabilidade e opacidade na córnea dos peixes que, foram diagnosticadas com N. melleni na superfície corporal. Todos os peixes foram tratados com duas aplicações de praziquantel 2 mg L-1 em intervalo de quatro dias. Sete dias após o início do tratamento, um novo exame parasitológico foi realizado o qual não revelou a presença dos parasitos, sendo liberados para comercialização.

Hard coral (Porites lobata) extracts and homarine on cytochrome P450 expression in Hawaiian butterflyfishes with different feeding strategies.

Dietary specialists tend to be less susceptible to the effects of chemical defenses produced by their prey compared to generalist predators that feed upon a broader range of prey species. While many researchers have investigated the ability of insects to detoxify dietary allelochemicals, little research has been conducted in marine ecosystems. We investigated metabolic detoxification pathways in three species of butterflyfishes: the hard coral specialist feeder, Chaetodon multicinctus, and two generalist feeders, Chaetodon auriga and Chaetodon kleinii. Each species was fed tissue homogenate of the hard coral Porites lobata or the feeding deterrent compound homarine (found in the coral extract), and the expression and catalytic activity of cytochrome P450 (CYP) 3A-like and CYP2-like enzymes were examined after one-week of treatment. The P. lobata homogenate significantly induced content and catalytic activity of CYP2-like and CYP3A-like forms, by 2-3 fold and by 3-9 fold, respectively, in C. multicinctus. Homarine caused a significant decrease of CYP2-like and CYP3A-like proteins at the high dose in C. kleinii and 60-80% mortality in that species. Homarine also induced CYP3A-like content by 3-fold and catalytic activity by 2-fold in C. auriga, while causing non-monotonic increases in CYP2-like and CYP3A-like catalytic activity in C. multicinctus. Our results indicate that dietary exposure to coral homogenates and the feeding deterrent constituent within these homogenates caused species-specific modulation of detoxification enzymes consistent with the prey selection strategies of generalist and specialist butterflyfishes.

Limited contemporary gene flow and high self-replenishment drives peripheral isolation in an endemic coral reef fish.

Extensive ongoing degradation of coral reef habitats worldwide has lead to declines in abundance of coral reef fishes and local extinction of some species. Those most vulnerable are ecological specialists and endemic species. Determining connectivity between locations is vital to understanding recovery and long-term persistence of these species following local extinction. This study explored population connectivity in the ecologically-specialized endemic three-striped butterflyfish (Chaetodon tricinctus) using mt and msatDNA (nuclear microsatellites) to distinguish evolutionary versus contemporary gene flow, estimate self-replenishment and measure genetic diversity among locations at the remote Australian offshore coral reefs of Middleton Reef (MR), Elizabeth Reef (ER), Lord Howe Island (LHI), and Norfolk Island (NI). Mt and msatDNA suggested genetic differentiation of the most peripheral location (NI) from the remaining three locations (MR, ER, LHI). Despite high levels of mtDNA gene flow, there is limited msatDNA gene flow with evidence of high levels of self-replenishment (≥76%) at all four locations. Taken together, this suggests prolonged population recovery times following population declines. The peripheral population (NI) is most vulnerable to local extinction due to its relative isolation, extreme levels of self-replenishment (95%), and low contemporary abundance.

Toxic coral gobies reduce the feeding rate of a corallivorous butterflyfish on Acropora corals.

The obligate coral-dwelling gobiid genus Gobiodon inhabits Acropora corals and has developed various physiological, morphological and ethological adaptations towards this life habit. While the advantages of this coral-fish association are well documented for Gobiodon, possible fitness-increasing factors for the host coral are unknown. This study examines the influence of coral-dwelling gobies on the feeding behaviour of obligate corallivorous butterflyfishes. In an aquarium experiment using video observation, the corallivorous butterflyfish Chaetodon austriacus fed significantly less on corals inhabited by two Gobiodon species compared to unoccupied coral colonies of similar size. The more agonistic species G. histrio, which mostly displayed directed movements towards butterflyfishes, decreased butterflyfish bite rate by 62-98 % compared to uninhabited colonies. For Gobiodon sp. 3, which mostly displayed undirected movements in response to visits by C. austriacus, bite rate reduction was 64-68 %. The scale-less skin of Gobiodon spp. is covered by mucus that is toxic and multi-functional by reducing predation as well as affecting parasite attachment. A choice flume experiment suggests that the highly diluted skin mucus of Gobiodon spp. also functions as a corallivore repellent. This study demonstrates that Gobiodon spp. exhibit resource defence against coral-feeding butterflyfishes and also that coral colonies without resident Gobiodon suffer higher predation rates. Although the genus Gobiodon is probably a facultative corallivore, this study shows that by reducing predation on inhabited colonies by other fishes, these obligate coral-dwellers either compensate for their own fitness-decreasing impact on host colonies or live in a mutualistic association with them.

Persistence of self-recruitment and patterns of larval connectivity in a marine protected area network.

The use of marine protected area (MPA) networks to sustain fisheries and conserve biodiversity is predicated on two critical yet rarely tested assumptions. Individual MPAs must produce sufficient larvae that settle within that reserve's boundaries to maintain local populations while simultaneously supplying larvae to other MPA nodes in the network that might otherwise suffer local extinction. Here, we use genetic parentage analysis to demonstrate that patterns of self-recruitment of two reef fishes (Amphiprion percula and Chaetodon vagabundus) in an MPA in Kimbe Bay, Papua New Guinea, were remarkably consistent over several years. However, dispersal from this reserve to two other nodes in an MPA network varied between species and through time. The stability of our estimates of self-recruitment suggests that even small MPAs may be self-sustaining. However, our results caution against applying optimization strategies to MPA network design without accounting for variable connectivity among species and over time.